Average Mutation Research Articles

Peto's "Paradox" and Six Degrees of Cancer Prevalence.

Peto's paradox and the epidemiologic observation of the average six degrees of tumor prevalence are studied and hypothetically solved. A simple consideration, Petho's paradox challenges our intuitive understanding of cancer risk and prevalence. Our simple consideration is that the more a cell divides, the higher the chance of acquiring cancerous mutations, and so the larger or longer-lived organisms have more cells and undergo more cell divisions over their lifetime, expecting to have a higher risk of developing cancer. Paradoxically, it is not supported by the observations. The allometric scaling of species could answer the Peto paradox. Another paradoxical human epidemiology observation in six average mutations is necessary for cancer prevalence, despite the random expectations of the tumor causes. To solve this challenge, game theory could be applied. The inherited and random DNA mutations in the replication process nonlinearly drive cancer development. The statistical variance concept does not reasonably describe tumor development. Instead, the Darwinian natural selection principle is applied. The mutations in the healthy organism's cellular population can serve the species' evolutionary adaptation by the selective pressure of the circumstances. Still, some cells collect multiple uncorrected mutations, adapt to the extreme stress in the stromal environment, and develop subclinical phases of cancer in the individual. This process needs extensive subsequent DNA replications to heritage and collect additional mutations, which are only marginal alone. Still, together, they are preparing for the first stage of the precancerous condition. In the second stage, when one of the caretaker genes is accidentally mutated, the caused genetic instability prepares the cell to fight for its survival and avoid apoptosis. This can be described as a competitive game. In the third stage, the precancerous cell develops uncontrolled proliferation with the damaged gatekeeper gene and forces the new game strategy with binary cooperation with stromal cells for alimentation. In the fourth stage, the starving conditions cause a game change again, starting a cooperative game, where the malignant cells cooperate and force the cooperation of the stromal host, too. In the fifth stage, the resetting of homeostasis finishes the subclinical stage, and in the fifth stage, the clinical phase starts. The prevention of the development of mutated cells is more complex than averting exposure to mutagens from the environment throughout the organism's lifetime. Mutagenic exposure can increase the otherwise random imperfect DNA reproduction, increasing the likelihood of cancer development, but mutations exist. Toxic exposure is more challenging; it may select the tolerant cells on this particular toxic stress, so these mutations have more facility to avoid apoptosis in otherwise collected random mutational states.

Mutational analysis and assessment of its impact on proteins of SARS-CoV-2 genomes from India.

Mutational status of SARS-CoV-2 genomes from India along with their impact on proteins was ascertained through multiple tools including MEGA, Genome Detective, SIFT, PROVEAN and ws-SNPs&GO. Excluding gaps and ambiguous sequences, 493 variable sites (152 parsimony informative and 341 singleton) were observed. NSP3 had the highest incidence of 101 sites followed by S protein (74), NSP12b (43) and ORF3a (31). Average mutations per sample for males and females was 2.56 and 2.88 respectively. Non-uniform geographical distribution of mutations suggests that sequences in some regions are mutating faster than others. There were 281 mutations (198 Neutral and 83 Disease) affecting amino acid sequence. NSP13 has a maximum of 14 Disease variants followed by S protein and ORF3a with 13 each. Disease mutations in genomes from asymptomatic people was mere 11% but those from deceased patients was at 38% indicating contribution of these mutations to the pathophysiology of the SARS-CoV-2.

Mutation spectrum of EGFR in 21,324 patients with non-small cell lung cancer successfully tested by multiple platforms in a CAP-accredited laboratory.

e13637 Background: Genotyping epidermal growth factor receptor ( EGFR) gene in patients with advanced non-small cell lung cancers (NSCLC) is essential to identify those patients who may benefit from targeted treatments. However, the testing methodology is still a puzzle for many clinicians. This study retrospectively reviewed the EGFR data from testing patients with NSCLC in our laboratory in 10 years. Methods: A total of 21,324 NSCLC cases successfully underwent EGFR genotyping in our laboratory for clinical therapeutic purpose, including 5,244 cases tested by Sanger sequencing, 13,329 cases tested by real-time PCR, and 2,751 tested by next generation sequencing-NGS. Results: 54.6%, 20% and 2.3% samples were recorded with tumor cell content (TCC) of < 40%, < 20% and 1% respectively. The average EGFR mutation rate was 45.1%, with 40.3% identified by Sanger sequencing, 46.5% by real-time PCR and 47.5% by NGS. Comparable EGFR mutation rates were observed in cases with 1%-20% TCC from real-time PCR and NGS platforms. In these cases with EGFR mutations, 93.3% of them carried a single EGFR mutation (92.1% with a classical mutation as 19del or L858R, and 7.9% with an uncommon mutation) and 6.7% harbored complex EGFR mutations. 65.3% of the cases carrying EGFR mutations identified by NGS also have tumor related variants in other genes. 26.5% more cases were found to have oncogenic driver mutations with therapeutic significance beyond EGFR by NGS. Conclusions: NGS may be the most optimal methodology to detect EGFR mutations in patients with NSCLC compared with Sanger sequencing and real-time PCR platforms from accuracy and comprehensiveness aspects, but some other issues, such as Turn Around Time (TAT), still should be considered in clinical practice.

