Familial Esophageal Squamous Cell Carcinoma Research Articles

RAD50 Loss of Function Variants in the Zinc Hook Domain Associated with Higher Risk of Familial Esophageal Squamous Cell Carcinoma.

Simple SummaryTwo deleterious RAD50 loss-of-function germline mutations were identified from the blood DNA of a cohort of 3289 Henan individuals by next-generation sequencing. These rare loss-of-function RAD50 variants were associated with a substantial increased risk of familial esophageal squamous cell carcinoma in high-risk Northern China. A functional study suggested that the RAD50 mutations may affect DNA repair and cell survival upon replication stress. Our preliminary functional study provided novel insight and the potential clinical implication that patients with heterozygous RAD50L1264F and RAD50Q672X status may have a potential synthetic lethal therapeutic option with CHK1 inhibitors. Further study is warranted for validation of the implicated genetic susceptibility role of the RAD50 Zinc Hook mutants.Unbiased whole-exome sequencing approaches in familial esophageal squamous cell carcinoma (ESCC) initially prioritized RAD50 as a candidate cancer predisposition gene. The combined study with 3289 Henan individuals from Northern China identified two pathogenic RAD50 protein truncation variants, p.Q672X and a recurrent p.K722fs variant at the zinc hook domain significantly conferring increased familial ESCC risk. Effects of ~10-fold higher familial ESCC risk were observed, when compared to East Asians from the gnomAD database. Functional characterization suggested that the RAD50Q672X mutation contributes a dominant-negative effect in DNA repair of double-stranded breaks. Overexpression of the RAD50Q672X and RAD50L1264F missense mutation also sensitized cell death upon replication stress stimuli induced by formaldehyde treatment and the CHK1 inhibitor, AZD7762. Our study suggested the novel insight of the potential for synthetic lethal therapeutic options for RAD50Q672X and the East-Asian-specific RAD50L1264F variants and CHK1 inhibitors. Our study also suggested the association of RAD50 LOF variants in the zinc hook domain with a higher risk of familial ESCC in Chinese.

Genetic Heterogeneity of Esophageal Squamous Cell Carcinoma with Inherited Family History.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common malignant tumor with significant geographical variation and familial aggregation. However, the potentially different mechanisms underlying tumorigenesis in patients with ESCC with and without a family history of the disease remain unclear. In this study, the genes mutated in familial and nonfamilial ESCC were analyzed. Further, we aimed to explore the genes related to ESCC and attempt to identify potential patients in families with a history of ESCC.MethodsNext-generation sequencing technology was used to examine germline mutations and mutation profiles in 36 matched tumor-normal ESCC specimens. Additionally, tumor mutational burden (TMB) values were measured in two cohorts.ResultsWe identified four novel germline mutations in patients with familial ESCC, in BAX (c.121dupG: p.E41G), CDKN2A (c.374dupA: p.D125E), TP53 (c.856G>A: p.E286K), and CHEK1 (c.923+1G>A). Mutation profiles revealed that patients with and without a family history of ESCC had similar high-frequency gene mutation profiles, among which TP53 was the most commonly mutated gene. Additionally, tumor-specific mutated genes in patients with a positive family history of ESCC were APC, AKT3, DPYD, EP300, NFE2L2, PPP2R1A, RUNX1, and VEGFA, while those in patients without a family history of ESCC were CXCR4, PIK3R2, SMARCA4, and TTF1. Moreover, patients with positive family history had significantly higher TMB values (7.8 ± 4.1 vs 5.0 ± 2.4, for patients with and without a family history, respectively; P = 0.038).ConclusionOur results identified mutation profiles in patients with familial and nonfamilial ESCC, and identified germline mutations in patients with positive history. TMB values may be informative for immunotherapy approaches in familial ESCC.

Germline BRCA2 Truncating Mutation in Familial Esophageal Squamous Cell Carcinoma: A Case Controlled Study in China.

