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Supplementary Figures and Tables

Supplementary Table 1. Linkage disequilibrium patterns of the tag-SNPs used for genotyping the 6q23.3 locus. Supplementary Table 2. Cytogenetic and molecular information on AML patients used for functional studies (n=12). Supplementary Table 3. French-American-British (FAB) classification35 and molecular information on AML patients used to determine miR-3662's abundance (n=8). Supplementary Table 4. Canonical pathway analysis of the miR-3662-associated gene expression signature. Supplementary Figure 1. Transcription factor binding according to the transcription factor chip data from ENCODE. Supplementary Figure 2. Electrophoretic mobility shift assay comparing the binding affinity of the alleles of rs66650371 and rs9483788. Supplementary Figure 3. Endogenous miR-3662 expression levels of hematopoietic progenitor (HP) cells during differentiation, total bone marrow aspirate of three non-leukemic donors (total BM 1-3), different populations of differentiated peripheral blood cells, and three AML cell lines. Supplementary Figure 4. Top panel, Macroscopic pictures of the spleens of three mice of the scramble and miR-3662-infected groups (organs harvested post-mortem). All mice had a massive splenomegaly compared to the un-injected, sacrificed control mouse. Bottom panel, images of spleen histologies (40x enlargement). Slides were stained for CD45 to proof MV4-11 origin of the leukemia. Supplementary Figure 5. Endogenous abundance of miR-3662 and IKBKB in patient samples and cell lines. Supplementary Figure 6. Comparison of the relative miR-3662 abundance of AML patient blasts and AML cell lines before (black) and after (red) forced miR-3662 expression with the lentiviral expression construct.

Related Article from The Search for Unaffected Individuals with Lynch Syndrome: Do the Ends Justify the Means?

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with cytogenetic and molecular factors and patient demographics (e.g., age and race). We compared survival of 25,523 non-Hispanic Black and White adults with AML using Surveillance Epidemiology and End Results (SEER) Program data and performed mutational profiling of 1,339 patients with AML treated on frontline Alliance for Clinical Trials in Oncology (Alliance) protocols. Black patients had shorter survival than White patients, both in SEER and in the setting of Alliance clinical trials. The disparity was especially pronounced in Black patients NPM1 and more IDH2 mutations in younger Black patients. Overall survival of younger Black patients was adversely affected by IDH2 mutations and FLT3-ITD, but, in contrast to White patients, was not improved by NPM1 mutations.Significance:We show that young Black patients have not benefited as much as White patients from recent progress in AML treatment in the United States. Our data suggest that both socioeconomic factors and differences in disease biology contribute to the survival disparity and need to be urgently addressed.See related commentary by Vyas, p. 540.This article is highlighted in the In This Issue feature, p. 521

Monosomy of chromosome 7 is the most frequent autosomal monosomy in acute myeloid leukemia (AML), where it associates with poor clinical outcomes. However, molecular features associated with this sole monosomy subtype (-7 AML), which may give insights into the basis for its poor prognosis, have not been characterized. In this study, we analyzed 36 cases of -7 AML for mutations in 81 leukemia/cancer-associated genes using a customized targeted next-generation sequencing panel (Miseq). Global gene and miRNA expression profiles were also determined using paired RNA and small RNA sequencing data. Notably, gene mutations were detected in all the major AML-associated functional groups, which include activated signaling, chromatin remodeling, cohesin complex, methylation, NPM1, spliceosome, transcription factors, and tumor suppressors. Gene mutations in the chromatin remodeling groups were relatively more frequent in patients SMARCA2 gene. In patients ≥60 years of age, the presence of spliceosome mutations associated with a lower complete remission rate (P = 0.03). RNA sequencing revealed distinct gene and miRNA expression patterns between the sole -7 and non -7 AML cases, with reduced expression, as expected, of many genes and miRNAs mapped to chromosome 7, and overexpression of ID1, MECOM, and PTPRM, among others. Overall, our findings illuminate a number of molecular features of the underlying aggressive pathobiology in -7 AML patients. Cancer Res; 77(1); 207–18. ©2016 AACR.

Supplementary Methods, Supplementary References Supplementary Table S1: Inherited regions of homozygosity in >1% of 1,798 non-leukemic individuals Supplementary Table S2: Acquired uniparental disomies detected in 425 cytogenetically normal AML patients Supplementary Table S3. Associations between recurrent UPDs with pretreatment patient characteristics for patients with cytogenetically normal acute myeloid leukemia Supplementary Table S5. Variant allele fraction of gene mutations that co-occurred with UPDs Supplementary Table S6. Allelic ratio of FLT3 internal tandem duplications that co-occurred with UPDs Supplementary Table S7: Copy number gains and losses in 425 patients with cytogenetically normal acute myeloid leukemia