Compensated pathogenic variants in coagulation factors VIII and IX present complex mapping between molecular impact and hemophilia severity

Compensated pathogenic deviations (CPDs) are sequence variants that are pathogenic in humans but neutral in other species. In recent years, our molecular understanding of CPDs has advanced substantially. For example, it is known that their impact on human proteins is generally milder than that of average pathogenic mutations and that their impact is suppressed in non-human carriers by compensatory mutations. However, prior studies have ignored the evolutionarily relevant relationship between molecular impact and organismal phenotype. Here, we explore this topic using CPDs from FVIII and FIX and data concerning carriers’ hemophilia severity. We find that, regardless of their molecular impact, these mutations can be associated with either mild or severe disease phenotypes. Only a weak relationship is found between protein stability changes and severity. We also characterize the population variability of hemostasis proteins, which constitute the genetic background of FVIII and FIX, using data from the 1000 Genome project. We observe that genetic background can vary substantially between individuals in terms of both the amount and nature of genetic variants. Finally, we discuss how these results highlight the need to include new terms in present models of protein evolution to explain the origin of CPDs.

Abstract 5372: The mutational landscape of primary cutaneous melanoma

Abstract Large-scale melanoma genome profiling studies to date have mainly focused on metastatic tumors. To better understand the link between genomic changes and outcome, primary tumors must be studied. We have designed a comprehensive targeted genomic panel to study genetic alterations in melanoma, and applied this to a large collection of 536 primary melanomas, part of the Leeds Melanoma Cohort. Patients were invited to participate 3 months after diagnosis of primary melanoma. 1% received targeted therapies or immunotherapy and thus, our analysis is reflective of the landscape of primary melanoma in the treatment naïve setting. The sequencing panel allowed us to study genetic alterations in over 500 genes, including more than 150 genes frequently mutated in melanoma and an additional 250 genes linked to other solid tumor types. Both tumor and germline DNA were analyzed for most samples. The average somatic mutation rates we observed was 15 mutations/MB (0 to 236 mutations/MB), while the nonsynonymous mutational load ranged from 0 to 57 mutations/MB (average 4 mutations/MB). We found that a high mutational burden (highest 20%, >45 nonsynonymous mutations/Mb) positively correlated with better survival (Cox proportional hazards model with age, sex and AJCC stage as additional covariates). Among the top recurrently mutated genes were well established melanoma driver genes such as BRAF (44%), NRAS (28%), NF1 (10%), CDKN2A (15%), TP53 (11%) and ARID2 (8%). We also found hotspot mutations in less well documented melanoma genes, including RQCD1 p.P131L (4.5%). Altogether, our preliminary data shows a strong correlation between survival and high mutational load in primary melanomas. Our data also suggests a similar landscape of driver mutations in primary and metastatic melanoma, with some variants/genes possibly reflecting the differences in disease stage. We will further investigate the mutational landscape in our cohort of primary melanomas, including analyses of gene co-occurrence and mutual exclusivity. Additionally, we will use extensive clinical parameters and transcriptomics to examine the possibility of predicting survival and other clinically relevant parameters in our cohort based on patient mutational profiles. Citation Format: Sofia Y. Chen, Rashid Mamunur, Jon Laye, D. Timothy Bishop, Julia A. Newton-Bishop, David J. Adams. The mutational landscape of primary cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5372.

Ems and gamma radiation induced mutation in grasspea (Lathyrus sativus L.)

Present investigation has been undertaken to study the induction of mutation by individual and combination treatments of EMS and gamma rays in three diverged genotypes of grasspea viz. Nirmal, Biol-212 and Berhampur Local. Combination treatments caused the more biological damage (lethality, injury and sterility) followed by gamma rays and EMS alone. The LD50 dose of gamma rays for Nirmal, Biol-212 and Berhampur Local corresponded to 590 Gy, 630 Gy and 539 Gy respectively. Above 0.5% EMS for Nirmal and Biol-212 and above 1% EMS in case of Berhampur Local was found to be lethal. Average total mutation frequency of three varieties together was highest (6.32 %) in gamma radiation followed by both EMS and combination treatment (5.8%). The most efficient dose/concentration of gamma rays and EMS were found to be 400 Gy and 0.5% EMS respectively. Mutagenic efficiency increases and effectiveness decreases with increasing concentration of EMS but the consequence was showed almost inverse relationship in case of gamma rays. Overall, irrespective of genotypes, gamma rays and EMS respectively were found to be most efficient and effective mutagen.

