Number Of Mutational Events Research Articles

Abstract PO-005: Spatio-temporal geographical molecular mapping of primary papillary thyroid carcinomas and paired distant metastases: Clinical relevance for monitoring and targeting tumor heterogeneity

Abstract Approximately 15% of papillary thyroid carcinomas (PTCs) develop distant metastases (DMs). Little is known about the genetic alterations that underlie the metastatic spread. Despite DMs may differ from primary tumors (Pts) at the molecular level, in most patients with metastatic PTCs genetic alterations are often exclusively assessed in Pt samples and the results correlated by means of statistical analyses with a known deleterious prognostic parameter as it is the presence or not of DMs. The involvement of intratumor genetic heterogeneity (ITGH) or clonal status of actionable driver events in DMs of PTCs nowadays is still puzzling. Dynamic changes in mutation distribution through space and time have not been in deep characterized. So far no one has performed a geographical multiregional mapping of paired primary PTCs and DMs using genetic alteration profiling at different time points or different evolutionary stages In the attempt to shed some light in the complex aforementioned issues, in this study we performed a comprehensive, multiregional genotyping of 11 of the most distinctive driver genes associated with progression and aggressiveness in thyroid cancer (BRAF,HRAS,NRAS,KRAS, PIK3CA, TERTp, TP53, EIF1AX, MED12, KIT and 5’UTR of TBC1D12) in 13 cases of PTCs with matched DMs. A total of 54 tumor samples, including different areas across space and time within the Pts and the DMs were characterized by means of PCR+SSCP or DS Twelve cases (92%) were mutated in at least one of the genes screened [TERTp 69%, BRAF 54%, KRAS 23%, NRAS 23%, HRAS 15% and PIK3CA 15%, KIT 8%]. No mutations were seen at TP53, EIF1AX, MED12 and 5’UTR of TBC1D12. Among the mutated cases 67% exhibited ITGH. In 62.5% of the cases with ITGH coexisted at least 3 genes mutated. Two cases displayed four mutated genes. The genes more frequently altered in Pts with ITGH were TERTp (80%) and BRAF (80%) followed by RAS (60%). Within DMs with ITGH the genes more commonly activated were TERTp (86%) and RAS (86%) followed by BRAF (71%). In all the cases in which more than 1 area of DM, across space and time, was analyzed, the mutations at TERTp, KRAS and HRAS resulted clonal, whereas BRAF mutations tended to be subclonal. De novo mutations at DMs were seen in 7 cases. Within Pts, subclonality was as follows TERTp 30%, RAS 25%, BRAF 30%, PIK3CA 100% The number of mutational events in PTCs with DM is strikingly higher than in PTCs without DMs. Our results do not support a role for MED12, EIF1AX, TP53 and 5’UTR of TBC1D12 mutations in the aggressiveness of PTCs and development of DMs. In approximately 33% of the metastatic PTCs regional pressures, most likely exerted by the homing microenvironment at DMs, lead to the appearance of advantageous “de novo” mutations. DMs show an enrichment in TERTp, RAS, PIK3CA and KIT mutations and a decrease in BRAF mutations. The clonal status of advance stage PTCs evolves with metastatization. ITGH may reflect the early expression of an invasive phenotype in PTCs, such that PTCs with high ITGH could probably be labelled as “born to be noxious / harmful.” Citation Format: Sara Gil, José Javier Estébanez, Noa Féas, Joaquín Fra, Ginesa María Garcia-Rostán. Spatio-temporal geographical molecular mapping of primary papillary thyroid carcinomas and paired distant metastases: Clinical relevance for monitoring and targeting tumor heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-005.

Comprehensive study of genetic features of pancreatic neuroendocrine tumors.

