Hybridization Capture-based Next-generation Sequencing Assay Research Articles

Clinical and Technical Validation of OncoIndx® Assay-A Comprehensive Genome Profiling Assay for Pan-Cancer Investigations.

Comprehensive next-generation sequencing (NGS) assays enable the identification of clinically relevant mutations, enhancing the capability for targeted therapeutic interventions. In addition, genomic alterations driving the oncogenic roadmap and leading to resistance mechanisms are reshaping precision oncology. We report the workflow and clinical and technical validation of the OncoIndx® NGS platform-a comprehensive genomic profiling (CGP)-based assay for pan-cancer investigation. We evaluated the concordance between the OncoIndx® test findings and clinically established hotspot detection using SeraSeq reference standards. OncoIndx is a hybridization capture-based NGS assay for the targeted deep sequencing of all exons and selected introns of 1080 cancer-related genes. We show the outcome in the form of tier I and tier II single nucleotide variants (SNVs), copy number alterations (CNAs), and specific gene fusions. OncoIndx® also informs genome-wide tumor mutational burden (TMB), microsatellite instability (MSI), homologous recombination deficiency (HRD), and genomic loss of heterozygosity (gLOH). A total of 63 samples were utilized for validation with reference standards, clinical samples, and orthogonal assessment for genomic alterations. In addition, 49 cross-laboratory samples were validated for microsatellite instability (MSI), and for the tumor mutation burden (TMB), 18 samples as reference standards, 6 cross-laboratory samples, and 29 TCGA samples were utilized. We show a maximum clinical sensitivity of 98% and a positive predictive value (PPV) of 100% for the clinically actionable genomic variants detected by the assay. In addition, we demonstrate analytical validation with the performance of the assay, limit of detection (LoD), precision, and orthogonal concordance for various types of SVs, CNAs, genomic rearrangements, and complex biomarkers like TMB, MSI, and HRD. The assay offers reliable genomic predictions with the high-precision detection of actionable variants, validated by established reference standards.

Prospective Clinical Genomic Profiling of Ewing Sarcoma: ERF and FGFR1 Mutations as Recurrent Secondary Alterations of Potential Biologic and Therapeutic Relevance.

PURPOSEEwing sarcoma (ES) is a primitive sarcoma defined by EWSR1-ETS fusions as the primary driver alteration. To better define the landscape of cooperating secondary genetic alterations in ES, we analyzed clinical genomic profiling data of 113 patients with ES, a cohort including more adult patients (> 18 years) and more patients with advanced stage at presentation than previous genomic cohorts.METHODSThe data set consisted of patients with ES prospectively tested with the US Food and Drug Administration–cleared Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets large panel, hybrid capture-based next-generation sequencing assay. To assess the functional significance of ERF loss, we generated ES cell lines with increased expression of ERF and lines with knockdown of ERF. We assessed cell viability, clonogenic growth, and motility in these ES lines and performed transcriptomic and epigenetic analyses. Finally, we validated our findings in vivo using cell line xenografts.RESULTSNovel subsets were defined by recurrent secondary alterations in ERF, which encodes an ETS domain transcriptional repressor, in 7% of patients (five truncating mutations, one deep deletion, and two missense mutations) and in FGFR1 in another 2.7% (one amplification and two known activating mutations). ERF alterations were nonoverlapping with STAG2 alterations. In vitro, increased expression of ERF decreased tumor cell growth, colony formation, and motility in two ES cell lines, whereas ERF loss induced cellular proliferation and clonogenic growth. Transcriptomic analysis of cell lines with ERF loss revealed an increased expression of genes and pathways associated with aggressive tumor biology, and epigenetic, chromatin-based studies revealed that ERF competes with EWSR1-FLI1 at ETS-binding sites.CONCLUSIONOur findings open avenues to new insights into ES pathobiology and to novel therapeutic approaches in a subset of patients with ES.

Percutaneous Image-Guided Biopsy for a Comprehensive Hybridization Capture-Based Next-Generation Sequencing in Primary Lung Cancer: Safety, Efficacy, and Predictors of Outcome

IntroductionTo evaluate factors associated with successful comprehensive genomic sequencing of image-guided percutaneous needle biopsies in patients with lung cancer using a broad hybrid capture-based next-generation sequencing assay (CHCA). MethodsWe conducted a single-institution retrospective review of image-guided percutaneous transthoracic needle biopsies from January 2018 to December 2019. Samples with confirmed diagnosis of primary lung cancer and for which CHCA had been attempted were identified. Pathologic, clinical data and results of the CHCA were reviewed. Covariates associated with CHCA success were tested for using Fisher’s exact test or Wilcoxon ranked sum test. Logistic regression was used to identify factors independently associated with likelihood of CHCA success. ResultsCHCA was requested for 479 samples and was successful for 433 (91%), with a median coverage depth of 659X. Factors independently associated with lower likelihood of CHCA success included small tumor size (OR = 0.26 [95% confidence interval (CI): 0.11–0.62, p = 0.002]), intraoperative inadequacy on cytologic assessment (OR = 0.18 [95% CI: 0.06–0.63, p = 0.005]), small caliber needles (≥20-gauge) (OR = 0.22 [95% CI: 0.10–0.45, p < 0.001]), and presence of lung parenchymal abnormalities (OR = 0.12 [95% CI: 0.05–0.25, p < 0.001]). Pneumothorax requiring chest tube insertion occurred in 6% of the procedures. No grade IV complications or procedure-related deaths were reported. ConclusionsPercutaneous image-guided transthoracic needle biopsy is safe and has 91% success rate for CHCA in primary lung cancer. Intraoperative inadequacy, small caliber needle, presence of parenchymal abnormalities, and small tumor size (≤1 cm) are independently associated with likelihood of failure.

