DOI: 10.5061/dryad.jq2bvq8gh
title: Data from: Lorcaserin-induced rat mammary mutants quantified by CarcSeq

description: Lorcaserin, a drug for weight management, is a selective agonist of the
 serotonin (5-hydroxytryptamine) 2C receptor. Although lorcaserin is a
 non-genotoxic rat carcinogen, FDA approval was granted in part based on
 dose extrapolation considerations. A post-marketing study, CAMELLIA-TIMI,
 designed to detect potential cardiovascular effects of lorcaserin therapy
 detected excess cancer risk in the lorcaserin treatment arm. Consequently,
 a study of lorcaserin-treated rats was conducted to elucidate the
 mechanism of lorcaserin-induced carcinogenesis and facilitate detection of
 other carcinogens operating through the same mechanism in the future.
 Another study goal was to characterize CarcSeq utility in detecting the
 neoplasia-related effects of a non-genotoxic carcinogen. CarcSeq is an
 error-corrected next-generation sequencing method for quantitation of
 panels of hotspot cancer driver mutations (CDMs) and can detect mutations
 with mutant fractions (MFs) ≥10-4. Female Sprague Dawley rats were treated
 by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating the tumor
 bioassay doses but with shorter duration treatments of 12 or 24 weeks.
 Lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions,
 terminal plasma and terminal liver samples using ultra high-performance
 liquid chromatography-electrospray tandem mass spectrometry.
 N-nitroso-lorcaserin was not detected. Mammary DNAs (n = 6/group) were
 used to synthesize PCR products from genes containing known hotspot CDMs
 (Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53) and
 variant MFs were quantified by CarcSeq. Considering MFs in all targets, no
 significant effects of lorcaserin treatment were observed. However,
 significant induction of Pik3ca H1047R mutation was observed after 12 and
 24 weeks of treatment (ANOVA, P&lt;0.05), with greater numbers of
 mutants and mutants with higher MFs observed in 24-week samples compared
 to 12-week samples. Given that Pik3ca H1047R mutation can be detected in
 normal tissues, is the most prevalent mutation in human breast cancer, and
 occurs in several other cancers, these results suggest lorcaserin-induced
 carcinogenesis involves promoting the outgrowth of spontaneously-occurring
 Pik3ca H1047R mutant clones. The underlying mechanism(s) of promotion are
 under investigation. This study provides the first demonstration that
 CarcSeq can identify the carcinogenic impact of a non-genotoxic
 carcinogen, doing so within a shorter timeframe than is needed to measure
 a tumor response.
description: <strong>Study Rationale</strong>
 There is a need to improve the assessment of carcinogenic risk
 associated with exposure to various test articles, including
 pharmaceuticals intended for chronic use. The need is particularly acute
 in terms of detecting the carcinogenic impacts of non-genotoxic
 carcinogens. The CarcSeq error corrected sequencing method was developed
 as an approach to address this need. CarcSeq was designed to quantify
 mutant fraction (MF, mutant bases per total number of bases characterized
 at a given position) of mutants in segments of DNA encompassing hotspot
 cancer driver mutations (CDMs), including segments of the <em>Apc,
 Braf,</em> <em>Hras</em>, <em>Kras</em>,
 <em>Nfe2l2</em>, <em>Pik3ca</em>,
 <em>Setbp1</em>, <em>Stk11</em>, and
 <em>Tp53 </em>genes. Using CarcSeq, it was shown that CDMs
 with MFs greater than or equal to
 10&lt;sup&gt;-4&lt;/sup&gt; are present in normal human
 tissues [1], as well as untreated tissues (mammary and lung) of rat and
 mouse [2,3]. Given that spontaneously occurring CDMs are obligatory for
 carcinogenesis induced by a non-genotoxic carcinogen, this study
 investigated whether CarcSeq could detect the early clonal expansion of
 CDMs induced by the non-genotoxic carcinogen, lorcaserin. Lorcaserin is an
 agonist of the G protein-coupled 5-hydroxytryptamine (serotonin) receptor
 subtype 2c (5-HT2c)[4], which was intended to reduce appetite and body
 weight as the active pharmaceutical ingredient in the diet drug Belviq
 [5]. Significant tumor findings were observed in a rat bioassay of
 lorcaserin (mammary adenocarcinoma/fibroadenoma in male and female rats,
 along with additional tumors in male rats)[5]. Further, total
 malignancies, patients with malignancy, multiple primary tumors,
 metastatic malignancies and cancer deaths were increased in the lorcaserin
 treatment arm of the CAMELLIA-TIMI trial relative to the placebo arm [6].
