Germline Single Nucleotide Polymorphisms Research Articles

Mechanisms Underlying Augmented Lymphomagenesis by LUBAC; Possible Accumulation of AID-Mediated Somatic Mutations

Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is characterized by constitutive activation of NF-κB signaling, where protein ubiquitination is involved in various steps. The linear ubiquitin chains specifically generated by the linear ubiquitin assembly complex (LUBAC) has recently drawn attention in NF-κB signaling. LUBAC is known to be involved in NF-κB activation and cell death protection by conjugating linear chains onto several substrates including NEMO. We have reported increased frequency of rare germline single-nucleotide polymorphisms (SNPs) of HOIP, the catalytic subunit of LUBAC in ABC-DLBCL patients (Yang et al., Cancer Discov, 2014). Although the SNPs enhanced ligase activity of LUBAC, precise mechanisms underlying augmented lymphomagenesis by LUBAC remains unknown.In order to consolidate the roles of LUBAC in the pathogenesis of ABC-DLBCL, a mouse strain, in which wild-type murine HOIP is constitutively expressed in B cells from as early as the pre-B cell stage (CD19-cre-HOIP) is generated. In CD19-cre-HOIP, the increase of HOIP elevated the amount of trimeric LUBAC composed of HOIP, HOIL-1L and SHARPIN, augmenting NF-κB activation mildly in B cells. TLR-induced proliferation is more prominent in CD19-cre-HOIP B cells than littermate control, indicating that MYD88-dependent signaling is facilitated by LUBAC. Although some of the aged CD19-cre-HOIP mice showed splenomegaly, no CD19-cre-HOIP mice developed lymphomas as far as examined. These results indicated that enhanced expression of HOIP leads to augmentation of TLR-mediated signaling and proliferation in B cells, but it cannot induce lymphomagenesis by itself.Importantly, majority of ABC-DLBCL found in patients with the HOIP SNPs had oncogenic MYD88 L265P mutation. To assess synergistic effects of LUBAC and MYD88 L265P, we generated two mice strains, expressing MYD88 L252P, an equivalent mutation of human L265P in mice alone (CD19-cre-MYD88LP) or MYD88 L252P and HOIP (CD19-cre-HOIP/MYD88LP) in a B-cell specific manner.In CD19-cre-MYD88LP B cells, enhanced phosphorylation of IRAK4, degradation of IκBα, and expression of NF-κB target genes were observed as a result of constitutive activation of MYD88 signaling. Constitutive MYD88 signaling in B cells also led to the expansion of B cell population and splenomegaly since early age. As reported previously (Knittel et al., Blood, 2016), B-cell specific expression of MYD88 L252P resulted in a reduced survival and introduction of a HOIP transgenic allele significantly shortened survival of mice expressing MYD88 L252P. Since clonal B-cell lymphomas displaying characteristics of DLBCL could be found in both CD19-cre-MYD88LP and CD19-cre-HOIP/MYD88LP mice, the augmented LUBAC activity promotes active MYD88-mediated lymphomagenesis of B cells.To clarify the mechanisms underlying LUBAC-mediated acceleration of B lymphomagenesis, whole-exome-sequencing analyses of lymphomas developed in these mice were performed. Many of the genes mutated in these lymphomas recapitulated those reported in human DLBCL, and alignment analyses suggested that majority of these mutations were identified as targets of aberrant somatic hypermutations induced by AID, which were especially prominent in the tumors developed in CD19-cre-HOIP/MYD88LP mice. These findings strongly implied that LUBAC enhances AID-mediated somatic mutations.Induced expression of AID was unlikely the major reason for increased mutation loads by LUBAC, since AID expression was elevated in B cells of both CD19-cre-HOIP/MYD88LP, and CD19-cre-MYD88LP mice almost equally. LUBAC has been reported involved in the protection of DNA damage-induced apoptosis. AID is known to induce double strand DNA break, and we hypothesized that LUBAC may protect cells from DNA damage-induced cell death caused by AID. To probe the effect of enhanced expression of LUBAC on protection from genotoxin-mediated apoptosis, we treated Jurkat cells with cisplatin and found that enforced expression of HOIP protected cells from cisplatin-induced cell death, implying that cells with augmented LUBAC activity have higher tolerance to genotoxic stress.In summary, the present results indicated that protection of B-cells from cell death underlies accelerating lymphomagenesis caused by augmented LUBAC activity via partial overcoming AID-mediated DNA damage, which leads to accumulation of somatic mutations. DisclosuresNishikori:Ono Pharmaceuticals: Research Funding; Eisai: Research Funding. Takaori-Kondo:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria; Pfizer: Honoraria.

Cancer-specific expression quantitative loci are affected by expression dysregulation.