Purpose:Uniparental disomy (UPD) is a way cancer cells duplicate a mutated gene, causing loss of heterozygosity (LOH). Patients with cytogenetically normal acute myeloid leukemia (CN-AML) do not have microscopically detectable chromosome abnormalities, but can harbor UPDs. We examined the prognostic significance of UPDs and frequency of LOH in patients with CN-AML.Experimental Design: We examined the frequency and prognostic significance of UPDs in a set of 425 adult patients with de novo CN-AML who were previously sequenced for 81 genes typically mutated in cancer. Associations of UPDs with outcome were analyzed in the 315 patients with CN-AML younger than 60 years.Results:We detected 127 UPDs in 109 patients. Most UPDs were large and typically encompassed all or most of the affected chromosome arm. The most common UPDs occurred on chromosome arms 13q (7.5% of patients), 6p (2.8%), and 11p (2.8%). Many UPDs significantly cooccurred with mutations in genes they encompassed, including 13q UPD with FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and 11p UPD with WT1 mutations (P = 0.02). Among patients younger than 60 years, UPD of 11p was associated with longer overall survival (OS) and 13q UPD with shorter disease-free survival (DFS) and OS. In multivariable models that accounted for known prognostic markers, including FLT3-ITD and WT1 mutations, UPD of 13q maintained association with shorter DFS, and UPD of 11p maintained association with longer OS.Conclusions:LOH mediated by UPD is a recurrent feature of CN-AML. Detection of UPDs of 13q and 11p might be useful for genetic risk stratification of patients with CN-AML.

Supplementary Figure S1. Unsupervised clustering of the sole -7 AML cohort and the complete TCGA AML patient data set. Supplementary Figure S2. SMARCA2 mutations in AML patients with sole -7. Supplementary Figure S3. Heatmap depicting the differential gene expression of AML patients with sole -7. Supplementary Figure S4. Heatmap visualizing the gene ontology (GO) analyses of the predicted target genes of the miRs upregulated in AML patients with sole -7. Supplementary Figure S5. Heatmap visualizing the gene ontology (GO) analyses of the predicted target genes of the miRs downregulated in AML patients with sole -7. Tables: Supplementary Table S1. Gene mutations detected in 36 patients with acute myeloid leukemia (AML) and sole -7 (DNA sequencing of 81 genes). Supplementary Table S2. Mutation analysis in paired germline-leukemic samples of 10 sole -7 patients. Supplementary Table S3. Genes significantly downregulated in 31 AML patients with sole -7 compared with gene expression of 136 AML patients from the TCGA cohort (14) who had other chromosome abnormalities that did not include abnormalities of chromosome 7 or a complex karyotype. Supplementary Table S4. Genes significantly upregulated in 31 AML patients with sole -7 compared with gene expression of 136 AML patients from the TCGA cohort (14) who had other chromosome abnormalities that did not include abnormalities of chromosome 7 or a complex karyotype. Supplementary Table S5. MicroRNAs (miRs) significantly downregulated in 31 AML patients with sole -7 compared with gene expression of 136 AML patients from the TCGA cohort who had other chromosome abnormalities that did not include abnormalities of chromosome 7 or a complex karyotype. Supplementary Table S6. MicroRNAs (miRs) significantly upregulated in 31 AML patients with sole -7 compared with gene expression of 136 AML patients from the TCGA cohort who had other chromosome abnormalities that did not include abnormalities of chromosome 7 or a complex karyotype. Supplementary Table S7. Gene ontogeny analyses of significantly downregulated genes located on chromosome 7 in 31 AML patients with sole -7.

Materials and Methods: Participating institutions. List of all institutions that participated in the study. Patients and treatments. Detailed information about patients included in the study and their treatments. Mutational profiling. Detailed information about the techniques used to perform the mutational profiling. Definition of clinical endpoints. Statistical definition of the used end point terms . Univariable outcome analysis. Listing of all parameters that were included in the univariable outcome analysis.

Mutations detected in 425 CN-AML patients

Supplementary Table 1 from Origins and Prevalence of the American Founder Mutation of MSH2

Supplementary Table 2 from A Susceptibility Locus for Papillary Thyroid Carcinoma on Chromosome 8q24

Supplementary Figure 1 from A Susceptibility Locus for Papillary Thyroid Carcinoma on Chromosome 8q24

Supplementary Figure 1 from Tgfbr1 Haploinsufficiency Is a Potent Modifier of Colorectal Cancer Development

Supplementary Table 2 from Screening for Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer) among Endometrial Cancer Patients

Supplementary Figure 2 from A Susceptibility Locus for Papillary Thyroid Carcinoma on Chromosome 8q24

Endometrial cancer is the most common cancer in women with Lynch syndrome. The identification of individuals with Lynch syndrome is desirable because they can benefit from increased cancer surveillance. The purpose of this study was to determine the feasibility and desirability of molecular screening for Lynch syndrome in all endometrial cancer patients. Unselected endometrial cancer patients (N = 543) were studied. All tumors underwent microsatellite instability (MSI) testing. Patients with MSI-positive tumors underwent testing for germ line mutations in MLH1, MSH2, MSH6, and PMS2. Of 543 tumors studied, 118 (21.7%) were MSI positive (98 of 118 MSI high and 20 of 118 MSI low). All 118 patients with MSI-positive tumors had mutation testing, and nine of them had deleterious germ line mutations (one MLH1, three MSH2, and five MSH6). In addition, one case with an MSI-negative tumor had abnormal MSH6 immunohistochemical staining and was subsequently found to have a mutation in MSH6. Immunohistochemical staining was consistent with the mutation result in all seven truncating mutation–positive cases but was not consistent in two of the three missense mutation cases. We conclude that in central Ohio, at least 1.8% (95% confidence interval, 0.9-3.5%) of newly diagnosed endometrial cancer patients had Lynch syndrome. Seven of the 10 Lynch syndrome patients did not meet any published criteria for hereditary nonpolyposis colorectal cancer, and six of them were diagnosed at age >50. Studying all endometrial cancer patients for Lynch syndrome using a combination of MSI and immunohistochemistry for molecular prescreening followed by gene sequencing and deletion analysis is feasible and may be desirable. (Cancer Res 2006; 66(15): 7810-7)

