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Familial atypical multiple mole melanoma (FAMMM) syndrome is a hereditary cancer syndrome that results from mutations in several genes, including the CDKN2A gene. In addition to melanoma, certain other malignancies such as pancreatic cancer are known to occur more frequently in family members who carry the mutation. However, as these families have been followed over time, additional cancers have been observed in both carriers and noncarriers. We sought to determine whether these additional cancers occur at higher frequencies in carriers than noncarriers. We performed survival analyses using 10 FAMMM syndrome families (N = 1,085 individuals) as well as a mixed effects Cox regression, with age at last visit to the clinic or age at cancer diagnosis as our time variable. This analysis was done separately for the known FAMMM-related cancers and “other” cancer groups. The survival curves showed a significant age effect with carriers having a younger age at cancer onset than noncarriers for FAMMM-related cancers (as expected) as well as for newly associated cancers. The Cox regression reflected what was seen in the survival curves, with all models being highly significant (P = 7.15E−20 and P = 5.00E−13 for the FAMMM-related and other cancers, respectively). These analyses support the hypothesis that CDKN2A mutation carriers in FAMMM syndrome families have increased risk for early onset of several cancer types beyond the known cancers. Therefore, these individuals should be screened for additional cancers, and mutation screening should be extended to more than first-degree relatives of an index carrier patient. Significance: This study shows that carriers of mutations in the CDKN2A gene in FAMMM syndrome are at increased risk for early onset of several cancer types beyond the known cancers.

Lung cancer (LC) is the most common cause of cancer mortality and the third most common cancer by incidence in the United States. While environmental factors play an important role in its development, LC risk also exhibits a high degree of heritability. Linkage analyses and genome-wide association studies have identified multiple loci associated with increased LC risk; however, much of the heritability has yet to be explained by these loci. Structural mutations, including copy number variations (CNVs), contribute to phenotypic diversity through dosage and/or cis-regulatory effects. Unbiased CNV mapping is only possible with the use of high-depth, short-read sequencing and remains much more complicated and prone to false positives than SNP detection. As the tools necessary for CNV detection have improved, both somatic and germline CNVs have been shown to play an important role in disease, especially cancer, and often they account for a significant proportion of pathogenic variants. Little has been done to understand the role of germline CNVs in the biological pathways of hereditary lung cancer (HLC). The goal of the current project is to utilize the whole exome sequencing (WES) data in identifying the CNVs in the HLC families (≥3 LC/family) recruited by the Genetic Epidemiology of Lung Cancer Consortium (GELCC). This work uses germline WES data from 203 individuals (60 with a LC diagnosis) from 25 HLC families (≥3 LC affected/family). We limited our investigation to CNVs called by two independent tools using read depth to infer copy number changes and genomic breakpoints: GATK 4 (https://gatk.broadinstitute.org) and XHMM (https://atgu.mgh.harvard.edu/xhmm/). We have identified CNVs (deletions) in the following protein coding genes: KIR2DL1 (19q13.42; OR=1.77), DMBT1 (10q26.13; OR=1.56), SIBPB1 (20p13; OR=1.53), LILRA6 (20p13; OR=1.53), and HLA-DRB5 (6p21.32; OR=1.64) in ≥3 families in at least two individuals/family. All of these genes with the exception of HLA-DRB5 were previously reported in LC studies; HLA-DRB5 was reported in other cancer studies. DMBT1 was reported to be a candidate tumor suppressor gene and frequent loss of heterozygosity (LOH) was observed in several human tumors. It is highly expressed in lung and loss of expression was reported in LC cell lines. All CNVs identified in our study are consistent with Mendelian expectations and appear to be uncommon in the general population. CNVs in these genes may contribute to the lung cancer risk in the HLC families. Further validation of the identified variants is ongoing. We will consider overall CNV burden and disease association with identified deletions and duplications. The enrichment of rare variants in oncogenically associated genes that co-segregate with LC provides specific mutations for future work and improves our understanding of the inheritance and pathogenesis of LC. Citation Format: John Waldron, Kirsten W. Termine, Anthony M. Musolf, Mariza de Andrade, Colette Gaba, Ramaswamy Govindan, Ping Yang, Ming You, Ellen Jaeger, Angelle F. Bencaz, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Joan E. Bailey-Wilson, Diptasri M. Mandal. Analyses of whole exome sequencing (WES) data of hereditary lung cancer families identify germline copy number variations (CNVs) in multiple genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1452.

Introduction: Mutations within the BRCA1 gene have been linked to up to an 80% lifetime risk of breast cancer as well as increased risk for ovarian, pancreatic and melanoma cancers. In this study we examined families with known germline mutations in BRCA1 after long-term follow-up to determine whether carriers experience higher rates of other cancers that have not yet been associated with germline mutations in the BRCA1 gene. Methods: We studied 127 Hereditary Breast and Ovarian Cancer (HBOC) syndrome families (N = 23,078 individuals who have been followed at Creighton University) in which a causal mutation in the BRCA1 gene was identified. We performed survival analysis and a mixed effects cox regression with age at follow-up or cancer event as our time variable and presence or absence of BRCA1-related or other cancers (separate analyses) as our indicator variable. Results: The survival curves showed a significant age effect with carriers having a younger age at cancer onset for BRCA1-related (as expected) as well as other cancers than that of non-carriers. The cox regression models were also highly significant (P = 1.77E-37 and P = 1.04E-07 for the BRCA1-related and other cancers, respectively). Of the cancers with enough samples to do stratified analyses, uterine, skin, lymphoma and colon cancers occurred at higher rates and at earlier ages in mutation carriers. Conclusions: These analyses support the hypothesis that the BRCA1 mutations carriers of HBOC syndrome have increased risk for early onset of several additional cancer types, especially cancers that arise in estrogen-influenced tissues. Citation Format: Candace Middlebrooks, Kenzhane Pantin, Mark Stacey, Carrie Snyder, Murray J. Casey, Trudy Shaw, Joan E. Bailey-Wilson, Henry T. Lynch. Deleterious germline mutations in the BRCA1 gene are associated with increased risk for cancers of the female reproductive system other than breast and ovarian as well as other cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2300.

