Targeted Capture Next-generation Sequencing Research Articles

Abstract 2424: Detection of prostate cancer in urine by fragment-level DNA methylation analysis

Abstract Introduction: Screening for prostate cancer (PCa) with molecular markers is hindered by low tumor allele fraction (TAF) in liquid biopsy samples such as blood or urine. Others have suggested that tumor allele enrichment can be achieved by evaluating fragment-level methylation patterns generated by next generation sequencing (NGS). Here, we test this concept using target capture NGS on cfDNA from prediagnostic collected urine samples to discriminate PCa from otherwise healthy men. Methods: Buffered urines were collected from 23 patients with biopsy confirmed PCa (Gleason score 6-9) and 24 otherwise healthy age matched cancer-free controls. cfDNA was isolated and enzymatic methyl-seq libraries were prepared and enriched by hybrid capture using probes mapping to 197 differentially methylated regions (DMRs) from a previous PCa/normal tissue marker discovery study in our lab. These DMRs were subsequently vetted independently using a methylome-wide dataset from a cohort of 36 clean and disease control urines to ensure low non-cancer background signal (<1%). Indexed libraries were sequenced at 560X deduplicated coverage and fragment-level CpG methylation information was extracted from individual sample .bam files. Mapped sequencing reads exhibiting concordant methylation were used to derive a quantitative α-score. Reads had to contain at minimum 5 CpGs and have an α-value (% 5mC per read) > 60%. Positivity for the 197 markers required an α-score greater than 6 sigma (σ) above the mean of the control arm. Values were variance-adjusted to ensure equal weighting. Beta (β) analysis (mean % 5mC across all reads) of the data was also performed for comparison. Results: Of the 197 markers, 187 were positive for one or more PCa patients. Tumor-derived fragments were detected in 22 of 23 cases, which included 8 G-6, 9 G-7, 2 G-8, and 3 G-9 patients. 1 G-7 was missed, however the sequencing coverage for this sample was significantly lower than the others due to poor library enrichment. 2 of the 24 healthy controls were positive, one with extremely high fragment counts. Upon re-review of the full control cohort, this patient had been diagnosed with bladder cancer 2 years after urine collection. The 2nd patient had was diagnosed with lung cancer in a similar timeframe. The other 22 confirmed cancer-free controls had α-scores below the 6σ threshold. Using a co-captured reference gene (B3GALT6) to mimic genomic equivalents, the TAFs ranged from 0.015% to 8%. β analysis of the data detected only 13 PCa patients (>6σ) and marker positivity was lower than the fragment-level approach. Conclusion: Target capture NGS coupled with fragment-level analysis is a rational approach for sensitive and specific detection of low tumor-derived DNA in liquid biopsy samples. For PCa assay in urine, we were able to uncover positivity in cancer patients approaching 1 in 10,000 ctDNA dilution levels. These results warrant further validation studies with this and other cancers. Citation Format: Tibor Bedekovics, William R. Taylor, Zhifu Sun, Saurabh Baheti, Xiaoming Cao, Kelli N. Burger, Karen A. Doering, Patrick H. Foote, Benjamin Gochanour, Douglas, W. Mahoney, Matthew T. Gettman, John B. Kisiel. Detection of prostate cancer in urine by fragment-level DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2424.

Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing.

Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

Validation of Circulating Tumor DNA As an Alternative to Tumor Tissue Biopsy in Genotyping across Multiple Lymphoma Subtypes