Supplementary Table 1 from A Susceptibility Locus for Papillary Thyroid Carcinoma on Chromosome 8q24

Supplementary Figure 1 from Origins and Prevalence of the American Founder Mutation of MSH2

Supplementary Table 2 from Origins and Prevalence of the American Founder Mutation of MSH2

Supplementary Table 3 from A Susceptibility Locus for Papillary Thyroid Carcinoma on Chromosome 8q24

Large germline deletions within the mismatch repair gene MSH2 account for a significant proportion (up to 20%) of all deleterious mutations of this gene which are associated with Lynch syndrome. An exons 1 to 6 deletion of MSH2, originally reported in nine families, has been associated with a founding event within the United States, which genealogic studies had previously dated to 1727, and the number of present day carriers was estimated to be 18,981. Here, we report the development of a robust multiplex PCR which has assisted in the detection of 32 new families who carry the MSH2 American Founder Mutation (AFM). By offering testing to family members, 126 carriers of the AFM have been identified. Extensive genealogic studies have connected 27 of the 41 AFM families into seven extended pedigrees. These extended families have been traced back to around the 18th century without any evidence of further convergence between them. Characterization of the genomic sequence flanking the deletion and the identification of a common disease haplotype of between 0.6 and 2.3 Mb in all probands provides evidence for a common ancestor between these extended families. The DMLE+2.2 software predicts an age of ∼500 years (95% confidence interval, 425–625) for this mutation. Taken together, these data are suggestive of an earlier founding event than was first thought, which likely occurred in a European or a Native American population. The consequences of this finding would be that the AFM is significantly more frequent in the United States than was previously predicted. [Cancer Res 2008;68(7):2145–53]

PURPOSE Hereditary cancer syndromes infer high cancer risks and require intensive surveillance. Identification of high-risk individuals among patients with colorectal cancer (CRC) needs improvement. METHODS Three thousand three hundred ten unselected adults who underwent surgical resection for primary invasive CRC were prospectively accrued from 51 hospitals across Ohio between January 1, 2013, and December 31, 2016. Universal Tumor screening (UTS) for mismatch repair (MMR) deficiency was performed for all, and pathogenic germline variants (PGVs) were identified using multigene panel testing (MGPT) in those who met at least one inclusion criterion: MMR deficiency, diagnosed < 50 years, multiple primary tumors (CRC or endometrial cancer), or with a first-degree relative with CRC or endometrial cancer. RESULTS Five hundred twenty-five patients (15.9%) had MMR deficiency. Two hundred thirty-four of 3,310 (7.1%; 16% of the 1,462 who received MGPT) had 248 PGVs in cancer susceptibility genes. One hundred forty-two (4.3%) had a PGV in an MMR gene, and 101 (3.1%) had a PGV in a non-MMR gene. Ten with Lynch syndrome (LS) also had a non-MMR PGV and were included in both groups. Two (0.06%) had constitutional MLH1 hypermethylation. Of unexplained MMR-deficient patients, 88.4% (76 of 86) had double somatic MMR mutations. Testing for only MMR genes in MMR-deficient patients would have missed 18 non-MMR gene PGVs (7.3% of total PGVs identified). Had UTS been the only method used to screen for hereditary cancer syndromes, 38.6% (91 of 236) would have been missed, including 6.3% (9 of 144) of those with LS. These results have treatment implications as 5.3% (175 of 3,310) had PGVs in genes with therapeutic targets. CONCLUSION UTS alone is insufficient for identifying a large proportion of CRC patients with hereditary syndromes, including some with LS. At a minimum, 7.1% of individuals with CRC have a PGV and pan-cancer MGPT should be considered for all patients with CRC.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. The molecular characteristics of histologically normal appearing tissue adjacent to the tumor (NAT) from PTC patients are not well characterized. The aim of this study was to characterize the global gene expression profile of NAT and compare it with those of normal and tumor thyroid tissues. We performed total RNA sequencing with fresh frozen thyroid tissues from a cohort of three categories of samples including NAT, normal thyroid (N), and PTC tumor (T). Transcriptome analysis shows that NAT presents a unique gene expression profile, which was not associated with sex or the presence of lymphocytic thyroiditis. Among the differentially expressed genes (DEGs) of NAT vs N, 256 coding genes and 5 noncoding genes have been reported as cancer genes involved in cell proliferation, apoptosis, and/or tumorigenesis. Bioinformatics analysis with Ingenuity Pathway Analysis software revealed that “Cancer, Organismal Injury and Abnormalities, Cellular Response to Therapeutics, and Cellular Movement” were major dysregulated pathways in the NAT tissues. This study provides improved insight into the complexity of gene expression changes in the thyroid glands of patients with PTC.