More Americans die every year of lung cancer than any other cancer. While the environmental risks for lung cancer are well understood, the genetic risk factors for this disease are not, even though it has been shown that lung cancer risk aggregates in families. Segregation analyses have confirmed the likelihood of rare, highly penetrant variants affecting lung cancer. Family studies offer a unique opportunity to identify such rare risk variants. We have previously identified a genome-wide significant risk locus for lung cancer using multipoint linkage analysis in highly aggregated lung cancer families. In this study, we performed targeted sequencing on this 6q23-6q27 region on 75 individuals from the 9 most strongly linked families. Parametric two-point linkage analysis using an autosomal dominant model with 10% penetrance for carriers and a 1% phenocopy rate and disease allele frequency of 1% was performed for each family. While we did not find any genome-wide significant results in any of the individual families, we did observe several interesting potential rare risk variants. First, we observed that the significant signal previously observed on 6q is recapitulated in these data but the location of the peak LOD is highly heterogeneous across families and it is likely that each family has a different risk gene. While most of the highest LOD scores were in noncoding variants, we did observe exonic variants with the highest LOD scores in two families (44 and 42). Both variants were rare, with a minor allele frequency (MAF) of approximately 1% and were in LPA and GPR126 respectively. Both these genes have been implicated somatically in lung cancer, however this is the first time that they have been implicated in the germline. While the other families have their highest LOD scores in noncoding variants, some of these genes are also good potential candidate genes including PARK2 (family 30), GRM1 (family 47), and PDE10A (family 102). All three genes have been implicated in lung cancer in some capacity, and this is the first time that GRM1 and PDE10A have been implicated as germline mutations. Again, all these variants are rare (MAF ⇐ 0.01) in the general population, which makes sense for a potential highly penetrant risk variant. In this study, we have elucidated that a previously identified risk locus on 6q25 is heterogeneous in the nine most strongly linked families, with different families appearing to be carrying different risk variants. Many of the top variants in each family are rare variants that are in good potential causal genes that have been identified for the first time here as germline risk variants for lung cancer. Further functional annotation is underway for these variants and some additional linkage analyses using other penetrance matrices may also be performed. Citation Format: Anthony M. Musolf, Claire L. Simpson, Bilal A. Moiz, Mariza de Andrade, Diptasri Mandal, Colette Gaba, Ping Yang, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Joan E. Bailey-Wilson. Highly aggregated lung cancer families reveal a heterogeneous cause for a previous linkage signal on 6q [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 37.

Familial atypical multiple mole melanoma (FAMMM) syndrome is a hereditary cancer syndrome that results from mutations in several genes, including the

Lung cancer (LC) kills more people in the United States each year than any other cancer. While it is well known that a variety of environmental factors (particularly tobacco smoke) strongly increase the risk of LC, there are multiple associated genetic variants with small contributions to risk. High aggregation of LC within rare individual families suggest that there are high-risk genetic variants as well. However, these genetic risk factors for LC are under studied due to the rapid fatality of LC. We studied 28 highly aggregated extended high-risk familial lung cancer (HRFLC) families collected from eight different sites across the US. Whole exome sequencing was performed on 290 individuals from these families to identify potential risk variants for HRFLC using genetic linkage analysis. Quality control was performed on the sequence data, filtering on parameters such as read depth, genotype quality, missingness, and Mendelian inconsistencies. Identity-by-descent (IBD) values were also calculated to verify correct familial relationships. Quality control procedures left approximately 400,000 SNVs and indels for analysis.Parametric two-point linkage analysis was performed assuming an autosomal dominant mode of inheritance. Disease allele frequency was set to 1% with a penetrance of 80% for carriers and 1% phenocopy rate. While we did not identify any genome-wide significant variants across the 28 families, multiple suggestive variants were identified. The largest cluster of suggestive variants was located at 14q32 in the CATSPERB gene. Given the likely locus heterogeneity in LC (combined with the lack of power in some families), it is not surprising that none of the variants were significant across the families; looking at the individual family results proved more informative. Long haplotypes linked to LC risk were identified in multiple families. These long runs of positive linkages, which have little to no negative linkage evidence across them, are characteristic of true linkage signals in these types of analysis. Two of the most interesting linked regions were at 7p36.1 and 4q21.23-28.23. The 7p signal (observed in a single family) was genome-wide suggestive and located within the SSPO gene. SSPO has been implicated in breast and skin cancer (melanoma); it is a novel lung cancer signal. The 4q linkage (again observed in a single family) covers a large chunk of 4q and contains multiple potential candidate genes, however, the best candidate gene is PTPN13, a gene implicated in lung cancer but never in familial lung cancer. We are currently evaluating the individual family results of several other pedigrees and plan to perform additional analyses to confirm these linkages. Citation Format: Anthony Musolf, Haiming Sun, Bilal A. Moiz, Claudio W. Pikielny, Mariza de Andrade, Diptasri Mandal, Colette Gaba, Ping Yang, Yafang Li, Ming You, Richard K. Wilson, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Ramaswamy Govindan, Joan E. Bailey-Wilson. Familial lung cancer exhibits multiple novel linked haplotypes within pedigrees [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4176.

