Microsatellite Instability Events Research Articles

FAT1 mutations in colorectal cancer patients are associated with the therapeutic benefit of immunotherapy.

e15523 Background: Immunotherapy is one of the main treatment modalities for advanced colorectal cancer. Although microsatellite instability (MSI) is widely applied as an effective immune efficacy prediction biomarker in clinical practice, the majority of the colorectal patients are microsatellite stable (MSS). This study is designed to identify new biomarkers for immune efficacy in colorectal cancer. Methods: Targeted sequencing with a 425 gene panel was performed with the tumor tissues from a total of 161 colorectal cancer (CRC) patients (discovery cohort) for mutation analysis. The association between FAT1 mutations and immunotherapy markers MSI and TMB was investigated using an external database of 727 CRC patients from cBioportal (validation cohort). Results: The discovery cohort consisted of 95(59%) males and 66(41%) females with a median age of 59 years old. There were 2(1.2%) stage II, 5(3.1%) stage III, 24(14.9%) stage IV, and 130(80.8%) unknown stage patients. The majority of the discovery cohort was MSS (154, 95.7%), which was similar to the validation cohort (518, 71.2%). FAT1 mutations were present around similar ratios in both discovery cohort (12.4%) and validation cohort (11.6%). In both cohorts, the co-occurrence analysis revealed that MSI events were often co-occurred with FAT1 mutations and patients with FAT1 mutations had a higher mutational load compared to the FAT1 wild-type patients. Analysis with survival data from the validation cohort showed that OS was significantly higher in patients with FAT1-mutated tumors than in ones with FAT1 wild-type. Compared to the FAT1 wild-type tumors, the higher infiltration rate of immune cell identified in the colorectal tumors with FAT1 mutation may also explain the good immune efficacy of patients with FAT1 mutation. Conclusions: Colorectal cancer patients with FAT1 mutations may benefit from immunotherapy. The increased immunogenicity in patients with FAT1 mutations may be due to increased immune cell infiltration.

Mismatch Repair Deficiency and Microsatellite Instability in Triple-Negative Breast Cancer: A Retrospective Study of 440 Patients.

PurposeTo investigate the status of mismatch repair (MMR) and microsatellite instability (MSI) in triple-negative breast cancer (TNBC) and to examine correlations between MMR/MSI status and clinicopathological parameters.MethodsWe retrospectively collected tissue samples from 440 patients with TNBC and constructed tissue microarrays. Protein expression of MLH1, MSH2, MSH6, and PMS2 was detected by immunohistochemistry (IHC). We also analyzed 195 patient samples using MSI polymerase chain reaction (PCR) testing. Correlations between MSI status and clinicopathological parameters and prognosis were analyzed.ResultsThe median age of the cohort was 49 years (range: 24–90 years) with a median follow-up period of 68 months (range: 1–170 months). All samples were positive for MLH1, MSH2, MSH6, and PMS2, except for one sample identified as MMR-deficient (dMMR) by IHC, with loss of MSH2 and intact MSH6 expression. MSI PCR revealed no case with high-frequency MSI (MSI-H), whereas 14 (7.2%) and 181 (92.8%) samples demonstrated low-frequency and absence of MSI events, respectively. The dMMR sample harbored low-frequency instability, as revealed by MSI PCR, and a possible EPCAM deletion in the tumor, as observed from next-generation sequencing. No correlations were detected between MMR or MSI status and clinicopathological parameters, programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression, or survival.ConclusionsThe incidence of dMMR/MSI-H is extremely low in TNBC, and rare discordant MSI PCR/MMR IHC results may be encountered. Moreover, MMR/MSI status may be of limited prognostic value. Further studies are warranted to explore other predictive immunotherapy biomarkers for TNBC.