e16699 Background: Pancreatic neuroendocrine tumors (panNETs) are a heterogeneous, relatively rare type of neoplasm which distinguishes a long clinical course in a large number of cases, that leads to the fact that the prevalence rate of NETs is one of the highest. The purpose of this study was a research the mutational profiles of panNETs of various grades of malignancy and to search for new candidate genes involved in the oncogenic processes of this pathology. Methods: The study included a retrospective group of 24 patients (10 men, 14 women, median age - 60 years old) with a diagnosis of panNET, treated in the RRIO from 2011 to 2018. Comprehensive cancer panel (409 genes) were used for NGS procedure on the NextSeq 550 platform(Illumina, USA). To work with raw data, we used the standard pipeline proposed by BaseSpace (Illumina, USA). Interpretation of the identified variants was carried out in accordance with the recommendations of the Association for Molecular Pathology, American Society of Clinical Oncology and College of American Pathologists. Results: 119 mutations in 54 genes were detected in samples of panNETs. 26 genetic variations were characterized as new, not previously described for panNETs. 18% of the mutations were activating, 35% of the variants led to a loss of function of the encoded protein, 52% were not classified. Candidate genes that have not been previously described in the literature and which are potentially involved in pancreatic oncogenesis are identified: CREB1, TCF12, PRKAR1A, BCL11A and BUB1B. The largest number of mutational events occurred in the genes responsible for histone methylation; in addition, 38% described genetic alterations in the regulation of the SMAD2/3 signaling pathway. Conclusions: According to the NGS study of panNETs of low and moderate malignancy in the Russian cohort, a unique spectrum of 119 mutations was obtained, of which 26 were not previously described. The discovered molecular genetic changes in the signaling pathways underline the importance of impaired epigenetic control of transcription in pancreatic oncogenesis and open up new prospects for targeted therapy.

Fraction genome altered (FGA) to regulate both cell autonomous and non-cell autonomous functions in prostate cancer and its effect on prostate cancer aggressiveness.

347 Background: Our ability to distinguish lethal from non-lethal forms of prostate cancer (PC) is limited. Given prostate tumors’ genetic heterogeneity, is it unlikely that a single somatic variant is prognostic. Herein we investigated fraction of genome altered (FGA; percentage of copy number altered chromosome regions out of measured regions; cBioportal) and tumor mutational count (TMC; number of mutational events per case) harbored by the primary tumor as two tumor-specific factors posited to influence disease aggressiveness or responsiveness to certain therapeutic agents. Methods: We used the TCGA data (n= 490 primary PC) and MSKCC-IMPACT (n=717, Zehir et al 2017) PC datasets to analyze the correlation between FGA and TMC in PC. GSEA was performed with transcriptomes used to identify signaling pathways associated with these two measures. We then categorized 490 primary PC patients from TCGA dataset into 4 groups based on FGA and TMC levels (based on the median values) to assess associations with outcomes. Results: Primary PC patients who harbor FGAhighTMClow exhibited shorter disease-free survival (High Risk). We observed attenuation of the androgen signaling pathway and induction of cell proliferation pathways associated with this aggressive form of disease. We used results from CIBERSORT algorithm and deep learning methods of TCGA data and observed that quantities of tumor infiltrating lymphocytes was higher in the FGAhighTMClow group (p=0.038). However, we also observed significantly reduced immune effector signaling-pathway signaling in this high-risk FGAhighTMClow group suggesting the presence of immune-suppressive networks in primary disease associated with a high risk of progression. Conclusions: A greater understanding of molecular features of aggressive primary PC (FGA/TMC) will be important in developing management strategies. Based on our preliminary analyses, we hypothesize that patients whose primary PC harbors FGAhighTMClow have a higher likelihood of aggressive disease due to their impact on PC cell proliferation and dedifferentiation (cell autonomous), and subdued immune responses (non-cell-autonomous).

Abstract 2534: A comparative analysis of fraction genome altered vs tumor mutational count in prostate cancer