Expression quantitative trait loci (eQTLs) have been touted as the missing piece that can bridge the gap between genetic variants and phenotypes. Over the past decade, we have witnessed a sharp rise of effort in the identification and application of eQTLs. The successful application of eQTLs relies heavily on their reproducibility. The current eQTL databases such as Genotype-Tissue Expression (GTEx) were populated primarily with eQTLs deriving from germline single nucleotide polymorphisms and normal tissue gene expression. The novel scenarios that employ eQTL models for prediction purposes often involve disease phenotypes characterized by altered gene expressions. To evaluate eQTL reproducibility across diverse data sources and the effect of disease-specific gene expression alteration on eQTL identification, we conducted an eQTL study using 5178 samples from The Cancer Genome Atlas (TCGA). We found that the reproducibility of eQTLs between normal and tumor tissues was low in terms of the number of shared eQTLs. However, among the shared eQTLs, the effect directions were generally concordant. This suggests that the source of the gene expression (normal or tumor tissue) has a strong effect on the detectable eQTLs and the effect direction of the eQTLs. Additional analyses demonstrated good directional concordance of eQTLs between GTEx and TCGA. Furthermore, we found that multi-tissue eQTLs may exert opposite effects across multiple tissue types. In summary, our results suggest that eQTL prediction models need to carefully address tissue and disease dependency of eQTLs. Tissue-disease-specific eQTL databases can afford more accurate prediction models for future studies.

Mutations of genes including DNMT3A detected by next-generation sequencing in thyroid cancer

ABSTRACT More than 90% of thyroid cancer belongs to the papillary and follicular thyroid carcinomas based on pathological subtypes. Papillary and follicular thyroid carcinoma are generally associated with a good prognosis. In contrast, other pathological subtypes such as poorly-differentiated and anaplastic thyroid carcinoma (PDTC and ATC) have a poor clinical outcome with a short life expectancy. To identify the genetic variations and biomarkers that may potentially distinguish the aggressive form of thyroid cancer, we performed a retrospective analysis of the formalin-fixed paraffin-embedded tumor samples from 50 patients who mainly displayed aggressive thyroid cancer using next-generation sequencing of 416 solid tumor-related genes. We adopted extensive bioinformatic analysis to vigorously remove germline single-nucleotide polymorphism and systematic sequencing errors, and report here that mutation in DNMT3A gene was significantly enriched in patients with PDTC or ATC.

Efficient identification of somatic mutations in acute myeloid leukaemia using whole exome sequencing of fingernail derived DNA as germline control

Recent advances in next-generation sequencing have made it possible to perform genome wide identification of somatic mutation in cancers. Most studies focus on identifying somatic mutations in the protein coding portion of the genome using whole exome sequencing (WES). Every human genome has around 4 million single nucleotide polymorphisms (SNPs). A sizeable fraction of these germline SNPs is very rare and will not be found in the databases. Thus, in order to unambiguously identify somatic mutation, it is absolutely necessary to know the germline SNPs of the patient. While a blood sample can serve as source of germline DNA from patients with solid tumours, obtaining germline DNA from patients with haematological malignancies is very difficult. Tumor cells often infiltrate the skin, and their DNA can be found in saliva and buccal swab samples. The DNA in the tips of nails stems from keratinocytes that have undergone keratinization several months ago. DNA was successfully extracted from nail clippings of 5 probands for WES. We were able to identify somatic mutations in one tumor exome by using the nail exome as germline reference. Our results demonstrate that nail DNA is a reliable source of germline DNA in the setting of hematological malignancies.

Abstract IA08: Early detection of high-risk localized prostate cancer

Abstract Despite prostate cancer being the second leading cause of cancer death amongst U.S. men and prevalent use of serum prostate specific antigen (PSA) as an FDA-approved biomarker, PCa screening and early detection is highly controversial. Widespread PSA screening and prostate biopsy led to a profound stage migration, with the majority of newly diagnosed cases being clinically localized and low grade. Thus PSA-screening programs, typically using a threshold of 4.0ng/mL for biopsy referral, result in substantial overdiagnosis, estimated at 15-84%. Additionally, although active surveillance (AS) is an emerging management option for men with presumed indolent, low-grade (Gleason score 6, grade group I) PCa, most U.S. men with such disease still undergo curative treatment via radical prostatectomy (RP) or radiation therapy (RT), which have well-documented side effects that substantially impact quality of life. Hence, serum PSA-based screening has also led to PCa overtreatment, where screening-detected tumors that never would have caused clinical symptoms are treated definitively. Lastly, as a result of PSA screening, an estimated ~1,000,000 prostate biopsies are performed annually, which are associated with infection requiring antibiotics in ~6% of patients and hospitalization in ~1% of biopsied patients. Despite appeals to lower the commonly used cutoff for biopsy in the U.S. or the development of multivariate models that incorporate PSA and other clinical factors to provide individualized PCa risk estimates, PSA is still commonly used as a dichotomous test, with referral for biopsy if PSA is > 4.0ng/mL. Several modifications of serum PSA, including free PSA, PSA velocity, various PSA isoforms, and related proteins including KLK2, have been proposed as biomarkers and show clinical utility to further define an individual’s risk for PCa. More recently, urine-based biomarkers, such as the long noncoding RNA (lncRNA) transcript PCA3, as well as germline single-nucleotide polymorphisms (SNPs) that are associated with increased PCa risk, have also been proposed as biomarkers. Likewise, advances in multiparametric MRI are also enabling the noninvasive detection and targeted biopsy of prostate cancer. Lastly, numerous tissue-based prognostic assays are available to identify undersampled or unsampled aggressive prostate cancer from diagnostic material. Here, I will review the underlying challenges inherent in the early detection of aggressive, high-risk localized prostate cancer, focusing on both the high prevalence of disease as well as multifocality. In addition, I will review the clinical utility of available approaches, as well as implementation issues. Importantly, enormous advances have been made in our understanding of the prostate cancer genome and transcriptome, which have been incompletely leveraged in early detection strategies. These findings will be discussed in the context of improved detection of high-risk localized prostate cancer, which has the potential to dramatically impact the lives of millions of U.S. men. Citation Format: Scott A. Tomlins. Early detection of high-risk localized prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA08.

