CLIA Laboratory Research Articles

#75: Microbial Cell-free DNA Sequencing for Prediction of Culture-Negative Infection Events in Children with Cancer

Abstract Background New culture-independent diagnostics are being investigated for diagnosis and prediction of infection. One method is microbial cell-free DNA sequencing (mcfDNA-seq), which can detect a wide range of pathogens directly from plasma. Immunocompromised children who develop febrile neutropenia (FN) without documented bloodstream infection (BSI) may have an undiagnosed bacterial infection, but identification of this subset is difficult. The percentage of episodes of FN caused by bacterial pathogens that did not grow in culture is unknown, as is the relative contribution of other specific etiologies such as viruses, fungi, immunotherapies, and immune reconstitution or graft vs. host disease. The value of mcfDNA-seq in FN patients is also unknown. As part of a larger study evaluating the ability of mcfDNA-seq to predict BSI in a cohort of pediatric patients with relapsed or refractory leukemia, we analyzed mcfDNA-seq results in a sample of cases of FN for which the definitive etiology was unknown. Methods Eligible participants were <25 years of age and undergoing treatment for cancer. Remnant plasma from clinical testing was prospectively obtained and stored. Samples collected on the day of onset of FN (Day 0) and the day prior (Day –1) underwent mcfDNA-seq by Karius Inc. in a CLIA and CAP-accredited laboratory. Pathogen detection results were reported in molecules per microliter (MPM) of plasma. Negative control samples from study participants without impending or recent fever or infection were also obtained. Testing was batched and blinded, so results were not clinically available. Results mcfDNA-seq results were obtained from eight episodes of FN in seven patients. Five episodes occurred in participants awaiting engraftment after HCT and three were receiving chemotherapy only. All participants receiving chemotherapy were receiving antibacterial prophylaxis with vancomycin and ciprofloxacin, antifungal prophylaxis with micafungin or voriconazole, and PJP prophylaxis with TMP-SMX or pentamidine. No HCT recipients were receiving antibacterial prophylaxis, but all received PJP prophylaxis, antifungal prophylaxis or treatment, and antiviral prophylaxis or treatment. Of 8 FN episodes, 4 (50%) had a common bacterial pathogen identified by mcfDNA-seq on Day 0 (Table 1). In 2 (50%) of these cases, the same organism was also identified on Day –1, at a lower concentration. One fungal pathogen was identified prior to and at the onset of FN. A common bacterial pathogen was identified in 3/64 (5%) control samples from the study population. Culture-negative sepsis was the final diagnosis in one episode; in this case, Streptococcus mitis, an important cause of sepsis in neutropenic patients, was identified in both Day 0 and Day –1 samples. In another episode where E. coli was identified, antibiotics were discontinued after 48 hours, but the patient was re-admitted within 24 hours for recurrent FN. Conclusions In this sample of culture-negative FN episodes in pediatric patients with relapsed or refractory leukemia, mcfDNA-seq identified a common bacterial pathogen in 50% of cases. The same organism was identifiable on the day prior to FN in 50% of cases, suggesting that predictive testing might be feasible. More data regarding sensitivity and specificity of mcfDNA-seq to diagnose and predict FN are needed.

Impact of COVID-19 on prevalence of community pharmacies as CLIA-Waived facilities.