Abstract 3538: Microsatellite instability (MSI) and homologous recombination deficiency (HRD) are mutually exclusive mechanisms of genomic instability

Abstract Background: Genomic instability refers to the tendency to accumulate genomic alterations. MSI-High (MSI-H) tumors are characterized by a mismatch repair deficiency triggering hypermutation, whereas HRD tumors display genomic lesions that result in genomic loss of heterozygosity (gLOH). These mechanistic differences have critical implications for treatment strategies, where immune checkpoint blockade is often efficacious in MSI-H tumors and PARP inhibition is linked to response in HRD tumors. We investigated the patterns of co-occurrence or mutual exclusivity between these two mechanisms of genomic instability. Methods: Comprehensive genomic profiling was performed on >130K tumors to coding exons and select introns of up to 465 genes. MSI was determined on up to 114 loci and high gLOH (≥14%) was used as a surrogate of HRD (PMID: 27908594). Results: In this cohort, HRD was most prevalent in triple-negative breast and fallopian tube cancers and absent in cancers such as skin neuroendocrine cancers. HRD was strongly associated with biallelic loss of homologous recombination (HR) pathway genes and not monoallelic loss of these genes. High MSI was most frequently found in endometrial and small intestine cancers, and absent in cancers such as fallopian tube cancers and GIST. MSI-H tumors and HRD tumors were mutually exclusive (6.5% of MSI-H tumors were HRD vs 25.7% of tumors that were not MSI-H; OR = 5.0; p<1e-108). Conclusions: We show that high MSI and HRD events are mutually exclusive across tumor types, suggesting that co-occurrence of these genomic instability mechanisms may be evolutionarily disadvantageous. High gLOH is associated with biallelic loss of HR pathway genes, supporting its use as a surrogate for HRD. The genomic differences between HRD and MSI-H may underlie differential susceptibility to targeted therapeutics. Gene% Biallelic loss in gLOH-H% Biallelic loss in gLOH-LowBiallelic loss p-value% Monoallelic loss in gLOH-H% Monoallelic loss in gLOH-LowMonoallelic loss p-valueATM2.45%1.54%1.24E-251.15%1.83%1.12E-17BARD10.25%0.03%5.15E-290.25%0.28%0.40BRCA15.15%0.23%<1.00E-1000.49%0.57%0.10BRCA25.32%0.76%<1.00E-1000.74%1.11%4.62E-09BRIP10.36%0.12%1.97E-160.41%0.49%0.07CDK120.49%0.31%3.95E-060.30%0.52%1.31E-07CHEK20.65%0.44%8.20E-060.54%0.86%1.92E-09FANCA0.51%0.25%1.36E-120.38%0.47%0.04FANCC0.22%0.07%2.03E-100.11%0.26%1.64E-07FANCG0.10%0.03%1.08E-050.08%0.12%0.04RAD51B0.39%0.15%5.36E-150.04%0.09%0.01RAD51C0.32%0.05%2.66E-320.10%0.12%0.41RAD51D0.18%0.02%2.52E-230.08%0.11%0.20 Citation Format: Dexter X. Jin, Ethan S. Sokol, Sally E. Trabucco, Garrett M. Frampton, Luciana Molinero. Microsatellite instability (MSI) and homologous recombination deficiency (HRD) are mutually exclusive mechanisms of genomic instability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3538.

Impact of age on N-methyl-N-nitrosourea induced microsatellite instability in young and old C57BL/6J mice

Age is an important factor in the evaluation of chemical toxicology. Chemical carcinogenic compounds can induce genomic mutations. However, few studies have been conducted on the association between genomic mutation frequency, such as microsatellite instability (MSI), and the age of mice treated with a nitrosourea mutagen. In the current work, we treated young (6 weeks) and old (10 months) mice with N-methyl-N-nitrosourea (MNU) for 4 months; the MSI frequency was then measured using polymerase chain reaction (PCR) and short tandem repeat (STR) scanning. The percentage of animals with MSI in the old group was significantly higher than that in the young group (100% and 75%). The frequency of MSI events was significantly different between the two groups as well (15.8% for old and 9.4% for young). The ratio of MSI loci displayed no obvious difference between the two groups. In addition, a few loci, including D15Mit5 and D8Mit14 exhibited the highest frequency of MSI events. Since specific loci showed increased MSI in the present study and a higher frequency in previous studies, these loci could be regarded as “hot spot”. These results suggested that old mice would be more susceptible to this mutagen, and prone to accrue MSI. The hot spot microsatellite loci are potentially useful markers for genomic instability analysis.