Abstract 1227: Somatic genome alterations in cancer as compared to inferred patient ancestry

Abstract Background The frequency of targetable somatic genomic alterations is known to differ based on a patient's ancestry; for example, in lung adenocarcinoma EGFR mutations are more common in East Asians than Europeans. Accurately characterizing these differences will help to illuminate differences in the genetic etiology of cancer between populations. Methods We inferred estimated population ancestry for more than 100,000 de-identified patients from comprehensive genomic profiling (CGP) of tumor specimens assessed in the course of routine clinical care. More than 40,000 germline single nucleotide polymorphisms (SNPs) are sequenced as part of our CGP assay which were also characterized in the publicly available 1000 Genomes data. We used the 1000 Genomes data to train and validate a classifier using these overlapping SNPs to classify individuals into one of five inferred population groups, estimated to be of predominantly African, European, Central and South American, South Asian, or East Asian ethnic origin. We then performed statistical testing to identify differences in tumor somatic genomic properties between the five inferred ancestry groups for each cancer type. Results We found that the inference of population ancestry from tumor sequence data and its comparison to somatic mutations recapitulated known differences between populations. Specifically, in lung adenocarcinoma, EGFR mutations were significantly more common in tumor specimens from individuals with inferred East Asian SNP-inferred ancestry (rate=0.52) compared to individuals with inferred European ancestry (rate=0.17, p < 10-119), and KRAS mutations were more common in tumor specimens from individuals with inferred European ancestry (rate=0.40) than individuals with inferred East Asian ancestry (rate=0.14, p < 10-60). In lung adenocarcinoma, we also found that tumor mutational burden (TMB), a biomarker for immunotherapy response, differed significantly between populations. Tumor specimens from patients with inferred East Asian ancestry had the lowest TMB (4.5 mutations/Mb median, 4.7% >20), then from patients with inferred European ancestry (6.3 median, 12.8% >= 20), and inferred African ancestry the highest (9.0 median, 21.2% >=20). Finally, we found that in glioblastoma, TERT promoter mutations were more common in specimens from individuals with inferred European ancestry (rate=0.69) compared to specimens from individuals with inferred East Asian ancestry (rate=0.44). Based on the statistical power provided by this large cohort, novel ancestry based differences in gene alteration rates will be presented across multiple cancer types. Conclusions Clear differences in genomics based on SNP-inferred ancestry were observed. In particular, we found that TMB differs significantly between populations in lung adenocarcinoma, suggesting that the likelihood of individuals benefiting from immunotherapy may differ between populations. Citation Format: Caitlin F. Connelly, Jian Carrot-Zhang, Philip J. Stephens, Garrett M. Frampton. Somatic genome alterations in cancer as compared to inferred patient ancestry [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 1227.

Abstract 236: Identification of novel cancer target genes by combining data from the cancer genome-wide association studies (GWAS), regulatory DNA elements and The Cancer Genome Atlas (TCGA)

Abstract Unlike tumors that develop because of somatic genetic alterations such as frequent coding mutations or chromosomal deletions, amplifications or translocations, some tumors are addicted to expression of certain genes for their sustained proliferation and survival - for example, LIM domain only 1 (LMO1) expression in neuroblastoma (NB). Numerous genome-wide association studies (GWAS) have identified significant associations between germline single-nucleotide polymorphisms (SNPs), many in the non-coding genome, and cancer. A robust genome-wide association has been reported previously between an intronic LMO1 SNP and NB susceptibility. By further sequencing and epigenetic fine mapping of the SNP locus, it was demonstrated that the causal SNP is part of a transcription factor binding site within an enhancer element, regulates expression of LMO1 and in turn increases the tumorigenic potential. To identify additional examples of regulatory SNPs as cancer drivers, we overlaid published genome-wide significant cancer associations with active chromatin marks from Encyclopedia of DNA Elements and searched for SNPs that resided within gene regulatory elements. To map these SNPs to candidate genes and determine direction of effect, we co-localized GWAS signals with expression quantitative trait (eQTL) signals from the Genotype-Tissue Expression (GTEx) Consortium database. Lastly, we checked for gene amplification and/or overexpression of the mapped genes in the Cancer Genome Atlas (TCGA) data to identify a set of target genes that not only exhibit significant cancer association in GWAS, but also have evidence for epigenetic regulation and propensity for amplification and/or overexpression in tumors. We identified more than 25 novel cancer-target pairs with strong germline, regulatory and somatic evidence. A look up through synthetic lethality screen data available in-house suggested that several of these targets are self-lethal, further underscoring their importance for cancer cell proliferation and survival. Additional in vitro experiments are being planned to further validate the targets. Citation Format: Diptee A. Kulkarni, Karl Guo, Junping Jing, Mugdha Khaladkar, Kijoung Song, Coco Dong, David Cooper, Benjamin Schwartz. Identification of novel cancer target genes by combining data from the cancer genome-wide association studies (GWAS), regulatory DNA elements and The Cancer Genome Atlas (TCGA) [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 236.

