NRAS Codon Research Articles

Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8×10−4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches.

Prognostic impact of mutation profiling in patients with stage II and III colon cancer.

Development of colorectal cancer (CRC) associates with accumulation of genetic mutations include the epidermal growth factor receptor (EGFR) signaling pathway. However, whether mutations in KRAS together with downstream factors BRAF, PIK3CA and NRAS impact prognosis is still unclear for stage II-III colon cancer. In the present study a total of 228 stage II-III colon cancer samples were retrospectively collected, KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons 12, 13 and 61) status was detected by Sanger sequencing, 37.89% (86/227) tumors harbored a KRAS mutation, 7.02% (16/228) harbored a BRAF mutation, 13.18% (29/220) harbored a PIK3CA mutation and 0.89% (2/224) harbored a NRAS mutation. NRAS mutations existed only in stage II colon cancer. Older groups harbored a higher KRAS and BRAF mutation (P < 0.05), PIK3CA (exon9) mutations appeared more common in worse differentiation tumors (P = 0.032). Moreover, PIK3CA (E545K) mutation was significantly associated with tumor recurrence (P = 0.031) and acted independently prognostic for poor OS (P = 0.044), while only in stage III colon cancer. KRAS, BRAF and NRAS mutations do not have major prognostic value in stage II and III colon cancer, subtypes of gene mutations should be further investigated for a better understanding in CRC.

Immunohistochemical detection of NRASQ61R protein in follicular-patterned thyroid tumors

The NRAS(A182G) mutation, which results in the NRAS(Q61R) protein, is a major driver mutation in follicular-patterned thyroid neoplasms. Although new immunohistochemistry (IHC) for NRAS(Q61R) is now available, its sensitivity, specificity, and diagnostic utility for thyroid tumors are not yet established. We performed IHC for NRAS(Q61R) and direct sequencing for NRAS codon 61 in 4 thyroid cancer-derived cell lines and 98 follicular-patterned thyroid tumors that included 22 follicular thyroid adenomas (FTAs), 35 follicular thyroid carcinomas (FTCs), and 41 cases of nodular hyperplasia (NH). In the tumors with NRAS(Q61R), the expression of BRAF(V600E) was further evaluated immunohistochemically. Two cell lines with NRAS(A182G) showed selective immunoreactivity for NRAS(Q61R). In tumor tissues, NRAS(Q61R) IHC was positive in 18% (4/22), 29% (10/35), and 2% (1/41) of FTAs, FTCs, and NH samples, respectively. The frequencies of the NRAS(Q61R) in FTAs and FTCs were significantly higher than that in NH (P=.046 and P=.001, respectively). All tumors with NRAS(Q61R) expression exhibited uniform cytoplasmic positivity with or without accumulation in their cell membranes. Of the 15 tumors with NRAS(Q61R) expression, 13 cases showed NRAS(A182G) in direct sequencing, whereas all of the tumors without NRAS(Q61R) expression were negative for the mutation. There were no tumors with overlapping expression of NRAS(Q61R) and BRAF(V600E). In reference to the direct sequencing, sensitivity and specificity of the NRAS(Q61R) IHC were 100% and 98%, respectively. In conclusion, NRAS(Q61R) IHC is a highly sensitive and specific tool that is useful for differentiating follicular-patterned thyroid tumors.

