Sequencing of Lynch syndrome tumors reveals the importance of epigenetic alterations

Genomic instability and epigenetic aberrations are important classifiers of human tumors, yet, their interrelations are poorly understood. We used Lynch syndrome (LS) to address such relationships. Forty-five tumors (11 colorectal adenomas, 18 colorectal carcinomas, and 16 ovarian carcinomas) were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations. All tumors showed high-degree microsatellite instability. Panel sequencing of 578 cancer-relevant genes revealed the average number of 1433, 1124, and 657 non-synonymous somatic mutations per colorectal adenoma, colorectal carcinoma, and ovarian carcinoma, respectively. Genes harboring mutations with allele frequency 25 % or higher in at least 31 % of tumors were regarded to be possible drivers. Among 72 and 10 such genes identified in colorectal and ovarian tumors, respectively, the most frequently mutated genes BRD4 and MLL2 (62 % of colorectal tumors) and ARID1A (50 % of ovarian carcinomas) are involved in epigenetic regulation. The total number of somatic mutations or mutant genes per tumor were significantly associated with CIMP. Our results suggest that even in an inherited disease, tumor type-specific epigenetic changes are significant and may result from regulatory changes (CIMP) or structural events (mutations of epigenetic regulatory genes). The findings are clinically relevant since many of the affected pathways can be therapeutically targeted.Peer reviewe

Does breast carcinoma belong to the Lynch syndrome tumor spectrum? - Somatic mutational profiles vs. ovarian and colorectal carcinomas

: ; ; ; ;Inherited DNA mismatch repair (MMR) defects cause predisposition to colorectal, endometrial, ovarian, and other cancers occurring in Lynch syndrome (LS). It is unsettled whether breast carcinoma belongs to the LS tumor spectrum. We approached this question through somatic mutational analysis of breast carcinomas from LS families, using established LS-spectrum tumors for comparison. Somatic mutational profiles of 578 cancer-relevant genes were determined for LS-breast cancer (LS-BC, n = 20), non-carrier breast cancer (NC-BC, n = 10), LS-ovarian cancer (LS-OC, n = 16), and LS-colorectal cancer (LS-CRC, n = 18) from the National LS Registry of Finland. Microsatellite and MMR protein analysis stratified LS-BCs into MMR-deficient (dMMR, n = 11) and MMR-proficient (pMMR, n = 9) subgroups. All NC-BCs were pMMR and all LS-OCs and LS-CRCs dMMR. All but one dMMR LS-BCs were hypermutated (> 10 non-synonymous mutations/Mb; average 174/Mb per tumor) and the frequency of MMR-deficiency-associated signatures 6, 20, and 26 was comparable to that in LS-OC and LS-CRC. LS-BCs that were pMMR resembled NC-BCs with respect to somatic mutational loads (4/9, 44%, hypermutated with average mutation count 33/Mb vs. 3/10, 30%, hypermutated with average 88 mutations/Mb), whereas mutational signatures shared features of dMMR LS-BC, LS-OC, and LS-CRC. Epigenetic regulatory genes were significantly enriched as mutational targets in LS-BC, LS-OC, and LS-CRC. Many top mutant genes of our LS-BCs have previously been identified as drivers of unselected breast carcinomas. In conclusion, somatic mutational signatures suggest that conventional MMR status of tumor tissues is likely to underestimate the significance of the predisposing MMR defects as contributors to breast tumorigenesis in LS.Peer reviewe

Molecular Basis of Mismatch Repair Protein Deficiency in Tumors from Lynch Suspected Cases with Negative Germline Test Results

