Targeted Exome Sequencing Research Articles

Four novel compound heterozygous mutations in C5orf42 gene in patients with pure and mild Joubert syndrome.

Joubert syndrome (JS) is a rare clinically and genetically heterogeneous disease. Using whole or targeted exome sequencing, we identified four novel compound heterozygous mutations in chromosome 5 open reading frame 42 gene (C5orf42), including c.2876C>T (missense mutation) and c.3921+1G>A (splicing mutation), c.2292 -2delA (splicing mutation) and c.4067C>T (missense mutation), c.6997_6998insT (frameshift mutation) and c.8710C>T (nonsense mutation), c.3981G>C (nonsense mutation) and c.230 _233del (frameshift mutation), in four Chinese JS families. They were all inherited from their heterozygosis parents in the autosomal recessive inheritance mode. Pure JS clinical manifestations and mild neuroimaging findings were found in these patients. These verified the previous findings that C5orf42 mutations generally resulted in a purely neurological Joubert phenotype, and neuroimaging findings were mild in JS with C5orf42 mutations. Our report analyzed these C5orf42 mutations-associated phenotypes and neuroimaging findings in JS and updated the genetic variation spectrum of JS caused by C5orf42.These will help clinicians and geneticists reach a more accurate diagnosis for JS.

MITF variants cause nonsyndromic sensorineural hearing loss with autosomal recessive inheritance

MITF is a known gene underlying autosomal dominant hearing loss, Waardenburg syndrome (WS). Biallelic MITF mutations have been found associated with a rare hearing loss syndrome consisting eye abnormalities and albinism; and a more severe type of WS whose heterozygous parents were affected with classic WS in both cases. The aims of this study were to identify a new candidate gene causing autosomal recessive nonsyndromic hearing loss (ARNSHL) and confirm its causation by finding additional families affected with the candidate gene and supporting evidences from functional analyses. By using whole exome sequencing, we identified a homozygous c.1022G>A: p.Arg341His variant of MITF, which co-segregated with the hearing loss in five affected children of a consanguineous hearing couple. Targeted exome sequencing in a cohort of 130 NSHL individuals, using our in-house gene panel revealed a second family with c.1021C>T: p.Arg341Cys MITF variant. Functional studies confirmed that the Arg341His and Arg341Cys alleles yielded a normal sized MITF protein, with aberrant cytosolic localization as supported by the molecular model and the reporter assay. In conclusion, we demonstrate MITF as a new cause of ARNSHL, with heterozygous individuals free of symptoms. MITF should be included in clinical testing for NSHL, though it is rare.

Targeted exome sequencing designed for blood group, platelet, and neutrophil antigen investigations: Proof-of-principle study for a customized single-test system.

Immunohematology reference laboratories provide red blood cell (RBC), platelet (PLT), and neutrophil typing to resolve complex cases, using serology and commercial DNA tests that define clinically important antigens. Broad-range exome sequencing panels that include blood group targets provide accurate blood group antigen predictions beyond those defined by serology and commercial typing systems and identify rare and novel variants. The aim of this study was to design and assess a panel for targeted exome sequencing of RBC, PLT, and neutrophil antigen-associated genes to provide a comprehensive profile in a single test, excluding unrelated gene targets. An overlapping probe panel was designed for the coding regions of 64 genes and loci involved in gene expression. Sequencing was performed on 34 RBC and 17 PLT/neutrophil reference samples. Variant call outputs were analyzed using software to predict star allele diplotypes. Results were compared with serology and previous sequence genotyping data. Average coverage exceeded 250×, with more than 94% of targets at Q30 quality or greater. Increased coverage revealed a variant in the Scianna system that was previously undetected. The software correctly predicted allele diplotypes for 99.5% of RBC blood groups tested and 100% of PLT and HNA antigens excepting HNA-2. Optimal throughput was 12 to 14 samples per run. This single-test system demonstrates high coverage and quality, allowing for the detection of previously overlooked variants and increased sample throughput. This system has the potential to integrate genomic testing across laboratories within hematologic reference settings.

Detection of somatic TP53 mutation in surgically resected small-cell lung cancer by targeted exome sequencing: association with longer relapse-free survival