BackgroundGermline mutations of BRCA2 have been reported in various malignancies. We investigated BRCA2 germline mutations in familial clusters with esophageal squamous cell carcinoma (ESCC).Material/MethodsWe screened the DNA of familial ESCC patients for BRCA2 germline mutations with whole gene sequencing. Multiple BRCA2 mutations including one novel splice variant, c.426-2A>G were identified. Other family members, sporadic ESCC patients, and controls were also assessed for the novel mutation.ResultsThe mutation c.426-2A>G was found in 2 affected ESCC sisters and 7 other family members. The splice variant mutation results in exon 5 skipping with a frame shift leading to a premature stop codon in exon 6 and truncation. Novel mutation tracking ruled out single nucleotide polymorphism (SNP) in 100 chromosomes of healthy individuals.ConclusionsBRCA2 germline mutation in ESCC patients may play a role in genetic susceptibility to familial ESCC. Genetic analysis of BRCA2 in patients with familial ESCC could provide opportunities for targeted therapies.

Differential Cumulative Risk of Genetic Polymorphisms in Familial and Nonfamilial Esophageal Squamous Cell Carcinoma.

To explore the relationship between family history of esophageal cancer, SNPs, and the risk of esophageal squamous cell carcinoma (ESCC), we performed a population-based case-control study and developed a genetic family history-related risk (GFR) score and non-family history-related risk (GnFR) score to quantify the cumulative number of risk genotypes carried by each individual. We used data of 700 patients with nonfamilial ESCC, 341 patients with familial ESCC, 1,445 controls without a family history of esophageal cancer, and 319 controls with a family history. We genotyped 87 genetic variants associated with the risk for ESCC, and constructed GFR and GnFR scores for cases and controls. Our results show that ESCC risk increased with higher GFR score (P trend = 0.0096). Among the familial subgroup, we observed a nearly 7-fold [95% confidence interval (CI), 1.92-24.77] higher risk of ESCC in the highest GFR score group. The corresponding estimate was only 2-fold (95% CI, 1.41-3.93) higher risk of ESCC, in the stratum without a reported family history of esophageal cancer. Certain cell signaling pathways and immune-related pathways were enriched, specifically in familial ESCC. Results from a reconstructed cohort analysis demonstrated that cumulative risk to get esophageal cancer by age 75 years was 13.3%, 10.2%, 8.2%, and 5.1%, respectively, in four subgroups as defined by first-degree relatives of cases or controls with high or low genetic risk score. In particular, the cohort of relatives of ESCC cases with low genetic risk score exhibit a higher cumulative risk than the cohort of relatives of controls with high genetic risk score. It demonstrates that environmental factors play a major role in esophageal cancer. Further studies are warranted to dissect the mechanisms of shared environmental and genetic susceptibility affecting the risk of getting ESCC. Our study highlights that the need of preventive strategies to screen certain genetic polymorphisms, especially in individuals whose relatives had ESCC.

BRCA2 loss-of-function germline mutations are associated with esophageal squamous cell carcinoma risk in Chinese.

Esophageal squamous cell carcinoma (ESCC) occurs with highest frequency in China with over 90% mortality, highlighting the need for early detection and improved treatment strategies. We aimed to identify ESCC cancer predisposition gene(s). Our study included 4,517 individuals. The discovery phase using whole-exome sequencing (WES) included 186 familial ESCC patients from high-risk China. Targeted gene sequencing validation of 598 genes included 3,289 Henan and 1,228 moderate-risk Hong Kong Chinese. A WES approach identified BRCA2 loss-of-function (LOF) mutations in 3.23% (6/186) familial ESCC patients compared to 0.21% (9/4300) in the ExAC East Asians (odds ratio [OR] = 15.89, p = 2.48 × 10-10 ). BRCA2 LOF mutation frequency in the combined Henan cohort has significantly higher prevalence (OR = 10.55, p = 0.0035). Results were independently validated in an ESCC Hong Kong cohort (OR = 10.64, p = 0.022). One Hong Kong pedigree was identified to carry a BRCA2 LOF mutation. BRCA2 inactivation in ESCC was via germline LOF mutations and wild-type somatic allelic loss via loss of heterozygosity. Gene-based association analysis, including LOF mutations and rare deleterious missense variants defined with combined annotation dependent depletion score ≥30, confirmed the genetic predisposition role of BRCA2 (OR = 9.50, p = 3.44 × 10-5 ), and provided new evidence for potential association of ESCC risk with DNA repair genes (POLQ and MSH2), inflammation (TTC39B) and angiogenesis (KDR). Our findings are the first to provide compelling evidence of the role of BRCA2 in ESCC genetic susceptibility in Chinese, suggesting defective homologous recombination is an underlying cause in ESCC pathogenesis, which is amenable to therapeutic options based on synthetic lethality approaches such as targeting BRCA2 with PARP1 inhibitors in ESCC.