Abstract Prostate cancer is the second leading cause of cancer related death among men in the United States. Although 5 new life-prolonging agents have been FDA-approved for castration resistant prostate cancer in the past 8 years, drug resistance limits the durability of responses to these agents for most patients. We hypothesized that biomarkers of genomic alterations, defined as fraction genome altered (% of copy number altered chromosome regions out of measured regions) and mutational count (number of mutational events per cases), were prognostic biomarkers which might provide insights into resistance mechanisms. We used The Cancer Genome Atlas (TCGA; n= 490 primary prostate cancer) dataset to analyze fraction of genome altered and tumor mutational count in prostate tumors. Patients whose primary prostate cancer harbored a high frequency of fraction genome altered exhibited significant disease progression in TCGA cohort . In contrast, we did not observe a significant association between high tumor mutational count and disease progression. Fraction genome altered was more significantly associated with Gleason grade compare to tumor mutation count. Interestingly, our preliminary observation showed that primary prostate cancer patients who harbor high fraction genome altered (>6%), but low tumor mutational count (<30/cases) exhibited shorter disease-free survival, indicating that those primary prostate cancer patients who harbor high fraction genome altered but low mutational count may have more aggressive prostate cancer. We have also developed a transcriptional signature which is associated with high fraction genome altered. . A detailed understanding of these molecular features will help us to design appropriate therapeutic approaches for different subsets of the disease. We hypothesize that cases with a high fraction of genome altered are associated with aggressive disease and those with lower mutational count may be associated with a diminished immunoresponsiveness. Tumor mutational count and fraction genome alteration in various stages TCGA prostate cancerDisease Status (Gleason unadjusted)p valueGleasonp value (trend)Disease FreeRecurred/ Progressed6 (n=45)7 (n=244)>=8 (n=198)Fraction Genome Altered (Mean + SEM)0.08248 ± 0.00490.1254 ± 0.01120.00030.02504 ±0.00370.05985 ± 0.00390.1425 ± 0.0087< 2.2e-16Mutational Count (Mean + SEM)35.05 ± 1.35439.88 ± 2.6550.394429.42 ± 1.749,34.69 ± 0.9089,38.58 ± 1.272,=9.971e-05 (7 vs >=8; p value 0.0445) Citation Format: Goutam Chakraborty, Mohammad Atiq, Subhiksha Nandakumar, Ying Z. Mazzu, Joshua Armenia, Yuki Yoshikawa, Nabeela Khan, Gwo-Shu M. Lee, Lorelei Mucci, Philip W. Kantoff. A comparative analysis of fraction genome altered vs tumor mutational count in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2534.

Abstract 2770: Spatio-temporal genetic heterogeneity and clonal evolution in advanced papillary thyroid carcinomas and matched distant metastases

Abstract Papillary Thyroid Carcinoma (PTC) represents 65-80% of all thyroid cancers. Though the vast majority of PTCs are indolent tumors, around 5-15% behave aggressively, developing blood-borne metastases, which cause patient's death. The molecular mechanisms underlying metastatic spread are poorly understood. Little it is known about the contribution of intratumor molecular heterogeneity to distant metastases (DM). Dynamic changes in mutation distribution through space and time have not been in deep characterized. In this study, by genotyping 13 cases of matched primary tumors (PrT) and DMs, we sought to determine the prevalence of mutations in genes that have been associated with tumor progression and aggressiveness in follicular thyroid carcinogenesis (TERTp, MED12, BRAF, NRAS, KRAS, HRAS, PIK3CA). To asses the contribution of intratumor heterogeneity and clonal evolution to DMs, 54 tumor areas, including different areas across space and time within the PrTs and the DMs were characterized. Mutational analysis was approached by means of PCR and SSCP or direct sequencing. Twelve cases (92%) were mutated in at least one of the genes screened [TERTp=9 cases (69%), BRAF=7 cases (54%), KRAS=3 cases (23%), NRAS=3 cases (23%), HRAS=2 cases (15.4%) and PIK3CA=2 cases (15.4%)]. No mutations were seen at MED12. Among the mutated cases 67% exhibited more than 1 gene activated. Three mutated genes co-existed in 62.5% of the cases with concomitant mutations [3 cases (60%) TERTp+RAS+BRAF and 2 cases (40%) TERTp+BRAF+PIK3CA]. Concurrent activation of TERTp+RAS or TERTp+BRAF was seen in 5 cases each event (62.5%). Simultaneous activation of BRAF and RAS was found in 4 cases (50%). In all the cases in which more than 1 area of DM, across space and time, was analyzed, the mutations at TERTp, KRAS and HRAS resulted clonal. De novo mutations at DM, not present in the PrT, were seen in 3 cases mutated at TERTp, 2 cases mutated at NRAS, 1 case mutated at KRAS. Among the mutated cases, in which more than one area of PrT was analyzed, clonality was seen in 80% of the cases mutated at TERTp and subclonality was seen in 80% of the cases mutated at BRAF. The activation of RAS within the PrT tend to be a clonal event. Conclusions: The number of mutational events in PTC with DM is strikingly higher than in in PTC without DM. While TERTp and RAS mutations tend to be clonal within the PrT and the DMs, BRAF mutations tend to be subconal. TERTp and RAS mutations may appear the novo at DM. PTC with DM display a much higher rate of genetic heterogeneity (67%). The coexistence of mutations in different genes is in agreement with the hypothesis that tumor progression relies on progressive accumulation of genetic alterations. MED12 does not play a role in aggressive papillary thyroid carcinomas. Concurrent mutations at TERTp, and different PI3K/AKt and MAPK pathway genes are common in poorly differentiated and anaplastic carcinomas. Note: This abstract was not presented at the meeting. Citation Format: Sara Gil-Bernabe, Noa Feas Rodríguez, Miriam Vega Herrero, Jose Javier Estébanez García, Ginesa M. Garcia-Rostan. Spatio-temporal genetic heterogeneity and clonal evolution in advanced papillary thyroid carcinomas and matched distant metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2770.

