Additional Driver Mutations Research Articles

Spatial evolution of tumors with successive driver mutations.

We study the influence of driver mutations on the spatial evolutionary dynamics of solid tumors. We start with a cancer clone that expands uniformly in three dimensions giving rise to a spherical shape. We assume that cell division occurs on the surface of the growing tumor. Each cell division has a chance to give rise to a mutation that activates an additional driver gene. The resulting clone has an enhanced growth rate, which generates a local ensemble of faster growing cells, thereby distorting the spherical shape of the tumor. We derive formulas for the abundance and diversity of additional driver mutations as function of time. Our model is semi-deterministic: the spatial growth of cancer clones is deterministic, while mutants arise stochastically.

Abstract 2987: Next generation sequencing analysis of genetically engineered mouse models of human cancers

Abstract Genetically engineered mouse models (GEMM) of cancer are invaluable tools for oncology research. They recapitulate critical aspects of tumor etiology and allow for unique interrogation of the tumor stroma interactions during tumorigenesis. In addition, these models are poised for assessing therapeutic drug efficacy and resistance mechanisms. Although these models are widely used, the mutational landscape and transcriptional profile of GEMMs have yet to be thoroughly characterized. In this study we sought to investigate the precise mutational landscape of four well-validated GEMMs representing three types of cancers, non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC) and melanoma. Using next generation sequencing techniques, we profiled one adenovirally-induced Kras mutant NSCLC model (KrasLSL.G12D; p53frt/frt), two Kras mutant models of spontaneous PDAC (KrasLSL.G12D; p16/p19fl/fl; Pdx1.Cre and KrasLSL.G12D; p16/p19fl/wt; p53LSL.R270H/wt; Pdx1.Cre) and one Braf mutant melanoma model (BrafV600E/wt; PTENfl/fl; TyrCreER). All four GEMMs incorporate mutations that are well documented to occur in the human patient population. Additionally these models have been documented to approximate histological and phenotypic aspects of their human correlate. We carried out whole exome sequencing for each model (NSCLC n = 120; PDAC n = 73 and n = 40, respectively; melanoma n = 17) to comprehensively characterize the baseline genomes. We also looked for additional recurrent driver mutations beyond those engineered within each model. Interestingly, both intra and intertumoral heterogeneity was evident. Furthermore, we performed RNA sequencing and identified significantly affected genes and gene sets. Finally, we compared our GEMMs to human tumors from TCGA (LUAD, LUSC, PAAD and SKCM) at the genomic and transcriptomic level. Taken together, the results we will present provide a clearer understanding of the genomic and transcriptional landscape of these GEMMs and their relationship with human patients. More importantly, these analyses will allow us to better interpret results from previous and future experiments using these GEMMs of cancer. Citation Format: Wei-Jen Chung, Jason Long, Jason Cheng, Chris Tran, Anwesha Dey, Anneleen Daemen, Melissa Junttila. Next generation sequencing analysis of genetically engineered mouse models of human cancers. [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 2987. doi:10.1158/1538-7445.AM2015-2987

Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics.

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n = 412) to determine if these alterations could be used to distinguish GCT from other osteoclast‐rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast‐rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast‐rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

Identification of constrained cancer driver genes based on mutation timing.

Cancer drivers are genomic alterations that provide cells containing them with a selective advantage over their local competitors, whereas neutral passengers do not change the somatic fitness of cells. Cancer-driving mutations are usually discriminated from passenger mutations by their higher degree of recurrence in tumor samples. However, there is increasing evidence that many additional driver mutations may exist that occur at very low frequencies among tumors. This observation has prompted alternative methods for driver detection, including finding groups of mutually exclusive mutations and incorporating prior biological knowledge about gene function or network structure. Dependencies among drivers due to epistatic interactions can also result in low mutation frequencies, but this effect has been ignored in driver detection so far. Here, we present a new computational approach for identifying genomic alterations that occur at low frequencies because they depend on other events. Unlike passengers, these constrained mutations display punctuated patterns of occurrence in time. We test this driver–passenger discrimination approach based on mutation timing in extensive simulation studies, and we apply it to cross-sectional copy number alteration (CNA) data from ovarian cancer, CNA and single-nucleotide variant (SNV) data from breast tumors and SNV data from colorectal cancer. Among the top ranked predicted drivers, we find low-frequency genes that have already been shown to be involved in carcinogenesis, as well as many new candidate drivers. The mutation timing approach is orthogonal and complementary to existing driver prediction methods. It will help identifying from cancer genome data the alterations that drive tumor progression.

A Novel Xenograft Model of Cytogenetically Normal Acute Myeloid Leukemia Harboring RUNX1 and ASXL1 Mutations Demonstrates Residual Disease after Anthracycline/Cytarabine-Based Chemotherapy

Introduction: Cytogenetically normal acute myeloid leukemia (CN-AML) patients harboring RUNX1 mutations have a poor prognosis with standard, anthracyline/cytarabine-based chemotherapy and novel therapeutic approaches are urgently needed. Development of novel therapeutic approaches in RUNX1-mutated, CN-AML is hindered by a lack of adequate in vivo models. Munker et al. have generated a RUNX1-mutated, CN-AML cell line (termed CG-SH) from an AML patient and characterized its properties in vitro [Leukemia Research 33 (2009) 1405-1408; PMID: 19414191]; however, its potential to model the disease in vivo has not previously been explored. Moreover, CG-SH has not been comprehensively examined for additional AML driver mutations that might contribute to its biological properties. Our hypothesis was that CG-SH cells would efficiently engraft immune-deficient mice and demonstrate residual disease after anthracycline/cytarabine-based chemotherapy, rendering it a robust, in vivo platform to explore novel therapeutic approaches targeting RUNX1-mutated CN-AML.Methods: CG-SH cells were generously provided to us by Dr. Reinhold Munker of LSU-Shreveport and cultured in RPMI containing 12% FCS + P/S. For xenograft transplantation studies, CG-SH were injected via tail vein into 8-10 week old NOD/SCID/IL2Rg null (NSG) mice. Engraftment of CG-SH in NSG mice was determined by quantifying the percentage of human CD45+ (hCD45+) cells in bone marrow, spleen, and peripheral blood by flow cytometry. Whole exome sequencing and identification of sequence variations in genes known to be recurrently mutated in AML was done in conjunction with the Genomics Research Center at the University of Rochester Medical Center.Results: In initial experiments, CG-SH were confirmed to harbor the previously reported mutation in exon 8 of RUNX1 (c.1213_1214insCCCC) by Sanger Sequencing and to be cytogenetically normal by conventional karyotyping. Since our goal was to assess response of CG-SH-engrafted NSG mice to AML-like chemotherapy, and mice pre-conditioned with irradiation do not tolerate this therapy, we first tested the ability of CG-SH to engraft non-irradiated NSG mice. We found that tail vein injection of 1e6 CG-SH cells directly from culture into non-irradiated NSG mice resulted in high-level engraftment; at 6 weeks, mean engraftment in bone marrow, spleen, and peripheral blood was 71 +/- 9%, 49 +/- 12%, and 35 +/- 16%, respectively. Since CG-SH cells grow slowly in culture, we wanted to know the fewest number of cells necessary to establish leukemia in non-irradiated NSG mice. Limiting dilution analysis demonstrated that as few as 1000 CG-SH cells were sufficient to establish leukemia in non-irradiated NSG mice. At an equivalent cell dose, CG-SH engraftment levels were approximately 100-fold greater than those of M9-ENL cells, a different leukemia line commonly used for engraftment studies in NSG mice. Given the chemotherapy resistance demonstrated by patients with RUNX1-mutated, CN-AML, we hypothesized that mice xenografted with CG-SH cells would demonstrate residual disease after anthracycline/cytarabine-based chemotherapy. Mice xenografted with CG-SH cells were allowed to develop leukemia and then treated with a 5-day regimen of doxorubicin and cytarabine adapted for NSG mice [Wunderlich et al. Blood 2013; 121(12):e90-e97; PMID: 23349390]. Disease response was assessed 4 days after completion of chemotherapy (Figure 1A). Chemotherapy treatment resulted in eradication of CG-SH cells from the spleen and peripheral blood (data not shown), but persistence of a low-level of disease in the bone marrow (Figure 1B). To better understand the spectrum of AML driver mutations in CG-SH, we conducted whole exome sequencing. In addition to the RUNX1 and NRAS mutations previously reported, we identified mutations in ASXL1, which frequently co-occur with RUNX1 mutations in CN-AML, and CEBPA.Conclusions: Tail vein injection of CG-SH cells directly from culture into non-irradiated NSG mice efficiently generates leukemia in recipient animals that is not eradicated with AML-like chemotherapy. As such, these xenografts are a robust, in vivo platform to explore mechanisms of chemotherapy resistance and novel therapeutic approaches in CN-AML with RUNX1 and ASXL1 mutations. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New Insights into Potential Driver Mutations in Pediatric Burkitt Lymphoma

IntroductionBurkitt Lymphoma (BL) is the most common subtype of Non-Hodgkin Lymphoma (NHL) in childhood and adolescence. On the molecular level translocations involving the MYC oncogene are known as the hallmark of BL. However, as this event is not sufficient for BL pathogenesis, the search for additional driver mutations is ongoing. Recently published next-generation sequencing studies introduced various recurrently mutated genes in BL and reported the finding of mutations in Inhibitor of DNA-binding 3 (ID3) (Richter et al., Nat Genet 2012; Schmitz et al., Nature 2012; Love et al., Nat Genet 2012). Within the same pathway also the functional partner of ID3, TCF3, and the downstream target CCND3 were shown to harbor recurrent mutations. Functional analyses of the altered proteins hint at increased cellular growth and proliferation, among others by activating PI3K and CDK4/6.MethodsTo characterize the frequency and clinical relevance we investigated these candidate genes in a well-defined cohort of 63 pediatric BL that were uniformly diagnosed and treated according to NHL-BFM protocols. Also we screened the second most common pediatric B cell malignancies, precursor B cell leukemias (pB-ALL) and Diffuse Large B-cell Lymphoma (DLBCL), for mutations within this pathway. Initial pretreatment tumor samples from 63 BL (including 14 Burkitt leukemias), 15 DLBCL, 6 cases with features intermediate between BL and DLBCL (B-NHL nfc), 96 pB-ALL and an extended group of another 10 relapsed BL were available for analysis. Tumor cell content for DNA extraction was estimated to be at least 60%. Sanger sequencing was performed for the full coding region of ID3 in all entities. Additionally, TCF3 exon 16 and CCND3 exon 5 were investigated in all BL, DLBCL and B-NHL nfc cases. Clinical data were available from the NHL-BFM and ALL-BFM study center. This study was approved by the Ethical Advisory Board of the University of Giessen (A89/11 Amendment 2013).ResultsMutation frequencies for the 60/63 MYC translocation positive BL were 48 (80%) for ID3, TCF3 11 (13%) and CCND3 22 (37%) respectively. Most of the cases presented with more than one ID3 mutation and all affected cases had at least one mutation affecting the functional binding helices of ID3. At least one of the three candidate genes was altered by a potential protein-changing mutation in 53/60 cases (88%). In the group of 15 DLBCL three presented with ID3 mutations. Remarkably, in two of these cases a translocation involving MYC was reported in the study database. There were no ID3 mutations in 96 pB-ALL cases, but two cases presented with non-protein changing variants.With respect to clinical characteristics and patient outcome there were no significant differences with respect to ID3, TCF3 and CCND3 mutation status. To further analyze whether ID3, TCF3 and CCND3 mutations at initial diagnosis have prognostic impact on outcome, ten additional cases of initial tumor samples from patients who suffered relapse were analyzed. However, the ID3, TCF3 and CCND3 mutation status at diagnosis did not differ significantly among the 19 relapsed and the 52 non-relapsed BL patients.DiscussionThe aim of this study was to determine the relevance of ID3, TCF3 and CCND3 gene mutations in a well-defined cohort of pediatric BL. Most interestingly we found ID3 mutations in BL with a significantly higher frequency than previously published in the index studies including patients of all age groups by Richter et al., Schmitz et al. and Love et al., with frequencies ranging between 34 and 68%.The finding that ID3 mutations were virtually exclusive for MYC translocation positive tumors offers the prospect of the ID3 mutation status as a criterion of interest for analysis of intermediate cases between BL and DLBCL.As expected, no ID3 mutations were found in pB-ALL cases, supporting the assumption of exclusive occurrence within the pathogenesis of mature B cell lymphoma.While there was no evidence for a certain role with respect to clinical manifestations and patient outcome, the high frequency of 88% BL cases with at least one mutation in ID3, TCF3 or CCND3 leads to the proposal that this pathway plays an emerging role especially in pediatric BL and it might represent the second hit in BL pathogenesis. This is of special clinical interest as already available kinase inhibitors might successfully address the downstream targets PI3K and CDK4/6. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5590: Molecular characterization of pancreatic tumors arising in the background of germline BRCA mutations

Abstract Background: BRCA1 and BRCA2 are tumor suppressor genes which play a role in the repair of DNA double stranded breaks and the maintenance of genomic stability. Inherited BRCA mutations increase the risk of development of pancreatic cancer. In sporadic tumors BRCA mutations occur late in pancreatic tumorigenesis. Few studies to date have examined molecular heterogeneity within pancreatic tumors with respect to founder and progressor mutations. This is the first study to examine heterogeneity of mutations in pancreatic carcinomas associated with germline BRCA mutations. Design: Cases of pancreatic tumors arising in four patients with previously confirmed germline BRCA1 or BRCA2 mutations were identified from our case files. Representative sections of formalin-fixed, paraffin embedded tissue from surgical resection specimens were reviewed and 3 sections of tumor from different geographic regions and 1 section of non-tumor tissue were selected for molecular analysis for each case. Samples were manually macrodissected and genomic DNA was isolated. Genomic DNA was used for target capture and amplification of 212 amplicons targeting mutational hotspots in 48 genes using the TruSeq Amplicon Cancer Panel. Next generation sequencing was performed on the MiSeq Benchtop Sequencer and analyzed with NextGENe software. Results: In all cases concordant results were observed in all three tumor samples, with no evidence of intratumoral heterogeneity for the genes tested. In one patient with a BRCA1 germline mutation, TP53 and SMAD4 mutations were identified. In two patients with germline BRCA2 mutations, two additional driver mutations were identified involving KRAS, TP53, or SMAD4. In one case with BRCA2 germline mutation, no additional mutations were identified. Conclusion: Pancreatic tumors arising in the background of germline BRCA mutations had between 1-3 driver mutations per tumor, which is consistent with the reported numbers of driver mutations in sporadically arising pancreatic tumors. No mutual exclusivity among the mutated genes was observed. Mutations observed include KRAS codon 12 mutation, TP53 mutation (within the DNA binding domain), and SMAD4 mutation. Taken together, these results suggest that pancreatic tumors occurring in BRCA patients show similar mutational profiles of key oncogenes and tumor suppressor genes as seen in sporadic tumors with little heterogeneity with respect to these driver mutations. Citation Format: Susan Hsiao, Anne Koehne de Gonzalez, Mahesh M. Mansukhani, Robert L. Fine, Helen E. Remotti. Molecular characterization of pancreatic tumors arising in the background of germline BRCA mutations. [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 5590. doi:10.1158/1538-7445.AM2014-5590

Occurrence and Characteristics of Keap1-Mutations in Patients with Non-Small Cell Lung Cancer (Nsclc)

ABSTRACT Aim: The Kelch-like ECH-associated protein 1 (KEAP1) suppresses the Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) under physiological conditions and therefore the antioxidative and anti-inflammatory effect during oxidative stress. Mutations in KEAP1 are described for diverse tumor entities with a relatively high frequency. Limited data is available on the clinical relevance, the exact frequency of occurrence and the association with specific characteristics of patients. This study was performed to characterize KEAP1-mutated NSCLC clinically and genetically. Methods: Tumor tissue collected from 446 patients within a regional screening network was analyzed for KEAP1 mutations using next-generation sequencing (NGS). Clinical, pathological and genetic characteristics of these patients are described and compared with a control group of patients without KEAP1 mutation. Results: So far, we identified 33 patients with KEAP1 mutations. Among these we found at least 19 different mutations, of which the majority was not previously described. KEAP1 mutations were not restricted to a special exon, and in 30 patients (90.9%), additional driver mutations in KRAS, FGFR1, STK11, ALK, DDR2, and NRAS could be detected, as well as mutations and polymorphisms in TP53. KEAP1 mutations occurred in both adenocarcinoma and squamous cell carcinoma histology. Results of the ongoing clinical characterization of the patients as well as the prognostic and predictive impact of KEAP1 mutations in a prospective cohort will be presented. Conclusions: Our data suggest a role of KEAP1 mutations as a cofactor in addition to classical driver mutations underlying the malignant phenotype of lung cancer cells. Further data analysis will reveal the role of these mutations for the outcome of these patients. Disclosure: All authors have declared no conflicts of interest.

Genomic Changes in Lung Cancer Patients with Multiple Lung Relapses

ABSTRACT Aim: Patients with early stage non-small cell lung cancer (NSCLC) undergo surgical removal of their tumors. However, in some individuals tumors can recur and are re-removed multiple times. We identified patients (pts) who underwent multiple serial surgeries for early stage NSCLC and evaluated genetic changes in their tumors over time. Methods: Using an institutional database, we identified 15 pts that underwent surgical resection for stage I-II NSCLC with 2 or more recurrent tumors without intervening therapy. Among these, we selected 20 tumors from 6 pts with available tissue (median tumors/pt = 3 [3-4]) and their matched germline DNA. All pts provided informed consent. DNA extracted from archival FFPE samples and paired blood was analyzed by massively parallel sequencing (MPS) on an Illumina HiSeq 2500 using a targeted hybrid capture approach for 504 cancer-related genes. Sequencing was performed at high depth with a mean target coverage of 186x across all samples. We compared the somatic mutations and copy number variations (CNV) in the recurrent specimens to those in the primary tumor. We performed hierarchical clustering to compare the relapsed tumors to the primary tumors. Results: In 2/6 pts, the genetic changes confirmed that all the tumors were related among relapses, while in 4/6 pts some tumors showed distinctive genetic changes consistent with a different lung cancer. In pts with recurrent tumors, the subsequent relapsed tumors contained either additional mutations or increased CNV, suggesting genomic evolution over time. In pts with 3 or more surgically removed tumors, the time to relapse shortened with each subsequent tumor. In 3 pts with EGFR mutant NSCLC, the relapsed tumors contained additional potential driver mutations including EGFR T790M, PIK3CA E542K, and TP53 C176F, suggesting that these genetic changes can evolve even in the absence of drug treatment. Conclusions: Genomic changes in tumors from NSCLC pts with multiple recurrences evolve over time in the absence of systemic therapy. MPS helps to classify relapses vs. new primaries and reveals new insights in the biology and natural history of EGFR mutant lung cancer. Disclosure: All authors have declared no conflicts of interest.

Role of DNMT3A, TET2, and IDH1/2 mutations in pre-leukemic stem cells in acute myeloid leukemia

Aberrant changes in the epigenome are now recognized to be important in driving the development of multiple human cancers including acute myeloid leukemia. Recent advances in sequencing technologies have led to the identification of recurrent mutations in genes that regulate DNA methylation including DNA methyltransferase 3A (DNMT3A), ten-eleven translocation 2 (TET2), and isocitrate dehydrogenase 1 (IDH1) and IDH2. These mutations have been shown to promote self-renewal and block differentiation of hematopoietic stem/progenitor cells. Acquisition of these mutations in hematopoietic stem cells can lead to their clonal expansion resulting in a pre-leukemic stem cell (pre-LSC) population. Pre-LSCs retain the ability to differentiate into the full spectrum of mature daughter cells but can become fully transformed with the acquisition of additional driver mutations. Here, we review the effects of mutations in DNMT3A, TET2, and IDH1/2 on mouse and human hematopoiesis, the current understanding of their role in pre-LSCs, and therapeutic strategies to eliminate this population which may serve as a cellular reservoir for relapse.

Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome

AbstractSome neonates with Down syndrome (DS) are diagnosed with self-regressing transient myeloproliferative disorder (TMD), and 20% to 30% of those progress to acute megakaryoblastic leukemia (AMKL). We performed exome sequencing in 7 TMD/AMKL cases and copy-number analysis in these and 10 additional cases. All TMD/AMKL samples contained GATA1 mutations. No exome-sequenced TMD/AMKL sample had other recurrently mutated genes. However, 2 of 5 TMD cases, and all AMKL cases, showed mutations/deletions other than GATA1, in genes proven as transformation drivers in non-DS leukemia (EZH2, APC, FLT3, JAK1, PARK2-PACRG, EXT1, DLEC1, and SMC3). One patient at the TMD stage revealed 2 clonal expansions with different GATA1 mutations, of which 1 clone had an additional driver mutation. Interestingly, it was the other clone that gave rise to AMKL after accumulating mutations in 7 other genes. Data suggest that GATA1 mutations alone are sufficient for clonal expansions, and additional driver mutations at the TMD stage do not necessarily predict AMKL progression. Later in infancy, leukemic progression requires “third-hit driver” mutations/somatic copy-number alterations found in non-DS leukemias. Putative driver mutations affecting WNT (wingless-related integration site), JAK-STAT (Janus kinase/signal transducer and activator of transcription), or MAPK/PI3K (mitogen-activated kinase/phosphatidylinositol-3 kinase) pathways were found in all cases, aberrant activation of which converges on overexpression of MYC.

Concurrent driver mutations in non-small cell lung cancer (NSCLC) patients (p) on targeted therapy uncovered by comprehensive molecular profiling.

11004 Background: NSCLC p with EGFR mutations respond initially to EGFR tyrosine kinase inhibitors (TKIs) but invariably develop acquired EGFR TKI resistance. Prior studies identified the EGFR T790M mutation and activation of MET, PI3K, AXL, HER2 and the MAPK pathway as drivers of acquired EGFR TKI resistance. To date, comprehensive molecular profiling to identify actionable modifiers of EGFR TKI response has not been conducted in NSCLC p on therapy. Methods: We performed next generation sequencing (NGS) using a 263-gene Nimblegen custom cancer panel on DNA isolated from primary patient lung adenocarcinoma FFPE specimens prior to initiating standard erlotinib treatment and upon the development of acquired erlotinib resistance after only 3 months of therapy. Results: In the pretreatment sample, we confirmed the presence of the EGFRL858R mutation in 95% of the sequencing reads and discovered a concurrent BRAF V600E mutation with a frequency of ~ 6%. NGS performed on the acquired erlotinib resistance sample revealed acquisition of the EGFR T790M mutation with a frequency of ~ 14%. Notably, the frequency of the BRAF V600E mutation increased 10-fold upon acquired erlotinib resistance from ~ 6% in the pretreatment tumor to ~ 60% in the recurrent tumor. We found that overexpression of BRAF V600E in H3255 human NSCLC, which harbor EGFR L858R (but not BRAF V600E) and are erlotinib sensitive, caused resistance to erlotinib treatment (10-fold increase in erlotinib IC50). BRAF V600E-mediated erlotinib resistance was reversed by treatment with the BRAF inhibitor vemurafenib. Additional functional studies are ongoing and the complete dataset will be presented. Conclusions: These results indicate that EGFR-mutant NSCLC can harbor additional oncogenic driver mutations in BRAF at low frequencies prior to therapy. EGFR TKI treatment can lead to expansion of BRAF V600E expressing tumor cells, resulting in acquired EGFR TKI resistance that can be reversed by BRAF inhibitor treatment. The data demonstrate the utility of routine molecular profiling of NSCLC p on targeted therapy and offer unprecedented insight into the genetic basis of therapeutic resistance.

Abstract 3806: Progressive biallelic loss of TP53 is associated with progression of pleuropulmonary blastoma initiated by germline loss and somatic mutation of DICER1.

Abstract Pleuropulmonary blastoma (PPB) is a rare, aggressive childhood cancer arising from lung mesenchymal cells. In early stages, these cells reside in the walls of epithelial-lined cysts and may remain dormant or progress to thickened cysts and then highly malignant masses over several years. Familial germline loss-of-function DICER1 variants have been described, although carriers may not be affected. Therefore, we sought to uncover additional genetic events underlying tumor development through whole exome sequencing of 15 tumor/normal pairs. In addition to known loss-of-function germline variants found throughout DICER1, this analysis identified somatic missense mutations clustered in the DICER1 RNase IIIb domain in nearly every case. While the somatic mutations impact novel amino acid residues, expression analysis demonstrated depletion of 5p-derived miRNAs similar to that described in other DICER1-mutant tumors. While mutation rates were relatively high compared to other pediatric solid tumors, most mutations, except those in DICER1 and TP53, appeared to be subclonal. Early stage tumors (PPB Type II) had predominantly single-copy deletion of TP53 whereas late stage tumors (PPB Type III) had secondary TP53 mutations. Several late stage tumors also had activating mutations in RAS pathway members, including NRAS and BRAF. Therefore, compound DICER1 loss appears to be initiating and synergistic with incremental loss of TP53 and acquisition of additional driver mutations. We subsequently characterized this molecular pattern using deep sequencing and immunohistochemistry in an extension cohort of 45 additional tumors. This step-wise series of germline and somatic alterations illustrates a genetic mechanism consistent with the progressive nature of pleuropulmonary blastoma. Citation Format: Trevor J. Pugh, Weiying Yu, Jiandong Yang, Lauren Ambrogio, Scott L. Carter, Adam Kiezun, Aaron McKenna, Petros Giannikopoulos, Gad Getz, Yoav Messinger, Matthew Meyerson, D. Ashley Hill. Progressive biallelic loss of TP53 is associated with progression of pleuropulmonary blastoma initiated by germline loss and somatic mutation of DICER1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3806. doi:10.1158/1538-7445.AM2013-3806

Mutations in SETD2 and genes affecting histone H3K36 methylation target hemispheric high-grade gliomas

Recurrent mutations affecting the histone H3.3 residues Lys27 or indirectly Lys36 are frequent drivers of pediatric high-grade gliomas (over 30 % of HGGs). To identify additional driver mutations in HGGs, we investigated a cohort of 60 pediatric HGGs using whole-exome sequencing (WES) and compared them to 543 exomes from non-cancer control samples. We identified mutations in SETD2, a H3K36 trimethyltransferase, in 15 % of pediatric HGGs, a result that was genome-wide significant (FDR = 0.029). Most SETD2 alterations were truncating mutations. Sequencing the gene in this cohort and another validation cohort (123 gliomas from all ages and grades) showed SETD2 mutations to be specific to high-grade tumors affecting 15 % of pediatric HGGs (11/73) and 8 % of adult HGGs (5/65) while no SETD2 mutations were identified in low-grade diffuse gliomas (0/45). Furthermore, SETD2 mutations were mutually exclusive with H3F3A mutations in HGGs (P = 0.0492) while they partly overlapped with IDH1 mutations (4/14), and SETD2-mutant tumors were found exclusively in the cerebral hemispheres (P = 0.0055). SETD2 is the only H3K36 trimethyltransferase in humans, and SETD2-mutant tumors showed a substantial decrease in H3K36me3 levels (P < 0.001), indicating that the mutations are loss-of-function. These data suggest that loss-of-function SETD2 mutations occur in older children and young adults and are specific to HGG of the cerebral cortex, similar to the H3.3 G34R/V and IDH mutations. Taken together, our results suggest that mutations disrupting the histone code at H3K36, including H3.3 G34R/V, IDH1 and/or SETD2 mutations, are central to the genesis of hemispheric HGGs in older children and young adults.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1095-8) contains supplementary material, which is available to authorized users.

The effect of one additional driver mutation on tumor progression

Tumor growth is caused by the acquisition of driver mutations, which enhance the net reproductive rate of cells. Driver mutations may increase cell division, reduce cell death, or allow cells to overcome density-limiting effects. We study the dynamics of tumor growth as one additional driver mutation is acquired. Our models are based on two-type branching processes that terminate in either tumor disappearance or tumor detection. In our first model, both cell types grow exponentially, with a faster rate for cells carrying the additional driver. We find that the additional driver mutation does not affect the survival probability of the lesion, but can substantially reduce the time to reach the detectable size if the lesion is slow growing. In our second model, cells lacking the additional driver cannot exceed a fixed carrying capacity, due to density limitations. In this case, the time to detection depends strongly on this carrying capacity. Our model provides a quantitative framework for studying tumor dynamics during different stages of progression. We observe that early, small lesions need additional drivers, while late stage metastases are only marginally affected by them. These results help to explain why additional driver mutations are typically not detected in fast-growing metastases.

Modelling the evolution of genetic instability during tumour progression.

The role of genetic instability in driving carcinogenesis remains controversial. Genetic instability should accelerate carcinogenesis by increasing the rate of advantageous driver mutations; however, genetic instability can also potentially retard tumour growth by increasing the rate of deleterious mutation. As such, it is unclear whether genetically unstable clones would tend to be more selectively advantageous than their genetically stable counterparts within a growing tumour. Here, we show the circumstances where genetic instability evolves during tumour progression towards cancer. We employ a Wright–Fisher type model that describes the evolution of tumour subclones. Clones can acquire both advantageous and deleterious mutations, and mutator mutations that increase a cell's intrinsic mutation rate. Within the model, cancers evolve with a mutator phenotype when driver mutations bestow only moderate increases in fitness: very strong or weak selection for driver mutations suppresses the evolution of a mutator phenotype. Genetic instability occurs secondarily to selectively advantageous driver mutations. Deleterious mutations have relatively little effect on the evolution of genetic instability unless selection for additional driver mutations is very weak or if deleterious mutations are very common. Our model provides a framework for studying the evolution of genetic instability in tumour progression. Our analysis highlights the central role of selection in shaping patterns of mutation in carcinogenesis.

IL2 - Cancer Research and Clinical Oncology for the Next Decade

ABSTRACT The next decade of cancer research will continue to focus on identification and effective targeting of somatic driver mutations. The first successfully targeted driver mutation in lung cancer is the mutated epidermal growth factor receptor (EGFR) discovered 8 years ago. Multiple prospective trials for patients with EGFR mutations comparing treatment with gefitinib and erlotinib versus combination chemotherapy have been reported. The prolongation in PFS and decreased toxicity with EGR-TKIs has led to their regulatory approval as initial systemic therapy in patients with non-small-cell lung cancer (NSCLC) and EGFR sensitizing mutations in many countries throughout the world. The second driver mutation successfully targeted is the ALK rearrangement in NSCLC. Five years have past since Dr. Mano et al. discovered a rearrangement between ALK and EML4 in adenocarcinomas of the lung that could transform NIH 3T3 cells. Further research has shown that additional ALK rearrangements can give rise to constitutively activated ALK tyrosine kinase in ∼5% of NSCLC patients. The ALK inhibitor, crizotinib, has been studied in NSCLC patients with prospectively identified ALK rearrangements in a series of phase I, II, and III trials. The prospective clinical studies administering crizotinib to NSCLC with ALK rearrangements have response rates of ∼60%, PFS of ∼10 months, and more than half of the patients are alive 2 years from starting crizotinib. The high response rates, prolongation in PFS and decreased toxicity with crizotinib compared with chemotherapy has led to the regulatory approval of crizotinib as systemic therapy in patients with NSCLC and ALK rearrangements in the United States in August 2011. The additional driver mutations targeted in NSCLC include KRAS, BRAF, HER2, and PIK3CA mutations, ROS1 and RET rearrangements as well as MET amplification. BRAF has been successfully targeted in melanoma and HER2 amplification in breast cancer. Multiplex genomic characterization efforts are identifying these driver mutations and multi-institutional trials with therapeutic agents directed against these genomic alterations are underway.

Highlights of This Issue

Activating mutations in PIK3CA have been identified in many solid tumors. To investigate PIK3CA-mutant lung adenocarcinomas, Chaft and colleagues examined the clinical and molecular characteristics of 1,125 patients, testing for driver mutations in PIK3CA and 8 other oncogenes. PIK3CA mutations were identified in 2% of lung adenocarcinoma specimens, potentially more than 3000 cases in the U.S. annually. Unlike the typical paradigm of driver-oncogene mutual exclusivity in lung adenocarcinomas, 70% tumors with a PIK3CA mutation harbored an additional driver mutation. These results support the necessity to comprehensively genotype patients enrolling in clinical trials testing agents that target the PIK pathway.Medulloblastoma is the most common malignant pediatric brain tumor. It is an invasive neoplasm with poorly understood etiology. Guessous and colleagues uncover previously unknown molecular and functional interactions between the tyrosine kinases c-Met and FAK/Pyk2 in medulloblastoma. They show that c-Met activates FAK and Pyk2 and that FAK and Pyk2 mediate the invasive and malignant effects of c-Met in medulloblastoma. They show for the first time that combined inhibition of c-Met and FAK/Pyk2 has experimental therapeutic advantage. Their study therefore establishes proof-of-principle for the usefulness of combining existing clinically applicable small molecule inhibitors of c-Met and FAK/Pyk2 in medulloblastoma therapy.Recently, trastuzumab has shown clinical benefit in HER2 amplified gastric cancer. To explore its therapeutic potential, Nam and colleagues evaluated the activity and mechanisms of PF00299804, an irreversible pan-HER inhibitor, in gastric cancer cells. PF00299804 showed significant antitumor effects in HER2 amplified gastric cancer cells through inhibition of HER family heterodimer formation. The combination of PF00299804 with cytotoxic chemotherapeutic agents or molecularly targeted agents, including trastuzumab, CP751871 (IGF1R inhibitor), PD0325901 (ERK1/2 inhibitor), and PF04691502 (PI3K/mTOR inhibitor), produced synergistic effects. These findings strongly suggest that PF00299804, alone or in combination with other agents, can be used as a targeted therapy for the treatment of HER2 amplified gastric cancer.

Abstract PL04-01: Improving patient outcomes in lung cancer by targeting different driver mutations

Abstract Multiple groups reported in 2004 on the association between clinical response to treatment with gefitinib and erlotinib and somatic mutations of the epidermal growth factor receptor (EGFR) in patients with lung cancer. Subsequent research showed these somatic sensitizing mutations of EGFR are also driver mutations. Somatic mutations of EGFR are predictive markers for higher response rates, prolonged time to progression, and a trend towards longer survival in patients with non-small cell lung cancer (NSCLC) treated with gefitinib or erlotinib rather than combination chemotherapy. The prospective clinical trials have led to approval of gefitinib for as initial treatment for patients with sensitizing mutations of EGFR or following relapse after conventional chemotherapy in Europe. Erlotinib is recommended as initial therapy for patients with sensitizing mutations of EGFR in the National Comprehensive Cancer Network (NCCN) guidelines. However, only 10% to 30% of patients with lung cancer have activating mutations of EGFR. Ongoing research is attempting to identify additional driver mutations in patients with lung cancer and other malignancies that can be targeted with existing or novel compounds. The activating mutations in both adenocarcinomas and squamous cell carcinomas of the lung are being systemically characterized through the Tumor Sequencing Project, The Cancer Genome Atlas projects (http://cancergenome.nih.gov/wwd/program), and other programs around the world. These projects have identified the most frequently mutated and/or activated genes in both lung adenocarcinoma and in squamous cell carcinoma of the lung. In addition to EGFR mutations, other driver mutations that can be effectively treated with existing targeted agents have been identified including EML4-ALK intrachromosomal rearrangements and BRAF mutations. Investigators from Japan discovered a gene that arose from a intrachromosomal rearrangement in adenocarcinomas of the lung which could transform NIH 3T3 cells. The transforming gene is a fusion of the ALK gene with echinoderm microtubule-associated protein-like 4 (EML4). The chromosomal rearrangement gives rise to a fusion gene in which the ALK tyrosine kinase is constitutively activated. Further studies have shown the EML4-ALK rearrangements are present in NSCLC arising in patients from the United States and Europe. The translocated gene can now be detected by using fluorescence in situ hybridization (FISH) in histological sections of the tumor. There are drugs that are directed against the ALK tyrosine kinase including PF2341066 or crizotinib. Crizotinib has been tested in patients with EML4-ALK intrachromosomal rearrangements in an expansion cohort of the phase I trials. Crizotinib has shown evidence of antitumor activity with response rates of approximately 57% and progression-free survival in excess of 6 months in patients with this rearrangement. Crizotinib is being tested in patients with relapsed NSCLC and EML4-ALK translocations randomized to either conventional therapy with pemetrexed or docetaxel versus crizotinib (ClinicalTrials.gov NCT01000025). Other potential therapeutic targets in patients with NSCLC include activating mutations in BRAF, present in 2% of patients with NSCLC. BRAF has been shown to be a driver mutations in melanoma and successfully targeted with PLX4032 {Bollag, #4046; Flaherty, #4045}. One thousand adenocarcinomas of the lung are now being characterized for 10 different mutations, gene amplification, and chromosomal rearrangements through the 14 institutions participating in the Lung Cancer Mutation Consortium (http://www.golcmc.com/). The Clinical Trials Committee within the Lung Cancer Mutation Consortium has established trials with drugs specifically directed at these driver mutations in different subsets of patients with lung cancer. The goal is to continue to expand the number of patients who can be treated with effective targeted therapy against these driver mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr PL04-01. doi:10.1158/1538-7445.AM2011-PL04-01