Most melanocytic nevi develop on sun-exposed skin during childhood and adolescence and commonly harbor BRAF mutations or, less frequently, NRAS mutations. A small subset of nevi is present at birth, and therefore must develop independently of UV light. To assess whether these nevi have a different mutation spectrum than those that develop on sun-exposed skin, we determined the BRAF and NRAS mutation frequencies in 32 truly congenital nevi. We found no BRAF mutations, but 81% (26/32) harbored mutations in NRAS. Consistently, seven of 10 (70%) proliferating nodules that developed early in life in congenital nevi showed mutations in NRAS. A separate set of nevi that displayed histological features frequently found in nevi present at birth ("congenital pattern nevi") but lacked a definitive history of presence at birth showed an inverse mutation pattern with common BRAF mutations (20/28 or 71%) and less frequent NRAS mutations (7/28 or 25%). Thus, nevi that develop in utero are genetically distinct from those that develop later, and histopathologic criteria alone are unable to reliably distinguish the two groups. The results are consistent with the finding in melanoma that BRAF mutations are uncommon in neoplasms that develop in the absence of sun-exposure.

BACKGROUND

Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors.

RESULTS

We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations.

CONCLUSIONS

Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Therapeutic antibodies blocking programmed death-1 and its ligand (PD-1/PD-L1) induce durable responses in a substantial fraction of melanoma patients. We sought to determine whether the number and/or type of mutations identified using a next-generation sequencing (NGS) panel available in the clinic was correlated with response to anti-PD-1 in melanoma. Using archival melanoma samples from anti-PD-1/PD-L1-treated patients, we performed hybrid capture-based NGS on 236-315 genes and T-cell receptor (TCR) sequencing on initial and validation cohorts from two centers. Patients who responded to anti-PD-1/PD-L1 had higher mutational loads in an initial cohort (median, 45.6 vs. 3.9 mutations/MB; P = 0.003) and a validation cohort (37.1 vs. 12.8 mutations/MB; P = 0.002) compared with nonresponders. Response rate, progression-free survival, and overall survival were superior in the high, compared with intermediate and low, mutation load groups. Melanomas with NF1 mutations harbored high mutational loads (median, 62.7 mutations/MB) and high response rates (74%), whereas BRAF/NRAS/NF1 wild-type melanomas had a lower mutational load. In these archival samples, TCR clonality did not predict response. Mutation numbers in the 315 genes in the NGS platform strongly correlated with those detected by whole-exome sequencing in The Cancer Genome Atlas samples, but was not associated with survival. In conclusion, mutational load, as determined by an NGS platform available in the clinic, effectively stratified patients by likelihood of response. This approach may provide a clinically feasible predictor of response to anti-PD-1/PD-L1. Cancer Immunol Res; 4(11); 959-67. ©2016 AACR.

Recent evidence highlights the potential prognostic and predictive value of BRAF and K-RAS gene alterations in patients with colorectal cancer. However, a comprehensive evaluation of BRAF and K-RAS mutations and their specific clinicopathological features, histomorphological presentation and effect on protein expression have not been systematically analyzed. The aim of this study was to characterize the clinicopathological, histomorphological and protein expression profiles of BRAF- and K-RAS-mutated colorectal cancers and determine their impact on patient survival. Molecular analysis for microsatellite instability (MSI), K-RAS and BRAF was carried out on paraffin-embedded samples from 404 patients with primary colorectal cancer. Using tissue microarrays, 36 tumor-associated and 14 lymphocyte/inflammatory-associated markers were evaluated by immunohistochemistry. BRAF mutation was associated with right-sided tumor location (p < 0.001), higher tumor grade (p = 0.029), absence of peritumoral lymphocytic inflammation (p = 0.026) and MSI-H (p < 0.001). In right-sided tumors, loss of CDX2 expression was observed in 23 of 24 cases (95.8%). BRAF mutation was a poor prognostic indicator in patients with right-sided disease (p = 0.01). This result was maintained in multivariable analysis (p < 0.001; HR = 2.82; 95% CI: 1.5-5.5) with pT, pN and vascular invasion and independent of CDX2 expression. K-RAS mutation, in contrast, was not associated with any of the features analyzed. BRAF gene mutation is an adverse prognostic factor in right-sided colon cancer patients independent of MSI status and, moreover, in patients with lymph node-negative disease. These results indicate that molecular analysis for BRAF may be a useful biomarker for identifying patients with right-sided colon cancer with poor outcome who may benefit from a more individualized course of therapy.