PO-249 Phylogenetic relationships between paired primary papillary thyroid carcinomas and distant metastasis: intra-tumour molecular heterogeneity and clonal evolution

IntroductionThough the vast majority of Papillary Thyroid carcinomas (PTC) are indolent tumours, around 5%–15% behave aggressively, developing distant metastases (DM), which cause patient´s death. The molecular events underlying metastatic spread are poorly understood. Little it is known about the contribution of intratumor molecular heterogeneity to DM.Material and methodsIn this study, by genotyping 13 cases of matched primary tumours (PrT) and DMs, we sought to determine the prevalence of mutations in genes that have been associated with progression and aggressiveness in thyroid cancer (TERTp, BRAF, NRAS, KRAS, HRAS, PIK3CA). To asses the contribution of intratumor heterogeneity and clonal evolution to DMs, 54 tumour areas, including different areas across space and time within the PrTs and DMs, were characterised. Mutational analysis was done by means of PCR and SSCP or direct sequencing.Results and discussionsTwelve cases (92%) were mutated in at least one of the genes screened [TERTp=9 cases (69%), BRAF=7 cases (54%), KRAS=3 cases (23%), NRAS=3 cases (23%), HRAS=2 cases (15.4%) and PIK3CA=2 cases (15.4%). Among the mutated cases 67% exhibited more than 1 gene activated. Three mutated genes coexisted in 62.5% of the cases bearing several mutations [3 cases (60%) TERTp +RAS + BRAF and 2 cases (40%) TERTp +BRAF+PIK3CA]. Concurrent activation of TERTp +RAS or TERTp +BRAF was seen in 5 cases each combination(62.5%). Parallel activation of BRAF and RAS was found in 4 of the cases (50%) with genetic heterogeneity. In all of the cases in which more than 1 area of DM, across space and time, was analysed, the mutations at TERTp, KRAS and HRAS resulted clonal. ’De novo’ mutations at DM, not present in the PrT, were seen in 3 cases mutated at TERTp, 2 cases mutated at NRAS and 1 case mutated at KRAS. Among the mutated cases, in which more than one area of PrT was analysed, TERTp mutations were clonal in 80% of the cases and BRAF mutations subclonal in 80% of the cases.The activation of RAS within the PrT tended to be a clonal event.ConclusionThe number of mutational events in PTC with DM is strikingly higher than in in PTC without DM. While TERTp and RAS mutations tend to be clonal within the PrT and the DMs, BRAF mutations tend to be subconal. TERTp and RAS mutations may appear the novo at DM. PTC with DM display a much higher rate of genetic heterogeneity (67%). The coexistence of mutations in different genes is in agreement with the hypothesis that tumour progression relies on progressive accumulation of genetic alterations.

Abstract 5288: Dynamic genomic indexing enables accurate somatic variant detection from cancer genome sequencing without sequence alignment limitations