Objective Lynch syndrome is the most common cause of inherited endometrial cancer, attributable to germline pathogenic variants (PV) in mismatch repair (MMR) genes. Tumor microsatellite instability (MSI-high) and MMR IHC abnormalities are characteristics of Lynch syndrome. Double somatic MMR gene PV also cause MSI-high endometrial cancers. The aim of this study was to determine the relative frequency of Lynch syndrome and double somatic MMR PV. Methods 341 endometrial cancer patients enrolled in the Ohio Colorectal Cancer Prevention Initiative at The Ohio State University Comprehensive Cancer Center from 1/1/13–12/31/16. All tumors underwent immunohistochemical (IHC) staining for the four MMR proteins, MSI testing, and MLH1 methylation testing if the tumor was MMR-deficient (dMMR). Germline genetic testing for Lynch syndrome was undertaken for all cases with dMMR tumors lacking MLH1 methylation. Tumor sequencing followed if a germline MMR gene PV was not identified. Results Twenty-seven percent (91/341) of tumors were either MSI-high or had abnormal IHC indicating dMMR. As expected, most dMMR tumors had MLH1 methylation; (69, 75.8% of the dMMR cases; 20.2% of total). Among the 22 (6.5%) cases with dMMR not explained by methylation, 10 (2.9% of total) were found to have Lynch syndrome (6 MSH6, 3 MSH2, 1 PMS2). Double somatic MMR PV accounted for the remaining 12 dMMR cases (3.5% of total). Conclusions Since double somatic MMR gene PV are as common as Lynch syndrome among endometrial cancer patients, paired tumor and germline testing for patients with non-methylated dMMR tumor may be the most efficient approach for LS screening.

Background: AML is a clinically and molecularly heterogeneous disease associated with poor survival. Multiple disease-related factors including cytogenetic findings and gene mutations, as well as patient-related factors, such as demographics and African American (AA) heritage, have been identified that impact on pt outcomes. However, with recent improved survival it is unknown whether racial health disparities persist. Moreover, we are not aware of a large study that assessed possible race-associated molecular differences. Thus, the goals of our study were to 1) analyze the outcomes of adult AML pts in a nationwide population study, including possible impacts of sociodemographic, financial and racial disparities and 2) characterize molecular features of AA compared with those of Caucasian AML pts. Methods: For a nationwide population analysis, the Surveillance Epidemiology End Results (SEER) Program of the National Cancer Institute was used to identify 11,190 adults aged 18-60 years (y) diagnosed with AML (excluding acute promyelocytic leukemia) between 1986 and 2015. To characterize molecular features we performed targeted sequencing of 81 genes in 1,339 AML pts treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology (Alliance) protocols based on standard intensity cytarabine/anthracycline induction followed by consolidation between 1986 and 2016. No Alliance pt received an allogeneic stem cell transplant in 1st complete remission (CR). Results: The associations between demographic parameters and risk of death among SEER registry AML pts are shown in Table 1. While there was a slightly higher risk of death for men (HR 1.09) and a lower risk of death for pts with a higher median household income (>79.6k vs Conclusion: Self-reported AA race is the most important pt-associated factor associated with poor survival in AML pts < 60 y of age based on SEER. Survival analyses in Alliance pts identify AA race as independent poor survival prognosticator in AML pts besides established molecular markers. . This disparity must be urgently addressed to ensure improved outcomes for AA AML pts, and larger studies to establish molecular risk profiles are needed. Support: U10CA180821, U10CA180882 U24CA196171, https://acknowledgments.alliancefound.org; Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224 Disclosures Bhatnagar: KaryoPharm Therapuetics: Research Funding; Cell Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. Mims:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Agios: Consultancy. Walker:Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company. Powell:Genentech: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Pfizer: Research Funding; Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Novartis: Research Funding. Kolitz:Magellan: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Stone:Biolinerx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Other; Aztra-Zeneca: Consultancy; Jazz: Consultancy; Argenix: Other; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Pfizer: Consultancy; Gemoab: Consultancy; Syndax: Consultancy, Research Funding; Takeda: Other: DSMB; Macrogenics: Consultancy; Trovagene: Consultancy; Syntrix: Other: DSMB; Abbvie: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Arog: Consultancy, Research Funding; Syros: Consultancy; Stemline: Consultancy. Byrd:Trillium: Research Funding; Leukemia and Lymphoma Society: Other; Acerta Pharma: Research Funding; Syndax: Research Funding; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Kartos Therapeutics: Research Funding; Vincera: Research Funding; Novartis: Research Funding. Eisfeld:Karyopharm: Current Employment, Current equity holder in publicly-traded company; Vigeo Therapeutics: Consultancy.

BackgroundPapillary thyroid carcinoma (PTC) demonstrates high heritability and a low somatic mutation burden relative to other cancers. Therefore, the genetic risk predisposing to PTC is likely due to a combination of low penetrance variants. A recent genome-wide association study revealed the association of PTC with a missense variant, rs6793295, at 3q26 in a gene called Leucine Repeat Rich Containing 34 (LRRC34).MethodsWe report the mechanisms of PTC risk at 3q26 using a combination of overexpression, mass spectroscopy, knockdown, transcriptome profiling, migration assays and genetic analysis.ResultsWe observed differential binding of wild-type and missense LRRC34 to RANBP1. Overexpression of missense LRRC34 reduced RanGTP levels and increased apoptosis. We also identified a second linkage disequilibrium (LD) block upstream of LRRC34 containing regulatory variants with allele-specific expression. Transcriptome profiling of LRRC34 knockdown cells showed changes in genes involved with cellular movement. LRRC34 knockdown reduced the migration of thyroid cancer cell lines. Lastly, we assessed the relative contribution of PTC risk from each locus using haplotype analysis.ConclusionsOur study demonstrates two separate mechanisms, one in G protein signalling and the other in transcriptional control, dictating PTC risk at 3q26 using both biochemical and genetic techniques.

