Q61L Mutation Research Articles

Path Sampling Simulations Reveal How the Q61L Mutation Alters the Dynamics of KRas.

Flexibility is essential for many proteins to function, but can be difficult to characterize. Experiments lack resolution in space and time, while the time scales involved are prohibitively long for straightforward molecular dynamics simulations. In this work, we present a multiple state transition path sampling simulation study of a protein that has been notoriously difficult to characterize in its active state. The GTPase enzyme KRas is a signal transduction protein in pathways for cell differentiation, growth, and division. When active, KRas tightly binds guanosine triphosphate (GTP) in a rigid state. The protein-GTP complex can also visit more flexible states, in which it is not active. KRas mutations can affect the conversion between these rigid and flexible states, thus prolonging the activation of signal transduction pathways, which may result in tumor formation. In this work, we apply path sampling simulations to investigate the dynamic behavior of KRas-4B (wild type, WT) and the oncogenic mutant Q61L (Q61L). Our results show that KRas visits several conformational states, which are the same for WT and Q61L. The multiple state transition path sampling (MSTPS) method samples transitions between the different states in a single calculation. Tracking which transitions occur shows large differences between WT and Q61L. The MSTPS results further reveal that for Q61L, a route to a more flexible state is inaccessible, thus shifting the equilibrium to more rigid states. The methodology presented here enables a detailed characterization of protein flexibility on time scales not accessible with brute-force molecular dynamics simulations.

HRAS Q61L Mutation as a Possible Target for Non-Small Cell Lung Cancer: Case Series and Review of Literature

Introduction: Assessment of actionable gene mutations and oncogene fusions have made a paradigm shift in treatment strategies of non-small cell lung cancer (NSCLC). HRAS mutations involved around 0.2–0.8% of NSCLC patients, mostly on codon 61. For these patients, few data are available regarding clinical characteristics and response to therapies. Methods: Next-Generation Sequencing (NGS) done routinely at Nantes University Hospital was used to identify HRAS molecular alterations in NSCLC patients. We identified and described four HRAS p.GlnQ61Leu mutated patients. Literature of previously HRAS-mutant NSCLC cases was reviewed, and available data in solid tumour with the most advanced H-Ras specific inhibitor, tipifarnib, were presented. Results: Of 1614 patients diagnosed with advanced NSCLC from January 2018 to December 2020, four (0.25%) had HRAS p.Gln61Leu mutation. Three of them died during the first-line systemic therapy. Furthermore, three additional cases were identified in literature. All cases were current or former smokers, most of them had pleural or pericardial effusion at diagnosis. Conclusions: The clinical course of patients with HRAS-mutant NSCLC remains unclear. Furthers cases should be identified in order to clarify prognosis and response to therapies. Tipifarnib, a farnesyl transferase inhibitor, is a promising candidate to target HRAS-mutant tumours and should be explored in NSCLC patients.

NMR 1H, 13C, 15N backbone resonance assignments of the T35S and oncogenic T35S/Q61L mutants of human KRAS4b in the active, GppNHp-bound conformation

RAS proteins cycling between the active-form (GTP-bound) and inactive-form (GDP-bound) play a key role in cell signaling pathways that control cell survival, proliferation, and differentiation. Mutations at codon 12, 13, and 61 in RAS are known to attenuate its GTPase activity favoring the RAS active state and constitutively active downstream signaling. This hyperactivation accounts for various malignancies including pancreatic, lung, and colorectal cancers. Active KRAS is found to exist in equilibrium between two rapidly interconverting conformational states (State1–State2) in solution. Due to this dynamic feature of the protein, the 1H–15N correlation cross-peak signals of several amino acid (AA) residues of KRAS belonging to the flexible loop regions are absent from its 2D 1H–15N HSQC spectrum within and near physiological solution pH. A threonine to serine mutation at position 35 (T35S) shifts the interconverting equilibrium to State1 conformation and enables the emergence of such residues in the 2D 1H–15N HSQC spectrum due to gained conformational rigidity. We report here the 1HN, 15N, and 13C backbone resonance assignments for the 19.2 kDa (AA 1–169) protein constructs of KRAS-GppNHp harboring T35S mutation (KRAST35S/C118S-GppNHp) and of its oncogenic counterpart harboring the Q61L mutation (KRAST35S/Q61L/C118S-GppNHp) using heteronuclear, multidimensional NMR spectroscopy at 298 K. High resolution NMR data allowed the unambiguous assignments of 1H–15N correlation cross-peaks for all the residues except for Met1. Furthermore, 2D 1H–15N HSQC overlay of two proteins assisted in determination of Q61L mutation-induced chemical shift perturbations for select residues in the regions of P-loop, Switch-II, and helix α3.

