Hypermutator Phenotype Research Articles

Distinct features between MLH1-methylated and unmethylated colorectal carcinomas with the CpG island methylator phenotype: implications in the serrated neoplasia pathway.

The presence or absence of MLH1 methylation may critically affect the heterogeneity of colorectal carcinoma (CRC) with the CpG island methylator phenotype (CIMP). Here, we investigated the differential characteristics of CIMP-high (CIMP-H) CRCs according to MLH1 methylation status. To further confirm the MLH1-dependent features in CIMP-H CRC, an independent analysis was performed using data from The Cancer Genome Atlas (TCGA). In our CIMP-H CRC samples, MLH1-methylated tumors were characterized by older patient age, proximal colonic location, mucinous histology, intense lymphoid reactions, RUNX3/SOCS1 promoter methylation, BRAF mutations, and microsatellite instability-high (MSI-H) status. By contrast, MLH1-unmethylated tumors were associated with earlier age of onset, increased distal colorectal localization, adverse pathologic features, and KRAS mutations. In the TCGA dataset, the MLH1-silenced CIMP-H CRC demonstrated proximal location, MSI-H status, hypermutated phenotype, and frequent BRAF mutations, but the MLH1-non-silenced CIMP-H CRC was significantly associated with high frequencies of KRAS and APC mutations. In conclusion, the differential nature of CIMP-H CRCs depends primarily on the MLH1 methylation status. Based on the current knowledge, the sessile serrated adenoma/polyp may be the major precursor of MLH1-methylated CIMP-H CRCs, whereas MLH1-unmethylated CIMP-H CRCs may develop predominantly from KRAS-mutated traditional serrated adenomas and less commonly from BRAF-mutated traditional serrated adenomas and/or sessile serrated adenomas/polyps.

Levels of oxidative stress and telomeres in Lynch syndrome-associated malignancies.

582 Background: Lynch Syndrome (LS) is caused by germline mutations in mismatch repair genes (MMR) genes which are critical in maintaining cellular integrity. Failure of the MMR pathway in LS culminates in the hypermutable phenotype of Microsatellite Instability. LS confers an increased risk of malignancy of which colorectal cancer (CRC) is most common. Carriers exhibit significant phenotypic variation in the age of onset of malignancy which cannot be predicted. Telomere length attrition is considered an early step in carcinogenesis and may be accelerated by oxidative stress. We investigated an association between relative telomere length (RTL) and levels of DNA oxidative damage in LS affected carriers (AC), unaffected carriers (UAC) and in patients with MMR- proficient colorectal cancer (MPC). Methods: Peripheral blood mononuclear cells were isolated from patients within each group. DNA was extracted and RTL measured by quantitative polymerase chain reaction (PCR). Real-Time PCR was used to quantitate expression levels of TERT, TERC and DKC1 (telomerase components) from RNA. Serum levels of 8-hydroxydeguanosine (8-OHdG) were measured from patients using the ELISA technique. Pearson’s correlation was used to compare mean RTL, telomerase levels and 8-OHdG between groups. Results: RTL and telomerase components were measured in 27 AC (median age 50yrs) 27 UAC (median age 40yrs) and 27 MPC (median age 66yrs). Corresponding RTLs were 0.89, 0.91 and 1.69 respectively. AC had significantly shorter RTL compared to UAC (p = 0.03) and MPC (p < 0.0001). There we no differences in the mean expression of TERT, TERC or DKC between groups. Younger age of tumour onset was associated with shorter telomere length in both AC (p = 0.0006) and MPC (p < 0.0001). 8-OHdG levels have been measured in 17 AC, 19 UAC and 14 MPC. The mean levels of AC and UAC were not statistically different. However the mean MPC level was significantly less than UAC (p = 0.03) and AC (p < 0.0001). Conclusions: Shortened telomere length is an important step in carcinogenesis. Affected LS patients have shorter telomeres and evidence of higher levels of DNA oxidative stress than patients with MMR-proficient CRC.