Abstract 3425: Exome-sequencing derived mutations of endocrine treated ER-positive early breast cancer

Abstract Anti-hormonal treatment of ER-positive early breast cancer with tamoxifen and/or aromatase inhibitors (AI) significantly improves patient outcome, however there is a lack of markers that predict the one third of treated patients who relapse or die. Although ER positive (luminal) tumors are known to be genetically highly diverse, the relevance of somatic mutations with regards to endocrine treatment outcome and course of the patients' disease is not fully understood. We conducted a prospective observational clinical trial for the investigation of treatment outcome determinants including 1219 tamoxifen and/or AI-treated postmenopausal ER-positive early breast cancer patients (DRKS00000605, recruitment between 2005-2010, median follow up 5.1 years). Of these, 40 tumors divided into 20 patients each with and without disease recurrence matched by prognostic variables (age, tumor size, grade, nodal status, treatment) were analyzed for their mutation pattern. We performed whole-exome sequencing of tumor and normal DNA (Agilent SureSelect, Illumina) for the identification of single nucleotide variants and small indels. Somatic mutations were identified following subtraction of germline single nucleotide polymorphisms (SNP), filtering based on raw-read quality parameters, in silico predicted biological function, and disease relatedness using the Ingenuity variant analysis software package (Qiagen). Clinical outcome associations were tested either on gene or on variant level. Somatic mutations with predicted deleterious effects were identified in 18 out of 40 known mutation-driver genes including PIK3CA, GATA3, MAP3K1, and TP53 (45%). Thirteen patients carried a mutation in the most frequently affected PIK3CA (32.5%). Of note, three out of four patients with a E542K mutation belonged to the breast cancer recurrence group, while 5 of 6 tumors with the H1047R mutation originated from patients without recurrence suggesting differential effects of these PIK3CA hotspot mutations. Common germline SNPs at IL17RB, ANK2, POM121, PGR, PTPRB, RAD51D, RFPLA4, and SUMO3 provided first hints for a possible association with relapse status (p<0.05). We will report on the extended genotype-outcome-correlations to further evaluate the relevance of somatic mutations for endocrine treatment outcome of early ER-positive breast cancer. Citation Format: Werner Schroth, Siarhei Kandabarau, Peter Fritz, Thomas Mürdter, Liza Bacchus, German Tamoxifen Study Group, Matthias Schwab, Hiltrud Brauch. Exome-sequencing derived mutations of endocrine treated ER-positive early breast 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 3425.

Abstract 1080: The effect of germline polymorphisms on somatic hotspot mutations in TP53 for the treatment of high-grade serous ovarian carcinoma

Abstract Introduction: Due to frequent recurrences and acquired resistance to platinum-based chemotherapy in high-grade serous ovarian cancer (HGS OvCa), it is important to identify novel targets and pathways to overcome drug resistance and improve the outcome of patients with ovarian cancer. TP53, which encodes the transcription factor p53, is mutated in 96% of HGS OvCa . Majority of the studies that establish the relationship between mutations in TP53 and cancer have focused on hotspot somatic mutations. In addition to somatic mutations, nonsynonymous germline single nucleotide polymorphisms (SNPs) at codon 72 play a role in altering the function of p53 and a cancer susceptibility. However, the effect of codon 72 (P72R) polymorphism on somatic mutations in TP53 is not well characterized with regard to the Epithelial to Mesenchymal phenotype and cancer progression. Objective: -Characterize the effect of somatic mutations in TP53 in the presence of the P72R SNP on cellular morphology and the Epithelial to Mesenchymal transition (EMT) transcriptional network. -Determine the effect of somatic mutations in TP53 in the presence of the P72R SNP on proliferation and the cell cycle checkpoint transcriptional network Experimental Procedures: We generated 8 pairs of hotspot mutations in TP53 in the context of the P72 or R72 SNP into p53-null SKOV3 cells. Cell morphology was recorded by fluorescence microscopy. Using CHIP-seq, RNA-seq and immunoblotting, we studied the effect of the polymorphism + mutation on EMT markers like BMP1, CALD1, CAMK2N1, CDH2 that are up-regulated during EMT and genes like CAV2, CDH1, DSP, FGFBP1, IL1RN, KRT19, MITF that are down-regulated during EMT. We used techniques like chromatin immunoprecipitation ChIP-seq, gene expression profiling, and computational methods to construct blueprints of the transcription factors like Cyclin D1, p21 that play a role in cell cycle checkpoint regulation which help understand compromised pathways due to the presence of somatic mutations in combination with germline polymorphisms. Summary: - Hotspot mutants with the R72 SNP cause changes in cell morphology resembling mesenchymal cells whereas mutants with the P72 SNP resemble epithelial cells. This effect can be accounted for by changes in the EMT network. - The hotspot missense mutants with R72 have a greater growth potential in comparison to their P72 counterpart which could explain the Gain of Function (GOF) effect of the former. Conclusions: Germline polymorphisms at codon 72 in the p53 protein plays a significant role in regulating the Gain-of-Function (GOF) effect of TP53 somatic hotspot mutations. This is significant because it will be useful in designing a treatment plan for patients with specific somatic mutations and germline polymorphisms. Citation Format: Cristabelle Madona N. De Souza. The effect of germline polymorphisms on somatic hotspot mutations in TP53 for the treatment of high-grade serous ovarian 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 1080.