Abstract 2000: Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts

Abstract Background: Growing evidence points towards the concept that not all RAS allelic variances are equal. Different RAS mutations produce distinct variations in signal transduction to sustain their proliferative needs accordingly. This is an especially important concept to consider as medicine shifts towards a more personalized approach. In accordance with this notion, through this study, we aim to explore the network dynamics and pinpoint disparities in signaling events associated with seven common KRAS allelic variances in non-small cell lung carcinoma. Methods: We used Mouse Embryonic Fibroblasts (MEF) provided by the RAS initiative (https://www.cancer.gov/research/key-initiatives/ras/outreach/reference-reagents/cell-lines). MEF were engineered to selectively harbor and express either wild type KRAS (4A and 4B WT) or one of seven following mutations: 4B G12D, G12C, G12R, G12V, G13D, Q61R and Q61L. Using high throughput immunoassay, Reverse Phase Protein Microarray (RPPA), we explored broad changes in signaling dynamics across our models. Specifically, we captured expression and/or activation levels of 68 proteins and phosphoproteins including: Receptor Tyrosine Kinases, MAPK, AKT/mTOR, JNK/STAT. Data were analyzed using unsupervised hierarchical clustering analysis using the Ward methods and the Kruskal Wallis test. P values <0.05 were considered significant. Results: Based on the unsupervised clustering analysis, Q61R and Q61L mutations showed the least activated signaling when compared to all other models. Activation of Raf was increased in cells harboring mutations of the G12 residues compared to those with mutations of the G13 and Q61 residues. Furthermore, the five G12 mutations clustered together with an overall higher active signaling compared to other cell lines. However, among the group of G12 mutants, unique signal transduction profiles were identified. We report distinct significant differences that divide the clustered G12 mutants into high versus low activation of specific signaling proteins. For example, ERK T202/Y204 (p=0.011) and S6 Ribosomal Protein S235/236 (p=0.001) phosphorylation were significantly higher in G12R compared to G12C and G12D models. On the contrary, STAT1 and STAT3 protein phosphorylation at residues Y701 and Y705 respectively were decreased in the G12R model. G12C has the lowest activation levels of p90RSK S380 (p=0.029), NF-KappaB p65 S536 (p=0.027) and P70 S6 Kinase S371 (p=0.013). Conclusions: In alignment with other findings, we show that based on RAS mutation, individual signaling components are affected differently, producing extensive heterogeneity in signaling dynamics. Even within grouped mutations presenting an overall similar net effect on downstream signaling, each individual variant produced discrete alterations. Citation Format: Emna El Gazzah, Elisa Baldelli, Chamodya Ruhunsiri, Emanuel F. Petricoin, Mariaelena Pierobon. Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2000.