OBJECTIVE

Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAF(V600E)) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAF(V600E) in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAF(V600E) and wild-type BRAF MA.

METHODS

BRAF(V600E) mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAF(V600E) MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition in vitro and in vivo.

RESULTS

BRAF(V600E) mutations were identified in 11 and 10% of MAs from two distinct series of tumors (six of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAF(V600E) was identified in four instances. Using the BRAF(V600E)-specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAF(V600E) mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we show that PLX4720 treatment decreases tumor growth and increases overall survival in mice-bearing BRAF(V600E) mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF.

CONCLUSIONS

Our results indicate a 10% incidence of activating BRAF(V600E) among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAF(V600E)-specific inhibitors for treating BRAF(V600E) MA patients.

Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition.

OBJECTIVE

Categorization of colon cancers into distinct subtypes using a combination of pathway-based biomarkers could provide insight into stage-independent variability in outcomes.

METHODS

We used a polymerase chain reaction-based assay to detect mutations in BRAF (V600E) and in KRAS in 2720 stage III cancer samples, collected prospectively from patients participating in an adjuvant chemotherapy trial (NCCTG N0147). Tumors deficient or proficient in DNA mismatch repair (MMR) were identified based on detection of MLH1, MSH2, and MSH6 proteins and methylation of the MLH1 promoter. Findings were validated using tumor samples from a separate set of patients with stage III cancer (n = 783). Association with 5-year disease-free survival was evaluated using Cox proportional hazards models.

RESULTS

Tumors were categorized into 5 subtypes based on MMR status and detection of BRAF or KRAS mutations which were mutually exclusive. Three subtypes were MMR proficient: those with mutations in BRAF (6.9% of samples), mutations in KRAS (35%), or tumors lacking either BRAF or KRAS mutations (49%). Two subtypes were MMR deficient: the sporadic type (6.8%) with BRAF mutation and/or or hypermethylation of MLH1 and the familial type (2.6%), which lacked BRAF(V600E) or hypermethylation of MLH1. A higher percentage of MMR-proficient tumors with BRAF(V600E) were proximal (76%), high-grade (44%), N2 stage (59%), and detected in women (59%), compared with MMR-proficient tumors without BRAF(V600E) or KRAS mutations (33%, 19%, 41%, and 42%, respectively; all P < .0001). A significantly lower proportion of patients with MMR-proficient tumors with mutant BRAF (hazard ratio = 1.43; 95% confidence interval: 1.11-1.85; Padjusted = .0065) or mutant KRAS (hazard ratio = 1.48; 95% confidence interval: 1.27-1.74; Padjusted < .0001) survived disease-free for 5 years compared with patients whose MMR-proficient tumors lacked mutations in either gene. Disease-free survival rates of patients with MMR-deficient sporadic or familial subtypes was similar to those of patients with MMR-proficient tumors without BRAF or KRAS mutations. The observed differences in survival rates of patients with different tumor subtypes were validated in an independent cohort.

CONCLUSIONS

We identified subtypes of stage III colon cancer, based on detection of mutations in BRAF (V600E) or KRAS, and MMR status that show differences in clinical and pathologic features and disease-free survival. Patients with MMR-proficient tumors and BRAF or KRAS mutations had statistically shorter survival times than patients whose tumors lacked these mutations. The tumor subtype found in nearly half of the study cohort (MMR-proficient without BRAF(V600E) or KRAS mutations) had similar outcomes to those of patients with MMR-deficient cancers.

