Share Give access Share full text access. Share full text access. Please review our Terms and Conditions of Use and check box below to share full-text version of article. Citing Literature. Volume 49 , Issue 4 21 October Pages Related Information. Close Figure Viewer. Browse All Figures Return to Figure. Previous Figure Next Figure. Email or Customer ID. Forgot password? Old Password. Analogously to human cancer genome projects, genome-scale mutational signatures can be extracted from highly controlled carcinogen exposure experiments using mammalian cells and animal models, in combination with advanced computational methods Olivier et al.
By integrating massively parallel sequencing and DNA adduct analysis in a mammalian cell clonal expansion model Olivier et al. S1 suitable for massively parallel sequencing Olivier et al.
No TP53 mutations were observed in the Spont clones. The detection of TP53 mutations in three out of seven ACR clones and in one out of five GA clones Table 1 provided a sound rationale for extended sequencing at the exome scale.
Summary of cell lines, treatment conditions, and TP53 mutation status. This background mutation type appears related to the culture conditions used for the MEF immortalization assay, and its consistent formation has been observed previously Olivier et al. Thus, higher SBS counts owing to GA treatment may selectively promote the senescence bypass and the selection, with a decreased functional contribution of indels, whereas an inverse scenario is plausible for the Spont and ACR clones, consistent with a previous report based on the Big Blue mouse embryonic fibroblasts and cII transgene Besaratinia and Pfeifer Analysis of the mutation patterns derived from experimental exome sequencing data.
PCA was computed using as input the mutation count matrix of the clones that immortalized spontaneously Spont or were derived from exposure to acrylamide ACR or glycidamide GA. Each sample is plotted considering the value of the first and second principal components Dim1 and Dim2. The percentage of variance explained by each component is indicated within brackets on each axis.
C Transcription strand bias analysis for the six mutation types in GA-exposed clones. For each mutation type, the number of mutations occurring on the transcribed T and nontranscribed N strand is shown on the y -axis. These observations suggest early effects of the GA exposure, reproducible contribution of the induced mutations to senescence bypass, and their clonal propagation during the immortalization stage.
This signature has been linked to cell culture conditions Behjati et al.
S6, S7. S8A ; Supplemental Table S4. S7, S9. Thus, the mutation patterns with a three-class strand bias generated by the GA treatment render the resulting mutational signature unique and novel. Comparison of GA signature to known signatures.
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C Transcription strand bias analysis for the six mutation types underlying the signatures in panel B. For each mutation type using the pyrimidine convention , the number of mutations occurring on the transcribed T and nontranscribed N strand is shown on the left y -axis. The significance is expressed as —log 10 P -value indicated on the right y -axis. S8 ; Alexandrov et al. S8, S10 ; Supplemental Table S4. Next, we interrogated the PCAWG data for the presence of the experimentally defined, class strand-biased GA and B[a]P signatures in tumors of 19 cancer types from 14 organ sites Fig.
The stringency of the process was controlled by determining the P -value and the false-discovery rate FDR for the signature presence test and the reconstruction accuracy Supplemental Table S6 and by modeling false-positive rates FPRs and FDRs of the experimental signature detection using synthetic tumors as described in the Methods and in Supplemental Tables S7 through S In the subset of PCAWG-7 cancers known to carry SBS4 signature adenocarcinomas and squamous cell carcinomas of the lung, hepatocellular carcinomas of the liver and head, and neck squamous cell carcinomas , we compared the GA and B[a]P signatures to estimated levels of SBS4 and found that in the lung and head and neck cancers, a combination of the GA and B[a]P signatures accounted for very similar numbers of mutations as SBS4, suggesting that SBS4 represents combined and highly correlated exposure to GA and B[a]P Fig.
Moreover, we identified the GA signature in additional 15 cancer types without SBS4, including clear cell renal cell carcinoma 78 GA-positive of analyzed tumors , papillary renal cell carcinoma 26 GA-positive out of 32 , biliary adenocarcinoma 20 GA-positive out of 35 , colorectal adenocarcinomas 24 GA-positive out of 60 , stomach adenocarcinoma 17 GA-positive out of 75 , bladder transitional cell carcinoma six GA-positive out of 23 , and uterine adenocarcinoma 10 GA-positive out of 51 Fig. AdenoCA Lung adenocarcinoma, Lung.