ObjectivesFew reports have explored clinical biomarkers, including those identified by targeted exome sequencing (TES) of surgically resected small-cell lung cancer (SCLC) and correlation with patient survival. Patients and methodsWe collected formalin-fixed paraffin-embedded tumor samples from 127 patients with SCLC who had undergone surgery and analysed nonsynonymous somatic gene mutation profiles by TES of 26 cancer-related genes using next-generation sequencing (NGS) and web databases (UMIN Registration No. 000010117). ResultsWe detected 38 nonsynonymous somatic tumor protein p53 (TP53) mutations in 43 (54.4%) patients. Among these TP53 lesions, we identified clinically relevant mutations including those encoding Y220C, R248W, R249M, M237I, and R273L substitutions in the p53 protein. These mutations have been reported to be associated with certain clinical outcomes or biology in other types of malignancies but not in SCLC. Moreover, nonsynonymous somatic mutations of TP53 were positively associated with relapse-free survival (RFS) (median, 17.33 months [95% confidence interval (CI), 3.86–30.79] in a mutation-positive group vs 10.39 months (6.96–13.82) in a mutation-negative group, p = 0.042). Multivariate analysis revealed that nonsynonymous somatic TP53 mutation was an independent factor of prolongation of RFS (hazard ratio: 0.51, 95% CI: 0.29–0.89, p = 0.019) but not overall survival (OS). ConclusionThese data suggested that TES may play a critical role for promoting reverse-translational studies, including investigations of the biology of TP53 mutations in different stages of SCLC. Accumulation of the data using cancer panels with a broader range of genes, including TP53, is expected to be useful for future clinical applications for patients with SCLC.

Abstract PR08: Noninvasive epidermal sampling as a means of genomic UV dosimetry

Abstract It is now well accepted that normal-appearing, but chronically UV-exposed skin, can harbor tremendous mutational loads while maintaining homeostatic function. Even so, clones of keratinocytes harboring tumorigenic mutations can be readily observed using targeted and whole-exome sequencing. Therefore, it may be possible to objectively measure chronic, cumulative UV exposure as a means of predicting skin cancer risk and measuring molecular responses to chemoprevention by identifying UV-driven mutations or damage in epidermal keratinocytes. However, such a methodology requires the ability to noninvasively sample normal, nonlesional skin. We have previously optimized a novel surfactant mixture that, when coupled to ultrasound or mechanical abrasion, can noninvasively sample epidermis without scarring. This process can be tuned to selectively remove epidermis only. Within hours of application, a fibrinous exudate appears over the wound and this area heals completely within 2 weeks without scarring. Using a Hairless UV-driven model of squamous cell carcinoma, we have been able to sample and perform both exome and RNA sequencing with recovery rates of about 2.5-3.5 μg /cm2 of DNA and RNA. Using this model, we demonstrated a dose-dependent increase in mutational load in normal UV-exposed skin that reflects what is seen in humans. Mice were sampled and the DNA exome was sequenced (150X) following exposure to UV. Among the 28 most mutated genes across the exome, mice sampled after 1 month of UV exposure harbored variants in an average of 12 genes. After 3 months of UV exposure, followed by 2 months of no exposure, variants were observed in an average of 22 genes (80%) of the 28, suggesting continuing evolution of clones harboring mutations in exposed epidermis. Interestingly, at this relatively low coverage, none of the genes is strongly associated with skin cancer; instead, many are not expressed, reflecting the expansion of mutational burdens without strong selection on these alterations. We then extended our efforts into humans by sampling immunosuppressed solid organ transplant recipients with markedly elevated risk for skin cancer. Sun-exposed areas had an average of 4.5 times the number of variants than sun-protected areas across targeted exome sequencing of 348 cancer-related genes at a depth of ~5000X. Over 24 genes (including TP53, ATM, NOTCH, and KMT2 family members) were identified with nonsense C-T transitions, suggesting the presence of detectable (and potentially selected) mutational patterns in cancer-related genes that correlate with clinically relevant exposure. Our results collectively suggest that noninvasive sampling and genomic interrogation of normal-appearing epidermis is feasible and potentially useful as a risk prediction and chemoprevention assessment tool. This approach is easily extended to other surface epithelia, and we are currently enhancing the sensitivity of this assay based on identification of hotspots and nontranscribed genes as identified in our model. This abstract is also being presented as Poster A32. Citation Format: Kenneth Y. Tsai, Elnara Muradova, Sandy S. Ojeda, Charles H. Adelmann, Kimberly Nguyen, Samir Mitragotri, Nishit Patel. Noninvasive epidermal sampling as a means of genomic UV dosimetry [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr PR08.

Novel SERPING1 Small Deletion Mutation in an Indian Family with Autosomal Dominant Hereditary Angioedema-TypeI

Hereditary Angioedema (HAE) is characterized by recurrent attacks of submucosal or subcutaneous edema and is caused by mutations in gene SERPING1. We identified a novel heterozygous small deletion in SERPING1 using targeted exome sequencing in an HAE patient from an Indian family. The mutation c.491_502delAGACCAACATGG,p. Glu164_ Met167del, in exon 3 of SERPING1 gene is reported in the proband and her father but was not found in proband’s unaffected sibling and control samples. The mutation was classified as ‘Likely Pathogenic’ according to ACMG guidelines for interpretation of sequence variants. To the best of our knowledge, this is the first documented report for SERPING1 mutation in HAE type1 patients from India.

