Abstract
Purpose:
Defective DNA damage response (DDR) is a hallmark of cancer leading to genomic instability and is associated with chemosensitivity. While the mismatch repair system has been extensively studied, the clinical implications of other mechanisms associated with DDR alterations in patients with colorectal cancer (CRC) remain unclear. This study aimed to understand DDR pathways’ alterations and their association with common clinical features in CRC patients.
Experimental Design:
Next-generation sequencing and whole transcriptome sequencing were conducted using formalin-fixed paraffin-embedded samples submitted to a commercial CLIA-certified laboratory. Samples with pathogenic or presumed pathogenic mutations in 29 specific DDR-related genes were considered as DDR-mutant (DDR-MT) and the remaining samples as DDR-wild-type (DDR-WT).
Results:
Of 9321 CRC patients, 1290 (13.8%) were DDR-MT. The frequency of DDR-MT was significantly higher in MSI-high (MSI-H) cases than in microsatellite stable cases (76.4% vs. 9.5%). The DDR-MT genotype was higher in the right-sided, RAS-wild, BRAF-mutant, and CMS1 subgroups. However, these associations were primarily confounded by the distribution of MSI status. Compared with the DDR-WT tumors, the DDR-MT tumors had a higher mutational burden and gene expression levels in the immune-related pathway, which were independent of MSI status.
Conclusions:
We characterized a distinct subgroup of CRC patients with tumors harboring mutations in the DDR-related genes. These patients more commonly had MSI-H tumors and exhibited an activated immune signature regardless of their tumor’s MSI status. These findings warrant further investigations to develop personalized treatment strategies in this significant subgroup of CRC patients.
Introduction
DNA damage response (DDR) is an autonomous cellular response against endogenous and exogenous DNA damage which can cause detrimental replication stress. Multiple pathways handling key DDR components are governed by numerous genes relevant to DNA repair, DNA damage checkpoints, transcriptional response, and apoptosis. These repair mechanisms permit cells to respond to and repair the myriad of DNA errors that can arise during DNA replication.(1) Despite the physiological function of the DDR system in maintaining human genomic stability, its dysregulation is a hallmark of cancer.(2)
Despite similar histologic appearances under the microscope, colorectal cancers (CRCs) are molecularly heterogeneous tumors.(3) Two distinct molecular mechanisms, which are mutually exclusive, lead to genomic instability in CRC. One is microsatellite instability (MSI) caused by the inactivation of the DNA mismatch repair (MMR) system, which corrects replicative mismatches that escape DNA polymerase proofreading.(4) Compared with microsatellite stable (MSS) tumors, MSI tumors, accounting for approximately 15% of all sporadic CRC and less than 5% of metastatic cases, are associated with higher tumor mutation burden (TMB) and better response to treatment with immune checkpoint inhibitors.(5–9) The other mechanism leading to CRC is chromosomal instability (CIN), which is observed in up to 85% of sporadic cases.(10,11) Given the preclinical evidence showing that DNA damage that occurs prior to a cell entering the mitotic phase induces whole chromosome mis-segregation,(12) inadequate DDR and/or excessive replication stress during the pre-mitotic phase can be considered the major drivers of CIN.(13) Oncogene-induced hyper-replication stress activates the DDR pathways by leading to collapse of the DNA replication forks and, in turn, DNA double-strand breaks (DSBs).(14) While cells that can facilitate accurate DDR to the oncogene-induced DNA damage undergo an irreversible cell-cycle arrest (known as oncogene-induced senescence),(15) some cells with a faulty DDR system develop mutations and chromosome aberrations that are not lethal, leading in some cases to tumorigenesis.(16) Several studies have shown that an activating RAS mutation, a key oncogenic alteration associated with CIN-positive CRC, can cause DNA replication stress leading to genomic instability.(17–19) These findings show that the landscape of the DDR pathways may be diverse among CRC patients, which is partly due to the various oncogenic origins of individual tumors. However, besides MMR, the clinical implications of other DDR pathways that are altered in CRC patients remain poorly characterized.
Presently, the DDR pathway is of clinical interest because of a plethora of data linking defects in this pathway to chemosensitivity.(20) One example of an aberrant DDR pathway with potential clinical implications is homologous recombination deficiency (HRD, commonly referred to as “BRCAness”). Defects in this repair mechanism are indicative of insufficient DSB repair and tumors arising through this pathway exhibit sensitivity to platinum agents and/or PARP inhibitors in ovarian, breast, and pancreatic cancers.(21–24) Because HRD has strong implications for therapeutic strategies in cancers regardless of their organ of origin, a better understanding of molecular associations of DDR pathways can facilitate further exploration of predictive biomarkers and superior strategies in the treatment of a subset of patients with CRC. Therefore, we conducted a large-scale molecular investigation using a comprehensive tumor profiling platform to address genomic and transcriptomic alterations in key DDR pathways and their association with common clinical features in CRC patients.
Materials and methods
Samples collected from the participants
Physicians worldwide submitted formalin-fixed paraffin-embedded (FFPE) samples to a commercial CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ) from February 2015 to July 2019. These samples were analyzed for molecular profiles. This study was conducted in accordance with guidelines of the Declaration of Helsinki, Belmont Report, and U.S. Common Rule. In keeping with 45 CFR 46.101 (b) (4), this study was performed utilizing retrospective, deidentified clinical data from 9,321 CRC patients. Therefore, this study was considered IRB exempt and no patient consent was necessary from the subject.