Some 10–50% of Lynch-suspected cases with abnormal immunohistochemical (IHC) staining remain without any identifiable germline mutation of DNA mismatch repair (MMR) genes. MMR proteins form heterodimeric complexes, giving rise to distinct IHC patterns when mutant. Potential reasons for not finding a germline mutation include involvement of an MMR gene not predicted by the IHC pattern, epigenetic mechanism of predisposition, primary mutation in another DNA repair or replication-associated gene, and double somatic MMR gene mutations. We addressed these possibilities by germline and tumor studies in 60 Lynch-suspected cases ascertained through diagnostics (n = 55) or research (n = 5). All cases had abnormal MMR protein staining in tumors but no point mutation or large rearrangement of the suspected MMR genes in the germline. In diagnostic practice, MSH2/MSH6 (MutS Homolog 2/MutS Homolog 6) deficiency prompts MSH2 mutation screening; in our study, 3/11 index individuals (27%) with this IHC pattern revealed pathogenic germline mutations in MSH6. Individuals with isolated absence of MSH6 are routinely screened for MSH6 mutations alone; we found a predisposing mutation in MSH2 in 1/7 such cases (14%). Somatic deletion of the MSH2-MSH6 region, joint loss of MSH6 and MSH3 (MutS Homolog 3) proteins, and hindered MSH2/MSH6 dimerization offered explanations to misleading IHC patterns. Constitutional epimutation hypothesis was pursued in the MSH2 and/or MSH6-deficient cases plus 38 cases with MLH1 (MutL Homolog 1)-deficient tumors; a primary MLH1 epimutation was identified in one case with an MLH1-deficient tumor. We conclude that both MSH2 and MSH6 should be screened in MSH2/6- and MSH6-deficient cases. In MLH1-deficient cases, constitutional epimutations of MLH1 warrant consideration

Epidemiological, clinical and molecular characterization of Lynch-like syndrome : A population-based study

Colorectal carcinomas that are mismatch repair (MMR)-deficient in the absence of MLH1 promoter methylation or germline mutations represent Lynch-like syndrome (LLS). Double somatic events inactivating MMR genes are involved in the etiology of LLS tumors. Our purpose was to define the clinical and broader molecular hallmarks of LLS tumors and the population incidence of LLS, which remain poorly characterized. We investigated 762 consecutive colorectal carcinomas operated in Central Finland in 2000-2010. LLS cases were identified by a stepwise protocol based on MMR protein expression, MLH1 methylation and MMR gene mutation status. LLS tumors were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations in 578 cancer-relevant genes. Among 107 MMR-deficient tumors, 81 (76%) were attributable to MLH1 promoter methylation and 9 (8%) to germline mutations (Lynch syndrome, LS), leaving 14 LLS cases (13%) (3 remained unclassified). LLS carcinomas were diagnosed at a mean age of 65 years (vs. 44 years in LS, p <0.001), had a proximal to distal ratio of 1:1, and all were BRAF V600E-negative. Two somatic events in MMR genes were identifiable in 11 tumors (79%). As novel findings, the tumors contained an average of 31 nonsynonymous somatic mutations/Mb and 13/14 were CIMP-positive. In conclusion, we establish the epidemiological, clinical and molecular characteristics of LLS in a population-based study design. Significantly more frequent CIMP-positivity and lower rates of somatic mutations make a distinction to LS. The absence of BRAF V600E mutation separates LLS colorectal carcinomas from MLH1-methylated colorectal carcinomas with CIMP-positive phenotype.Peer reviewe

Converging endometrial and ovarian tumorigenesis in Lynch syndrome : Shared origin of synchronous carcinomas