Noninvasive Assessment of Epidermal Genomic Markers of UV Exposure in Skin

The measurement of UV-induced DNA damage as a dosimeter of exposure and predictor of skin cancer risk has been proposed by multiple groups. Although UV-induced mutations and adducts are present in normal-appearing UV-exposed epidermis, sampling normal nonlesional skin requires noninvasive methods to extract epidermal DNA for analysis. Here, we demonstrate the feasibility of such an approach, termed surfactant-based tissue acquisition for molecular profiling. Sampling in patients was performed using a felt-tip pen soaked in a mixture of surfactants (Brij-30/N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). In mice, we show that the epidermis can be selectively removed without scarring, with complete healing within 2 weeks. We exposed hairless mice to low-dose UV radiation over a period of 3 months and serially sampled them through up to 2 months following the cessation of UV exposure, observing a progressive increase in a UV signature mutational burden. To test whether surfactant-based tissue acquisition for molecular profiling could be applied to human patients, samples were collected from sun-exposed and sun-protected areas, which were then subjected to high-depth targeted exome sequencing. Extensive UV-driven mosaicism and substantially increased mutational loads in sun-exposed versus sun-protected areas were observed, suggesting that genomic measures, as an integrated readout of DNA damage, repair, and clonal expansion, may be informative markers of UV exposure.

Correlation of pathogenic POLE mutations with clinical benefit to immune checkpoint inhibitor therapy.

3008 Background: Mutations in DNA polymerase epsilon ( POLE) may induce DNA replication errors, increasing neoantigen load and potentially enhancing clinical benefit to immune checkpoint inhibitors (ICI). We present a clinicopathologic analysis of patients (pts) with advanced cancers harboring POLE mutations and their response to ICI therapy at MD Anderson Cancer Center. Methods: We used targeted exome sequencing via CLIA-certified next generation sequencing assays to identify pts with POLE-aberrant tumors and their co-occurring mutations. The pathogenicity of each POLE mutation was annotated utilizing InterVar and ClinVar databases. Chi-square analysis was performed. Results: Tumors from 12,947 pts were analyzed and 448 (3.5%) pts had a mutation or copy number variation in POLE (3.5%), comparable to the TCGA PanCancer Atlas (4.0%). Clinical data were available for 293 pts; the most common cancers were colorectal (14.7%), non-small cell lung (13.7%), cholangiocarcinoma (13.3%) and melanoma (10.2%). There were 267 unique co-mutations, including KRAS (23.0%), ARID1A (21.5%), BRCA2 (18.7%), ATM (18.4%), CDKN2A (17.5%), BRAF (15.3%), EGFR (15.3%), ATRX (12.6%), CREBBP (11.7%), APC (11.3%), ATR (11.0%), BRCA1 (11.0%) and CDK12 (10.4%). POLE variants were annotated in all pts: pathogenic/likely pathogenic (n = 34, 11.6%), benign/likely benign (61, 20.8%), and variant of unknown significance (198, 67.6%). 104 (35.8%) of 293 pts with POLE mutations received PD-1/L1 inhibitors as monotherapy or in combination. 93 (88.4%) of 104 pts were evaluable for response: Radiological CR 4.3% (n = 4), PR 26.9% (n = 25), SD 22.6% (n = 21), PD 46.2% (n = 43), for a clinical benefit rate (CR + PR + SD) of 53.8%. Pathogenic status of POLE mutation was associated with clinical benefit to PD-1/L1 inhibitors (p = 0.04). TMB (p = 0.44), PD-L1 (p = 0.11), and MSI (p = 0.66) status were not associated with pathogenic status. MSI-H status was not over-represented in pts with ICI clinical benefit (p = 0.36). Conclusions: Pathogenic POLE mutations were associated with clinical benefit to ICI therapy. Further studies are warranted to validate POLE mutations as a predictive biomarker. Multiple co-occurring DNA damage response mutations were found, which may contribute to ICI clinical benefit.

Clinically relevant genomic alterations identified by targeted exome sequencing in U.S. veterans with prostate cancer.

e17599 Background: Amongst male United States Veterans, prostate cancer accounts for one third of cancer diagnoses and is the second leading cause of cancer death. Prostate cancers in the Veteran population may have differing frequencies of clinically relevant genomic alterations than the general population owing to chemical exposures, military service, or other unknown factors. Veterans, however, have been underrepresented in large-scale prostate cancer sequencing cohorts to date. Methods: Archival or fresh prostate cancer tissue from Veterans cared for by our team within the VA Greater Los Angeles Healthcare System (a VA Prostate Cancer Center of Excellence) undergo targeted sequencing as part of routine clinical care through the VA Precision Oncology Program. Sequencing covers over 181 genes frequently mutated in cancers. Prostate cancers from 81 Veterans (76 primary tumors, 5 metastases) have been sequenced through the Personalis ACE CancerPlus platform. Results: 43% of Veterans had primary tumors with clinically relevant genomic alterations, including 6.2% with activating mutations in MAPK pathway members (KRAS, ERBB2, or BRAF), 3.7% with somatic mutations in DDR genes (BRCA2 or ATR), 7.3% with mutations in TP53 or RB1, 4.9% in APC, 1.2% in the WNT pathway (CTNNB1), 3.7% with mutations in the PI3K/AKT pathway (PIK3R1 or AKT1), 3.7% with PTEN deletions, and 22.2% had alterations involving an AR regulated gene (SLC45A3 or TMPRSS). Of the five metastatic tumors sequenced, one had a mutation in TP53 and another had an ETS gene fusion detected. Half of the Veterans who underwent tumor sequencing consented for screening for, or enrollment on, active clinical trials at VA Greater LA. Conclusions: Large-scale sequencing of prostate cancers within VA is feasible and may facilitate enrollment in precision oncology trials specifically designed for Veterans. These data suggest that clinically relevant genomic alterations within the primary tumors may differ between Veterans and the general population; larger data sets along with more robust sequencing platforms are required for clarification.