Molecular Profiling in Extramedullary Acute Myeloid Leukemia Reveals Distinct Subgroups Defined By Mutations in KIT and RUNX1

Background: Between 2 to 23% of patients with acute myeloid leukemia manifest extramedullary involvement including myeloid sarcoma. While the genomic landscape of AML and the respective clinical and outcome correlations are well characterized, our understanding of the molecular underpinnings of extramedullary AML remains limited. Various cytogenetic abnormalities have been described in association with extramedullary AML, including core binding factor (CBF) translocations t(8;21) and inv(16). Recent studies have also applied targeted gene sequencing approaches (21 to 39 genes) in small patient cohorts (6-18) to generate limited mutational profiles of extramedullary AML [Li et al. Leukemia 2015; Pastoret et al. Leukemia & Lymphoma 2016; Kashofer et al. Leukemia & Lymphoma]. Here we utilize a comprehensive next generation sequencing (NGS) panel interrogating 585 genes implicated in hematological malignancies in 17 patients and provide the most comprehensive mutational profile of extramedullary AML to date.Methods: 17 pathologically confirmed cases of extramedullary AML were identified. Genomic DNA extraction was performed from formalin-fixed, paraffin-embedded (FFPE) tumor samples. Isolated DNA was sent for sequencing using the HemePACT panel, a custom hybridization capture-based NGS assay encompassing 585 genes related to hematologic malignancies. All HemePACT data were compared to an unmatched normal sample. Data were passed to standard pipelines used to detect candidate substitutions and indels (Caveman and pindel respectively). Candidate variants were manually reviewed to eliminate single nucleotide polymorphisms and artifacts and to find a subset of high-confidence putative oncogenic variants. Cytogenetic data was obtained from standard clinical karyotyping and fluorescence in-situ hybridization assays from bone marrow aspirates from patients with bone marrow AML involvement.Results: Demographic data for sequenced extramedullary AML patients is provided in Table 1. We identified a total of 80 mutations. The median number of mutations /sample is 4 (range:1-11). The most frequently mutated genes are KIT found in 7/17 patients (41%) and RUNX1 found in 6/17 (35%) patients. Intriguingly, KIT and RUNX1 mutations are mutually exclusive(p<0.05), and 13/17 (76.5%) sequenced extramedullary AML samples have mutations in either KIT or RUNX1 . (Figure 1) DNMT3A mutations are found in 3/17 (17%) patients. FLT3-tyrosine kinase domain (TKD) mutations are found in 4/17 (23%) of patients. DNMT3A and FLT3-TKD mutations co-segregate with RUNX1 (2/3 and 2/4 respectively) but not with KIT mutations. As only 2/17 patients underwent cytogenetic evaluation from extramedullary AML, mutational data from extramedullary AML was largely integrated with cytogenetic data from bone marrow aspirates. 7/17 (41.1%) of sequenced patients had CBF translocations (6/7 with inv(16)/t(16;16), and 1/7 with t(8;21) present in bone marrow cytogenetic evaluation by karyotype or FISH. Out of 7 patients with CBF translocations, 6/7 (85.7%) have KIT mutations present in extramedullary AML. 1 patient with inv(16) found by karyotyping from myeloid sarcoma did not have a KIT mutation in extramedullary AML. No patients with RUNX1 mutations in extramedullary AML (0/6) have CBF translocations. Kaplan-Meier analysis from this small cohort did not show a statistically significant difference in overall survival between patients with KIT and RUNX1 mutations (p=0.57).Overall, our data suggest the potential for distinct and specific mutational profiles associated with AML extramedullary involvement.Conclusions: Our study provides the most comprehensive mutational profiling of extramedullary AML to date. Combined molecular and cytogenetic profiling demonstrate that extramedullary AML is characterized by a distinct repertoire of acquired mutations enriched for mutations in KIT and RUNX1. Importantly our data identify two distinct and non-overlapping molecular subgroups. One subgroup is defined by CBF alterations and KIT mutations, while the second most prominent subtype is defined by mutations in RUNX1. Recognition of these subtypes and their co-mutations may form the basis for genotype-clinical correlations, as well as experimental models to explore the biology of extramedullary AML. Additional extramedullary AML patient samples are being sequenced and will be presented. [Display omitted] DisclosuresGoldberg:Genentech: Research Funding; Pfizer: Research Funding; ADC Therapeutics: Research Funding. Levine:Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Reduced PCR-generated errors from a hybrid capture-based NGS assay for HLA typing

Next generation sequencing (NGS) assays are state of the art for HLA genotyping. To sequence on an Illumina sequencer, the DNA of interest must be enriched, fragmented, and bookended with known oligonucleotide sequences, a process known as library construction. Many HLA genotyping assays enrich the target loci by long-range PCR (LR-PCR), prior to fragmentation. This PCR step has been reported to introduce errors in the DNA to be sequenced, including inaccurate replication of repeated sequences, and the in vitro recombination of alleles encoded on separate chromosomes. An alternative library construction method involves fragmentation of genomic DNA, followed by hybrid-capture (HC) enrichment of target HLA loci. This HC-based method involves PCR, but with far fewer cycles. Consequently, the HC method had significantly fewer PCR-induced errors, including more faithful replication of repeated sequences, and the near elimination of recombinant sequences. These improvements likely produce more accurate NGS sequencing data of HLA loci.

Abstract 5742: Comparison of variants identified by next-generation sequencing panels: A 48-gene panel for myeloid neoplasms and a 35-gene panel for hematologic neoplasms