Abstract IA20: Targeting hyperactive Ras signaling in acute leukemia

Abstract Ras proteins are molecular switches that cycle between active guanosine triphosphate (GTP) and inactive guanosine diphosphate (GDP)-bound conformations (Ras-GTP and Ras-GDP). Guanine nucleotide exchange factors (GNEFs) stimulate nucleotide exchange on Ras, resulting in increased Ras-GTP levels. Upon GTP binding, the switch I and switch II domains of Ras undergo a conformational change that allows them to interact productively with downstream effectors including Raf, phosphatidylinositol 3-kinase (PI3K), and Ral-GDS to activate kinase signaling cascades that regulate cell proliferation, differentiation, and survival. Mutational, biochemical, and cell biologic studies of human cancers and experiments in mouse models strongly implicate deregulated signaling through the PI3K/Akt/mTOR and Raf/MEK/ERK cascades in cancer initiation and maintenance. Ras-GTP is hydrolyzed to Ras-GDP through an intrinsic GTPase activity. This slow “off” reaction is greatly augmented by GTPase activating proteins (GAPs). Thus, the competing activities of GNEFs and GAPs regulate Ras-GTP levels in vivo. Neurofibromin, the protein encoded by NF1, and p120 GAP are the predominant Ras GAPs in mammalian cells. Codons 12, 13, and 61 of RAS genes are the most common targets of dominant mutations in human cancer, with NRAS codon 12 mutations predominating in AML. Substitutions in these residues result in constitutively elevated levels of Ras-GTP due to reduced intrinsic GTP hydrolysis and resistance to GAPs. RAS and NF1 mutations occur in pediatric patients with a spectrum of hematologic malignancies including juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL). Because oncogenic Ras proteins are exceedingly challenging biochemical targets, most recent drug discovery efforts have focused on inhibiting downstream signaling molecules such as Raf, MEK, and Akt with many compounds. We have engineered and exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human acute leukemias and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable acute leuekmias characterized by hyperactive Ras signaling. This system provides a novel in vivo forward genetic strategy for introducing cooperating mutations and for generating clonal heterogeneity. Leukemia cells can be manipulated ex vivo and transplanted into congenic recipients. This experimental flexibility provides an opportunity to establish cohorts of mice that are engrafted with the same primary cancers for conducting preclinical testing and for investigating mechanisms of intrinsic and acquired drug resistance. I will discuss biologic and preclinical therapeutic studies in which we treated primary Nras, Kras, and Nf1 mutant AMLs characterized by hyperactive Ras signaling with targeted agents in vivo, isolated drug resistant clones at relapse, and compared drug sensitive and resistant clones to discover genes that modulate resistance. This latter strategy represents a potent and unbiased strategy to address mechanisms of resistance to kinase inhibitors, which has emerged as a major clinical problem in cancer therapeutics, and for investigating therapeutic strategies that might be effective in human cancers in which oncogenic RAS is a driver mutation. Our results in these robust AML models support testing drug combinations that include Raf/MEK/ERK pathway inhibitors and other targeted and conventional agents. Citation Format: Kevin M. Shannon. Targeting hyperactive Ras signaling in acute leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA20.

Diffuse sclerosing variant of papillary thyroid carcinoma: major genetic alterations and prognostic implications.

Diffuse sclerosing variant of papillary thyroid carcinoma (DSV-PTC) is an uncommon variant of PTC, and its prognostic significance remains controversial. The aim of this study was to investigate the major genetic alterations of DSV-PTC and their prognostic implications. We included 37 patients with DSV-PTC who underwent thyroid surgery and had formalin-fixed paraffin-embedded samples. We tested for a panel of genetic alterations, including BRAF(V) (600E) , NRAS codon 61, HRAS codon 12/13/61 and KRAS codon 12/13 point mutations as well as RET/PTC1, RET/PTC3 and PAX8/PPARγ rearrangements using reverse transcription real-time polymerase chain reaction (PCR). All genetic alterations found on PCR were confirmed by Sanger sequencing. Associations between the identified genetic alterations and clinicopathological characteristics were evaluated. Among 37 cases of DSV-PTC, 17 were positive for RET/PTC1 (46%), six for RET/PTC3 (16%) and nine for BRAF(V) (600E) (24%). All mutations/rearrangements were mutually exclusive. The remaining five cases had none of the above genetic alterations. DSV-PTC with RET/PTC3 rearrangement was associated with advanced-stage disease, including T4 and distant metastasis (P < 0.05). Patients with RET/PTC3 showed a higher frequency of persistent disease (P < 0.01). In contrast, DSV-PTC with RET/PTC1 was associated with a higher prevalence of disease remission (P < 0.05) and coexistent Hashimoto's thyroiditis (P < 0.01). Taken together, RET/PTC rearrangement was the major genetic alteration seen in patients with DSV-PTC, and the RET/PTC3 rearrangement was associated with advanced stage at diagnosis and poor clinical outcome.

Genetic Alterations in Differentiated Thyroid Cancer Patients with Acromegaly.