BackgroundThe Clinical Laboratory Improvement Amendments of 1988 (CLIA) enabled greater access to low-risk tests by allowing their use in facilities with a Certificate of Waiver in the U.S. Recently, the 2019 novel coronavirus (COVID-19) pandemic has shined a spotlight on CLIA-waived diagnostic testing. To meet this increased patient demand for diagnostic testing, the U.S. Department of Health and Human Services (HHS) authorized licensed pharmacists to order and administer FDA authorized COVID-19 tests.ObjectiveThis study aims to update the previous national benching report and examine both the number of pharmacies in the United States with CLIA Certificates of Waiver before and after the SARS-CoV-2 pandemic and the state-by-state differences in the percentage of pharmacies with CLIA Certificates of Waiver.MethodsData were collected from the U.S. Centers for Disease Control and Prevention CLIA Laboratory Search website May 3rd, 2015, August 4th, 2019 and November 26th, 2020. The website allows for exportation of demographic data on all CLIA-waived facilities by state.ResultsPharmacies exhibited the largest growth both in number (4865 new locations) and by percent (45%) of CLIA-waived facilities between 2015 and 2020. The total number of pharmacies with a CLIA-waiver grew from 10,626 (17.94%) locations in 2015 to 12,157 (21.43%) locations in 2019, to 15,671 (27.63%) locations in 2020. States demonstrated considerable variability in the percentage of pharmacies with a CLIA-waiver, with a range of 2.92%–56.52%.ConclusionsPharmacies have become an increasingly important location for patients to access CLIA-waived tests in the United States, now serving as the second largest provider of CLIA-waived tests by the total number of locations. Most of this growth occurred between 2019 and 2020 due to the COVID-19 pandemic, and concentrated efforts will be necessary to sustain this momentum.

Metagenomic sequencing and evaluation of the host response in the pediatric aerodigestive population.

To assess the diagnostic utility of metagenomic sequencing in pediatric aerodigestive clinic patients being evaluated for chronic aspiration. We hypothesize that using a metagenomics platform will aid in the identification of microbes not found on standard culture. Twenty-four children referred to an aerodigestive clinic were enrolled in a prospective, single-site, cross-sectional cohort study. At the time of clinical evaluation under anesthesia, two samples were obtained: an upper airway sample and a sample from bronchoalveolar lavage (BAL). Samples were sent for routine culture and analyzed using Explify® Respiratory, a CLIA Laboratory Developed Test which identifies respiratory commensals and pathogens through RNA and DNA sequencing. Since RNA was sequenced in the course of the metagenomic analysis to identify organisms (RNA viruses and bacteria), the sequencing approach also captured host derived messenger RNA during sample analysis. This incidentally obtained host transcriptomic data were analyzed to evaluate the host immune response. The results of these studies were correlated with the clinical presentation of the research subjects. In 10 patients, organisms primarily associated with oral flora were identified in the BAL. Standard culture was negative in three patients where clinical metagenomics led to a result with potential clinical significance. Transcriptomic data correlated with the presence or absence of dysphagia as identified on prior videofluoroscopic evaluation of swallowing. Clinical metagenomics allows for simultaneous analysis of the microbiota and the host immune response from BAL samples. As the technologies in this field continue to advance, such testing may improve the diagnostic evaluation of patients with suspected chronic aspiration.

Universal Sensitive, Accurate and Precise Microchimerism Surveillance Solution for Allogeneic Hematopoietic Cell Transplant