Purpose: Prostate cancer is the most common cancer in males, but also exhibits a ∼1.5-2-fold higher incidence in African American men compared with whites. Epidemiologic evidence supports a large heritable contribution to prostate cancer, with over 100 susceptibility loci identified to date that can explain ∼33 % of the familial risk. A portion of the undefined risk may be due to rare susceptibility variants. The African American Hereditary Prostate Cancer (AAHPC) Study, established in 1997, enrolled 77 AA families from seven clinical sites across the United States (Ann Epidemiol, 2000). The aim of this study is to identify rare, predictive, deleterious variants through exome sequencing of 99 cases from 26 families selected from the AAHPC families (2 to 3 affected men sequenced per family) and three female 1000 Genome controls. Methods: To explore the contribution of rare variation in coding regions of 38 known cancer-causing genes to prostate cancer risk (PCa), we sequenced the exomes of 99 AAHPC cases at a mean coverage of 30x. Post-variant calling quality control (QC) was implemented using Golden Helix SVS 8 software with filters set for removal of variants with Read Depth >10, Quality Score >10, and Quality Score: Read Depth Ratio > 0.5. Mendelian inconsistency was checked using PLINK. Prioritization of all candidate genes/variants were evaluated using online databases including 1000 Genomes and ANNOVAR for non-reference allele frequency and predictions of functional impact. Conclusions. Through exome sequencing of 99 AAHPC cases and 3 female 1000 Genome controls, we identified 37 non-synonymous single nucleotide variants that are considered damaging by at least one predictive scoring tool in our 38 candidate genes. However, most of these variants were common and thus unlikely to be causal. We observed three rare variants that were considered damaging by three predictive scoring tools in two known PCa genes, PCNT and ZFHX3. These 3 variants were each observed in a single different family (in 1 of 3 affected individuals). Interesting rare candidate variants (MAF ≤ 0.01) rs190517526 & rs114692729 were found in known cancer susceptibility loci (CD82 and EZH2). The CD82 variant was observed in all 3 sequenced affecteds in one family and the EZH2 variant was observed in 2 of 3 sequenced affecteds in a different family. These predicted damaging variants in these four genes represent potentially novel causal candidates for some of the 26 AAHPC families sequenced here. Future work will carefully analyze the remainder of the genome in the other 21 sequenced families including planned sequencing of additional family members. Citation Format: Deyana D. Lewis, Shukmei Wong, Angela S. Baker, Isaac Powell, John D. Carpten, Joan E. Bailey-Wilson, Cheryl Cropp. Rare candidate variants shared among affected family members in the African American Hereditary Prostate Cancer Study families [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4240.

Lung cancer (LC) is the primary cause of cancer-related deaths in the United States. Whereas smoking and other environmental factors strongly increase LC risk, multiple genetic variants also contribute to risk in smokers. Furthermore, among smokers, some families have been identified with an abnormally high prevalence of LC, suggesting that unknown genetic factors can greatly increase LC risk in smokers. The typically short survival after LC diagnosis impedes collection of detailed genotypic information on any single large family pedigree, impairing the identification of putative high-risk factors. Therefore, the Genetic Epidemiology of LC Consortium has collected epidemiological and genetic data from a number of families with high numbers of LC cases from eight different sites across the US. In this study, we have obtained whole exome sequences (WES) from 290 members of 28 families, including 66 LC cases. We used a gene-based approach to allow for the possibility that different families may contain different variants of the same gene. Variants were filtered for i) allele frequency, ii) functional effect using combined annotation-dependent depletion (CADD), and, iii) affecting a gene with either a known or suspected role in cancer. We further selected variants based on their segregation in family pedigrees in a pattern consistent with a large effect on LC risk. Candidate LC risk genes were then identified as those represented in at least two families by the same or different variants. We further culled the list of genes by requiring the presence of at least one rare, functional variant enriched in the WES of 1060 cases relative to 899 controls from the Transdisciplinary Research on Cancer of the Lung consortium. This analysis narrowed our results to two genes, one being E2A, a member of the E family of bHLH transcription factors. Whereas loss-of-function mutations in E2A drive lymphoid cancers, the E2A protein also participates in an oncogenic heterodimer with TWIST1 that promotes the epithelial-mesenchymal transition and is implicated in multiple cancer types. Furthermore, the E2A/TWIST1 heterodimer is the primary TWIST1-containing complex implicated in oncogenesis, and silencing of E2A in KRAS-mutant non-small cell lung cancer (NSCLC) cell lines results in oncogene-stimulated senescence and apoptosis. Our data identified three distinct E2A variants present in all 10 sequenced LC cases in the 5 families in which those variants are found. Finally, two of these E2A variants are located only 57 nucleotides from each other, immediately adjacent to sequences encoding a transcription activation domain, suggesting that both variants alter the same specific protein function. These data identify E2A variants as likely high risk factors for LC in smokers and validate our general approach for identifying genetic factors with a large impact on LC risk. Citation Format: Claudio W. Pikielny, Anthony M. Musolf, Mariza de Andrade, Diptasri Mandal, Colette Gaba, Ping Yang, yafang Li, Ming You, Richard Wilson, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Ambrose I. Granizo-Mackenzie, Yanhong Liu, Ramaswamy Govindan, James McKay, Rayjean Hung, John K. Field, David C. Christiani, Joan E. Bailey-Wilson, Christopher I. Amos. Familial studies identify variants in the E2A transcription factor as putative risk factors for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-053.