Background: Our genome sequencing analysis revealed a frameshift mutation in the shelterin gene TINF2 in a large family with individuals affected with papillary thyroid carcinoma (PTC) and melanoma. Here, we further characterized the mutation and screened for coding variants in the 6 shelterin genes in 24 families. Methods: Sanger sequencing was performed to screen for the TINF2 mutation in the key family. Quantitative reverse transcription-polymerase chain reaction (PCR) was used for TINF2 gene expression analysis. Exogenous expression and co-immunoprecipitation techniques were used for assessing TINF2 binding to TERF1. Relative telomere length (RTL) was quantified in DNAs from lymphocytes by using quantitative real-time PCR. Whole exome sequencing (WES) was performed in seven families with individuals affected with PTC and other cancer types. Screening for DNA variants in shelterin genes was performed by using whole genome sequencing data from 17 families and WES data from 7 further families. Results: The TINF2 mutation (TINF2 p.Trp198fs) showed complete co-segregation with PTC and melanoma in the key family. The mutation is not reported in databases and not identified in 23 other families we screened. The expression of TINF2 was borderline reduced in individuals with the mutation. The truncated TINF2 protein showed abolished binding to TERF1. The RTL in the individuals with the mutation was significantly longer when compared with those without the mutation from the same family as well as compared with 62 healthy controls. Among the 24 families, we identified 3 missense and 1 synonymous variant(s) in 2 shelterin genes (TINF2 and ACD). Conclusions: The rare frameshift mutation in the TINF2 gene and the associated longer telomere length suggest that dysregulated telomeres could be a mechanism predisposing to PTC and melanoma. DNA coding variants in shelterin genes are rare. Further studies are required to evaluate the roles of variants in shelterin genes in thyroid cancer and melanoma.

Accurate colorectal cancer (CRC) risk prediction models are critical for identifying individuals at low and high risk of developing CRC, as they can then be offered targeted screening and interventions to address their risks of developing disease (if they are in a high-risk group) and avoid unnecessary screening and interventions (if they are in a low-risk group). As it is likely that thousands of genetic variants contribute to CRC risk, it is clinically important to investigate whether these genetic variants can be used jointly for CRC risk prediction. In this paper, we derived and compared different approaches to generating predictive polygenic risk scores (PRS) from genome-wide association studies (GWASs) including 55,105 CRC-affected case subjects and 65,079 control subjects of European ancestry. We built the PRS in three ways, using (1) 140 previously identified and validated CRC loci; (2) SNP selection based on linkage disequilibrium (LD) clumping followed by machine-learning approaches; and (3) LDpred, a Bayesian approach for genome-wide risk prediction. We tested the PRS in an independent cohort of 101,987 individuals with 1,699 CRC-affected case subjects. The discriminatory accuracy, calculated by the age- and sex-adjusted area under the receiver operating characteristics curve (AUC), was highest for the LDpred-derived PRS (AUC = 0.654) including nearly 1.2 M genetic variants (the proportion of causal genetic variants for CRC assumed to be 0.003), whereas the PRS of the 140 known variants identified from GWASs had the lowest AUC (AUC = 0.629). Based on the LDpred-derived PRS, we are able to identify 30% of individuals without a family history as having risk for CRC similar to those with a family history of CRC, whereas the PRS based on known GWAS variants identified only top 10% as having a similar relative risk. About 90% of these individuals have no family history and would have been considered average risk under current screening guidelines, but might benefit from earlier screening. The developed PRS offers a way for risk-stratified CRC screening and other targeted interventions.

Purpose:Uniparental disomy (UPD) is a way cancer cells duplicate a mutated gene, causing loss of heterozygosity (LOH). Patients with cytogenetically normal acute myeloid leukemia (CN-AML) do not have microscopically detectable chromosome abnormalities, but can harbor UPDs. We examined the prognostic significance of UPDs and frequency of LOH in patients with CN-AML.Experimental Design: We examined the frequency and prognostic significance of UPDs in a set of 425 adult patients with de novo CN-AML who were previously sequenced for 81 genes typically mutated in cancer. Associations of UPDs with outcome were analyzed in the 315 patients with CN-AML younger than 60 years.Results:We detected 127 UPDs in 109 patients. Most UPDs were large and typically encompassed all or most of the affected chromosome arm. The most common UPDs occurred on chromosome arms 13q (7.5% of patients), 6p (2.8%), and 11p (2.8%). Many UPDs significantly cooccurred with mutations in genes they encompassed, including 13q UPD with FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and 11p UPD with WT1 mutations (P = 0.02). Among patients younger than 60 years, UPD of 11p was associated with longer overall survival (OS) and 13q UPD with shorter disease-free survival (DFS) and OS. In multivariable models that accounted for known prognostic markers, including FLT3-ITD and WT1 mutations, UPD of 13q maintained association with shorter DFS, and UPD of 11p maintained association with longer OS.Conclusions:LOH mediated by UPD is a recurrent feature of CN-AML. Detection of UPDs of 13q and 11p might be useful for genetic risk stratification of patients with CN-AML.