We report a comprehensive molecular characterization of pheochromocytomas and paragangliomas (PCCs/PGLs), a rare tumor type. Multi-platform integration revealed that PCCs/PGLs are driven by diverse alterations affecting multiple genes and pathways. Pathogenic germline mutations occurred in eight PCC/PGL susceptibility genes. We identified CSDE1 as a somatically mutated driver gene, complementing four known drivers (HRAS, RET, EPAS1, and NF1). We also discovered fusion genes in PCCs/PGLs, involving MAML3, BRAF, NGFR, and NF1. Integrated analysis classified PCCs/PGLs into four molecularly defined groups: a kinase signaling subtype, a pseudohypoxia subtype, a Wnt-altered subtype, driven by MAML3 and CSDE1, and a cortical admixture subtype. Correlates of metastatic PCCs/PGLs included the MAML3 fusion gene. This integrated molecular characterization provides a comprehensive foundation for developing PCC/PGL precision medicine.

For more than three decades, postoperative chemotherapy-initially fluoropyrimidines and more recently combinations with oxaliplatin-has reduced the risk of tumor recurrence and improved survival for patients with resected colon cancer. Although universally recommended for patients with stage III disease, there is no consensus about the survival benefit of postoperative chemotherapy in stage II colon cancer. The most recent adjuvant clinical trials have not shown any value for adding targeted agents, namely bevacizumab and cetuximab, to standard chemotherapies in stage III disease, despite improved outcomes in the metastatic setting. However, biomarker analyses of multiple studies strongly support the feasibility of refining risk stratification in colon cancer by factoring in molecular characteristics with pathologic tumor staging. In stage II disease, for example, microsatellite instability supports observation after surgery. Furthermore, the value of BRAF or KRAS mutations as additional risk factors in stage III disease is greater when microsatellite status and tumor location are taken into account. Validated predictive markers of adjuvant chemotherapy benefit for stage II or III colon cancer are lacking, but intensive research is ongoing. Recent advances in understanding the biologic hallmarks and drivers of early-stage disease as well as the micrometastatic environment are expected to translate into therapeutic strategies tailored to select patients. This review focuses on the pathologic, molecular, and gene expression characterizations of early-stage colon cancer; new insights into prognostication; and emerging predictive biomarkers that could ultimately help define the optimal adjuvant treatments for patients in routine clinical practice.

Most gastrointestinal stromal tumors (GISTs) are driven by KIT or PDGFRA-activating mutations, but a small subset is associated with loss of function of the succinate dehydrogenase (SDH) complex of mitochondrial inner membrane proteins. This occurs by germline mutations of the SDH subunit genes and hitherto unknown mechanisms. SDH-deficient GISTs especially include pediatric GISTs and those associated with Carney triad (CT) or Carney-Stratakis syndromes (CSSs); the latter 2 also include paraganglioma as a component. SDH-deficient GISTs were identified in this study on the basis of immunohistochemical loss of succinate dehydrogenase subunit B (SDHB), which signals functional loss of the SDH complex. We found 66 SDH-deficient GISTs among 756 gastric GISTs, with an estimated frequency of 7.5% of unselected cases. Nearly, all gastric GISTs in patients <20 years, and a substantial percentage of those in patients <40 years, but only rare GISTs in older adults were SDH deficient. There was a female predominance of over 2:1. Two patients each had either pulmonary chondroma or paraganglioma (CT), but none of the examined cases had SDH germline mutations (CSS) or somatic KIT/PDGFRA or BRAF mutations. SDH-deficient GISTs were often multiple and typically showed plexiform muscularis propria involvement and epithelioid hypercellular morphology. They were consistently KIT-positive and DOG1/Ano 1-positive and almost always smooth muscle actin negative. Tumor size and mitotic activity varied, and the tumors were somewhat unpredictable with low mitotic rates developing metastases. Gastric recurrences occurred in 11 patients, and peritoneal and liver metastases occurred in 8 and 10 patients, respectively. Lymph node metastases were detected in 5 patients, but lymphovascular invasion was present in >50% of cases studied; these 2 were not related to adverse outcome. Seven patients died of disease, but many had long survivals, even with peritoneal or liver metastases. All 378 nongastric GISTs and 34 gastric non-GIST mesenchymal tumors were SDHB positive. SDH-deficient GISTs constitute a small subgroup of gastric GISTs; they usually occur in children and young adults, often have a chronic course similar to that of pediatric and CT GISTs, and have potential association with paraganglioma, necessitating long-term follow-up.