Acromegaly is associated with increased thyroid cancer risk. We aimed to analyze the frequency of point mutations of BRAF and RAS genes, and RET/PTC, PAX8/PPARγ gene rearrangements in patients with acromegaly having differentiated thyroid cancers (DTC) and their relation with clinical and histological features. 14 acromegalic patients (8 male, 6 female) with DTC were included. BRAF V600E and NRAS codon 61 point mutations, RET/PTC1, RET/PTC3, and PAX8/PPARγ gene rearrangements were analyzed in thyroidectomy specimens. We selected 14 non-acromegalic patients with DTC as a control group. 2 patients (14.3%) were detected to have positive BRAF V600E and 3 patients (21.4%) were detected to have NRAS codon 61 mutation. NRAS codon 61 was the most frequent genetic alteration. Patients with positive mutation had aggressive histologic features more frequently than patients without mutations. Comparison of the acromegalic and non-acromegalic patients with DTC revealed that BRAF V600E mutation was more frequent in non-acromegalic patients with DTC (14.2% vs. 64.3%, p=0.02). RET/PTC 1/ 3, PAX8/PPARγ gene rearrangements were not detected in any patient. None of the patients including the patients with positive point mutations had recurrence, and local and/or distant metastasis. NRAS codon 61 is the most frequent genetic alteration in this acromegaly series with DTC. Since acromegalic patients have lower prevalance of BRAF V600E mutation, BRAF V600E mutation may not be a causative factor in development of DTC in acromegaly. Despite the relation of BRAF V600E and NRAS codon 61 mutations with aggresive histopathologic features, their impact on tumor prognosis remains to be defined in acromegaly in further studies.

BRAF codons 594 and 596 mutations identify a new molecular subtype of metastatic colorectal cancer at favorable prognosis

BackgroundWhile the negative prognostic role of BRAF V600E mutation in metastatic colorectal cancer (mCRC) is well established, the impact of BRAF codons 594 and 596 mutations, occurring in <1% of CRCs, is completely unknown. The present work aims to describe clinical, pathological and molecular features and prognosis of BRAF codons 594 and 596 mutant mCRCs, compared with BRAF V600E mutant and wild-type ones. Patients and methodsPatients treated for mCRC at three Italian Institutions between October 2006 and October 2014, with available KRAS and NRAS codon 12, 13, 59, 61, 117 and 146 and BRAF codon 594, 596 and 600 mutational status, as detected by means of direct sequencing or matrix assisted laser desorption ionization time-of-flight MassArray, were included. ResultsTen patients bearing BRAF codons 594 or 596 mutated tumors were identified and compared with 77 and 542 patients bearing BRAF V600E mutated and BRAF wild-type tumors, respectively. While BRAF V600E mutated tumors were more frequently right-sided, mucinous and with peritoneal spread, BRAF 594 or 596 mutated were more frequently rectal, nonmucinous and with no peritoneal spread. All BRAF 594 or 596 mutated tumors were microsatellite stable. Patients with BRAF codons 594 or 596 mutated tumors had markedly longer overall survival (OS) when compared with BRAF V600E mutated [median OS: 62.0 versus 12.6 months; hazard ratio: 0.36 (95% confidence interval 0.20–0.64), P = 0.002], both at univariate and multivariate analyses. ConclusionsBRAF codon 594 or 596 mutated mCRCs are different from BRAF V600E ones in terms of molecular features, pathological characteristics and clinical outcome. This is consistent with preclinical evidences of a kinase inactivating effect of these mutations. The role of CRAF in transducing the intracellular signal downstream BRAF 594 or 596 mutated proteins opens the way to further preclinical investigation.

Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔTm (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5–3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity—all of the qualities required for clinical genotyping.