Oncogenic mutations Q61L and Q61H confer active form-like structural features to the inactive state (state 1) conformation of H-Ras protein

GTP-bound forms of Ras proteins (Ras•GTP) assume two interconverting conformations, “inactive” state 1 and “active” state 2. Our previous study on the crystal structure of the state 1 conformation of H-Ras in complex with guanosine 5’-(β, γ-imido)triphosphate (GppNHp) indicated that state 1 is stabilized by intramolecular hydrogen-bonding interactions formed by Gln61. Since Ras are constitutively activated by substitution mutations of Gln61, here we determine crystal structures of the state 1 conformation of H-Ras•GppNHp carrying representative mutations Q61L and Q61H to observe the effect of the mutations. The results show that these mutations alter the mode of hydrogen-bonding interactions of the residue 61 with Switch II residues and induce conformational destabilization of the neighboring regions. In particular, Q61L mutation results in acquirement of state 2-like structural features. Moreover, the mutations are likely to impair an intramolecular structural communication between Switch I and Switch II. Molecular dynamics simulations starting from these structures support the above observations. These findings may give a new insight into the molecular mechanism underlying the aberrant activation of the Gln61 mutants.

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the “insert region” (residues 122–135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.

Abstract 419: Single cell-driven secreted proteins as a surrogate biomarker for Cetuximab therapy in colorectal cancer

Abstract Epidermal growth factor receptor (EGFR) blockade is efficacious in KRAS wild-type of colorectal cancer, but acquired resistance is inevitably generated. Here, we established the 5 acquired resistant single cell models (CR cell lines) to cetuximab and investigated the mechanism of acquired resistance using single cell-based whole-exome sequencing and transcriptional profiling. Of these, 4 cell lines have KRAS Q61L mutation and the other has BRAF V600E mutation. From gene enrichment analysis (GSEA), we found that epithelial to mesenchymal transition (EMT) occurs and CXCL chemokine family, which are known as secreted proteins, are overexpressed in the CR cell lines compared with parental cells. Interestingly, we found that CXCL chemokines were strongly secreted/expressed in KRAS/BRAF mutant cell lines and plasma/serum samples from colorectal cancer patients who acquired KRAS/BRAF mutation during cetuximab therapy. CXCL chemokines are regulated coordinately by Slug and NF-B transcription factors and trans-activate EGFR through CXCR2/MMP1/sHB-EGF axis. This process activates MAPK pathway via autocrine. To overcome the resistance to cetuximab, combination treatment of cetuximab and MEK/BRAF inhibitor had a larger antitumor effect compared to the single agents, decreasing the expression of MAPK pathway and secretion of CXCL chemokines. This finding highlights that CXCL chemokines, as a secreted proteins circulating in the bloodstream, show correlation with KRAS/BRAF mutation and could be clinical biomarkers for predicting resistance to anti-EGFR therapy in CRC. Citation Format: Ye-Lim Park, Hwang-Phill Kim, Jun Kyu Kang, Yoo Joo Lim, Sang-Hyun Song, Sae-Won Han, Tae-You Kim. Single cell-driven secreted proteins as a surrogate biomarker for Cetuximab therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 419.

Conjunctival Melanoma Targeted Therapy: MAPK and PI3K/mTOR Pathways Inhibition.

To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells. The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining. A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested. The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.

Corrigendum: A GAL4-inducible transgenic tool kit for the in vivo modulation of Rho GTPase activity in zebrafish.

DEVELOPMENTAL DYNAMICS 245:844–853, 2016; DOI: 10.1002/DVDY.24412 It has been brought to our attention by the authors that au68 (Cdc42CA) and au72 (Rac1CA) were incorrectly listed in Figure 1B as containing the Q61L mutation when in fact they contain the G12V mutation. The corrected Figure 1 appears below. The authors apologize for this error.

Oncogenic function and clinical implications of SLC3A2-NRG1 fusion in invasive mucinous adenocarcinoma of the lung.