BRAF is a serine/threonine kinase that receives a mitogenic signal from RAS and transmits it to the MAP kinase pathway. Recent studies have reported that mutations of the BRAF gene were detected with varying frequencies in several cancers, notably more than 60% in melanoma. We analysed mutations of BRAF and RAS genes in 100 cases of thyroid carcinoma to investigate genetic aberrations in the RAS/RAF/MEK/MAP kinase pathway. BRAF mutations were detected exclusively in papillary carcinomas (40 in 76 cases: 53%), and were exclusively V599E, a mutation frequently observed in other carcinomas. NRAS mutation was observed in six cases (6%), all in histological types other than papillary carcinoma, and was exclusively Q61R. No mutations were found in KRAS or HRAS. Our results suggest that BRAF mutations may play a critical role in the carcinogenesis of papillary carcinoma of the thyroid.

OBJECTIVE

Brain metastases are frequent in patients with metastatic melanoma, indicating poor prognosis. We investigated the BRAF kinase inhibitor vemurafenib in patients with advanced melanoma with symptomatic brain metastases.

METHODS

This open-label trial assessed vemurafenib (960mg twice a day) in patients with BRAF(V600) mutation-positive metastatic melanoma with non-resectable, previously treated brain metastases. The primary end-point was safety. Secondary end-points included best overall response rate, and progression-free and overall survival.

RESULTS

Twenty-four patients received vemurafenib for a median treatment duration of 3.8 (0.1-11.3) months. The majority of discontinuations were due to disease progression (n=22). Twenty-three of 24 patients reported at least one adverse event (AE). Grade 3 AEs were reported in four (17%; 95% confidence interval [CI], 4.7-37.4%) patients and included cutaneous squamous cell carcinoma in four patients. Median progression-free survival was 3.9 (95% CI, 3.0-5.5) months, and median survival was 5.3 (95% CI, 3.9-6.6) months. An overall partial response (PR) at both intracranial and extracranial sites was achieved in 10 of 24 (42%; 95% CI, 22.1-63.4) evaluable patients, with stable disease in nine (38%; 95% CI, 18.8-59.4) patients. Of 19 patients with measurable intracranial disease, seven (37%) achieved >30% intracranial tumour regression, and three (16%; 95% CI, 3.4-39.6%) achieved a confirmed PR. Other signs of improvement included reduced need for corticosteroids and enhanced performance status.

CONCLUSIONS

Vemurafenib can be safely used in patients with advanced symptomatic melanoma that has metastasised to the brain and can result in meaningful tumour regression.

OBJECTIVE

In part A, the aim was to define the maximum tolerated dose (MTD) of the hydrogen sulfate (Hyd-Sulfate) oral capsule formulation of the mitogen-activated protein kinase kinase inhibitor AZD6244 (ARRY-142886). In part B, the aim was to compare the pharmacokinetic profile of the new Hyd-Sulfate capsule with the initial AZD6244 free-base suspension and further characterize the pharmacodynamic profile and efficacy of the new formulation.

METHODS

In part A, 30 patients received escalating doses of AZD6244 Hyd-Sulfate twice daily. In part B, 29 patients were randomized to a single dose of the Hyd-Sulfate capsule or free-base suspension, followed by a washout, then a single dose of the alternative formulation. Patients received the Hyd-Sulfate capsule twice daily at MTD of part A thereafter.

RESULTS

The MTD of the Hyd-Sulfate capsule was 75 mg twice daily. Dose limiting toxicities were Common Terminology Criteria for Adverse Events grade 3 acneiform rash and pleural effusion. Fatigue (65.7%) and acneiform dermatitis (60.0%) were the most frequent adverse events at the MTD. Based on area under curve(0-24), exposure of the 75 mg Hyd-Sulfate capsule relative to the 100 mg free-base suspension was 197% (90% confidence interval, 161-242%). Pharmacodynamic analysis showed that inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced extracellular signal-regulated kinase phosphorylation in peripheral blood lymphocytes was related to plasma concentrations of AZD6244, with an estimated IC(50) of 352 ng/mL and maximum inhibition (E(max)) of approximately 91%, showing target inhibition. A patient with metastatic melanoma bearing a V600E BRAF mutation achieved a complete response persisting after 15 months of therapy.