Genome and transcriptome analyses
Next-generation sequencing (NGS) on a custom-designed panel enriching 592 gene targets (Caris MI TumorSeek panel) and whole transcriptome sequencing (WTS) were conducted using DNA and RNA isolated from FFPE samples, respectively (Supplementary Methods). The NGS data of all patients (n = 9321) were available. However, only 1529 patients had WTS data because the WTS platform was launched during the middle of the research period (February 2019).
Definition of DDR-mutant and DDR-wild type
In this study, we focused on 29 specific DDR-related genes that are known to have roles in five distinct pathways, that are included in the Caris MI TumorSeek Panel. These pathways were homologous recombination (HR, 14 genes), non-homologous end joining (NHEJ, one gene), DNA damage checkpoints (CP, four genes), Fanconi anemia (FA, seven genes), and nucleotide excision repair (NER, three genes). This gene set was determined based on the available data in the literature regarding the suspected core functionality in the pathways handling cellular responses to covalent alterations in DNA structure. The MMR pathway was not included because it processes noncovalent anomalous DNA structures.(1) The detailed gene list is depicted Table 1. The samples with pathogenic or presumed pathogenic mutations, categorized according to the American College of Medical Genetics and Genomics standards, in any of the 29 DDR-related genes were considered DDR-mutant (DDR-MT) and the remaining samples as DDR-wild type (DDR-WT).
Table 1:
Gene set of DDR pathway
DDR pathways | HR pathway | BAP1 |
BARD1 | ||
BLM | ||
BRCA1 | ||
BRCA2 | ||
BRIP1 | ||
CDK12 | ||
MRE11 | ||
NBN | ||
PALB2 | ||
RAD50 | ||
RAD51 | ||
RAD51B | ||
WRN | ||
NHEJ pathway | PRKDC | |
CP pathway | ATM | |
ATR | ||
CHEK1 | ||
CHEK2 | ||
FA pathway | FANCA | |
FANCC | ||
FANCD2 | ||
FANCE | ||
FANCF | ||
FANCG | ||
FANCL | ||
NER pathway | ERCC1 | |
XPA | ||
XPC |
Abbreviations: CP, DNA damage checkpoints; DDR, DNA damage response; FA, Fanconi anemia; HR, homologous recombination; NER, nucleotide excision repair; NHEJ, non-homologous end joining;
Consensus molecular subtype classification
The Caris consensus molecular subtype (CMS) classifier was developed using RNA sequencing data from the WTS platform (Supplementary Methods).
Assessment of immunotherapy-related biomarkers
MSI/MMR status was assessed with a combination of methods using immunohistochemistry (IHC), fragment analysis, and NGS, with resulting status defined as either MSI-high (MSI-H)/MMR-deficient (dMMR) or MSS/MMR-proficient (pMMR) (Supplementary Methods).
TMB was measured by counting all nonsynonymous missense mutations found per tumor (592 genes and 1.4 megabases [Mb] sequenced/tumor). Two cutoff values of TMB were set to determine TMB-high (TMB-H): 17 mutations/Mb, based on the report of TMB exhibiting high concordance with MSI-H in CRC(25,26); and 10 mutations/Mb, based on the result of KEYNOTE-158 trial showing clinical activity of pembrolizumab in tumors harboring a TMB ≥10 (TMB-H) across a variety of previously treated solid tumors.(27)
PD-L1 expression was tested via IHC using SP142 antibody (Spring Biosciences). The staining intensity on the tumor cell membrane was assessed on a semiquantitative scale: 0 for no staining, 1+ for weak staining, 2+ for moderate staining, and 3+ for strong staining. Tumors exhibiting ≥5% of tumor cells stained as 2+ or 3+ were considered PD-L1 positive.
Statistical analysis
The clinical and molecular features between DDR-MT and DDR-WT were compared. The alteration frequencies of DDR-related genes/pathways were compared between subgroups based on common clinical features. The nonparametric Wilcoxon rank-sum test was used to compare age and TMB distribution between DDR-MT and DDR-WT. Other categorical data were assessed using the chi-square or Fisher’s exact test, where appropriate. Proximal tumors occurring in the cecum, ascending colon, or transverse colon, and distal tumors occurring in the descending colon, sigmoid colon, or rectum were defined as right- and left-sided tumors, respectively. Mutual exclusivity/co-occurrence of DDR gene mutations was tested by calculating the log2 odds ratio for each DDR gene pair, in which values > 0 indicated a tendency of co-occurrence, while values ≤ 0 indicated a tendency of mutual exclusivity. Statistical significance was tested by one-sided Fisher’s exact tests. The Microenvironment Cell Population-counter (MCP-counter) was used for the quantification of the abundance of immune and stromal cell populations using WTS data, as previously described.(28) The median gene expression levels were compared between each subgroup, and the fold change was calculated. Patients with any missing data were not included in the analysis. To adjust p values for multiple hypothesis testing, the q values were calculated using the Benjamini–Hochberg method. All statistical analyses were two-sided at a significance level set to 0.05 and conducted with SPSS v23 (IBM SPSS Statistics).
Results
Characteristics of the participants
The patient characteristics are shown in the Table S1. The analyzed samples were collected from 5059 (54.3%) primary lesions and 4252 (45.6%) metastatic lesions, and 10 samples (0.1%) lacked information about the location of tumor sampling. Of 9321 patients, 1290 (13.8%) were DDR-MT. Compared with DDR-WT patients, DDR-MT patients had a higher median age and frequency of female sex, right-sided tumor, and MSI-H/dMMR. Compared with MSI-H/dMMR patients, MSS/pMMR patients had a higher frequency of RAS mutation (54.1% vs. 28.5%, p < .001) and a lower frequency of BRAF mutation (6.9% vs. 41.2%, p < .001) (Table S2).