Accurately estimating the length distributions of genomic micro-satellites by tumor purity deconvolution

BackgroundGenomic micro-satellites are the genomic regions that consist of short and repetitive DNA motifs. Estimating the length distribution and state of a micro-satellite region is an important computational step in cancer sequencing data pipelines, which is suggested to facilitate the downstream analysis and clinical decision supporting. Although several state-of-the-art approaches have been proposed to identify micro-satellite instability (MSI) events, they are limited in dealing with regions longer than one read length. Moreover, based on our best knowledge, all of these approaches imply a hypothesis that the tumor purity of the sequenced samples is sufficiently high, which is inconsistent with the reality, leading the inferred length distribution to dilute the data signal and introducing the false positive errors.ResultsIn this article, we proposed a computational approach, named ELMSI, which detected MSI events based on the next generation sequencing technology. ELMSI can estimate the specific length distributions and states of micro-satellite regions from a mixed tumor sample paired with a control one. It first estimated the purity of the tumor sample based on the read counts of the filtered SNVs loci. Then, the algorithm identified the length distributions and the states of short micro-satellites by adding the Maximum Likelihood Estimation (MLE) step to the existing algorithm. After that, ELMSI continued to infer the length distributions of long micro-satellites by incorporating a simplified Expectation Maximization (EM) algorithm with central limit theorem, and then used statistical tests to output the states of these micro-satellites. Based on our experimental results, ELMSI was able to handle micro-satellites with lengths ranging from shorter than one read length to 10kbps.ConclusionsTo verify the reliability of our algorithm, we first compared the ability of classifying the shorter micro-satellites from the mixed samples with the existing algorithm MSIsensor. Meanwhile, we varied the number of micro-satellite regions, the read length and the sequencing coverage to separately test the performance of ELMSI on estimating the longer ones from the mixed samples. ELMSI performed well on mixed samples, and thus ELMSI was of great value for improving the recognition effect of micro-satellite regions and supporting clinical decision supporting. The source codes have been uploaded and maintained at https://github.com/YixuanWang1120/ELMSIfor academic use only.

Mutational signatures and mutagenic impacts associated with betel quid chewing in oral squamous cell carcinoma.

Betel quid (BQ) chewing is a prevailing risk for oral squamous cell carcinoma (OSCC) in Southeast Asia. Yet, the detailed mechanisms by which BQ chewing damages the genome are still not fully understood. Through exome sequencing of tumor-normal pairs from 196 male patients with OSCC, including 95 habitual BQ chewers and 101 non-BQ users, we conducted a quantitative survey of mutational signatures and genomic aberrations and explored their association with BQ chewing. We found that BQ-associated elevation in mutation rate was seen in cancers of the tongue, but not in overall OSCC. Additionally, we identified a mutational signature that is enriched in tumors from BQ users. Moreover, the numbers of small insertions and deletions (INDELs) and breakpoints derived from structural variations (SV) were increased, whereas the extent of loss of heterozygosity was decreased in BQ-related OSCC genomes. However, neither the number of base substitutions and microsatellite instability events nor the extent of copy-number alterations differed between BQ-related and -unrelated OSCC. In conclusion, consistent with the proposition that BQ chewing increases OSCC risk as a mutagen, our results unveil a BQ-associated mutational signature and indicate mutagenic impacts of BQ chewing on preferentially eliciting INDELs and SV-related breakpoints in OSCC genomes.

Abstract LB-215: CMSI: A Bayesian model for estimating clonal micro-satellites instability from NGS data