Acute Inhibition of MEK Suppresses Congenital Melanocytic Nevus Syndrome in a Murine Model Driven by Activated NRAS and Wnt Signaling

Congenital melanocytic nevus (CMN) syndrome is the association of pigmented melanocytic nevi with extra-cutaneous features, classically melanotic cells within the central nervous system, most frequently caused by a mutation of NRAS codon 61. This condition is currently untreatable and carries a significant risk of melanoma within the skin, brain, or leptomeninges. We have previously proposed a key role for Wnt signaling in the formation of melanocytic nevi, suggesting that activated Wnt signaling may be synergistic with activated NRAS in the pathogenesis of CMN syndrome. Some familial pre-disposition suggests a germ-line contribution to CMN syndrome, as does variability of neurological phenotypes in individuals with similar cutaneous phenotypes. Accordingly, we performed exome sequencing of germ-line DNA from patients with CMN to reveal rare or undescribed Wnt-signaling alterations. A murine model harboring activated NRASQ61K and Wnt signaling in melanocytes exhibited striking features of CMN syndrome, in particular neurological involvement. In the first model of treatment for this condition, these congenital, and previously assumed permanent, features were profoundly suppressed by acute post-natal treatment with a MEK inhibitor. These data suggest that activated NRAS and aberrant Wnt signaling conspire to drive CMN syndrome. Post-natal MEK inhibition is a potential candidate therapy for patients with this debilitating condition.

RAS Mutations in AUS/FLUS Cytology: Does it Have an Additional Role in BRAFV600E Mutation-Negative Nodules?

The object of this study is to evaluate the additional role of RAS mutation in detecting thyroid malignancy among BRAFV600E mutation-negative nodules diagnosed as atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) on cytology.From December 2009 to December 2011, 202 BRAFV600E mutation-negative thyroid nodules diagnosed as AUS/FLUS cytology in 201 patients were included in this study. RAS mutation analysis was performed using residual material from ultrasonography-guided fine needle aspiration (US-FNA) cytology testing for K-RAS, N-RAS, and H-RAS codons 12/13 and 61 point mutations. The authors evaluated the association between RAS mutation status and cytopathologic characteristics.Of the 202 BRAFV600E mutation-negative thyroid nodules with AUS/FLUS cytology, 4 were considered insufficient for mutation analysis. Of the 198 thyroid nodules, 148 (74.7%) were confirmed as benign and 50 (25.3%) as malignant. Thirty-one (15.7%) of the 198 thyroid nodules were positive for any RAS mutation, 4 positive for K-RAS 12/13, 26 for N-RAS 61, and 1 positive for H-RAS 61. Seven (22.6%) of the RAS mutation positive nodules were malignant, 1 with K-RAS 12/13, 6 with N-RAS 61. Twenty-four (77.4%) of the 31 nodules positive for K-RAS 12/13 (N = 3), N-RAS 61 (N = 20), or H-RAS 61 (N = 1) mutations were proven benign. None of the 198 thyroid nodules were positive for K-RAS 61, N-RAS 12/13, or H-RAS 12/13 mutations.N-RAS 61 mutation is the most common mutation detected among BRAFV600E mutation-negative nodules with AUS/FLUS cytology. RAS mutation has limited value in predicting malignancy among BRAFV600E mutation-negative thyroid nodules with AUS/FLUS cytology and further, investigation is anticipated to evaluate the true role of RAS mutation in thyroid malignancy.

Analysis of RAS mutation and PAX8/PPARγ rearrangements in follicular-derived thyroid neoplasms in a Korean population: frequency and ultrasound findings.

To investigate the frequency and ultrasonography (US) findings of RAS mutations and PAX8/PPARγ rearrangements between follicular thyroid adenomas (FTAs) and follicular thyroid carcinomas (FTCs) in a Korean population. RAS mutations and PAX8/PPARγ rearrangements in 56 FTAs and 35 FTCs were analyzed. We also analyzed the US findings of FTCs and FTAs. 16 nodules of 35 FTCs (45.7 %) and 19 nodules of 56 FTAs (33.9 %) harbored RAS mutations. Three FTCs and three FTAs showed two point mutations simultaneously. K-RAS codon 12-13 (n = 6, 31.6 %), N-RAS codon 61 (n = 5, 26.3 %), H-RAS codon 61 (n = 4, 21.1 %), K-RAS codon 61 (n = 3, 15.8 %), and N-RAS codon 12-13 (n = 1, 5.3 %) were found in FTCs, and N-RAS codon 61 (n = 10, 45 %), K-RAS codon 12-13 (n = 5, 22.7 %), H-RAS codon 61 (n = 5, 22.7 %), K-RAS codon 61 (n = 1, 4.5 %), and N-RAS codon 12-13 (n = 1, 4.5 %) were observed in FTAs. 4 of 56 (7.1 %) FTAs and 1 of 35 (2.9 %) FTCs represented PAX8/PPARγ rearrangements, respectively (P = 0.645). The absence of a hypoechoic rim (P = 0.021) and presence of calcifications (P = 0.049) were significantly associated with FTCs compared with FTAs. RAS mutation frequency targeting the Korean population showed a 45.7 % in FTCs and 35.7 % in FTAs, and PAX8/PPARγ rearrangements were more frequently showed in FTAs. K-RAS codon 12-13 was the most common RAS mutation in FTCs, whereas N-RAS codon 61 was more frequent in FTAs. The presence of calcifications and absence of a hypoechoic rim showed more frequently in FTCs.