Abstract Background: Identification of genomic variants by molecular testing plays an important role in classification, risk stratification, and management of myeloid neoplasms. Next-generation sequencing (NGS) assays that target frequently mutated genes are commonly used in clinical settings. Owing to the complexity of NGS workflow, the performance requirements of each NGS assay must be validated for the intended use. However, the scarcity of optimal validation samples challenges efficient validation of NGS assays. We previously reported development and validation of a 48-gene NGS panel for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). In this study, we compared the variant calls of a clinically validated 48-gene NGS panel for myeloid neoplasms to those of an independently developed and clinically validated 35-gene NGS panel for hematologic neoplasms. Method: The 48-gene NGS assay is a hybrid capture-based NGS assay using an Illumina NextSeq 500; it was developed to detect single nucleotide variants (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). The 35-gene NGS assay HemeSEQ (performed by med fusion, Lewisville, TX) uses Illumina TruSeq custom-amplicon library preparation chemistry, which is sequenced on an Illumina MiSeq. A total of 33,812 bp in 26 genes overlapped between the 2 assays; genes included ASXL1, CALR, FLT3, IDH1, IDH2, JAK2, NPM1, RUNX1, SF3B1, TET2, and TP53. For this study, 141 specimens harboring at least 1 pathogenic mutation within the overlapping genes were selected and sequenced by the 2 assays in CLIA-certified and CAP-accredited laboratories. Detected variants included those for which a targeted therapy or an experimental drug is available. Results: In total, 4.8 million individual base calls (33,812 bp X 141 specimens) were compared. A total of 1,094 variants (278 unique) were concordantly detected by both assays: 1,007 (219 unique) SNVs, and 87 (59 unique) indels; no calls were discordant. Thus, sensitivity was 100% (95% CI: 99.7-100) and specificity was 100% (95% CI: 99.9-100) for overlapping genes. In addition, agreement in variant allele frequency between the 2 assays was good (R2=0.986). The 48-gene assay detected an additional 36 pathogenic mutations from 26 specimens in 14 non-overlapping genes including PTPN11, ATM, BCOR, NF1, and STAG2. Conclusions: Of 4.8 million base calls compared, variant calls were 100% concordant between the 2 NGS assays, and variant allele frequency was in good agreement. The 48-gene assay detected an additional 36 pathogenic mutations in 14 myeloid neoplasm related genes. This study highlights the reliability of robustly validated NGS assays in detection of potentially actionable mutations in clinical settings. Citation Format: Sun Hee Rosenthal, Hansook Chong, Charles Ma, Daniel Sugganth, Renius Owen, Joseph Catanese, Christopher Elzinga, Andrew Grupe, Tracey Freitas, Jennifer Graham, Kristen Champion, Amanda Larsen, Frederick Racke, Felicitas Lacbawan. Comparison of variants identified by next-generation sequencing panels: A 48-gene panel for myeloid neoplasms and a 35-gene panel for hematologic neoplasms [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5742.

Immunohistochemical analysis of estrogen receptor in breast cancer with ESR1 mutations detected by hybrid capture-based next-generation sequencing

Estrogen receptor-α (ER-α), encoded by ESR1, is detected by immunohistochemistry in approximately 70% of invasive breast cancers and serves as a strong predictive biomarker. ESR1-activating mutations in the ligand-binding domain have been reported in up to 35–40% of ER-positive metastatic breast cancers and are associated with endocrine therapy resistance and disease progression. At present, it is unclear whether ESR1 mutations alter the immunohistochemical detection of ER performed in routine clinical practice. In this study, ESR1 mutations in breast cancer were identified utilizing Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a Food and Drug Administration-approved hybridization capture-based next-generation sequencing assay. Five hundred and eighty-six breast cancers from patients with locally advanced or metastatic disease were analyzed using MSK-IMPACT in the study period. ESR1 somatic alterations were identified in 67 breast cancer samples from 66 patients. Immunohistochemical analysis of ER, progesterone receptor, and human epidermal growth factor receptor 2 was performed on the primary and treated breast cancers from these patients at the time of diagnosis. Twenty unique ESR1 mutations were identified involving the ligand-binding domain, all in breast cancer samples from patients previously treated with endocrine therapy. The most frequent mutations were D538G (n = 22), Y537S (n = 7), and E380Q (n = 7). All breast cancer samples with an ESR1 mutation were ER-positive by immunohistochemistry. Review of the ER immunohistochemistry in the paired untreated primary tumor and treated tumor from 34 patients showed no detectable change in the ER-positive immunohistochemical status (median percentage of invasive tumor cells with nuclear staining: untreated primary tumor 90%, treated tumor 95%). We conclude that ESR1 mutations do not appreciably diminish ER-positive staining by immunohistochemistry. In addition to standard biomarker testing by immunohistochemistry, the assessment of ESR1 mutations by molecular testing can help guide the clinical management of patients with ER-positive breast cancer in the setting of endocrine resistance and progression of disease.

GNAQ Mutations in Diffuse and Solitary Choroidal Hemangiomas

GNAQ mutations have been identified in port wine stains (both syndromic and nonsyndromic) and melanocytic ocular neoplasms. This study investigates the presence of GNAQ mutations in diffuse (those associated with Sturge-Weber syndrome [SWS]) and solitary choroidal hemangiomas. Tissue from 11 patients with the following diagnoses: port wine stain (n= 3), diffuse choroidal hemangioma (n= 1), solitary choroidal hemangioma (n= 6), and choroidal nevus (n= 1). Ten specimens were interrogated with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets, a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 468 key cancer genes in formalin-fixed, paraffin-embedded tumors. Digital polymerase chain reaction was used to detect GNAQ Q209 mutation in 1 specimen. Detection of GNAQ codon-specific mutation. Activating somatic GNAQ mutations (c.547C > T; p.Arg183Cys) were found in 100% (3 of 3) of the port wine stain and in the diffuse choroidal hemangioma. Somatic GNAQ mutations (c.626A > T; p.Gln209Leu) were found in 100% (6 of 6) of the solitary choroidal hemangiomas and (c.626A > C; p.Gln209Pro) in the choroidal nevus. GNAQ mutations occur in both diffuse and solitary hemangiomas, although at distinct codons. An R183 codon is mutant in diffuse choroidal hemangiomas, consistent with other Sturge-Weber vascular malformations. By contrast, solitary choroidal hemangiomas have mutations in the Q209 codon, similar to other intraocular melanocytic neoplasms.

A Next-Generation Sequencing-Based Karyotyping Algorithm Reveals the Genomic Structure of Acute Myeloid Leukemia