Panel-based targeted exome sequencing reveals novel candidate susceptibility loci for age-related cataracts in Chinese Cohort.

BackgroundAge‐related cataracts (ARC) is the most common blinding eye disease worldwide, and its incidence tend to become younger. However, the relationship between genetic factors and mechanisms is not fully understood. The aim of the study was to further clarify the relationship between ARC and genetic mechanisms in East Asian populations and to elucidate the pathogenesis.MethodsThe study collected 191 sporadic cataracts and 208 healthy people from the eastern provinces of China, with an average age of about 60 years. All participants were subjected to a comprehensive ophthalmic clinical examination and peripheral blood samples were collected and their genomic DNA was extracted. Mutations were screened among 792 candidate genes to enhance understanding of the disease through targeted capture and high‐throughput sequencing.ResultsWe identified novel candidate susceptibility gene, which may serve as a potential susceptibility factor leading to an increase in the incidence of age‐related cataracts. Three novel loci are associated with age‐related cataracts significant significance: rs129882 in DBH (p = 5.27E‐07, odds ratio = 3.9), rs1800280 in DMD (p = 2.85E‐06, odds ratio = 1.4) and rs2871776 in ATP13A2 (p = 4.18E‐05, odds ratio = 0.04). Gene–gene interaction analysis revealed that the most significant interactions between genes include the interaction between DBH and TUB (rs17847537 in TUB, rs129882 in DBH, p‐value = 2.12E‐14), and the interaction between DBH and DMD (rs1800280 in DMD, rs129882 in DBH, p‐value = 2.12E‐14). Pathway analysis shows that the most significant processes are concentrated in response to light stimulation (adjusted p‐Value = 5.56E‐03), response to radiation (adjusted P‐Value = 5.56E‐03), abiotic stimulus (adjusted p‐Value = 5.56E‐03). eQTL analysis shows that DBH rs129882 could regulate the expression of DBH mRNA in various tissues including retina.ConclusionOur study indicates rs129882 and rs1800280 loci are associated with age‐related cataracts, which enlarge the gene map of age‐related cataracts.

A novel missense mutation of CRYBB1 causes congenital cataract in a Chinese family.

To identify the pathogenic gene and mutation site of a Chinese family with congenital cataract. Eight family members and 100 controls were employed, and targeted exome sequencing was used to identify the genetically pathogenic factor of the proband. Targeted next-generation sequencing identified a novel missense mutation c.209A>C (p.Q70P) of CRYBB1 gene in the family. Sanger sequencing results showed that this heterozygous mutation was a causative mutation, which was not found in unaffected family members and healthy controls. Bioinformatics predicts that the effect of this mutation on protein function is probably harmful. We demonstrate that c.209A>C of CRYBB1 gene is a pathogenic mutation in the family of congenital nuclear cataract in this study. This is the first report that this mutation leads to congenital nuclear cataract, which broadens the mutation spectrum of CRYBB1 gene in congenital nuclear cataract.

Targeted Exome Sequencing Provided Comprehensive Genetic Diagnosis of Congenital Anomalies of the Kidney and Urinary Tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. The search for genetic causes of CAKUT has led to genetic diagnosis in approximately 5–20 % of CAKUT patients from Western countries. In this study, genetic causes of CAKUT in Korean children were sought using targeted exome sequencing (TES) of 60 genes reported to cause CAKUT in human or murine models. We identified genetic causes in 13.8% of the 94 recruited patients. Pathogenic single nucleotide variants of five known disease-causing genes, HNF1B, PAX2, EYA1, UPK3A, and FRAS1 were found in 7 cases. Pathogenic copy number variations of 6 patients were found in HNF1B, EYA1, and CHD1L. Genetic abnormality types did not significantly differ according to CAKUT phenotypes. Patients with pathogenic variants of targeted genes had syndromic features more frequently than those without (p < 0.001). This is the first genetic analysis study of Korean patients with CAKUT. Only one-seventh of patients were found to have pathogenic mutations in known CAKUT-related genes, indicating that there are more CAKUT-causing genes or environmental factors to discover.