CONCLUSIONS

The AZD6244 Hyd-Sulfate capsule formulation has shown a favorable toxicity, pharmacokinetic, and pharmacodynamic profile, and is being taken forward in ongoing clinical trials.

OBJECTIVE

To assess the immunologic effects of dabrafenib and trametinib in vitro and to test whether trametinib potentiates or antagonizes the activity of immunomodulatory antibodies in vivo.

METHODS

Immune effects of dabrafenib and trametinib were evaluated in human CD4(+) and CD8(+) T cells from healthy volunteers, a panel of human tumor cell lines, and in vivo using a CT26 mouse model.

RESULTS

Dabrafenib enhanced pERK expression levels and did not suppress human CD4(+) or CD8(+) T-cell function. Trametinib reduced pERK levels, and resulted in partial/transient inhibition of T-cell proliferation/expression of a cytokine and immunomodulatory gene subset, which is context dependent. Trametinib effects were partially offset by adding dabrafenib. Dabrafenib and trametinib in BRAF V600E/K, and trametinib in BRAF wild-type tumor cells induced apoptosis markers, upregulated HLA molecule expression, and downregulated certain immunosuppressive factors such as PD-L1, IL1, IL8, NT5E, and VEGFA. PD-L1 expression in tumor cells was upregulated after acquiring resistance to BRAF inhibition in vitro. Combinations of trametinib with immunomodulators targeting PD-1, PD-L1, or CTLA-4 in a CT26 model were more efficacious than any single agent. The combination of trametinib with anti-PD-1 increased tumor-infiltrating CD8(+) T cells in CT26 tumors. Concurrent or phased sequential treatment, defined as trametinib lead-in followed by trametinib plus anti-PD-1 antibody, demonstrated superior efficacy compared with anti-PD-1 antibody followed by anti-PD-1 plus trametinib.

CONCLUSIONS

These findings support the potential for synergy between targeted therapies dabrafenib and trametinib and immunomodulatory antibodies. Clinical exploration of such combination regimens is under way.

OBJECTIVE

The purpose of this study was to examine the prognostic and oxaliplatin predictive value of mismatch repair (MMR) status and common hot spot mutations, which we previously identified in stage II and III colon cancer.

METHODS

Mutations in BRAF, KRAS, NRAS, MET, and PIK3CA were profiled in 2,299 stage II and III colon tumors from National Surgical Adjuvant Breast and Bowel Project (NSABP) clinical trials C-07 (n = 1,836) and C-08 (n = 463) with Type Plex chemistry and mass spectrometry. C-07 tested the worth of adding oxaliplatin to 5-fluorouracil plus leucovorin, and C-08 tested the worth of adding bevacizumab to FOLFOX. Cox proportional hazard models were used to assess prognostic or oxaliplatin predictive value of mutations for tumor recurrence, overall survival (OS), and survival after recurrence (SAR).

RESULTS

BRAF mutations were associated with MMR-deficient tumors (P < 0.0001), poor OS [HR, 1.46; 95% confidence interval (CI), 1.20-1.79; P ≤ 0.0002], and poor SAR (HR, 2.31; 95% CI, 1.83-2.95; P < 0.0001). Mutations in KRAS, NRAS, MET, and PIK3CA were not associated with recurrence, OS, or SAR. MMR-deficient tumors were associated with an improved prognosis based on recurrence (HR, 0.48; 95% CI, 0.33-0.70; P < 0.0001). Mutations and MMR status were not predictive for oxaliplatin benefit.

CONCLUSIONS

This study shows that BRAF mutations profiled from stage II and III colon cancer tumors were associated with poor SAR and validates and explains, at least in part, previous observations associating it with poor OS. Profiling of all of these mutations is warranted for future clinical trials testing new targeted therapies that block relevant signaling pathways. Such clinical trials are under development at NSABP.

