Mutations In NRAS Genes Research Articles

Abstract 615: Noninvasive diagnosis of actionable mutations by deep sequencing of circulating tumor DNA in multiple myeloma

Abstract Background: Genome sequencing of multiple myeloma (MM) tumors has revealed recurrent mutations that serve as a fertile ground for targeted therapies. Indeed, activating mutations of KRAS, NRAS and BRAF have been reported in approximately 27%, 24% and 4% of MM cases. Based on this observation, we initiated a Phase II NCI-CTEP sponsored clinical trial of trametinib in patients with MM (PHL-9460). Detection of mutations currently require bone marrow aspirates that are invasive and can yield suboptimal samples. Targeted, ultra-deep sequencing of circulating tumor DNA (ctDNA) is a promising tool for accessing the tumor genome that has not been well studied in MM. We set out to determine the feasibility of detecting ctDNA in MM and of identifying actionable mutations and mutational load using liquid biopsy through ctDNA analysis. Methods: MM patients enrolled onto PHL-9460 or those with heavy tumor burden were identified and consented to have their peripheral blood drawn for analysis. Where possible, matched tumor DNA was also obtained. Cell-free DNA was extracted from 7-15 mL of plasma isolated within ¬1 hour of blood draw using the QIAamp Circulating Nucleic Acid Kit and tagged with barcoded sequencing adapters for subsequent pooling. All exons of KRAS, NRAS, BRAF, PIK3CA and EGFR genes were isolated using a custom hybrid capture panel (IDT xGen Lockdown) and sequenced on an Illumina HiSeq 2000. Reads were aligned to the human genome reference (hg19) using bwa and somatic mutations were detected using muTect. Results: We have collected 25 samples from 23 patients, 7 from patients on PHL-9460, 5 newly diagnosed, and 13 from relapsed patients having received 3.3 median prior lines of therapy (range 1-7). To date, 11 samples from 10 patients have been sequenced. The sample with the lowest DNA yield failed due to low library complexity (range 16.6-3872 ng, median yield: 197 ng). From the remaining 10 samples, the mean target coverage ranged from 31,500 to 32,500. Somatic mutations in KRAS, NRAS, or PIK3CA genes were present in 5 of 9 patients with mutant allele frequencies ranging from 1.1% to 32% (3 KRAS and 2 NRAS of which 2 cases also had a low frequency PIK3CA mutation). We did not uncover mutations in BRAF or EGFR. For patients with matched tumor DNA, mutations in ctDNA concurred with those found in tumor DNA sequencing (4 of 4 tumors with known genotypes). Two patients with NRAS mutations enrolled onto PHL-9460 have responded to trametinib (1 partial and 1 minor response) and remain on therapy. Conclusion: ctDNA analysis in this cohort has identified key mutations in MM. The rate of RAS or RAF mutations in ctDNA compared to matched tumors and correlation to response to trametinib is ongoing and will be presented. Preliminary data suggest that ctDNA may be a reliable method of detecting mutations in MM and an alternative to bone marrow biopsy. Citation Format: Rayan Kaedbey, Olena Kis, Arnavaz Danesh, Mark Dowar, Tiantian Li, Zhihua Li, Jessica Liu, Mark Mansour, Mahadeo Sukhai, Tong Zhang, Suzanne Kamel-Reid, Trevor J. Pugh, Suzanne Trudel. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating tumor DNA in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 615. doi:10.1158/1538-7445.AM2015-615

Molecular spectrum of BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias: implication for MEK-ERK pathway activation.

Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell (PC) malignancy. Whole-exome sequencing has identified therapeutically targetable mutations such as those in the mitogen-activated protein kinase (MAPK) pathway, which are the most prevalent MM mutations. We used deep sequencing to screen 167 representative patients with PC dyscrasias [132 with MM, 24 with primary PC leukemia (pPCL) and 11 with secondary PC leukemia (sPCL)] for mutations in BRAF, NRAS and KRAS, which were respectively found in 12%, 23.9% and 29.3% of cases. Overall, the MAPK pathway was affected in 57.5% of the patients (63.6% of those with sPCL, 59.8% of those with MM, and 41.7% of those with pPCL). The majority of BRAF variants were comparably expressed at transcript level. Additionally, gene expression profiling indicated the MAPK pathway is activated in mutated patients. Finally, we found that vemurafenib inhibition of BRAF activation in mutated U266 cells affected the expression of genes known to be associated with MM. Our data confirm and extend previous published evidence that MAPK pathway activation is recurrent in myeloma; the finding that it is mediated by BRAF mutations in a significant fraction of patients has potentially immediate clinical implications.