Abstract A35: Elucidating mechanisms involved in long-term response to platinum-based chemotherapy in high-grade serous ovarian cancer.

Abstract Introduction: Approximately eighty percent of women with stage III high-grade serous ovarian cancer (HGSOC) develop recurrent disease despite initial surgery and platinum-based combination chemotherapy. Clinico-pathological factors do not allow precise prediction of outcome and hence initial treatment is not stratified. Potential genomic and immunologic predictive factors of response/resistance to platinum-based chemotherapy could be identified by comparing and contrasting long-term with short-term survivors. Methods: Patients with stage III HGSOC and upfront debulking surgery followed by platinum-based chemotherapy were selected from the Princess Margaret Cancer Centre registry. Retrospectively, 25 patients were identified as long-term responders (alive 10 years or more from the time of diagnosis) and 24 patients as short-term responders (dead within 2 years of diagnosis and primary platinum resistant). Available archival snap frozen or formalin-fixed paraffin-embedded tumor and matched normal tissue sections from identified patients were obtained through the University Health Network (UHN) Biobank. Pathology was confirmed by an expert gynecological pathologist. Each archival specimen was characterized by histological, molecular, genomic and immunologic patterns at presentation of disease. We performed whole exome sequencing (WES) and transcriptome sequencing (RNAseq) from total DNA and RNA extracted from archival tumor tissues to characterize genetic alterations and gene expression profiles. We profiled the tumor tissue protein expression of p53, Ki67, p16, Rb1, cyclin E, cyclin D1, CD3, CD8, CD68, PDL-1, FoxP3, and the Bcl-2 family proteins (Bcl-xL, Bcl-2, MCL-1, Bax, Bak, Bim, Noxa, Puma) through immunohistochemistry (IHC) analyses. Results: Preliminary genomic data were analyzed from the initial set of 3 long-term survivors and 7 short-term survivors. 8/10 tumors were characterized by TP53 mutations. All long-term survivors harbor germline mutations in BRCA1 or 2. Somatic mutations in ADAMTS family genes associated with chemo-sensitivity occur at a higher frequency in long-term survivors. One patient with a long-term response had a hyper-mutated phenotype. Preliminary IHC analyses on 21 patients (10 long- and 11 short-term survivors) examined to date showed that apoptosis-regulating genes—such as p53, Bcl-2, Bcl-xL, Bax, which regulate cancer cell sensitivity/resistance—do not have a significant difference in protein expression at baseline. Purity estimates generated from WES shows that tumors from long survivors have reduced purity as compared to short-term survivors. Overall levels of immune and stromal infiltration for each tumor by applying the ESTIMATE algorithm to the quantile-normalized gene expression profiles suggest higher immune and stromal scores in long surviving compared to short surviving HGSOC. Conclusions: The initial results indicate a potential predictive signature for long-term response in HGSOC. Citation Format: Stephanie Lheureux, Cindy Yang, Patricia Shaw, Blaise Clarke, Julissa Tsao, Katherine Karakasis, Marcus Butler, Noor Salman, Mark Dowar, Jing Xu, Laurent Poulain, Pamela S. Ohashi, Trevor Pugh, Amit Oza. Elucidating mechanisms involved in long-term response to platinum-based chemotherapy in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A35.

Pilot study of clinical targeted next generation sequencing for prostate cancer: Consequences for treatment and genetic counseling.