Abstract Cancer genome sequencing is a widely adopted approach for identifying critical genetic variants and increasingly, for diagnostic application in precision medicine. Accurate analysis of cancer genome sequencing is essential, yet remains challenging - the reliable and accurate identification of somatic variants continues to be a significant issue. One source of issues involves the mapping/alignment of sequences. It relies entirely on the human reference genome, a static data consisting of character strings of chromosome. However, the current reference is derived from few individuals and thus, does not account for the full diversity or complex structure of human genomes. Critical features of cancer genomes are missed or called with errors. As a solution, we developed dynamic genomic indexing (DGI), which identifies somatic variants by using the frequency of short sequences that are 20∼200 bases in length (20∼200mer) from paired tumor/normal sequencing data. Any somatic variants will affect the frequency of short sequences. In this study, DIG relies on the presence of neo-20mers, derived by point mutations and insertions/deletions (indels), that are different in frequency between normal and tumor sequence reads. These 20mer are unique sequences that arise from cancer genetic variants. First, the DGI identifies the four groups of differential 20mers that their frequencies are significantly different between tumor and normal; tumor specific (TS), normal specific (NS), tumor dominant (TD), and normal dominant (ND). Second, DIG clusters differential 20mers with neighbor 20mers sharing 19 bp in the middle within each group. Third, clustered tumor specific 20mers are matched to clustered NS or ND 20mers. The number of clustered 20mers represents the number of mutational events and ratio between frequency of TS 20mers and matched ND 20mers implies allelic depth. All calculations are conducted in comparison to matched normal samples. Thus, the reference genome is never required throughout the analysis process. Furthermore, these 20mers can be searched easily in RNA-Seq to see if mutations are expressed. We evaluated the performance of DGI using simulated whole exome sequencing (WES) data and 24 WES from the Cancer Genome Atlas project. We identified many of reported somatic mutations (>90%) from WES as well as novel mutations. We observed DGI performs as good as other variant callers for substitutions while outperforms for indels. Our results support the principle of non-alignment sequencing analysis. DGI provides simple, but accurate matrices of somatic variants for cancer sequencing data for any population and can be easily scaled to handle population-based studies. This study provides small variant detection (<50bp) as a first step toward in a broader effort to develop non-alignment sequencing analysis for structural variation such as copy number and chromosomal rearrangement. Citation Format: HoJoon Lee, Jacob J. Parker, John Bell, Hanlee P. Ji. Dynamic genomic indexing enables accurate somatic variant detection from cancer genome sequencing without sequence alignment limitations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5288.

Molecular Predictors of Response in Patients with Myeloid Neoplasms Treated with Lenalidomide

