ctDNA Abundance Research Articles

Abstract CT331: “BEECH”, a phase I/II study of the AKT inhibitor AZD5363 combined with paclitaxel in patients with advanced or metastatic breast cancer: results from the dose-finding study, including quantitative assessment of circulating tumor DNA as a s

Abstract Background: The PI3K/AKT/MTOR pathway is frequently de-regulated in breast cancer. AZD5363 (AZD) is a potent selective inhibitor of AKT1-3. We report the dose-finding phase assessing the safety/tolerability of two intermittent weekly dosing schedules of AZD combined with paclitaxel (P) in HER2 negative breast cancer. The study incorporates prospective assessment of whether circulating tumour DNA (ctDNA) analysis can predict response/resistance earlier than conventional RECIST. Design: AZD capsules were dosed p.o. in one of two schedules: “4/3” (4 days on-treatment followed by 3 days off, starting 360 mg twice daily (bd)) or “2/5” (2 days on-5 days off, starting 560 mg bd). Both schedules comprised 3 weeks P 90 mg/m2 IV on Day 1 with AZD started on Day 2, and 1 week off-treatment. Patients who stopped P were able to continue AZD alone at investigators’ discretion. Escalating doses of AZD were evaluated in cohorts of 3-6 evaluable patients treated until disease progression, unacceptable toxicity, or withdrawal of consent. A safety review committee reviewed safety data and dose limiting toxicities (DLTs) prior to dose modification. Plasma samples for ctDNA extraction were collected at baseline, weekly in cycle 1, day 1 of all subsequent cycles, and were analysed by digital PCR. Female patients aged ≥18 years with HER2 negative MBC and up to two prior chemotherapy courses for advanced cancer were recruited. Results: 37 patients have received AZD. DLTs in 2/6 patients at 480 mg 4/3 comprised one patient with maculo-papular rash, and another with immune allergic reaction (both CTCAE grade 3). No DLT was observed in the 400 mg 4/3 cohort, which was defined as the maximum tolerated dose (MTD) in combination with P. DLTs in 2/6 patients at 640 mg 2/5 comprised one patient with prolonged neutropaenia (grade 4), and another with diarrhoea (grade 3). No DLTs were observed in 11 patients on the 560 mg 2/5 cohort. In all patients median progression free survival was 8.2 months, despite pre-treatment with taxane chemotherapy in 62% patients and a median of 2 prior lines of chemotherapy. A patient with Cowden's syndrome (PTEN germline mutation) had a partial response maintained for 18 months, including 12 months on AZD5363 alone. ctDNA assessment suggests that lack of a fall in ctDNA abundance may predict for early progression. Conclusions: AZD5363 400mg bd 4/3 in combination with paclitaxel was determined to be well tolerated in patients with MBC, with investigation of the 2/5 schedule continuing. Two international double-blind randomised studies have commenced to assess AZD5363 400 mg bd 4/3 schedule combined with paclitaxel versus paclitaxel plus placebo in ER positive HER2 negative MBC stratified by PIK3CA mutation status (BEECH Part B), and in triple negative breast cancer (PAKT). Citation Format: Nicholas C. Turner, Mafalda Oliveira, Anne Armstrong, Marie-Paule Sablin, José A. Perez-Fidalgo, Sarah Herebien, Isaac Garcia-Murillas, Stan Johnson, Andrew Foxley, Adnan Mahmood, Justin P. Lindemann. “BEECH”, a phase I/II study of the AKT inhibitor AZD5363 combined with paclitaxel in patients with advanced or metastatic breast cancer: results from the dose-finding study, including quantitative assessment of circulating tumor DNA as a s [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT331. doi:10.1158/1538-7445.AM2015-CT331

Abstract 2402: A research approach for the detection of somatic mutations at 0.5% frequency from cfDNA and cTc DNA using a multiplex sequencing assay targeting 2000 tumor mutations