FOLFOXIRI plus bevacizumab versus FOLFIRI plus bevacizumab as initial treatment for metastatic colorectal cancer (TRIBE study): Updated survival results and final molecular subgroups analyses.

3510 Background: The phase III TRIBE study met its primary endpoint by demonstrating that first-line FOLFOXIRI plus bev significantly prolongs PFS, as compared to FOLFIRI plus bev (Loupakis et al, N Eng J Med 2014). At a median follow-up of 32.2 months a preliminary OS analysis indicated a borderline OS improvement with FOLFOXIRI plus bev (HR = 0.79, p = 0.054) with a consistent effect across RAS (KRAS and NRAS codons 12, 13, 61) and BRAFV600E molecular subgroups. Methods: 508 pts were randomized to either FOLFIRI plus bev (Arm A, N = 256) or FOLFOXIRI plus bev (Arm B, N = 252). On available samples from RAS and BRAF wild-type (wt) pts (N = 129), also KRAS and NRAS codons 59, 117 and 146 were analysed by means of Sequenom MassArray, identifying a new “all wt” population (N = 93). Results: At a median follow-up of 48.1 months, in the ITT population, updated median OS for Arm B vs Arm A was 29.8 vs 25.8 months (HR = 0.80, 95%CI, 0.65-0.98, p = 0.030). Estimated 5-years OS rate were: Arm B, 24.9% vs Arm A, 1...

FOLFOX4 plus cetuximab treatment and RAS mutations in colorectal cancer

BackgroundThe OPUS study demonstrated that addition of cetuximab to 5-fluorouracil, folinic acid and oxaliplatin (FOLFOX4) significantly improved objective response and progression-free survival (PFS) in the first-line treatment of patients with KRAS exon 2 wild-type metastatic colorectal cancer (mCRC). In patients with KRAS exon 2 mutations, a detrimental effect was seen upon addition of cetuximab to FOLFOX4. The current study reports outcomes in subgroups defined by extended RAS testing. Patients and methodsSamples from OPUS study KRAS exon 2 wild-type tumours were reanalysed for other RAS mutations in four additional KRAS codons (exons 3–4) and six NRAS codons (exons 2–4) using BEAMing. A cutoff of ⩾5% mutant/wild-type sequences was selected to define RAS status; we also report an analysis using a cutoff based on the technical lower limit for mutation identification (0.1%). ResultsOther RAS mutations were detected in 31/118 (26%) evaluable patients. In the extended analysis of RAS wild-type tumours (n=87), objective response was significantly improved by addition of cetuximab to FOLFOX4 (58% versus 29%; odds ratio 3.33 [95% confidence interval 1.36–8.17]; P=0.0084); although limited by population size, there also appeared to be trends favouring the cetuximab arm in terms of PFS and overall survival in the RAS wild-type group compared with the RAS evaluable group. There was no evidence that patients with other RAS mutations benefited from cetuximab, but small numbers precluded precise estimations of treatment effects. In the combined population of patients with any RAS mutation (KRAS exon 2 or other RAS), a clear detrimental effect was associated with addition of cetuximab to FOLFOX4. ConclusionPatients with RAS-mutant mCRC, as defined by mutations in KRAS and NRAS exons 2–4, derive no benefit and may be harmed by the addition of cetuximab to FOLFOX4. Restricting cetuximab administration to patients with RAS wild-type tumours will further tailor therapy to maximise benefit.