 This resulted in Belviq being voluntarily withdrawn from the US market
 [7]. To investigate whether CarcSeq could detect early clonal expansion of
 CDMs, potentially indicative of future tumor development, a repeat-dose
 rat study was conducted in which female Sprague Dawley rats were treated
 by gavage daily with 0, 30, or 100 mg/kg of lorcaserin hydrochloride
 hemihydrate, for 12 and 24 weeks. These doses were chosen because they
 were the mid and high bioassay doses that induced mammary tumors in female
 Sprague Dawley rats.  <strong>Study
 Procedures</strong> Cancer driver mutation (CDM)
 levels were measured in mammary DNA of lorcaserin treated rats using
 CarcSeq and six rats per treatment group. Six different treatment groups
 were examined (three dose groups each, for the 12- and 24-week
 treatments), using a total of 36 rats. Genomic DNA was isolated from the
 flash-frozen mammary tissue of each rat and high-fidelity PCR
 amplification was performed to amplify 15 gene segments encompassing
 hotspot CDMs from each rat. These PCR reactions were carried out using
 primers synthesized with eight bases of degenerate sequence at their
 5'ends. After PCR amplification, these degenerate sequences
 constitute a 16-basepair unique molecular identifier sequence (eight
 basepairs from each end) that can be used for error correction by
 consensus. The DNA concentrations of the 15 individual PCR products
 generated from each rat were quantified, then appropriate amounts of the
 amplicons from each rat were pooled and used in library preparation.
 Libraries were constructed using Illumina TruSeq ChIP kits (Illumina, San
 Diego, CA). One step in library preparation is the ligation of adapters
 that include an index sequence to discriminate reads from different
 samples sequenced on the same flow cell. The final PCR step in libary
 preparation was carried out using a fraction of each library that
 contained 22.5 million copies, as determined using the QX200 Droplet
 Digital PCR (ddPCR) System and ddPCR Library Quantification Kits for
 Illumina TruSeq (Bio-Rad, Hercules, CA). Finally, libraries were sequenced
 using 151-cycle, paired-end sequencing and NSQ 500/550 Mid Output (300
 CYS) flow cells on an Illumina NextSeq 500. Two independent CarcSeq
 analyses were performed using genomic DNA from rats treated for 24 weeks,
 allowing the reproducibility of the CarcSeq MF measurements to be
 assessed.  <br>   <br><strong>Error Correction and
 Post-Error Correction Filtering</strong> The
 fastq files derived from sequencing the genomic DNA of each rat were run
 through the Kennedy error correction pipeline [8]. For each position,
 consensus among reads with the same UMI was used to distinguish correct
 base calls from PCR or sequencing errors. At least two reads with the same
 UMI and 90% identity among base calls were required to create one single
 strand consensus sequence (SSCS) and between 300,000 and 600,000 SSCSs
 were analyzed across 1077 sequenced positions for each rat. The output of
 the Kennedy pipeline is a mutpos (mutant position) file, which identifies
 the number of mutant SSCSs, their genomic location, mutation type, and
 total number of SSCSs recovered at each position. For each type of
 mutation, MF was quantified as mutant SSCSs/total SSCSs at a specific
 position. To reach a minimum of 300,000 SSCSs, in some cases libraries
 were re-sequenced, the fastq files were combined with those from previous
 sequencing runs, and the combined fastq files were re-run through the
 error correction pipeline. The error corrected output (mutpos.txt file)
 for each rat analyzed was transferred to a Microsoft Excel template
 worksheet (containing preset functions) within an Excel workbook. The
 mutpos output was filtered in three ways to obtain the final data. For any
 mutant call to be considered further, three or more mutant SSCSs had to
 have been recovered. Also, only mutants with MFs greater than or equal to
 10<sup>-4</sup> were considered further. For each mutation, MF
 was calculated and the two filters were applied using Excel worksheet
 functions. Because variability across samples has been identified as a
 measure indicative of clonal expansion [2, 3, 9, 10], mutants at positions
 with invariant MFs were removed from the dataset, as the third filtering
 step. Specifically, mutants (of a particular type and at a particular
 position) with a coefficient of variation less than 60% considering all
 samples in a dataset were filtered out. A custom R script
 (CS-ecNGS-spreadsheet-processing-08-08-2023v2.Rmd) was used to filter the
 data as described. An Excel workbook (with one worksheet corresponding to
 the mutpos output of one rat and a worksheet for each rat in the dataset)
 was used as input for the R script. The R script reports the error
 corrected and filtered mutant output in several useful forms (MF values
 and mutant counts, reported and searchable by amplicon, chromosome,
 genomic position, and mutation type). The R script also generates a report
 on SSCS number recovery, identifies the minimum SSCS number recovered in a
 dataset, and calculates the ratio (quotient) of recovered SSCS number for
 each amplicon of each sample divided by the minimum SSCS recovery of each
 amplicon across dataset samples. This ratio is used to calculate the
 number of mutants that are randomly downsampled, and the R script performs
 the random downsampling of mutants to normalize the output relative to the
 rat sample with the lowest number of SSCSs recovered. The R script reports
 MFs of mutants recovered before and after downsampling. Downsampled data,
 normalized relative to the sample with the lowest SSCS recovery for each
 amplicon was used in all statistical analyses. 