Whole-exome sequencing of familial esophageal squamous cell carcinoma identified rare pathogenic variants in new predisposition genes.

Esophageal squamous cell carcinoma (ESCC) is one of the most important causes of mortality in the developing world. Although hereditary forms arise from germ-line mutations in TP53, Rb, and the mismatch repair genes, many familial cases present with an unknown inherited cause. The new theory of rare, high-penetrance mutations in less known genes is a likely explanation for the underlying predisposition in some of these familial cases. Exome sequencing was performed in 9 patients with esophageal squamous cancer from 9 families with strong disease aggregation without mutations in known hereditary esophageal cancer genes. Data analysis was limited to only really rare variants (0-0.01%), producing a putative loss of function and located in genes with a role compatible with carcinogenesis. Twenty-two final candidate variants were selected and validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in CDK11A, ARID1A, JMJD6, MAML3, CDKN2AIP, and PHLDA1. Together, we identified new potential esophageal squamous cancer predisposition variants in genes which may have a role in cancer and are involved in chromatin remodeling and cell-cycle pathway, which could increase the risk of ESCC.

Abstract 3465: FANCD2 depletion suppresses tumor growth in esophageal squamous cell carcinoma

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) has a remarkably high incidence in Northern China. Our next-generation sequencing analysis on familial ESCC samples suggests that members of the Fanconi Anemia (FA) pathway play important roles in ESCC development. Fanconi anemia complementation group D2 (FANCD2) as a key player of FA pathway has multiple cellular functions. It is well-known for its role in DNA damage repair through the FA pathway. Patients with germline FANCD2 mutations are prone to tumor formation. Identifying roles of FANCD2 in ESCC development may enhance our foundation for future development of specific treatment regimens for this deadly disease. Methods and Results: To study the function of FANCD2 in ESCC, we applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to inhibit FANCD2 protein expression by functional knock-out (fKO). As expected, we observed increased DNA damage in FANCD2 fKO cells as shown by Comet assay, consistent with the classic role of FANCD2 in genome integrity. We also observed that the DNA damage by irradiation takes longer to be repaired in FANCD2 fKO cells, indicating that FANCD2 enhances irradiation-induced DNA damage repair but is not necessary. Similarly, cells with FANCD2 fKO are more sensitive to irradiation, as indicated by cell cycle arrest at S/G2 phase. Surprisingly, without inducing any DNA damage, FANCD2 fKO cells also show cell cycle arrest at S/G2 phase. The MTT cell proliferation assay also show that FANCD2 fKO reduces proliferation and colony-forming ability. Consistent with the in vitro data, FANCD2 fKO cells also form smaller tumors in vivo by the nude mouse tumorigenicity assay. Conclusion: These results indicate that FANCD2 plays important roles in ESCC development and suggest a pro-oncogenic feature of wildtype FANCD2 in ESCC. Further investigation is ongoing to examine the functional role and molecular mechanisms of the oncogenic FANCD2, ultimately aiming at improving ESCC disease management. Acknowledgement: We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative Research Fund (C7031.15G) and the Asian Fund for Cancer Research to M.L.L. Citation Format: Chan Lei, Zhuoyou Yu, Lvwen Ning, Mun-Yee Ko, Lidong Wang, Maria Li Lung. FANCD2 depletion suppresses tumor growth in esophageal squamous cell carcinoma [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 3465.