The success of hematopoietic cell transplantation (HCT) is limited by a high rate of disease relapse. Post-HCT chimerism analysis is typically used to evaluate donor cell engraftment and success of therapy in allogeneic HCT. The detection of microchimerism, especially in the early period after allogeneic HCT, is an important high-risk factor for disease relapse (Ahci et al, 2017). However, common approaches based on semi-quantitative fluorescent PCR of short tandem repeats (STRs) suffer from low sensitivity and high result variability and often fail to detect microchimerism and predict patient outcomes (Schuurhuis et al, 2018). Here, we describe development and characterization of AlloHemeTM, a novel sensitive and precise chimerism surveillance solution with high accuracy for detection and quantification of low levels of patient cells which ultimately can lead to clinical disease recurrence. The method is compatible with chimerism assessment in whole blood, bone marrow, or specific cell subtypes. CD3, CD15, CD33 and CD34 markers are used to separate T cells, myeloid progenitors and granulocytes from blood and hematopoietic stem cells from bone marrow, respectively. A minimum of 8ng genomic DNA is used to amplify hundreds of single nucleotide polymorphisms (SNPs) using target specific primers in a single multiplex reaction. Because there is no need to select genetic variants that differ for specific donor-patient pair, the method is universally applicable to any allogeneic HCT. Relative contribution of SNP alleles is measured by Next Generation Sequencing (NGS) and % chimerism of post-HCT samples is calculated using an analysis pipeline incorporating open source and custom bioinformatics tools. To evaluate reproducibility of cell subtype separation, blood from 3 healthy volunteers was subjected to cell separation for CD3, CD15 and CD33 cells at four different timepoints after draw (24, 48, 72 and 96 hours). Flow cytometry analysis using non-competing antibodies targeting same cell populations demonstrated that the cell subtype isolation step consistently yields sub-populations with an average purity of 96% across all cell subtypes and timepoints evaluated (93-99% range). To evaluate assay sensitivity, accuracy and precision, genomic DNA samples (gDNA, Coriell Institute) were used to prepare 2 panels, A and B, each containing different mixtures of gDNAs to simulate post-HCT samples with varying contribution of patient's cells. Panel A contained mixtures of gDNA samples from unrelated individuals ranging from 0.01-50% and panel B from related (parent-child) ranging from 0.01-75%. Each mixture was tested in 6 (Panel A) or 5 (Panel B) independent replicates. The results show that the assay is accurate from 0.03% through 50% for panel A and 0.1% through 75% for panel B with minimal proportional and systematic bias (Figure 1). The assay precision is high, with a mean coefficient of variation (CV) of 2.6% for panel A and 6.1% for panel B across the entire linear range. For sample mixtures with chimerism levels greater than 1%, mean CV was 0.76% for panel A and 1.62% for panel B. AlloHeme results show that the test can detect microchimerism down to 0.03%. High result reproducibility is achieved through the use of a proprietary NGS process and automated data analysis and workflow, generating results in the CLIA lab with a turnaround time of 3 days. The test was developed with rigorous QC steps to ensure high purity and accuracy for each cell population analyzed, increasing confidence and sensitivity of results. Given previously published data on clinical value of microchimerism, AlloHeme has the potential for early relapse detection post-HCT. Future studies will evaluate the utility of AlloHeme in clinical setting with the goal to improve patient outcomes and survival rates. In summary, we have developed a sensitive, accurate and precise method for the universal detection and quantification of chimerism and potential early disease recurrence detection for allogeneic HCT patient surveillance. Because the method provides high sensitivity and consistent results for microchimerism detection, it may be particularly suited to help identify recurrence of primary disease in hematologic cancer patients with or without known cancer-associated genetic determinants. Disclosures Egidio: CareDx: Current Employment, Current equity holder in publicly-traded company. Janakiraman:CareDx: Current Employment. Mao:CareDx: Current Employment. Gulbahce:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Wong:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Marchis:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Ghosh:CareDx: Current Employment. Grskovic:CareDx: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.

Changes in genetic variant results over time in pediatric cardiomyopathy and electrophysiology.

Genetic testing for cardiac disorders continues to change. Our objective was to assess trends in variant classification in pediatric arrhythmia and cardiomyopathy. We conducted a retrospective review of patients tested for genetic arrhythmia and cardiomyopathy disorders from 2006-2017. Variants were classified by CLIA laboratories. Trends were assessed by the Spearman correlation. There were 914 variants in 583 patients from 337 families. The total number of tests ordered increased over time, accelerating after 2012. There was a strong positive correlation between the average number of genes tested per panel and year of testing (r=.97, p<.001) and a weak correlation between the year and a decrease in the percentage of clinically actionable variants (r=-.20, p=.005). By 2011, VUS represented >50% of variants reported on panels. Over 12years, 203 genes were interrogated; one or more variants were reported in 91 of 203 genes (45%). 32% of patients had at least one clinically actionable variant; 28% had at least one VUS. Reclassification is an important long-term issue, with 21.5% variants changing clinical interpretation. We observed an increase over time in three areas: total number of tests ordered, average number of genes/panel, and percentage of VUS. Providers may need to interpret results from 90+genes, and ongoing education is critical. Due to their specific training in test result interpretation, we recommend the inclusion of a genetic counselor in pediatric electrophysiology and cardiomyopathy teams.

Analytical validation of digital cytometry (iSort) for leukocyte enumeration using stored blood.