 <strong>Study Results</strong>
 Many mutants with MFs greater than or equal to
 10<sup>-4</sup> were recovered in this study (15 to 55 mutants
 per rat, with a total of 565 mutants recovered in the replicate analysis
 of the 24 week samples). Significantly greater MFs were observed in
 amplicons corresponding to known rat mammary-specific driver genes
 (<em>Hras</em>, <em>Pik3ca</em>, and
 <em>Tp53</em>), as compared to the remaining amplicons.
 Importantly, in both the 12 and 24 week datasets, significant
 dose-dependent increases in <em>Pik3ca </em>H1047R MFs and
 numbers of samples with <em>Pik3ca </em>H1047R mutants were
 observed. This was interpreted as evidence of clonal expansion. Also, a
 greater number of samples with measureable <em>Pik3ca
 </em>H1047R MFs and greater clonal expansion (identified by larger
 MF values) were observed in the 24 week samples relative to the 12 week
 samples. Replicate measurements of <em>Pik3ca </em>H1047R MFs
 in the 24 weeks samples were highly concordant. The median absolute
 deviation in MFs observed among the dose groups after 24 weeks of
 lorcaserin treatment correlated significantly with mammary tumor incidence
 (i.e., number of mammary tumors per number of female rats tested in each
 dose group). Lorcaserin-induced clonal expansion of <em>Pik3ca
 </em>H1047R mutants in rat was considered relevant to human because
 <em>Pik3ca </em>H1047R is the most prevalent CDM reported to
 occur in human breast cancer. In conclusion, CarcSeq detected the
 carcinogenic effect of a non-genotoxic carcinogen using a duration of
 treatment as short as three months. [1] Harris, K. L.,
 Walia, V., Gong, B., McKim, K. L., Myers, M. B., Xu, J. and Parsons, B. L.
 (2020). Quantification of cancer driver mutations in human breast and lung
 DNA using targeted, error-corrected carcseq. <em>Environ Mol
 Mutagen</em> 61, 872-889.<br>[2] McKim, K. L., Myers, M. B.,
 Harris, K. L., Gong, B., Xu, J. and Parsons, B. L. (2021). Carcseq
 measurement of rat mammary cancer driver mutations and relation to
 spontaneous mammary neoplasia. <em>Toxicol Sci</em> 182,
 142-158.<br>[3] Harris, K. L., McKim, K. L., Myers, M. B., Gong, B.,
 Xu, J. and Parsons, B. L. (2021). Assessment of clonal expansion using
 carcseq measurement of lung cancer driver mutations and correlation with
 mouse strain- and sex-related incidence of spontaneous lung neoplasia.
 <em>Toxicol Sci</em> 184, 1-14.<br>[4] Gustafson, A.,
 King, C. and Rey, J. A. (2013). Lorcaserin (belviq): A selective serotonin
 5-HT2c agonist in the treatment of obesity. <em>P T</em> 38,
 525-534.<br>[5] U.S. Food and Drug Administration. 2012. Center for
 Drug Evaluation and Research Application Number: 022529orig1s000
 pharmacology review(s) [accessed May 15, 2023].