Abstract 327: Functional characterization of RAD50 germline deleterious variants identified from deep targeted sequencing study of familial esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC), a histological subtype of esophageal cancer, is a highly prevalent malignancy in Southeast Asia with poor prognosis and high mortality rate. From previous Next-Generation Sequencing (NGS) studies that sampled familial ESCC cases from Henan, a hotspot of ESCC cases in China, rare germline variants of RAD50 homolog, double strand break repair protein (RAD50) were identified and predicted to be deleterious by in silico models. We hypothesized that ESCC patients carrying dominant-negative germline or somatic RAD50 alterations with disrupted MRN (MRE11-RAD50-NBS1) functions may show improved prognosis, as tumor cells are sensitized to genotoxic agents such as cisplatin and ionizing radiation(IR). The current study aims to characterize if these identified germline loss-of-function (LOF) mutants of RAD50 exert dominant-negative impacts on double-strand break (DSB) repair and ATM/ATR signalling. A stop-gain mutation at the zinc-hook domain (RAD50-SG1), and a missense mutation at the C-terminal ATP-binding cassette (RAD50-MS1) were chosen for functional characterization. These mutations were introduced into RAD50 ORF by site-directed mutagenesis. The wildtype (RAD50-WT), RAD50-SG1 and RAD50-MS1 were expressed alongside a vector-alone control (VA) in two cell lines, U2OS and KYSE70(ESCC), using a lentiviral system. DSB repair efficiency was assessed by γH2AX foci recovery. Expression of RAD50-WT reduced the number of γH2AX foci-(+) cells compared to VA at 6 hours post-IR, hinting of an improved DSB repair efficiency while RAD50-MS1 showed no reduction in γH2AX foci-(+) cells. Intriguingly, the number of γH2AX foci-(+) cells was significantly elevated in RAD50-SG1, indicating a reduced DSB repair efficiency in a dominant-negative manner as U2OS expresses wildtype RAD50 endogenously. RAD50-SG1 expressed U2OS was sensitized to Chk-inhibitor AZD7762, when compared to RAD50-WT, RAD50-MS1 and VA control, as measured by MTT assay. Details about the downstream ATM/ATR signalling for reduced phosphorylation of ATM/ATR substrates for impaired checkpoint signalling, data of dominant-negative functional impacts of the RAD50-SG1 mutant regarding cell proliferation in the presence of DSB stimuli (cisplatin treatment/γ irradiation), DSB repair efficiency and sensitivity towards PARP inhibitor will be presented. We expect our study to provide insight into the potential therapeutic impacts of RAD50 LOF mutants with PARP inhibition or Chk inhibition. It should provide a strategy for design of new therapeutic regimens for combined targeted disruption of MRN function with PARPi or Chki, which is expected to benefit ESCC patients, who show poor responses to conventional chemotherapy and radiotheapy. Citation Format: Shiu Yeung Lam, Lvwen Ning, Hoi Yan Ng, Maria Li Lung, Josephine Mun-Yee Ko. Functional characterization of RAD50 germline deleterious variants identified from deep targeted sequencing study of familial esophageal squamous cell carcinoma [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 327.

Abstract 1357: Functional characterization of FANCD2 in esophageal squamous cell carcinoma

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in Northern China, where there is evidence for a significant familial association. We performed targeted next-generation sequencing (NGS) analysis on familial ESCC germline samples compared to non-cancer controls from the same high-risk region and compiled a list of candidate cancer predisposition genes. Interestingly, genes related to the Fanconi Anemia (FA) - BRCA pathway are enriched in the list. Among these FA-BRCA genes, Fanconi anemia complementation group D2 (FANCD2) was one of the top candidates, as it also had a high frequency of somatic mutations in ESCC tumor specimens. Therefore, we aim to characterize the role of FANCD2 in tumor development and explore its translational value. Methods: We knocked out the FANCD2 gene in ESCC cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to evaluate its potential oncogenic function in ESCC. Cell proliferation was measured by a MTT 2D clonogenic assay in vitro. Subcutaneous injection of the FANCD2 knockout ESCC cells into BALB/c-nude mice in vivo was performed to assess its functional impact on tumorigenesis. The single cell gel electrophoresis/comet assay was used to investigate the genome stability. Results: The FANCD2 knockout efficiency was confirmed by western blotting. Surprisingly, in vitro functional analyses showed that ESCC cells with FANCD2 knockout survive, with a greatly reduced growth rate and colony-forming ability. Consistent with the in vitro data, ESCC cells with FANCD2 knockout form significantly smaller subcutaneous tumors in nude mice. By applying the comet assay to examine the genome integrity, ESCC cells with FANCD2 knockout show significantly greater damage to the genome. Conclusion: These results suggest that FANCD2 plays an important role in supporting ESCC tumor growth. We attribute this to its core function in DNA repair ability and genome integrity maintenance. Acknowledgement: We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative Research Fund (C7031.15G to M.L.L.). Citation Format: Lisa Chan Lei, Valen Zhuoyou Yu, Lvwen Ning, Josephine Mun-Yee Ko, Li Dong Wang, Maria Li Lung. Functional characterization of FANCD2 in esophageal squamous cell carcinoma [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 1357.