Compelling evidence supports a genetic component to prostate cancer susceptibility and aggressiveness. Recent genome-wide association studies have identified more than 30 single-nucleotide polymorphisms associated with prostate cancer susceptibility. It remains unclear, however, whether such genetic variants are associated with disease aggressiveness—one of the most important questions in prostate cancer research today. To help clarify this and substantially expand research in the genetic determinants of prostate cancer aggressiveness, the first National Cancer Institute Prostate Cancer Genetics Workshop assembled researchers to develop plans for a large new research consortium and patient cohort. The workshop reviewed the prior work in this area and addressed the practical issues in planning future studies. With new DNA sequencing technology, the potential application of sequencing information to patient care is emerging. The workshop, therefore, included state-of-the-art presentations by experts on new genotyping technologies, including sequencing and associated bioinformatics issues, which are just beginning to be applied to cancer genetics. Cancer Res; 71(10); 3442–6. ©2011 AACR.

Lung cancer is the deadliest cancer in the United States, contributing approximately 25% of all cancer deaths. Lung cancer risk is well-documented to increase in response to environmental factors, particularly tobacco smoking. As one might expect for a complex trait, there is also a significant genetic risk component in lung cancer, though it is not very well studied. We obtained whole exome sequencing (WES) genotype data from Washington University on 204 subjects from 25 extended families. These individuals were recruited from families with a history of lung cancer and previous analyses showed these 25 families be informative. The purpose of this study is to identify potential risk variants for lung cancer by performing genetic linkage analysis. Quality control was performed on the sequence data, filtering on parameters such as depth (less than 10), genotype quality (less than 10), missingness, and Mendelian inconsistencies. Identity-by-descent (IBD) values were also calculated to verify correct familial relationships. Quality control procedures left approximately 500,000 SNVs and indels for analysis. We performed two-point parametric linkage analysis assuming an autosomal dominant mode of inheritance with a disease allele frequency of 1%, a 10% penetrance for carriers and a 1% penetrance for non-carriers. Two discrete sets of linkage analyses were performed. One was a variant-based analysis, which evaluated linkage between the phenotype and individual SNVs or indels. The second was gene-based analysis, which created a multi-allelic pseudomarker corresponding to a gene from haplotypes of rare variants (minor allele frequency While the variant-based analysis did not identify any genome-wide significant results, several were identified by the gene-based analysis. The highest HLOD scores were both greater than the genome-wide significance level of HLOD = 3.3 and were located on two regions of chromosome 1q: 1q42.13-43 and 1q21.2-21.1. In both regions, the significant HLOD scores clustered around known cancer genes. At the 1q42.13-43 region, signals centered on the cancer-implicated genes OBSCN and RYR2 (also a known mesothelioma gene), while at 1q21.2-21.1 the signals centered on five genes in the neuroblastoma breakpoint family (NBPF) genes, a cluster of recently duplicated genes. NBPF genes have previously been implicated in a variety of different cancers including lung cancer. We are currently performing additional analyses to corroborate the significant results, as well as examining the individual families to determine which families are driving the significant signals. Citation Format: Anthony M. Musolf, Haiming Sun, Bilal A. Moiz, Diptasri Mandal, Mariza de Andrade, Colette Gaba, Ping Yang, Yafang Li, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Joan E. Bailey-Wilson. Whole exome sequencing identifies significantly linked regions on multiple chromosomes in families with a history of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3276.

Recent genome-wide association studies have linked the chromosome 15q24-25.1 locus to nicotine addiction and lung cancer susceptibility. To refine the 15q24-25.1 locus, we performed a haplotype-based association analysis of 194 familial lung cases and 219 cancer-free controls from the Genetic Epidemiology of Lung Cancer Consortium (GELCC) collection, and used proliferation and apoptosis analyses to determine which gene(s) in the 15q24-25.1 locus mediates effects on lung cancer cell growth in vitro. We identified two distinct subregions, hapL (P = 3.20 × 10−6) and hapN (P = 1.51 × 10−6), which were significantly associated with familial lung cancer. hapL encompasses IREB2, LOC123688, and PSMA4, and hapN encompasses the three nicotinic acetylcholine receptor subunit genes CHRNA5, CHRNA3, and CHRNB4. Examination of the genes around hapL revealed that PSMA4 plays a role in promoting cancer cell proliferation. PSMA4 mRNA levels were increased in lung tumors compared with normal lung tissues. Down-regulation of PSMA4 expression decreased proteasome activity and induced apoptosis. Proteasome dysfunction leads to many diseases including cancer, and drugs that inhibit proteasome activity show promise as a form of cancer treatment. Genes around hapN were also investigated, but did not show any direct effect on lung cancer cell proliferation. We concluded that PSMA4 is a strong candidate mediator of lung cancer cell growth, and may directly affect lung cancer susceptibility through its modulation of cell proliferation and apoptosis. [Cancer Res 2009;69(19):7844–50]

In this study, we observed loss of heterozygosity (LOH) in human chromosomal fragment 6q25.1 in sporadic lung cancer patients. LOH was observed in 65% of the 26 lung tumors examined and was narrowed down to a 2.2-Mb region. Single-nucleotide polymorphism (SNP) analysis of genes located within this region identified a candidate gene, termed p34. This gene, also designated as ZC3H12D, C6orf95, FLJ46041, or dJ281H8.1, carries an A/G nonsynonymous SNP at codon 106, which alters the amino acid from lysine to arginine. Nearly 73% of heterozygous lung cancer tissues with LOH and the A/G SNP also exhibited loss of the A allele. In vitro clonogenic and in vivo nude mouse studies showed that overexpression of the A allele exerts tumor suppressor function compared with the G allele. p34 is located within a recently mapped human lung cancer susceptibility locus, and association of the p34 A/G SNP was tested among these families. No significant association between the less frequent G allele and lung cancer susceptibility was found. Our results suggest that p34 may be a novel tumor suppressor gene involved in sporadic lung cancer but it seems not to be the candidate familial lung cancer susceptibility gene linked to chromosomal region 6q23-25. [Cancer Res 2007;67(1):93–9]