Mutation frequency in DDR-related genes
Among the 29 DDR-related genes, the highest mutation rate was identified in the ATM (4.5%), BRCA2 (2.7%), PRKDC (1.6%), and CHEK2 (1.2%) genes. The frequency of alterations in each DDR pathway was 8.1% in HR, 5.5% in CP, 1.9% in FA, and 1.6% in the NHEJ pathways. No pathogenic or presumed pathogenic mutations were observed in the NER pathway (Figure 1). Among 1290 DDR-MT patients, 959 (74.3%) had single DDR gene mutations, and 331 (25.7%) had two or more genes mutations (Table S3). There was an overall tendency for co-occurrence of DDR gene mutations (Table S4).
Figure 1. The frequency of DDR pathways alteration.
Abbreviations: CP, DNA damage checkpoints; DDR, DNA damage response; FA, Fanconi anemia; HR, homologous recombination; NER, nucleotide excision repair; NHEJ, non-homologous end joining.
Frequency of DDR-MT between the clinical subgroups
The frequency of DDR-MT was significantly higher in MSI-H/dMMR patients than in MSS/pMMR patients (76.4% [456/597] vs. 9.5% [829/8702], both p and q < .001). Consistently, all pathways, except for NER, were more commonly altered in MSI-H/dMMR patients (Figure 2, Table S5). Among MSS/pMMR patients with POLE mutation (N = 42), DDR-MT was 37 patients (88.1%), which accounted for 4.5% (37/829) of all DDR-MT patients in MSS/pMMR subgroup (Figure S1).
Figure 2. The frequency of DDR pathways alteration by MSI/MMR status.
Significant difference is indicated by **q < .05.
Abbreviations: CP, DNA damage checkpoints; DDR, DNA damage response; dMMR, mismatch repair deficient; FA, Fanconi anemia; HR, homologous recombination; NER, nucleotide excision repair; NHEJ, non-homologous end joining; MSI-H, microsatellite instability high; MSS, microsatellite stable; pMMR, mismatch repair proficient.
Right-sided tumors had a significantly higher DDR-MT frequency than left-sided tumors (20.9% vs. 10.8%, both p and q < .001). However, when compared individually in subgroups based on MSI status, DDR-MT frequency was not significantly associated with the sidedness in either the MSS/pMMR (10.6% vs. 9.1%; p = 0.055, q > .99) or the MSI-H/dMMR (79.6% vs. 72.5%; p = .11, q > .99) subgroups (Figure S2, Table S6).
The RAS-mutant (RAS-MT) tumors had a significantly lower DDR-MT frequency than RAS-wild type (RAS-WT) tumors (12.2% vs. 15.6%, both p and q < .001). However, there was no significant difference in either the MSS/pMMR (9.8% vs. 9.2%, p = .29, q = .72) or the MSI-H/dMMR (77.1% vs. 76.1%, p = .81, q > .99) subgroups (Figure S3, Table S7). Conversely, the BRAF-mutant-type (BRAF-MT) tumors had a significantly higher DDR-MT frequency than the BRAF-wild-type (BRAF-WT) tumors (31.1% vs. 12.1%, both p and q < .001). However, this difference was not significant in either the MSS/pMMR (11.0% vs. 9.4%; p = 0.188, q > .99) or the MSI-H/dMMR (80.4% vs. 73.7%; p = .06, q = .40) subgroups (Figure S4, Table S8).
In total, 1529 samples were available for the CMS classifier analysis. The frequencies of DDR-MT in each subtype were 34.8% (CMS1: MSI immune), 7.1% (CMS2: Canonical), 15.2% (CMS3: Metabolic), and 11.8% (CMS4: Mesenchymal). A pairwise comparison revealed that the CMS1 had the highest frequency of DDR-MT, whereas the CMS2 had the lowest. In MMS/pMMR cases, the frequencies of DDR-MT were 12.2% (CMS1), 7.1% (CMS2), 9.2% (CMS3), and 9.8% (CMS4). However, any significant differences were not identified based on a pairwise comparison (Figure S5, Table S9).
DDR gene expression levels between the clinical subgroups
The left-sided tumors had higher expression levels of most DDR-related genes than the right-sided tumors. This trend was consistent in both the MSS/pMMR and MSI-H/dMMR subgroups (Figure 3, Table S10). The RAS-MT tumors had lower expression levels of most DDR genes than the RAS-WT tumors in the MSS/pMMR subgroup. Conversely, in the MSI-H/dMMR subgroup, the RAS-MT tumors had higher expression levels of the DDR genes than the RAS-WT tumors. Compared with the BRAF-WT tumors, the BRAF-MT tumors had inconsistent relative levels of DDR gene expression in the MSS/pMMR subgroup. Notably, in the MSI-H/dMMR subgroup, the BRAF-MT tumors had lower expression levels of most DDR genes than the BRAF-WT tumors (Figure 3, Table S10).
Figure 3. Comparison of DDR gene expression levels between clinical subgroups.
Heatmap indicates fold-change (subgroup A/ subgroup B) in median gene expression levels. Red color means higher expression levels in subgroup A compared to subgroup B, while blue color means the opposite directions. Significant difference is indicated by *p < .05 (and q ≥ .05) and **q < .05.
Abbreviations: DDR, DNA damage response; MSI-H, microsatellite instability high; MSS, microsatellite stable; MT, mutant; WT, wild type.