Genetic and clinical findings of panel-based targeted exome sequencing in a northeast Chinese cohort with retinitis pigmentosa.

BackgroundPanel‐based targeted exome sequencing was used to analyze the genetic and clinical findings of targeted genes in a cohort of northeast Chinese with retinitis pigmentosa.MethodsA total of 87 subjects, comprising 23 probands and their family members (total patients: 32) with confirmed retinitis pigmentosa were recruited in the study. Panel‐based targeted exome sequencing was used to sequence the patients and family members, all subjects with retinitis pigmentosa underwent a complete ophthalmologic examination.ResultsOf the 23 probands, the clinical manifestations include night blindness, narrowing of vision, secondary cataracts, choroidal atrophy, color blindness, and high myopia, the average age of onset of night blindness is 12.9 ± 14 (range, 0–65; median, 8). Posterior subcapsular opacities is the most common forms of secondary cataracts (nine cases, 39.1%), and peripheral choroidal atrophy is the most common form of secondary choroidal atrophy (12 cases, 52.2%). Of these probands with complication peripheral choroidal atrophy, there were eight probands (66.7%, 8/12) caused by the pathogenic variation in USH2A gene. A total of 17 genes and 45 variants were detected in 23 probands. Among these genes, the commonest genes were USH2A (40%; 18/45), RP1 (15.6%; 7/45), and EYS (8.9%; 4/45), and the top three genes account for 56.5% (13/23) of diagnostic probands. Among these variants, comprising 22 (48.9%) pathogenic variants, 14 (31%) likely pathogenic variants, and nine (20%) uncertain clinical significance variants, and 22 variants was discovered first time. Most of the mutations associated with RP were missense (53.3%, 24/45), and the remaining mutation types include frameshift (35.6%, 16/45), nonsense (6.7%, 3/45), and spliceSite (4.4%, 2/45). Among the probands with mutations detected, compound heterozygous forms was detected in 13 (56.5%, 13/23) probands, and digenic inheritance (DI) forms was detected in five (21.7%, 5/23) probands.ConclusionPanel‐based targeted exome sequencing revealed 23 novel mutations, recognized different combinations forms of variants, and extended the mutational spectrum of retinitis pigmentosa and depicted common variants in northeast China.

Rare Variants in Genes Linked to Appetite Control and Hypothalamic Development in Early-Onset Severe Obesity.

Context: The hypothalamic circuit has an essential role in the regulation of appetite and energy expenditure. Pathogenic variants in genes involved in the hypothalamic leptin–melanocortin pathway, including melanocortin-4-receptor (MC4R), have been associated with monogenic obesity.Objective: To determine the rate and spectrum of rare variants in genes involved in melanocortin pathway or hypothalamic development in patients with severe early-onset obesity (height-adjusted weight >60% before age 10 years).Methods: We used a custom-made targeted exome sequencing panel to assess peripheral blood DNA samples for rare (minor allele frequency <0.5%), pathogenic/likely pathogenic variants in 24 genes related to the hypothalamic circuit in 92 subjects (51% males, median age 13.7 years) with early-onset severe obesity (median body mass index (BMI) Z-score + 4.0).Results: We identified a novel frameshift deletion in MC4R (p.V103Afs5*) in two unrelated patients and a previously reported MC4R variant (p.T112M) in one patient. In addition, we identified rare heterozygous missense variants in ADCY3 (p.G1110R), MYT1L (p.R807Q), ISL1 (p.I347F), LRP2 (p.R2479I, and p.N3315S) and a hemizygous missense variant in GRPR (p.L87M) (each in one patient), possibly contributing to the obesity phenotype in these patients. Altogether 8 % (7/92) of the subjects had rare pathogenic/likely pathogenic variants in the studied genes.Conclusions: Rare genetic variants within the hypothalamic circuit are prevalent and contribute to the development of severe early-onset obesity. Targeted exome sequencing is useful in identifying affected subjects. Further studies are needed to evaluate the variants' clinical significance and to define optimal treatment.

Rationale for the advancement of PI3K pathway inhibitors for personalized chordoma therapy.