RAS status in Korean patients with stage III and IV colorectal cancer.

KRAS mutations are common and clearly contribute to malignant progression. The frequency of NRAS mutations and their relationship to clinical, pathologic, and molecular features remains unclear. We evaluated 130 colorectal tumors for mutations in KRAS and NRAS gene. We tested for mutations in codons 61 and 146 of KRAS and codons 12, 13, 59, 61 and 146 of NRAS. Mutation status was determined by targeted dideoxy sequencing. Among the analyzed primary tumors, 36.2% had KRAS mutation. Of the 83 KRAS codon 12 and 13 wild-type patients, 7.2% had KRAS codon 61, 146 or NRAS. 40.7% harbored any RAS mutation. The frequency of other RAS (NRAS and KRAS exon 3, 4) activating mutations in colorectal cancers is relatively low in Korean colorectal cancer patients.

Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.

Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in Colorectal Cancer.

Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies.

Mutations in the RAS and PI3K pathways are associated with metastatic location in colorectal cancers.

Identification of mutations in the downstream epidermal growth factor receptor (EGFR) signaling pathway could provide important insights of EGFR-targeted therapies in colorectal cancers. We analyzed the mutation spectra of the PI3K/PTEN/AKT and RAS/RAF/MAPK pathways in colorectal cancers and the associations of these mutations with sites of metastases or recurrence. The study population comprised 1,492 retrospectively collected stages I-IV colorectal cancer specimens. Tissue was obtained between 2000 and 2010 at a single hospital. We analyzed 61 hot spots using MALDI-TOF mass spectrometry for nucleic acid analysis. Mutations were found in the RAS pathway in 47.3% of patients and in the PI3K pathway in 14.3% of patients, with 9.2% of patients carrying mutations in both pathways. Both the RAS and PI3K pathway mutations were significantly associated with proximal tumors, mucinous tumors, and microsatellite instability. Tumors carrying a RAS pathway mutation exhibited a higher frequency of lung and peritoneal metastasis than did tumors with a wild-type gene (P = 0.025 and 0.009, respectively). NRAS gene mutation was significantly associated with lung metastasis (P = 0.001). Somatic mutations in the RAS pathway of the primary tumor in colorectal cancer can influence patterns of metastasis and recurrence.

Detection of KRAS,NRAS and BRAF gene mutations in colorectal carcinoma

To investigate mutations frequencies of KRAS,NRAS and BRAF genes in colorectal carcinoma. Tissue specimens from 200 colorectal cancer patients at diagnosis were collected and subject to KRAS,NRAS and BRAF mutation analyses by PCR-based direct DNA sequencing targeting exons 2, 3 and 4 of KRAS gene, exons 2, 3 and 4 of NRAS gene and exon 15 of BRAF gene. Activating mutations were detected in KRAS (44%, 88/200), NRAS (2%, 4/200) and BRAF (5%, 10/200) in this study cohort.Among KRAS mutations, 64.8% (57/88) occurred in codon 12 and 12.5% (11/88) occurred in codon 13. KRAS gene mutation in exon 3 mainly involved codons 59 and 61. KRAS gene mutation in exon 4 mainly involved codons 117 and 146. Mutations at exon 2 of KRAS gene have the highest frequency in colorectal carcinoma. Expanding the detection sites of KRAS gene combined with NRAS and BRAF genes may help to identify patients who will most likely benefit from targeted therapies.

RAS mutations vary between lesions in synchronous primary colorectal cancer: testing only one lesion is not sufficient to guide anti-EGFR treatment decisions.

Mutations in KRAS and NRAS genes are negative predictors of anti-EGFR therapies response in metastatic colorectal cancer. There are few reports on RAS testing in synchronous primary colorectal cancer (SP-CRC) and a lack of recommendations on which tissue should be tested for the mutation in this disease. This study analyzed the RAS status of both lesions in SP-CRC patients and in their metastasis. DNA was obtained from formalin-fixed-paraffin-embedded tissue, and mutations were analyzed by pyrosequencing. RAS status was heterogeneous in 6 (75%) of 8 SP-CRC patients between primary lesions. Five showed heterogeneity regarding RAS mutational status, and from these, four presented with metastasis: 3 cases (75%) had WT metastatic tissue, and 1 case (25%) had mutated metastatic tissue. One patient showed divergence regarding RAS mutation type. RAS mutations vary significantly between SP-CRC lesions, and the status of the metastasis is unpredictable. Testing for RAS mutations in only 1 of the primary lesions can misguide clinical decisions and hind the predictive potential of anti-EGFR treatment. A more appropriate approach in metastatic SP-CRC is to test the metastatic tissue or both primary lesions for providing more accurate mutation scenery and support more assertive clinical decisions.