SCC lung squamous cell carcinoma, Liver. HCC liver hepatocellular carcinoma Head. SCC head squamous cell carcinoma. AdenoCA and Lung. SCC and, to an extent, in Head. The lines in GA versus B[a]P scatter plots have a slope of 0. Assignments were performed using mSigAct positivity was determined by the signature.
AdenoCA two of , 1. AdenoCA two of , 0.
Proportion indicates percentage of GA-positive tumors within each listed cancer type. C The dot plot shows the proportion of mutations assigned to GA signature among other identified signatures see Supplemental Material in individual tumors of cancer types not showing the direct effects of tobacco smoking i.
This is supported by further mechanistic studies showing that lung tissue from mice exposed to ACR and GA displays comparable DNA adduct patterns, as well as similar mutation frequencies in the cII transgene Manjanatha et al. Similar observations were made in the context of in vitro mutagenicity of ACR in human and mouse cells, suggesting the key role for the epoxide metabolite GA to form premutagenic DNA adducts Besaratinia and Pfeifer The observation that ACR itself is not efficiently metabolized by MEFs is consistent with similar differences reported by previous animal carcinogenicity studies.
We addressed the lack of ACR activation by the addition of human S9 fraction, yet the assessment of DNA adducts suggested limited metabolic activation of ACR with adduct levels substantially lower compared with the direct GA exposure. However, a robust mutational signature in the experimental setting was generated exclusively by exposing the cells directly to GA.
Single reporter gene studies had previously linked ACR and GA exposure to multiple different mutation types. This could reflect differences in DNA repair efficiency concerning the individual guanine and adenine adduct species or the fact that the resulting clones are derived from single cells that selectively immortalized but do not accurately represent the bulk exposed primary cell population in which the GA-DNA adduct levels were measured after exposure. It is also plausible that the excessive and possibly highly cytotoxic N7-GA-Gua adduct burden leads to negative selection of a large number of affected cells.
The established animal models Beland et al. Next, genome-scale sequencing of human tumors and adduct analysis of normal tissues collected in well-designed molecular epidemiological studies focusing on ACR intake are warranted to provide further evidence that the GA signature mutations identified in various cancer types indeed correlate with the exposure to ACR.
Here we show that a new pattern can be identified in a large subset of pan-cancer tumors when experimentally modeled signatures are combined with sophisticated computational signature reconstruction methods while considering the extended features, such as TSB supported by premutagenic adduct analysis. Such integrated approaches can thus lead to future identification of yet unrecognized carcinogen signatures that may be eluding the solely computation-based analyses of the pan-cancer data. The quest for understanding the contribution of ACR to cancer development is reflected by recent accumulation of mechanistic data on the compound's mutagenicity and carcinogenicity in experimental models.
Our findings related to the reconstruction of signature SBS4 by the experimental signatures of GA and B[a]P, together with the detection of the GA signature in lung and liver cancer, are relevant given the established high content of ACR in tobacco smoke. However, we cannot exclude a possibility that in the human tissues directly exposed to tobacco smoke the adenine residues can be targeted by carcinogens such as B[a]P derivatives or nitrosamines. Overall, our findings offer new insights into the thus-far tenuous association of ACR with human carcinogenesis.
Primary human-p53 knock-in Hupki MEFs were isolated from The mice had been tested for specific pathogen-free SPF status. The derived primary cells were genotyped for the human TP53 codon 72 polymorphism Table 1 to authenticate the embryo of origin. Cells from three different embryos E, E, and E were used for the exposure experiments Table 1. All subsequent cell cultures were routinely tested at all stages for the absence of mycoplasma. Exposed and untreated control primary cells were cultured until they bypassed senescence and immortalized clonal cell populations could be isolated Todaro and Green Cells were seeded in well plates and treated as indicated.