Abstract Background: Availability of effective blood screening for tracking of recurrence and resistance of tumors may improve outcomes. Research studies suggest that virtually all tumors carry somatic DNA mutations, and these may serve as biomarkers that may be tracked from blood. The two well-characterized sources of tumor DNA in blood are circulating tumor cells (cTc) and cell-free tumor DNA (ctDNA). The abundance of cTc and/or ctDNA in blood may be very low at critical stages such as early recurrence or development of resistance. Hence there is great interest in being able to detect biomarkers at very low frequency from blood, and in characterizing the relationship between somatic mutations present in the tumor and those in cTc or ctDNA. Method: We present a research use only analysis workflow that facilitates researchers in detecting low frequency variants in blood. We developed an analysis algorithm, using statistical modeling of next generation sequencing reads, and optimizing parameters and filters to enable sensitive and specific detection of somatic mutations to 0.5% allele ratio. We demonstrate the analysis on a blood sample split into 3 sub-samples comprising germline blood cells, cTc, and ctDNA. We barcode the 3 sub-samples and run them on a single Ion PGM 318 sequencing chip using Ion AmpliSeq CHPv2 cancer hot-spot panel, that enables very deep (∼10,000x coverage) and accurate sequencing. This panel allows interrogation of ∼2000 relevant biomarkers from COSMIC and FDA actionable databases, and de-novo variant detection at ∼20,000 genomic positions. Mutations were annotated using the Oncomine database in Ion Reporter software. The assay requires a small amount of input DNA (∼10ng), and has a fast turn around time from sample to variants of less than 24 hr. Results: We tested the limits of variant detection in a dilution series; in cTc; and in ctDNA. First, we diluted an engineered DNA sample from Acrometrix into a control sample (NA12878) down to 0.5% frequency. The Acrometrix sample contains ∼500 common cancer mutations from COSMIC and FDA actionable databases. We achieved >99% sensitivity and specificity for variants present at frequency above 0.5%. Next, we spiked cTc cells from cancer cell lines into normal blood samples at ratio 1:1,000,000, obtaining 40% purity of cTc after enrichment, and demonstrated > 99% sensitivity and specificity of variant detection. Finally, we performed analytical validation of variant detection performance in ctDNA using a dilution series of two normal blood samples, and we were able to detect in all 20 variants present in either sample in ctDNA when their frequency was above 0.5%. Conclusions: The analysis workflow may facilitate researchers to study biomarkers in DNA from germline, cTc, and ctDNA, all available from a single blood sample, and to explore the relationship between biomarkers observed in solid tumor and those in blood. Citation Format: Dumitru Brinza, Dalia Dhingra, Charles Scafe, Richard Chien, Fiona Hyland. A research approach for the detection of somatic mutations at 0.5% frequency from cfDNA and cTc DNA using a multiplex sequencing assay targeting 2000 tumor mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2402. doi:10.1158/1538-7445.AM2015-2402

Abstract 4930: Next generation sequencing performance for the detection of mutations in plasma cell free DNA

Abstract The identification of mutations in circulating tumor DNA (ctDNA) can serve as a biomarker for non-invasive diagnosis and real time therapeutic monitoring. Although next generation sequencing (NGS) is a promising technology, the intrinsic low abundance of ctDNA in the high background of wild-type cell free DNA (cfDNA) makes the detection and quantification of such mutations in plasma a challenging task. This study aims to measure the performance of NGS analysis when applied to the identification of somatic mutations in plasma samples. Plasma samples from two healthy controls were used to isolate cfDNA. Constitutional genomic DNA from these controls was previously genotyped using the Human OmniExpress BeadChip. A custom multiplex PCR panel was designed to target 420 unique variants. To cover a broad range (0.5%-100%) of minor allele frequencies (MAF), as expected for somatic mutations in the plasma of cancer patients, the cfDNA samples from the two controls were mixed at five different rations (7:25, 1:6, 1:8, 1:20 and 1:100). Because sequence coverage is a key driver of accuracy, 5 different coverage profiles (100x, 500x, 1000x, 2000x and 5000x) were established for each of the cfDNA pools. On total 25 libraries were generated, pooled and sequenced with the Ion PGM system. Ion Reporter software was used for data analysis. The observed MAFs were compared with expected results for all the variants of each cfDNA pool. Median exon sequencing coverage of 133x, 650x, 1312x, 2628x and 5466x was obtained for each of the 5 cfDNA pools, with >99% of the amplicons covered. Overall base substitution and indel detection performance was high (>95% of the variants were detected across the different coverage levels). A direct correlation between expected and observed MAFs for the known variants was observed. For variants present at MAF ≥5%, a sensitivity of 91.4% and 100% was achieved at 100x and ≥500x coverage levels, respectively. For variants with 5% ≤ MAF ≤ 10% at 100x of coverage, the average quality score was <Q20. For variants occurring at low MAF (<5%) the detection sensitivity and the quality score dropped as coverage decreased. At 1000x coverage and above, more than 94% of the variants present at MAF <5% were successfully detected with an average quality score >Q30. Overall, positive predictive value remained high (>98%), with an average of 5 false-positive calls across the full coverage range. To validate our approach we analyzed 10 pairs of primary tumour/plasma samples. All cases carried known mutations of the EGFR gene detected during routine analysis. We were able to detect the corresponding mutation in the plasma samples in all cases. MAF in plasma samples ranged from 3% to 85%. Here we demonstrate that NGS can be used to detect mutations in cfDNA. Our approach with controlled data sets also enabled us to define allele frequency, allele depth, and allele quality score cutoffs for a reliable detection of low MAF variants in ctDNA. Citation Format: Ana Justino, Gabriela Fernandes, Ana Barroso, Barbara Parente, Venceslau Hespanhol, Jose C. Machado, Jose L. Costa. Next generation sequencing performance for the detection of mutations in plasma cell free DNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4930. doi:10.1158/1538-7445.AM2015-4930