Neuroblastoma RAS viral oncogene homolog is a commonly mutated oncogene in melanoma, and therapeutic targeting of neuroblastoma RAS viral oncogene homolog has proven difficult. We characterized the expression and phenotypic functions of five recently discovered splice isoforms of neuroblastoma RAS viral oncogene homolog in melanoma. Canonical neuroblastoma RAS viral oncogene homolog (isoform-1) was expressed to the highest degree and its expression was significantly increased in melanoma metastases compared to primary lesions. Isoform-5 expression in metastases showed a significant, positive correlation with survival and tumours over-expressing isoform-5 had significantly decreased growth in a xenograft model. In contrast, over-expression of any isoform resulted in enhanced proliferation, and invasiveness was increased with over-expression of isoform-1 or isoform-2. Downstream signalling analysis indicated that the isoforms signalled differentially through the mitogen-activated protein kinase and PI3K pathways and A375 cells over-expressing isoform-2 or isoform-5 showed resistance to vemurafenib treatment in vitro. The neuroblastoma RAS viral oncogene homolog isoforms appear to play varying roles in melanoma phenotype and could potentially serve as biomarkers for therapeutic response and disease prognosis.

Background: Thyroid cancer patients with radioiodine-refractory (RAI-R) disease, resulting from insufficient RAI delivery and/or RAI resistance, have increased mortality and limited treatment options. To date, studies have largely focused on tumor mutations associated with different stages of disease, which could provide prognostic value for RAI-R disease. It was hypothesized that germline variants contributing to intrinsic differences in iodine metabolism, tumor microenvironment, and/or immune surveillance are associated with RAI-R disease. Methods: Whole-genome genotyping data analysis was performed on 1145 Caucasian (CAU) patients, 244 of whom were RAI-R, and 55 African American (AA) patients, nine of whom were RAI-R. Germline-variant association studies were conducted using candidate genes involved in iodine metabolism or DNA-damage repair, as well as genome-wide association analysis. Initial data indicated several notable variants in a small number of patients (n = 7), who were later determined to be AA patients of >80% African ancestry (n = 37). This led to the study focusing on germline single nucleotide polymorphisms uniquely associated with RAI-R AA patients. Sanger sequencing was performed to validate risk alleles and identify the incidence of the common somatic mutations BRAF(V600E), NRAS(Q61R), and HRAS(Q61R) in AA patients whose primary tumor samples were available (28/55). Results: TG, BRCA1, and NSMCE2 haplotypes were identified as being uniquely associated with RAI-R AA patients of >80% African ancestry. All patients with the TG haplotype (n = 4) had a biochemical incomplete response to RAI therapy. Patients with the NSMCE2 haplotype (n = 4) were diagnosed at a young age (13, 17, 17, and 26 years old) with distant metastatic disease at initial diagnosis. The BRCA1 haplotype co-occurred in three out of four patients with the NSMCE2 haplotype. The incidence of BRAF(V600E) appears lower in papillary thyroid carcinomas from AA patients of >80% African ancestry (3/14; 21%) than in AA patients of

BackgroundPatients with colorectal cancer (CRC) with mismatch repair-deficient (dMMR) tumours without MLH1 methylation or germline MMR pathogenic variants (PV) were previously thought to have Lynch syndrome (LS). It is now appreciated that they can have double somatic (DS) MMR PVs. We explored the clinical characteristics between patients with DS tumours and LS in two population-based cohorts.MethodsWe included patients with CRC from Ohio 2013–2016 and Iceland 2000–2009. All had microsatellite instability testing and/or immunohistochemistry (IHC) of MMR proteins, and MLH1 methylation testing when indicated. Germline next-generation sequencing was performed for all with dMMR tumours; tumour sequencing followed for patients with unexplained dMMR. Clinical characteristics of DS patients and patients with LS were compared.ResultsOf the 232 and 51 patients with non-methylated dMMR tumours in the Ohio and Iceland cohorts, respectively, 57.8% (n=134) and 45.1% (n=23) had LS, 32.8% (n=76) and 31.4% (n=16) had DS PVs, 6% (n=14) and 9.8% (n=5) were unexplained and 4.3% (n=10) and 13.7% (n=7) had incorrect IHC. Age of diagnosis for DS patients was older than patients with LS (p=3.73×10−4) in the two cohorts. Patients with LS were more likely to meet Amsterdam II criteria (OR=15.81, p=8.47×10−6) and have multiple LS-associated tumours (OR=6.67, p=3.31×10−5). Absence of MLH1/PMS2 was predictive of DS PVs; isolated MSH6 and PMS2 absence was predictive of LS in both cohorts.ConclusionsIndividuals with LS are 15× more likely to meet Amsterdam II criteria and >5× more likely to have multiple cancers as compared with those with DS tumours. Furthermore, isolated loss of MSH6 or PMS2 protein predicts LS.

Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (p 0 center dot 0001 for each of the three three continents). These familial risk factors resulted in a wide within-gene variation in the risk of colorectal cancer for men and women from each continent who all carried pathogenic variants in the same gene or the MSH2 c.942+3A T variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and onlyBackground Existing clinical practice guidelines for carriers of pathogenic variants of DNA mismatch repair genes (Lynch syndrome) are based on the mean age-specific cumulative risk (penetrance) of colorectal cancer for all carriers of pathogenic variants in the same gene. We aimed to estimate the variation in the penetrance of colorectal cancer between carriers of pathogenic variants in the same gene by sex and continent of residence. Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero. Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (pT variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and only 10-19% having a penetrance of 40-60%. Interpretation Our study findings highlight the important role of risk modifiers, which could lead to personalised risk assessments for precision prevention and early detection of colorectal cancer for people with Lynch syndrome. Funding National Health and Medical Research Council, Australia. Copyright (c) 2021 Elsevier Ltd. All rights reserved.Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero.

International audience; Background: The literature on associations of circulating concentrations of minerals and vitamins with risk of colorectal cancer is limited and inconsistent. Evidence from randomized controlled trials (RCTs) to support the efficacy of dietary modification or nutrient supplementation for colorectal cancer prevention is also limited. Objectives: To complement observational and RCT findings, we investigated associations of genetically predicted concentrations of 11 micronutrients (β-carotene, calcium, copper, folate, iron, magnesium, phosphorus, selenium, vitamin B-6, vitamin B-12, and zinc) with colorectal cancer risk using Mendelian randomization (MR). Methods: Two-sample MR was conducted using 58,221 individuals with colorectal cancer and 67,694 controls from the Genetics and Epidemiology of Colorectal Cancer Consortium, Colorectal Cancer Transdisciplinary Study, and Colon Cancer Family Registry. Inverse variance-weighted MR analyses were performed with sensitivity analyses to assess the impact of potential violations of MR assumptions. Results: Nominally significant associations were noted for genetically predicted iron concentration and higher risk of colon cancer [ORs per SD (ORSD): 1.08 95% CI: 1.00, 1.17 P value = 0.05] and similarly for proximal colon cancer, and for vitamin B-12 concentration and higher risk of colorectal cancer (ORSD: 1.12 95% CI: 1.03, 1.21 P value = 0.01) and similarly for colon cancer. A nominally significant association was also noted for genetically predicted selenium concentration and lower risk of colon cancer (ORSD: 0.98 95% CI: 0.96, 1.00 P value = 0.05) and similarly for distal colon cancer. These associations were robust to sensitivity analyses. Nominally significant inverse associations were observed for zinc and risk of colorectal and distal colon cancers, but sensitivity analyses could not be performed. None of these findings survived correction for multiple testing. Genetically predicted concentrations of β-carotene, calcium, copper, folate, magnesium, phosphorus, and vitamin B-6 were not associated with disease risk. Conclusions: These results suggest possible causal associations of circulating iron and vitamin B-12 (positively) and selenium (inversely) with risk of colon cancer.

Background We studied a large family with 22 individuals affected with autosomal dominant hereditary spherocytosis (HS). Methods Genome‐wide linkage, whole‐genome sequencing (WGS), Sanger sequencing, RT‐PCR, and ToPO TA cloning analyses were performed. Results We revealed a heterozygous G>A transition in the 14q23 locus, at position +1 of the intron 8 donor splice site of the spectrin beta, erythrocytic (SPTB) gene. This splice variant (SPTB c.1064+1G>A) was confirmed by Sanger sequencing and showed complete co‐segregation with HS in the family. Further RT‐PCR reactions and sequencing analysis indicated that the variant leads to the exclusion of exon 8 and subsequent frameshift in exon 9 and a premature stop codon in SPTB. Translation of the altered allele would lead to a truncation with a loss of all spectrin repeat domains in SPTB protein. Conclusion This variant is novel and has not been found in any databases. We propose that this splice variant explains the spherocytosis phenotype observed in this large family., We studied a large family with 22 individuals affected with autosomal dominant hereditary spherocytosis (HS). Genome‐wide linkage and whole‐genome sequencing analyses revealed a heterozygous G>A transition in the 14q23 locus, at position +1 of the intron 8 donor splice site of the spectrin beta, erythrocytic (SPTB) gene.

In this brief article, we celebrate the life and numerous scientific achievements of Dr. Albert de la Chapelle, a pioneer in the fields of human genetics and cytogenetics.

Incidence of differentiated thyroid carcinoma (DTC) varies considerably between ethnic groups, with particularly high incidence rates in Pacific Islanders. Here, we conducted a genome-wide association study (GWAS) involving 1,554 cases/1,973 controls of European ancestry and 301 cases/348 controls of Oceanian ancestry from the EPITHYR consortium. Our results confirmed the association with the known DTC susceptibility loci at 2q35, 8p12, 9q22.33 and 14q13.3 in the European ancestry population and suggested two novel signals at 1p31.3 (rs334729) and 16q23.2 (rs16950982), which were associated with TSH levels in previous GWAS. We additionally replicated an association with 5p15.33 reported previously in Chinese and European populations. Except at 1p31.3, all associations were in the same direction in the population of Oceanian ancestry. The frequency of risk alleles at 2q35, 5p15.33 and 16q23.2 were significantly higher in Oceanians than in Europeans and may explain part of the highest DTC incidence observed in Oceanians.Competing Interest StatementThe authors have declared no competing interest.Funding StatementINCA (grant number 9533) and ARC (grant number PGA120150202302), Ligue Nationale Contre le Cancer, Agence Nationale pour la Recherche (ANR), the Direction Generale de la Sante, the Agence Francaise de Securite Sanitaire de l alimentation, de lenvironnement et du travail (ANSES), CHILDTHYR EEC program, and the Fondation de France. JG and CX were the recipient of a PhD fellowship from Region Ile-de-France.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:All participants provided informed consent and each study was approved by their governing ethics committee.All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. (Less)