Epigenetic alterations of a novel antioxidant gene SLC22A3 predispose susceptible individuals to increased risk of esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) occurs with the highest frequency in China, especially in the high-risk Northern Chinese. Recent studies have reported that SLC22A3 is significantly downregulated in non-tumor (NT) esophageal tissues from familial ESCC patients compared with those from sporadic ESCC. However, the mechanism of how SLC22A3 regulates familial ESCC remains unknown. In this study, post hoc genome-wide association studies (GWAS) in 496 cases with a family history of upper gastrointestinal tract cancers and 1056 controls were performed and the results revealed that SLC22A3 is a novel susceptibility gene for familial ESCC. Reduced expression of SLC22A3 in NT esophageal tissues from familial ESCC patients significantly correlates with its promoter hypermethylation. Moreover, case-control study of Chinese descendants from different risk areas of China revealed that the methylation of the SLC22A3 gene in peripheral blood leukocyte (PBL) DNA samples could be a risk factor for developing ESCC in this high-risk population. Functional studies showed that SLC22A3 is a novel antioxidant gene, and deregulation of SLC22A3 facilitates heat stress-induced oxidative DNA damage and formation of γ-H2AX foci in normal esophageal epithelial cells. Collectively, we show that epigenetic alterations of SLC22A3 predispose susceptible individuals to increased risk of esophageal cancer.

Familial Esophageal Squamous Cell Carcinoma with damaging rare/germline mutations in KCNJ12/KCNJ18 and GPRIN2 genes

In Iran, esophageal cancer is the fourth common cancers in women and sixth common cancers in men. Here we evaluated the importance of familial risk factors and the role of genetic predisposition in Esophageal Squamous Cell Carcinoma (ESCC) using Whole-Exome Sequencing (WES). Germline damaging mutations were identified in WES data from 9 probands of 9 unrelated ESCC pedigrees. Mutations were confirmed with Sanger sequencing and evaluated amplification-refractory mutation system-Polymerase Chain Reaction (ARMS-PCR) in 50 non-related ethnically matched samples and in complete genomics database. Sixteen candidate variants were detected in ESCC 9 probands. Four of these 16 variants were rare damaging mutations including novel mutations in KCNJ12/KCNJ18, and GPRIN2 genes. This WES study in Iranian patients with ESCC, provides insight into the identification of novel germline mutations in familial ESCC. Our data suggest an association between specific mutations and increased risk of ESCC.

RNA editing of SLC22A3 drives early tumor invasion and metastasis in familial esophageal cancer

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.

Novel candidate genes may be possible predisposing factors revealed by whole exome sequencing in familial esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma is one of the deadliest of all the cancers. Its metastatic properties portend poor prognosis and high rate of recurrence. A more advanced method to identify new molecular biomarkers predicting disease prognosis can be whole exome sequencing. Here, we report the most effective genetic variants of the Notch signaling pathway in esophageal squamous cell carcinoma susceptibility by whole exome sequencing. We analyzed nine probands in unrelated familial esophageal squamous cell carcinoma pedigrees to identify candidate genes. Genomic DNA was extracted and whole exome sequencing performed to generate information about genetic variants in the coding regions. Bioinformatics software applications were utilized to exploit statistical algorithms to demonstrate protein structure and variants conservation. Polymorphic regions were excluded by false-positive investigations. Gene-gene interactions were analyzed for Notch signaling pathway candidates. We identified novel and damaging variants of the Notch signaling pathway through extensive pathway-oriented filtering and functional predictions, which led to the study of 27 candidate novel mutations in all nine patients. Detection of the trinucleotide repeat containing 6B gene mutation (a slice site alteration) in five of the nine probands, but not in any of the healthy samples, suggested that it may be a susceptibility factor for familial esophageal squamous cell carcinoma. Noticeably, 8 of 27 novel candidate gene mutations (e.g. epidermal growth factor, signal transducer and activator of transcription 3, MET) act in a cascade leading to cell survival and proliferation. Our results suggest that the trinucleotide repeat containing 6B mutation may be a candidate predisposing gene in esophageal squamous cell carcinoma. In addition, some of the Notch signaling pathway genetic mutations may act as key contributors to esophageal squamous cell carcinoma.