251 Background: Targeted next generation sequencing (NGS) panels for identification of actionable mutations are increasingly used in oncology for therapeutic decision-making, but have yet to be widely used for prostate cancer.To determine the utility of applying a targeted next generation sequencing to prostate cancer management, we conducted a pilot study using the clinical molecular diagnostic assay, UW-OncoPlex, on primary and metastatic tumors from patients with prostate cancer. Methods: Patients receiving treatment for prostate cancer by a medical oncologist were eligible. After providing informed consent, tumor DNA was extracted from formalin-fixed, paraffin-embedded (FFPE)primary tumors and/or metastatic biopsies. Extracted DNA was analyzed using UW-OncoPlex, a targeted NGS panel designed to assess genomic alterations in 234 cancer-associated genes (PMID: 24189654). Results were discussed at a monthly multidisciplinary precision tumor board prior to communicating results to patients. Results: Forty patients consented to the study and 31 (78%) had reportable results. Findings included frequently observed prostate cancer genomic aberrations, including: TMPRSS2-ERG fusions and copy-number alterations of PTEN, TP53, FOXA1, AR, and SPOP. We also observed potentially actionable alterations in BRAF, MAP2K1 (MEK1), PIK3R1, MET, FGFR1, and FGFR3, which inform future treatment and clinical trial considerations. Notably, 8/31 (25%) patients had suspected high penetrance germline mutations, including in BRCA2 (N = 3), TP53 (N = 2), ATM (N = 1), and CHEK2 (N = 2). These 8 individuals were referred to medical genetics and 7 of 8 mutations were confirmed to be germline. One patient was found to have a hypermutated phenotype associated with bi-allelic MSH6 mutation and microsatellite instability. Conclusions: Targeted NGS panel testing may be useful in informing future treatment approaches and clinical trial selection for patients with prostate cancer. In addition, we observed a high proportion of cases with suspected high-penetrance germline mutations and immediate actionability through referral to medical genetics.

Whole-Exome Sequencing Analyses of Inflammatory Bowel Disease−Associated Colorectal Cancers

A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer. Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors. We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and colorectal cancer (15 with ulcerative colitis, 14 with Crohn's disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the microdissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exome-sequencing studies. Two specimens had somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1, and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for noncanonical WNT signaling in development of colorectal tumors in patients with IBD. Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and colorectal cancer.

Approaches to diagnose DNA mismatch repair gene defects in cancer.

The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind by the replicative polymerases and results in increased mutation load at the genome. The realization that defective MMR leads to a hypermutation phenotype and increased risk of tumorigenesis highlights the relevance of this pathway for human disease. The association of MMR defects with increased risk of cancer development was first observed in colorectal cancer patients that carried inactivating germline mutations in MMR genes and the disease was named as hereditary non-polyposis colorectal cancer (HNPCC). Currently, a growing list of cancers is found to be MMR defective and HNPCC has been renamed Lynch syndrome (LS) partly to include the associated risk of developing extra-colonic cancers. In addition, a number of non-hereditary, mostly epigenetic, alterations of MMR genes have been described in sporadic tumors. Besides conferring a strong cancer predisposition, genetic or epigenetic inactivation of MMR genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance of MMR gene alterations in human disease.

Genetic and epigenetic aberrations occurring in colorectal tumors associated with serrated pathway.

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high‐methylation CRC. The methylation levels of six Group‐1 and 14 Group‐2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group‐1 and Group‐2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high‐methylation CRC. But TSA showed low‐methylation levels of Group‐1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high‐methylation CRC. High‐methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p < 0.0001) and insertion/deletions (p = 0.002). MLH1‐methylated SSA/P showed lower methylation level of MLH1 compared with high‐methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1‐methylated and MLH1‐unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF‐β and BMP signaling (but not in TP53 signaling) were significantly involved in high‐methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.