Immune signature in DDR-MT and DDR-WT tumors
The mean TMB was significantly higher in DDR-MT than in DDR-WT (20.9/Mb vs. 7.7/Mb, p < .001). This finding was consistent in both the MSS/pMMR (13.7/Mb vs. 7.6/Mb, p = .02) and the MSI-H/dMMR (54.5/Mb vs. 27.8/Mb, p < .001) subgroups. The frequency of TMB-H was higher in DDR-MT than in DDR-WT, using both cutoff values (17/Mb; and 10/Mb), in all (38.1% vs. 2.1%, p < .001; 52.3% vs. 20.7%, p < .001), MSS/pMMR (5.6% vs. 0.6%, p < .001; 26.3% vs. 19.4%, p < .001), and MSI-H/dMMR (97.1% vs. 84.1%, p < .001; 99.3% vs. 94.2%, p < .001) cases. Furthermore, PD-L1 positivity was higher in DDR-MT than in DDR-WT in all (10.1% vs. 2.7%, p < .001) and MSS/pMMR (4.8% vs. 2.4%, p < .001) cases. However, in MSI-H/dMMR, no difference was observed in terms of PD-L1 positivity between DDR-MT and DDR-WT (Table 2). In MSS/pMMR patients without POLE mutations, DDR-MT showed higher mean TMB (8.0/Mb vs. 7.6/Mb, p = .03), frequency of TMB-H (1.2% vs. 0.5%, p = .04, with cutoff of 17/Mb; 22.8% vs. 19.4%, p = .02, with cutoff of 10/Mb), and PD-L1 positivity (4.4% vs. 2.4%, p < .001) than DDR-WT (Table S11).
Table 2.
Comparison of immunotherapy-related markers between DDR-MT and DDR-WT
All | MSS/pMMR | MSI-H/dMMR | |||||||
---|---|---|---|---|---|---|---|---|---|
DDR-MT (N = 1290) |
DDR-WT (N = 8031) |
P-value | DDR-MT (N = 829) |
DDR-WT (N = 7873) |
P-value | DDR-MT (N = 456) |
DDR-WT (N = 141) |
P-value | |
TMB (Mean) | 20.9/Mb | 7.7/Mb | < .001 | 13.7/Mb | 7.6/Mb | .02 | 54.5/Mb | 27.8/Mb | < .001 |
TMB-H (≥17/Mb) |
38.1% | 2.1% | < .001 | 5.6% | 0.6% | < .001 | 97.1% | 84.1% | < .001 |
TMB-H (≥10/Mb) |
52.3% | 20.7% | < .001 | 26.3% | 19.4% | < .001 | 99.3% | 94.2% | < .001 |
PD-L1 ≥5% | 10.1% | 2.7% | < .001 | 4.8% | 2.4% | < .001 | 19.8% | 20.4% | .87 |
Abbreviations: DDR, DNA damage response; dMMR, mismatch repair deficient; MSI-H, microsatellite instability high; MSS, microsatellite stable; MT, mutant; pMMR, mismatch repair proficient; TMB, tumor mutation burden; TMB-H, tumor mutation burden high; WT, wild type.
An MCP-counter analysis revealed that DDR-MT had higher levels of all immune cell and fibroblast populations than DDR-WT regardless of MSI status. Consistently, the gene expression levels of all immune checkpoints were higher in DDR-MT than in DDR-WT, except for LAG3, CTLA4, and PD-L1 with lower levels in DDR-MT in the MSI-H/dMMR subgroups (Figure 4, Table S12).
Figure 4. Comparison of immune-related gene signature between DDR-MT and DDR-WT.
Heatmap indicates fold-change (DDR-MT/ DDR-WT) in median gene expression levels by the MCP-counter analysis. Red color means higher expression levels in DDR-MT compared to DDR-WT, while blue color means the opposite directions. Significant difference is indicated by *p < .05 (and q ≥ .05) and **q < .05.
Abbreviations: DDR, DNA damage response; MSI-H, microsatellite instability high; MSS, microsatellite stable; MT, mutant; WT, wild type.
Discussion
To the best of our knowledge, this is the largest study that investigated the DDR pathway’s alterations in CRC patients. We have characterized a distinct subgroup of patients whose tumors harbor mutations in DDR-related genes. These patients were strongly enriched in MSI-H and associated with activated immune signature both in MSS and MSI-H subgroups.
DDR is a highly complex and redundant system coordinated by different DNA repair pathways. Thus, we extensively focused on several types of DDR pathway, and 29 specific genes were selected in the present study. Our gene set mostly overlapped with the published gene set determining “BRCAness”. However, it covered a wider spectrum of DDR pathways.(29,30) Our results showed that 13.8% of CRC patients harbored pathogenic or presumed pathogenic mutations in the DDR pathways even though each gene was mutated in a small subset of patients.
One of the main findings of this study was the significant association between DDR-MT and MSI-H. As previously reported, MSI-H tumors frequently develop secondary mutations, including both driver and passenger mutations.(31,32) Although well-established MSI target genes include ACVR2A and TGFBR2 in CRC, a high mutation rate was reported in DDR-related genes. Specifically, Miquel et al. reported that mutations in the MRE11 gene were observed in 74% of MSI-H CRC tumors in a small study (n = 39).(33) Meanwhile, the present study showed that the mutations in the DDR pathways were noted in 76.4% of the MSI-H CRC patients (n = 597), which was significantly higher than the mutational rate in MSS patients (9.5%). Almost all DDR genes were consistently mutated at a higher rate in MSI-H tumors than in MSS, implying the MSI status broadly contributed to secondary mutations in the DDR pathway. Because we only counted pathogenic and presumed pathogenic mutations, our findings revealed that MSI-H tumors commonly harbor functional mutations in the DDR pathways that may have clinical relevance. Several preclinical studies showed that MMR proteins recognize DNA adducts formed by 5-fluorouracil (FU) metabolite and cisplatin (but not oxaliplatin) and, in turn, lead to ultimate cell-cycle arrest and apoptosis via the futile cycling of mismatches.(34–37) These findings have been supported by some clinical studies showing that FU-based adjuvant therapy was not effective in patients with stage II or III CRC exhibiting MSI-H.(38–40) Our results further provide important evidence that MSI-H tumors imply a functional defect in the DDR system independent of the MMR system. This may favor chemosensitivity, which is in the opposite direction to the loss of MMR function as the detector of DNA adduct. These indicative two-faced mechanisms inherent in MSI tumors may be important in determining responsiveness to chemotherapy. Thus, the therapeutic implication of alterations in the DDR pathways in MSI-H CRC should be assessed in future clinical trials.
