Data from Hotspot Mutation Panel Testing Reveals Clonal Evolution in a Study of 265 Paired Primary and Metastatic Tumors
Rashmi S. GoswamiKeyur P. PatelRajesh R. SinghFunda Meric‐BernstamScott KopetzVivek SubbiahRicardo H. ÁlvarezMichael A. DaviesKausar J. JabbarSinchita Roy‐ChowdhuriAlexander J. LazarL. Jeffrey MedeirosRussell R. BroaddusRajyalakshmi LuthraMark J. Routbort
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<div>Abstract<p><b>Purpose:</b> We used a clinical next-generation sequencing (NGS) hotspot mutation panel to investigate clonal evolution in paired primary and metastatic tumors.</p><p><b>Experimental Design:</b> A total of 265 primary and metastatic tumor pairs were sequenced using a 46-gene cancer mutation panel capable of detecting one or more single-nucleotide variants as well as small insertions/deletions. Mutations were tabulated together with tumor type and percentage, mutational variant frequency, time interval between onset of primary tumor and metastasis, and neoadjuvant therapy status.</p><p><b>Results:</b> Of note, 227 of 265 (85.7%) tumor metastasis pairs showed identical mutation calls. Of the tumor pairs with identical mutation calls, 160 (60.4%) possessed defining somatic mutation signatures and 67 (25.3%) did not exhibit any somatic mutations. There were 38 (14.3%) cases that showed at least one novel mutation call between the primary and metastasis. Metastases were almost two times more likely to show novel mutations (<i>n</i> = 20, 7.5%) than primary tumors (<i>n</i> = 12, 4.5%). <i>TP53</i> was the most common additionally mutated gene in metastatic lesions, followed by <i>PIK3CA</i> and <i>SMAD4</i>. <i>PIK3CA</i> mutations were more often associated with metastasis in colon carcinoma samples.</p><p><b>Conclusions:</b> Clinical NGS hotspot panels can be useful in analyzing clonal evolution within tumors as well as in determining subclonal mutations that can expand in future metastases. <i>PIK3CA</i>, <i>SMAD4</i>, and <i>TP53</i> are most often involved in clonal divergence, providing potential targets that may help guide the clinical management of tumor progression or metastases. <i>Clin Cancer Res; 21(11); 2644–51. ©2015 AACR</i>.</p></div>Keywords:
Mutation frequency
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Summary: The ability of somatic mutation to modify the course of an immune response is well documented. However, emphasis has been placed almost exclusively on the ability of somatic mutation to improve the functional characteristics of representative antibodies. The harmful effects of somatic mutation, its dark side, have been far less well characterized. Yet evidence suggests that the number of B cells directed to wastage pathways as a result of harmful somatic mutation probably far exceeds the number of cells whose antibodies have been improved. Here we review our recent findings in understanding the structural and functional consequences of V‐region mutation.
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Koana, T., Takashima, Y., Okada, M. O., Ikehata, M., Miyakoshi, J. and Sakai, K. A Threshold Exists in the Dose– Response Relationship for Somatic Mutation Frequency Induced by X Irradiation of Drosophila. Radiat. Res. 161, 391– 396 (2004).The dose–response relationship of ionizing radiation and its stochastic effects has been thought to be linear without any thresholds. The basic data for this model were obtained from mutational assays in the male germ cells of the fruit fly Drosophila melanogaster. However, it is more appropriate to examine carcinogenic activity in somatic cells than in germ cells. Here the dose–response relationship of X irradiation and somatic mutation was examined in Drosophila. A threshold at approximately 1 Gy was observed in DNA repair-proficient flies. In the repair-deficient siblings, the threshold was smaller and the inclination of the dose–response curve was much steeper. These results suggest that the dose–response relationship between X irradiation and somatic mutation has a threshold and that the DNA repair function contributes to its formation.