Chordomas are rare and serious tumors with few effective treatments outside of aggressive surgery and radiation. Targeted therapies may present a more effective option for a subset of patients with lesions possessing certain genetic biomarkers. A small molecule inhibitor library was tested in patient-derived UM-Chor1 cells to identify targeted therapies with potential efficacy. Targeted exome sequencing of UM-Chor1 and UM-Chor2 cells was performed to investigate genetic aberrations in relevant pathways. Chordoma cell lines were treated with inhibitors of the phosphotidylinositol 3-kinase (PI3K), epidermal growth factor receptor (EGFR), and cyclin dependent kinase (CDK) pathways, and responses were determined using resazurin cell viability assays, Annexin V apoptosis assays, and western blotting. Pan-PI3K inhibitor BKM120 was also tested in five chordoma xenograft models. Unbiased small molecule profiling nominated PI3K-AKT-mTOR pathway inhibitors as a promising therapy in chordoma, and genetic analyses of UM-Chor1 and UM-Chor2 cell lines revealed aberrations in PTEN, EGFR, and CDKN2A. Treatment of UM-Chor1 and UM-Chor2 with targeted PI3K, EGFR, and CDK inhibitors inhibited growth and proliferation and induced apoptosis more robustly than imatinib, a currently used chordoma therapy. Furthermore, BKM120 significantly inhibited tumor growth in a subset of the xenograft models tested. Targeted therapies, especially those inhibiting PI3K, display promising effects in multiple chordoma cell line and xenograft models. Nevertheless, the limited effects of PI3K, EGFR, and CDK targeting agents in other models reveal the presence of resistance mechanisms, which motivates future research to both identify biomarkers of response and develop combination therapies.

Abstract P4-01-10: Targeted exome sequencing of circulating tumor cells in breast cancer

Abstract Background: Next-generation sequencing (NGS) has provided the means to comprehensively analyze somatic mutations in solid tumors. The major challenge is the accurate detection of somatic variants in low-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues and circulating tumor cells (CTCs) as white blood cells (WBCs) or other background cell populations confound variant identification. The aim of our study was to evaluate the feasibility of combining whole genome amplification (WGA) with hybridization capture-based, targeted exome sequencing in identifying clinically relevant variants in low-quality and limited input metastatic breast cancer (MBC) samples. Methods: For clinical samples, we collected 7.5 mL of blood in Streck tubes and FFPE biopsies from 5 newly diagnosed MBC patients. The CTCs were isolated with the ANGLE Parsortix using a 10um cassette within 24h of blood draw. WBCs from the same patients were profiled to eliminate germline contaminants. We used spiked cell lines (CTC mimics) as a positive control. MDA-MB-231 cells (n=50) were spiked into 7.5ml healthy donor whole-blood. We used peripheral blood not processed on the Parsortix as a negative control (no spiked cells). We performed exome sequencing on a total of 20 samples (experimental samples: spike-in MDA-MB-231 samples (S1 and S2, n=2), positive control bulk MDA-MB-231 cells (P1 and P2, n=2) and negative control WBCs (WL1 and WL2, n=2); clinical samples: CTCs (n=5), MBC (n=4) and WBCs (n=5)). We used the Repli-G WGA method and the SeqCap EZ MedExome Probes that targets the human exome for ~4600 medically relevant genes, followed by sequencing using the Illumina HiSeq2500. Somatic mutations were called with GATK MuTect2 in clinical samples and Samtools using Partek Flow for experimental samples. The variant annotation was performed using SnpEff. Results: The sequencing quality of all 6 spike-in experimental samples showed that &amp;gt;75% of the reads were overlapping in genomic features. The total number of variants identified in P1 and P2 were 69,954 and 69,848, respectively. The number of variants identified in S1 and S2 was 51,359 and 50,798 respectively. We found a substantial overlap of 67% (47,201/ 69,954) common variants in P1 and S1. Likewise, an overlap of 64% common variants (45,392/69,848) was detected in P2 and S2. We analyzed the pattern of somatic variants in both coding and noncoding regions, resulting in the identification of 8786 variants in MBC (n=4) and 638 variants in CTCs (n=5). With further filtering steps, we detected 1959 coding variants in MBC and 118 variants in CTCs. SnpEff predicted that 74% of these variants in CTCs (88/118) and 14% in MBC (274/1959) had a high impact on protein function. A total of 25 common genes were also identified in CTCs and MBC in which variants were found in protein-coding regions. We matched a list of genes having coding variants in CTCs and MBC with the actionable gene panel of a commercial genomic testing platform (FoundationOne) and a precision oncology database (OncoKB) cancer gene list. The CTCs (3/5 samples) harbored alterations in a total of 11 FoundationOne genes and 9 OncoKB genes. We identified 54 actionable genes that showed overlap with FoundationOne and 104 genes with OncoKB in all 4 MBC samples. The copy number alterations (CNAs) analysis showed a larger number of amplifications in CTCs and deletions in MBC. Conclusion: We established the feasibility of exome sequencing of medically relevant genes in detecting known and unknown variants in a breast cancer cell line spike in experiment. This workflow has further been validated on CTCs and MBC samples by sequencing both low-input material and low-quality FFPE samples. This approach has a high potential for the identification of the complete repertoire of mutations along with CNAs and for monitoring the presence of actionable alterations throughout the course of treatment. Citation Format: Pushpinder K. Bains, Daniel Campo, Tania B. Porras, Janice Lu, Julie E. Lang. Targeted exome sequencing of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-10.