Enhanced FHL2 and TGF-β1 Expression Is Associated With Invasive Growth and Poor Survival in Malignant Melanomas

This study examines the expression and the role of four-and-a-half LIM domains protein 2 (FHL2) and transforming growth factor β1 (TGF-β1) in human malignant melanoma. It is determined whether both proteins influence melanoma survival time. We analyzed the immunohistochemical staining intensities of FHL2 and TGF-β1 in normal skin and in 50 malignant melanomas with different mutation status (BRAF-V600E, NRAS codon 61 mutation, and wild type). Survival data were available for 45 cases. In melanocytes of nonneoplastic human skin, FHL2 expression was absent. In contrast, 38 (76%) of 50 melanomas showed strong cytoplasmic and partly nuclear FHL2 expression. At the invasion front, cytoplasmic TGF-β1 staining was observed in 32 (64%) of 50 melanomas, and a correlation of FHL2 and TGF-β1 staining intensities was detectable. In follow-up analyses, enhanced FHL2 and TGF-β1 staining intensities in the tumor invasion front were associated with poor survival. Enhanced FHL2 and TGF-β1 expression is correlated with poor survival in human malignant melanoma. Protumorigenic effects of autocrine TGF-β1 secretion might be exerted by induction of FHL2 expression in melanoma cells. Since melanomas treated with targeted therapies often do not show sufficient response rates, inhibition of FHL2 and/or TGF-β1 might be a promising therapeutic approach.

The nationwide genomic screening project for gastrointestinal cancer in Japan (GI-SCREEN): Simultaneous identification of KRAS, NRAS, BRAF, and PIK3CA mutation in advanced colorectal cancer (aCRC) (GI-SCREEN 2013-01).

578 Background: Recent studies confirmed that minor KRAS or NRAS mutations are associated with the resistance to anti-EGFR therapy for advanced colorectal cancer (aCRC). Although the impact of BRAF or PIK3CA mutation on efficacy of anti-EGFR therapy is still controversial, targeting agents for these mutations are under developing. Efficient screening systems for these relatively minor mutations with short turnaround time are necessary for the successful development of targeted therapies. Methods: This study was initiated in February 2014 as one of new nationwide genomic screening projects for advanced gastrointestinal cancer patients in Japan. Patients with aCRC who are planned to receive systemic chemotherapy were eligible. A total of 36 mutations of KRAS codon 61, 146, NRAS codon 12, 13, 61, BRAF codon 600, PIK3CA codon 542, 545, 546 and 1047 in genomic DNA of cancer cells were simultaneously analyzed at a quality-controlled central laboratory using Luminex (xMAP) technology in a single reaction using 50 ng of DNA. Results: As of August 31, 2014, this study is ongoing with the participation of 16 major cancer centers in Japan. A total of 437 aCRC patients were enrolled to this study and 361 tumor samples has been analyzed with success rate for genomic analysis of 100%. Among the 237 patients with KRAS exon 2 wild type, 28 patients (11.8%) had other RAS mutations and 15 patients (6.3%) had BRAF mutation. Twenty-seven of 437 patients had PIK3CA mutations (6.2%). One patient with BRAF mutation was enrolled in early clinical trials of BRAF inhibitors in combination with anti-EGFR antibody and most of other patients are still treated with standard chemotherapies, which may become future candidates for early clinical trials. Conclusions: From our preliminary results, this nationwide screening system enabled to detect rare mutations using limited amounts of samples from aCRC, which may facilitate the enrollment of patients in IND registration trials for targeted therapies as well as optimal individualized treatment.

Preoperative RAS mutational analysis is of great value in predicting follicular variant of papillary thyroid carcinoma.