Abstract Genomic micro-satellites are the genomic regions consisting of short and repetitive DNA motifs. Micro-satellite region usually exposes intrinsic polymorphism in terms of the numbers of repetitive motifs, which is often described as a probability distribution of the numbers of repeats. In cancer genomics, for any micro-satellite region, it is considered as a micro-satellite instability (MSI) event, if the probability distribution sampled from tumor tissue is significantly different from the distribution sampled from the corresponding normal tissue. Since recent studies have emphasized the importance of micro-satellite instability events in cancer diagnosis and treatments, a series of computational approaches have been developed to detect MSI events from the sequencing data. However, the existing methods suffer an accuracy loss when clonal micro-satellites exist, which are recently observed in some TCGA/ICGC samples. For a clonal micro-satellite, different sub-clones may carry different distributions, while the observed “distribution” from the sequencing data is actually a convolution of the sub-clonal ones. In this case, a sub-clonal distribution may present a true MSI event, but the convolutional one dilutes the data signal and misleads the detection algorithm to report a micro-satellite stability (MSS) event, which introduces type-I error. In addition, a comprehensive understanding of the micro-satellite distribution of each sub-clone is also quite informative for downstream analyses. Thus, to overcome the potential weakness of existing approaches and further improve the computational model, here, we proposed a probabilistic framework, named CMSI, to identify the MSI events under tumor heterogeneous structure. Similar to other approaches, CMSI works on the next generation sequencing data. The proposed framework follows the assumption that the probability density function of the numbers of repeats of a micro-satellite region usually follows a normal distribution. Then, when clonal micro-satellite exists, the convolution distribution observed from the sequencing data should obey a Gaussian mixture distribution. CMSI establishes a variational Bayesian mixture model for the Gaussian distribution calculated from the sequencing reads. This mixture model clusters the reads by the numbers of repeats they bring or infer, and further provide a probabilistic assignment to each read by maximizing the global posterior distribution. By solving this computational model by an EM algorithm, CMSI estimates the number of sub-clones, the proportion of each sub-clone and the parameters of each distribution. Finally, each sub-clonal distribution is examined by statistical test by weighting the clonal proportion, and CMSI outputs the MSI events of sub-clones. To verify the performance of the proposed framework, we conducted several experiments on both simulation datasets and real datasets, where CMSI effectively identified an acceptable percentage of the preset MSI events. Note: This abstract was not presented at the meeting. Citation Format: Yixuan Wang, Xuanping Zhang, Yi Huang, Tao Liu, Xiao Xiao, Jiayin Wang. CMSI: A Bayesian model for estimating clonal micro-satellites instability from NGS data [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 LB-215.

Abstract 421: Improved detection and identification of microsatellite instability features in colorectal cancer: Implications for immunotherapy

Abstract Colorectal cancers with loss of DNA mismatch repair display microsatellite instability (MSI). These tumors have a better prognosis and an improved response rate to immune check point therapies compared to non-MSI tumors. MSI-positive tumors demonstrate a surprising range of somatic alterations in repetitive sequence tracts. For example, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that may be related to functional loss of MSH3. Current MSI tests are based on PCR and capillary electrophoresis. This conventional approach is suboptimal because i) only a small number of microsatellite markers are interrogated per assay, and ii) sensitivity is insufficient for low allelic fraction alleles in heterogeneous tumors. Next generation sequencing permits the inclusion of more microsatellite markers, but these NGS method still rely on PCR methods, which is the major source of artifacts that obscure the true somatic alterations. In this study, we analyzed a series of colorectal cancers using a new sequencing technology that sequences in parallel more than 200 microsatellite regions - we evaluate mono-, di- , tri- and tetranucleotide repeats to provide a broader spectrum of MSI events. This deep sequencing technology provides greater than 2,000X coverage, uses in vitro CRISPR-Cas9 segmentation, and eliminates the need for PCR amplification. Because every read corresponded to a single DNA molecule, we eliminated the artifacts introduced by PCR that obscured true MSI events. Moreover, we improved the sensitivity of detecting minor allele fractions to as low as 0.1% by measuring microsatellite haplotypes. Included in our analysis was new method for somatic copy number changes in 82 cancer genes, by which we can infer chromosomal instability (CIN) status. Among our cases, 10 cancers had MSI when considering both mononucleotide and tetranucleotide repeats. Interestingly, diagnostic testing of two of these tumors were false negatives and were demonstrated to be MSI-high. There were three major types among the 10 MSI cancers: MSI-high, EMAST, and the cancers having both. However, such types were distinguishable only by microsatellites with intermediate length (10 - 20 bp). MSI-high tumors (n = 3) showed no sign of CIN while all the EMAST tumors (n = 4) also showed CIN phenotype. Our results suggest that the current gold standard MSI diagnostic test may have issues with both false negatives and false positives, indicating that NGS approaches are better suited for evaluating MSI status of tumors and consideration of immunotherapy. Citation Format: Giwon Shin, HoJoon Lee, Susan M. Grimes, Matthew A. Kubit, Hanlee P. Ji. Improved detection and identification of microsatellite instability features in colorectal cancer: Implications for immunotherapy [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 421.

Correlation between gastric carcinoma and ZAC gene-associated microsatellite instability and loss of heterozygosity.