GENO-15IDENTIFICATION AND GENOMIC ANALYSIS OF HYPER-MUTATED AND ULTRA-MUTATED GBMS

BACKGROUND: Somatic mutations on mismatch repair genes (MMR) or DNA polymerases define distinct molecular subgroups in many solid tumors with hyper or ultra mutated phenotypes. These subgroups, as recently reported, carry clinical significance by presenting favorable response to immunotherapies. METHODS: We have performed whole exome sequencing on 118 primary and secondary gliomas with matching blood samples. We analyzed our cohort based on level of mutation rate, mutation signature, clinical and histological attributes to identify the hyper/mutated gliomas. RESULTS: We have identified 7 outlier samples, with increased mutation burden compared to the rest of the cohort (mean = 2404 vs. 32.81, p.value= 0.03). 2/7 samples were primary GBMs and carried deleterious somatic mutations on the exonucelase domain of POLE gene. They were ultra-mutated (mean = 6253), with increased C > A transversions, had multi-nucleated giant cell histology, were younger at the time of diagnosis (average age, 35.5 vs 58y; p.value= 0.005) and had better prognosis. 5/7 of the outlier samples carried somatic mutations on the MMR genes (MSH6, MLH1 and MSH2) and displayed a hyper-mutated phenotype (mean = 864.4) with increased C > T transitions. 4/5 of the MMR deficient cases were previously treated, which might have induced the somatic mutations on the MMR genes. One of these hyper-mutated, MMR deficient GBM cases was later treated with targeted immunotherapy based on genomic analysis findings and had favorable results. CONCLUSION: Recent studies revealed that some solid cancers such as lung and colorectal cancer with increased mutation burden identified a subgroup that benefited from immunotherapy. We have identified and analyzed 2 sub-classes of gliomas with distinctive molecular attributes such as increased mutation burden, shifted mutation signature together with clinical attributes. As in the presented case of the hyper-mutated phenotype patient who underwent personalized immunotherapy, these subgroups can benefit from targeted therapies such as immunotherapy.

MTR-16MISMATCH REPAIR DEFECTS PREDICT CLINICAL OUTCOME OF PRIMARY AND RECURRENT MALIGNANT GLIOMAS

INTRODUCTION: Temozolomide (TMZ), a standard drug for glioblastoma (GBM), induces inactivation of the DNA mismatch repair (MMR) system and hence causes hypermutator phenotype in the glioma especially with methylated O6-methylguanine-DNA methyltransferase (MGMT) promoter. MMR defects are also found in primary gliomas and are associated with resistance to TMZ and malignant progression to secondary GBM. However, the association between alteration of MMR system and clinical outcome of both primary and recurrent GBMs was yet to be elucidated. METHODS: We analyzed protein expression of MLH1, MSH2, MSH6, and PMS2 as well as MGMT methylation in a set of 117 primary GBMs and 34 paired sample of initial and recurrent gliomas. Protein expression was analyzed by Western blotting and/or immunohistochemistry. MGMT methylation was determined by methylation-specific PCR. Progression-free and overall survival times according to each molecular status were reviewed. RESULTS: Loss of MMR protein expression (MLH1, MSH2, MSH6, and PMS2) was observed in 12%, 10%, 50%, and 16% of the primary samples, respectively. Inactivation of MSH6 during the recurrence was associated with MGMT methylation at recurrence (p = 0.02), while MGMT methylation of the initial samples did not predict the changes of any MMR protein. Malignant progression from lower grade glioma was associated with inactivation of MSH6 (p = 0.035). Loss of MMR protein expression, as well as unmethylated MGMT promoter, was associated with shorter overall survival in patients with primary GBMs. In contrast, MMR inactivation was associated with longer survival in patients with recurrent GBMs, while MGMT methylation was not. CONCLUSION: Inactivation of MMR protein was associated with resistance to TMZ for primary GBMs, malignant progression of lower grade gliomas, and was predictive factor for survival of the patients with recurrent GBMs. Further investigation to define optimal treatment for glioma according to MGMT and MMR status is warranted.

Acinetobacter baumannii Repeatedly Evolves a Hypermutator Phenotype in Response to Tigecycline That Effectively Surveys Evolutionary Trajectories to Resistance.