Mutational landscape of metastatic colorectal cancer: Aggregate insights from a molecular tumor board.

837 Background: The mutational landscape of metastatic colorectal cancer (CRC) is being elucidated by next-generation sequencing of both primary tumors and metastatic foci. In this study, we examined the results of all stage IV CRC cases submitted to our molecular tumor board (MTB). Methods: We performed a retrospective analysis of all patients who had next-generation sequencing performed between January 2015 and August 2017 on either their primary tumor and/or metastasis. Cases were presented at a MTB convened twice monthly. For the purposes of this study only pathogenic mutations were notated, not variants of unknown significance (VUS). Results: Eighty-seven unique patients had 97 specimens sequenced (28 primary tumors and 69 metastases). The primaries averaged 3 mutations per specimen (range: 1-6) whereas the metastases averaged 4 (range: 1-14, p=.25). The most common anatomic sites of submitted metastatic tissue were the liver (n=35, average mutations=4), followed by the lungs (n=10, average mutations=3) and the omentum/peritoneum (n=8, average mutations=3). Two patients had both a primary tumor and a metastasis sequenced, with a 33% rate of concordance in inter-specimen mutations. Five patients had multiple metastases sequenced, with a 53% rate of concordance in inter-specimen mutations; in every case of longitudinal sequencing, mutational burden increased in metastases over time. The most common mutation was apc (21% of all mutations), followed by p53 (16%) and then kras (13%). Candidacy for EGFR-directed therapy was found in 8 cases, and mismatch repair defects were detected in 5 cases. Conclusions: In stage IV CRC cases sent to our MTB, tissue from metastases was more commonly submitted for analysis than from the primary tumors. There is not necessarily concordance between mutations in primary tumors and metastases, nor among multiple metastases in the same patient. Sequencing at multiple time points in the disease course may allow observation of clonal evolution and dynamic adaptation of therapeutic targets.

Chloroplast genomes of Lilium lancifolium, L. amabile, L. callosum, and L. philadelphicum: Molecular characterization and their use in phylogenetic analysis in the genus Lilium and other allied genera in the order Liliales.

Chloroplast (cp) genomes of Lilium amabile, L. callosum, L. lancifolium, and L. philadelphicum were fully sequenced. Using these four novel cp genome sequences and five other previously sequenced cp genomes, features of the cp genomes were characterized in detail among species in the genus Lilium and other related genera in the order Liliales. The lengths and nucleotide composition showed little variation. No structural variation was found among the cp genomes in Liliales. Gene contents were conserved among four newly sequenced cp genome in Lilium species, the only differences being in two pseudogenes. We identified 112 genes in 13 functional categories, 18 of which carried introns that were conserved among the species in Liliales. There were 16–21 SSR loci (>12 bp, >3 repeats) in the cp genomes in Lilium and the genomic locations of these loci were highly variable among the species. Average mutations were 15 SNPs per 1kb and 5 indels per 1kb, respectively, in the cp genomes of the newly sequenced four Lilium species. Phylogenetic classifications revealed some discrepancies between trees based on the cp genomes and previous classifications based on the morphology and geographic distributions.

Abstract 4371: Genomic characterization of oropharyngeal squamous cell carcinoma in Korean population