Abstract 5234: Rare deleterious germline mutations of candidate genes associated with risk of familial esophageal squamous cell carcinoma (ESCC) by targeted sequencing

Abstract Background and aims: Esophageal cancer (EC) is a deadly disease with poor survival rates of only ∼10%. The EC incidence remains highest in the Shanxi and Henan regions near the Tang Hang Mountain area in Northern China. In endemic esophageal squamous cell carcinoma (ESCC) regions, common familial clustering and historical migration patterns suggest an inherited ESCC genetic susceptibility. We hypothesize that cancer predisposition gene(s) (CPGs) carrying rare deleterious variants play a crucial role in ESCC genetic susceptibility and aim to identify the underlying CPGs of familial ESCC from Henan by applying the targeted sequencing approach. Methods: Our discovery set utilized NGS technology to capture a comprehensive gene panel critical for ESCC by targeted re-sequencing of 1271 selected genes (11 Mb) and sequenced for the germline variants in the blood DNA from 222 familial ESCC individuals and 124 Henan non-cancer controls. Familial ESCC was defined as at least two family members developed ESCC. The custom designed panel contains a cancer panel of 578 gene list (4Mb) and a custom list of 693 (7Mb) ESCC-specific genes derived from various approaches including microarray profiling in FH+ ESCC primary tissues, homozygosity-by-descent (HBD), SNP array-based analysis by both GWAS and candidate gene approaches of our own set of MassArray SNP association data. The data from 346 individuals were processed using the analysis pipeline following the GATK guideline. Briefly, raw fastq reads were mapped using the Burrows-Wheeler Aligner (BWA). PCR duplicates are marked using Picard. The raw variants are called by GATK HaplotypeCaller using a joint genotyping of all samples. Results: Preliminary data analysis for the 346 samples resulted in the final call set comprised of 52328 rare variants. There are 26486 rare variants present in the 222 FH+ ESCC cases, but absent in the 124 controls. The average sequencing coverage was 88X. Principal component analysis (PCA) and multidimensional scaling (MDS) indicated a homogenous population structure between the case and the control populations. Targeted re-sequencing results of the 1271 selected genes in 222 familial ESCC individuals identified a recurrent missense variant in AIM1L (Absent in Melanoma 1-like) in 14/222 (6.3%) of FH+ ESCC patients but absence in the 124 non-cancer control. Data obtained by a melting curve-based genotyping platform by LC480 and LightSNiP assay for high throughput validation of this recurrent SNP in larger sample cohort are underway and will be presented. Conclusions: A SNP in AIM1L may be associated with higher risk for ESCC in Henan and validation in larger cohort is required. The data may provide valuable insight for understanding genetic susceptibility to this deadly cancer. Acknowledgements: We acknowledge the Asian Fund for Cancer Research for grant support. Citation Format: Josephine Mun Yee Ko, Lu wen Ni, Sheyne, Sta Ana Choi, Lisa Chan Lei, Li Dong Wang, Maria Li Lung. Rare deleterious germline mutations of candidate genes associated with risk of familial esophageal squamous cell carcinoma (ESCC) by targeted sequencing. [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 5234.