The evolution of hypermutators in response to antibiotic treatment in both clinical and laboratory settings provides a unique context for the study of adaptive evolution. With increased mutation rates, the number of hitchhiker mutations within an evolving hypermutator population is remarkably high and presents substantial challenges in determining which mutations are adaptive. Intriguingly however, hypermutators also provide an opportunity to explore deeply the accessible evolutionary trajectories that lead to increased organism fitness, in this case the evolution of antibiotic resistance to the clinically relevant antibiotic tigecycline by the hospital pathogen Acinetobacter baumannii. Using a continuous culture system, AB210M, a clinically derived strain of A. baumannii, was evolved to tigecycline resistance. Analysis of the adapted populations showed that nearly all the successful lineages became hypermutators via movement of a mobile element to inactivate mutS. In addition, metagenomic analysis of population samples revealed another 896 mutations that occurred at a frequency greater than 5% in the population, while 38 phenotypically distinct individual colonies harbored a total of 1712 mutations. These mutations were scattered throughout the genome and affected ~40% of the coding sequences. The most highly mutated gene was adeS, a known tigecycline-resistance gene; however, adeS was not solely responsible for the high level of TGC resistance. Sixteen other genes stood out as potentially relevant to increased resistance. The five most prominent candidate genes (adeS, rpsJ, rrf, msbA, and gna) consistently re-emerged in subsequent replicate population studies suggesting they are likely to play a role in adaptation to tigecycline. Interestingly, the repeated evolution of a hypermutator phenotype in response to antibiotic stress illustrates not only a highly adaptive strategy to resistance, but also a remarkably efficient survey of successful evolutionary trajectories.

Hypermutator Salmonella Heidelberg induces an early cell death in epithelial cells

We have previously described that a strain of Salmonella Heidelberg with a hypermutator phenotype, B182, adhered strongly to HeLa cells. In this work, we showed that this hypermutator Salmonella strain invaded HeLa epithelial cells and induced cytoskeleton alteration. Those changes lead to HeLa cell death which was characteristic of apoptosis. For the first time, we showed that this hypermutator strain induced apoptosis associated with the activation of caspases 2, 9 and 3. Complementation of B182 strain showed a decrease in cells death induction. In the presence of other Salmonella Heidelberg with a normomutator phenotype, such as WT and SL486, cell death and caspase 3 were undetectable. These results suggested that early apoptosis and caspase 3 activation were specific to B182. Besides, B182 induced LDH release and caspase 3 activation in CaCo-2 and HCT116 cells. Heat-treated B182 and diffusible products failed to induce this phenotype. Epithelial cells treatment with cytochalasin D caused the inhibition of B182 internalisation and caspase 3 activation. These results showed that this cell death required active S. Heidelberg B182 protein synthesis and bacterial internalisation. However sipB and sopB, usually involved in apoptosis induced by Salmonella were not overexpressed in B182, contrary to fimA and fliC. Comparative genome analysis showed numerous mutations as in rpoS which would be more investigated. The role of the hypermutator phenotype might be suspected to be implicated in these specific features. This result expands our knowledge about strong mutators frequently found in bacterial organisms isolated from clinical specimens.

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity.

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild-type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild-types attained similar population densities. Given the importance of the methyl-directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild-type phenotype.

Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing.

Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53 and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention.

Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype

Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.

Clonal architectures and driver mutations in metastatic melanomas.

To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2). Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3′ base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.

Germline variants in POLE are associated with early onset mismatch repair deficient colorectal cancer.

Germline variants affecting the exonuclease domains of POLE and POLD1 predispose to multiple colorectal adenomas and/or colorectal cancer (CRC). The aim of this study was to estimate the prevalence of previously described heterozygous germline variants POLE c.1270C>G, p.(Leu424Val) and POLD1 c.1433G>A, p.(Ser478Asn) in a Dutch series of unexplained familial, early onset CRC and polyposis index cases. We examined 1188 familial CRC and polyposis index patients for POLE p.(Leu424Val) and POLD1 p.(Ser478Asn) variants using competitive allele-specific PCR. In addition, protein expression of the POLE and DNA mismatch repair genes was studied by immunohistochemistry in tumours from POLE carriers. Somatic mutations were screened using semiconductor sequencing. We detected three index patients (0.25%) with a POLE p.(Leu424Val) variant. In one patient, the variant was found to be de-novo. Tumours from three patients from two families were microsatellite instable, and immunohistochemistry showed MSH6/MSH2 deficiency suggestive of Lynch syndrome. Somatic mutations but no germline MSH6 and MSH2 variants were subsequently found, and one tumour displayed a hypermutator phenotype. None of the 1188 patients carried the POLD1 p.(Ser478Asn) variant. POLE germline variant carriers are also associated with a microsatellite instable CRC. POLE DNA analysis now seems warranted in microsatellite instable CRC, especially in the absence of a causative DNA mismatch repair gene germline variant.