Introduction: The BeatAML master trial is evaluating the use of precision medicine via genomically guided therapy in acute myeloid leukemia (AML). A variety of assays are used to test patient samples including cytogenetic karyotyping, fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS)-based approaches to analyze genomic alterations. We developed an algorithm to extract karyotype information from NGS-based genomic sequencing data in the BeatAML cohort. We also utilized this algorithm to retrospectively study 1603 unique AML samples collected during routine clinical care.Methods: Comprehensive genomic profiling (CGP) was performed using a CLIA/CAP/NYS approved DNA and RNA hybrid capture-based next-generation sequencing assay that sequences 426 genes in DNA and 265 genes to capture fusions by RNA. We analyzed aneuploidy by comparing the log ratio of read counts in tumor DNA to a process matched normal control and developed signal to noise metrics to measure chromosome arm copy number. A chromosome arm was considered altered if >50% of the arm was altered. Chromosome (chr) 5q was also considered lost if only chr5q31 (containing EGR1) was lost. Based on the noise metrics of each sample, we also calculated a per sample limit of detection, which was defined as the lowest percentage of cells that could have a single copy gain or loss and be detected. We validated chr5q loss, chr7q loss, and chr8 gain using FISH results obtained from available BeatAML samples as the gold standard. To better understand the genomic landscape of AML, we also included 1603 samples run in standard clinical practice. False discovery rate (FDR) was assessed using the Bonferroni method.Results: We developed a method to analyze chr5q loss, chr7q loss and chr8 gain that had 95% concordance across all chromosomes compared to the FISH gold standard. The median limit of detection was 10.1% (percentage of cells required to have a single copy gain/loss to be detected). Overall concordance adjusted for limit of detection was 97% with 92% sensitivity (55/60 chromosomes) and 99% specificity (162/164 chromosomes) with similar performance across chromosomes. For chr7q loss and chr8 gain, over 50% of the chromosomal region was altered. However, the size of the loss on chr5q varied widely from the entire arm to a single gene (EGR1). We next applied the algorithm to 1603 unique patient samples analyzed by CGP during routine clinical care. We detected loss of chr5q in 10.9% (174), loss of chr7q in 10.4% (166), and gain of chr8 in 9.2% (147) of AML samples. Frequently altered genes in this cohort included FLT3 (21%, 337), RUNX1 (20.5%, 328), DNMT3A (17.5% 280), and TP53 (17.5%, 280). To further explore the genomic landscape, we analyzed the cooccurrence of each chromosomal alteration with all genes assayed. All three chromosomal aberrations exhibited significant cooccurrence with each other and TP53 (FDR P < 0.05). TP53 was altered in 75% (130/174) of chr5q loss samples 49% (82/166) of chr7q loss samples and 37% (55/147) of chr8 gain samples. Loss of chr5q also exhibited significant mutual exclusivities with FLT3, DNMT3A, NPM1, SRSF2, ASXL1, CEBPA, and WT1 (FDR P < 0.05). Loss of chr7q exhibited a slightly different mutual exclusivity profile with FLT3, NPM1, and PTPN11 (FDR P < 0.05). The difference in the cooccurrence profile between chr5q and chr7q was not a matter of p-value cutoffs since the magnitude of effect varied widely between groups.Conclusions: We demonstrated an NGS-based algorithm to determine karyotype that has a high concordance with FISH results. Limit of detection was equivalent to karyotyping by metaphase analysis but slightly worse than FISH assays on freshly prepared samples. We identified a previously described genomic stratification of TP53-aneuploidy since chromosomal aberrations and TP53 exhibited significant cooccurrence. However, only 50% of samples with loss of chr5q exhibited loss of chr7q and vice versa. While both were mutually exclusive with FLT3 and NPM1, loss of chr5q was mutually exclusive with several genes of prognostic significance including chromatin factors (DNMT3A) and splice factors (SRSF2). This study shows how NGS-based approaches can capture both chromosome arm level copy number alterations as well as all four categories of genomic alterations, thereby enabling risk stratification and identification of potential treatment options as defined in current NCCN guidelines. DisclosuresPavlick:Foundation Medicine Inc: Employment. Montesion:Foundation Medicine Inc: Employment. Severson:Foundation Medicine Inc: Employment. Brennan:Foundation Medicine Inc: Employment. Frampton:Foundation Medicine Inc: Employment, Other: Employee and stockholder. He:Foundation Medicine Inc: Employment. Levine:Roche: Consultancy, Research Funding; Epizyme: Patents & Royalties; Celgene: Consultancy, Research Funding; Isoplexis: Equity Ownership; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; C4 Therapeutics: Equity Ownership; Imago: Equity Ownership; Loxo: Consultancy, Equity Ownership. Vergilio:Foundation Medicine Inc: Employment. Albacker:Foundation Medicine Inc: Employment; Foundation Medicine Inc: Employment.

Myeloid/Histiocytic Neoplasms Associated with Germ Cell Tumors Harbor Shared Genetic Abnormalities Indicating a Clonal Relationship, Unique Molecular Pathogenesis and Cellular Origin

IntroductionNeoplasms of different lineages that arise in the same patient and show a clonal relationship are exceedingly rare, but have been described in instances of histiocytic and myeloid/lymphoid neoplasms (Durham BH, et al. Blood 2017;130(2):176-80; Ansari J, et al. Eur J Haematol 2016;97(1):9-16). The clonal relationships can be demonstrated by shared genetic abnormalities such as translocations, somatic mutations, and other chromosomal level alterations. Nevertheless, a clonal link between germ cell tumors (GCT), a group of solid tumors derived from primitive stem cells, and hematologic malignancies has been less well-characterized. Relevant literature has mostly reported associations of GCT with acute leukemias, without a comprehensive assessment of genetic alterations (Mukherjee S et al. Ann Hematol 2017;96: 1435-39). To further characterize this phenomenon, we identified a small cohort of patients diagnosed with both GCT and any myeloid/histiocytic neoplasm. We evaluated their molecular and cytogenetic alterations, identifying shared and unique abnormalities, providing evidence of a clonal relationship between these two groups of neoplasms, which traditionally represent different cellular origins.MethodsA search of the pathology database at a major referral center (Memorial Sloan-Kettering Cancer Center) was performed to identify patients diagnosed with GCT between 2012-2018, and had at least 1 prior or subsequent bone marrow biopsy. The medical records were reviewed for details of clinical presentations and evidence of myeloid neoplasm, with corresponding morphologic, cytogenetic, and molecular findings. The findings were correlated with the genetic alterations detected in the GCT during diagnostic work-up. Cytogenetic analyses include karyotyping, fluorescence in situ hybridization (FISH) studies for common abnormalities among myeloid neoplasms, and single nucleotide polymorphism (SNP) array for copy number gain/loss and copy neutral-loss of heterozygosity (CN-LOH). Molecular analyses include an amplicon capture-based next generation sequencing (NGS) assay for 49 genes relevant for hematologic malignancies, and MSK-IMPACTTM, a hybrid capture-based NGS assay for mutation and copy number alteration in 400+ genes. Somatic nature of the identified variants was confirmed on MSK-IMPACTTM by germline variant filtering with the aid of appropriate normal control samples (blood or nail).Results8 patients with GCT diagnoses showed marrow findings consistent with or suspicious for involvement by myeloid/histiocytic neoplasms, with clinical presentations not typical for therapy-related myeloid neoplasms. The patients were predominantly male (n=6) and young (age range: 13-55, median=26.5). The GCT included mixed GCT (n=6), mature teratoma (n=1) and immature teratoma (n=1). The primary sites of involvement were mediastinum (n=4), testes (n=2), and ovaries (n=2). In all cases, the myeloid/histiocytic neoplasms and GCT were diagnosed in close proximity in time (range: <1 to 24 months). So far, cytogenetic and molecular data in 4 patients with mediastinal-based tumors showed shared alterations between the GCT and myeloid/histiocytic neoplasms, including isochromosome 12p in 3 patients, an alteration characteristic of GCT. Other shared alterations observed included identical mutations in TP53, PIK3CD, KRAS, BCOR; trisomy 1, 8, 21; gain of 21q; loss of 13q; and CN-LOH of 2q, 5q, 17p. Interestingly, in all 4 patients, the GCT and myeloid/histiocytic neoplasm each showed additional unique genetic alterations.ConclusionThese findings suggest that a subset of patients with GCT can develop clonally-related myeloid/histiocytic neoplasms, with a relatively short latency period. The genetic changes seen in our cases are relatively less common in conventional myeloid neoplasms, suggesting unique molecular pathogenesis. Yolk sac component in the GCT, which has been described to harbor hematopoietic precursor cells, may contribute to pathogenesis (Orazi A, et al Cancer 1993;71(12):3873-81). Although the detailed pathogenic mechanism remains uncertain, we observed that in the cohort, the GCT and myeloid/histiocytic neoplasm each harbored additional unique genetic changes. This suggests the presence of a common neoplastic progenitor giving rise to neoplasms of different lineages, which subsequently accumulated additional alterations. [Display omitted] DisclosuresHo:Invivoscribe, Inc.: Honoraria. Yabe:Y-mAbs Therapeutics: Consultancy. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria.