Clinical analysis of seven cases with primary hyperoxaluria type 1 in children

Objective: To investigate the clinical, imaging and molecular characteristics of primary hyperoxaluria type 1 (PH1) in children and to sum up existing evidence for further understanding the phenotype-genotype correlation of infantile PH1. Methods: This retrospective analysis was based on the medical records of children with PH1 diagnosed by gene test in the Department of Nephrology, Guangzhou Women and Children's Medical Center from June 2016 to May 2019. Targeted exome sequencing was performed on tubular disease-related genes of the probands and Sanger sequencing was conducted to validate suspected pathogenic variants of family members. Logistic regression analysis of NC and CCr was adopted to show the relation between NC and renal function. The literature review was conducted, and the clinical, imaging and molecular biogenetic characteristics of the disease were analyzed and summarized. Results: A total of 7 children from 6 families were enrolled. The median age of onset was 5 months. The median age of diagnosis was 8 months. Five cases had progressed to end-stage renal disease (ESRD), one case had chronic kidney disease (CKD) stage 1, and the other one had CKD stage 2. Four cases died, one case maintained on hemodialysis, and the other two non-dialysis cases were followed up. Among the 7 cases, 4 patients had infantile PH1, 1 patient had child and adolescent type, 1 patient had family type and the other one had unknown classification. There were two siblings (the younger brother had uremia and the sister had normal renal function) who had the delayed diagnosis for 5 and 3 years respectively. All patients in this cohort had proteinuria and microscopic hematuria, but no patients had gross hematuria. Three cases had hypercalciuria. Comprehensive diagnostic imaging evaluation include CT scan, MR scan, radiography and ultrasound led to the diagnosis of nephrocalcinosis (NC) in 5 cases, including 4 cases of simple NL and 1 case of NC with nephrolithiasis (NL), 1 case of multiple NL and 1 case of microcrystal deposition in renal medulla. However, only one case of NC was identified by ultrasound, the other 4 cases of NC were identified by radiograph examination. In the logistic regression analysis involving NC and creatinine clearnce rate (CCr), the results showed that NC was an independent risk factor for renal dysfunction (OR 2.5, 95%CI 0.7-1.2, P<0.05). All the 7 cases had AGXT gene variant, including homozygous variant in 4 cases and compound heterozygous variant in 3 cases. A total of 9 variant genotypes were found, and exon 6 variants were found in 4 children. Among them, there were 3 cases with c.679_680delAA. To our knowledge, both c.679_680delAA and c.190A>T in the cohort have not been reported previously. Conclusions: Infantile PH1 is the most common type of PH1 in children, which progresses rapidly or even begins with renal failure, with poor prognosis. It is also highly heterogeneous in phenotype and genotype. NC is an independent risk factor leading to renal failure. Radiograph examination showed high specificity for the diagnosis of NC. At present, the misdiagnosis and delayed diagnosis of PH1 are still common in China. It is of great significance to carry out quantitative determination of uric oxalate in order to reduce the misdiagnosis rate and enhance follow-up technologies for evaluating the therapeutic effect.

Comprehensive Profiling of Poor-Risk Paired Primary and Recurrent Triple-Negative Breast Cancers Reveals Immune Phenotype Shifts.

Emerging data suggest immune checkpoint inhibitors have reduced efficacy in heavily pretreated triple-negative breast cancers (TNBC), but underlying mechanisms are poorly understood. To better understand the phenotypic evolution of TNBCs, we studied the genomic and transcriptomic profiles of paired tumors from patients with TNBC. We collected paired primary and metastatic TNBC specimens from 43 patients and performed targeted exome sequencing and whole-transcriptome sequencing. From these efforts, we ascertained somatic mutation profiles, tumor mutational burden (TMB), TNBC molecular subtypes, and immune-related gene expression patterns. Stromal tumor-infiltrating lymphocytes (stromal TIL), recurrence-free survival, and overall survival were also analyzed. We observed a typical TNBC mutational landscape with minimal shifts in copy number or TMB over time. However, there were notable TNBC molecular subtype shifts, including increases in the Lehmann/Pietenpol-defined basal-like 1 (BL1, 11.4%-22.6%) and mesenchymal (M, 11.4%-22.6%) phenotypes, and a decrease in the immunomodulatory phenotype (IM, 31.4%-3.2%). The Burstein-defined basal-like immune-activated phenotype was also decreased (BLIA, 42.2%-17.2%). Among downregulated genes from metastases, we saw enrichment of immune-related Kyoto Encyclopedia of Genes and Genomes pathways and gene ontology (GO) terms, and decreased expression of immunomodulatory gene signatures (P < 0.03) and percent stromal TILs (P = 0.03). There was no clear association between stromal TILs and survival. We observed few mutational shifts, but largely consistent transcriptomic shifts in longitudinally paired TNBCs. Transcriptomic and IHC analyses revealed significantly reduced immune-activating gene expression signatures and TILs in recurrent TNBCs. These data may explain the observed lack of efficacy of immunotherapeutic agents in heavily pretreated TNBCs. Further studies are ongoing to better understand these initial observations.See related commentary by Savas and Loi, p. 526.