OP01 * MISMATCH REPAIR DEFICIENCY CONFERS INCREASED SENSITIVITY TO IONISING RADIATION (XR) IN GLIOBLASTOMA

INTRODUCTION: Combination chemo-radiotherapy, including the alkylating agent temozolomide (TMZ) is standard of care for patients with glioma. Increased TMZ use has lead to sustained interest in the mechanisms underlying acquired resistance. Amongst these, deficiency in mismatch repair (MMR) is best described, with TMZ treatment promoting loss of functional MMR proteins most frequently through point mutations in MSH2/6, which may then lead to a hyper-mutation phenotype. Since patients are increasingly exposed to TMZ as first-line treatment with subsequent radiotherapy we set out to investigate how MMR status may influence response to XR and/or radiosensitising agents. METHOD: Effects of MMR protein deficiency on response to XR in paired isogenic glioma cell lines; U251 (MMR proficient) and U251.TR3 (MMR deficient, TMZ-induced MSH6mut) lines determined by clonogenic assay and immunofluorescence to detect γH2AX foci. MMR proteins were down-regulated by siRNA transfection. MMR protein expression was determined by western blotting and MMR deficient cell lines were further characterised by sequencing. RESULTS: We have shown that U251.TR3 cells (MMR deficient) are more sensitive to radiation-induced cell death than MMR proficient U251 cells in clonogenic assays (surviving fraction at 2Gy 0.41 ± 0.03 and 0.71 ± 0.04 respectively, p < 0.001). This sensitisation is accompanied by an increase in gH2AX foci (28 ± 3 and 50 ± 6 foci per cell in U251 and U251.TR3 cells respectively). On-going work is investigating whether this effect is due to MMR deficiency per se or secondary mutations driven by a hyper-mutation phenotype. CONCLUSION: Detection of MMR proteins following TMZ exposure may serve as a tool to stratify patients for treatment.

Abstract 5172: Whole exome mutation landscape of 70 commonly used colorectal cancer cell lines

Abstract Human colorectal cancer (CRC) cell lines are an important model system for studying the biology of this malignancy as well as therapeutic and biomarker discovery. However, data on the degree to which established CRC cell lines reflect the somatic diversity of primary cancers at the genome level are limited. Using whole exome sequencing and SNP microarray analysis we show that the profile of mutations and DNA copy-number alterations in 70 widely-used CRC cell lines closely resembles that of primary colorectal tumors published by The Cancer Genome Atlas Network. We demonstrate presence of at least two hypermutation phenotypes, consistent with defective DNA mismatch repair and DNA polymerase epsilon (POLE) proof-reading deficiency, and similar mutation signatures in the WNT, MAPK, PI3K, TGF-beta and TP53 pathways. Paired cell lines derived from the same tumor are found to exhibit substantial mutation and DNA copy-number differences, with in silico simulations suggesting that these largely reflect pre-existing tumor cell heterogeneity, but these differences do not obscure known driver genes. Our genome level data indicate that CRC cell lines are representative models of the main molecular subtypes of primary tumors, and will facilitate informed selection of molecularly-defined models for preclinical investigations. Citation Format: Oliver Sieber, Dmitri Mouradov, Clare Sloggett, Robert Jorissen, Chris Love, Shan Li, Antony Burgess, Diego Arango, Robert Strausberg, Daniel Buchanan, Samuel Wormald, Liam O'Connor, Jenny Wilding, David Bicknell, Ian Tomlinson, Walter Bodmer, John Mariadason. Whole exome mutation landscape of 70 commonly used colorectal cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5172. doi:10.1158/1538-7445.AM2014-5172

Abstract 2833: Mutation analysis of cancer drivers and DNA repair genes in chemosensitive versus resistant ovarian cancers