Follicular variant of papillary thyroid carcinoma (FVPTC), particularly the encapsulated subtype, often causes a diagnostic dilemma. We reconfirmed the molecular profiles in a large number of FVPTCs and investigated the efficacy of the preoperative mutational analysis in indeterminate thyroid nodules. BRAF V600E/K601E and RAS mutational analysis was performed on 187 FVPTCs. Of these, 132 (70.6%) had a point mutation in one of the BRAF V600E (n = 57), BRAF K601E (n = 11), or RAS (n = 64) genes. All mutations were mutually exclusive. The most common RAS mutations were at NRAS codon 61. FNA aspirates from 564 indeterminate nodules were prospectively tested for BRAF and RAS mutation and the surgical outcome was correlated with the mutational status. Fifty-seven and 47 cases were positive for BRAF and RAS mutation, respectively. Twenty-seven RAS-positive patients underwent surgery and all except one patient had FVPTC. The PPV and accuracy of RAS mutational analysis for predicting FVPTC were 96% and 84%, respectively. BRAF or RAS mutations were present in more than two-thirds of FVPTCs and these were mutually exclusive. BRAF mutational analysis followed by N, H, and KRAS codon 61 mutational analysis in indeterminate thyroid nodules would streamline the management of patients with malignancies, mostly FVPTC.

Whole exome sequencing identifies a recurrent RQCD1 P131L mutation in cutaneous melanoma.

Melanoma is often caused by mutations due to exposure to ultraviolet radiation. This study reports a recurrent somatic C > T change causing a P131L mutation in the RQCD1 (Required for Cell Differentiation1 Homolog) gene identified through whole exome sequencing of 20 metastatic melanomas. Screening in 715 additional primary melanomas revealed a prevalence of ~4%. This represents the first reported recurrent mutation in a member of the CCR4-NOT complex in cancer. Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations. There was no association with nodal disease (p = 0.3). Mutually exclusive mutations of other members of the CCR4-NOT complex were found in ~20% of the TCGA melanoma dataset suggesting the complex may play an important role in melanoma biology. Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes. From thirteen patients with mutant RQCD1, an anti-tumor CD8⁺ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.

Association of MicroRNA-31-5p with Clinical Efficacy of Anti-EGFR Therapy in Patients with Metastatic Colorectal Cancer.

Gene mutations in the pathway downstream of epidermal growth factor receptor (EGFR) are considered to induce resistance to anti-EGFR therapy in colorectal cancer (CRC). We recently reported that microRNA-31 (miR-31)-5p may regulate BRAF activation and play a role in the signaling pathway downstream of EGFR in CRC. Therefore, we hypothesized that miR-31-5p can be a useful biomarker for anti-EGFR therapy in CRC. We evaluated miR-31-5p expression and gene mutations [KRAS (codon 61 or 146), NRAS (codon 12, 13, or 61), and BRAF (V600E)] in the EGFR downstream pathway in 102 CRC patients harboring KRAS (codon 12 or 13) wild-type who were treated with anti-EGFR therapeutics. Progression-free survival (PFS) and overall survival (OS) were evaluated. KRAS (codon 61 or 146), NRAS, and BRAF mutations were detected in 6.9, 6.9, and 5.9% patients, respectively. Compared with CRCs with at least one mutation (n=20), significantly better PFS (P=0.0003) but insignificantly better OS were observed in CRCs harboring all wild-type genes (KRAS, NRAS, and BRAF). High miR-31-5p expression was identified in 11% (n=11) patients and was significantly associated with shorter PFS (P=0.003). In CRCs carrying all wild-type genes, high miR-31-5p was associated with shorter PFS (P=0.027). High miR-31-5p expression was associated with shorter PFS in patients with CRC treated with anti-EGFR therapeutics. Moreover, in CRCs carrying all wild-type genes, high miR-31-5p was associated with shorter PFS, suggesting that it may be a useful and additional prognostic biomarker for anti-EGFR therapy.

Frequent silencing of RASSF1A via promoter methylation in follicular thyroid hyperplasia: a potential early epigenetic susceptibility event in thyroid carcinogenesis.