Activating PIK3CA mutations coexist with BRAF or NRAS mutations in a limited fraction of melanomas.

BackgroundActivated PI3K-AKT pathway may contribute to decrease sensitivity to inhibitors of key pathogenetic effectors (mutated BRAF, active NRAS or MEK) in melanoma. Functional alterations are deeply involved in PI3K-AKT activation, with a minimal role reported for mutations in PIK3CA, the catalytic subunit of the PI3K gene. We here assessed the prevalence of the coexistence of BRAF/NRAS and PIK3CA mutations in a series of melanoma samples.MethodsA total of 245 tumor specimens (212 primary melanomas and 33 melanoma cell lines) was screened for mutations in BRAF, NRAS, and PIK3CA genes by automated direct sequencing.ResultsOverall, 110 (44.9%) samples carried mutations in BRAF, 26 (10.6%) in NRAS, and 24 (9.8%) in PIK3CA. All identified PIK3CA mutations have been reported to induce PI3K activation; those detected in cultured melanomas were investigated for their interference with the antiproliferative activity of the BRAF-mutant inhibitor vemurafenib. A reduced suppression in cell growth was observed in treated cells carrying both BRAF and PIK3CA mutations as compared with those presenting a mutated BRAF only. Among the analysed melanomas, 12/245 (4.9%) samples presented the coexistence of PIK3CA and BRAF/NRAS mutations.ConclusionsOur study further suggests that PIK3CA mutations account for a small fraction of PI3K pathway activation and have a limited impact in interfering with the BRAF/NRAS-driven growth in melanoma.

BRAF, KIT, NRAS, GNAQ and GNA11 mutation analysis in cutaneous melanomas in Turkish population.

KIT and mitogen-activated protein kinase cascade are important for melanomagenesis. In the present study, we analyzed the frequency of BRAF, NRAS, KIT, GNAQ and GNA11 gene mutations and investigated their association with clinicopathological features of melanomas in Turkish population. Forty-seven primary cutaneous melanomas were included in our study. Sanger sequencing method was used for mutation analysis in all cases. Mean age was 62.1 (29-101) years. Female:male ratio was 17:30. Among 47 melanomas, 14 (29.8%) BRAF, 10 (21.3%) NRAS, 4 (8.5%) KIT and 1(2.1%) GNAQ gene mutations were detected. Two of the KIT mutations were found in acral lentiginous melanoma (ALM). In the head and neck region, mutation frequency was significantly lower than in other locations (P = 0.035). The only GNAQ gene mutation (p.Q209L) was detected in a melanoma arising from blue nevus located on the scalp. None of the melanomas harbored NRAS exon 2, KIT exon 13/17/18, GNAQ exon 4 and GNA11 exon 4/5 mutations. Overall mutation frequency did not show significant difference between metastatic (8/14, 57.1%) and nonmetastatic (18/33, 54.5%) patients. We did not observe any significant association between mutation status and gender or age of various patients. Our results support that BRAF and NRAS gene mutations are common in cutaneous melanomas. The activating mutations of KIT gene are rare and especially seen in ALM. GNAQ and GNA11 mutations are infrequent in cutaneous melanomas and may be associated only with melanomas arising from blue nevus.