Introduction Genotyping plays an important role in the diagnosis, risk stratification and treatment response surveillance of lymphoma, a disease with considerable heterogeneity in clinical and pathological characteristics. To date, tumor tissue biopsy based genotyping is still considered as gold standard in lymphoma, but its application is hampered by the invasive nature and imperfect performance in capturing spatial and temporal tumor heterogeneity. Circulating tumor DNA (ctDNA), which is shed into peripheral blood by apoptotic or necrotic tumor cells, has shown the potential as a noninvasive alternative to tumor tissue DNA (tDNA) for molecular profiling. Many efforts have been made to investigate the feasibility of ctDNA based on next-generation sequencing (NGS) in lymphoma, but most of the studies were conducted in Caucasian population and limited to B-cell lymphoma patients. In the present study, we evaluated the performance of plasma-derived ctDNA against tDNA based genotyping across multiple lymphoma subtypes. Methods Between 4/2021 and 5/2023, a total of 110 patients were enrolled in this study. Matched tumor tissue biopsies and peripheral blood were collected at diagnosis. Both tDNA and ctDNA were extracted and subjected to a targeted capture NGS assay covering the entire coding region or recurrently mutated region of 196 genes. The mean coverage depth was 2431x and 2513x for tDNA and ctDNA, respectively. The limit of detection (LOD) was 1% and 0.5% for tDNA and ctDNA, respectively. Results The enrolled cohort comprised of 63 (57.3%) male and 47(42.7%) female patients, with a median age of 58 years old (range 1.25-89). Among the 110 patients, the most prevalent subtype is diffuse large B-cell lymphoma (DLBCL，n=45), following by angioimmunoblastic T-cell lymphoma (AITL, n=16), anaplastic large cell lymphoma (ALCL, n=13) , mantle cell lymphoma (MCL, n=10), follicular lymphoma (FL, n=9), primary mediastinal large B-cell lymphoma (PMBL, n=9), extranodal natural killer/T cell lymphoma (ENKTL, n=4), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS, n=4). For further analysis, DLBCL, MCL, FL and PMBL were categorized as B-cell lymphoma group (n=73), while AITL, ALCL, ENKTL and PTCL-NOS were categorized as T-cell lymphoma group (n=37). Mutations were detected in 109（99.10%）tDNA and 110（100%）ctDNA, respectively. A total of 1586 mutations involving 177 genes were detected in tDNA, and the median number of mutations per sample was 12 (range 0-62), with KMT2D (39.09%), KMT2C (30.90%), IGLL5 (30.00%), TET2 (25.45%) and TP53 (20.90%) being the top 5 mutated genes. For ctDNA, a total of 1750 mutations involving 178 genes were detected and the median number of mutations per sample was 14 (range 2-63), with KMT2D (37.27%), KMT2C (29.09%), IGLL5 (27.27%), TET2 (25.45%) and DNMT3A (27.27%) being the top 5 mutated genes. To determine the concordance of mutations detected in tDNA and ctDNA, sensitivity was calculated utilizing tDNA as gold standard. The overall sensitivity of ctDNA based genotyping was 85.39% (95%CI: 82.36%-88.41%), with B-cell lymphoma showing a slightly higher sensitivity than T-cell lymphoma (86.17%, 95%CI: 83.51%-88.83% vs. 83.84%, 95%CI: 76.30%-91.32%). In terms of subtypes, PTCL-NOS exhibited the highest sensitivity (89.58%, 95%CI: 56.43%-122.73%, while AITL exhibited the lowest sensitivity (78.53%, 95%CI: 64.09%-92.97%) (Figure 1A). In addition, the difference of mutational spectrum between B-cell lymphoma and T-cell lymphoma was precisely depicted by ctDNA based genotyping. We discovered mutations in TET2, RHOA, and PLCG1 were significantly present in T-cell lymphoma, while mutations in MYD88, CD79B, CARD11, CREBBP, MYC, PIM1, IGLL5, ATM, SOCS1 and HIST1HIE were significantly enriched in B-cell lymphoma (Figure 1B). Conclusion In the present study, recurrent mutations across a variety of lymphoma subtypes were identified by ctDNA based genotyping, which were in high concordance with tDNA based genotyping. These data demonstrated that ctDNA based genotyping is a reliable noninvasive alternative to tumor tissue biopsy for molecular profiling of lymphoma patients, especially for patients whose tissues are not accessible for genotyping.

Differences of RAS/BRAF mutations between ISCC and RSCC in a Chinese population.

e15636 Background: According to the location and different clinical manifestations, colon cancer can be divided into left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC). There are significant differences in morphology, molecular biology, and drug sensitivity between these two type. In addition, colorectal cancer is associated with the mutation of many essential tumor suppressor genes, such as proto-oncogene (KRAS), serine/threonine protein kinase (BRAF), etc. At that point, this study retrospectively analyzed the RAS/BRAF mutation landscape and studied the gene expression differences between RAS/BRAF-mutant and wild-type patients respectively in LSCC and RSCC, to provide some reference for potential treatment plans. Methods: We retrospectively profiled the somatic mutations of 497 patients with colon cancer from the General Hospital of Southern Theatre Command and the TCGA database using 808-gene-panel target-capture next-generation sequencing. Tumor mutation burdens (TMB) were calculated using an Acornmed panel with 808 cancer-related genes. Results: Among 415 LSCC, 48.7% (n = 202) patients harbored RAS mutations, of which 183 carried KRAS mutations and 19 had NRAS mutations. Five main subtypes of KRAS mutation were detected including G12D (28.7%), G12V (17.5%), G13D (13.2%), A146T(8.5%), G12C (7.9%). 3% (11) of LSCC patients were found to have BRAF mutation, including BRAF V600E(63.6%), D594G(18.2%), G466E(9.1%), K601E(9.1%). We explored differentially expressed genes between RAS/BRAF-mutant and wild-type patients. The top5 mutant genes found only in LSCC RAS/BRAF-mutant cohort include JAK2(3%), TERT(2%), MSH6(2%), KMT2A(1%), NOTCH1(1%). In contrast, genes found only in LSCC patients with RAS/BRAF wild type include LATS2(2%), SMARCA4(2%), CBL(1%), SLX4(1%), CDKN2A(1%). Among 82 RSCC, 50% (41) patients harbored RAS mutations, of which 39 carried KRAS mutations and 2 carried NRAS mutations. 9% (39) of RSCC patients were found BRAF mutation, and the BRAF V600E was the only subtype found in the RSCC. In terms of differentially expressed genes, the top5 mutant genes found only in RAS/BRAF-mutant RSCC include NSD1(6%), ASXL2(4%), BARD1(4%), PPP2R1A(4%), SPRED1(4%), while genes found only in RAS/BRAF wild-type RSCC include ERBB2(17.6%), DICER1(11.8%), EP300(11.8%), ARID2(9%), BRIP1(9%).The TMB of RAS/BRAF wild-type RSCC patients was 33.2 mut/Mb, significantly higher than that of RAS/BRAF wild-type LSCC patients (7.0 mut/Mb, P = 0.01). Conclusions: RAS mutation and subtype were almost the same in LSCC and RSCC cohorts. BRAF mutation mainly occurred in RSCC, and the mutation subtype in RSCC was found only V600E, but we found three other BRAF subtypes D594G, G466E, and K601E in the LSCC cohort. Considering the differential expression genes in RAS/BRAF-mutant and wild-type cohorts in RSCC and LSCC patients, there may be more options for genetic targets in the future for LSCC and RSCC treatment.