Follicular thyroid hyperplasia (FTH) refers to enlargement of the thyroid gland due to cellular hyperplasia. It is frequently encountered in clinical practice in nontoxic uninodular or multinodular goiter. The genetic and epigenetic events associated with the origin and malignant potential of FTH are poorly understood. To analyze FTH samples for known recurrent genetic and epigenetic driver events in thyroid neoplasms such as activating mutations in proto-oncogenes BRAF and NRAS and promoter hypermethylation of tumor suppressor genes CDKN2A, PTEN, and RASSF1A. Clinical characteristics and thyroid specimens were prospectively obtained from 43 patients who underwent thyroid surgery at Yale-New Haven Hospital. Presence of BRAF(V600E) and NRAS codon 61 mutations were assessed in FTH. Methylation status of CDKN2A, PTEN, and RASSF1A gene promoters in FTH, follicular thyroid adenoma, and follicular thyroid carcinoma was quantified. Regulation of RASSF1A messenger RNA (mRNA) and protein expression and its potential neoplastic role in FTH were examined. An exploratory cohort of FTH (n = 10) was negative for BRAF(V600E) and NRAS codon 61 mutations. In contrast, epigenetic analysis displayed significant promoter hypermethylation of the tumor-suppressor gene RASSF1A in 6 FTH samples (60%) compared with their adjacent normal tissue (P = .01). The overall genome CpG methylation and promoter methylation of PTEN and CDKN2A were unaffected in the lesions. Further analysis of an expanded cohort of patients with FTH (n = 23), follicular thyroid adenoma (n = 10), and follicular thyroid carcinoma (n = 10) showed RASSF1A promoter hypermethylation in 14 (61%), 9 (90%), and 7 (70%), respectively (P < .001). The overall hypermethylation level in FTH showed a statistically significant inverse correlation with RASSF1A mRNA expression (P = .005). Immunohistochemistry demonstrated minimal or no protein expression in most FTH samples studied. To explore the potential neoplastic contribution of RASSF1A downregulation, we analyzed the expression pattern of thyroid proliferation markers Ki-67 and NF-κB in representative samples. Although Ki-67 expression was undetectable, similar to normal tissue, FTH samples expressed high levels of NF-κB, similar to the expression levels in thyroid tumors. We demonstrate silencing of tumor suppressor RASSF1A in a subset of FTH in the absence of other known thyroid cancer-associated genetic and epigenetic changes. Silencing of RASSF1A and concurrent NF-κB activation demonstrate that a subset of FTH shares epigenetic changes and downstream signaling events associated with malignant lesions, suggesting that FTH may have the potential to be a premalignant lesion.

Abstract 4009: N-ras codon 61 mutation is associated with distant metastasis in patients with follicular thyroid carcinoma

Abstract Distant metastasis is a main cause of death in follicular thyroid carcinoma (FTC) patients. Factors related to distant metastasis in FTC had been known as age, primary tumor size, and invasiveness. Ras mutations were also supposed to be associated with poor clinical outcomes. We analyzed Ras mutations in FTC with a distant metastasis (FTC M1, n=28), size matched-FTC without a distant metastasis (FTC M0, n=28), follicular adenoma (FA, n=17), and nodular hyperplasia (NH, n=12) to figure out the roles of Ras mutations in follicular thyroid carcinogenesis and metastasis. In addition, we assess the relationship between Ras mutations and clinical outcomes in FTC patients. NRAS, HRAS, and KRAS mutations were assessed using direct sequencing method. Among 85 patients, 39 patients (46%) had Ras mutations. NRAS codon 61 mutation (n=21; 25%) was the most common point mutation. HRAS codon 61, KRAS codon 12/13, and KRAS codon 61 mutations were found in 7, 6, and 4 patients, respectively. NRAS codon 12/13 mutation was found in only 1 patient, and HRAS codon 12/13 mutation was not found. Ras mutations were significantly more common in the FTC than FA or NH groups. Especially, NRAS codon 61 mutation was associated with distant metastasis in patients with FTC. However, there was no significant difference in survival between the Ras mutation positive-FTC and Ras mutation negative-FTC patients. Ras mutation, especially NRAS codon 61 mutation, was significantly associated with the presence of distant metastases. NRAS codon 61 mutation status might be a potential prognostic factor in FTC patients. Citation Format: MinJi Jeon, Eun Kyung Jang, Dong Eun Song, So Young Sim, Eui Young Kim, Yun Mi Choi, Ji Min Han, Won Gu Kim, Tae Yong Kim, Young Kee Shong, Won Bae Kim. N-ras codon 61 mutation is associated with distant metastasis in patients with follicular thyroid carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4009. doi:10.1158/1538-7445.AM2014-4009