Our results showed that DDR-MT was enriched in right-sided, RAS-WT, BRAF-MT, and CMS1 tumors. However, all these findings were primarily confounded by the distribution of MSI status. In fact, when individually analyzed in the MSS and MSI-H subgroups, no significant associations were observed between the frequency of DDR-MT and each clinical feature. However, our transcriptomic research revealed that several clinical and oncogenic characteristics could affect the DDR signature. Notably, left-sided CRC tumors had a more activated DDR signature than right-sided CRC regardless of MSI status. Meanwhile, the RAS and BRAF mutational statuses might have a different impact on the DDR signature based on MSI status. In MSS tumors, RAS-MT had a lower DDR signature than RAS-WT. Furthermore, in MSI-H tumors, RAS-MT had a higher DDR signature than RAS-WT, and BRAF-MT had a lower DDR signature than BRAF-WT. These findings indicate the different levels of DDR signature as determined based on the side of the tumor’s origin, RAS/BRAF mutation, and MSI status, which may affect chemosensitivity in individual patients.
POLE mutation is known to be accompanied with numerous secondary mutations leading to hypermutated tumors.(41) Our results showed POLE mutation was strongly associated with a DDR-MT genotype with a finding that 88.1% of patients with POLE mutation were DDR-MT in MSS subgroup. However, these patients were a small subset (4.5%) of DDR-MT and MSS.
Furthermore, the present study showed that DDR-MT CRC had higher TMB and gene expression levels in the immune-related pathway than DDR-WT independent of MSI status. Even after excluding patients with POLE mutation, DDR-MT CRC showed higher TMB and PD-L1 positivity than DDR-WT. Our results are supported by previous studies showing that genomic instability caused by deficient DDR increases immunogenicity in the tumor microenvironment. Specifically, preclinical data revealed that the DDR-deficient tumor cells have increased cytosolic DNA and, in turn, constitutive activation of the cGAS/STING/TBK/IRF3 pathway, which activates chemokine expression and T cell recruitment.(42,43) Consistently, clinical evidence revealed that breast, urothelial, and lung tumors with a DDR-deficient phenotype exhibit higher neoantigen loads, tumor-infiltrating lymphocyte, TMB, and PD-L1 expression.(43–46) Furthermore, DDR alterations were significantly associated with response to immune checkpoint inhibitors in patients with metastatic urothelial carcinoma.(47) However, data on this issue in CRC are limited. A large study focusing on 10 DDR-related genes showed that gastrointestinal cancers with DDR alterations had a higher TMB than those without DDR alterations.(48) However, this finding was believed to be affected by a deficient MMR rather than DDR. The present study first showed that a deficient DDR increases immune activity in the tumor microenvironment of CRC both in MSS and MSI-H cases. Given a recent study showing TMB is predictive of response to immune checkpoint inhibitors in MSI-H metastatic CRC, our results indicate DDR-MT may be a predictive biomarker for immunotherapy in MSI-H CRC.(49) However, in MSS cases, it seems to be doubtful whether immunotherapy exerts a clinically meaningful efficacy in DDR-MT patients, because the difference of mean TMB was small compared to DDR-WT, especially for patients without POLE mutations. Thus, the predictive values of DDR alterations in immunotherapy warrant further validations in prospective clinical trials.
The present study had several limitations. Given the nature of the study that assessed the tumor tissue, whether a given mutation was a somatic or germline mutation or a bi-allelic or mono-allelic mutation was not determined. A previous pan-cancer analysis revealed that bi-allelic alterations in HR-related genes were more associated with the genomic features of HRD than mono-allelic mutations.(50) Hence, future studies that assess the additional level of details on allelic status must be conducted. Furthermore, because the core DDR-related genes were manually curated based on available studies in the literature, our gene set and classification were not validated. However, a high number of genes are associated with the DDR system, and several genes overlap in several DDR pathways. The published lists of genes significantly differed, and they have not yet been standardized for determining DDR-deficient or HRD phenotype.(29,30,50–52) Thus, to date, it may be difficult to make up the solid panel to detect DDR alterations. However, given the failure in predicting treatment outcomes based on a single gene or protein alteration in the DDR pathway,(53,54) assessing multiple genes will be helpful for accurate treatments in future medicine targeting the DDR pathway. Thus, further validation and refinement may be needed for making up such a panel. Finally, this study lacked data showing phenotypic effects of DDR-MT genotype. Further translational researches are warranted to address whether the DDR alterations contribute to carcinogenesis, reflect a BRCAness molecular phenotype, and link to clinical outcomes.
In conclusion, the landscape of the DDR pathway is highly diverse in individual tumors, and alterations in this pathway are significantly associated with MSI-H in CRC. DDR-MT CRCs have activated immune profiles regardless of MSI status. These findings may be used in testing future treatment strategies targeting the DDR pathways and/or immune checkpoints in CRC patients.