Accurate colorectal cancer (CRC) risk prediction models are critical for identifying individuals at low and high risk of developing CRC, as they can then be offered targeted screening and interventions to address their risks of developing disease (if they are in a high-risk group) and avoid unnecessary screening and interventions (if they are in a low-risk group). As it is likely that thousands of genetic variants contribute to CRC risk, it is clinically important to investigate whether these genetic variants can be used jointly for CRC risk prediction. In this paper, we derived and compared different approaches to generating predictive polygenic risk scores (PRS) from genome-wide association studies (GWASs) including 55,105 CRC-affected case subjects and 65,079 control subjects of European ancestry. We built the PRS in three ways, using (1) 140 previously identified and validated CRC loci; (2) SNP selection based on linkage disequilibrium (LD) clumping followed by machine-learning approaches; and (3) LDpred, a Bayesian approach for genome-wide risk prediction. We tested the PRS in an independent cohort of 101,987 individuals with 1,699 CRC-affected case subjects. The discriminatory accuracy, calculated by the age- and sex-adjusted area under the receiver operating characteristics curve (AUC), was highest for the LDpred-derived PRS (AUC = 0.654) including nearly 1.2 M genetic variants (the proportion of causal genetic variants for CRC assumed to be 0.003), whereas the PRS of the 140 known variants identified from GWASs had the lowest AUC (AUC = 0.629). Based on the LDpred-derived PRS, we are able to identify 30% of individuals without a family history as having risk for CRC similar to those with a family history of CRC, whereas the PRS based on known GWAS variants identified only top 10% as having a similar relative risk. About 90% of these individuals have no family history and would have been considered average risk under current screening guidelines, but might benefit from earlier screening. The developed PRS offers a way for risk-stratified CRC screening and other targeted interventions.

ObjectiveAn understanding of the etiologic heterogeneity of colorectal cancer (CRC) is critical for improving precision prevention, including individualized screening recommendations and the discovery of novel drug targets and repurposable drug candidates for chemoprevention. Known differences in molecular characteristics and environmental risk factors among tumors arising in different locations of the colorectum suggest partly distinct mechanisms of carcinogenesis. The extent to which the contribution of inherited genetic risk factors for sporadic CRC differs by anatomical subsite of the primary tumor has not been examined.DesignTo identify new anatomical subsite-specific risk loci, we performed genome-wide association study (GWAS) meta-analyses including data of 48,214 CRC cases and 64,159 controls of European ancestry. We characterized effect heterogeneity at CRC risk loci using multinomial modeling.ResultsWe identified 13 loci that reached genome-wide significance (P−8) and that were not reported by previous GWAS for overall CRC risk. Multiple lines of evidence support candidate genes at several of these loci. We detected substantial heterogeneity between anatomical subsites. Just over half (61) of 109 known and new risk variants showed no evidence for heterogeneity. In contrast, 22 variants showed association with distal CRC (including rectal cancer), but no evidence for association or an attenuated association with proximal CRC. For two loci, there was strong evidence for effects confined to proximal colon cancer.ConclusionGenetic architectures of proximal and distal CRC are partly distinct. Studies of risk factors and mechanisms of carcinogenesis, and precision prevention strategies should take into consideration the anatomical subsite of the tumor.Significance of this studyWhat is already known about this subject?Heterogeneity among colorectal cancer (CRC) tumors originating at different locations of the colorectum has been revealed in somatic genomes, epigenomes, and transcriptomes, and in some established environmental risk factors for CRC.Genome-wide association studies (GWAS) have identified over 100 genetic variants for overall CRC risk; however, a comprehensive analysis of the extent to which genetic risk factors differ by the anatomical sublocation of the primary tumor is lacking.What are the new findings?In this large consortium-based study, we analyzed clinical and genome-wide genotype data of 112,373 CRC cases and controls of European ancestry to comprehensively examine whether CRC case subgroups defined by anatomical sublocation have distinct germline genetic etiologies.We discovered 13 new loci at genome-wide significance (P−8) that were specific to certain anatomical sublocations and that were not reported by previous GWAS for overall CRC risk; multiple lines of evidence support strong candidate target genes at several of these loci, including PTGER3, LCT, MLH1, CDX1, KLF14, PYGL, BCL11B, and BMP7.Systematic heterogeneity analysis of genetic risk variants for CRC identified thus far, revealed that the genetic architectures of proximal and distal CRC are partly distinct.Taken together, our results further support the idea that tumors arising in different anatomical sublocations of the colorectum may have distinct etiologies.How might it impact on clinical practice in the foreseeable future?Our results provide an informative resource for understanding the differential role that genes and pathways may play in the mechanisms of proximal and distal CRC carcinogenesis.The new insights into the etiologies of proximal and distal CRC may inform the development of new precision prevention strategies, including individualized screening recommendations and the discovery of novel drug targets and repurposable drug candidates for chemoprevention.Our findings suggest that future studies of etiological risk factors for CRC and molecular mechanisms of carcinogenesis should take into consideration the anatomical sublocation of the colorectal tumor.