Abstract Objective: Oropharyngeal squamous cell carcinoma (OPSCC) is a subtype of head and neck squamous cell carcinoma (HNSCC). Environmental risk factors such as tobacco, alcohol, and especially, human papillomavirus (HPV) are known to be associated with HNSCC. Also, large-scale sequencing studies identified several HNSCC-associated genes such as TP53, EGFR and PIK3CA. However, still the initiation and progression of OPSCC and mutational profiles of OPSCC in Korean population are unclear. Methods: Frozen non-tumor mucosa, tumor tissues and blood were collected from nine OPSCC patients; 6 HPV positive and 3 negative. Adjacent non-tumor tissues were obtained about 1 cm apart from the tumor tissue and were histologically confirmed as non-neoplastic. We then performed whole exome sequencing with Agilent SureSelect Human All Exon Kit v4+UTR, yielding average on-target coverage of 100X. Results: First, we compared somatic alterations of adjacent non-tumor and tumor tissue to understand the genomic status of non-tumor mucosa. Comparison of genome-wide copy number alterations (CNA) between non-tumor and OPSCCs showed that majority of somatic CNA (SCNA) was occurred after tumor progression. Also, non-tumor mucosa rarely harbored genomic changes, showing average nonsilent mutations rate of 0.07 per Mb. In contrast, OPSCC showed 1.0 mutations per Mb with few shared mutations with non-tumor. These results indicate occurrence of new distinct subclones after tumor progression. Second, somatic mutations and CNA analysis of 9 OPSCC identified two distinct groups divided by the HPV status of tumor. HPV(+) tumors showed lower mutation burden than HPV(-) tumors (0.63 and 1.73 mutations per Mb, respectively). And TP53 and CDKN2A mutations were detected exclusively in HPV(-) group, accounting for 66.6% of HPV(-) tumors. Furthermore, HPV(-) tumors showed greater degree of chromosomal alterations compared to HPV(+) tumors. Conclusions: Although there are many studies to understand the relationships between premalignant and malignant tissue, there is a lack of understanding for genetic changes further from non-neoplastic mucosa. Our data show that genetic profile of normal crypt mucosa rarely changes and tumor develops in distinct subclones. This would provide a glimpse into the fundamental cellular changes of non-tumor cells of tonsillar crypts and oral cavity. Also, we provide a mutational landscape in HNSCC of Korean population. Although our OPSCC showed recurrent mutations and copy number alterations of genes related to RTK/PI(3)K- and differentiation-signaling pathways, similar as western, mutation burden in tumors was relatively lower, and somatic mutations on well-known HNSCC-associated genes such as PIK3CA, FAT1 and HRAS weren’t detected. Further investigation and molecular experiments are required to understand the mechanism of carcinogenesis and to discover early driver mutations of HNSCC. Citation Format: Jeesoo Chae, Weon Seo Park, Dongwan Hong, Jong-Il Kim, Yuh-Seog Jung. Genomic characterization of oropharyngeal squamous cell carcinoma in Korean population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2017-4371

Abstract 2994: Clinical applications of comprehensive genome analysis of papillary renal cell carcinoma, type II

Abstract Purpose Papillary renal cell carcinoma (PRCC) is the most common type of non-clear cell renal cell carcinoma (NCCRCC). Of PRCC, PRCC,type2(PRCC2) is known to have highly aggressiveness and relative drug resistance. In contrast of clear cell renal cell carcinoma (CCRCC), we poorly understand genetic alterations to drive PRCC2. Moreover, though many tyrosine kinase inhibitors (TKIs) are now available to treat PRCC, the predictive marker of TKIs is not in existence. Therefore, we aimed to analyze genetic alteration of PRCC2. Patients and Methods We performed whole-exome sequencing (WES) of DNA from 9 PRCC2 samples and matched normal tissue. In addition, whole-transcriptome sequencing (WTS) was conducted for 7 of these samples. We analyzed somatic mutations, copy number alterations, germ line mutations using WES data, pathway alterations and gene fusions through RNA expression data. We investigated the relationship between clinical prognosis and genetic alterations. Results Of nine samples, average non-silent mutation rate is 1.13 mutations per Mb (range, 0.49 to 2.14 mutations per Mbs). Forty-seven somatic mutations including SRRD(four), PKD1L2(three) and MX1(three) are discovered in more than 2 specimens. The genes associated to histone modification and ubiqutination are frequently altered. We found FH somatic mutation, a potent target of VEGFR TKI, in young female PRCC2 patient. Her tumor is extremely shrunk after 6cycles treatment and after that she received pazopanib for 52weeks maintaining effective response. Conclusion We suggest the possibility of translational research from sequencing data to real medical treatment of PRCC2. This pivotal study demonstrates that novel somatic FH mutation is the therapeutic target of VEGFR TKI. Therefore, further large scaled comprehensive genetic analysis of PRCC2 is required to propose personalized medicine for conquering papillary renal cell carcinoma, type 2. Note: This abstract was not presented at the meeting. Citation Format: Ji-Yeon Kim, Se-Hoon Lee, Soojin Cha, Kyung Chul Moon, Jong-Il Kim, Dae Seog Heo. Clinical applications of comprehensive genome analysis of papillary renal cell carcinoma, type II. [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 2994. doi:10.1158/1538-7445.AM2015-2994