Spectrum of oncogene mutations is different in melanoma subtypes

Melanoma is the most lethal malignancy of skin, which is comprised of clinically relevant molecular subsets defined by specific "driver" mutations in BRAF, NRAS, and KIT genes. Recently, the better results in melanoma treatment were obtained with the mutation-specific inhibitors that have been developed for clinical use and target only patients with particular tumor genotypes. The aim of the study was to characterize the spectrum of "driver" mutations in melanoma subtypes from 137 patients with skin melanoma and 14 patients with mucosal melanoma. In total 151 melanoma cases, the frequency of BRAF, NRAS, KIT, PDGFRA, and KRAS mutations was 55.0, 10.6, 4.0, 0.7, and 0.7%, respectively. BRAF mutations were found in 69% of cutaneous melanoma without UV exposure and in 43% of cutaneous melanoma with chronic UV exposure (p=0.045), rarely in acral and mucosal melanomas. Most of melanomas containing BRAF mutations, V600E (92%) and V600K (6.0%) were potentially sensitive to inhibitors vemurafenib and dabrafenib. NRAS mutations were more common in cutaneous melanoma with chronic UV exposure (26.0%), in acral and mucosal melanomas; the dominant mutations being Q61R/K/L (87.5%). KIT mutations were found in cutaneous melanoma with chronic UV exposure (8.7%) and mucosal one (28.6%), but not in acral melanoma. Most of KIT mutations were identified in exon 11; these tumors being sensitive to tyrosine kinase inhibitors. This is the first monitoring of BRAF, NRAS, KIT, PDGFRA, and KRAS hotspot mutations in different subtypes of melanoma for Russian population. On the base of data obtained, one can suppose that at the molecular level melanomas are heterogeneous tumors that should be tested for "driver" mutations in the each case for evaluation of the potential sensitivity to target therapy. The obtained results were used for treatment of melanoma patients.

Setting up a wide panel of patient-derived tumor xenografts of non-small cell lung cancer by improving the preanalytical steps.

With the ongoing need to improve therapy for non–small cell lung cancer (NSCLC) there has been increasing interest in developing reliable preclinical models to test novel therapeutics. Patient-derived tumor xenografts (PDX) are considered to be interesting candidates. However, the establishment of such model systems requires highly specialized research facilities and introduces logistic challenges. We aimed to establish an extensive well-characterized panel of NSCLC xenograft models in the context of a long-distance research network after careful control of the preanalytical steps. One hundred fresh surgically resected NSCLC specimens were shipped in survival medium at room temperature from a hospital-integrated biobank to animal facilities. Within 24 h post-surgery, tumor fragments were subcutaneously xenografted into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical, aCGH and next-generation sequencing approaches. For this model system, the tumor take rate was 35%, with higher rates for squamous carcinoma (60%) than for adenocarcinoma (13%). Patients for whom PDX tumors were obtained had a significantly shorter disease-free survival (DFS) compared to patients for whom no PDX tumors (P = 0.039) were obtained. We established a large panel of PDX NSCLC models with a high frequency of mutations (29%) in EGFR, KRAS, NRAS, MEK1, BRAF, PTEN, and PI3KCA genes and with gene amplification (20%) of c-MET and FGFR1. This new patient-derived NSCLC xenograft collection, established regardless of the considerable time required and the distance between the clinic and the animal facilities, recapitulated the histopathology and molecular diversity of NSCLC and provides stable and reliable preclinical models for human lung cancer research.

Abstract PR09: Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes

Abstract Mouse models have proven their utility in establishing causal roles and addressing genetic interactions of candidate alterations in tumorigenesis. Previously, our cross-species comparison of human and mouse melanoma genomes led to the identification of NEDD9 (Neural precursor cell Expressed, Developmentally Down-regulated 9) as a novel melanoma metastasis gene that is up-regulated in 35~50% of metastatic melanomas. We have shown that NEDD9 enhances proliferation and drives invasion in vitro and lung seeding in vivo of melanoma cells. In this study, to explore the biological roles of NEDD9 in melanoma tumorigenesis, we have generated tet-inducible NEDD9 mouse model that carries a doxycycline-responsive, melanocyte-targeted NEDD9. Two independent transgenic founder lines (line A and D) were obtained and genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes have been tested. Melanoma displays frequent activation of NRAS and BRAF and inactivation of CDKN2A (INK4A/ARF) and PTEN. First, we asked whether NEDD9 cooperates with RAS/RAF/MAPK pathway activation by crossing Nedd9 alleles onto the non-metastatic Tyr-RAS* Ink4a/Arf-/- melanoma model to establish a colony of compound mutant mice designated as “iNEDD9” (e.g. Tyr-HRAS*;Tyr-rtTA/Tet-NEDD9;Ink4a/Arf-/-). These mice developed spontaneous cutaneous nodular type melanomas and ocular melanomas indistinguishable from the ones developed in Tyr-RAS* Ink4a/Arf-/- model. In these mice, NEDD9 expression in melanocytes via doxycycline administration led to decreased tumor latency (Median latency: 88 (NEDD9 ON) vs. 120 (NEDD9 OFF) days (p=0.041, log-rank test)) and increased tumor initiation without signs of metastasis. Median melanoma-free survival of iNEDD9 mice was 111 (line A, p=0.009, log-rank test) and 114.5 (line D, p=0.032) days, lower than 166 days observed in control mice without NEDD9. This data supports the cooperation of NEDD9 with RAS/RAF/MAPK pathway activation in melanoma tumorigenesis. Secondly, we addressed whether NEDD9 can synergize with loss of PTEN and INK4a/ARF in vitro and in vivo. We observed that NEDD9 cooperated with PTEN loss in Ink4a/Arf-/- melanocytes without activating RAS or RAF mutation and enhanced proliferation, anchorage independent growth, and invasion in vitro and tumor growth in nude mice. NEDD9 expression correlated with increased phosphorylation of SFKs (Src family kinases), AKT2, and STAT3. To analyze the biological consequence of NEDD9 expression in vivo, we generated a mouse model based on loss of PTEN and CDKN2A (INK4A/ARF) with or without NEDD9 up-regulation designated as “NIP” (e.g. Tyr-CreERT2;Tyr-rtTA/Tet-NEDD9;cInk4a/ArfL/L;cPtenL/L). These mice developed cutaneous and ocular melanomas following tamoxifen treatment, which are under characterization. This study will generate a body of knowledge for the in vivo roles of NEDD9 in melanoma tumorigenesis and invaluable melanoma models driven by genetic lesions observed in human melanoma patients. Since most of the current mouse melanoma models are driven by activating mutations in NRAS or BRAF genes, our model may provide a novel opportunity to understand melanomas without these mutations. This abstract is also presented as Poster B04. Citation Format: Hyeran Sung, Jong Woo Lee, Gretchen Alicea, Ji-Hyun Lee, Jane Messina, Scott Granter, Lynda Chin, Minjung Kim. Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR09.

Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells

Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma.

Low Incidence of Oncogenic EGFR, HRAS, and KRAS Mutations in Seborrheic Keratosis

Seborrheic keratosis (SK) represents a frequent epidermal skin tumor. Although lacking a malignant potential, these tumors reveal multiple oncogenic mutations. A previous study identified activating mutations in 89% of SK, particularly in FGFR3 and PIK3CA genes. The aim of this study was to identify further oncogenic mutations in human SK. Therefore, we screened for mutations in EGFR, FGFR2, PIK3R1, HRAS, KRAS, and NRAS genes using both Sanger sequencing of selected exons and a multiplex SNaPshot assay in 58 SK of 14 patients. We identified a somatic EGFR p.L858R mutation in 1 SK. Furthermore, the HRAS mutations p.G13R (2/58 SK) and p.Q61L (2/58 SK) were found. These mutations have not been described in human SK yet. In addition, 1 SK revealed the KRAS p.G12V mutation, which has already been reported in SK. No mutations were detected in FGFR2, PIK3R1, and NRAS genes. The results of this study suggest that activating mutations of EGFR, HRAS, and KRAS contribute to the pathogenesis of human SK, although at a lower frequency than FGFR3 and PIK3CA mutations. FGFR2, PIK3R1, and NRAS mutations obviously do not have a significant role in the development of SK.

Mutation analysis of key genes in RAS/RAF and PI3K/PTEN pathways in Chinese patients with hepatocellular carcinoma.

The RAS/RAF and PI3K/PTEN signaling pathways play central roles in hepatocarcinogenesis. KRAS, NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN are key cancer-related genes in the RAS/RAF and PI3K/PTEN signaling pathways. Genetic alterations in these genes often lead to the dysregulation of the two cascades. Little is known regarding the frequency of hotspot mutations in these critical components among Chinese patients with hepatocellular carcinoma (HCC). In the current study, 57 somatic hotspot mutations in 36 HCCs samples collected from Chinese patients using direct DNA sequencing method were examined. Two cases of KRAS somatic mutations (KRAS codon 61; Gln to His) were identified among 36 HCCs (5.6%). However, no mutations were found in the NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN genes. These findings indicated that point mutations in the KRAS gene, but not mutations in NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN genes, at a somatic level contribute to the abnormal activation of the RAS/RAF and PI3K/PTEN pathways in HCC.

TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA.

The telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T have been found in many malignancies, including in thyroid carcinomas. However, it is unclear how early these mutations occur in thyroid tumorigenesis. The study included primary tumors from 58 patients initially diagnosed with follicular thyroid adenoma (FTA), a benign entity, 18 with atypical FTA (AFTA) having an uncertain malignant potential, and 52 with follicular thyroid carcinoma (FTC). Sanger sequencing was used to investigate the mutational status of the TERT promoter. Telomere length and TERT messenger RNA (mRNA) expression were determined using quantitative polymerase chain reaction (PCR). Telomerase activity was assessed using a Telomerase PCR enzyme-linked immunosorbent assay kit. The C228T mutation was identified in 1 of 58 FTA (2%) and 3 of 18 AFTA (17%) samples. These 4 tumors all expressed TERT mRNA and telomerase activity, whereas the majority of C228T-negative adenomas lacked TERT expression (C228T versus wild-type, P = .008). The C228T mutation was associated with NRAS gene mutations (P = .016). The patient with C228T-mutated FTA later developed a scar recurrence and died of FTC, whereas none of the remaining 57 patients with FTA had recurrence. No recurrence occurred in 3 patients with AFTA who carried C228T during the follow-up period (36-285 months). Nine of the 52 FTCs (17%) exhibited the TERT mutation (8 of 9 C228T and 1 of 9 C250T), and the presence of the mutation was associated with shorter patient survival. TERT promoter mutations may occur as an early genetic event in thyroid follicular tumors that have not developed malignant features on routine histopathological workup.

Evolutionary trajectories of hyperdiploid ALL in monozygotic twins.

Identical twins have provided unique insights on timing or sequence of genetic events in acute lymphoblastic leukaemia (ALL). To date, this has mainly focused on ALL with MLL or ETV6-RUNX1 fusions, with hyperdiploid ALL remaining less well characterised. We examined three pairs of monozygotic twins, two concordant and one discordant for hyperdiploid ALL, for single-nucleotide polymorphism (SNP)-defined copy number alterations (CNAs), IGH/L plus TCR gene rearrangements and mutations in NRAS, KRAS, FLT3 and PTPN11 genes. We performed whole exome sequencing in one concordant twin pair. Potential 'driver' CNAs were low, 0-3 per case, and all were different within a pair. One patient had an NRAS mutation that was lacking from leukaemic cells of the twin sibling. By exome sequencing, there were 12 nonsynonymous mutations found in one twin and 5 in the other, one of which in SCL44A2 was shared or identical. Concordant pairs had some identical IGH/L and TCR rearrangements. In the twin pair with discordant hyperdiploid ALL, the healthy co-twin had persistent low level hyperdiploid CD19+ cells that lacked a CNA present in the ALL cells of her sibling. From these data, we propose that hyperdiploid ALL arises in a pre-B cell in utero and mutational changes necessary for clinical ALL accumulate subclonally and postnatally.

Evolution of heterogeneous mechanisms of acquired resistance to cetuximab-based therapy in colorectal cancer.

3526 Background: Anti-EGFR drug resistance is a major challenge in metastatic colorectal cancer (mCRC). The aim of this study was to elucidate molecular mechanisms of acquired resistance in mCRC patients treated with cetuximab-based therapy. Methods: 37 mCRC patients with acquired resistance to cetuximab-based therapy with paired tumor samples (prior and at progression to treatment) were prospectively included. In three patients, multiple biopsies were obtained over the course of the disease. Mutations in EGFR, KRAS, NRAS, BRAF and PIK3CA genes were evaluated by next-generation pyrosequencing. Gene amplification was evaluated by FISH. Mutations were also assessed in plasma in 7 representative cases. Results: Eighty-one percent of patients harbored a molecular event (comprising mutations and amplifications) in post-treatment biopsies. In one third of patients, multiple molecular events (range 2-5) coexisted in the same sample. RAS mutations were the most frequent event (40% of patients) and mostly affected...

Compound HRAS/PIK3CA Mutations in Chinese Patients with Alveolar Rhabdomyosarcomas

The rhabdomyosarcoma (RMS) is the most common type of soft tissue tumor in children and adolescents; yet only a few screens for oncogenic mutations have been conducted for RMS. To identify novel mutations and potential therapeutic targets, we conducted a high-throughput Sequenom mass spectrometry-based analysis of 238 known mutations in 19 oncogenes in 17 primary formalin-fixed paraffin-embedded RMS tissue samples and two RMS cell lines. Mutations were detected in 31.6% (6 of 19) of the RMS specimens. Specifically, mutations in the NRAS gene were found in 27.3% (3 of 11) of embryonal RMS cases, while mutations in NRAS, HRAS, and PIK3CA genes were identified in 37.5% (3 of 8) of alveolar RMS (ARMS) cases; moreover, PIK3CA mutations were found in 25% (2 of 8) of ARMS specimens. The results demonstrate that tumor profiling in archival tissue samples is a useful tool for identifying diagnostic markers and potential therapeutic targets and suggests that these HRAS/ PIK3CA mutations play a critical role in the genesis of RMS.