The neuregulin 1 (NRG1) fusion is a recently identified novel driver oncogene in invasive mucinous adenocarcinoma of the lung (IMA). After identification of a case of SLC3A2-NRG1 in a patient with IMA, we verified this fusion gene in a cohort of 59 patients with IMA. Targeted cancer panel sequencing and RT-PCR identified the possible coexistence of other driver oncogenes. Among 59 IMAs, we found 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Of 16 patients with NRG1 fusions, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusions. When comparing overall survival (OS) according to the presence of NRG1 fusions showed that patients harboring NRG1 fusions had significantly inferior OS than those without NRG1 fusions (hazard ratio = 0.286; 95% confidence interval, .094 to .865). Ectopic expression of the SLC3A2-NRG1 fusion in lung cancer cells increased cell migration, proliferation and tumor growth in vitro and in xenograft models, suggesting oncogenic function for the fusion protein. We found that the SLC3A2-NRG1 fusion promoted ERBB2-ERBB3 phosphorylation and heteroduplex formation and activated the downstream PI3K/AKT/mTOR pathway through paracrine signaling. These findings suggested that the SLC3A2-NRG1 fusion was a driver in IMA with an important prognostic impact. SLC3A2-NRG1 should be considered a therapeutic target for patients with IMA.

Abstract 2376: Clonal evolution of skin carcinomas during tumor progression and metastasis

Abstract Tumor heterogeneity and the ongoing evolution of tumor subpopulations have presented considerable challenges to the success of cancer therapies, highlighting a critical need for greater understanding of these phenomena. We used a K5CreER-Confetti reporter mouse in combination with the DMBA (dimethylbenzanthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate) skin carcinogenesis model to study the genetics, clonality, and evolutionary behavior of tumor subpopulations from tumor initiation to metastasis. Treating the skin of K5CreER-Confetti mice with tamoxifen results in widespread labeling of the epidermis and hair follicles, which persists for at least 18 months. Papillomas that develop from Confetti-activated skin are of clonal origin, evidenced by each tumor being distinctly dominated by a single Confetti color (RFP, YFP, GFP, or CFP; or uncolored). Twelve weeks after initiation, the majority of tumors remain single-colored, with only 22% (36 of 133) showing any evidence of a secondary color population present. By 20 weeks after initiation, however, nearly all tumors (78%, 42 of 54) have incorporated one or more secondary populations, readily identifiable as streaks of a distinct color. FACS separation and sequencing of these different colored populations reveals these populations are genetically distinct: in preliminary analysis, the primary color population typically carries the expected driver Hras Q61L mutation while the secondary populations do not. The tumor thus appears to be incorporating pseudo-normal tissue from its microenvironment during early growth. Activation of the Confetti reporter in tumors post-initiation in papillomas results in labeling of single cells with all 4 Confetti colors, allowing subpopulations and their evolution to be studied at later stages. These highly multi-color papillomas subsequently give rise to single-color carcinomas and metastases, suggesting that the conversion from a benign to malignant tumor is clonally driven. The Confetti mouse is a valuable tool for studying the clonality and genetics of subpopulations present in tumors, which need to be better understood in order to improve the treatment of heterogeneous human tumors. Citation Format: Melissa Q. McCreery, Allan Balmain. Clonal evolution of skin carcinomas during tumor progression and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2376.

HRAS mutation prevalence and associated expression patterns in pheochromocytoma.

Pheochromocytomas (PCC) and abdominal paragangliomas (PGL) display a highly diverse genetic background and recent gene expression profiling studies have shown that PCC and PGL (together PPGL) alter either kinase signaling pathways or the pseudo‐hypoxia response pathway dependent of the genetic composition. Recurrent mutations in the Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been verified in sporadic PPGLs. In order to further establish the HRAS mutation frequency and to characterize the associated expression profiles of HRAS mutated tumors, 156 PPGLs for exon 2 and 3 hotspot mutations in the HRAS gene was screened, and compared with microarray‐based gene expression profiles for 93 of the cases. The activating HRAS mutations G13R, Q61R, and Q61K were found in 10/142 PCC (7.0%) and a Q61L mutation was revealed in 1/14 PGL (7.1%). All HRAS mutated cases included in the mRNA expression profiling grouped in Cluster 2, and 21 transcripts were identified as altered when comparing the mutated tumors with 91 HRAS wild‐type PPGL. Somatic HRAS mutations were not revealed in cases with known PPGL susceptibility gene mutations and all HRAS mutated cases were benign. The HRAS mutation prevalence of all PPGL published up to date is 5.2% (49/950), and 8.8% (48/548) among cases without a known PPGL susceptibility gene mutation. The findings support a role of HRAS mutations as a somatic driver event in benign PPGL without other known susceptibility gene mutations. HRAS mutated PPGL cluster together with NF1‐ and RET‐mutated tumors associated with activation of kinase‐signaling pathways. © 2016 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