Lung cancer (LC) is the leading cancer killer of Americans; an estimated 158,000 people will die in the U.S. from LC in 2016. While it is well-known that LC risk is affected by the environment, particularly tobacco smoking, there is a substantial genetic risk to LC also. We examined genotype data from Illumina HumanCore-12v1-0 array (297,000 SNPs) on 175 individuals in 25 extended families with a strong history of LC recruited by the Genetic Epidemiology of Lung Cancer Consortium. The purpose of this study is to determine which families are segregating a high-penetrance genetic risk haplotype so that the most informative families can be selected for DNA sequencing studies. Quality control was performed to remove SNPs and individuals with greater than 1% missingness as well as monomorphic SNPs. SNPs with Mendelian errors in more than one family were dropped. Identity-by-descent values were calculated to confirm correct familial relationships; 1 individual was dropped. After quality control we were left with approximately 245,000 SNPs for analysis. We performed three types of parametric linkage analyses using an autosomal dominant model with 40% penetrance in carriers and 1% penetrance in non-carriers. A disease allele frequency of 0.01 was used. Standard single variant two-point analysis between the disease and each marker was performed using TwoPointLods. Multipoint linkage analysis was performed using SimWalk2. We also performed regional-based linkage analyses using SEQLinkage and MERLIN. SEQLinkage builds a multiallelic regional marker (similar to a microsatellite) that corresponds to a gene or a portion of a gene. Two-point linkage analyses were then performed on the regional markers using MERLIN. We identified five loci that were genome-wide significant (HLOD score ≥ 3.3) from the regional-based linkage analyses on 18p11.23 (HLOD = 4.1), 2p22.2 (3.9), 14q13.1 (3.7), 16p13.1 (3.4), and 20q13.11 (3.4). It is particularly exciting that the scores centered on prospective cancer genes. Our highest score was centered on PTPRM, a protein tyrosine phosphatase on chromosome 18 that has been implicated as a LC oncogene. The signal on 20q13 also centered on another protein tyrosine phosphatase (PTPRT) that has been shown to be mutated in LC cells. The source of the signal on 16p13 was RNA binding protein RBFOX1, which has been shown to be deleted in LC cell lines and the source of the signal on 2p22 was LRP1B, a LDL receptor-related protein that is often inactivated in LC cells. The source of the final signal on 14q13 was NPAS3, a transcription factor that is a tumor suppressor in brain tumors. It should be noted that all previous evidence linking these genes to cancer was based on somatic mutations; this is the first time any of these genes has been shown to be significantly linked to germline disease risk in a family-based study. We plan to perform targeted sequencing on the linked regions to elucidate the exact causal variant. Citation Format: Joan E. Bailey-Wilson, Anthony M. Musolf, Claire L. Simpson, Mariza de Andrade, Diptasri Mandal, Colette G. Gaba, Ping Yang, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos. Familial lung cancer is significantly linked to cancer-associated genes on five chromosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4268. doi:10.1158/1538-7445.AM2017-4268

Lung cancer is the leading cancer-related cause of death in the United States. We had previously performed multipoint linkage analysis on families with a strong family history of lung cancer and found significant linkage to the 6q25 region. In order to find the source of that linkage signal, we performed targeted sequencing 6q23-6q27 on 75 individuals from the 9 most highly linked families. We performed two types of parametric linkage analysis, using an autosomal dominant mode of inheritance with 10% penetrance for carriers and a 1% phenocopy rate using a disease allele frequency of 1%. The first type was a two-point analysis using an Elston-Stewart algorithm. This approach did not lead to any genome-wide significant results but demonstrated significant heterogeneity throughout the nine families.In an effort to recover the power from the original multipoint analysis, we performed a regional linkage analysis using SEQLinkage and MERLIN. SEQLinkage built regional haplotypes that corresponded to a gene or an intergenic region - the regions were based on a customized map of our design. The regional markers were multiallelic which allowed for greater information content and were similar to the microsatellites that were used in the original multipoint analysis. The regional markers were then analyzed in a two-point linkage analysis via MERLIN. This allowed us to identify two genome-wide significant signals at PACRG-AS1 at 6q26 (HLOD = 3.4) and SAMD5 at 6q24 (3.3). The PACRG-AS1 is novel, though it is associated with the known lung cancer gene PARK2. SAMD5 may be involved in lung cancer cell proliferation. The heterogeneity of the two signals was particularly interesting. The PACRG-AS1 was driven primarily by two families. The SAMD5 marker was not being strongly driven by any of the families and appears to be a small cumulative effect across the nine families. In addition, several families have large, but non-significant LOD scores at other loci across the region. This further reinforces the locus heterogeneity within the region, and it is likely that our two significant signals here are not the only variants affecting the phenotype. We further attempted to localize the signals by running SEQLinkage using a custom map where genes are broken into exons and introns. Though this resulted in no significant markers, the highest signal was located in the intronic region of SAMD5 (HLOD = 3) and several other suggestive signals were localized to intronic regions of good candidate genes like SASH1 and ARID1B. Examination of predicted effects of the candidate regulatory variants using eQTL databases is ongoing. This is a region full of promising candidates, and it is likely that the two significant signals found here are just part of many that could be affecting lung cancer risk. We plan to do further analysis within the individual families to elucidate any more genes affecting this signal. Citation Format: Anthony M. Musolf, Claire L. Simpson, Bilal A. Moiz, Candace Middlebrooks, Mariza de Andrade, Diptasri Mandal, Colette Gaba, Ping Yang, Yafang Li, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Joan E. Bailey-Wilson. A study in locus heterogeneity: Targeted sequencing analysis of 6q reveals multiple significant loci as the source of a previous linkage peak in familial lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4290. doi:10.1158/1538-7445.AM2017-4290