Abstract 3788: Antigen presentation by MHC-I molecule and immune escape in acute myeloid leukemia with high burden of genomic aberrations

Abstract Background: Antigen presentation is crucial for tumor immune response and MHC-I plays a key role. Solid and hematological tumors with high load of newly tumor associated antigens develop evading immune surveillance. A subset of patients with acute myeloid leukemia (AML), precisely ones with complex karyotype and TP53 alterations have a high load of mutation or genomic aberrations. In our study, we screened the entire genome of a large set of newly diagnosed AML to discover if MHC I antigen presentation is altered in the set of AML with a high neo-antigen load. Methods: We screened 300 samples of AML bone marrow at diagnosis from 3 hematological Institutions. We analyzed 636 single nucleotide polymorphism (SNP) arrays to detect karyotype aberration at genomic level; germline SNP arrays were performed in 114/300 patients, the remaining 186/300 were normalized with a reference pool of 222 healthy donors. Of the 300 patients, we collected clinical data and we tested TP53, IDH1, IDH2, FLT3, NPM1, DNMT3A and RUNX1 mutations according ordinary practice. Genes in MHC class I antigen presentation pathway were selected basing on Reactome. We considered MHC-I antigen presentation altered if pathway's alteration rate (altered genes in a pathway/gene in a pathway ratio) scored in the top 25% between all pathways. Results: Twenty out of 300 patients (6.7%) had loss of genes in MHC I antigen presentation (group A). Patients had alterations in one or more genes; overall, 53 genes were founded altered in our set. Patients in group A did not have differences in age or in white blood count at diagnosis compared with other patients in our set (group B). Group A was enriched for patients with of complex karyotype (14/20 patients, p<.001), isolated del(5q) (3/20) and for patients harboring TP53 mutation (11/20 patients, p<.001). In 280 patients (20 not tested), 70 patient had complex karyotype; 14/70 (20%, p<.001) patients with complex karyotype were from group A. Group A had a higher median burden of genomic than patients in group B (0.08 vs 0.04 alterations per pathway, p<.001). Patients in group A had a lower median overall survival (OS) than patients in group B (3.0 months vs 15.7 months, p<.001). Conclusion: We demonstrated that patients with MHCI alterations have a high chance to be TP53 mutated or to have complex karyotype; patients with complex karyotype have a high chance to harbor concomitant alterations in MHC I antigen presentation. In the set of AML with high burden of aberrations, MHC I alterations could be permissive for leukemia development toward immune escape. Our approach is a good candidate for further testing inside clinical trials to identify a cohort of patients that are unlikely to respond to immune check-point inhibitors within patients with larger sets of patients with high burden of alterations. Acknowledgements: ELN, AIL, AIRC, Regione-Università 2010-12, FP7 NGS-PTL, HARMONY, Fondazione del Monte BO e RA. Citation Format: Giovanni Marconi, Maria Chiara Fontana, Cristina Papayannidis, Mariachiara Abbenante, Antonella Padella, Giorgia Simonetti, Michele Cavo, Giovanni Martinelli. Antigen presentation by MHC-I molecule and immune escape in acute myeloid leukemia with high burden of genomic aberrations [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 3788.

Comprehensive genomic transcriptomic tumor-normal gene panel analysis for enhanced precision in patients with lung cancer.

A CMS approved test for lung cancer is based on tumor-only analysis of a targeted 35 gene panel, specifically excluding the use of the patient’s normal germline tissue. However, this tumor-only approach increases the risk of mistakenly identifying germline single nucleotide polymorphisms (SNPs) as somatically-derived cancer driver mutations (false positives). 621 patients with 30 different cancer types, including lung cancer, were studied to compare the precision of tumor somatic variant calling in 35 genes using tumor-only DNA sequencing versus tumor-normal DNA plus RNA sequencing. When sequencing of lung cancer was performed using tumor genomes alone without normal germline controls, 94% of variants identified were SNPs and thus false positives. Filtering for common SNPs still resulted in as high as 48% false positive variant calling. With tumor-only sequencing, 29% of lung cancer patients had a false positive variant call in at least one of twelve genes with directly targetable drugs. RNA analysis showed 18% of true somatic variants were not expressed. Thus, sequencing and analysis of both normal germline and tumor genomes is necessary for accurate identification of molecular targets. Treatment decisions based on tumor-only analysis may result in the administration of ineffective therapies while also increasing the risk of negative drug-related side effects.

Phase III trial of adjuvant sunitinib in patients with high-risk renal cell carcinoma: Exploratory pharmacogenomic analysis.