Distribution and concordance of PD-L1 expression by routine 22C3 assays in East-Asian patients with non-small cell lung cancer

BackgroundCurrently, programmed death ligand-1 (PD-L1) expression has been widely applied in clinical trials and real-world clinical practice as a major biomarker for the efficacy of immune-checkpoint inhibitors. The purpose of this study is to reveal the distribution and concordance of PD-L1 expression in a large-scale consecutive cohort from East-Asian patients with non-small cell lung cancer (NSCLC).MethodsPD-L1 testing was conducted using 22C3 assays, and cases were categorized into the high, low, and no expression of PD-L1 based on the tumor proportion score (TPS). Target-capture next-generation sequencing was used to identify molecular events.ResultsA total of 4550 patients and 4622 tests of PD-L1 expression were enrolled. There were 3017 (66.3%) patients with no PD-L1 expression (TPS < 1%), 1013 (22.3%) with low PD-L1 expression (TPS 1–49%), 520 (11.4%) with high PD-L1 expression (TPS ≥ 50%). Higher proportions of positive PD-L1 expression (TPS ≥ 1%) were observed in smokers, males, squamous cell carcinoma, and high-grade lung adenocarcinoma. Further analyses revealed fair agreement in primary and metastatic lesions (kappa = 0.533), poor agreement in multi-focal primary tumors (kappa = 0.045), and good agreement in biopsy and resection samples (kappa = 0.662) / two biopsy samples (kappa = 0.711). Mutational analyses revealed association between high PD-L1 expression (TPS ≥ 50%) and EGFR wild-type, KRAS mutation, ALK rearrangement, and TP53 mutation.ConclusionThe study reveals the unique distribution pattern of PD-L1 expression in a large-scale East-Asian cohort with NSCLC, the concordance of multiple PD-L1 tests, and the association between PD-L1 expression and molecular events. The results shed a light on the optimization of PD-L1 testing in clinical practice.

Genetic analysis of a child with XYY syndrome in conjunct with 3-methylglutaenedioic aciduria type I

To explore the genetic basis for a child with mental retardation. The child was subjected to chromosomal microarray analysis (CMA) and targeted capture next-generation sequencing for the exons of genes related to genetic and metabolic diseases. Candidate variants were verified by Sanger sequencing of the child and his parents. CMA suggested that the child has a 47,XYY karyotype. Next-generation sequencing revealed that the child has harbored compound heterozygous variants of the AUH gene, including c.677G>A (p.R226H) and c.373C>T (p.R125W), which were respectively inherited from his parents. Based on the American college of Medical Genetics and Genomics (ACMG) standards and guidelines, the c.677G>A (P.r226h) variant was predicted as variant of uncertain significance (PM2+PP4+PP3), whilst the c.373C>T (P.R125W) variant was predicted as likely pathogenic (PM1+PM2+PP3+PP4). The child had XYY syndrome in conjunct with 3-methylglutaenedioic aciduria type I due to biallelic pathogenic variants of the AUH gene.