Up to 70% of patients with del(5q) MDS may respond to Lenalidomide (LEN). However, the success rates in non-del(5q) cases, while substantial, are much lower (ranging from 20-40% depending on selection criteria). Aside from the presence of del(5q), up front identification of potentially responsive patients is difficult, particularly as the mechanistic underpinnings of LEN response are still under investigation. Initial attempts to prospectively predict LEN sensitivity resulted in a description of response expression signatures, but they have not been robust enough to serve as an actionable diagnostic test. In the outset of this study, we stipulated that apart of clinical selection (low risk MDS, transfusion-dependence, normal/low risk cytogenetics, etc.), analyses of molecular lesions including somatic mutations and chromosomal defects may help to predict LEN responsiveness. To that end, we performed deep targeted NGS (using multiamplicon panel of the top most commonly mutated genes in MDS). In total we analyzed 143 cases of myeloid neoplasms (MDS, MDS/MPN, or MPN) treated with LEN (median duration 6 months) for whom annotated clinical outcomes were available (83 responders vs. 60 refractory cases). Clinical parameters including IPSS-R, cytogenetics (FISH, SNP-array or metaphase cytogenetics) were used to characterize patients, whose responses were assessed by 2006 IWG criteria.Initially, in a combined analysis, we included both del(5q) (N =37) and non-del(5q) patients (N =106). Very low/low, intermediate, high/very high IPSS-R scores were found in 47%, 23%, 34% cases, respectively. Of 143 LEN-treated patients, regimens included LEN (80%), or LEN+5-Aza (20%). Any hematologic improvement (HI), partial response (PI), and complete response (CR) were achieved in 44%, 14% and 42%, respectively. Responses were associated with a better survival (median survival time 6.2 yrs. vs. 3.7 yrs. in refractory cases; P =.003). Non-responders showed significantly lower platelet levels compared to responders (median 169 vs. 89 K/uL; P =.007) but intricate analysis of clinical parameters (age, other blood counts, blasts and IPSS-R score) failed to identify other factors that would help to select potential responders. As expected, when sub-analysis of patients with del(5q) was performed, combined response was achieved in 78% (OR 13.14 [4.34-39.75]; PR 16%, CR 35%) of patients, respectively, while in non-del(5q) the responses were as predicted lower at 51% (P =.004). Of note is that both del(5q) involving and excluding commonly retained regions (CRR; q11.1-q14.2 and/or q34-qter) also was associated with sensitivity (CRR affected; OR=9.9 [1.4-102], vs. CRR not affected; OR=6.3 [1.3-37.6]). When we analyzed impact of karyotype on LEN sensitivity, -7/del(7q), -20/del(20q), complex karyotype and normal cytogenetics did not correlate with response, but in addition to del(5q); the presence of +8 (7/10 responded; OR 12.25 [1.33-113.06]) was significantly associated with responsiveness. Using targeted deep NGS, we confirmed 168 somatic mutations in responders vs. 142 mutations in non-responders (OR .85; .67-1.07). The number of mutational events per patient did not correlate with responses (P =.38).Among genes sequenced mutations in DDX41 (100% vs. 0%; OR infinity [6.7-infinity]) and RUNX1 mutation+deletion (75% vs. 25%; OR=8.1 [1.1-84.6]) were overrepresented in responders vs. refractory cases while in U2AF1 mutationswere more common among non-responders (20% vs. 80%; OR=.075 [.004-.76]). When reverse analysis was performed DDX41 mutations correlated with LEN response (10% mutant cases among responders vs. 0% in refractory; P =.009), while mutations in U2AF1 correlated with LEN failure (2.4% vs. 13.3% of mutant cases in responders and refractory, respectively; P =.02). The presence of all combined or any of the other spliceosomal mutations (SRSF2, SF3B1, ZRSR2, LUC7L2, and PRPF8) did not influence the results of the therapy. All TP53 mutations were found with complex karyotype with del(5q) and 5/7 (71%) TP53 mutant cases were treatment failures (OR .19 [.01-2.41]).In conclusion, in addition to the presence of del(5q), low platelet count and the presence of various molecular lesions (+8 and RUNX1, DDX41 mutations or wild type status of U2AF1) may help to predict responses to LEN. DisclosuresSekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