Clinical utility of tumor genomic profiling in patients with high plasma circulating tumor DNA burden or metabolically active tumors

BackgroundThis retrospective study was undertaken to determine if the plasma circulating tumor DNA (ctDNA) level and tumor biological features in patients with advanced solid tumors affected the detection of genomic alterations (GAs) by a plasma ctDNA assay.MethodCell-free DNA (cfDNA) extracted from frozen plasma (N = 35) or fresh whole blood (N = 90) samples were subjected to a 62-gene hybrid capture-based next-generation sequencing assay FoundationACT. Concordance was analyzed for 51 matched FoundationACT and FoundationOne (tissue) cases. The maximum somatic allele frequency (MSAF) was used to estimate the amount of tumor fraction of cfDNA in each sample. The detection of GAs was correlated with the amount of cfDNA, MSAF, total tumor anatomic burden (dimensional sum), and total tumor metabolic burden (SUVmax sum) of the largest ten tumor lesions on PET/CT scans.ResultsFoundationACT detected GAs in 69 of 81 (85%) cases with MSAF > 0. Forty-two of 51 (82%) cases had ≥ 1 concordance GAs matched with FoundationOne, and 22 (52%) matched to the National Comprehensive Cancer Network (NCCN)-recommended molecular targets. FoundationACT also detected 8 unique molecular targets, which changed the therapy in 7 (88%) patients who did not have tumor rebiopsy or sufficient tumor DNA for genomic profiling assay. In all samples (N = 81), GAs were detected in plasma cfDNA from cancer patients with high MSAF quantity (P = 0.0006) or high tumor metabolic burden (P = 0.0006) regardless of cfDNA quantity (P = 0.2362).ConclusionThis study supports the utility of using plasma-based genomic assays in cancer patients with high plasma MSAF level or high tumor metabolic burden.

Analytical Validation of a Hybrid Capture–Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture–based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode–based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%–99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%–95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%–71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments–certified/College of American Pathologists–accredited/New York State–approved laboratory.

Abstract 5359: An ultrasensitive and highly reproducible hybridization capture-based Next-Generation Sequencing clinical assay to enable precision oncology in patients with solid tumors

Abstract Sequencing whole cancer exome and whole genome provides useful information to study cancer evolution. However, cost and amount of data may become overwhelming. Enrichment capture-based methods to design custom targeted gene panel have rapidly evolved recently in cancer genomics arena. Custom targeted gene sequencing entails several advantages (i.e., better quality, ethical, affordable, technically suitable, personalized and reimbursable) for cancer patients over whole exome and whole genome sequencing. With this in mind, and to enable precision oncology in patients with solid tumors, we developed ICG100 2.0 panel, a hybridization capture-based next-generation sequencing assay for targeted sequencing of all exons and flanking introns of 162 commonly mutated cancer genes in formalin-fixed, paraffin-embedded (FFPE) tumors. To establish ICG100 2.0 panel, we compared three commercially available capture based technologies and evaluated reproducibility, sensitivity, specificity and the detection limit for low-frequency variants using internally developed bioinformatics pipeline. Cell lines, reference standards/synthetic DNA and solid tumor samples with the known genetic information was utilized in this analysis. Results were derived from MiSeq and NextSeq platforms and cross-compared on other platforms, including MassArray and ddPCR for establishing concordance and uniformity. Intra and interrun replicates were utilized to assess the quality, precision and reproducibility of variant calling. Mean depth of coverage was observed at &amp;gt;300X with &amp;gt;99% sensitivity and specificity. These findings and observations will guide other clinical laboratories to establish new assay that require less DNA input, enzyme based fragmentation and reduced preparation time. While we show that the three capture based methods (after comparison) had an overall accuracy in SNP and CNV detection similar to each other with minor differences, we describe an approach to assess and establish the best assay from the clinical standpoint to guide treatment decisions and match cancer patients to the most appropriate clinical trials. Citation Format: Moises Hernandez, Sara King, Sharanya Raghunath, Christopher Johnson, David Loughmiller, Lincoln Nadauld, Pravin J. Mishra. An ultrasensitive and highly reproducible hybridization capture-based Next-Generation Sequencing clinical assay to enable precision oncology in patients with solid tumors [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 5359. doi:10.1158/1538-7445.AM2017-5359

Abstract 3732: Analytical performance of TruSight® Tumor 170 on small nucleotide variations and gene amplifications using DNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples

Abstract Expanding the paradigm of solid tumor profiling from single-gene testing to comprehensive panels presents many challenges. One such challenges is the ability of these panels to detect genetic alterations from FFPE samples, where the DNA is of low abundance and often heavily compromised. Despite these challenges, next-generation sequencing (NGS) offers the ability to assess multiple variants simultaneously in an ever-expanding list of relevant tumor genes. To that end, Illumina developed a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes that is FFPE optimized. The assay consists of a DNA workflow for the identification of single and multiple nucleotide variants (SNVs, MNVs), small insertions and deletions (indels), gene amplifications, as well as a RNA workflow for the identification of splice variants and gene fusions. Following sequencing on the NextSeq® or HiSeq® instruments, the analytical pipeline initiates variant calling. The DNA aligner and variant callers were first optimized against the simulated read data from &amp;gt;40,000 COSMIC[1] mutations reported in the exons of the 170 genes. To reduce false positive variant calling due to systematic errors, each variant call was evaluated against its locus specific background error distribution. This distribution was compiled from a panel of FFPE normal samples and was also used to normalize against systematic bias in read coverage to increase the accuracy of amplification calling. Furthermore, gene amplification calling was improved by the addition of enhancer probes to the hybrid capture pool. The analytical sensitivity and specificity of TruSight® Tumor 170* was assessed on a large collection of FFPE samples and reference material. A panel of 72 cancer samples, including multiple tissue types, reference standards, and cell line and FFPE mixes were used to evaluate the limit of detection. The samples contained 533 SNVs, 80 indels including deletions up to 30 base pairs and insertions up to 31 base pairs, 4 MNVs, and 31 gene amplifications, characterized by orthogonal testing methods. Using 40 ng DNA input, detection sensitivity of the &amp;gt;1000 variants (including replicates) tested at variant allele frequencies down to ~5% was at 99.6%, while detection sensitivity of gene amplifications as low as 1.45x to 2.2x was at 98%. For limit of blank samples, a panel of 24 normal samples was used. Again using 40 ng DNA input, we show &amp;gt;99% specificity for small variant calling and &amp;gt;95% specificity for gene amplification calling. These data demonstrates the TruSight® Tumor 170 is able to detect multiple variant types within a single sample at low nucleic acid input, while exhibiting high analytical sensitivity and specificity for low allele fraction detection. [1] Forbes, et al. (2015) *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Danny Chou, Xiao Chen, Austin Purdy, Li Teng, Byron Luo, Chen Zhao, Laurel Ball, Allan Castaneda, Katie Clark, Brian Crain, Anthony Daulo, Manh Do, Tingting Du, Sarah Dumm, Yonmee Han, Michael Havern, Chia-Ling Hsieh, Tingting Jiang, Suzanne Johansen, Scott Lang, Rachel Liang, Jennifer S. LoCoco, Jaime McLean, Yousef Nassiri, Jason Rostron, Jennifer Silhavy, June Snedecor, Natasha Talago, Kevin Wu, Clare Zlatkov, Ali Kuraishy, Karen Gutekunst, Sohela De Rozieres, Matthew Friedenberg, Han-Yu Chuang, Anne C. Jager. Analytical performance of TruSight® Tumor 170 on small nucleotide variations and gene amplifications using DNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples [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 3732. doi:10.1158/1538-7445.AM2017-3732

PUB053 Panel Based Hybrid Capture Sequencing Assay to Correlate Mutational Load with Response to Immunotherapy

The use of immune checkpoint inhibitors has shown promise in lung cancer as well as several other tumor types. However, two of their limitations are the overall relatively low response rate as well as the lack of powerful predictors for response. Recently, several studies have shown that stratification of patients according to the load of somatic mutations can provide predicitive information for either ‘mono’ or ‘combinational’ immunotherapy. Here, we describe the use of a hybrid-capture based next-generation sequencing assay, NEOplus, to determine the somatic mutational load in clinical samples. NEOplus is a hybrid-capture based next-generation sequencing assay that covers clinically relevant genomic alterations in a panel of more than 90 tumor-associated genes, including point mutations, small insertions and deletions, copy number alterations and gene fusions. First, the correlation between number of mutations observed in the NEOplus exonic territory and response to immune checkpoint inhibitors was analyzed in silico based on published data. Second, tumors of multiple histological types with available outcome data were subjected to NEOplus analysis to assess mutational load. We were able to correlate the amount of mutations detected within the exonic territory covered by the NEOplus assay and clinical response to immune checkpoint inhibitors. Furthermore, we confirmed that hybrid-capture based next-generation sequencing assays, covering sub-fractions of the exome, can reliably detect somatic mutation load in diagnostic patient samples. We show correlations between mutational load and patient response to immunotherapy and are able to indicate how to stratify patients who would most likely benefit from treatment.

Next-Generation Assessment of Human Epidermal Growth Factor Receptor 2 (ERBB2) Amplification Status: Clinical Validation in the Context of a Hybrid Capture-Based, Comprehensive Solid Tumor Genomic Profiling Assay

Establishing ERBB2 [human epidermal growth factor receptor 2 (HER2)] amplification status in breast and gastric carcinomas is essential to treatment selection. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) constitute the current standard for assessment. With further advancements in genomic medicine, new clinically relevant biomarkers are rapidly emerging and options for targeted therapy are increasing in patients with advanced disease, driving the need for comprehensive molecular profiling. Next-generation sequencing (NGS) is an attractive approach for up-front comprehensive assessment, including ERBB2 status, but the concordance with traditional methods of HER2 assessment is not well established. The Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay, a hybrid capture-based NGS assay interrogating the coding regions of 410 cancer-related genes, was performed on manually macrodissected unstained sections from formalin-fixed, paraffin-embedded breast (n=213) and gastroesophageal (n=39) tumors submitted for clinical mutation profiling. ERBB2 status was assessed using a custom bioinformatics pipeline, and NGS results were compared to IHC and FISH. NGS ERBB2 amplification calls had an overall concordance of 98.4% (248/252) with the combined IHC/FISH results in this validation set. Discrepancies occurred in the context of low tumor content and HER2 heterogeneity. ERBB2 amplification status can be reliably determined by hybridization capture-based NGS methods, allowing efficient concurrent testing for other potentially actionable genomic alterations, particularly in limited material.