A pancancer analysis of KRAS alterations in Chinese population.

e15127 Background: Kirsten rat sarcoma viral oncogene homologue (KRAS) is the most frequently mutated oncogene in human cancers. Sotorasib (AMG 510) has shown promising anticancer activity in patients with heavily pre-treated KRAS p.G12C mutant solid tumors. Here, we provide an overview of genomic diversity of KRAS alterations in a large cohort of Chinese patients. Methods: Molecular profiles of 16,654 tumor specimens were obtained using gene-panel target-capture next-generation sequencing, and classified based on the presence and subtypes of KRAS mutation. Tumor mutation burdens (TMB) were calculated using an Acornmed panel with 808 cancer-related genes. Results: In total,15.04% (2505/16654) of pan-cancer patients harbored RAS mutations, and 13.58% (2262/16654) patients carried KRAS mutations in the current study. Five main subtypes of KRAS mutation were detected including G12D (642, 28.38%), G12V (392, 17.33%), G12C (354, 15.65%), G13 (220, 9.73%), G12A (101, 4.47%). In our cohort, the highest KRAS G12C mutation frequency tumor type was lung cancer (12.33%) followed by colorectal cancer (1.99%), which is comparable with the previously published data. Among all patients with KRAS G12C mutation, 77.40% (274/354) of the patients were male and 22.60% (80/354) were female. Tumor mutation burdens (TMB) were calculated for 151 samples of all the 352 KRAS G12C mutation samples. The median TMB was 10.15 mut/Mb with 50.99%≥10 mut/Mb and 14.57%≥20 mut/Mb. However, according to the results of CodeBreaK 100 exploratory analysis at the 2021 ASCO annual meeting, among all patients with KRAS G12C mutation, 17.86% of the patients had ≥ 10 mut /Mb, which was defined as TMB-high. Therefore, it can be found that the TMB of domestic and foreign patients is different. Conclusions: Genomic alterations in KRAS are widespread among Chinese pan-cancer patients. Results of CodeBreaK 100 exploratory analysis presented at the 2021 ASCO Annual Meeting showed no significant difference (40% vs 42%) in objective response rates between TMB high (≥ 10 mut/Mb) and TMB low (&lt; 10 mut/Mb). Based on our data, the threshold of TMB high in the Chinese population should be higher. The molecular marker exploration of AMG 510 efficacy in the Chinese population needs to be further carried out and confirmed.

Targeted-Capture Next-Generation Sequencing in Diagnosis Approach of Pediatric Cholestasis.

Background: Cholestasis is a frequent and severe condition during childhood. Genetic cholestatic diseases represent up to 25% of pediatric cholestasis. Molecular analysis by targeted-capture next generation sequencing (NGS) has recently emerged as an efficient diagnostic tool. The objective of this study is to evaluate the use of NGS in children with cholestasis. Methods: Children presenting cholestasis were included between 2015 and 2020. Molecular sequencing was performed by targeted capture of a panel of 34 genes involved in cholestasis and jaundice. Patients were classified into three categories: certain diagnosis; suggested diagnosis (when genotype was consistent with phenotype for conditions without any available OMIM or ORPHANET-number); uncertain diagnosis (when clinical and para-clinical findings were not consistent enough with molecular findings). Results: A certain diagnosis was established in 169 patients among the 602 included (28.1%). Molecular studies led to a suggested diagnosis in 40 patients (6.6%) and to an uncertain diagnosis in 21 patients (3.5%). In 372 children (61.7%), no molecular defect was identified. Conclusions: NGS is a useful diagnostic tool in pediatric cholestasis, providing a certain diagnosis in 28.1% of the patients included in this study. In the remaining patients, especially those with variants of uncertain significance, the imputability of the variants requires further investigations.

Phenotype and genetic variant analysis of seven pedigrees affected with blepharophimosis syndrome

To analyze the clinical manifestations and gene variants of patients with blepharophimosis, ptosis and epicanthus inversus syndrome (BPES). Clinical data of 7 pedigrees affected with BPES were collected, and genomic DNA was extracted from peripheral blood samples of the probands and their relatives. All exons of the FOXL2 gene were subjected to Sanger sequencing. Those with negative findings were further screened by targeted capture and next generation sequencing (NGS) and microarray analysis. Pathogenicity of candidate variants were predicted by search of PubMed and related databases, and the impact of the variants was interpreted by protein prediction software. Diagnosis was confirmed by clinical phenotype, medical history and mutation analysis. A pathogenic variant was identified in six of the 7 pedigrees, which included four known pathogenic variants and one novel FOXL2 c.299dupA variant. A heterozygous 3q22.3q23 deletion, which encompassed the FOXL2 gene, was identified in another pedigree.As predicted, the c.299dupA frameshift mutation of FOXL2 gene can lead to the premature termination of protein translation, which is pathogenic. A novel and 5 known pathogenic variants have been identified in six pedigrees affected with BPES by the combined Sanger sequencing, target capture NGS and microarray analysis. Above findings have enabled genetic counseling and prenatal diagnosis for these pedigrees.