 https://www.accessdata.fda.gov/drugsatfda_docs/nda/2012/022529Orig1s000PharmR.pdf.<br>[6] Sharretts, J., Galescu, O., Gomatam, S., Andraca-Carrera, E., Hampp, C. and Yanoff, L. (2020). Cancer risk associated with lorcaserin — the FDA’s review of the CAMELLIA-TIMI 61 trial. <em>N Engl J Med</em> 383, 1000-1002.<br>[7] U.S. Food and Drug Administration. 2021. Determination that belviq (lorcaserin hydrochloride) tablets, 10 milligrams, and belviq xr (lorcaserin hydrochloride) extended-release tablets, 20 milligrams, were withdrawn from sale for reasons of safety or effectiveness. Federal Register. College Park, MD: The National Archives and Records Administration. p. 12697-12698.<br>[8] Kennedy, S. R., Schmitt, M. W., Fox, E. J., Kohrn, B. F., Salk, J. J., Ahn, E. H., Prindle, M. J., Kuong, K. J., Shen, J.-C., Risques, R.-A. et al. (2014). Detecting ultralow-frequency mutations by duplex sequencing. <em>Nat Protoc</em> 9, 2586-2606.<br>[9] Parsons, B. L., McKim, K. L. and Myers, M. B. (2017). Variation in organ-specific &lt;i&gt;Pik3ca&lt;/i&gt; and &lt;i&gt;Kras&lt;/i&gt; mutant levels in normal human tissues correlates with mutation prevalence in corresponding carcinomas. <em>Environ Mol Mutagen</em> 58, 466-476.<br>[10] Parsons, B. L. (2018). Modern conception of carcinogenesis creates opportunities to advance cancer risk assessment. <em>Curr Opin Toxicol</em> 11-12, 1-9.
description: # Data from: Lorcaserin-induced rat mammary mutants quantified by CarcSeq
 ## Description of the data and file structure Although three datasets were
 generated in the conduct of this study, based upon dataset size
 considerations, only one dataset is provided to enable independent
 re-analysis of CarcSeq output. Specifically, the data obtained in the
 replications of the CarcSeq analysis of the 24-week samples is provided
 (24 week replicate 2, Rep 2). The data is provided in the form of one
 compressed folder for the sequencing output of each rat treated for 24
 weeks. More precisely, each compressed folder contains all the fastq files
 produced by the CarcSeq analysis of genomic DNA from one rat that were run
 through the Kennedy error correction pipeline [8] and post-error
 correction filtering steps to obtain the final data. The folder naming
 convention uses M to signify mammary tissue as the source of genomic DNA,
 incorporates the rat identifier number (ID#, between 19 and 36 for the
 rats treated for 24 weeks), and Rep 2 to denote the replicated 24 week
 dataset (i.e., M19_Rep 2.zip to M36_Rep2.zip). For rats treated with
 lorcaserin for 24 weeks, rats with ID#s M19 to M24 were treated with 0
 mg/kg, rats M25 to M30 were treated with 30 mg/kg, and rats M31 to M36
 were treated with 100 mg/kg. Two additional items/files are provided. The
 excel template file used to collect the mutpos data is provided and
 identified as: EC-NGS Template. Also, the custom R script used to filter
 the raw mutpos data to obtain the final data used in statistical analyses
 is provided as: CS-ecNGS-spreadsheet-processing-08-08-2023v2.Rmd. ##
 Sharing/Access information A manuscript describing the results of this
 study has been published in Toxicological
 Sciences [https://doi.org/10.1093/toxsci/kfae070](https://doi.org/10.1093/toxsci/kfae070) ## Code/Software Error correction was performed on each sample independently, by running all the fastq files for a particular sample through the Kennedy pipeline [8]. The output of the pipeline was a mutpos file. An Excel workbook was assembled for each dataset, with one worksheet capturing the mutpos data from one sample. The assembled Excel workbooks were used, separately, as input for a custom R script (provided as: CS-ecNGS-spreadsheet-processing-08-08-2023v2.Rmd). The custom R script was written by Dr. Page B. McKinzie (US Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR). The script accomplishes several filtering steps and delivers the mutant data in several useful forms (MF values and mutant counts, reported and searchable by amplicon, chromosome, genomic location, and mutation type). The R script processes the mutation data, such that three conditions are met for mutants to be considered in the final data: 1) three or more mutant SSCSs were recovered, 2) mutant SSCSs/total SSCSs was greater than or equal to 10<sup>-4</sup>, and 3) across the samples in a dataset/workbook, the coefficient of variation of MFs at a particular position for a particular type of mutation was greater than or equal to 60%. The R script also performs random downsampling of mutants to normalize the output relative to the sample with the lowest number of SSCSs recovered for each amplicon. The R script reports MFs of mutants recovered before and after downsampling. The R script generates plots in which the panel position of each mutant is plotted on the X axis (arraryed from lowest chromosome number/position to highest) and the mutant's log<sub>10</sub> MF is plotted on the Y axis. Further, the R script has functionality that allows the user to identify which amplicons are considered tissue-specific drivers and reports can be generated that parse the data accordingly.