Several papers have implicated mutations in the AT-rich interactive domain containing protein 1B (ARID1B) in carcinoma formation in multiple tissue types, including the lung. The ARID1B protein functions as part of SWI/SNF, a multi-subunit chromatin remodeling complex that repositions nucleosomes and may act as a tumor suppressor in cancer. ARID1B is located in a region in 6q that was previously shown to have strong evidence of linkage with lung cancer risk. As it is one of the more promising candidates within the target region, this study presents the viability of ARID1B as a candidate gene for familial lung cancer. Here, we chose individuals from families in our previous linkage study that showed the strongest linkage signal on 6q. 75 individuals from 9 families were sequenced for 37Mb of chromosome 6 from 130Mb to 167Mb using Illumina technology and a custom Agilent kit. Genotypes were imported into GoldenHelix SVS 7 for quality control and dropped if read depth < 10, quality score < 10 or a quality score to read depth ratio < 0.5. Variants with more than 1 Mendelian error were dropped. Variants with a single Mendelian inconsistency were dropped only for that family. Two-point parametric linkage analysis at theta = 0 was performed using R and an implementation of the Elston-Stewart algorithm in the paramlink package using a penetrance model of 0.01/0.1/0.1 (dd/Dd/DD) and a disease allele (D) frequency of 0.01. Family 102 showed strong evidence for linkage near ARID1B. The highest LOD score (1.34) out of all sequenced variants was located near this gene in a known conserved intergenic region between ARID1B and NOX3. Four other LOD scores > 0.7 were found in the intergenic region between these two genes. Two are transcription factor binding sites; two are conserved genomic elements. We also observed two linked intronic variants of ARID1B in this family; an enhancer (LOD = 0.89) and a transcribed region (LOD = 0.73). Two other families (47 and 59) displayed evidence of linkage in ARID1B. Here, the interest is not the value of the LOD scores (top scores were 0.64 and 0.54), but the centering of the linked haplotype around the gene. Family 47 has 60 variants with LODs between 0.64 and 0.61; 45 located within ARID1B or known regulatory regions. Family 59 has 61 variants with LODs between 0.54 and 0.51; 29 located within ARID1B or known regulatory regions The variants appear to be spread evenly throughout ARID1B, suggesting the presence of linked haplotypes. Analyses are currently underway to reconstruct possible haplotypes in these families. The results of this study show that variants in the ARID1B gene at least partially account for the linkage signal on 6q in three of the nine most strongly linked families in our cohort and thus we believe that ARID1B is a plausible candidate gene for further studies involving familial lung cancer. Replication studies using 27 new extended families are currently underway to confirm this initial result. Citation Format: Anthony M. Musolf, Claire L. Simpson, Mariza de Andrade, Diptasri Mandal, Colette Gaba, Ping Yang, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Joan E. Bailey-Wilson. Chromatin remodeling gene ARID1B is linked to familial lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2542.

Cigarette smoking is the major cause for lung cancer, but genetic factors also affect susceptibility. We studied families that included multiple relatives affected by lung cancer. Results from linkage analysis showed strong evidence that a region of chromosome 6q affects lung cancer risk. To characterize the effects that this region of chromosome 6q region has on lung cancer risk, we identified a haplotype that segregated with lung cancer. We then performed Cox regression analysis to estimate the differential effects that smoking behaviors have on lung cancer risk according to whether each individual carried a risk-associated haplotype or could not be classified and was assigned unknown haplotypic status. We divided smoking exposures into never smokers, light smokers (

Several studies have identified common genetic factors that influence susceptibility to lung cancer but no large studies have systematically studied familial lung cancers. The Genetic Epidemiology of Lung Cancer Consortium completed a genome-wide association study (GWAS) using samples from 757 cases and 796 controls, all of European American ancestry, and frequency matched on age and sex. Genotyping was performed by the Center for Inherited Disease Research (CIDR) using an Illumina Human OmniExpressExome-8v1 array. The most significantly associated SNPs were identified in the cases as a whole, and were also stratified by histology (adenocarcinoma and squamous cell carcinoma) and family history (0-1 and 2-5 affected family members). Analysis of all cases and controls identified a large number of SNPs around the CFTR gene that are strongly associated with lung cancer risk. Upon imputation with European Ancestry 1000 genomes data, the most significant SNP in this region is rs43034 (p = 3.89E-07, OR = 0.657) in the promoter region. In addition, an exonic missense SNP, rs213950 (p = 6.32E-07, OR = 0.663), and an intronic SNP with a possible splicing effect, rs213935 (p = 5.20E-07, OR = 0.660) were also identified as possible protective alleles. Analysis by histological subtype identified several rare and exonic SNPs that were specific by histology. We highlight two SNPs, rs114719990 (p = 1.194E-04, OR = 336.8, SETD2, c.3229A>G, p.Thr1077Ala) and rs199710487 (p = 1.124E-04, OR = 366, NTN5, c.656G>A, p.Arg219Gln), that were identified as strongly associated SNPs in cases with histologically confirmed adenocarcinoma, and two SNPs, rs144465058 (p = 1.029E-04, OR = 484.6, ANK3, c.6555G>T, p.Gln2185His) and rs140216112 (p = 1.087E-04, OR = 476, GRIK4, c.1013C>G, p.Ala338Gly), that were identified as top SNPs in cases with histologically confirmed squamous cell carcinoma. The SETD2 gene is thought to be a tumor suppressor gene and variants in this gene have previously been associated with leukemia and glioma (Zhu X et al. 2014, Fontebasso AM et al. 2013), and may now have implications in lung cancer, specifically adenocarcinoma development. In conclusion, we identified SNPs in the CFTR gene that may have a protective effect on lung cancer and several rare and exonic variants that were specific to histology subtype. SNPChrPositionGeneGenomic/ Nucleotide/ Amino acid changeOdds Ratiop-valueCFTR Protective Allelesrs430347117108449CFTRg.117108449T>C; 2.4kb upstream0.6573.89×10−7rs2139507117199533CFTRc.1408G>A; p.Val470Met0.6636.32×10−7rs2139357117178208CFTRc.869+1481A>G0.6605.20×10−7 Citation Format: Diana M. Toledo, Susan M. Pinney, Diptasri Mandal, Mariza de Andrade, Elena Kupert, Jennifer Franks, Colette Gaba, Claire L. Simpson, Ming You, Marshall W. Anderson, Joan E. Bailey-Wilson, Christopher I. Amos, Ann Schwartz. Genetic Epidemiology of Lung Cancer Consortium: genome-wide association study of familial lung cancer cases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-189. doi:10.1158/1538-7445.AM2015-LB-189