3542 Background: Blood leukocyte enumeration is a cornerstone for clinical diagnosis and immune monitoring of diverse cancers and immunotherapies. Existing methods rely on intact/living cells and can thus be limiting due to handling time constraints and need for predefined antibody panels. While indirect cytometry methods including digital cytometry can overcome this limitation on archival specimens, their clinical performance has not been extensively characterized. We developed iSort, a novel transcriptome deconvolution method based on CIBERSORTx. Here, we comprehensively evaluated iSort and validated it against established diagnostic standards. Methods: We recruited 36 healthy adult blood donors and characterized their blood leukocyte profiles. We used several established clinical cytometry methods requiring intact cells in a CLIA laboratory including Complete Blood Count [CBC] and 6-color TBNK [TBNK]. We also immunophenotyped leukocytes by a research flow cytometry panel (FACS) and by mass cytometry (CyTOF). We then used these techniques as standards for validating leukocyte populations enumerated by iSort. iSort was performed on whole blood through deconvolution of 22 subsets from RNA-Seq. We assessed iSort’s analytical detection performance by spiking purified lymphocyte subsets into lymphodepleted human blood and by simulating blood mixtures using defined leukocyte mixtures within latin square designs. We assessed iSort concordance with an FDA approved IVD assay (TBNK) comparing distinct RNA-Seq library preparation chemistries. Results: iSort was highly correlated with TBNK/CBC across CD4 T, CD8 T, B cells, NK cells, monocytes, and neutrophils (r≥0.95). When comparing correlations to TBNK/CBC, we found no significant differences between iSort, CyTOF, and FACS, nor between RNA-Seq library chemistries. iSort demonstrated high linearity at low abundance levels (0.1 - 1%, r = 0.99, B-cells spiked into lymphodepleted blood samples after Rituximab) and at higher abundance levels (0.5 - 90%, r &gt; 0.99) across lymphoid and myeloid subsets. iSort also showed high reproducibility among triplicate blood tubes for each population (median CV = 11%). Conclusions: iSort digital cytometry achieves highly accurate and robust leukocyte enumeration for diverse hematopoietic subsets. Given its favorable performance against existing clinical standards that require intact/living cells, iSort is a promising approach for the development of immunotherapy biomarkers.

Protein Biomarker Quantification by Immunoaffinity Liquid Chromatography-Tandem Mass Spectrometry: Current State and Future Vision.

Immunoaffinity-mass spectrometry (IA-MS) is an emerging analytical genre with several advantages for profiling and determination of protein biomarkers. Because IA-MS combines affinity capture, analogous to ligand binding assays (LBAs), with mass spectrometry (MS) detection, this platform is often described using the term hybrid methods. The purpose of this report is to provide an overview of the principles of IA-MS and to demonstrate, through application, the unique power and potential of this technology. By combining target immunoaffinity enrichment with the use of stable isotope-labeled internal standards and MS detection, IA-MS achieves high sensitivity while providing unparalleled specificity for the quantification of protein biomarkers in fluids and tissues. In recent years, significant uptake of IA-MS has occurred in the pharmaceutical industry, particularly in the early stages of clinical development, enabling biomarker measurement previously considered unattainable. By comparison, IA-MS adoption by CLIA laboratories has occurred more slowly. Current barriers to IA-MS use and opportunities for expanded adoption are discussed. The path forward involves identifying applications for which IA-MS is the best option compared with LBA or MS technologies alone. IA-MS will continue to benefit from advances in reagent generation, more sensitive and higher throughput MS technologies, and continued growth in use by the broader analytical community. Collectively, the pursuit of these opportunities will secure expanded long-term use of IA-MS for clinical applications.

Deep Sequencing of Viral Cell-Free DNA for Noninvasive Detection of Immunosuppression-Related Lymphoid Malignancies