Abstract LB-134: A novel model to investigate Kras mutation specificity in the skin

Abstract The selection and specificity of mutations occurring in genes that drive cancer is a highly complex process that remains poorly understood for even the most well studied oncogenes. The specific pattern of RAS mutations across different cancers is thought to be modulated by differences in carcinogen exposure and metabolism, local DNA features and repair mechanisms, tissue-specific expression patterns and functional differences of the RAS isoforms1. Less is known about functional differences between the various activating mutations in RAS that may lead to selection of particular mutants in mouse or human cancers. Studies of mouse cancer models have provided evidence for strong genetic background effects on allele-specificity of Ras mutations2 as well as on selection of particular mutations at Kras codon 61 in lung adenomas3. We have also recently demonstrated a major role of germline Kras status in mutation selection during initiation, where carcinogen-induced lung tumors from Kras WT mice carry mostly (94%) Q61R Kras mutations, while those from Kras heterozygous mice carry mostly (92%) Q61L mutations4. In a M. Musculus (Mm) x M. Spretus (Ms) backcrossed population of HrasKO mice treated with DMBA/TPA, a model of Kras-driven tumorigenesis in the skin, we observed a wide spectrum of Kras mutations. Interestingly, genotype at the Kras locus significantly influenced Kras mutation specificity, with a switch from predominantly G13R mutations in Mm/Mm carcinomas to G12 and Q61L mutations in Mm/Ms carcinomas. Importantly, total Kras expression driven by the Mm Kras allele is higher than that driven by the Ms allele in the skin, and Kras mutations occur with high specificity for the Mm allele, suggesting that Q61L and G12 mutations may be suppressed by higher expression of the WT Kras allele in Kras Mm/Mm animals. We hypothesize that the ability of WT Kras to suppress certain mutants may be dependent on upstream activation by EGFR, and that this phenomenon may underlie the divergent responses to EGFR inhibition of colorectal cancer patients harboring Kras G12 versus G13 mutations5. Experiments to test this in mice by extended EGFR inhibition following carcinogen treatment are ongoing.

Abstract 614: Clinical implications of NRG1 fusion in invasive mucinous adenocarcinoma of the lung

Abstract Background: We investigated the clinical implications of NRG1 fusion, a newly identified oncogenic rearrangement, in surgically resected invasive mucinous adenocarcinoma (IMA) of the lung. Patients and Methods: We performed RNA sequencing on 7 lung adenocarcinomas that had no known driver oncogenes and identified a novel fusion, SLC3A2-NRG1 in a case of IMA. The newly detected NRG1 fusion was verified in an IMA cohort of 59 cases using reverse transcription polymerase chain reaction (RT-PCR), direct sequencing and fluorescence in situ hybridization (FISH) analysis. Targeted caner panel sequencing and RT-PCR were performed to identify the possible coexistence of NRG1 fusions and other genetic alterations. Results: After screening 59 IMA, we found a total of 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Among the 16 cases with NRG1 fusion, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusion. When comparing survival according to the presence of an NRG1 fusion, patients harboring NRG1 fusion showed inferior overall survival (OS) compared to those without NRG1 fusion (median 51.9 months [mo] vs. not reached [NR], P = 0.019). They also showed a trend toward shorter disease-free survival (DFS) (median 21.9 vs. 79.4 mo, P = 0.113). In the stage I IMA, patients with NRG1 fusion showed significantly inferior OS (median 48.1 mo vs. NR, P = 0.009) and DFS (median 18.9 vs. 91.1 mo, P = 0.013) compared to those without NRG1 fusion. Conclusion: NRG1 fusion is an important prognostic factor in IMA and should be considered a novel therapeutic target for IMA. Citation Format: Ji-Youn Han, Yeon-Su Lee, Dong Hoon Lee, Dong Wan Hong, Seung Hyun Hong, Jung-Ah Hwang, Byung Il Lee, Hye Jin You, Dong Hoon Shin, Geon Kook Lee. Clinical implications of NRG1 fusion in invasive mucinous adenocarcinoma of the lung. [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 614. doi:10.1158/1538-7445.AM2015-614

The mutational landscapes of genetic and chemical models of Kras-driven lung cancer.

SUMMARYNext-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding factors that induce driver mutations, and the processes that shape their selection during tumourigenesis. We performed whole-exome sequencing (WES) on adenomas from three mouse models of non-small cell lung cancer (NSCLC), induced by exposure to carcinogens (Methylnitrosourea (MNU) and Urethane), or by genetic activation of Kras (KrasLA2). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the KrasLA2 model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single nucleotide variants (SNVs), compared to only 6 in tumours from the KrasLA2 model. In contrast, the KrasLA2 tumours exhibited a significantly higher level of aneuploidy and copy number alterations (CNAs) compared to the carcinogen-induced tumours, suggesting that carcinogen and genetically-engineered models adopt different routes to tumour development. The wild type (WT) allele of Kras has been shown to act as a tumour suppressor in mouse models of NSCLC. We demonstrate that urethane-induced tumours from WT mice carry mostly (94%) Q61R Kras mutations, while those from Kras heterozygous animals carry mostly (92%) Q61L mutations, indicating a major role of germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C>T mutations at CpG sites. These data provide a basis for understanding the conclusions from human tumour genome sequencing that identified two broad categories based on relative frequency of SNVs and CNAs1, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.

KRAS and BRAF Mutation Analysis in Colorectal Adenocarcinoma Specimens with a Low Percentage of Tumor Cells

Background and ObjectiveThe rapid development of molecular biology techniques allows for the introduction of real-time polymerase chain reaction (PCR) methods with a limit of mutation detection at 1 % in a background of wild-type DNA. Analysis of KRAS mutations in codons 12, 13, and 61, together with analysis of BRAF mutations in codon 600, are predictive biomarkers for anti-epidermal growth factor receptor (EGFR) treatment in colorectal cancer. Our aim was to compare PCR methods for KRAS mutations and BRAF mutation analysis using DNA isolated from tissue samples previously evaluated for presence of tumor cells using a quantitative scale and the percentage of tumor cells (PTC) scale. We addressed the question of whether a low number of tumor cells can be qualified for somatic mutation testing.ResultsOur study showed that PTC as low as 10 % was good enough to detect KRAS G12D, G13D, and Q61L mutations in formalin-fixed paraffin-embedded (FFPE) material. Furthermore, our results indicate that up to 20 % of colorectal cancer may carry mutations in the KRAS codon 61 and BRAF codon 600, which suggests the value of these mutation analyses because patients carrying them are unlikely to respond to cetuximab or panitumumab. A low level of KRAS somatic mutation detection has not been studied in depth in the context of clinical outcomes in patients; therefore, we compared new PCR methods, (KRBR-RT 50 Entrogen; ViennaLab StripAssay) and re-evaluated KRAS and BRAF status in patients with relapse after targeted therapy.ConclusionsThe importance of molecular results was confirmed by clinical observation of a patient with relapse who had qualified for targeted therapy with KRAS WT status (but was diagnosed by less sensitive single-stranded conformation polymorphisms method). Interestingly, during anti-EGFR treatment, it came to the selection of cells with KRAS G12C mutation which were present from the beginning in the tumor but at a low level (detected by PCR methods) only and led consequently to the metastasis. Taking into consideration the limit of detection, labor time, and assay cost, the real-time PCR method seems to be very promising especially for FFPE material with the PTC below 15 %.