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Effect of caffeine on the frequency of 8-azaguanine (8AG) resistant mutation in Chinese hamster cells (V79 cells) treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was investigated. The results showed that the effects of caffeine appeared to be different in manner depending on the time and duration of caffeine exposure after MNNG treatment. (1) When the cells were exposed to caffeine during the first half of mutation expression time (0-24h after MNNG treatment), the mutation frequency was slightly enhanced compared with MNNG treatment alone. (2) On the contrary, mutation frequency was reduced if caffeine was present during the latter half of expression time (24-48h after MNNG treatment). (3) Caffeine drastically reduced the frequency of mutation if the cells were exposed to caffeine for the whole mutation selection period from 48 to 240h after MNNG treatment.
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In order to establish a sensitive assay system for the mutagens and to understand the nature of the differences in the mutation frequency among various somatic mammalian cell lines at various genetic markers reported by many workers, we examined the frequency of the forward and reverse mutations induced by X- and UV-irradiation, by using 8-azaguanine (8AG)-sensitive cells (prototrophic CH-hai Cl 23 cells) isolated from an original Chinese hamster hai cell line and 8AG-resistant cells isolated from CH-hai Cl 23 cells. However, both the mutation systems, especially the forward mutation system used here, were less sensitive as the assay system of the mutations induced by X-rays or UV, compared with the nutritional forward mutation system used previously (Ban et al. 1976). These results underline the complexity of the study of mutagenesis using cultured mammalian cells, particularly because the induced mutation frequency in cultured cells is affected by the factors of the mutation expression time, the size of the cell inoculum, the concentration of the selective agent, etc.
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Mutations are the substrate of cancer. Yet, little is known about the degree and nature of mutations in tumors because measurement of mutation load in tumors and normal tissues was generally not possible until the advent of transgenic mouse mutation detection systems. Herein, we present the first analysis of mutation frequency and pattern in thymic tumors from a mouse model of Li-Fraumeni syndrome (p53+/− murine model) using the Big Blue ® assay with sequencing of all mutants. We also make the first characterization of mutation frequency and pattern in p53-deficient extra-thymic cancers. The data more than triple the literature on all non-mismatch repair deficient tumors for which mutations are identified by sequence analysis, allowing mutation frequency and pattern to be determined. Most tumors had a normal mutation frequency and a normal mutation pattern. Five tumors showed modest increases in mutation frequency (2.3-fold or less). Alterations in mutation patterns were uncommon, tumor-specific and not necessarily associated with increases in mutation frequency. Given the data from two spontaneous tumors (normal mutation frequency with an abnormal pattern in a p53−/− mouse and low mutation frequency in a p53+/+ control mouse), we hypothesize that tumors sometimes can carry a low mutation load. The study was not without certain caveats: mutation load could not be compared between tumor and normal tissue from the same animal; sample sizes for extra-thymic tumor types were small, and only point mutations and deletions, insertions and indels up to 2 kb were detected. However, the data clearly show key differences in tumors from p53+/− mice compared with mismatch repair deficient tumors; a lack of dramatic increase in mutation frequency and absence of a signature of mutation.
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In mutation breeding, mutation frequencies are expressed as the proportion of X1 ears which segregate mutant progeny. Since mutations occur as sectors in X1 ear and the size of mutated sectors varies with increasing dose, the procedure used every ear for calculation of the mutation frequency might result in underestimates of mutation frequency. Using the method of the segregation frequency of mutants in mutated X1-ear-branch progenies, it could be shown that the primary branch was traced back to a single cell of rice seed embryo. Mutation frequencies were measured on the X1 ear, X2 plant or X1 ear-branch basis. The deviation from the linear relationship between dose and the frequency of mutations seemed to be less on the X1 ear-branch basis. Consequently, it was proposed to measure the mutation frequency per original treated cell as the proportion of primary branches of X1 ear which segregated mutant progeny. Furthermore, the mutation frequencies and the segregation ratios were less in the lower primary branches of X1 ear. It was supposed that even in haplontic selection the course of the elimination process was dependent on the environment in which the X2 seeds were produced.
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