Abstract 3087: Whole genome sequencing of rhabdoid tumors of the kidney

Abstract Rhabdoid tumors (RT) of the kidney (RTK) are aggressive pediatric solid tumors that predominantly affect infants. There is no effective chemotherapy and the overall 4-year survival rate is 23%. RT has a characteristic loss of SMARCB1 function, found in >90% of the patients. SMARCB1 is a conserved core subunit of the SWI/SNF chromatin-remodeling complex, which in turn is responsible for proper chromatin assembly and dynamic regulation of gene expression. Previous studies showed a remarkable paucity of mutations in coding regions of genomes and a highly penetrant cancer susceptibility in a conditional knockout mouse model. These findings support the interpretation that SMARCB1 is a tumor suppressor whose inactivation is the primary driver in RT and that RT follows a tumorigenesis model in which cancer is driven by aberrant epigenetic regulation and gene expression instead of accumulation of somatic mutations. Characterizing interplays of mutations, gene expression and epigenetic regulation will be important in understanding RT development and biology. To achieve this goal, we will comprehensively characterize genetic and epigenetic aberrations in RT using HiSeq sequencing technology. Our research efforts include profiling whole genome, whole transcriptome, promoter methylation and histone modification in 40 primary RTK samples. Here, we report preliminary results from whole genome analyses. Using an amplification-free library construction method, we sequenced whole genomes of 40 RTK and matched normal cases to an average haploid coverage of 39.4X. The RTK genomes were mostly diploid, but we found 35 loci that are either recurrently focally amplified or deleted using GISTIC 2.0 at FDR ≤0.05. Using the Trans-ABySS de novo short-read assembler, we assembled the RT cases’ whole genomes and identified a total of 19 genes that were recurrently rearranged in 8 out of 40 cases. Eleven of the genes were either known tumor suppressors (e.g. CABIN1, BCR) or associated with developmental or neurodegenerative diseases (e.g. UPB1, SPECC1L). The genome-wide single nucleotide variant and indel analysis showed an average somatic mutation rate at 0.37 per Mb in RTK, comparable to the previous finding of 0.19 per Mb in AT/RT. Approximately 99% of the somatic mutations occurred in non-genic regions. SMARCB1 had homozygous loss of function in 83% of cases by somatic homozygous deletion, or heterozygous deletion or truncating point mutations followed by loss of heterozygosity. The remaining cases appeared to have at least 1 copy of the gene unaffected and analyses are ongoing to investigate the inactivation mechanism in these cases. Citation Format: Hye-Jung E. Chun, Kelsey Zhu, Jenny Q. Qian, Karen L. Mungall, Yussanne Ma, Yong-Jun Zhao, Andrew J. Mungall, Richard A. Moore, Jacquie E. Schein, Daniela S. Gerhard, Elizabeth J. Perlman, Marco A. Marra. Whole genome sequencing of rhabdoid tumors of the kidney. [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 3087. doi:10.1158/1538-7445.AM2014-3087

Investigation on Eutrophication Level for Gucheng Lake by the Model of Butterfly Catastrophe

Based on the field measured results in the typical periods of flood season and dry season of Gucheng Lake, the present eutrophication levels of the lake were investigated by the Model of Butterfly Catastrophe, in which series of the environmental factors were considered including the ecological indicator of chlorophylla, the nutrient indicator of total nitrogen and total phosphorus, and the physical index of water transparency. The results showed that: the water eutrophication levels of Gucheng Lake were mal-distributed in space and time; the lake eutrophication tendency in flood seasons were more obvious than that in dry seasons; the eutrophication levels in the south part of the lake were relatively lower than that in the north and the average comprehensive mutation of fuzzy membership value in the south was about 0.3006 in the dry seasons. Due to the increased pollution load and the hydrodynamic conditions, the eutrophication degree in the estuary areas were higher and the fuzzy membership value was close to 0.235, achieving the medium eutrophication level.

The genomic relationship among matched prostate cancer foci.

5028 Background: Cancer management is influenced by how one views progression and how one calculates the risk of metastases and death. For prostate cancer, this is based largely on histologic appearance, or Gleason score. Cancers with a Gleason score of 6 exhibit indolent behavior and are often considered low risk. Despite recommendations supporting active surveillance for Gleason 6 prostate cancer, the vast majority of American patients receive aggressive local therapy, in part based on a presumption that low grade cancer progresses to high grade, lethal disease. Methods: To assess the genomic relationship between low and high grade disease, laser capture microdissection was used to isolate concurrent cancer foci from prostates with multifocal disease, and somatic mutations were identified using exome sequencing. The relationship between a Gleason 6 focus and a concurrent Gleason 8 or higher focus was determined for four subjects, and a lymph node metastasis was examined for two of those subjects. Results: We obtained an average of 41-fold median coverage of the exome, with an average high confidence mutation rate of 0.8/Mb. Seventy of 79 (0.886) high confidence somatic mutations in low grade disease were private to the low grade foci. For the cases for which a metastatic focus was available, 15 of 80 (0.188) high confidence somatic mutations in the high grade focus were private. Seven of the 80 (0.088) were shared with low grade foci, and 65 (0.813) were shared with metastatic foci. Conclusions: The pattern of shared versus private mutations is consistent with early divergence between Gleason 6 and Gleason 8 or 9 disease, and late divergence between Gleason 8 disease and lymph node metastases. These data support a model of parallel evolution of lower and higher Gleason score disease, rather than progression from Gleason 6 to higher Gleason scores.