576 Background: In the phase III S-TRAC trial, adjuvant sunitinib (SU) prolonged disease-free survival (DFS) vs placebo (PBO) in patients with locoregional renal cell carcinoma at high risk of recurrence after nephrectomy (median 6.8 vs 5.6 y; hazard ratio [HR] 0.76, 95% confidence interval [CI] 0.59–0.98; P= 0.03). An exploratory analysis evaluated associations between single nucleotide polymorphisms (SNPs) in angiogenesis-related genes and clinical outcomes in S-TRAC. Methods: Prospectively collected blood samples were genotyped for 10 SNPs and 1 insertion/deletion mutation with TaqMan assays. DFS was compared with a log-rank test for each SNP genotype in SU vs PBO arms and between SNP genotypes within each arm. P-values are unadjusted for multiplicity comparison. Results: Of 615 patients, 286 (142 SU; 144 PBO) were analyzed. There were generally no genotype frequency deviations from the Hardy-Weinberg equilibrium, but linkage disequilibrium was seen between VEGFA rs699947 and rs833061 on chromosome 6 (D′ = 1.000, r2 = 0.979). Longer DFS was observed with SU vs PBO for VEGFR1 rs9554320 C/C (median: not reached [NR] vs 5.56 y; HR 0.44, 95% CI 0.21–0.91; P= 0.023), VEGFR2 rs2071559 T/T (median: NR vs 4.47 y; HR 0.46, 95% CI 0.23–0.90; P= 0.020), and eNOS rs2070744 T/T (median: 7.07 vs 3.44 y; HR 0.53, 95% CI 0.30–0.94; P= 0.028), with a trend for VEGFR1 rs9582036 A/A (median: NR in both arms; P= 0.054) and SH3GL2 rs10963287 C/T (median: NR vs 5.35 y; P= 0.088). Shorter DFS was observed for VEGFR1 rs9582036 C/A vs C/C in the SU, PBO, and combined arms ( P< 0.05); for A/A vs common, the association was only seen in the SU arm ( P= 0.022). VEGFR1 rs9554320 A/C was associated with shorter DFS vs A/A in the PBO ( P= 0.038) and combined arm ( P= 0.006), with a trend in the SU arm ( P= 0.051). VEGFR2 rs1870377 T/T was associated with longer DFS vs A/A in the combined arms, but not in the PBO arm (n = 7 with A/A genotype in the SU arm precluded statistical tests). Conclusions: Correlations between common VEGFR1 and VEGFR2 SNPs and longer DFS with SU suggest germline SNPs are predictive of improved outcomes with adjuvant SU. Due to the exploratory nature of this analysis, prospective validation studies are needed to confirm these findings. Clinical trial information: NCT00375674.

A computational approach to distinguish somatic vs. germline origin of genomic alterations from deep sequencing of cancer specimens without a matched normal.

A key constraint in genomic testing in oncology is that matched normal specimens are not commonly obtained in clinical practice. Thus, while well-characterized genomic alterations do not require normal tissue for interpretation, a significant number of alterations will be unknown in whether they are germline or somatic, in the absence of a matched normal control. We introduce SGZ (somatic-germline-zygosity), a computational method for predicting somatic vs. germline origin and homozygous vs. heterozygous or sub-clonal state of variants identified from deep massively parallel sequencing (MPS) of cancer specimens. The method does not require a patient matched normal control, enabling broad application in clinical research. SGZ predicts the somatic vs. germline status of each alteration identified by modeling the alteration’s allele frequency (AF), taking into account the tumor content, tumor ploidy, and the local copy number. Accuracy of the prediction depends on the depth of sequencing and copy number model fit, which are achieved in our clinical assay by sequencing to high depth (>500x) using MPS, covering 394 cancer-related genes and over 3,500 genome-wide single nucleotide polymorphisms (SNPs). Calls are made using a statistic based on read depth and local variability of SNP AF. To validate the method, we first evaluated performance on samples from 30 lung and colon cancer patients, where we sequenced tumors and matched normal tissue. We examined predictions for 17 somatic hotspot mutations and 20 common germline SNPs in 20,182 clinical cancer specimens. To assess the impact of stromal admixture, we examined three cell lines, which were titrated with their matched normal to six levels (10–75%). Overall, predictions were made in 85% of cases, with 95–99% of variants predicted correctly, a significantly superior performance compared to a basic approach based on AF alone. We then applied the SGZ method to the COSMIC database of known somatic variants in cancer and found >50 that are in fact more likely to be germline.

LncRNA CDKN2BAS rs2157719 genetic variant contributes to medulloblastoma predisposition.

How germline single nucleotide polymorphisms are involved in the etiology of medulloblastoma remans poorly understood. We hypothesized that CCDKN2A/B rs1063192 and rs4977756 and also the long noncoding RNA (lncRNA) CDKN2BAS rs2157719 glioma susceptibility polymorphisms identified by genome-wide association studies may contribute to medulloblastoma predisposition. To test this hypothesis, we genotyped these genetic variants among 160 medulloblastoma patients and 443 health controls in a Chinese population. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. We found that only the lncRNA CDKN2BAS rs2157719 T>C genetic polymorphism was significantly associated with an increased medulloblastoma risk (C allele: OR = 1.85, 95% CI = 1.32-2.58; p = 2.7 × 10-4 ). The stratified analyses showed an elevated risk of pediatric medulloblastoma associated with CDKN2BAS rs2157719 CC or TC genotype (both p < 0.05). Moreover, the association between the CDKN2BAS rs2157719 polymorphism and medulloblastoma risk is more pronounced in males (OR = 2.22, 95% CI = 1.36-3.62; p = 0.001). The findings of the present study provide important insights into the genetic complexities and predisposition of medulloblastoma in Chinese, especially at the lncRNA germline variation level.