Supplementary Material
Translational relevance.
Despite defect in DNA damage response (DDR) being associated with tumorigenesis and chemosensitivity, most of its clinical implication in colorectal cancer (CRC) remains unclear. This large-scale genomic and transcriptomic profiling project investigated the molecular and clinical characteristics of CRC exhibiting alterations in DDR pathways. Results showed that there was a significant association between alterations in the DDR pathway and microsatellite instability-high (MSI-H). Furthermore, DDR-mutant CRCs had activated immune profiles compared to DDR-wild type CRCs, both in MSI-H and microsatellite stable cases. Transcriptomic analyses indicated the different levels of DDR signature as determined based on the primary tumor sidedness, RAS/BRAF mutation, and MSI status. These findings highlight the landscape of the DDR pathway is highly diverse in individual tumors, and suggest future personalized treatment strategies targeting the DDR pathways and/or immune checkpoints in a distinct subpopulation in CRC.
Acknowledgment:
We thank all patients who contributed to this study.
Funding:
This work was supported by the National Cancer Institute [P30CA 014089 to H.-J.Lenz], Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Victoria and Philip Wilson Research Fund, San Pedro Peninsula Cancer Guild, and Daniel Butler Research Fund.
Footnotes
Conflict of interest statement: Goldberg RM reports consulting/advisory role for Merk, Taiho Pharmaceutical, Merck KGaA, and Novartis, research funding from Bristol-Myers Squibb, and travel paid by Merck KGaA and Merck; Weinberg BA reports speaker for Bayer, Lilly, Taiho, and Sirtex, and consulting/advisory role for Bayer; Sohal D reports consulting/advisory role for Perthera and Ability Pharmaceuticals, honoraria by Foundation Medicine, speakers bureau for Incyte, and research funding at institution by Amgen, Apexigen, Bristol-Meyers Squibb, Celgene, FibroGen, Genentech, Medimmune, Merck, OncoMed, and Rafael; Hwang JJ reports speakers bureau for Amgen, Bayer, Bristol Myers, Boehringer Ingelheim, Celgene, Eisai, Incyte, Lilly, and Roche, and consulting/advisory role for Genentech and Taiho; Marshall JL reports conflicts with Caris Life Sciences, Indivumed, Merck, Anger, Taiho, Bayer, Celgene, and Ipsen; Cremolini C reports honoraria by Roche, Merck, Amgen, Servier, Bayer, and MSD, and research grant by Merck and Bayer; Moretto R reports honoraria by Roche; Elliott A, Xiu J, Stafford P, Zhang J, and Korn WM are employed by Caris Life Sciences. All remaining authors have declared no conflicts of interest.
Data availability statement:
De-identified datasets analyzed in the current study are available from the corresponding author on reasonable request.
References
- 1.Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004;73:39–85 doi 10.1146/annurev.biochem.73.011303.073723. [DOI] [PubMed] [Google Scholar]
- 2.Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646–74 doi 10.1016/j.cell.2011.02.013. [DOI] [PubMed] [Google Scholar]
- 3.Dienstmann R, Vermeulen L, Guinney J, Kopetz S, Tejpar S, Tabernero J. Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer. Nature reviews Cancer 2017;17(2):79–92 doi 10.1038/nrc.2016.126. [DOI] [PubMed] [Google Scholar]
- 4.Sinicrope FA, Sargent DJ. Molecular pathways: microsatellite instability in colorectal cancer: prognostic, predictive, and therapeutic implications. Clin Cancer Res 2012;18(6):1506–12 doi 10.1158/1078-0432.CCR-11-1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Cancer Genome Atlas N Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012;487(7407):330–7 doi 10.1038/nature11252. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med 2015;372(26):2509–20 doi 10.1056/NEJMoa1500596. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz H-J, Morse MA, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. The Lancet Oncology 2017;18(9):1182–91 doi 10.1016/s1470-2045(17)30422-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Overman MJ, Lonardi S, Wong KYM, Lenz HJ, Gelsomino F, Aglietta M, et al. Durable Clinical Benefit With Nivolumab Plus Ipilimumab in DNA Mismatch Repair-Deficient/Microsatellite Instability-High Metastatic Colorectal Cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2018;36(8):773–9 doi 10.1200/JCO.2017.76.9901. [DOI] [PubMed] [Google Scholar]
- 9.Le DT, Kim TW, Van Cutsem E, Geva R, Jager D, Hara H, et al. Phase II Open-Label Study of Pembrolizumab in Treatment-Refractory, Microsatellite Instability-High/Mismatch Repair-Deficient Metastatic Colorectal Cancer: KEYNOTE-164. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2020;38(1):11–9 doi 10.1200/JCO.19.02107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Pino MS, Chung DC. The chromosomal instability pathway in colon cancer. Gastroenterology 2010;138(6):2059–72 doi 10.1053/j.gastro.2009.12.065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Carethers JM, Jung BH. Genetics and Genetic Biomarkers in Sporadic Colorectal Cancer. Gastroenterology 2015;149(5):1177–90 e3 doi 10.1053/j.gastro.2015.06.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Bakhoum SF, Kabeche L, Murnane JP, Zaki BI, Compton DA. DNA-damage response during mitosis induces whole-chromosome missegregation. Cancer Discov 2014;4(11):1281–9 doi 10.1158/2159-8290.CD-14-0403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Burrell RA, McClelland SE, Endesfelder D, Groth P, Weller MC, Shaikh N, et al. Replication stress links structural and numerical cancer chromosomal instability. Nature 2013;494(7438):492–6 doi 10.1038/nature11935. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damage model for cancer development. Science 2008;319(5868):1352–5 doi 10.1126/science.1140735. [DOI] [PubMed] [Google Scholar]