Abstract 2640: Clonal hematopoiesis identified by matched-normal blood sequencing of solid tumor patients without hematologic malignancy is common and is associated with decreased overall survival

Abstract Background: Recent genomic studies have identified somatic mutations in leukemia genes in asymptomatic individuals without hematologic malignancy. A subset of patients (pts) with clonal hematopoiesis (CH) subsequently develops hematologic malignancies. However, the incidence of CH in pts with solid tumors has not been extensively studied. We sought to assess for CH and its associated clinical impact in pts with solid tumors who were profiled using paired tumor/blood sequencing. Methods: This study included pts who were consented on protocol NCT01775072. All had tumor and blood genomic profiling using the MSK-IMPACT hybridization capture-based next-generation sequencing assay, encompassing all protein-coding exons of 410 cancer-associated genes. In matched blood, we investigated for hotspot mutations from COSMIC database (v71) and non-hotspot mutations in leukemia-associated genes and genes reported in prior CH studies. Mutations were scored as present if variant allele frequency (VAF) in blood was greater than 2% and at least twice the VAF seen in tumor. For cases where at least one mutation exceeded these thresholds, we reduced the VAF threshold in blood to 1% to detect subsequent events. Mutations with VAF &amp;gt;35% in both blood and tumor were excluded as likely germline events. Results: We analyzed 2,146 pts, including 239 pts (11%) with CH. Most commonly identified mutations were in DNMT3A, TET2, and TP53, seen in 167, 52, and 18 pts, respectively. The mean age at time of testing was 62.6 years in pts with CH and 55.4 years in pts without CH (p&amp;lt; 0.001). When comparing baseline blood parameters, there were no statistically significant differences except for higher MCV in CH vs. non-CH pts (91 vs. 90, p = 0.047). 51% of pts with CH had previous radiation therapy compared to 45% of pts without clonal mutations in their blood (p = 0.057). There was no statistically significant difference in the proportion of pts who had received previous chemotherapy (71% of pts in each group). On prospective follow up of pts with CH, no pts have progressed to develop overt hematologic malignancy, with limited follow up (median 13.1 months). Overall survival (OS) was estimated for pts without CH (n = 1907; 1-yr OS: 0.70), CH with 1 mutation (n = 184; 1-yr OS: 0.64), and CH with &amp;gt;1 mutation (n = 55; 1-yr OS: 0.59). The univariate hazard ratios were 1.16 (p = 0.269) and 1.63 (p = 0.015) for 1 mutation and &amp;gt;1 mutation, respectively, when compared to pts without CH. After adjusting for age and sex, a marginal OS association persisted for &amp;gt;1 mutation compared to no CH (HR: 1.46, p = 0.060) Conclusions: CH is common among solid tumor pts without known hematologic malignancy, occurring in 11% of pts, and appears to be associated with decreased OS. Analyses will be replicated in a larger cohort with longer follow up to determine the clinical impact of these mutations in cancer pts. Citation Format: Catherine Coombs, Ahmet Zehir, Sean Devlin, Sumit Middha, Donavan Cheng, Ashwin Kishtagari, David Hyman, David Solit, Mark Robson, Jose Baselga, Maria Arcila, Martin Tallman, Ross Levine, Michael Berger. Clonal hematopoiesis identified by matched-normal blood sequencing of solid tumor patients without hematologic malignancy is common and is associated with decreased overall survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2640.

Abstract 2419: Anastomotic recurrences are clonally related to primary tumors in sporadic colorectal carcinoma

Abstract Background: Anastomotic recurrences occur in 2-10% of colorectal carcinoma (CRC) cases after surgical resection of the primary tumor. To date there are no molecular data investigating their genetic profile and multiple theories exist as to their pathogenesis including re-growth of tumor cells present within local lymphatics after initial surgery, seeding of anastomosis by metastatic tumor cells and a second primary tumor. The aim of our study was to compare the genomic profile of anastomotic recurrences to that of matched primary tumors and, where available, to that of distant metastases. Design: Thirty-six tumors from 14 patients were genotyped using a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes. All patients had resection of their primary tumor with clear margins and recurred either at the anastomotic line or in the peri-anastomotic area 1.1 to 7.0 years following resection of their primary tumor. In 3 patients 2 consecutive anastomotic recurrences were sequenced, while in 6 patients a distant metastasis that occurred 1.2 to 3 years prior to the anastomotic recurrence was also analyzed. All tumors were microsatellite stable except in one patient with genetically confirmed Lynch syndrome. Results: A total of 254 somatic mutations were detected including 140 mutations in the microsatellite stable cases. The most commonly mutated genes (mutated in &amp;gt; 3 patients) were APC, KRAS, TP53, PIK3CA, ATM and PIK3R1. In 13/14 patients the anastomotic recurrence(s) and primary tumor shared between 50-100% of mutations, including mutations in key driver genes such as APC, KRAS and TP53, consistent with these tumors being clonally related. In the patient with the Lynch syndrome the 2 tumors showed distinct somatic mutations suggestive of independent primaries. We identified eleven genetic events present in a distant metastasis and not in a primary tumor and none of these events was detected in the anastomotic recurrence arguing against re-seeding of the anastomotic site by a metastatic clone. All five patients with isolated anastomotic recurrence were free of disease 0.9 to 6.1 years following resection of their recurrent CRC. Conclusions: Anastomotic recurrences appear to be clonally related to the primary tumor in sporadic CRC cases. Our data also provide molecular data in support of the treatment of anastomotic recurrences as localized disease. Citation Format: Efsevia Vakiani, Ronak H. Shah, Christine Iacobuzio-Donahue, David B. Solit, Martin R. Weiser. Anastomotic recurrences are clonally related to primary tumors in sporadic colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2419.