Abstract 1280: Germline PALB2 genetic variations of familial esophageal squamous cell carcinoma (ESCC) in Henan, a high risk ESCC region

Abstract Background and aims: ESCC is often a deadly cancer diagnosed at late stage with a 5-year survival rate less than 10% in advanced cancers. It is important to elucidate the molecular genetic basis for this deadly cancer to achieve the ultimate goal of early cancer detection and improved clinical management. Our aim is to understand the genetic basis of inherited ESCC by deciphering PALB2 mutation status in high risk northern China. By investigation of PALB2 germline mutations and variants, as compared to healthy individuals, the genetic basis and genomic risk factor associations with inherited EC will be clarified. Methods: Blood was collected from high-risk Henan familial history-positive (FH+) individuals. Blood DNAs were extracted for Sanger sequencing analysis. Results: By Sanger sequencing with primers covering more than 90% of the PALB2 coding sequence, PALB2 variants were detected in exon 4 for 4% (2/50) FH+ ESCC patients from Henan, but no germline protein truncation variations were observed. Since mutations were only observed in exon 4, further mutation screening were confined to exon 4 of PALB2 with an additional 300 FH+ Henan ESCC, 283 FH- Henan ESCC, and 364 Henan non-ESCC control individuals. However, no mutations were detected in the validation samples. Conclusions: Infrequent PALB2 germline mutations were detected in Henan FH+ ESCC patients, suggesting PALB2 may be involved in a small proportion of familial ESCC in Henan. Further study is necessary to clarify if the identified mutations are pathogenic. Citation Format: Josephine Mun Yee Ko, Li Dong Wang, Maria Lung. Germline PALB2 genetic variations of familial esophageal squamous cell carcinoma (ESCC) in Henan, a high risk ESCC region. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1280. doi:10.1158/1538-7445.AM2014-1280

Interrogation of Chromosome 13q12-14 in Esophageal Squamous Cell Carcinoma

Previous studies of esophageal squamous cell carcinoma (ESCC) suggested chromosome region 13q12-14 harbors a familial ESCC gene. DNA sequencing of the BRCA2 gene, located on 13q12, showed evidence of both germline and tumor specific alterations but the frequency of changes was low and did not fit the classic Knudsen two-hit gene inactivation model. To further investigate chromosome 13q12-14 in ESCC, quantitative expression measurements were performed on BRCA2 and 11 neighboring genes in matched normal epithelium and tumor from 17 cases. Transcript analysis showed normal levels of five genes, tumor down-regulation of two genes (TNFRS19 and TPT1), and tumor up- regulation of five genes, including BRCA2. No evidence of BRCA2 loss-of-function was detected based on reduced mRNA in tumor cells. Between 13q12.3 (KATNAL1) and 13q12.3-q13 (CCNA1) five adjacent genes showed increased mRNA expression raising the possibility of a DNA amplicon; however, qPCR analysis showed normal DNA amounts in this region. CCNA1 transcript was significantly up-regulated in tumors and was thus further interrogated at the protein level by immunohistochemistry. CCNA1 staining was restricted to normal basal epithelium and was not expressed in more superficial, differentiated regions. In contrast, the CCNA1 protein was ubiquitously and highly expressed throughout tumor foci. Overall, these data from a relatively small number of cases (17) suggest that TNFRS19 and TPT1 deserve further investigation as candidate tumor suppressor genes in esophageal cancer in a larger patient series; BRCA2 mRNA is increased in the tumors, likely as a compensatory response to the marked DNA damage that is present in these lesions; and, CCNA1 was identified as a novel up-regulated gene in ESCC.

Novel germ line BRCA2 mutations in familial esophageal squamous cell carcinoma

Novel germ line BRCA2 mutations in familial esophageal squamous cell carcinoma

Early age of onset, multiple primary malignancies and poor prognosis are indicative of an inherited predisposition to esophageal squamous cell carcinoma in familial rather than sporadic disease — An update based on 14- to 23-year follow-up

OBJECTIVE To demonstrate the effects of an inherited predisposition to familial esophageal squamous cell carcinoma (ESCC) through the comparison and analysis of the clinicopathologic di ff erences between familial and sporadic ESCC cases. METHODS Differences in age of onset, prevalence rates of double primary ESCC, and survival rates between familial ESCC ( n = 476) and sporadic ESCC cases ( n = 1226) were analyzed. RESULTS Overall, familial ESCC cases showed a signifi cantly younger age of onset (51.9 ± 8.2 vs. 53.4 ± 8.0, Pt-test = 0.00), a significantly higher prevalence rate for double ESCC (2.73 % vs. 1.22%, adjusted with TNM: χMH2 = 4.029, P = 0.045), and a lower survival rate than in sporadic cases (Pwald = 0.04). The familial cases showed both a younger age of onset and poorer survival in most subgroups, and the differences were more marked in early-stage rather than in the late-stage disease groups. CONCLUSION Theses fi ndings confirm the existence of familial as opposed to sporadic ESCC. By the theory of the “two-hit” origin of cancer, these fi ndings also suggest that the “ first hit”, a genetic predisposition, can affect the age of onset, number of primary carcinomas, and the prognosis for familial ESCC patients.