The present study explored the association between loss of heterozygosity (LOH) or microsatellite instability (MSI) of the zinc finger regulator of apoptosis and cell-cycle arrest (ZAC) gene and the clinicopathological factors of gastric cancer. Samples of cancer and cancer-adjacent normal tissue from 30 patients with gastric cancer were collected. The genomic DNA was extracted from each and amplified with primers specific to ZAC microsatellite mutations, then run on a polyacrylamide gel for analysis. The CA197 microsatellite locus exhibited LOH in cancer sample 4. There was LOH in the 15AAAG locus in cancer sample 27 and cancer-adjacent tissue 23, and MSI at 15AAAG in cancer-adjacent tissue 27. There was MSI at the D6S1703 microsatellite locus in cancer-adjacent tissue 28. There was no LOH or MSI in the CA340 microsatellite locus in the gastric cancer or adjacent tissues analyzed. Thus, the frequency of LOH or MSI at ZAC gene-associated microsatellite loci for all patients was 13.3% (4/30). The present study has demonstrated that LOH and MSI events may contribute to the downregulation of ZAC; however, it is unlikely to be the primary cause, as it was only identified in 13.3% of cases.

A molecular portrait of microsatellite instability across multiple cancers

Microsatellite instability (MSI) refers to the hypermutability of short repetitive sequences in the genome caused by impaired DNA mismatch repair. Although MSI has been studied for decades, large amounts of sequencing data now available allows us to examine the molecular fingerprints of MSI in greater detail. Here, we analyse ∼8,000 exomes and ∼1,000 whole genomes of cancer patients across 23 cancer types. Our analysis reveals that the frequency of MSI events is highly variable within and across tumour types. We also identify genes in DNA repair and oncogenic pathways recurrently subject to MSI and uncover non-coding loci that frequently display MSI. Finally, we propose a highly accurate exome-based predictive model for the MSI phenotype. These results advance our understanding of the genomic drivers and consequences of MSI, and our comprehensive catalogue of tumour-type-specific MSI loci will enable panel-based MSI testing to identify patients who are likely to benefit from immunotherapy.

Microsatellite instability detected in tumor-related genes in C57BL/6J mice with thymic lymphoma induced by N-methyl-N-nitrosourea

Microsatellite instability (MSI) has been observed within tumors and found to be closely associated with the degree of malignancy and prognosis in tumors. However, whether MSI in tumor-related genes can be induced by a chemical and whether a connection exists between MSI and tumors remain unclear. In the present study, we detected MSI in the tissues of N-methyl-N-nitrosourea (MNU) treated mice by targeting to 5, 29, 30 microsatellite loci in 3 mismatch repair (MMR) genes, 1 DNA repair gene, and 5 tumor suppressor (TS) genes, respectively. Among 26 mice survived in the MNU-group, 18 (69%) mice presented thymic lymphomas. Moreover, 61% (11/18) of the tumors metastasized to the other organs, including the liver, spleen, and kidney. We examined 104 tissues from MNU-treated mice using the 64 loci, and found 8 MSI events involved 4 loci in 4 tissues types. The MSI incidence in MMR, DNA repair, and TS genes was 67% (2/3), 0% (0/1) and 40% (2/5), respectively. MSI occurrence in tumor and non-tumor tissues was 5.6% (1/18) and 0% (0/8) and that in metastasis and non-metastasis tissues was 7.1% (1/14) and 9.4% (6/64), showing no significant difference. MSI loci in intronic regions of Atm, Msh6 and p21 and MSI in the 3′UTR of Pms2 were detected in MNU-treated mice. Specifically, we found a loss of heterozygosity in intron of Atm (ATM-8) in one metastasis mouse. Four similar events occurred in p21 gene intron (P21-1) of another non-metastasis mouse. Another MSI was a heterozygous mutation existed in an Msh6 allele (MSH6-2) in metastasis mouse. We also found a homozygous 2-bp insertion in the 3′UTR of Pms2 in two non-metastasis mice. These results imply that MNU can induce MSI in MMR and TS genes in C57BL/6J mice. MSI frequency does not seem to be associated with tumorigenesis or metastasis.