Recently, Wilson et al. (doi:10.1038/onc.2013.396) found that EYA4 was frequently inactivated biallelically in sporadic lung cancer, displays tumor suppressor gene-like properties and affects DNA repair, and that 5 of 17 common SNPs in this 0.3Mb region showed nominal association to familial lung cancer (FLC, p The highest total LOD score (summed over all 9 families) in the entire 37Mb targeted region was not near EYA4. In only one family was there any variant in EYA4 that appeared on the linked haplotype with close to the highest family-specific LOD score: Family 30 had a LOD of 0.6514 at several EYA4 variants (max LOD across region 0.6519). However, many variants across the region had similar LODs. In the remaining families, all EYA4 variants had LODs much lower than the maximum family-specific LOD score in the region. Thus, EYA4 variants are unlikely to explain our 6q linkage peak in these 6q-linked families. The second highest total LOD score for the entire 37Mb targeted region was found near EYA4 at 133.9Mb (LOD = 3.82), in the middle of the TARID antisense RNA gene. This variant appears to be entirely novel, as it is not present in any of the online variant databases, such as NHLBI's ESP or 1000 Genomes. In two families, the family LOD score at this locus was at or close to the highest in the region: in Family 44 the LOD score was 1.206 (max LOD across entire region 1.207) and in Family 35 the LOD score was 0.3958 (max LOD across region 0.3959). Therefore this variant in the TARID antisense RNA is a candidate in some of our families most strongly linked to chromosome 6q. Citation Format: Joan E. Bailey-Wilson, Claire L. Simpson, Susan M. Pinney, Mariza de Andrade, Colette Gaba, Ping Yang, Ming You, Elena Y. Kupert, Ann G. Schwartz, Diptasri Mandal, Christopher I. Amos, Marshall W. Anderson. Evaluation of EYA4 as a candidate risk locus in familial lung cancer families linked to 6q. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2757. doi:10.1158/1538-7445.AM2015-2757

Background: Recent genome-wide association studies (GWAS) have identified 28 susceptibility loci for Lung cancer (LC), 32 loci for smoking behavior, 10 loci for chronic obstructive pulmonary disease (COPD), 63 loci for pulmonary function and related phenotypes, totaling 107 GWAS susceptibility loci (as of November 2014). Given the common variants that have been found to be associated with LC in GWAS, targeted-region exome sequencing provides a cost-effective approach for further investigation of high-priority regions of the genome and has the great potential to identify rare causal variants in GWAS loci. Patients and Methods: Using an extreme phenotype approach, we selected 48 sporadic LC patients reporting heavy smoking histories with the presence of severe COPD in whom environmental factors are considered overwhelming, and 54 familial LC cases from families with at least three first-degree relatives with LC who are likely enriched for genomic signal, to search for the disease-causing rare mutations within the target 107 GWAS loci using exome sequencing data. Results: By focusing on exome profiles of these target 107 loci, we identified two disease-causing rare mutations on 6p21.32 TNXB p. Arg504His and 10q25.1 CCDC147 p.Arg696Cys. The homozygous Arg504His mutation presented in four familial and one sporadic LC cases. Notably, the minor allele frequency (MAF) of this variant in Caucasians from 1000 Genomes is very rare ( Conclusion: Our target exome sequencing results demonstrate novel highly disruptive LC risk-conferring TNXB and CCDC147 mutations. Since the rare variants have appreciable effects on disease risk prediction, families and patients may warrant screening for mutations in TNXB and CCDC147 (if validated) to assess their potential LC risk. Citation Format: Yanhong Liu, Farrah Kheradmand, Michael Scheurer, Caleb Davis, David Wheeler, Edwin Silverman, Shete Sanjay, Spiridon Tsavachidis, Georgina Armstrong, Elena Kupert, Marshall Anderson, Yafang Li, Claudio Pikielny, Joan E. Bailey-Wilson, Ming You, Colette Gaba, Mariza DeAndrade, Diptasri Mandal, Claire Simpson, Susan Pinney, Christopher Amos, Margaret Spitz. Target exome sequencing for disease-causing rare mutations in familial and sporadic lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4600. doi:10.1158/1538-7445.AM2015-4600