Attempted replication of SNPs in RANKL and OPG with musculoskeletal adverse events during aromatase inhibitor treatment for breast cancer

Aromatase inhibitor (AI) therapy is highly efficacious in the treatment of estrogen receptor-positive breast cancer; however, in a subset of patients AI use is discontinued due to drug-induced musculoskeletal adverse events (MS-AE). Several studies have investigated the role of germline single nucleotide polymorphisms (SNPs) on patients’ risk of MS-AEs; however, no associations have yet to be validated for translation into clinical practice. This study attempted to replicate SNPs in RANKL (rs7984870) and OPG (rs2073618) on the risk of AI-induced MS-AEs and screen for secondary associations with MS-AE-related treatment discontinuation and serum and urine markers of bone health. Previously reported associations were not replicated with our primary hypothesis, change in MS-AE from baseline to 3 mo; however, patients homozygous for the G allele of rs7984870 in RANKL had lower risk of MS-AE-associated treatment discontinuation in analyses of secondary phenotypes without statistical correction.

Genomic risk prediction of aromatase inhibitor-related arthralgia in patients with breast cancer using a novel machine-learning algorithm.

Many breast cancer (BC) patients treated with aromatase inhibitors (AIs) develop aromatase inhibitor‐related arthralgia (AIA). Candidate gene studies to identify AIA risk are limited in scope. We evaluated the potential of a novel analytic algorithm (NAA) to predict AIA using germline single nucleotide polymorphisms (SNP) data obtained before treatment initiation. Systematic chart review of 700 AI‐treated patients with stage I‐III BC identified asymptomatic patients (n = 39) and those with clinically significant AIA resulting in AI termination or therapy switch (n = 123). Germline DNA was obtained and SNP genotyping performed using the Affymetrix UK BioBank Axiom Array to yield 695,277 SNPs. SNP clusters that most closely defined AIA risk were discovered using an NAA that sequentially combined statistical filtering and a machine‐learning algorithm. NCBI PhenGenI and Ensemble databases defined gene attribution of the most discriminating SNPs. Phenotype, pathway, and ontologic analyses assessed functional and mechanistic validity. Demographics were similar in cases and controls. A cluster of 70 SNPs, correlating to 57 genes, was identified. This SNP group predicted AIA occurrence with a maximum accuracy of 75.93%. Strong associations with arthralgia, breast cancer, and estrogen phenotypes were seen in 19/57 genes (33%) and were functionally consistent. Using a NAA, we identified a 70 SNP cluster that predicted AIA risk with fair accuracy. Phenotype, functional, and pathway analysis of attributed genes was consistent with clinical phenotypes. This study is the first to link a specific SNP/gene cluster to AIA risk independent of candidate gene bias.

Common, germline genetic variations in the novel tumor suppressor BAP1 and risk of developing different types of cancer.

BRCA1 associated protein-1 (BAP1) is a novel tumor suppressor that has recently been shown to be somatically mutated in several cancers. The BAP1 gene also carries rare germline mutations in families with a high incidence of several types of cancers, such as mesothelioma, uveal melanoma, lung adenocarcinoma, melanocytic neoplasms, and renal cell carcinoma. To test the hypothesis that common, germline genetic variants in BAP1 may also contribute to the risk of developing different types of cancer, we genotyped germline single nucleotide polymorphisms (SNPs) for BAP1 in a large population of patients with cancer, including 2,340 with colorectal cancer, 1,436 with bladder cancer, 3,313 with lung cancer, 1,325 with renal cell carcinoma, and 1,162 with esophageal cancer. We identified significant association of rs11708581 (P = 0.0034) and rs390802 (P = 0.015) with risk of renal cell carcinoma and rs12163565 (P = 0.038) with risk of lung cancer. Expression quantitative trait loci analysis in renal cell carcinoma using publicly available data from TCGA showed that the proxy SNPs for rs11708581 and rs390802 were negatively associated with the expression level of BAP1. Our study indicate that common germline genetic variants of BAP1 play a role in mediating the risk of developing renal cell carcinoma and lung cancer.

IL-6 variant is associated with metastasis in breast cancer patients.

IntroductionAlthough tumor metastases remain significant drivers of mortality, the genetic factors that increase the risks of metastases are not fully identified. Interleukin 6 (IL-6) has emerged as an important factor in breast cancer progression with IL-6 single nucleotide polymorphism (SNP) variants shown to affect survival. We hypothesized that SNPs of the IL-6 promoter at rs1800795 in breast cancer patients are associated with distant metastases.MethodsWe performed an initial case-control study using Vanderbilt University Medical Center’s BioVU, a genomic biobank linked to de-identified electronic medical records in the Synthetic Derivative database, to identify germline SNPs that may predict the development of metastatic disease to any site from any solid tumor including breast cancer. We identified a SNP in IL-6: rs1800795 to be of significance and evaluated this finding using a separate, matched-pair cohort of breast cancer patients with and without metastases from The Ohio State University Wexner Medical Center.ResultsThe initial study suggested that GG relative to CG at rs1800795 (OR 1.52; 95% CI 1.14–2.02; p = 0.004) was significantly associated with the development of metastases. This association was also observed in the Ohio State University cohort (OR 2.23; 95% CI 1.06–4.71; p = 0.001). There were no significant relationships between rs1800795 status and any patient or tumor characteristics, including estrogen receptor status.ConclusionsThese findings suggest that GG SNP at IL-6: rs1800795 may indicate an increased risk of metastasis of primary breast cancer. Further studies in larger population sets are warranted as advanced screening and prophylactic intervention might be employed in GG carriers.