Target capture sequencing of SARS-CoV-2 genomes using the ONETest Coronaviruses Plus

We introduce a target capture next-generation sequencing methodology, the ONETest Coronaviruses Plus, to sequence the SARS-CoV-2 genome and select loci of other respiratory viruses. We applied the ONETest on 70 respiratory samples (collected in Florida, USA between May and July, 2020), in which SARS-CoV-2 had been detected by a PCR assay. For 48 of the samples, we also applied the ARTIC protocol. Of the 70 ONETest libraries, 45 (64%) had a (near-)complete sequence (>29,000 bases and >90% covered by >9 reads). Of the 48 ARTIC libraries, 25 (52%) had a (near-)complete sequence. In 19 out of 25 (76%) samples in which both the ONETest and ARTIC yielded (near-)complete sequences, the lineages assigned were identical. As a target capture approach, the ONETest is less prone to loss of sequence coverage than amplicon approaches, and thus can provide complete genomic information more often to track and monitor SARS-CoV-2 variants.

Phenotype and genotype analysis of patients with severe factor XI deficiency in Shaanxi Province, China.

Congenital coagulation factor XI (FXI) deficiency is a rare bleeding disorder with a heterogeneous haemorrhagic phenotype and various hotspot gene mutations associated with race and geography. Studies on FXI deficiency in Shaanxi Province, China, are scarce. In this study, seven patients with severe FXI deficiency and several family members were analysed. The International Society on Thrombosis and Hemostasis-Bleeding Assessment Tool (ISTH-BAT) was applied to assess bleeding symptoms. FXI activity was determined using a one-stage method, and the FXI antigen was measured by enzyme-linked immunosorbent assay. Targeted capture next-generation sequencing and Sanger sequencing were applied to detect FXI gene mutations. The bleeding phenotype varied, although none of the participants had a history of spontaneous bleeding. One maternal received replacement therapy during the perinatal period, one female patient presented with menorrhagia, one male patient experienced severe postoperative bleeding and others were asymptomatic. Family members with heterozygous mutations were all asymptomatic. The FXI activity of all the patients ranged from less than 1 to 3.1 IU/dl, and a synchronous decrease in the FXI antigen was observed. Two missense mutations (p. Gly350Glu and p. Cys482Trp), one nonsense mutation (p. Gln384∗) and one novel frameshift mutation (p. Ser225Phefs∗16) were detected. The bleeding manifestations and severity of severe FXI deficiency varied and were not related to its activity. Three reported mutations and one novel frameshift mutation were identified, thus extending the mutation spectrum of FXI deficiency.

Targeted Deep Sequencing in Blast Phase Myeloproliferative Neoplasms

Background: We have previously published our observations regarding targeted next generation sequencing (NGS) in primary myelofibrosis (PMF), essential thrombocythemia (ET) and polycythemia vera (PV) (Blood Advances 2016 1:105; Blood Advances 2016 1:21). In the current study, we took a similar approach in order to examine the mutational repertoire in blast phase myeloproliferative neoplasms (MPN-BP) and explore its clinical correlates, including phenotype, treatment response and survival.Methods:Diagnoses of MPN, including PMF, PV, ET and MPN-BP were according to WHO criteria (Blood 2016;127:2391). The study population was selected on the basis of the availability of sufficient DNA and was not otherwise biased by any other selection criteria. Targeted capture assays were carried out on bone marrow or whole blood DNA, according to previously published methods (Blood Advances 2016 1:105). Statistical analyses considered clinical and laboratory data collected at the time of blast transformation. Survival was calculated from the date of leukemic transformation to the date of death or last contact. Treatment response in MPN-BP was assessed according to conventional criteria (JCO 2003;21:4642).Results:72 consecutive cases of MPN-BP underwent target capture NGS using a panel of 39 genes. •Mutational landscape, frequencies and co-segregation patternsDriver mutations were detected in 62 (86%) patients, including JAK2 only in 39 (54%), JAK2 + CALR 3 (4%), JAK2 + MPL 3 (4%), CALR only 10 (14%), CALR + MPL 2 (3%) and MPL only 5 (7%). Sixty-six (92%) patients displayed at least one mutation/DNA variant, other than JAK2 / CALR / MPL ; the number (%) of patients with 0, 1, 2, 3, 4, 5, 6 or 7 such mutations was 6 (8%), 9 (13%), 19 (26%), 19 (26%), 13 (18%), 5 (7%), 0 and 1 (1.4%). The most frequent mutations (incidence), other than JAK2 / CALR / MPL were ASXL1 (46%), RUNX1 (22%), TET2 (21%), TP53 (17%), EZH2 (15%), IDH1 (14%), SRSF2 (14%), FLT3 (13%), and SH2B3 (11%). JAK2 mutations clustered with ASXL1 (p=0.02); MPL with IDH1 (p=0.0004) and SF3B1 (p=0.008); ASXL1 with EZH2 (p=0.009); RUNX1 with SRSF2 (p=0.02) and SH2B3 (p=0.04); SRSF2 with IDH2 (p=0.002); IDH2 with SH2B3 (p=0.04); and EZH2 with DNMT3A (p=0.0007). •Clinical course and managementAt the time of this writing, complete clinical and follow-up data were available in 66 patients with MPN-BP (median age 65 years; 65% males). Induction chemotherapy for MPN-BP was given to 38 patients, resulting in complete remission (CR) or CR with incomplete recovery of counts (CRi) in 14 (37%) patients. Treatment for MPN-BP included allogenic stem cell transplant (ASCT) in 6 patients. Median follow-up from time of leukemic transformation was 3.9 months with 64 documented deaths (97%). •Impact of mutations on survival and treatment response ratesIn univariate analysis, adverse impact on survival was demonstrated for TET2 (p=0.03), DNMT3A (p=0.05), PTPN11 (p=0.008) and SETBP1 (p=0.01) mutations, which were subsequently grouped as “adverse mutations”. Twenty-one (32%) patients harbored at least one adverse mutation. Patients with or without adverse mutations were similar in terms of their age and gender distribution, clinical characteristics at time of leukemic transformation and karyotype (P>0.05 in all instances). Spectrum and frequency of adverse mutations were similar among specific driver mutations, including triple-negative cases (p=0.97).The presence of adverse mutations was strongly predictive of shortened survival (HR 2.8, 95% CI 1.6-5.0; p=0.0003; Figure 1) and the effect was independent of several other clinical and laboratory variables including age, karyotype, specific MPN-BP morphologic category, blast burden and treatment undertaken for MPN-BP. In multivariable analysis, survival was independently affected by presence of adverse mutations (HR 4.8, 95% CI 2.0-11.8) and achievement of CR/CRi (HR 0.4; 95% CI 0.2-0.8), but not by ASCT. The presence of adverse mutations did not affect response rate to induction chemotherapy (p=0.6).Conclusions:The current study identifies the presence of prognostically-adverse mutations in approximately a third of patients with blast phase MPN (MPN-BP). The survival effect of adverse mutations was independent of other disease features and treatment strategies, including whether or not patients achieved CR/CRi or received ASCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract P4-10-27: Genomic aberrations in circulating tumor DNAs associated with progression in palbociclib-treated metastatic breast cancer patients