Analysis of the relationship between microsatellite instability and thymic lymphoma induced by N-methyl-N-nitrosourea in C57BL/6J mice

Microsatellite instability (MSI) has been found to be closely associated with many types of human tumors and often shows strong correlations with specific tumor features. However, the relationship between MSI and tumors are still unclear. The aim of the present study is to explore the relationships between MSI, tumor formation under the mutagenic effects of N-methyl-N-nitrosourea (MNU). Mice were administered with either MNU (90mg/kg) or PBS and DMSO (control) at the beginning of the 1st week of the experiment. Of the 31 mice that survived the entire experimental time course, 19 (61.3%) mice developed thymic lymphomas. In addition, 52.6% (10/19) of the tumors had metastasized to the liver. We detected MSI in MNU-treated mice using a panel of 42 mutation-sensitive loci. Nineteen loci (45.2%) in six organs showed 70 MSI events. Locus D8Mit14 showed enhanced MSI compared with the other examined loci. MSI frequency in thymus was higher than in other organs. Interestingly, there was no significant difference observed between the metastatic and non-metastatic livers. The MSI frequency (4.6%, 23/(42×12)) in the MNU-treated thymus that had never developed tumor was significantly higher than this in the thymus that had developed lymphoma (0.5%, 4/(42×19)) (p<0.0001). These results indicate that, although thymic tumorigenesis is associated with MSI, it occurs with higher frequency in these that have not developed tumors upon the MNU-treatment. Our study provides additional insights into the relationship between MSI occurrence and tumorigenesis.

A genome-wide view of microsatellite instability: old stories of cancer mutations revisited with new sequencing technologies.

Microsatellites are simple tandem repeats that are present at millions of loci in the human genome. Microsatellite instability (MSI) refers to DNA slippage events on microsatellites that occur frequently in cancer genomes when there is a defect in the DNA-mismatch repair system. These somatic mutations can result in inactivation of tumor-suppressor genes or disrupt other noncoding regulatory sequences, thereby playing a role in carcinogenesis. Here, we will discuss the ways in which high-throughput sequencing data can facilitate genome- or exome-wide discovery and more detailed investigation of MSI events in microsatellite-unstable cancer genomes. We will address the methodologic aspects of this approach and highlight insights from recent analyses of colorectal and endometrial cancer genomes from The Cancer Genome Atlas project. These include identification of novel MSI targets within and across tumor types and the relationship between the likelihood of MSI events to chromatin structure. Given the increasing popularity of exome and genome sequencing of cancer genomes, a comprehensive characterization of MSI may serve as a valuable marker of cancer evolution and aid in a search for therapeutic targets.

The Landscape of Microsatellite Instability in Colorectal and Endometrial Cancer Genomes

Microsatellites-simple tandem repeats present at millions of sites in the human genome-can shorten or lengthen due to a defect in DNA mismatch repair. We present here a comprehensive genome-wide analysis of the prevalence, mutational spectrum, and functional consequences of microsatellite instability (MSI) in cancer genomes. We analyzed MSI in 277 colorectal and endometrial cancer genomes (including 57 microsatellite-unstable ones) using exome and whole-genome sequencing data. Recurrent MSI events in coding sequences showed tumor type specificity, elevated frameshift-to-inframe ratios, and lower transcript levels than wild-type alleles. Moreover, genome-wide analysis revealed differences in the distribution of MSI versus point mutations, including overrepresentation of MSI in euchromatic and intronic regions compared to heterochromatic and intergenic regions, respectively, and depletion of MSI at nucleosome-occupied sequences. Our results provide a panoramic view of MSI in cancer genomes, highlighting their tumor type specificity, impact on gene expression, and the role of chromatin organization.

Microsatellite instability in radiation-induced murine tumours; influence of tumour type and radiation quality

Purpose: To investigate microsatellite instability (MSI) in radiation-induced murine tumours, its dependence on tissue (haemopoietic, intestinal, mammary, brain and skin) and radiation type.Materials and methods: DNA from spontaneous, X-ray or neutron-induced mouse tumours were used in Polymerase Chain Reactions (PCR) with mono- or di-nucleotide repeat markers. Deviations from expected allele size caused by insertion/deletion events were assessed by capillary electrophoresis.Results: Tumours showing MSI increased from 16% in spontaneously arising tumours to 23% (P = 0.014) in X-ray-induced tumours and rising again to 83% (P ≪ 0.001) in neutron-induced tumours. X-ray-induced Acute Myeloid Leukaemias (AML) had a higher level of mono-nucleotide instability (45%) than di-nucleotide instability (37%). Fifty percent of neutron-induced tumours were classified as MSI-high for mono-nucleotide markers and 10% for di-nucleotide markers. Distribution of MSI varied in the different tumour types and did not appear random.Conclusions: Exposure to ionising radiation, especially neutrons, promotes the development of MSI in mouse tumours. MSI may therefore play a role in mouse radiation tumourigenesis, particularly following high Linear Energy Transfer (LET) exposures. MSI events, for a comparable panel of genome-wide markers in different tissue types, were not randomly distributed throughout the genome.