Analysis of the complete chloroplast genome of Castanea pumila var. pumila, the Allegheny chinkapin

The complete plastid genome of Castanea pumila L. Mill. var. pumila, the Allegheny chinkapin, was generated using next-generation sequencing of the leaf transcriptome of the species and fill-ins of missing regions via Sanger sequencing. The chloroplast (cp) genome of C. pumila showed high homology (99.8 %) to the genome of Castanea mollissima (the Chinese chestnut, 600,799 bp) in overall gene organization and size (160, 603 bp). Castanea dentata 454 sequence data ( www.fagaceae.org ) were used to generate a partial chloroplast genome of the American chestnut (93 % coverage). Comparisons of the cp genomes of the American and Chinese Castanea species indicated that the species shared 68.0 % of the mutations at coding and intronic regions, while 18.0 % of the mutations were specific to C. pumila and 13.9 % to C. dentata. The loss and nuclear localization of the rpl22 gene was similarly a feature of the genome of the American Castanea species. Since a higher number of mutational events separated C. pumila from C. mollissima, a more recent divergence of C. dentata from a common ancestor was hypothesized.

Abstract 5338: Inferring evolutionary models of tumor progression from single-cell heterogeneity data

Abstract We describe computational methods to compute likely evolutionary histories from tumor single-cell copy number data and next generation sequencing data and apply the methods to data collected from diverse types of tumors. Experimental techniques for assessing heterogeneity in tumor cell populations have undergone great advances, but these improvements have created a great need for more sophisticated computer algorithms capable of making sense of these data sources in terms of coherent models of tumor evolution. We have addressed this problem by developing computer algorithms for building phylogenetic trees describing evolution of individual tumors based on copy numbers of fluorescence in situ hybridization (FISH) probes from single cells in these tumors. These algorithms reconstruct evolutionary trees for observed cell populations so as to heuristically minimize the number of mutational events needed to explain the observed combinations of probe counts by evolution from a common diploid ancestral cell. We have extended this work from initial simple evolutionary models of evolution by single copy number changes to account for distinct mechanisms of evolution at the gene, chromosome, or whole-genome scale, with potentially different rates of evolution by mutation type. We have applied these algorithms to several FISH data sets, including cervical cancers probed for four genes (LAMP3, PROX1, PRKAA1 and CCND1) measured for up to 250 cells of paired primary and metastatic samples from 16 patients, head-and-neck cancers probed for four genes (TERC, CCND1, EGFR and TP53) measured on up to 250 cells per patient for 65 patients at four tumor stages, prostate cancers probed for six genes (TBL1XR1, CTTNBP2, MYC, PTEN, MEN1 and PDGFB) measured for up to 407 cells in 6 non-progressive and 7 progressive carcinomas, and breast cancers probed for eight genes (COX-2, MYC, CCND1, HER-2, ZNF217, DBC2, CDH1 and TP53) measured on up to 220 cells of paired of ductal carcinoma in situ and invasive ductal carcinoma samples from 13 patients. We have then applied statistical and machine learning analysis to examine the ability of these trees to classify tumors by stage or potential for progression. The evolutionary tree models reveal robust features of evolutionary processes distinguishing progression stages and predicting future progression that lead to improved classification accuracy relative to predictions from cellular heterogeneity data alone. Our software is freely available at ftp://ftp.ncbi.nlm.nih.gov/pub/FISHtrees. In continuing work, we are exploring extension of these approaches to better modeling and analysis of tumor evolution using single-cell sequencing data and to more detailed models of tumor evolution. Citation Format: Salim A. Chowdhury, Ayshwarya Subramanian, Alejandro A. Schäffer, Stanley E. Shackney, Darawalee Wangsa, Kerstin Heselmeyer-Haddad, Thomas Ried, Russell Schwartz. Inferring evolutionary models of tumor progression from single-cell heterogeneity data. [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 5338. doi:10.1158/1538-7445.AM2014-5338

Radiation Effects on Human Heredity

In experimental organisms such as fruit flies and mice, increased frequencies in germ cell mutations have been detected following exposure to ionizing radiation. In contrast, there has been no clear evidence for radiation-induced germ cell mutations in humans that lead to birth defects, chromosome aberrations, Mendelian disorders, etc. This situation exists partly because no sensitive and practical genetic marker is available for human studies and also because the number of people exposed to large doses of radiation and subsequently having offspring was small until childhood cancer survivors became an important study population. In addition, the genome of apparently normal individuals seems to contain large numbers of alterations, including dozens to hundreds of nonfunctional alleles. With the number of mutational events in protein-coding genes estimated as less than one per genome after 1 gray (Gy) exposure, it is unsurprising that genetic effects from radiation have not yet been detected conclusively in humans.

Contribution of Mutation, Recombination, and Gene Conversion to Chicken Mhc-B Haplotype Diversity

The Mhc is a highly conserved gene region especially interesting to geneticists because of the rapid evolution of gene families found within it. High levels of Mhc genetic diversity often exist within populations. The chicken Mhc is the focus of considerable interest because of the strong, reproducible infectious disease associations found with particular Mhc-B haplotypes. Sequence data for Mhc-B haplotypes have been lacking thereby hampering efforts to systematically resolve which genes within the Mhc-B region contribute to well-defined Mhc-B-associated disease responses. To better understand the genetic factors that generate and maintain genomic diversity in the Mhc-B region, we determined the complete genomic sequence for 14 Mhc-B haplotypes across a region of 59 kb that encompasses 14 gene loci ranging from BG1 to BF2. We compared the sequences using alignment, phylogenetic, and genome profiling methods. We identified gene structural changes, synonymous and non-synonymous polymorphisms, insertions and deletions, and allelic gene rearrangements or exchanges that contribute to haplotype diversity. Mhc-B haplotype diversity appears to be generated by a number of mutational events. We found evidence that some Mhc-B haplotypes are derived by whole- and partial-allelic gene conversion and homologous reciprocal recombination, in addition to nucleotide mutations. These data provide a framework for further analyses of disease associations found among these 14 haplotypes and additional haplotypes segregating and evolving in wild and domesticated populations of chickens.

Inference of genome-wide mutation rates and distributions of mutation effects for fitness traits: a simulation study.