Abstract Background: Palbociclib is the first-in-class cyclin dependent kinase (CDK) 4/6 inhibitor approved for the treatment of patients (pts) with hormone receptor (HR) positive/human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancer (MBC). Despite an initial response, pts eventually develop resistance to therapy; however, the underlying molecular mechanisms remain elusive. This study aimed to identify genomic aberrations in circulating tumor DNA (ctDNA) associated with treatment resistance and disease progression in MBC pts receiving palbociclib. Methods: Blood samples were collected from pts with HR+/HER2− MBC who were treated with palbociclib plus endocrine therapies (letrozole or fulvestrant). Circulating cell-free DNAs were isolated from plasma and assayed by targeted-capture next-generation sequencing using an in-house developed panel that covers the exons in 92 breast cancer-related genes. Unique molecular index (UMI)-based sequencing was performed using the Illumina HiSeq 4000 system. Progression free survival (PFS) differences between pts with different mutation status were compared using the log-rank test. The Cox proportional hazards model with time-dependent covariates was used to evaluate associations between mutations and PFS. Results: The study included 16 pts with a median age of 54.7 years (range: 32.6-74.8 years). A total of 42 blood samples were collected, including 16 baseline samples before the initiation of palbociclib +/- endocrine therapies (within 1.5 months prior to initiation), 10 samples during treatment (1.0-3.9 months after initiation), and 16 after treatment completion (within 1.2 months after completion). During a median follow-up of 23.7 weeks (range: 6.0-99.6 weeks), 8 pts had disease progression at initial blood collection after treatment initiation, and 11 had disease progression at final blood collection during follow-up. Among the 3 pts without progression, 2 were lost to follow-up and 1 had a change in regimen due to adverse events. Mutations were detected in 77 out of 92 genes. Mutations were identified for PIK3CA, ALK, ERBB2, NOTCH1, IDH1 in the baseline samples of 7, 3, 7, 6, and 12 patients, respectively. A shorter time to progression was observed in the pts with PIK3CA, ALK, ERBB2, or NOTCH1 mutations (log-rank P = 0.011, 0.016, 0.057, and 0.059, respectively), whereas those pts with IDH1 mutations had a prolonged PFS (log-rank P = 0.005). Similar results were obtained when the events and survival time at the final blood collection were used for analyses (log-rank P = 0.007, 0.004, 0.046, 0.023, and 0.001 for PIK3CA, ALK, ERBB2, NOTCH1, and IDH1 mutations, respectively). To analyze the association between a given mutation and disease progression, we separated pts into four risk groups according to the status change of the mutation prior to and after treatment: Group 1, persistent absence of the mutation; Group2, loss of the mutation after treatment; Group 3, acquisition of the mutation after treatment; Group 4, persistent presence of the mutation. Longitudinal analyses showed that PIK3CA E542K, ERBB2 P1035H, and NOTCH1 K1069Q mutations were associated with unfavorable survival. For PIK3CA E542K mutation, pts in Group 2 had an increased risk for progression, as compared with those in Group 1 (hazard ratio [HR] =9.90, P = 0.110). For ERBB2 P1035H and NOTCH1 K1069Q mutations, when compared with pts in Group 1, those in Group 4 had the highest risk of progression (HR = 17.24, P = 0.049; HR = 19.17, P = 0.043, respectively), whereas pts in Group 2 had a non-significant slightly increased risk. Conclusions: Results of this study show that genomic aberrations in ctDNA were associated with progression in MBC pts receiving palbociclib-containing regimens. Further research is needed to validate these findings in larger independent studies. Citation Format: Maysa M Abu-Khalaf, Chun Wang, Zhong Ye, Zhenchao Zhang, Daniel Silver, Frederick Fellin, Saveri Bhattacharya, Rebecca Jaslow, Terrence Cescon, AnaMaria Lopez, Ronald Myers, Qiang Wei, Bingshan Li, Hushan Yang. Genomic aberrations in circulating tumor DNAs associated with progression in palbociclib-treated metastatic breast cancer patients [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 P4-10-27.