Background: Immunocompromised patients have higher risk for virally-driven lymphomas associated with pathogens including Epstein-Barr Virus (EBV), Kaposi Sarcoma Herpesvirus (KSHV), HTLV-1, and HIV-1. Within this high-risk patient population, early cancer detection remains a critically unmet clinical need. Recently, analysis of viral cell-free DNA (cfDNA) has shown promise as a predictive biomarker for both immune status and cancer risk. In solid organ transplant recipients, immunosuppression is associated with expansion of a circulating family of viruses called Anelloviridae (De Vlaminck I, Cell, 2013). In screening for EBV-driven nasopharyngeal carcinomas (NPC), EBV cfDNA fragment length distributions distinguish normal adults with transient viremia from those with high risk of NPC (Lam WK, PNAS, 2018). We developed VirCAPP-Seq (Viral Cancer Personalized Profiling by Deep Sequencing) as a clinical virome capture approach for enriching and deeply sequencing circulating viral nucleic acids to elucidate virome composition, fragment length distribution, host genome integration sites, and viral polymorphisms. These features of viral cfDNA may clarify patient risk for malignancy and immunosuppression. Method: 180 viral species from 15 families were selected on the basis of 3 criteria: (1) Known oncoviruses (eg, EBV, Human Papillomavirus (HPV), Hepatitis B Virus (HBV)); (2) Commensal viruses associated with immune state (eg, Anelloviridae); (3) Common clinically relevant viruses (Adenovirus, CMV, BK, etc.). Viral reference genomes were segmented into continuous sliding 50mer windows, and compared to hg19 and a pan-viral reference database for homology using BLASTN. Non-unique regions were excluded. Remaining regions were clustered using CD-HIT at 85% homology to minimize redundant probes (figure 1A). We applied VirCAPP-Seq to plasma samples of patients with known levels of EBV viremia. To evaluate possible interactions of VirCAPP-Seq with human probes, we applied the viral panel alone and in combination with a CAPP-Seq lymphoma panel (Kurtz DM, JCO, 2018). Reads were aligned to a composite reference genome consisting of 180 viral reference genomes and hg19. For each targeted virus, plasma (GE) were estimated by computing the ratio of unique viral depth to human depth, and multiplying by the measured plasma DNA concentration and plasma volume. We correlated the sequencing-based GE estimate to viral titers determined by a clinical RT-PCR assay measured in a CLIA laboratory. Viral integration sites were evaluated with Virus-Clip (Ho DW, Oncotarget, 2015). Results: The final VirCAPP-Seq design consists of 2349 target regions across 180 species, spanning 2.097 Mb. A median of 95.9% of each targeted virus was covered by the panel (IQR 85.1% - 99.0%). Sequence-based phylogenetic analysis demonstrated clear relationships between members of each viral family. However, following in silico masking of non-unique regions, these phylogenetic relationships were no longer discernible, demonstrating that our design avoids targeting regions with interspecies ambiguity (figure 1B). Surprisingly, addition of VirCAPP-Seq to a human capture panel did not significantly impact the human on-target rate compared to human-only capture of lymphoma CAPP-Seq targets (Kurtz et al 2018 JCO), suggesting minimal non-specific interaction between viral probes and host DNA (figure 1C). Across EBV-positive samples, VirCAPP-Seq enriched EBV cfDNA a median of 214,000x compared to off-target EBV abundance in human-only capture (IQR 55,100x - 277,600x) (figure 1D). EBV-positive samples achieved a median EBV depth of 1015x (IQR 210x - 2802x) after enrichment. Additionally, viral GE estimates from read counts were highly correlated with viral titer estimates by clinical RT-PCR (r = 0.92, p &amp;lt; 0.001, n=9) (figure 1E). We observed several significant correlations between virome measurements across a range of immunosuppression states and lymphoma subtypes, with details to be presented at the meeting. Conclusions: VirCAPP-Seq enables simultaneous enrichment of clinically relevant viral and host cfDNA from plasma over several orders of magnitude. This framework holds promise for monitoring diverse features of viral cfDNA that capture risks for malignancy and immunosuppression, facilitating personalized diagnostic and therapeutic interventions. Disclosures Kurtz: Roche: Consultancy. Advani:Janssen: Research Funding; Kura: Research Funding; Merck: Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cell Medica, Ltd: Consultancy; Kyowa Kirin Pharmaceutical Developments, Inc.: Consultancy; Forty-Seven: Research Funding; Agensys: Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celmed: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc./Kite Pharma, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity Pharma: Research Funding; Millennium: Research Funding; Regeneron: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Stanford University: Employment, Equity Ownership; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees. Diehn:AstraZeneca: Consultancy; BioNTech: Consultancy; Quanticell: Consultancy; Novartis: Consultancy; Roche: Consultancy.