The properties and limitations of maximum likelihood (ML) inference of genome-wide mutation rates (U) and parameters of distributions of mutation effects are investigated. Mutation parameters are estimated from simulated experiments in which mutations randomly accumulate in inbred lines. ML produces more accurate estimates than the procedure of Bateman and Mukai and is more robust if the data do not conform to the model assumed. Unbiased ML estimates of the mutation effects distribution parameters can be obtained if a value for U can be assumed, but if U is estimated simultaneously with the distribution parameters, likelihood may increase monotonically as a function of U. If the distribution of mutation effects is leptokurtic, the number of mutation events per line is large, or if genotypic values are poorly estimated, only a lower limit for U, an upper limit for the mean mutation effect, and a lower limit for the kurtosis of the distribution can be given. It is argued that such lower (upper) limits are appropriate minima (maxima). Estimates of the mean mutational effect are unbiased but may convey little about the properties of the distribution if it is leptokurtic.

Complex evolution of a salmonid microsatellite locus and its consequences in inferring allelic divergence from size information

Sequence analysis of 78 alleles of a microsatellite locus was resolved at various taxonomic levels among salmonid fishes to test for congruence between size and sequence information. Allelic variability of this locus involved changes in number of dinucleotide repeats as expected for microsatellite loci. However, additional mutational events involving large indels and base substitutions also occurred frequently among and within species. These caused the evolution of different imperfect microsatellite variants, from an ancestral perfect one, which was undetectable from allelic size information only. Alleles of the same length resulting from different mutational events were also observed among and within species. Altogether, these results revealed the incongruence between size and number of mutation events, implying that it may be imprecise to interpret mutational rates and relationships on the basis of size information alone in interspecific comparisons and, to a lesser extent, within species.

Relaxation dynamics in Dyson's model for the origin of metabolism.

This paper discusses some aspects of the dynamical evolution of the system described by Dyson’s model for the origin of metabolism. First, the mean number of mutation events required to observe the origin of metabolism in the model is calculated. This number can span a large range depending upon the values of some critical parameters. Second, the dynamics of the relaxation to equilibrium is investigated by computing the correlation function of an on-off random function that defines the instantaneous state of the system. It was found that the dynamics of the system is well described by a double or a stretched exponential relaxation.

Suppressor genes in breast cancer: an overview.

It is apparent that multiple genetic events occur in the development and progression of breast cancer. From the limited data available, no consistent temporal pattern of mutational events is required. This conclusion is consistent with data in colorectal carcinoma, where the number of mutational events, and not the order, appears to be relevant. Several authors have questioned whether the multiple mutational events occur independently or whether significant associations were evident. Cropp et al. postulated that two sets of mutational events occurred simultaneously in a higher degree of breast tumors than expected based on chance: Set 1 consisted of deletions on 11p, 17p, 18q, and int-2 and myc amplifications; set 2 consisted of 17q, 1p, and 3p deletions. Sato et al., analyzing another tumor cohort for simultaneous mutations, noted a correlation of 17p and 16q deletions, 13q and 17p deletions, and 17p deletion with erbB-2 amplification. Clearly, concordant data on this issue will require the use of large breast tumor cohorts for a comprehensive set of probes. The reasons why mutations to specific genes on different chromosomes tend to occur coordinately is unknown, but may involve common flanking and/or intron sequences at high risk for certain types of mutational events. Another interesting question is the degree to which alterations, but not homozygous inactivation, of suppressor genes occur and its phenotypic consequences. In this chapter, evidence was presented for the amplification of a DCC allele in breast cancer and for variable RB protein expression in breast tumors as a consequence of allelic deletion. For many of the metastasis suppressor genes, a simple reduction in their expression, or an alteration in their expression over the normal cellular regulatory controls, may be sufficient to fuel the metastatic process. The data suggest a more complex regulation of the cancer phenotype by suppressor genes than by recessive inactivation alone. Why do many sporadic cancers, including breast cancer, appear to require alterations to multiple suppressor genes, as compared to diseases such as retinoblastoma, where a single suppressor gene appears to control the cancer phenotype? The answer to this question is unknown, but most theories are based on the hypothesis that suppressor genes act to control cellular responses to either other cells or signals in the microenvironment. In retinoblastoma all cells can carry a germ-line mutation. Cells carrying the RB mutation can interact with both the embryonic and differentiated microenvironments; the specific interaction of mutated cells with the embryonic retinal microenvironment may trigger the onset of retinoblastoma.(ABSTRACT TRUNCATED AT 400 WORDS)