AbstractObjective The diagnosis of carcinoma in both the uterus and the ovary simultaneously is not uncommon and raises the question of synchronous primaries vs. metastatic disease. Targeted sequencing of sporadic synchronous endometrial and ovarian carcinomas has shown that such tumors are clonally related and thus represent metastatic disease from one site to the other. Our purpose was to investigate whether or not the same applies to Lynch syndrome (LS), in which synchronous cancers of the gynecological tract are twice as frequent as in sporadic cases, reflecting inherited defects in DNA mismatch repair (MMR). Methods MMR gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nationwide registry. Endometrial (n = 35) and ovarian carcinomas (n = 23), including 13 synchronous carcinoma pairs, were collected as well as endometrial hyperplasias (n = 56) and normal endometria (n = 99) from a surveillance program over two decades. All samples were studied for MMR status, ARID1A and L1CAM protein expression and tumor suppressor gene promoter methylation, and synchronous carcinomas additionally for somatic mutation profiles of 578 cancer-relevant genes. Results Synchronous carcinomas were molecularly concordant in all cases. Prior or concurrent complex (but not simple) endometrial hyperplasias showed a high degree of concordance with endometrial or ovarian carcinoma as the endpoint lesion. Conclusions Our investigation suggests shared origins for synchronous endometrial and ovarian carcinomas in LS, in analogy to sporadic cases. The similar degrees of concordance between complex hyperplasias and endometrial vs. ovarian carcinoma highlight converging pathways for endometrial and ovarian tumorigenesis overall.Peer reviewe

Mutation accumulation in cancer genes relates to nonoptimal outcome in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm accounting for similar to 15% of all leukemia. Progress of the disease from an indolent chronic phase to the more aggressive accelerated phase or blast phase (BP) occurs in a minority of cases and is associated with an accumulation of somatic mutations. We performed genetic profiling of 85 samples and transcriptome profiling of 12 samples from 59 CML patients. We identified recurrent somatic mutations in ABL1 (37%), ASXL1 (26%), RUNX1 (16%), and BCOR (16%) in the BP and observed that mutation signatures in the BP resembled those of acute myeloid leukemia (AML). We found that mutation load differed between the indolent and aggressive phases and that nonoptimal responders had more nonsilent mutations than did optimal responders at the time of diagnosis, as well as in follow-up. Using RNA sequencing, we identified other than BCR-ABL1 cancer-associated hybrid genes in 6 of the 7 BP samples. Uncovered expression alterations were in turn associated with mechanisms and pathways that could be targeted in CML management and by which somatic alterations may emerge in CML. Last, we showed the value of genetic data in CML management in a personalized medicine setting.Peer reviewe

Systematic drug screening reveals specific vulnerabilities and co-resistance patterns in endocrine-resistant breast cancer

Abstract Background The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display de novo resistance to tamoxifen, and acquired resistance evolves in 40 % of patients who initially respond. Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains. Methods Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance. Results We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome- and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred. Conclusion This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns

Systematic drug screening reveals specific vulnerabilities and co-resistance patterns in endocrine-resistant breast cancer

Background: The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display de novo resistance to tamoxifen, and acquired resistance evolves in 40 % of patients who initially respond. Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains. Methods: Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance. Results: We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome-and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred. Conclusion: This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns.Peer reviewe

Identification of precision treatment strategies for relapsed/refractory multiple myeloma by functional drug sensitivity testing

Novel agents have increased survival of multiple myeloma (MM) patients, however high-risk and relapsed/refractory patients remain challenging to treat and their outcome is poor. To identify novel therapies and aid treatment selection for MM, we assessed the ex vivo sensitivity of 50 MM patient samples to 308 approved and investigational drugs. With the results we i) classified patients based on their ex vivo drug response profile; ii) identified and matched potential drug candidates to recurrent cytogenetic alterations; and iii) correlated ex vivo drug sensitivity to patient outcome. Based on their drug sensitivity profiles, MM patients were stratified into four distinct subgroups with varied survival outcomes. Patients with progressive disease and poor survival clustered in a drug response group exhibiting high sensitivity to signal transduction inhibitors. Del(17p) positive samples were resistant to most drugs tested with the exception of histone deacetylase and BCL2 inhibitors. Samples positive for t(4; 14) were highly sensitive to immunomodulatory drugs, proteasome inhibitors and several targeted drugs. Three patients treated based on the ex vivo results showed good response to the selected treatments. Our results demonstrate that ex vivo drug testing may potentially be applied to optimize treatment selection and achieve therapeutic benefit for relapsed/refractory MM.Peer reviewe