The MTT assay was performed in triplicate for each experimental condition. Briefly, primary MEFs were seeded on coverslips in well plates and, the following day, treated as indicated in duplicate for 24 h. Subsequent incubation with a fluorochrome-conjugated secondary antibody , Cell Signaling Technology was performed for 60 min at room temperature. Immunofluorescence images were captured using a Nikon Eclipse Ti. The DNA was isolated from the cells using standard digestion with Proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. The samples were filtered through Amicon 3K molecular-weight cutoff filters Merck Millipore to separate the adducts from the intact DNA.
The amplicon and sequencing primers are listed in the Supplemental Methods. Sequences were analyzed using the CodonCode Aligner version 7. Refer to the Supplemental Methods for detailed information on PCA, assessment of sequencing-related artifacts and damage, and computation of the TSB and its significance.
Mutation patterns were then deconvolved into mutational signatures using NMF Brunet et al. For details on estimates of the optimal number of signatures to extract, see the Supplemental Methods. The reconstruction error calculation evaluated the accuracy with which the deciphered mutational signatures describe the original mutation spectra of each sample by applying Pearson's correlation and cosine similarity. The samples used for this procedure are listed in the Supplemental Methods , and the results are summarized in Supplemental Figures S6 and S7. Cosine similarity values of more than 0.
See the Supplemental Methods for sample details. We used the mutational signature activity mSigAct v0. R software Ng et al. S8 ; Supplemental Table S4 were used to detect tumors with the experimentally defined signatures present, at high stringency achieved also by incorporating the same TSB information in the class reconstructions of each tumor. Only about cases have ever been recorded. Tired of being a guinea pig in the search for remission, in August Rose sent his excised larynx tumor to Champions.
After a seven-month wait, Rose got results from his TumorGraft mice in February The TumorGraft results pointed to one promising combination, which Rose began in March. By April, a scan showed an unprecedented image—his tumor had shrunk. He moved onto a second regimen predicted by his mice, but this time, the drugs did not work. Rose is currently awaiting results from a new round of mice in development using newer tumor cells. In a study published last April, Sidransky and colleagues created TumorGrafts for 29 patients with advanced metastatic sarcoma. Of the 22 patients whose tumors successfully grafted, six died before data from the mice were available, but in 13 of the remaining 16 cases, there was a positive correlation between mouse and human results.
Champions would like to conduct a prospective controlled study in which physician recommendations will be compared side by side with the recommendations derived from mouse avatars, says Paz.
Referencias del cáncer de cuello uterino
A head-to-head comparison would go a long way toward showing that PDX mice are not just a nice model for human cancer, but can actually improve patient care. Such data will be needed to convince a largely skeptical community of cancer researchers and physicians. Today, doctors have clear guidelines to treat tumors with specific drugs, and they are unlikely to deviate from those established procedures unless all else fails, says Fiebig.
In the end, many oncologists believe that PDX mice are a powerful means to an end—achieving successful, individualized treatment plans for cancer patients based on the genetic makeup of their tumor—but not the end itself, as developing PDX mice for every cancer patient is simply not practical. If researchers can use the mice to gather information about specific tumor responses to specific drugs, then they can bank that information to be used in the general population.
An obvious way to surmount those obstacles is to use a cheaper, faster-breeding organism. Enter the humble fruit fly. Next, they genetically modify a fruit fly to dial up or down the activity of those genes, and, if possible, do so in the location where the original human tumor was found. For example, to mimic a colorectal cancer, the team alters genes in cells of the fruit-fly gut. The result is a fly tumor: cells that proliferate, invade nearby tissues, and even metastasize to other parts of the body.
The tumor-riddled flies are then used in a unique high-throughput drug-screening process. A total of flies are grown in a plate with 96 wells. Ten embryos are hatched per well, each of which contains food laced with a different drug or drug combination. The young cancerous flies are very sick, but begin eating the food. The flies that survive are those treated with drugs that were effective against the tumor but not so toxic as to kill the fly.
The team creates and screens about , fly avatars, as Cagan fondly calls them, per patient in about six months, and for a fraction of the money it takes to make a PDX mouse. And while personalized fly models are not yet available to cancer patients, the process has proven useful to human disease. Based on data that included those from the fly, AstraZeneca took the drug into human clinical trials, and in , vandetanib became the first drug approved for late-stage medullary thyroid cancer.
But we may be seeing more of these flies soon.