Abstract 4594: Recurrent FGFR1 hotspot mutations represent a novel therapeutic target in childhood astrocytoma.

Abstract Introduction: Pilocytic astrocytoma (PA) is the most common childhood brain tumor. This tumor can occur throughout the central nervous system, with roughly 50% of cases arising outside of the cerebellum. Tumors in these non-cerebellar locations are often difficult to treat surgically, leading to multiple tumor recurrences. PA can therefore become a chronic disease, with patients experiencing substantial morbidities. Alterations in the MAPK pathway, particularly BRAF, have previously been identified in ∼80% of PAs. Interestingly, however, the majority of those cases without a recognized change are non-cerebellar. Methods: To investigate the full range of genetic alterations occurring in PA, we used Illumina HiSeq technologies to perform whole-genome sequencing of matched tumor and germline DNA from 47 patients, with corresponding RNA sequencing data for 35 tumors. Results: The average somatic mutation rate in PA was extremely low, at 0.065/Mb, with an average of only 1.8 non-synonymous coding single nucleotide variants (SNVs) per tumor - almost ten times lower than we have recently reported for medulloblastoma. We found several novel alterations in known PA-related genes, including two new oncogenic BRAF fusions. Most strikingly, however, we identified mutations at two hotspots in the FGFR1 receptor tyrosine kinase in 4/6 centrally located PAs lacking any other MAPK pathway change. Two of these cases also carried a mutation in a downstream adaptor protein, PTPN11 (Shp2). Interestingly, germline mutations of PTPN11 are associated with Noonan syndrome (NS), and there are case reports of NS patients developing PAs. Screening of additional non-cerebellar PAs revealed four further cases with an FGFR1 mutation. All PAs, regardless of MAPK pathway alteration, displayed highly elevated expression of FGF2, indicating a general role for ligand-mediated activation of the FGFR1/MAPK pathway in PA tumorigenesis. Notably, the same FGFR1 mutations were also identified in four midline pediatric glioblastomas (GBM), a highly malignant brain tumor, suggesting a possible common origin for a subset of these two entities, despite their dramatically different clinical course. Conclusion: Altogether, MAPK alterations were identified in 96% of PAs, with very few other changes, confirming the concept of PA as a single-pathway disease. Our results also suggest that a subset of centrally located, FGFR1-driven pediatric PAs and GBMs may share common origins. Most importantly, they reveal a novel therapeutic target in clinically relevant subsets of childhood glioma. Citation Format: David TW Jones, Barbara Hutter, Natalie Jäger, Andrey Korshunov, Marcel Kool, Sally R. Lambert, Dong Anh Khuong Quang, Adam M. Fontebasso, Marina Ryzhova, Hans-Jörg Warnatz, Thomas Zichner, Jan O. Korbel, Stephan Wolf, Marie-Laure Yaspo, Keith L. Ligon, Mark W. Kieran, Benedikt Brors, Jörg Felsberg, Guido Reifenberger, V. Peter Collins, Nada Jabado, Roland Eils, Peter Lichter, Stefan M. Pfister, ICGC PedBrain Tumor Project. Recurrent FGFR1 hotspot mutations represent a novel therapeutic target in childhood astrocytoma. [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 4594. doi:10.1158/1538-7445.AM2013-4594

The molecular relationship among concurrent prostate cancer foci.

43 Background: Prostate cancer management is influenced by how one views prostate cancer progression and how one calculates the risk of prostate cancer metastases and death. Currently this is based largely on histologic appearance, or Gleason score. Cancers with a Gleason score of 6 exhibit indolent behavior and are often considered low risk. NCCN guidelines endorse surveillance for many of these tumors. Malignancies with a Gleason score of 8-10 exhibit aggressive behavior and are considered high risk. Methods: Prostate cancer is usually multifocal, and these concurrent foci are often histologically distinct. In this study the molecular relationship between a Gleason 6 focus and a concurrent Gleason 8 or higher focus was determined for four subjects. In two subjects a lymph node metastasis was also examined. Laser capture microdissection was used to isolate these multiple matched foci, and somatic mutations were identified using exome sequencing. Results: We obtained an average of 40-fold median coverage of the exome, with an average high confidence mutation rate of 0.64/Mb, excluding nonsynonymous mutations. Seventy of 80 (0.875) high confidence somatic mutations in low grade disease were private to the low grade foci. For the cases for which a metastatic focus was available, 17 of 79 (0.215) high confidence somatic mutations in the high grade focus were private. Eight of the 79 (0.101) were shared with low grade foci, and 62 (0.785) were shared with metastatic foci. Conclusions: The pattern of shared versus private mutations was consistent with early divergence between Gleason 6 and Gleason 8 or 9 disease, and late divergence between Gleason 8 disease and lymph node metastases. These data support a model of parallel evolution of lower and higher Gleason score disease, rather than progression from Gleason 6 to higher Gleason scores.