Screening for Microsatellite Instability Identifies Frequent 3′-Untranslated Region Mutation of the RB1-Inducible Coiled-Coil 1 Gene in Colon Tumors

BackgroundCoding region microsatellite instability (MSI) results in loss of gene products and promotion of microsatellite-unstable (MSI-H) carcinogenesis. Recent studies have indicated that MSI within 3′-untranslated regions (3′UTRs) may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3′UTR microsatellites (MSs) in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae.Methodology/Principal FindingsIn order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites). All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r = 0.86, p = 7.2×10−13). 3′UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%), NUAK family SNF1-like kinase 1(NUAK1, 31.0%), and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%). An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3′UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3′UTR MSI (9.0-fold; p = 3.6×10−4).ConclusionsThis mutational survey of well-characterized short 3′UTR MSs confirms that MSI incidence in MSI-H colorectal tumors correlates with MS length, but not with sequence conservation level or distance from other genetic elements. This study also identifies RB1CC1 as a novel target of frequent mutation and aberrant upregulation in MSI-H colorectal tumors. The predicted loss of a microRNA-accessible structure in mutant RB1CC1 RNA fits the hypothesis that 3′UTR MSI involves in aberrant RB1CC1 posttranscriptional upregulation. Further direct assessments are indicated to investigate this possibility.

Microsatellite instability (MSI) and loss of heterozigocity (LOH) in the peripheral blood mononuclear cells (PBMC) of breast cancer (BC) patients receiving systemic chemotherapy

9628 Background: Despite the consistent improvement in overall survival afforded by systemic adjuvant chemotherapy, about 1% of Breast Cancer (BC) patients develop secondary leukemias and/or myelodisplasia probably as a consequence of the genotoxic effects of this type of treatment. Methods: In order to evaluate if systemic chemotherapy could produce microsatelite instability (MSI), we studied 119 sequential blood samples from 30 previously untreated BC patients, collected at every three months intervals, before, during and after receiving systemic chemotherapy. After PCR amplification of the extracted DNA with specific primers for 6 microsatelite loci (BAT-26, BAT-40, MFD-28, MFD-41, TP53, PCR15.1, tp53ALU), the PCR products were denatured, subjected to SSCP and for microssatelite instability (MSI) and loss of heterozigocity (LOH). We also evaluated the expression of PCNA, hMLH1, hPMS1, hPMS2, hMSH2 and P-53 proteins in the PBMC by immunocytochemistry. Results: We observed MSI in 40 of the 119 samples and 27 out of 30 patients had MSI observed in at least one of their collected samples. We observed appearance of LOH at the PCR15.1 locus in 6 patients. We observed a significant correlation between the number of MSI events per sample and the use of chemotherapy ( p = 0.005) specially if it contained alquilating agents (p < 0.0001) . We also observed an inverse correlation between the number of MSI events per sample and the percentage of positive cells for MSH2 by immunocytochemistry (p = 0,0001). The percentage of positive MSH2 cells by immunocytochemistry was inversely correlated with the use of chemotherapy regimens containing alquilating agents (p = 0.019). Conclusions: We conclude that alquilating agent chemotherapy may induce MSI and LOH in PBMC from BC patients possibly through chemotherapy induced decrease in the expression of MSH2. These findings may be related to the generation of secondary leukemias and may also contribute to the intensification of the genetic instability of tumors and further resistance to treatment. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration FAPESP

Microsatellite instability (MSI) and loss of heterozigocity (LOH) in the peripheral blood mononuclear cells (PBMC) of breast cancer (BC) patients receiving systemic chemotherapy