Early onset, multiple primary malignancies, and poor prognosis are indicative of an inherited predisposition to esophageal squamous cell carcinoma for the familial as opposed to the sporadic cases – An update on over 14-year survival

Background To demonstrate the effect of an inherited predisposition in familial Esophageal Squamous Cell Carcinoma (ESCC) as opposed to the sporadic cancer form. Methods Differences in age of onset, prevalence rates of double primary ESCC, and post-operative survival rates between ESCC cases with ( N = 476) and without ( N = 1226)a family history of upper gastrointestinal cancer (FHUGIC, defined as having one or more first- or second-degree relatives with cancer of the esophagus or gastric cardia) were analyzed. Results Overall, familial ESCC cases show a significantly earlier onset age (51.9 ± 8.2 versus 53.4 ± 8.0, P t -test = .000), a significantly higher prevalence rate of double primary ESCC (2.73% Versus 1.22%, adjusted with TNM: X MH 2 = 4.029, P = .045), and a worse prognosis than the sporadic cases ( P wald = .049). In subgroup analyses, the familial cases showed earlier onset and poor survival at most subgroups as opposed to the sporadic cases, and the difference was greater in early-stage rather than in late-stage groups (P t-test for difference in onset age in T is,1N 0M 0, T 2,3N 0M 0, and T 2,3,4N 1M 0 were .002, .006, and .081 respectively; and P wald for difference in survival in T is,1N 0M 0, T 2,3N 0M 0, and in T 2,3,4N 1M 0 were .010, .180, and .520 respectively). Conclusion These findings suggest the existence of familial as opposed to the sporadic ESCC. By the theory of “two-hit” origin of cancer, these findings also suggest that the “first hit”, a genetic predisposition, is inherited in familial ESCC.

Contribution of germ lineBRCA2sequence alterations to risk of familial esophageal cancer in a high-risk area of India

The incidence of esophageal squamous cell carcinoma (ESCC) is very high in the northeast region of India. An earlier study from China and Iran suggested that mutations in BRCA2 gene may play a role in the etiology of familial ESCC. However, the frequency of BRCA2 gene germ line mutations and its contribution to risk of familial aggregation of ESCC in high-risk region of India are not known. In the current study of 317 cases of esophageal cancer, 92 (29%) cases had a family history of esophageal and/or other cancers. Of these 92 patients, 45 (49%) patients had a family history of esophageal cancer. The risk of developing esophageal cancer was higher in cases where family history showed occurrence of cancers in first-degree relatives (odds ratio [OR]: 3.1; confidence interval [CI]: 1.9-5.3) than in second-degree relatives (OR: 1.3; CI: 0.25-3.2). Moreover, the risk of developing esophageal cancer was higher in subjects whose predegree suffered from esophageal cancer (OR: 2.4; CI: 1.1-4.1) than from any other cancers (OR: 1.1; CI: 0.32-3.3). The subjects with family history of cancer were more likely to develop ESCC if they were tobacco chewers (OR: 4.2; CI: 2.1-5.8) and betel quid users (OR: 3.6; CI: 1.8-4.6). Screening for mutations of the BRCA2 gene in the germ line DNA was carried out for 20 familial and 80 nonfamilial ESCC patients. One hundred unrelated healthy controls from the same population were included in this study. Nonsynonymous variants in exon 18 (K2729N) and exon 27 (I3412V) of BRCA2 gene were found in 3 of 20 patients with familial ESCC. No sequence alterations were found in 80 nonfamilial ESCC cases (P=0.01) and 100 healthy controls (P=0.0037), suggesting that germ line BRCA2 gene mutation may play a role in familial aggregation of ESCC in high-risk region of India.