Next-Generation Sequencing of Retinoblastoma Identifies Pathogenic Alterations beyond RB1 Inactivation That Correlate with Aggressive Histopathologic Features

To determine the usefulness of a comprehensive, targeted-capture next-generation sequencing (NGS) assay for the clinical management of children undergoing enucleation for retinoblastoma. Cohort study. Thirty-two children with retinoblastoma. We performed targeted NGS using the UCSF500 Cancer Panel (University of California, San Francisco, San Francisco, CA) on formalin-fixed, paraffin-embedded tumor tissue along with constitutional DNA isolated from peripheral blood, buccal swab, or uninvolved optic nerve. Peripheral blood samples were also sent to a commercial laboratory for germline RB1 mutation testing. Presence or absence of germline RB1 mutation or deletion, tumor genetic profile, and association of genetic alterations with clinicopathologic features. Germline mutation or deletion of the RB1 gene was identified in all children with bilateral retinoblastoma (n= 12), and these NGS results were 100% concordant with commercial germline RB1 mutation analysis. In tumor tissue tested with NGS, biallelic inactivation of RB1 was identified in 28 tumors and focal MYCN amplification was identified in 4 tumors (2 with wild-type RB1 and 2 with biallelic RB1 inactivation). Additional likely pathogenic alterations beyond RB1 were identified in 13 tumors (41%), several of which have not been reported previously in retinoblastoma. These included focal amplifications of MDM4 and RAF1, as well as damaging mutations involving BCOR, ARID1A, MGA, FAT1, and ATRX. The presence of additional likely pathogenetic mutations beyond RB1 inactivation was associated with aggressive histopathologic features, including higher histologic grade and anaplasia, and also with both unilateral and sporadic disease. Comprehensive NGS analysis reliably detects relevant mutations, amplifications, and chromosomal copy number changes in retinoblastoma. The presence of genetic alterations beyond RB1 inactivation correlates with aggressive histopathologic features.

Phase 2 Results of APR-246 and Azacitidine (AZA) in Patients with TP53 mutant Myelodysplastic Syndromes (MDS) and Oligoblastic Acute Myeloid Leukemia (AML)

Phase 2 Results of APR-246 and Azacitidine (AZA) in Patients with TP53 mutant Myelodysplastic Syndromes (MDS) and Oligoblastic Acute Myeloid Leukemia (AML)

Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

Association of plasma DNA repair deficient (DRD) status with clinical outcome of metastatic castration-resistant prostate cancer (mCRPC) patients (pts) treated with abiraterone acetate (AA) plus prednisone/dexamethasone (+P/D).