African-Americans (AAs) are disproportionately affected by Lung Cancer (LC), having a higher incidence and a lower 5-year survival rate than any ethnic group ((European-Americans (EA) have 2nd highest incidence)). While tobacco use accounts for 90% of lung cancer cases, excess risk of LC in AAs is unlikely due to differences in cigarette smoking, since AAs and EAs smoke at comparable rates (∼20%) and even among smokers, AAs smoke fewer cigarettes. While social and environmental factors contribute to ethnic disparities in LC, it is likely that ethnic differences in genetic susceptibility play a role. A major barrier to this field of research has been historically low enrollment rates of AA LC patients for clinical studies. The University of Maryland is uniquely poised to address these research questions with a large database of ∼1000 Non-Small Cell Lung Cancer (NSCLC) (90% of LC cases) that includes a biobank of tissue samples with extensive clinical information. Not only is this database enriched for AA patients (40%), but it includes both tumor and non-tumor tissue allowing for examination of heritable (germline) and acquired (somatic) mutation. To address ethnic disparities, we are assessing somatic variability in the exons of 26 oncogenic lung cancer genes with the Ion Ampliseq™ Comprehensive Cancer Panel (CCP) and characterizing mutational profile differences in tumor samples between AA and EA NSCLC patients. We hypothesize that AA NSCLC patients will carry an excess of known recurrent driver mutations, and a differing mutational profile of somatic lung cancer driver mutations as compared to EA patients. Of the 10 patients (6 AA, 4 EA) sequenced to date, we detected oncogenic driver mutations in 18 of the 26 LC genes examined. Of the 6 AAs, all had multiple driver mutations and as a group, mutations were detected in 17 of the 26 LC genes. For the 4 EA patients, 3 were found to have 1 driver mutation each and in separate LC genes (AKT1, EGFR, STK11). Although results should be considered cautiously due to sample size, we observe that AA NSCLC patients have more driver mutations overall and per individual as compared to EA NSCLC patients. Citation Format: Sarah H. Stephens, Patrice J. Fleming, Braxton D. Mitchell, Nicholas Ambulos, Joan E. Bailey-Wilson, Christy Chang, Josephine L. Feliciano, Edward A. Sausville, Martin J. Edelman. Somatic mutation profile differences of “driver” mutations in 26 oncogenic lung cancer genes between African American and European American non-small cell lung cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4599. doi:10.1158/1538-7445.AM2015-4599

Prostate cancer is the most common male cancer in developed countries. Previously, we reported a genome-wide linkage scan in 69 Finnish Hereditary Prostate Cancer (HPC) families, which replicated the HPC9 locus on 17q21-q22 and a locus on 2q37. We used ordered subset analysis (OSA) to detect other loci linked to HPC in subsets of families to detect other loci linked to HPC incorporating mean family age of onset as a trait-related covariate to address genetic heterogeneity. The overall mean age of onset across the families was 66.2±8.8 years while the range of individual onset ages ranged from 46-98 years and age of onset varies within families. The highest OSA LOD score was 2.876 (ΔLOD p=0.02) on 15q26.2-q26.3 in a subset of 40 families ascending by age at onset, suggesting that a subset of early age at onset families may be segregating a risk allele(s) at this locus. No other ΔLOD scores were significant after permutation testing. To better capture the effect of age on the linkage signals, we used LODPAL to perform a model-free linkage analysis in affected relative pairs, while adjusting for the age of each individual family member as a single covariate. Preliminary results revealed strong evidence of linkage to HPC on chromosome 15q (LOD=4.9, 132cM) and 8q (LOD=3.1, 157cM). Future analyses are planned which will use age of onset, PSA levels and other clinical characteristics to prioritize regions which may harbor genetic variants of large effect on PRCA. Citation Format: Cheryl D. Cropp, Claire L. Simpson, Tiina Wahlfors, Asha George, MaryPat S. Jones, Ursula Harper, Damaris Ponciano-Jackson, Teuvo Tammela, Johanna Schleutker, Joan E. Bailey-Wilson. Unraveling phenotype heterogeneity in prostate cancer susceptibility in Finland utilizing covariate-based analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3648. doi:10.1158/1538-7445.AM2013-3648

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #1040 Background: Although mutations in BRCA1 and BRCA2 represent the primary lesions in hereditary breast and ovarian cancer (HBOC) families, these mutations are marked by incomplete penetrance, as 15-20% of carriers never develop either cancer. In addition, the expressivity of these gene mutations varies with manifestation in breast, ovary, or both breast and ovary, suggesting other genes may modify the effects of BRCA1 and BRCA2 mutations. Methods: DNA was isolated from nine large HBOC families with known BRCA1 and BRCA2 mutations. SNP data was generated using the GeneChip Mapping 10K Array (Affymetrix, Santa Clara, CA). The data were checked for deviation from Hardy-Weinberg equilibrium and Mendelian errors. SNPs with low completion rates were not used in the analyses. Two-point parametric linkage analysis was performed under dominant and recessive models using VITESSE and GAS. Results: Genotypes were generated for 301 individuals from nine families and included mutation carriers, and non-carriers with and without cancer. When all families were considered together using a dominant model, LOD scores >3.0 were detected for SNPs on chromosomes 6q25.3 and 18q21.3 and >2.0 for chromosomes 2q24, 5p13 and 5q33. When families were segregated by mutation type, linkage was detected at chromosome 22q12 (LOD =2.65) in BRCA1 families, and at 2q24 (LOD=2.17) in BRCA2 families. Conclusions: Chromosomal regions harboring putative genetic modifiers have been identified that may contribute to pathogenesis in both BRCA1 and BRCA2 families. For example, frequent gain of 2q24 has been detected in BRCA1 carriers with ovarian cancer, loss of 5q in breast tumors from BRCA1 patients, and SNP [rs720321][1] is intragenic to the apoptosis gene BCL2 on chromosome 18q21, expression of which has been associated with BRCA2 tumors. Linkage to chromosome 22q12 was specific to BRCA1 families, of note, the loss of the CHEK2 gene, located on chromosome 22q12, may promote tumor progression in BRCA1 tumors. Further elucidation of the underlying genetic lesion from each of these chromosomal regions may improve the risk assessment and treatment regimens of women with BRCA1 and BRCA2 mutations. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1040. [1]: /lookup/external-ref?link_type=GEN&access_num=rs720321&atom=%2Fcanres%2F69%2F2_Supplement%2F1040.atom