- 15.Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 2006;444(7119):638–42 doi 10.1038/nature05327. [DOI] [PubMed] [Google Scholar]
- 16.Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434(7035):864–70 doi 10.1038/nature03482. [DOI] [PubMed] [Google Scholar]
- 17.Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nature reviews Cancer 2011;11(11):761–74 doi 10.1038/nrc3106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Margetis N, Kouloukoussa M, Pavlou K, Vrakas S, Mariolis-Sapsakos T. K-ras Mutations as the Earliest Driving Force in a Subset of Colorectal Carcinomas. In Vivo 2017;31(4):527–42 doi 10.21873/invivo.11091. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Ogrunc M, Di Micco R, Liontos M, Bombardelli L, Mione M, Fumagalli M, et al. Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation. Cell Death Differ 2014;21(6):998–1012 doi 10.1038/cdd.2014.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Goldstein M, Kastan MB. The DNA damage response: implications for tumor responses to radiation and chemotherapy. Annu Rev Med 2015;66:129–43 doi 10.1146/annurev-med-081313-121208. [DOI] [PubMed] [Google Scholar]
- 21.Hoppe MM, Sundar R, Tan DSP, Jeyasekharan AD. Biomarkers for Homologous Recombination Deficiency in Cancer. J Natl Cancer Inst 2018;110(7):704–13 doi 10.1093/jnci/djy085. [DOI] [PubMed] [Google Scholar]
- 22.Tan DS, Rothermundt C, Thomas K, Bancroft E, Eeles R, Shanley S, et al. “BRCAness” syndrome in ovarian cancer: a case-control study describing the clinical features and outcome of patients with epithelial ovarian cancer associated with BRCA1 and BRCA2 mutations. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2008;26(34):5530–6 doi 10.1200/JCO.2008.16.1703. [DOI] [PubMed] [Google Scholar]
- 23.Swisher EM, Lin KK, Oza AM, Scott CL, Giordano H, Sun J, et al. Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 Part 1): an international, multicentre, open-label, phase 2 trial. The Lancet Oncology 2017;18(1):75–87 doi 10.1016/s1470-2045(16)30559-9. [DOI] [PubMed] [Google Scholar]
- 24.Golan T, Hammel P, Reni M, Van Cutsem E, Macarulla T, Hall MJ, et al. Maintenance Olaparib for Germline BRCA-Mutated Metastatic Pancreatic Cancer. N Engl J Med 2019;381(4):317–27 doi 10.1056/NEJMoa1903387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Vanderwalde A, Spetzler D, Xiao N, Gatalica Z, Marshall J. Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients. Cancer medicine 2018;7(3):746–56 doi 10.1002/cam4.1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Stadler ZK, Battaglin F, Middha S, Hechtman JF, Tran C, Cercek A, et al. Reliable Detection of Mismatch Repair Deficiency in Colorectal Cancers Using Mutational Load in Next-Generation Sequencing Panels. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2016;34(18):2141–7 doi 10.1200/JCO.2015.65.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Marabelle A, Fakih M, Lopez J, Shah M, Shapira-Frommer R, Nakagawa K, et al. Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study. The Lancet Oncology 2020;21(10):1353–65 doi 10.1016/s1470-2045(20)30445-9. [DOI] [PubMed] [Google Scholar]
- 28.Becht E, Giraldo NA, Lacroix L, Buttard B, Elarouci N, Petitprez F, et al. Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression. Genome biology 2016;17(1):218 doi 10.1186/s13059-016-1070-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Pennington KP, Walsh T, Harrell MI, Lee MK, Pennil CC, Rendi MH, et al. Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas. Clin Cancer Res 2014;20(3):764–75 doi 10.1158/1078-0432.CCR-13-2287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Park W, Chen J, Chou JF, Varghese AM, Yu KH, Wong W, et al. Genomic Methods Identify Homologous Recombination Deficiency in Pancreas Adenocarcinoma and Optimize Treatment Selection. Clin Cancer Res 2020. doi 10.1158/1078-0432.CCR-20-0418. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Campbell BB, Light N, Fabrizio D, Zatzman M, Fuligni F, de Borja R, et al. Comprehensive Analysis of Hypermutation in Human Cancer. Cell 2017;171(5):1042–56 e10 doi 10.1016/j.cell.2017.09.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Kim TM, Laird PW, Park PJ. The landscape of microsatellite instability in colorectal and endometrial cancer genomes. Cell 2013;155(4):858–68 doi 10.1016/j.cell.2013.10.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Miquel C, Jacob S, Grandjouan S, Aime A, Viguier J, Sabourin JC, et al. Frequent alteration of DNA damage signalling and repair pathways in human colorectal cancers with microsatellite instability. Oncogene 2007;26(40):5919–26 doi 10.1038/sj.onc.1210419. [DOI] [PubMed] [Google Scholar]
- 34.Tajima A, Hess MT, Cabrera BL, Kolodner RD, Carethers JM. The mismatch repair complex hMutS alpha recognizes 5-fluorouracil-modified DNA: implications for chemosensitivity and resistance. Gastroenterology 2004;127(6):1678–84 doi 10.1053/j.gastro.2004.10.001. [DOI] [PubMed] [Google Scholar]
- 35.Fink D, Zheng H, Nebel S, Norris PS, Aebi S, Lin TP, et al. In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair. Cancer Res 1997;57(10):1841–5. [PubMed] [Google Scholar]