Abstract Background: The mechanisms underlying resistance to chemotherapy in ovarian cancer are incompletely understood. Identifying genetic alterations associated with treatment response is decisive in the determination of which patients may benefit from adjuvant chemotherapy. Methods: Biopsies were collected from twenty patients diagnosed with ovarian cancer who were subjected to post-operative taxane- and platinum-containing chemotherapy. Patients were selected for genetic analyses based on response to chemotherapy, determined as time to relapse (10 sensitive and 10 resistant patients). A panel of 620 genes, including known cancer driver genes, as well as genes involved in DNA repair were analysed by massively parallel sequencing. Alignment and mutation calling was performed using MiSeq Reporter, with further manual filtering of variants to exclude common SNPs. Validation of low quality mutation calls was done by Sanger sequencing. Results: A median of 6 genes (range: 3 - 45) per patient was found to harbour non-synonymous mutations. Among previously identified driver genes in ovarian cancer, we found mutations in TP53, BRCA1, CDK12, NF1 and CSMD3. These mutations were more common among patients with more advanced disease and higher grade. For example, TP53 mutations were found in 10 out of 12 patients with high grade, stage 3c or 4 disease, and in 2 out of 5 with lower stage and/or grade. One patient was found to have a tumor potentially of a hyper-mutator phenotype with 49 mutations in 45 genes identified within our gene panel. With respect to treatment efficacy, 73 and 40 genes were found to be mutated exclusively in patients with a good and poor response to treatment, respectively. Conclusion: We describe the profile of mutations in cancer driver genes and DNA repair genes among patients suffering from ovarian cancer according to treatment response. Citation Format: Einar Birkeland, Rakel Blaalid, Merete Bjørnslett, Anne Dørum, Per Eystein Lønning, Stian Knappskog. Mutation analysis of cancer drivers and DNA repair genes in chemosensitive versus resistant ovarian cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2833. doi:10.1158/1538-7445.AM2014-2833

Colon and Endometrial Cancers With Mismatch Repair Deficiency Can Arise From Somatic, Rather Than Germline, Mutations

Patients with Lynch syndrome carry germline mutations in single alleles of genes encoding the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2; when the second allele becomes mutated, cancer can develop. Increased screening for Lynch syndrome has identified patients with tumors that have deficiency in MMR, but no germline mutations in genes encoding MMR proteins. We investigated whether tumors with deficient MMR had acquired somatic mutations in patients without germline mutations in MMR genes using next-generation sequencing. We analyzed blood and tumor samples from 32 patients with colorectal or endometrial cancer who participated in Lynch syndrome screening studies in Ohio and were found to have tumors with MMR deficiency (based on microsatellite instability and/or absence of MMR proteins in immunohistochemical analysis, without hypermethylation of MLH1), but no germline mutations in MMR genes. Tumor DNA was sequenced for MLH1, MSH2, MSH6, PMS2, EPCAM, POLE, and POLD1 with ColoSeq and mutation frequencies were established. Twenty-two of 32 patients (69%) were found to have 2 somatic (tumor) mutations in MMR genes encoding proteins that were lost from tumor samples, based on immunohistochemistry. Of the 10 remaining tumors 3 had one somatic mutation in a MMR gene, with possible loss of heterozygosity that could lead to MMR deficiency, 6 were found to be false-positive results (19%), and 1 had only one mutation in a MMR gene and remained unexplained. All of the tumors found to have somatic MMR mutations were of the hypermutated phenotype (>12 mutations/megabase); 6 had mutation frequencies >200/megabase, and 5 of these had somatic mutations in POLE, which encodes a DNA polymerase. Some patients are found to have tumors with MMR defects during screening for Lynch syndrome, yet have no identifiable germline mutations in MMR genes. We found that almost 70% of these patients acquire somatic mutations in MMR genes, leading to a hypermutated phenotype of tumor cells. Patients with colon or endometrial cancers with MMR deficiency not explained by germline mutations might undergo analysis for tumor mutations in MMR genes to guide future surveillance guidelines.