Abstract 3624: Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations

Abstract Background: As our knowledge of how DNA alterations can drive cancer progression increases, assays that can simultaneously detect multiple types of variants in a simple and cost-effective manner are becoming increasingly crucial. This holds true of copy number variations (CNVs), where evaluation of this type of variant is an important and necessary feature of any solid tumor profiling assay. Conventional methods for detecting CNVs such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR) are limited to detecting only one gene amplification at a time. This can be a significant drawback with FFPE samples, where the DNA is of low abundance and often heavily degraded, while the number of gene amplifications known to be important in cancer continues to grow. Additionally, different tumor types may express amplifications at different rates and expression may be heterogeneous within the tumor, potentially with irregular staining patterns - all of which illustrate the need for new approaches to CNV detection. Methods: Next-generation sequencing (NGS) offers the ability to assess variants in multiple genes using one sample. To that end, Illumina is developing a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes for sequencing on the NextSeq1 platform. The assay consists of a DNA workflow for the identification of single-nucleotide variants (SNVs), small insertions and deletions (indels), as well as an RNA workflow for the identification of splice variants and gene fusions. In addition, using the DNA workflow, a novel analysis pipeline, and CNV caller, CNVs from 57 different genes can be simultaneously assessed all by sequencing of a single sample. Results: Here we present data on both cell lines and FFPE samples of SNVs and indels down to 5% allele fraction and CNVs down to ∼2-fold amplification, all from 40 ng of DNA. To demonstrate the accuracy and precision of our CNV detection method, we tested 7 samples for CNVs using orthogonal CNV detection methods. The Illumina NGS assay detected ERBB2 amplifications in 4 out of 7 samples. Of the 4 Illumina NGS positive samples, 3 samples were positive by FISH and all 4 were positive by droplet digital PCR (ddPCR) and the Illumina TruSight Tumor 15 panel. The 3 samples that were negative for ERBB2 amplifications by the Illumina NGS assay were also negative by both FISH and ddPCR. Within these samples we also found a previously unknown FGFR1 amplification. Conclusions: The novel Illumina NGS library preparation method is an innovative and useful tool to find multiple CNVs, along with other variant types, within a single sample. The assay can detect multiple CNVs within a single FFPE sample and identify previously uncharacterized CNVs that could be important in finding the correct treatment for a cancer patient. Citation Format: Chia-Ling Hsieh, Clare Zlatkov, Byron Luo, Chen Zhao, Kathryn Stephens, Han-Yu Chuang, Lisa Kelly, Katherine Chang, Rachel Liang, Jianli Cao, Scott Lang, Ashley Adams, Naseem Ajili, Laurel Ball, Glorianna Caves, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Sarah Dumm, Ridwana Ekram, Yonmee Han, Anne Jager, Suzanne Johansen, Li Teng, Jenn Lococo, Jaime McLean, Juli Parks, Jason Rostron, Jennifer Sayne, Jennifer Silhavy, June Snedecor, Mckenzi Toh, Stephanie Tong, Elizabeth Upsall, Paulina Walichiewicz, Xiao Chen, Amanda Young, Ali Kuraishy, Karen Gutekunst, Matt Friedenberg, Charles Lin. Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3624.

Next-Generation Sequencing of Matched Normal Blood Identifies Clonal Hematopoiesis in a Significant Subset of Solid Tumor Patients without Hematologic Malignancies

Background:Recent genomic studies have identified somatic mutations in leukemia genes in asymptomatic individuals without known hematologic disease. These mutations lead to clonal expansion of hematopoietic cells, in the absence of clinically overt hematologic transformation. This entity is thought to be analogous to other precursor conditions such as monoclonal gammopathy of undetermined significance (MGUS) and monoclonal B-cell lymphocytosis (MBL); consistent with this notion, a subset of patients with clonal hematopoiesis can subsequently develop myeloid malignancies, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, the incidence of clonal hematopoiesis in patients with solid tumors has not been extensively studied. Moreover, most genomic profiling platforms used to identify somatic mutations in epithelial tumors use blood as the matched normal control, which may lead to the identification of clonal hematopoiesis in a subset of cancer patients. We therefore sought to assess for clonal hematopoiesis in patients with solid tumors seen at Memorial Sloan Kettering Cancer Center (MSKCC) who were profiled using paired tumor/blood sequencing which is primarily designed to identify tumor-specific mutations.Methods:The study population included patients at MSKCC who were consented on protocol NCT01775072, “Tumor genomic profiling in patients evaluated for targeted cancer therapy.” All patients had tumor and blood genomic profiling sequenced using the MSK-IMPACT hybridization capture-based next-generation sequencing assay, which encompasses all protein-coding exons of 410 cancer-associated genes. For assessment of the incidence and distribution of blood mutations, 3,964 patients consented were evaluated. Patients with known active hematologic malignancies at time of sequencing were excluded from analyses. For comparisons regarding clinical correlations, 2,146 patients were included for whom detailed chart review has been performed.We investigated for the presence of “hotspot” mutations, collected from COSMIC database (v71), in matched blood DNA. Mutations were scored as present in the normal blood if they were detected in more than 8 reads with a variant allele frequency (VAF) greater than 2% which was at least twice the VAF seen in tumor. For cases where at least one mutation exceeded these thresholds, we reduced the VAF threshold in blood to 1% to detect secondary events. Mutations where VAF in the normal blood and the tumor sample were both greater than 30% were excluded as likely germline events.Results:Clonal hematopoiesis was identified in 108/3,964 solid tumor patients who had paired tumor/blood sequencing (2.7% of all patients); 7 patients had 2 unique mutations. DNMT3A mutations were most frequent, occurring in 34% of patients with clonal hematopoiesis (Figure 1). 107/115 mutations (93%) were in known leukemia-associated genes.We collected and analyzed detailed clinical parameters for 2,146 patients, including 42 patients (2.0%) with clonal hematopoiesis. The mean age at the time of testing was 62.1 years in patients with clonal hematopoiesis and 57.2 years in patients without evidence of clonal hematopoiesis (p = 0.015). When comparing baseline blood parameters, there were no statistically significant differences between the two groups. 62% of patients with clonal hematopoiesis had previous radiation therapy compared to 45% of patients without clonal mutations in their blood (p = 0.046). There was no statistically significant difference in the proportion of patients who had received previous chemotherapy (71% of patients in each group).On prospective follow up of patients with clonal hematopoiesis, no patients have progressed to develop overt MDS or AML, though median follow up was limited (median 11.7 months, range 5.5-16.7 months).Conclusions:Clonal hematopoiesis in solid tumor patients without known hematologic disease is common and is associated with increasing age and prior radiation therapy. In our cohort, no patients with clonal hematopoiesis progressed to overt MDS or AML though follow up is limited. Ongoing prospective observation of these patients is imperative to determine the clinical impact of these mutations after ongoing exposure to cytotoxic therapy. Further data including updated clinical and genomic correlates will be presented.Reference:Cheng DT et al. J Mol Diagn 2015 May; 17(3): 251-64. [Display omitted] DisclosuresHyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.