Abstract 1115: Germline single nucleotide polymorphisms in DNA repair genes in urothelial cancer patients

Abstract Introduction: Germline single nucleotide polymorphisms (SNPs) have been investigated in several cancers. Repair of DNA damage is a key cellular process involved in the development of chemotherapy-resistance. The role of germline SNPs in DNA damage repair genes (DRGs) in determining resistance to DNA-damaging agents in urothelial carcinoma (UC) patients is not completely understood. Methods: We examined a cohort of 53 UC patients (median age 67, 42 males) enrolled in our IRB-approved Precision Medicine program. Patients had histologically confirmed UC (43 bladder, 10 upper tract UC) and received treatment with platinum-based chemotherapy. We isolated germline DNA from peripheral blood lymphocytes or buccal swabs, and used whole exome sequencing (WES) to examine germline SNPs. As a reference for SNP frequencies we used the Exome Aggregation Consortium (ExAC) database, which represents the largest, randomly selected, germline WES database in the general population, including 60,706 individuals, among which 7,601 patients with multiple cancer types from the Tumor Cancer Genome Atlas (TCGA) cohort. Results: Twelve different DRG SNPs were identified in germline DNA samples from 53 patients, affecting genes involved in non-homologous end-joining (RECQL4, n = 18, 54.50%; POLQ, n = 2, 6%), nucleotide excision repair (ERCC6, n = 2, 6%; XPA, n = 1, 3%; CCNH, n = 1, 3%; POLK, n = 1, 3%), homologous recombination (RNF168, n = 1, 3%; RAD17, n = 1, 3%; POLE, n = 1, 3%), Fanconi anemia pathway (POLN, n = 1, 3%), mismatch repair (EXO1, n = 1, 3%) and mitochondrial DNA repair (POLG, n = 2, 6%). The frequency of rs11342077 of the DNA helicase RECQL4 was significantly higher in our cohort (18/53, 34%) compared to its frequency in the ExAC database (p&amp;lt;0.01%). There was no significant difference in overall survival (OS) between patients with and without DRG SNPs (log-rank p=0.46). There was no significant association between the most commonly identified SNP, rs11342077, and overall survival among UC patients with DRG SNPs (log-rank p=0.39). Overall, the presence of DRG SNPs in our cohort occurred at a significantly higher frequency (33/53, 62.3%) compared to the ExAC database (0.56%, chi-square p&amp;lt;0.01). Conclusions: Germline SNPs in DNA repair pathway genes are common in UC. Additional study of the role of these SNPs as potential biomarkers of response DNA damaging chemotherapeutic agents, including platinum-based chemotherapy or/and PARP inhibitors is needed in a larger cohort. Citation Format: Bishoy M. Faltas, Panagiotis J. Vlachostergios, Linda Lam, Tuo Zhang, Olivier Elemento, Mark A. Rubin. Germline single nucleotide polymorphisms in DNA repair genes in urothelial cancer patients [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 1115. doi:10.1158/1538-7445.AM2017-1115

Abstract 3397: Post-mortem examination of an aggressive case of medullary thyroid cancer characterised by catastrophic genomic abnormalities

Abstract Catastrophic genomic alterations may drive aggressive cancer phenotypes. We describe a diagnostically challenging and rapidly fatal case of medullary thyroid carcinoma (MTC) occurring in a young, morbidly obese man presenting with diffuse bone marrow involvement and disseminated intravascular coagulation. Whole-exome (WES) and shallow whole-genome sequencing (sWGS) were carried out for the primary tumour, adjacent normal, bone marrow tissues, multiple metastases and blood samples derived from the patient. We identified three germline single nucleotide polymorphisms (SNPs) within the RET proto-oncogene that remained undetected using routine hospital genetic testing procedures. One variant (L769L) has been previously reported to be associated with aggressive MTC presentation yet remains untested for in the routine diagnosis of MTC. Supported by findings from both WES and sWGS, we report for the first time in thyroid cancer on the occurrence of a “chromothripsis-like pattern”, which involved shattering of chromosome 4 leading to complete abrogation of normal chromosomal function, along with dramatic widespread copy number aberrations across both primary tumour and bone marrow samples. Based on the somatic variants identified, we describe the evolutionary pathway of the case, showing that bone marrow metastasis occurred separately to other metastatic sites. The presence of disease-associated SNPs within the RET proto-oncogene supports their inclusion as part of routine genetic testing for MTC cases. The copy number aberrations and chromothripsis-like pattern affected a much broader range of genes than single mutations and may have led to the widespread chromosomal instability evident, possibly contributing to the rapid fatal course of the case. These results provide a rationale for the application of comprehensive genomic analysis of cancers presenting with unusual and aggressive phenotypes to facilitate more appropriate therapeutic options and diagnoses. Citation Format: Darran P. O'Connor, Sudipto Das, Deirdre Kelly, Bruce Moran, Kathleen Han, Niall Mulligan, Ciara Barrett, Patrick Buckley, Peter McMahon, J McCaffrey, Henrik van Essen, Kate Connor, Bauke Ylstra, Diether Lambrechts, William M. Gallagher, Catherine M. Kelly. Post-mortem examination of an aggressive case of medullary thyroid cancer characterised by catastrophic genomic abnormalities [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 3397. doi:10.1158/1538-7445.AM2017-3397