519P - Initial results of lung cancer genomic screening project for individualized medicine in Asia: LC-SCRUM-Asia

BackgroundLC-SCRUM was established in 2013 to screen target genes in lung cancer to advance the development of new molecular targeted drugs and diagnostics. To date, with the cooperation of over 200 institutions across Japan, LC-SCRUM has performed genomic analysis of over 7000 lung cancer patients. Since Mar 2019, LC-SCRUM has expanded to Taiwan as genomic screening infrastructure with the cooperation of Asian countries. Here, we report initial results of Asian international genomic screening, LC-SCRUM-Asia. MethodsTumor samples of non-small cell lung cancer patients were analyzed for oncogenic alterations using targeted next-generation sequencing (NGS) with Oncomine Comprehensive Assay. The analysis was performed as central test at CLIA lab in Japan. ResultsAs of May 31th in 2019, the LC-SCRUM-Asia has the participation of 5 institutions in Taiwan and 161 in Japan. For the first 2 month, a total of 477 patients (23 from Taiwan and 454 from Japan) were enrolled in this study. Median age were 68 years (range, 45-84) in Taiwan cohort and 67 (28-87) in Japan. In Taiwan 65% of patients were women, 70% adenocarcinoma and 70% non-smoker, and in Japan 37% of patients were women, 79% adenocarcinoma and 29% non-smoker, respectively. Of 13 analyzable patients in Taiwan, targeted NGS showed that 9 (69%) had at least one targetable oncogenic alterations, including 4 EGFR mut, 2 KRAS mut, 2 ALK fusinos and 1 RET fusions plus EGFR mut. Of 423 patients in Japan, 196 (46%) had at least one targetable oncogenic alterations. Oncogenic alterations commonly found were EGFR mut (19%), KRAS mut (13%), ALK fusions (2%), RET fusions (1%) and others in Japan. ConclusionsAsian international genomic screening can be implemented between Taiwan and Japan settings. LC-SCRUM-Asia will contribute to further development of precision medicine for lung cancer patients in Asia. Updated results will be presented at the meeting. Legal entity responsible for the studyLC-SCRUM-Asia. FundingAstraZeneca, Ignyta, Kyowa Hakko Kirin, Daiichi Sankyo, Eisai, Taiho, Takeda, Merck Serono, Novartis, MSD, Pfizer, Lilly, Bristol-Myers Squibb, Chugai, Boehringer Ingelheim, Astellas, LOXO, Janssen, Thermo Fisher Scientific, Ono. DisclosureC-H.S. Kuo: Honoraria (self): AstraZeneca; Honoraria (self), Advisory / Consultancy: Boehringer Ingelheim; Honoraria (self): Roche; Honoraria (self): Pfizer; Honoraria (self): Eli Lilly; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self): ONO Pharma; Honoraria (self), Advisory / Consultancy: Merck; Advisory / Consultancy: Chugai; Advisory / Consultancy: Takeda. K. Yoh: Honoraria (self): Chugai; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Taiho; Research grant / Funding (institution): MSD; Honoraria (self): Novartis. Y. Zenke: Honoraria (self): Boehringer Ingelheim; Honoraria (self): AstraZeneca; Honoraria (self): Lilly; Honoraria (self): Chugai; Honoraria (self), Research grant / Funding (institution): MSD; Honoraria (self): Taiho; Honoraria (self): Ono pharmaceutical. S. Matsumoto: Honoraria (self): AstraZeneca; Honoraria (self), Research grant / Funding (institution): Novartis; Honoraria (self): Pfizer; Honoraria (self), Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Merck Serono; Research grant / Funding (institution): MSD; Honoraria (self), Research grant / Funding (institution): Chugai Pharma. K. Goto: Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (institution): Lilly; Honoraria (self), Research grant / Funding (institution): Chugai; Research grant / Funding (institution): Ignyta; Research grant / Funding (institution): Kyowa Hakko Kirin; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Eisai; Research grant / Funding (institution): Taiho; Research grant / Funding (institution): Takeda; Research grant / Funding (institution): Merck Serono; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Astellas; Research grant / Funding (institution): LOXO; Research grant / Funding (institution): Janssen; Research grant / Funding (institution): Thermo Fisher Scientific; Research grant / Funding (institution): Ono. All other authors have declared no conflicts of interest.