5066 Background: Somatic DRD deficiency in 15-30% of mCRPC pts have been observed and inhibition of enzyme poly ADP ribose polymerase (PARP) could prove beneficial. We aimed to define DRD status using plasma from pts treated on AA and evaluate associations with prospectively-collected outcome measures. Methods: Plasma DNA samples (128 baseline [BL], 134 cycle 2 day 1 [C2D1], 46 progression [PROG]) from chemotherapy-naïve mCRPC pts in a phase 2 study (NCT01867710) evaluating AA+P/D were subjected to custom target-capture next-generation sequencing. DRD assay was optimized and validated to detect pathogenic point mutations, small insertions/deletions, and copy number alternations (DRD+) in 8 DRD genes: BRCA1, BRCA2, FANCA, ATM, CHEK2, HDAC2, BRIP1, and PALB2. Analysis for genomic aberrations was a secondary exploratory objective. Associations with overall survival (OS), progression-free survival (PFS), and radiographic PFS (rPFS) were assessed using Cox regression models and Kaplan Meier analyses. Results: 11.7% of BL and 17.4% of PROG were DRD+. Bi-allelic was observed in 73.3% of BL DRD+ samples. Shorter PFS was observed in BL DRD+ vs DRD- (5.3 vs 15.5 mo; HR: 2.32; 95%CI:1.39-4.28; P &lt; 0.002). Median PFS for BL DRD biallelic + vs DRD biallelic- was 5.1 vs 15.4 mo (HR: 2.49; 95% CI: 1.23-4.38; P &lt; 0.0095). For multivariate analysis using DRD+, ALP, and LDH as covariates, DRD+ (HR: 2.1; 95% CI: 1.18-3.75; P &lt; 0.012) and high ALP (HR: 1.66; 95% CI: 1.08-2.56; P &lt; 0.021) were strongly associated with worse PFS. Median OS for BL DRD+ vs DRD- was 28.8 vs 41.3 mo (HR: 1.67; 95%CI:0.88-3.18; P = 0.116). Median rPFS for BL DRD+ vs DRD- was 16.2 vs 20.9 mo (HR: 1.64; 95%CI:0.83-3.21; P = 0.152). Of 39 Pts with BL, C2D1 and PROG samples, 3 were DRD+ (7.7%) at all 3 timepoints, 3 (7.7%) only at BL, 3 (7.7%) only at PROG (bi-allelic), 2 (5.1%) had extra deletion at PROG. Conclusions: Patients with mCRPC harboring DRD+ have worse outcomes with AA and represent a population with an unmet medical need.

Low-level parental mosaicism affects the recurrence risk of holoprosencephaly

De novo variants (DNVs) represent an important fraction of the pathogenic variantburden in holoprosencephaly (HPE). However, unexpected recurrences can occur, as evidenced by multiple affected children harboring the same apparently DNV. This study was performed to estimate the rate of parental mosaicism in a cohort of patients with HPE. We developed a targeted capture next-generation sequencing (NGS) panel of 153 genes with potential implication in HPE. Sequencing data from a cohort of 136 HPE family trios were analyzed to identify probands with apparently DNVs. DNVs were examined in the proband and their parents to detect any deviations from the expected ~50/50 allele ratio of true heterozygosity. Selected variants were confirmed by Droplet Digital™ polymerase chain reaction (ddPCR). We identified 28 high-confidence DNVs, 20 of which occurred in known HPE genes. Nineteen of the 20 variants (95%) were pathogenic or likely pathogenic. Sequence data analysis showed evidence of parental mosaicism in five cases, for an overall mosaicism rate of 26%. In addition, we found evidence for likely postzygotic events in four cases (50%). High sensitivity methods, such as high-depth NGS and ddPCR, are essential to providing an accurate assessment of recurrence risk in HPE families with apparently DNVs.

Correction: HER2-Overexpressing Breast Cancers Amplify FGFR Signaling upon Acquisition of Resistance to Dual Therapeutic Blockade of HER2

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or pertuzumab has been shown to be superior to single-agent trastuzumab. However, a significant fraction of HER2-overexpressing (HER2+) breast cancers escape from these drug combinations. In this study, we sought to discover the mechanisms of acquired resistance to the combination of lapatinib + trastuzumab.Experimental Design: HER2+ BT474 xenografts were treated with lapatinib + trastuzumab long-term until resistance developed. Potential mechanisms of acquired resistance were evaluated in lapatinib + trastuzumab-resistant (LTR) tumors by targeted capture next-generation sequencing. In vitro experiments were performed to corroborate these findings, and a novel drug combination was tested against LTR xenografts. Gene expression and copy-number analyses were performed to corroborate our findings in clinical samples.Results: LTR tumors exhibited an increase in FGF3/4/19 copy number, together with an increase in FGFR phosphorylation, marked stromal changes in the tumor microenvironment, and reduced tumor uptake of lapatinib. Stimulation of BT474 cells with FGF4 promoted resistance to lapatinib + trastuzumab in vitro Treatment with FGFR tyrosine kinase inhibitors reversed these changes and overcame resistance to lapatinib + trastuzumab. High expression of FGFR1 correlated with a statistically shorter progression-free survival in patients with HER2+ early breast cancer treated with adjuvant trastuzumab. Finally, FGFR1 and/or FGF3 gene amplification correlated with a lower pathologic complete response in patients with HER2+ early breast cancer treated with neoadjuvant anti-HER2 therapy.Conclusions: Amplification of FGFR signaling promotes resistance to HER2 inhibition, which can be diminished by the combination of HER2 and FGFR inhibitors. Clin Cancer Res; 23(15); 4323-34. ©2017 AACR.

Phase 1b/2 Combination Study of APR-246 and Azacitidine (AZA) in Patients with TP53 mutant Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Phase 1b/2 Combination Study of APR-246 and Azacitidine (AZA) in Patients with TP53 mutant Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)