Abstract 5230: Circulating tumor DNA as a ‘liquid biopsy’ in head and neck cancer

Abstract In cancer patients, a larger fraction of cell-free DNA is of tumor origin, released in circulating blood as a consequence of cell death and tissue trauma, ctDNA carries information on tumor-related genetic and epi¬genetic changes. Given the utility as a noninvasive marker ctDNA is commonly referred to as “liquid biopsy”. Some studies have correlated the abundance of ctDNA with tumor size and stage, with as much as 10% of ctDNA in advanced-stage cancers but as low as 0.1-1% in premalignant or early stage disease. Only one published study has explored the detection of ctDNA in Head and neck tumors in advanced cancers detecting mutations in 60% of the cases. Our primary goal was to provide a comprehensive evaluation of the presence of ctDNA in circulating blood fractions from a series of HNSCC cases at early and late stages. We also evaluated the extent to which measures of ctDNA in blood samples correlate with similar findings from paired tumor tissue and correlate with outcome. 48 cases (including plasma, tumor and germline samples) diagnosed as head and neck SCC were selected from a multi center case- control study (ARCAGE), conducted between 2002-2005 involving 10 countries across Europe. Stage distribution was as following: 40% stage IV, 23% stage II, 19% stage III, 15% stage I, 3% no Info. Additionally, 65 controls matched by center, age, sex, smoking and alcohol status were included to assess a threshold of the background noise after sequencing. Targeted sequencing was performed independently on both germline DNA and the tumor material using the Ion Proton™ System at an average depth of 250X. Sequencing included the entire coding region of 14 of the most frequently mutated genes in HNSCC: TP53, NOTCH1, CDKN2A, CASP8, PIK3CA, HRAS, FAT1, MLL2, NSD1, FBXW7, RB1, TP63, IRF6 and PTEN. Plasma samples were sequenced using an amplicon based approach. All 65 controls were also sequenced for all mutations detected. Plasma samples were sequenced at an average depth of 10000X. Mutation calls were based on a poisson distribution to distinguish true variants from background noise. Indels were manually inspected using IGV. Statistical tests were carried out in R statistical programming environment. 80% of tumor samples sequenced harboured a somatic mutation in at least one of the 14 genes, for a total 54 somatic mutations. 50% of these mutations including 2 indels were also detected in the corresponding plasma samples, allelic frequencies ranged from 0.005 to 0.30; cases were mostly stage IV although mutations were also detected in four early stage cases. Overall survival was lower for cases harboring TP53 mutations in both tumor and plasma. It is feasible to detect frequent mutations in circulating free DNA in both early and late stage HNSCC. Mutations detected in both tumor and plasma correlated with poor survival especially in oropharyngeal cases demonstrating that ctDNA might be included as a potential biomarker for patient monitoring and outcome prediction. Citation Format: Sandra Perdomo, Devasena Anantharaman, James Mckay, Paul Brennan. Circulating tumor DNA as a ‘liquid biopsy’ in head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5230. doi:10.1158/1538-7445.AM2015-5230