PTEN germline mutations in patients first tested for other hereditary cancer syndromes: Would use of risk assessment tools have reduced health care costs?

1516 Background: PTEN Hamartoma Tumor Syndrome (PHTS) includes patients with Cowden (CS) or Bannayan-Riley-Ruvalcaba syndrome (BRRS) with germline PTEN mutation. Breast, colon, endometrial cancer and GI polyposis are associated, creating overlap with Hereditary Breast and Ovarian Cancer (HBOC), Lynch (LS) and adenomatous polyposis syndromes (APS). We reviewed our PHTS patient series to find how often testing criteria for these syndromes were met or testing was pursued before PTEN. Methods: Patients were prospectively recruited by relaxed International Cowden Consortium criteria or presence of known germline PTEN mutation; mutations were identified by mutation scanning or MLPA analysis and confirmed by sequencing or quantitative PCR, respectively. Families were excluded if diagnosed with CS or BRRS before 1998, family history was unavailable, or proband was <18 yrs. Pedigree, genetic testing reports, and medical records were reviewed to determine if patients met HBOC testing criteria, Amsterdam II or Bethesda 2004 criteria, or had adenomatous polyps. Risk assessment was conducted for BRCA1/2 via the BRCAPRO, Myriad II, and Penn II models; for MLH1/MSH2/MSH6 via the PREMM1,2,6 and MMRPro models; and for PTEN via the Cleveland Clinic (CC) PTEN Risk Calculator. Results: 115 PTEN mutation-positive adult probands were identified among 3,405 patients. 53 (46.1%) met testing criteria for HBOC or LS and 34 (29.6%) underwent previous testing for HBOC, LS or APS. Average risk for BRCA1/2 and MLH1/MSH2/MSH6 mutations in tested patients were 7.5-16.9% and 33.1-41.4%, respectively; whereas PTEN mutation risks were 35.9% and 79.7%. No patient tested for APS (average PTEN mutation risk 73%) had >5 adenomas. An estimated $89,700 was spent on negative analyses for HBOC, LS and APS among patients ultimately found to have germline PTEN mutations. Conclusions: PHTS should be part of the differential diagnosis for patients referred for question of HBOC, LS or APS. Pathology review is key for patients with polyposis. Using the CC PTEN risk calculator enables clinicians to understand when PTEN mutation risk is high, allowing testing to proceed in a step-wise and cost-saving manner.

Abstract A84: Reduced LINE-1 methylation in peripheral blood mononuclear cells is associated with risk of melanoma in families segregating CDKN2A germline mutations