The classification of endometrial carcinomas is based on pathological assessment of tumour cell type; the different cell types (endometrioid, serous, carcinosarcoma, mixed, undifferentiated, and clear cell) are associated with distinct molecular alterations. This current classification system for high-grade subtypes, in particular the distinction between high-grade endometrioid (EEC-3) and serous carcinomas (ESC), is limited in its reproducibility and prognostic abilities. Therefore, a search for specific molecular classifiers to improve endometrial carcinoma subclassification is warranted. We performed target enrichment sequencing on 393 endometrial carcinomas from two large cohorts, sequencing exons from the following nine genes: ARID1A, PPP2R1A, PTEN, PIK3CA, KRAS, CTNNB1, TP53, BRAF, and PPP2R5C. Based on this gene panel, each endometrial carcinoma subtype shows a distinct mutation profile. EEC-3s have significantly different frequencies of PTEN and TP53 mutations when compared to low-grade endometrioid carcinomas. ESCs and EEC-3s are distinct subtypes with significantly different frequencies of mutations in PTEN, ARID1A, PPP2R1A, TP53, and CTNNB1. From the mutation profiles, we were able to identify subtype outliers, ie cases diagnosed morphologically as one subtype but with a mutation profile suggestive of a different subtype. Careful review of these diagnostically challenging cases suggested that the original morphological classification was incorrect in most instances. The molecular profile of carcinosarcomas suggests two distinct mutation profiles for these tumours: endometrioid-type (PTEN, PIK3CA, ARID1A, KRAS mutations) and serous-type (TP53 and PPP2R1A mutations). While this nine-gene panel does not allow for a purely molecularly based classification of endometrial carcinoma, it may prove useful as an adjunct to morphological classification and serve as an aid in the classification of problematic cases. If used in practice, it may lead to improved diagnostic reproducibility and may also serve to stratify patients for targeted therapeutics.

OBJECTIVE

Recent studies have revealed that the majority of pediatric low-grade astrocytomas (PLGA) harbor the BRAF-KIAA1549 (B-K) fusion gene resulting in constitutive activation of the RAS/MAPK pathway. However, the clinical significance of this genetic alteration is yet to be determined. We aimed to test the prognostic role of the B-K fusion in progression of incompletely resected PLGA.

METHODS

We retrospectively identified 70 consecutive patients with incompletely resected "clinically relevant" PLGA. We added 76 tumors diagnosed at our institution between 1985 and 2010 as controls. We examined BRAF alterations by reverse transcriptase PCR, FISH, and single-nucleotide polymorphism array analysis and correlated that with progression-free survival (PFS).

RESULTS

Overall, 60% of tumors were B-K fusion positive. All patients with B-K fused PLGA are still alive. Five-year PFS was 61% ± 8% and 18% ± 8% for fusion positive and negative patients, respectively (P = 0.0004). B-K fusion resulted in similarly significant favorable PFS for patients who received chemotherapy. Multivariate analysis revealed that B-K fusion was the most significant favorable prognostic factor in incompletely resected PLGA and was independent of location, pathology, and age. In vitro, BRAF overexpression resulted in growth arrest associated with DNA damage (γH2AX expression). Five-year PFS was 68% ± 15% and 0% for patients with B-K fused and γH2AX-expressing PLGA versus negative tumors (P = 0.001).

CONCLUSIONS

These data suggest that B-K fusion confers a less aggressive clinical phenotype on PLGA and may explain their tendency to growth arrest. Combined analysis of B-K fusion and γH2AX expression can determine prognosis and may be a powerful tool to tailor therapy for these patients.

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAF(V600E)/PTEN(null)-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer.

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.

Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS/RAF/MEK/ERK/MAP-kinase pathway. Activating mutations in BRAF have recently been identified in melanomas, colorectal cancers, and thyroid and ovarian tumours. In the present study, an extensive characterization of BRAF and KRAS mutations has been performed in 264 epithelial and non-epithelial ovarian neoplasms. The epithelial tumours ranged from adenomas and borderline neoplasms to invasive carcinomas including serous, mucinous, clear cell, and endometrioid lesions. It is shown that BRAF mutations in ovarian tumours occur exclusively in low-grade serous neoplasms (33 of 91, 36%); these included serous borderline tumours (typical and micropapillary variants), an invasive micropapillary carcinoma and a psammocarcinoma. KRAS mutations were identified in 26 of 91 (29.5%) low-grade serous tumours, 7 of 49 (12%) high-grade serous carcinomas, 2 of 6 mucinous adenomas, 22 of 28 mucinous borderline tumours, and 10 of 18 mucinous carcinomas. Of note, two serous borderline tumours were found to harbour both BRAF and KRAS mutations. The finding that at least 60% of serous borderline tumours harbour mutations in two members of the ERK-MAP-kinase pathway (BRAF 36%, KRAS 30%) compared with 12% of high-grade serous carcinomas (BRAF 0%, KRAS 12%) indicates that the majority of serous borderline tumours do not progress to serous carcinomas. Furthermore, no BRAF mutations were detected in the other 173 ovarian tumours in this study.