- 36.Vaisman A, Varchenko M, Umar A, Kunkel TA, Risinger JI, Barrett JC, et al. The role of hMLH1, hMSH3, and hMSH6 defects in cisplatin and oxaliplatin resistance: correlation with replicative bypass of platinum-DNA adducts. Cancer Res 1998;58(16):3579–85. [PubMed] [Google Scholar]
- 37.Hewish M, Lord CJ, Martin SA, Cunningham D, Ashworth A. Mismatch repair deficient colorectal cancer in the era of personalized treatment. Nat Rev Clin Oncol 2010;7(4):197–208 doi 10.1038/nrclinonc.2010.18. [DOI] [PubMed] [Google Scholar]
- 38.Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2005;23(3):609–18 doi 10.1200/JCO.2005.01.086. [DOI] [PubMed] [Google Scholar]
- 39.Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2010;28(20):3219–26 doi 10.1200/JCO.2009.27.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, et al. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 2003;349(3):247–57 doi 10.1056/NEJMoa022289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Rayner E, van Gool IC, Palles C, Kearsey SE, Bosse T, Tomlinson I, et al. A panoply of errors: polymerase proofreading domain mutations in cancer. Nature reviews Cancer 2016;16(2):71–81 doi 10.1038/nrc.2015.12. [DOI] [PubMed] [Google Scholar]
- 42.Sen T, Rodriguez BL, Chen L, Corte CMD, Morikawa N, Fujimoto J, et al. Targeting DNA Damage Response Promotes Antitumor Immunity through STING-Mediated T-cell Activation in Small Cell Lung Cancer. Cancer Discov 2019;9(5):646–61 doi 10.1158/2159-8290.CD-18-1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Parkes EE, Walker SM, Taggart LE, McCabe N, Knight LA, Wilkinson R, et al. Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer. J Natl Cancer Inst 2017;109(1) doi 10.1093/jnci/djw199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Teo MY, Bambury RM, Zabor EC, Jordan E, Al-Ahmadie H, Boyd ME, et al. DNA Damage Response and Repair Gene Alterations Are Associated with Improved Survival in Patients with Platinum-Treated Advanced Urothelial Carcinoma. Clin Cancer Res 2017;23(14):3610–8 doi 10.1158/1078-0432.CCR-16-2520. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Chae YK, Anker JF, Oh MS, Bais P, Namburi S, Agte S, et al. Mutations in DNA repair genes are associated with increased neoantigen burden and a distinct immunophenotype in lung squamous cell carcinoma. Sci Rep 2019;9(1):3235 doi 10.1038/s41598-019-39594-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Park S, Lee H, Lee B, Lee SH, Sun JM, Park WY, et al. DNA Damage Response and Repair Pathway Alteration and Its Association With Tumor Mutation Burden and Platinum-Based Chemotherapy in SCLC. J Thorac Oncol 2019;14(9):1640–50 doi 10.1016/j.jtho.2019.05.014. [DOI] [PubMed] [Google Scholar]
- 47.Teo MY, Seier K, Ostrovnaya I, Regazzi AM, Kania BE, Moran MM, et al. Alterations in DNA Damage Response and Repair Genes as Potential Marker of Clinical Benefit From PD-1/PD-L1 Blockade in Advanced Urothelial Cancers. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2018;36(17):1685–94 doi 10.1200/JCO.2017.75.7740. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Parikh AR, He Y, Hong TS, Corcoran RB, Clark JW, Ryan DP, et al. Analysis of DNA Damage Response Gene Alterations and Tumor Mutational Burden Across 17,486 Tubular Gastrointestinal Carcinomas: Implications for Therapy. The oncologist 2019;24(10):1340–7 doi 10.1634/theoncologist.2019-0034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Schrock AB, Ouyang C, Sandhu J, Sokol E, Jin D, Ross JS, et al. Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSI-high metastatic colorectal cancer. Annals of oncology : official journal of the European Society for Medical Oncology 2019;30(7):1096–103 doi 10.1093/annonc/mdz134. [DOI] [PubMed] [Google Scholar]
- 50.Riaz N, Blecua P, Lim RS, Shen R, Higginson DS, Weinhold N, et al. Pan-cancer analysis of bi-allelic alterations in homologous recombination DNA repair genes. Nat Commun 2017;8(1):857 doi 10.1038/s41467-017-00921-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Heeke AL, Pishvaian MJ, Lynce F, Xiu J, Brody JR, Chen WJ, et al. Prevalence of Homologous Recombination-Related Gene Mutations Across Multiple Cancer Types. JCO Precis Oncol 2018;2018 doi 10.1200/PO.17.00286. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Mateo J, Carreira S, Sandhu S, Miranda S, Mossop H, Perez-Lopez R, et al. DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer. N Engl J Med 2015;373(18):1697–708 doi 10.1056/NEJMoa1506859. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Bang YJ, Xu RH, Chin K, Lee KW, Park SH, Rha SY, et al. Olaparib in combination with paclitaxel in patients with advanced gastric cancer who have progressed following first-line therapy (GOLD): a double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Oncol 2017;18(12):1637–51 doi 10.1016/S1470-2045(17)30682-4. [DOI] [PubMed] [Google Scholar]
- 54.Parikh AR, Lee FC, Yau L, Koh H, Knost J, Mitchell EP, et al. MAVERICC, a Randomized, Biomarker-stratified, Phase II Study of mFOLFOX6-Bevacizumab versus FOLFIRI-Bevacizumab as First-line Chemotherapy in Metastatic Colorectal Cancer. Clin Cancer Res 2019;25(10):2988–95 doi 10.1158/1078-0432.CCR-18-1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
De-identified datasets analyzed in the current study are available from the corresponding author on reasonable request.