Genomic alterations in colitis-associated cancers in comparison to those found in sporadic colorectal cancer and present in precancerous dysplasia.

191 Background: Patients with inflammatory bowel disease (IBD) [Crohn’s disease (CD) and ulcerative colitis (UC)] are at increased risk for small bowel or colorectal cancers (Colitis Associated Cancers, CAC). Currently CAC is treated the same as sporadic colorectal cancer (CRC) with significantly shorter overall survival for advanced CAC compared to matched patients with CRC. In a pilot series, we found that tumors developing in IBD have distinct genomic alterations (GA) with potential implications for early detection and treatment. We now extend this analysis and characterize the relationship of GA in synchronous dysplasia and cancer. Methods: 104 CAC (54 UC-associated and 50 CD-associated) were sequenced with targeted-exome sequencing of &gt; 300 cancer-related genes. GA in CAC were compared to those reported for sporadic CRC. Whole exome sequencing was performed on paired mucosa, dysplasia and carcinoma samples obtained from 15 colectomy specimens; in these cases, expert pathology review confirmed normal appearing mucosa intervening between areas of dysplasia and CAC. Results: TP53 mutations (89%), MYC amplifications (24%), and cell cycle copy number alterations (20%) were significantly enriched in CAC compared to sporadic CRC, while APC alterations (21%) were significantly less common in CAC compared to sporadic CRC. Distinct GA in CAC consisted of IDH1 R132 mutations (7%) and FGFR pathway alterations (7%). While IDH1 R132 mutations and PI3K pathway alterations were more common in CD-associated CAC, MAPK ( BRAF/ MEK1) alterations were more common in UC-associated CAC. GA in CACs did not significantly vary by duration of preceding IBD. GA were often shared in dysplasia and carcinoma, despite normal-appearing intervening mucosa. GA involving TP53, APC, KRAS, and IDH1 mutations were identified in dysplasia and shared between CAC and dysplasia. Conclusions: CAC exhibit distinct GA compared to sporadic CRC with near universal TP53 mutation, increased copy number alterations involving transcription factors and cell cycle genes, and unique drivers. A field effect can be seen for GA between distant dysplasia and carcinoma, but many GA remain private to CAC.

Genomic characterization of rectal cancer and molecular determinants of response to neoadjuvant chemoradiotherapy.

192 Background: Rectal cancers are clinically different from colon cancers and have not yet been molecularly characterized due to the scarcity of pretreatment specimens. Discovery of molecular determinants of response to chemotherapy and radiation in patients with locally advanced rectal cancer (LARC, stage II and III) are needed to select those who can avoid surgery and benefit from watch-and-wait strategies. Methods: We profiled 371 pre-treatment specimens using targeted-exome sequencing of 468 cancer genes (n = 325), whole-exome sequencing (n = 100), and RNA-sequencing (n = 113). The targeted-sequencing cohort included patients with stage I (n = 44), II (n = 41), III (n = 176), and IV (n = 64) disease. Primary tumors were divided into lower (LR: 0-4 cm to anal verge, n = 62), middle (MR: 4-8 cm, n = 115), and upper rectum (UR: 8-12 cm, n = 107). We examined molecular determinants of complete response (CR) and relapse free survival (RFS) in LARC patients treated with chemoradiotherapy only (CRT: n = 39), induction chemotherapy + CRT (INCT: n = 87) and consolidation chemotherapy after CRT (CCNT: n = 63). Results: Among MSS cases, oncogenic gene and signaling pathway alterations did not vary by clinical stage. WNT pathway alterations, driven by APC mutations, were more frequent in the UR (89% UR v 86% MR v 60% LR, p &lt; 0.001) while RTK/RAS alterations were more frequent in the LR (54% UR v 69% MR v 72% LR, p &lt; 0.03). A set of genes enriched in mTOR signaling, G2M checkpoint, EMT transition, and DNA repair were overexpressed in the UR (FDR &lt; 0.1). The 5-yr RFS rate for LARC was 75% (CI: 68%-82%) and 24% of the cases had a CR (n = 45). MSI cases had a higher rate of CR compared to MSS cases (50% v 23%, p = 0.07) and none relapsed (n = 8). KRAS-altered MSS tumors exhibited worse RFS in cases treated with CNCT (5-yr: 74% v 97%, p = 0.01), but not in cases treated with INCT (5-yr: 67% v 72%, p = 0.7). Conclusions: WNT alterations are more frequent in the UR while RTK/RAS alterations are more frequent in the LR, suggesting differences in tumor biology between proximal and distal rectal cancer. Further, we report correlations between distinct molecular profiles and response to treatment paradigms that could guide the design of future clinical trials.