BACKGROUND

Molecular characterization of circulating tumor cells (CTCs) is pivotal to increasing the diagnostic specificity of CTC assays and investigating therapeutic targets and their downstream pathways on CTCs. We focused on epidermal growth factor receptor (EGFR) and genes relevant for its inhibition in patients with colorectal cancer (CRC).

METHODS

We used the CellSearch® system for CTC detection in peripheral blood samples from 49 patients with metastatic CRC (mCRC) and 32 patients with nonmetastatic CRC (nmCRC). We assessed EGFR expression in 741 CTCs from 27 patients with mCRC and 6 patients with nmCRC using a fluorescein-conjugated antibody with the CellSearch Epithelial Cell Kit. DNA of a single CTC isolated by micromanipulation was propagated by whole-genome amplification and analyzed by quantitative PCR for EGFR gene amplification and sequencing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B1), and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) mutations.

RESULTS

At least 2 CTCs were detected in 24 of 49 patients with mCRC and 7 of 32 patients with nmCRC. In 7 of 33 patients, CTCs with increased EGFR expression were identified. Heterogeneity in EGFR expression was observed between CTCs from the same patient. EGFR gene amplification was found in 7 of 26 CTCs from 3 patients. The investigated BRAF gene locus was not mutated in 44 analyzed CTCs, whereas KRAS mutations were detected in 5 of 15 CTCs from 1 patient and PIK3CA mutations in 14 of 36 CTCs from 4 patients.

CONCLUSIONS

Molecular characterization of single CTCs demonstrated considerable intra- and interpatient heterogeneity of EGFR expression and genetic alterations in EGFR, KRAS, and PIK3CA, possibly explaining the variable response rates to EGFR inhibition in patients with CRC.

Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) encodes the p110α subunit of the mitogenic signaling protein phosphoinositide 3-kinase (PI3K). PIK3CA mutations in the helical binding domain and the catalytic subunit of the protein have been associated with tumorigenesis and treatment resistance in various malignancies. Characteristics of patients with PIK3CA-mutant lung adenocarcinomas have not been reported. We examined epidermal growth factor receptor (EGFR), Kirsten rate sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), human epidermal growth factor receptor 2 (HER2), PIK3CA, v-akt murine thymoma vial oncogene homolog 1 (AKT1), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen-activated protein kinase kinase 1 (MEK1), and anaplastic lymphoma kinase (ALK) in patients with adenocarcinoma of the lung to identify driver mutations. Clinical data were obtained from the medical records of individuals with mutations in PIK3CA. Twenty-three of 1,125 (2%, 95% CI: 1-3) patients had a mutation in PIK3CA, 12 in exon 9 (10 E545K and 2 E542K), and 11 in exon 20 (3 H1047L and 8 H1047R). The patients (57% women) had a median age of 66 at diagnosis (range: 34-78). Eight patients (35%) were never smokers. Sixteen of 23 (70%, 95% CI: 49-86) had coexisting mutations in other oncogenes-10 KRAS, 1 MEK1, 1 BRAF, 1 ALK rearrangement, and 3 EGFR exon 19 deletions. We conclude that PIK3CA mutations occur in lung adenocarcinomas, usually concurrently with EGFR, KRAS, and ALK. The impact of PIK3CA mutations on the efficacy of targeted therapies such as erlotinib and crizotinib is unknown. Given the high frequency of overlapping mutations, comprehensive genotyping should be carried out on tumor specimens from patients enrolling in clinical trials of PI3K and other targeted therapies.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic.