Average Number Of Mutations Research Articles

Abstract 4899: Tumor T-cell receptor (TCR) diversity elucidates the immune response to genetic alterations of muscle-invasive bladder cancer

Abstract Background: Muscle-invasive bladder cancer (MIBC), unlike the more favorable low-grade papillary form, has a dismal prognosis with a significant risk of recurrence after surgical resection. Previous whole-exome sequencing of MIBC tumors performed by our lab demonstrated that carriers of mutations in any of six DNA mismatch repair genes (ATM, ERCC2, FANCD2, PALB2, BRCA1 or BRCA2) not only had a high somatic mutation burden, but also had enhanced recurrence-free survival compared to non-carriers of these mutations. We hypothesize that a high somatic mutation burden may generate a higher number of tumor-specific antigens that induce clonal expansion of tumor-infiltrating T-lymphocytes (TILs) to reduce a risk of recurrence and improve the survival. To investigate this hypothesis, we have sequenced the TCR cDNAs of TILs. Methods: Tumor and adjacent normal tissue (verified by a pathologist) were collected from patients whose tumors had previously been whole-exome sequenced. Library preparation was performed using previously described methods. Next-generation sequencing of TCRs was conducted on the Illumina platform (San Diego, CA) and CDR3 sequences were identified using a published algorithm. The diversity index of the TCRα and TCRβ chains was calculated using Simpson's diversity index, an ecological formula for calculating biodiversity based on evenness and richness. CD4+, CD8+, and FOXP3 gene expression levels were also examined with TaqMan Gene Expression Assays (Thermo Fisher Scientific, Carlsbad, CA). Results: TCR sequencing of ten tumors (5 non-recurrent (NR) with at least two years follow-up, 5 recurrent) has been performed. The average TCRα DI of five recurrent patients is 97±56.5 (SEM) compared to 23± 12.7 for five NR patients. In addition, we found an average DI of 13.8±0.77 in tumors with a high somatic mutation burden (average number of mutations 221), while it was 71.4±36.8 for tumors with a low somatic mutation burden (avg mutations 31). In samples with a relatively high DI, the three most abundant TCR clones represent less than 15% of the total TCR population, while in samples with a low DI, the top three clones comprise 35-90% of the total population. Finally, the CD8+/FOXP3 ratios were 10.1±2.1(SEM) and 5.6±2.9 for tumors with a low DI and those with a high DI, respectively (low vs. high defined by the average). Conclusions: We have elicited a correlating pattern where MIBC tumors with a higher number of somatic mutations have a lower TCR DI, indicating an oligoclonal expansion. Furthermore, in patients with recurrence, the TCR DI is higher, suggesting a lack of anti-tumor immune responses and non-specific T-cell expansion. The higher CD8+/FOXP3 ratio observed in tumors with low DI suggests that the clonal expansion is of CD8+ cells in tumors, which have been shown to be associated with favorable prognosis. Citation Format: Noura Choudhury, Kai Lee Yap, Kazuma Kiyotani, Poh Yin Yew, Alexa Campanile, Tatjana Antic, Gary Steinberg, Jae-Hyun Park, Peter H. O'Donnell, Yusuke Nakamura. Tumor T-cell receptor (TCR) diversity elucidates the immune response to genetic alterations of muscle-invasive bladder cancer. [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 4899. doi:10.1158/1538-7445.AM2015-4899

Abstract 3826: A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development

Abstract Background and aims: New generation sequencing and array technologies now allow the systematic investigation and comparison of genetic or epigenetic alterations in AD-CRC. The colorectal adenoma-dysplasia-cancer (AD-CRC) development is characterised by sporadic (<45% frequency) mutations in a limited number of genes. Recent epigenetic investigations showed that methylation of selected genes occurs in high frequency (>95%) in early stages of cancers already. Aims: Evaluation of the role of epigenetic and genetic alterations in the AD-CRC development using new generation sequencing and DNA methylation arrays. Materials and methods: DNA was isolated from fresh frozen biopsy specimens (20 normal; 33 adenomas; 17 CRCs). First, a multiplex PCR panel was designed to amplify mutation hot spots of 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, MSH6, NRAS, PIK3CA, SMAD2, SMAD4, TP53). Further DNA methylation analysis of 94 genes was performed on Human Colon Cancer EpiTect Methyl II Signature PCR Array (Qiagen) from the same DNA specimen. After RNA isolation whole genome expression analysis was performed with HGU133plus2 microarrays (Affymetrix) from 49 normal, 49 adenoma and 49 CRC specimens and on HT-29 cells with and without 5-aza-deoxycytidin treatment. Immunohistochemistry confirmation of expression changes on protein level was performed using tissue microarrays. Results: Mutations were found in 76% of adenomas and 78% of the cancer cases. The average number of mutations found in mutated samples was 1; 1,8; 1,9 and 2,3 in low grade adenomas, high grade adenomas, carcinomas and serrated adenomas, respectively. The APC suppressor gene was mutated in adenomas more frequently than in carcinomas (36% vs. 24%). The most frequently mutated genes were APC, TP53 and KRAS with 36%, 18% and 26% frequencies in adenomas and 24%, 47% and 45% frequencies in carcinomas. DNA methylation was found in 100% of the investigated colorectal adenoma and cancer specimens. Eight genes were found to be methylated in all of the cases. This gen set included SFRP1, MAL, SLIT2, SST, VIM; another set of genes (DKK1, SLI3, TMEFF2) was found to be hypermethylated in adenomas and cancers in >75% of the cases. In adenomas 56 genes, in dysplasias 40 in cancer 37 genes were methylated (>50% of the cases). The effect of methylation could be confirmed by decreased mRNA and protein expression. Demethylation treatment successfully restored the expression profile of the top methylated genes. Conclusion: Methylation of gene sets occurred in early premalignant stages followed by somatic mutations in increasing number through the AD-CRC development. Demethylation treatment could reverse the systematic, robust methylation alterations. Epigenetic alterations precede the somatic mutations of the AD-CRC and show higher significance in CRC development than genetic. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Barnabas Wichmann, Arpad V. Patai, Zsolt Tulassay. A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development. [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 3826. doi:10.1158/1538-7445.AM2015-3826

Abstract P2-03-05: The heterogeneous clinical behavior of luminal breast cancers is associated with different mutational landscapes

Abstract Background: Genomic studies in estrogen-receptor (ER) positive breast cancer showed few commonly mutated genes and suggested that tumors with higher background of mutation rate were enriched in intrinsic aromatase-inhibitor-resistant tumors (Ellis MJ Nature 2012). Different combinations of proliferation (PROL) and estrogen-receptor related genes (ERS) allow the prediction of the risk of recurrence and endocrine treatment sensitivity (Bianchini G Breast Cancer Res 2013). The highPROL/lowERS is enriched in intrinsic endocrine resistant tumors. We explored whether different genomic alterations could contribute to the different clinical behavior of groups defined by PROL and ERS. Method: We defined luminal breast cancers by PAM50 and retrieved gene expression and whole-exome sequencing data of 324 luminal breast cancer cases from the Cancer Genome Atlas (TCGA). PROL and ERS were calculated as previously reported. Low and high expression groups were defined by median cut-points (Bianchini G Breast Cancer Res 2013). As a metric of the background mutation rate of each sample, we used the overall mutation frequency in whole exome (Kandoth C Nature 2013). The most frequently mutated genes (TP53, PIK3CA, MAP2K4, MAP3K1, GATA3, CDH1) were also assessed for their association with PROL and ERS defined groups. Results: The average number of mutations (Nmut) was 44.4 (5-366). High and low proliferation groups had different mutation frequency (average Nmut 53.6 and 35.3, respectively; p=6.1E-06). LowERS group also had a higher Nmut (average Nmut 54.4 vs. 34.5 in high ERS; p=1.2E-06). Notably, neither high or low expression of the ER gene (p=0.89), nor positive and negative progesterone receptor by immunohistochemistry (p=0.69) were associated with Nmut. LowERS group was associated with higher Nmut in luminal A (p=0.005) and B (p=0.006) subtypes. The group with highPROL and lowERS had the highest Nmut (average 62.1), which was not significantly different from basal-like tumors. HighPROL/lowERS was significantly associated with higher frequency of mutTP53 (32.3%) and lower rates of mutPIK3CA (28.1%), mutMAP2K4 and/or mutMAP3K1 (7.2%) and mutCHD1 (3.1%). The lowPROL/highERS had the lowest Nmut (average 29.8), with the higher rate of mutPIK3CA (61.4%) and mutMAP2K4 and/or mutMAP3K1 (29.2%), and lower rate of mutTP53 (6.7%). Discussion: In ER-positive breast cancers, the highPROL/lowERS group, which is characterized by the highest risk of early recurrences despite endocrine treatment and chemotherapy, poor down-regulation of Ki67 after neoadjuvant aromatase-inhibitor (AI), but the highest rate of pCR after neoadjuvant chemotherapy, was also associated with the highest frequency of mutations, of the same order of magnitude of basal-like tumors. This data is in line with the observation that the number of mutations is linked to intrinsic resistance to neoadjuvant AIs (Ellis MJ Nature 2012). These data could be also informative for interpreting results of the combination of everolimus and endocrine therapy (exemestane or tamoxifen) that suggest higher efficacy in tumors with acquired endocrine resistance (lower mutation rate) compared to intrinsic or primary endocrine resistance (higher mutation rate). Citation Format: Giampaolo Bianchini, Eugenia Galeota, Tingting Jiang, Maurizio Callari, Milvia Zambetti, Takayuki Iwamoto, Thomas Karn, Christos Hatzis, Lajos Pusztai, Luca Gianni. The heterogeneous clinical behavior of luminal breast cancers is associated with different mutational landscapes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-05.

O2-9-5 - Gene Mutation Profile of Pancreatic Cancer in Japanese Patients and Its Association with Prognosis

Background: Activating KRAS mutations are the major driver gene aberrations of pancreatic cancer and are present in over 90% of patients with pancreatic cancer. On the other hand, aberrations of the CDKN2A, TP53, and SMAD4 genes are also frequent events in pancreatic carcinogenesis. We constructed a mutation profile of pancreatic cancer-associated genes. Additionally, we analyzed the relationship between gene mutations and clinicopathological factors.Methods: Genomic DNA samples were extracted from fresh frozen surgical specimens obtained from 100 patients (98 with pancreatic ductal adenocarcinoma, 2 with adenosquamous carcinoma) who underwent radical operations for pancreatic cancer at the National Cancer Center Hospital between March 2005 and June 2012. Next-generation sequencer-based targeted deep sequencing was performed using a Cancer Panel reagent that covers representative cancer-related genes (50 genes, 190 hot spots).Results: Mutations were detected in 97% of the cases, and the average number of mutations per tumor sample was 1.6. The most frequently mutated genes were KRAS (96%), TP53 (42%), SMAD4 (13%), and CDKN2A (7%) in our cohort. The known druggable mutations that were detected were GNAS (1%), PIK3CA (1%), and KIT (1%). Among the patients who underwent radical operations followed by adjuvant chemotherapy (71 patients), the survival of patients who had 0 to 2 mutations in the 4 major driver genes (KRAS, TP53, SMAD4, and CDKN2A) was significantly longer than that of patients who had 3 or more mutations (median overall survival, 40.0 months vs. 12.6 months, P = 0.0020). A multivariate Cox proportional hazard model analysis showed that a low number of mutations among these 4 genes was significantly associated with a better prognosis.Conclusions: KRAS, TP53, SMAD4, and CDKN2A were the most frequently mutated genes in Japanese patients with pancreatic cancer. The number of mutations among these 4 genes as detected using targeted deep sequencing may be a useful biomarker for the prediction of postoperative outcomes.

Abstract 4261: Prospective next generation sequencing (NGS) of rare or poor prognosis cancers

Abstract Background. Genomic screening for somatic alterations in individual tumors is informative for understanding tumor biology and identifying novel treatment approaches and research opportunities. We initiated a research study where patients with rare or poor prognosis cancers were enrolled and underwent targeted sequencing using a CLIA certified assay. Sequencing results were reviewed at a molecular tumor board and therapeutic strategies were suggested if appropriate. Preliminary results of this study are now presented. Experimental procedures. We analyzed the genomic profiles of formalin-fixed paraffin embedded tumor specimens from 72 patients using the FoundationOne™ (Foundation Medicine, Cambridge, MA) platform for targeted sequencing of the entire coding sequence of 236 genes and 47 introns of 19 genes involved in fusions in a CLIA-certified CAP accredited laboratory. A range of evaluated tumor subtypes of epithelial and mesenchymal origin were evaluated (20). The majority of cases were from rare or uncommon histologic classes, though recurrent/refractory cases of colon cancer and triple negative breast cancers were also enrolled. Both clinical history and sequencing data were presented at a multidisciplinary molecular tumor board for development of therapeutic recommendations. Results. At least one genomic alteration was found in 68 cases (95%) and none in four cases (6%). Serial specimens were obtained in six patients. Five had a gain of at least one mutation in serial specimens. The average number of mutations identified was 3.6 (range 0-10). The most common genomic alterations detected were in p53 (40%), KRAS (17%), PI3K (15%) and PTEN (10%). Alterations in the FGF pathway were surprisingly common (16%). A number of mutations occurred at low frequency but included an additional large set of potentially actionable genes: ALK, ERBB2, BRCA2, MET. The majority of cases had mutations for which our molecular tumor board had recommended action: either potential enrollment in clinical trial or off label use of approved therapy. NGS sequencing results led to implemented clinical action in ∼15% of cases. Conclusion. Use of a comprehensive clinical NGS assay in a diverse set of cancers identifies a number of actionable genomic changes associated with targeted therapies. An institutional molecular tumor board is an effective venue to systematically review sequencing data and generate clinical recommendations. Knowledge of genomic alterations has led to referral to clinical trials, off label use of targeted therapy, and planned trials of agents targeting identified pathways. Challenges to effectively implementing clinical recommendations included lack of practical access to clinical trials and limited availability of targeted agents. Expanding clinical trial portfolios, and increasing access to targeted agents is critical for successfully implementing genomic-guided precision cancer therapy. Citation Format: Kim M. Hirshfield, Siraj M. Ali, Vincent A. Miller, Philip J. Stephens, Vassiliki Karantza, Robert S. DiPaola, Lorna Rodriguez-Rodriguez, Shridar Ganesan. Prospective next generation sequencing (NGS) of rare or poor prognosis cancers. [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 4261. doi:10.1158/1538-7445.AM2014-4261

689P - Gene Mutation Profile of Pancreatic Cancer Obtained Using Targeted Deep Sequencing and Its Association with Prognosis

ABSTRACT Aim: Activating KRAS mutations are the major driver gene aberrations of pancreatic cancer and are present in over 90% of patients with pancreatic cancer. On the other hand, aberrations of the CDKN2A, TP53, and SMAD4 genes are also frequent events in pancreatic carcinogenesis. We constructed a mutation profile of pancreatic cancer-associated genes. Additionally, we analyzed the relationship between gene mutations and clinicopathological factors. Methods: Genomic DNA samples were extracted from fresh frozen surgical specimens obtained from 100 patients (98 with pancreatic ductal adenocarcinoma, 2 with adenosquamous carcinoma) who underwent radical operations for pancreatic cancer at the National Cancer Center Hospital between March 2005 and June 2012. Next-generation sequencer-based targeted deep sequencing was performed using a Cancer Panel reagent that covers representative cancer-related genes (50 genes, 190 hot spots). Results: Mutations were detected in 97% of the cases, and the average number of mutations per tumor sample was 1.6. The most frequently mutated genes were KRAS (96%), TP53 (42%), SMAD4 (13%), and CDKN2A (7%). The most common mutation types of KRAS were G12D (48%), G12V (32%), and G12R (10%) in our cohort. The known druggable mutations that were detected were GNAS (1%), PIK3CA (1%), and KIT (1%). Among the patients who underwent radical operations followed by adjuvant chemotherapy (71 patients), the survival of patients who had 0 to 2 mutations in the 4 major driver genes (KRAS, TP53, SMAD4, and CDKN2A) was significantly longer than that of patients who had 3 or more mutations (median overall survival, 40.0 months vs. 12.6 months, P = 0.0020). A multivariate Cox proportional hazard model analysis showed that a low number of mutations among these 4 genes was significantly associated with a better prognosis. Conclusions: KRAS, TP53, SMAD4, and CDKN2A were the most frequently mutated genes in Japanese patients with pancreatic cancer. The number of mutations among these 4 genes as detected using targeted deep sequencing may be a useful biomarker for the prediction of postoperative outcomes. Disclosure: All authors have declared no conflicts of interest.

The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing

To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.

Clinical “MUTATOME” Of Myelodysplastic Syndrome; Comparison To Primary Acute Myelogenous Leukemia

Chromosomal aberrations and somatic mutations constitute key elements of the pathogenesis of myelodysplastic syndromes (MDS), a clonal hematologic malignancy characterized by cytopenias, a dysplastic bone marrow and propensity to clonal evolution. Next generation sequencing (NGS) enables definition of somatic mutational patterns and clonal architecture as a discovery platform, and for clinical applications.We systematically applied NGS to 707 cases of MDS and MDS-related disorders. 205 cases (low-risk MDS: N=78, high-risk MDS: N=42, MDS/MPN: N=48 and sAML: N=37) were tested by whole exome sequencing (WES). For validation in an additional 502 patients (low-risk MDS: N=192, high-risk MDS: N=104, MDS/MPN: N=111 and sAML: N=95), targeted deep NGS was applied for 60 index genes which were most commonly affected in the cohort analyzed by WES. For NGS data analysis a statistical pipeline was developed to focus on: i) identification of the most relevant somatic mutations, and ii) minimization of false positive results. We studied serial samples from 21 exemplary informative patients. We also compared somatic mutational patterns to those seen in primary AML TCGA cohort (N=201). Given the size of the cohort, there was, for example, a 87% chance of seeing mutations at a frequency of 1% and a 98% of seeing those with a frequency of 2%. While focusing on the most common events, we observed 1117 somatic mutations in 199 genes. The 88 genes mutated mutated in >1% of cases with MDS carried 388 mutations in MDS+sAML (2.5/case), 128 in MDS/MPN (2.7/case) and 398 in pAML (2.0/case). The average number of mutations per case increased during progression (2.2 in lower-risk, 2.8 in higher-risk MDS, 3.4 in sAML). In MDS, the 30 most frequently affected genes were present at least once in 70% of patients. The 30 most frequently mutated genes in MDS/MPN were mutated in 82% of patients. Individual mutations were also sub-grouped according to their function. When we compared three MDS subcategories (lower-risk, higher-risk MDS and sAML) in a cross-sectional view, RTK family, RAS family, IDH family and cohesin family mutations were more frequently detected in the sAML group than in the MDS group. In contrast, the frequency of the DNMT family, TET2 and ASXL family gene mutations did not increase in frequency in the sAML cohort. In addition to better definition of mutational patterns of known genes, we have also defined new mutations, including in the RNA helicase family and the BRCC3pathway.Clonal architecture analysis indicates that mutations of TET2, DNMT3A, ASXL1, and U2AF1 most likely represent ancestral/originator events, while those of the IDH family, RTK family and cohesin family are typical secondary events. Establishment of mutational patterns may improve the precision of morphologically-based diagnosis. The comparison between MDS-related diseases (MDS+sAML) and pAML revealed a notably different mutational pattern suggestive of a distinct molecular derivation of these two disease groups. While RTK, IDH family and NPM1 mutations were more frequently observed in the pAML cohort, mutations of SF3B1 and SRSF2, were more common in MDS+sAML. With regard to the connections between individual mutation combinations, RTK mutations were strongly associated with DNMT, but not with RAS family mutations in the pAML cohort, while the mutual association between TET2 and PRC2 family, cohesin family and RUNX1were encountered in the MDS+sAML cohort.Individual mutations may have prognostic significance, including having an impact on survival, either within the entire cohort or within specific subgroups. In the combined MDS cohort, TP53 family mutations were associated with a poor prognosis (HR; 3.65, 95%CI; 1.90-7.01, P<.0001) by univariate analysis. Similar results were found for mutations in TCF4(HR; 7.98, 95%CI; 1.58-10.1, P<.0007). Such an individual approach does not allow for assessment of the impact of less common mutational events.In conclusion, our study continues to indicate the power of NGS in the molecular analysis of MDS. MDS and related disorders show a great deal of pathogenetic molecular overlap, consistent with their morphologic and clinical pictures, but also distinct molecular differences in mutational patterns. Some of the specific mutations are pathognomonic for specific subtypes while some may convey a prognostic rather than discriminatory value. Disclosures:Makishima:Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding. Polprasert:MDS foundation: Research Funding.

Mutational landscape and significance across 12 major cancer types

The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known (for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, Wnt/β-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the number of driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment.Supplementary informationThe online version of this article (doi:10.1038/nature12634) contains supplementary material, which is available to authorized users.

Mutations in the NS5A gene are associated with response to interferon+ribavirin combination therapy in patients with chronic hepatitis C virus 3a infection

The clinical correlation between hepatitis C virus (HCV) NS5A gene mutations and response to antiviral treatment in HCV-3a-infected patients is not as well known as that in HCV-1a-infected and HCV-1b-infected patients. This study attempted to determine mutations in the NS5A gene in isolates of HCV-3a obtained from infected Pakistani patients treated with interferon-α standard therapy and to determine its association with several host factors. A total of 71 consecutive chronically infected HCV patients [43 men and 28 women, mean age 38 years (range, 16-70 years)] without previous antiviral therapy, who fulfilled other criteria of the current study were enrolled for antiviral therapy. Of the 71 patients enrolled, 53 completed the course of antiviral therapy. Among them, 23 (43.3%) patients showed an end-of-treatment response, 20 (41.4%) showed a sustained virological response, and three (5.6%) patients were HCV-RNA negative at the end of treatment but developed a relapse thereafter. Only seven (13.2%) patients were virologic nonresponders (NR). Probable transmission risk factors were previous major/minor surgery (20%), transfusion of blood or blood products (2%), dental surgery (10%), or sporadic (unknown; 60%). The estimated duration of infection varied from 6 months to 20 years. The correlation between the number of mutations and several factors was also studied. The first factor that was associated with the number of mutations in the NS5A gene was age older than 40 years, where the difference was statistically significant between viral sequences of patients showing an end-of-treatment response and NR (P<0.001). Further, the difference in the average number of mutations was statistically significant between patients showing sustained virological response and NR (P<0.005). The second and third factors that were found to be associated with number of mutations in the NS5A gene were sex and viral load, respectively. Along with host factors such as age older than 40 years, female sex, and low baseline viral load, the less number of mutations in the NS5A gene of HCV-3a is associated with a positive outcome of treatment response in HCV patients receiving interferon plus ribavirin therapy.

Whole Exome Analysis Reveals Spectrum of Gene Mutations in Juvenile Myelomonocytic Leukemia

AbstractAbstract 170 Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in >70% affected children in a mutually exclusive manner. To define the molecular pathogenesis of JMML, we identified the full spectrum of gene mutations in 13 cases of JMML using whole exome sequencing of paired tumor-normal DNA. We also performed target-deep sequencing of relevant mutational targets in 92 cases of JMML. Patient and Methods:We evaluated 92 children (61 boys and 31 girls) with JMML, including 7 with Noonan syndrome-associated myeloproliferative disorder, who were diagnosed at institutions throughout Japan. The median age at diagnosis was 19 months (range, 1–160 months). Karyotypic abnormalities were detected in 15 cases, including 8 with monosomy 7. Fifty-six of the 92 (61%) cases received allogeneic hematopoietic stem cell transplantation.Exome capture from paired tumor-normal (CD3-positive T cell) DNA obtained from 13 cases of JMML was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA, USA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA, USA) according to the manufacturers’ protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate germline and somatic nucleotide changes were validated by Sanger/deep sequencing. A total of 92 JMML tumor specimens were screened for mutations in RAS pathway genes (PTPN11, NRAS, KRAS, c-CBL, and NF1) and 3 newly identified genes using deep sequencing. Results:For the current exome study, 10 missense and 1 nonsense single nucleotide variations were confirmed as nonsilent somatic mutations. The average number of mutations per sample (0.79; range, 0–4) was surprisingly low compared with those reported in other human cancers. Among the 11 somatic mutations, 6 involved the known RAS pathway genes (1 NF1, 1 NRAS, 2 KRAS, and 2 PTNP11 mutations) and included 5 mutations/deletions of either NF1 (n = 2), c-CBL (n = 1), or PTPN11 (n = 2) as detected in the germline samples. Nonoverlapping RAS pathway mutations were confirmed in 11 of the 13 discovered cases of JMML (85%). Five of the 11 somatic mutations were observed in 3 non-RAS pathway genes that have never been reported in JMML cases.Deep sequencing revealed RAS pathway mutations in 80 of 92 cases (87%) in a mutually exclusive manner; PTPN11 mutations were predominant (39/92 or 42%), followed by N/KRAS (24/92 or 26%), c-CBL (11/92 or 12%), and NF1 (6/92 or 6.5%) mutations. In agreement with previous reports, the majority of c-CBL (7/11) and NF1 (5/6) mutations were bi-allelic in the affected cases, showing compound heterozygous mutations or uniparental disomy of the mutant alleles, whereas most of the PTPN11 and N/KRAS mutations were heterozygous. In contrast, the remaining 12 (13%) cases were negative for known RAS pathway mutations. In addition, the 3 newly identified genes were recurrently in 18 cases (20%). Many of these mutations had lower allele frequencies compared to the accompanying RAS pathway mutations, indicating that they were responsible for disease progression rather than the establishment of JMML. The probability of 5-year transplantation-free survival (95% confidence interval) for the latter patients was significantly inferior to that of other cases (0% vs. 19% (8–34%), p < 0.001). Conclusion:Whole exome sequencing revealed the spectrum of gene mutations in cases of JMML. Together with a very high frequency of RAS pathway mutations, the paucity of non-RAS pathway mutations is a prominent feature of JMML. Somatic mutations of 3 newly identified genes were common among recurrent secondary events presumed to be involved in tumor progression and associated with poor clinical outcomes. Our findings provide an important clue that aids in understanding the pathogenesis of JMML and will help in the development of novel diagnostics and therapeutics for this type of leukemia. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding.

CD45RO enriches for activated, highly mutated human germinal center B cells

AbstractTo date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker.

CD45RO: A Marker for BCR‐mediated Selection

We previously showed that IgH sequence alone minimally influenced germinal centre (GC) B-cell survival fate. As end-stage effector B cells are typically more mutated than founder GC B cells, we worked to develop an assay that would enrich for populations of GC B cells with progressively increasing numbers of somatic mutations, which could potentially be used as an indicator of positive selection. We targeted CD45 as it has been shown to influence activation-induced cytidine deaminase (AID) expression. In this study, anti-CD77 and anti-CD45RO (RO) were used to subdivide CD19(+)IgD(-)CD38(+)CD77(+) centroblasts (CB) and CD19(+)IgD(-)CD38(+)CD77(-) centrocytes (CC) into three contiguous RO fractions (RO(-), RO(+/-) and RO(+)) and assessed whether mutation frequency and characteristics associated with selection varied with respect to increasing RO expression. Here, we show that the average number of mutations per IgV(H)4 transcript increased concordantly with RO for CC, but not for CB. CC also exhibited an RO-associated increase in replacement mutations. Comparative analysis of clonally related sequences revealed that increased mutations were not due to the exclusive persistence of surface RO on highly mutated cells. RO-expressing CC and CB pools showed increased signs of activation (CD69(+)) and were enriched for surface Ig(+) cells. BCR-crosslinking induced a significant increase in surface RO on total tonsillar and GC B cells, which collectively suggests that the RO-associated increase in mutations is attributable, at least in part, to the cycling of cells that may have recently undergone BCR-mediated selection, or are potentially in developmental transition between CC and CB stages.

Identifying the interacting positions of a protein using Boolean learning and support vector machines

It is known that in the three-dimensional structure of a protein, certain amino acids can interact with each other in order to provide structural integrity or aid in its catalytic function. If these positions are mutated the loss of this interaction usually leads to a non-functional protein. Directed evolution experiments, which probe the sequence space of a protein through mutations in search for an improved variant, frequently result in such inactive sequences. In this work, we address the use of machine learning algorithms, Boolean learning and support vector machines (SVMs), to find such pairs of amino acid positions. The recombination method of imparting mutations was simulated to create in silico sequences that were used as training data for the algorithms. The two algorithms were combined together to develop an approach that weighs the structural risk as well as the empirical risk to solve the problem. This strategy was adapted to a multi-round framework of experiments where the data generated in the present round is used to design experiments for the next round to improve the generated library, as well as the estimation of the interacting positions. It is observed that this strategy can greatly improve the number of functional variants that are generated as well as the average number of mutations that can be made in the library.

Genetic Analysis of the Structure and Function of Transfer Messenger RNA Pseudoknot 1

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

Why High-error-rate Random Mutagenesis Libraries are Enriched in Functional and Improved Proteins

The fraction of proteins that retain wild-type function after mutation has long been observed to decline exponentially as the average number of mutations per gene increases. Recently, several groups have used error-prone polymerase chain reactions (PCR) to generate libraries with 15 to 30 mutations per gene, on average, and have reported that orders of magnitude more proteins retain function than would be expected from the low-mutation-rate trend. Proteins with improved or novel function were isolated disproportionately from these high-error-rate libraries, leading to claims that high mutation rates unlock regions of sequence space that are enriched in positively coupled mutations. Here, we show experimentally that error-prone PCR produces a broader non-Poisson distribution of mutations consistent with a detailed model of PCR. As error rates increase, this distribution leads directly to the observed excesses in functional clones. We then show that while very low mutation rates result in many functional sequences, only a small number are unique. By contrast, very high mutation rates produce mostly unique sequences, but few retain function. Thus an optimal mutation rate exists that balances uniqueness and retention of function. Overall, high-error-rate mutagenesis libraries are enriched in improved sequences because they contain more unique, functional clones. Our findings demonstrate how optimal error-prone PCR mutation rates may be calculated, and indicate that "optimal" rates depend on both the protein and the mutagenesis protocol.

Emergence of the precore mutant late in chronic hepatitis B infection correlates with the severity of liver injury and mutations in the core region.

The reason that precore negative mutants (e-minus DNA) gradually become predominant in some patients during chronic hepatitis B virus infection is not clear. Theoretically, as long as both e-plus and e-minus DNA share the same epitopes in the core region, HBcAg-specific cytotoxic T lymphocytes (CTLs) cannot distinguish between the target peptides expressed by e-plus and e-minus DNA. Therefore, e-minus DNA may be accompanied by additional mutations in the core region, which may affect cytotoxic T lymphocyte recognition. To examine this possibility, the sequences of the precore and the entire core region of the hepatitis B virus genome were analyzed from paired serum samples in CH-B patients who experienced HBeAg to anti-HBe seroconversion (SC). Patients were divided into two groups. Group A patients (n = 17) genome-converted to e-minus DNA in the precore region, which abolished HBeAg secretion within 3-4 yr after SC. Group B patients (n = 16) retained precore wild-type DNA for more than 3-4 yr after SC. To investigate the impact of the emergence of precore mutant type DNA on liver injury, alanine aminotransferase (ALT) levels were also examined. ALT flares were more severe among patients in group A than in group B. The average mean ALT level during the HBeAg negative phase of chronic infection was 54 +/- 38 in group A and 28 +/- 24 in group B. The average maximal ALT level during the HBeAg negative phase was 235 +/- 249 in group A and 83 +/- 106 in group B. Furthermore, all 17 patients in group A developed new core mutants during genome conversion. The average number of mutations in the core gene was 0.9 +/- 1.2 before genome conversion (e-plus DNA dominant phase) and increased to 2.8 +/- 1.3 for the 3-4 yr during genome conversion (e-minus DNA dominant phase). In contrast, only 56% (nine of 16) of patients in group B developed new core mutations after the loss of HBeAg. The average number of mutations in the core gene was 1.8 +/- 1.3 before SC (HBeAg-positive and e-plus DNA dominant phase), and decreased to 1.1 +/- 1.1 for 3-4 yr after seroconversion (anti-HBe-positive and e-plus DNA dominant phase). These data indicate that the emergence of a predominant precore negative genotype late in chronic hepatitis B virus infection is associated with the selection of additional mutations in the core gene, as well as with liver injury.

Emergence of the precore mutant late in chronic hepatitis B infection correlates with the severity of liver injury and mutations in the core region

OBJECTIVE: The reason that precore negative mutants (e-minus DNA) gradually become predominant in some patients during chronic hepatitis B virus infection is not clear. Theoretically, as long as both e-plus and e-minus DNA share the same epitopes in the core region, HBcAg-specific cytotoxic T lymphocytes (CTLs) cannot distinguish between the target peptides expressed by e-plus and e-minus DNA. Therefore, e-minus DNA may be accompanied by additional mutations in the core region, which may affect cytotoxic T lymphocyte recognition. To examine this possibility, the sequences of the precore and the entire core region of the hepatitis B virus genome were analyzed from paired serum samples in CH-B patients who experienced HBeAg to anti-HBe seroconversion (SC). METHODS: Patients were divided into two groups. Group A patients (n = 17) genome-converted to e-minus DNA in the precore region, which abolished HBeAg secretion within 3–4 yr after SC. Group B patients (n = 16) retained precore wild-type DNA for more than 3–4 yr after SC. To investigate the impact of the emergence of precore mutant type DNA on liver injury, alanine aminotransferase (ALT) levels were also examined. RESULTS: ALT flares were more severe among patients in group A than in group B. The average mean ALT level during the HBeAg negative phase of chronic infection was 54 ± 38 in group A and 28 ± 24 in group B. The average maximal ALT level during the HBeAg negative phase was 235 ± 249 in group A and 83 ± 106 in group B. Furthermore, all 17 patients in group A developed new core mutants during genome conversion. The average number of mutations in the core gene was 0.9 ± 1.2 before genome conversion (e-plus DNA dominant phase) and increased to 2.8 ± 1.3 for the 3–4 yr during genome conversion (e-minus DNA dominant phase). In contrast, only 56% (nine of 16) of patients in group B developed new core mutations after the loss of HBeAg. The average number of mutations in the core gene was 1.8 ± 1.3 before SC (HBeAg-positive and e-plus DNA dominant phase), and decreased to 1.1 ± 1.1 for 3–4 yr after seroconversion (anti-HBe–positive and e-plus DNA dominant phase). CONCLUSIONS: These data indicate that the emergence of a predominant precore negative genotype late in chronic hepatitis B virus infection is associated with the selection of additional mutations in the core gene, as well as with liver injury.

Random Mutagenesis by PCR

Error-prone PCR (EP-PCR) is the method of choice for introducing random mutations into a defined segment of DNA that is too long to be chemically synthesized as a degenerate sequence. Using EP-PCR, the 5' and 3' boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly, it is possible to mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be controlled as a function of the number of EP-PCR doublings performed. The EP-PCR technique described here is for a 400-bp sequence, and an Alternate Protocol is for a library. EP-PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.

Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates with the same PFGE or ribotyping patterns in 1997 as in 1994, and ciprofloxacin had a two- to fourfold higher MIC for the isolates collected in 1997 than those from 1994. Genomic DNA was amplified for gyrA, parC, mexR, and nfxB by PCR and sequenced. Eleven isolates had mutations in gyrA, seven isolates had mutations at codon 83 (Thr to Ile), and four isolates had mutations at codon 87 (Asp to Asn or Tyr). Sixteen isolates had mutations in nfxB at codon 82 (Arg to Leu). Increased amounts of OprN were found in six isolates and OprJ in eight isolates as determined by immunoblotting. No isolates had mutations in parC or mexR. Six isolates had mutations in efflux pumps without gyrA mutations. The average number of mutations was higher in isolates from 1997 than in those from 1994. The results also suggested that efflux resistance mechanisms are more common in isolates from CF patients than in strains from urine and wounds from non-CF patients, in which mutations in gyrA and parC dominate (S. Jalal and B. Wretlind, Microb. Drug Resist. 4:257-261, 1998).