9628 Background: Despite the consistent improvement in overall survival afforded by systemic adjuvant chemotherapy, about 1% of Breast Cancer (BC) patients develop secondary leukemias and/or myelodisplasia probably as a consequence of the genotoxic effects of this type of treatment. Methods: In order to evaluate if systemic chemotherapy could produce microsatelite instability (MSI), we studied 119 sequential blood samples from 30 previously untreated BC patients, collected at every three months intervals, before, during and after receiving systemic chemotherapy. After PCR amplification of the extracted DNA with specific primers for 6 microsatelite loci (BAT-26, BAT-40, MFD-28, MFD-41, TP53, PCR15.1, tp53ALU), the PCR products were denatured, subjected to SSCP and for microssatelite instability (MSI) and loss of heterozigocity (LOH). We also evaluated the expression of PCNA, hMLH1, hPMS1, hPMS2, hMSH2 and P-53 proteins in the PBMC by immunocytochemistry. Results: We observed MSI in 40 of the 119 samples and 27 out of 30 patients had MSI observed in at least one of their collected samples. We observed appearance of LOH at the PCR15.1 locus in 6 patients. We observed a significant correlation between the number of MSI events per sample and the use of chemotherapy ( p = 0.005) specially if it contained alquilating agents (p < 0.0001) . We also observed an inverse correlation between the number of MSI events per sample and the percentage of positive cells for MSH2 by immunocytochemistry (p = 0,0001). The percentage of positive MSH2 cells by immunocytochemistry was inversely correlated with the use of chemotherapy regimens containing alquilating agents (p = 0.019). Conclusions: We conclude that alquilating agent chemotherapy may induce MSI and LOH in PBMC from BC patients possibly through chemotherapy induced decrease in the expression of MSH2. These findings may be related to the generation of secondary leukemias and may also contribute to the intensification of the genetic instability of tumors and further resistance to treatment. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration FAPESP

Systemic chemotherapy induces microsatellite instability in the peripheral blood mononuclear cells of breast cancer patients

IntroductionSystemic chemotherapy is an important part of treatment for breast cancer. We conducted the present study to evaluate whether systemic chemotherapy could produce microsatellite instability (MSI) in the peripheral blood mononuclear cell fraction of breast cancer patients.MethodsWe studied 119 sequential blood samples from 30 previously untreated breast cancer patients before, during and after chemotherapy. For comparison, we also evaluated 20 women who had no relevant medical history (control group).ResultsIn 27 out of 30 patients we observed MSI in at least one sample, and six patients had loss of heterozygosity. We found a significant correlation between the number of MSI events per sample and chemotherapy with alkylating agents (P < 0.0001). We also observed an inverse correlation between the percentage of cells positive for hMSH2 and the number of MSI events per sample (P = 0.00019) and use of alkylating agents (P = 0.019).ConclusionWe conclude that systemic chemotherapy may induce MSI and loss of heterozygosity in peripheral blood mononuclear cells from breast cancer patients receiving alkylating agents, possibly mediated by a chemotherapy-induced decrease in the expression of hMSH2. These effects may be related to the generation of secondary leukaemia in some patients, and may also intensify the genetic instability of tumours and increase resistance to treatment.

Effects of certain metal ions on DNA repair in mammalian cells : Paton, G. R., Department of Cell Pathology, Clinical Research Centre, Harrow, (Great Britain)

Microsatellite instability (MSI) has been found to be closely associated with many types of human tumors and often shows strong correlations with specific tumor features. However, the relationship between MSI and tumors are still unclear. The aim of the present study is to explore the relationships between MSI, tumor formation under the mutagenic effects of N-methyl-N-nitrosourea (MNU). Mice were administered with either MNU (90 mg/kg) or PBS and DMSO (control) at the beginning of the 1st week of the experiment. Of the 31 mice that survived the entire experimental time course, 19 (61.3%) mice developed thymic lymphomas. In addition, 52.6% (10/19) of the tumors had metastasized to the liver. We detected MSI in MNU-treated mice using a panel of 42 mutation-sensitive loci. Nineteen loci (45.2%) in six organs showed 70 MSI events. Locus D8Mit14 showed enhanced MSI compared with the other examined loci. MSI frequency in thymus was higher than in other organs. Interestingly, there was no significant difference observed between the metastatic and non-metastatic livers. The MSI frequency (4.6%, 23/(42 × 12)) in the MNU-treated thymus that had never developed tumor was significantly higher than this in the thymus that had developed lymphoma (0.5%, 4/(42 × 19)) (p < 0.0001). These results indicate that, although thymic tumorigenesis is associated with MSI, it occurs with higher frequency in these that have not developed tumors upon the MNU-treatment. Our study provides additional insights into the relationship between MSI occurrence and tumorigenesis.