Abstract Background: CDKN2A is a major susceptibility gene for familial melanoma, but its incomplete penetrance suggests that other factors may modify the effect of this gene in melanoma-prone families. Epigenetic changes involving reduced levels of global DNA methylation in blood have been associated with genomic instability and cancer risk. In addition, genetic background has the potential to influence epigenetic processes through cis- or trans-regulatory variation. Therefore, we hypothesize that the CDKN2A carrier mutation state may be an important factor influencing global DNA methylation in predisposed individuals and that aberrant methylation may be an indicator of modified risk in individuals from melanoma-prone families segregating CDKN2A germline mutations. Methods: We measured the overall level of genomic DNA methylation using bisulfite pyrosequencing at four CpG sites (CpG1, CpG2, CpG3 and CpG4) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in peripheral blood mononuclear cells (PBMCs) from 42 CDKN2A mutation positive cases (20 males and 22 females) and 73 controls (29 CDKN2A carrier controls [9 males and 20 females] and 44 non-carrier controls [19 males and 25 females]) in 26 families with CDKN2A mutations. LINE-1 methylation was reported as the average (of all 4 CpGs) and as site-specific percentage methylation. Odds' ratios (OR) and P-values were obtained by comparing melanoma cases to controls using unconditional logistic regression with adjustment for family correlation (using the generalized estimating equations [GEE] approach), age, sex, number of moles and with melanoma status as the outcome variable. Percentage LINE-1 methylation was categorized using tertiles with the highest tertile as the reference. Results: Male gender was significantly associated with higher average LINE-1 methylation (P=<0.001) and methylation at all CpG sites (P=<0.03). In contrast, age, solar injury, dysplastic nevi (DN) and number of moles were not associated with average LINE-1 methylation. Although mean levels of LINE-1 methylation were similar in melanoma cases, carrier controls, and non-carrier controls, after adjusting for family correlation, age, sex and number of moles, low average LINE-1 methylation (tertile 1) was significantly associated with increased risk of melanoma (OR=3.87, 95% confidence interval [CI]: 1.11–13.5, P=0.033, compared to all controls; OR=1.83, 95% CI: 1.09–11.7, P=0.035, compared to carrier controls; and OR=3.43, 95% CI: 1.17–10.0, P= 0.024, compared to non-carrier controls). This association was observed for most CpG sites with the most significant association seen for site 3 and risk of melanoma compared to all controls (OR=5.18, 95% CI: 1.54–17.4, P=0.008). We observed no significant association between average or CpG site-specific LINE-1 methylation and CDKN2A mutation status among controls although the comparison was limited by small sample size. Conclusions: Melanoma cases from families segregating CDKN2A germline mutations have significantly lower LINE-1 global methylation in their PBMCs compared to controls. Changes in LINE-1 methylation in PBMCs from CDKN2A mutation positive cases may reflect a potential genetic background influence on global DNA methylation in these individuals. Further studies are needed to clarify these preliminary observations. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A84.

Abstract 3931: Whole genome and whole exome sequencing of head and neck cancer

Abstract Head and neck cancer is responsible for an enormous cancer burden worldwide, with an estimated 500,000 new cases per year. However, knowledge of the spectrum of genetic alterations that drive this malignancy remains incomplete. Previous studies of this disease have identified recurrent somatic mutations in a few specific genes, but few systematic genome-scale studies have been performed. Accordingly, we have undertaken a pilot study of complete genome/exome characterization in this malignancy. We obtained genomic DNA from 40 head and neck tumors together with matched normal DNA from whole blood. To date, we have obtained whole exome sequencing of 28 tumor/normal pairs and whole genome sequencing of two tumor/normal pairs. The average base mutation rate is 3.1 × 10-6 (range = 2.9 × 10-7 to 8.8 × 10-6), consistent with rates observed in lung cancer and other tobacco-related malignancies. We also identified 24 high-confidence somatic rearrangements in one sample and 120 in the other sample (including 7 and 44 inter-chromosomal rearrangements, respectively). In addition to frequent mutations of TP53 (53%), HRAS, PIK3CA, and PTEN, we also observed other recurrently mutated genes that implicate several oncogenic pathways. Furthermore, layering of somatic mutation data onto known pathway information strengthens the evidence for several oncogenic processes not previously implicated in head and neck cancer. Altogether, these data should enhance our understanding of the mechanisms that drive head and neck carcinogenesis and progression and may also illuminate new therapeutic opportunities. 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 3931. doi:10.1158/1538-7445.AM2011-3931

Inverse Relationship between Viral Load and Genotypic Resistance Mutations in Korean Patients with Primary HIV Type 1 Infections

The transmission of antiretroviral-resistant HIV-1 strains is associated with suboptimal virological responses to initial antiretroviral therapy. However, certain types of resistance mutations are known to be associated with decreased viral fitness, which confers a lower replication capacity than that of the wild-type virus in the absence of antiretroviral drugs. Therefore, we evaluated the relationship between antiretroviral resistance mutations and viral replication in the primary HIV-1 infection (PHI) period. From January 2002 to March 2005, 52 PHI patients were identified in the Republic of Korea. HIV-1 RNA genotyping was performed, and the resistance mutation score was obtained from the HIV Drug Resistance Database of Stanford University. We defined the sum of the average resistance mutation scores (SARMS) for each antiretroviral drug class as a measure of the degree of resistance of any specific strain. The overall mean SARMS was 2.00 +/- 2.74, and the annual mean did not change significantly during the study period. No critical resistance mutation gene was identified in the study group. The SARMS showed a weak negative correlation with the viral load log10 during PHI, but without statistical significance (r = -0.274, p = 0.051). But the mean SARMS of patients with a viral load exceeding 100,000 copies/ml was significantly lower than that of patients with a viral load of less than 100,000 copies/ml (p = 0.03). Evaluation of the potency of antiretroviral resistance revealed a weak negative correlation with viral replication in the PHI period. This could be one reason why the transmission of resistant strains in PHI patients is not increasing significantly despite the widespread use of highly active antiretroviral therapy (HAART).