Mutational Profiling of Multiple Myeloma Bone Marrow Aspirates As a Clinical Tool for Personalized Treatment of Myeloma

Abstract 3990 Introduction:Multiple Myeloma (MM) is increasingly being recognized as a heterogeneous disease. Previous studies have reported various oncogenic mutations in myeloma. Recent genome sequencing studies have revealed activating mutations of BRAF kinase in 4% of patients, a potentially druggable target, underscoring the importance of identifying these and other potential oncogenic mutations. However, a validated methodology for clinical assessment of mutation profiles is lacking. Applying such technologies will be increasingly important to accelerate development of targeted therapies toward tumor-specific oncogenic pathways. Utilizing a high-throughput genotyping platform we evaluated the commonly described somatic mutations in bone marrow aspirates in patients diagnosed with MM. Methods:We developed a CLIA (Clinical Laboratory Improvement Amendments) validated, highly sensitive multiplexed PCR-based assay (SNaPshot) to simultaneously identify 70 genetic loci frequently mutated in 15 cancer genes. This assay has been used at our institution for over 3 years for tumor genotyping and to help guide therapeutic decisions for patients with solid malignancies. To test the value of this approach in patients with MM, we first validated the methodology on MM cell lines followed by testing patient derived bone marrow samples. Results:The detectable mutations in the 8 MM cell lines tested include: MMIR: KRAS 35G>C, G12A mutation; MMIS: KRAS 35G>C, G12A mutation; H929: NRAS 38G>A, G13D mutation; LR5: KRAS 35G>C, G12A mutation; RPMI8226: TP53 743G>A, R248Q mutation;U266: no mutations identified; OPM2: NRAS 182A>T, Q61L mutation; OPM1: TP53 524G>A, R175H mutation.Thirty six bone marrow aspirates from patients with MM have been analyzed to date. Tweny five percent of patients had a detectable mutation, with KRAS mutation in 17%, NRAS in 5% and TP53 in 3%. Additional data in a larger cohort is currently being analyzed and will be presented. Conclusion:Oncogenic driver mutations can be detected by SNaPshot from routinely-collected bone marrow aspirate samples. Experience from lung and breast cancer studies indicates that while the type and frequency of mutation varies somewhat by tumor phenotype, the common oncogenic mutations are often found in isolation. Broad tumor mutational analysis will be helpful to efficiently tailor personalized therapies based on individual tumor genetic profiles. Disclosures:Dias-Santagata:Bio-Reference Laboratories, Inc.: Consultancy. Borger:Bio-Reference Laboratories, Inc: Consultancy. Iafrate:Bio-Reference Laboratories, Inc: Consultancy. Raje:Eli-Lilly: Research Funding; Amgen: Research Funding; Acetylon: Research Funding; Millennium: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

PIK3CA, HRAS and KRAS Gene Mutations in Human Penile Cancer

The knowledge of somatic mutations that arise in penile cancer is limited. We examined the dysregulation of components in the phosphatidylinositol 3-kinase and Ras pathways. Using single stranded conformational analysis and direct sequencing we performed mutational analysis of the PIK3CA, PTEN, HRAS, KRAS, NRAS and BRAF genes in 28 penile tumors. We identified somatic missense mutations in 11 of the 28 penile cancer samples (39%). In the PIK3CA gene 8 mutations (29%) were identified that were E542K or E545K. In the HRAS gene a G12S and a Q61L mutation were found (7%). The KRAS gene contained 1 mutation (3%), that is a G12S change. PIK3CA mutations were found in all grades and stages, whereas HRAS and KRAS mutations were found in larger and more advanced tumors. The mutations were mutually exclusive, suggesting that dysregulation of either pathway is sufficient for the development and progression of penile carcinoma. The high frequency of mutations in the PIK3CA, HRAS and KRAS genes leads us to believe that dysregulation of the phosphatidylinositol 3-kinase or Ras pathway is significant for the development and progression of penile carcinoma.