Highlights of This Issue

The FGFR pathway is thought to drive oncogenic progression of many tumor types. Bahleda and colleagues report on the results of a first-in-human phase I trial of erdafitinib (JNJ-42756493), a potent, oral pan-FGFR tyrosine kinase inhibitor, conducted on patients with advanced-stage disease with known genetic activation of the FGFR pathway. This agent was well-tolerated and showed promising efficacy, especially in urothelial carcinoma and cholangiocarcinoma patients with FGFR alterations. These results have the potential to influence future treatment of patients with FGFR positive tumors.Myelofibrosis is morphologically characterized by the presence of atypical megakaryocytes and bone marrow fibrosis. To determine whether elimination of these abnormal megakaryocytes has therapeutic benefit for myelofibrosis patients, Gangat and colleagues conducted a phase 1 study of alisertib, an aurora kinase A inhibitor and megakaryocyte differentiation agent. Alisertib was well-tolerated and provided a modest clinical benefit for spleen size and symptom burden. Moreover, alisertib normalized megakaryocyte morphology and reduced fibrosis in a substantial number of patients. This study underscores the role of megakaryocytes in myelofibrosis and highlights the feasibility of targeting this lineage in this disease.Cancer vaccines have long been considered a promising tool for cancer therapy. However, many potential vaccines have been ineffective in clinical trials, in part due to the immunosuppressive cancer microenvironment. In a phase I trial, Gatti-Mays and colleagues examined BN-CV301, a modified cancer vaccine expressing MUC1 and CEA tumor antigens, as well as costimulatory molecules. Treatment-related adverse events were relatively minor. Patients with mutant KRAS responded particularly well to this regimen – five out of six patients showed either a partial response or stable disease after BN-CV301 vaccination. This improved vaccine shows promise for further clinical evaluation.Pancreatic ductal adenocarcinoma is a highly lethal cancer due, in part, to a high rate of recurrence. Groot and colleagues developed and validated an assay to detect KRAS hotspot mutations in circulating tumor DNA (ctDNA) in a CLIA laboratory setting. Detection of mutant KRAS in ctDNA at the time of surgery was associated with a greater risk of recurrence, while rising mutant KRAS levels in ctDNA provided an early prediction of recurrence. In several instances, the detection of mutant KRAS in ctDNA outperformed the measurement of CA 19-9 in serum. This liquid biopsy test for mutant KRAS shows great promise for monitoring disease in pancreatic cancer patients, potentially leading to earlier intervention.

Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease.

Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.

Abstract P2-08-57: A biologic signature to predict ipsilateral breast event risk at 10 years for early breast cancer

Abstract Background Outcomes for women with early breast cancer have continually improved. A biologic signature to identify those patients that have elevated ipsilateral breast event (IBE) risk after breast conserving surgery (BCS) treated with or without radiation therapy (RT) is needed. More aggressive systemic or surgical options may be warranted for patients with elevated risk while BCS alone may be an option for very low risk patients. We report early results for a biologic signature interrogating critical pathways. Material and Methods This study includes patients from Uppsala University Hospital and Västerås Hospital diagnosed with early breast cancer, 20mm or less, treated surgically between 1987 and 2004. Women with lymph node metastases or treated with mastectomy or chemotherapy were excluded. A panel of biomarkers (HER2, PR, Ki67, COX2, p16/INK4A, FOXA1 and SIAH2) were assayed and scored in PreludeDx's CLIA lab by board-certified pathologists. There were 171 eligible patients with biomarker data; 131 received RT and 9 received hormone therapy. Risk groups were calculated using biomarkers and clinical factors age and size. Absolute 10-year IBE risk was assessed using Kaplan-Meier survival analysis. Hazard ratios (HR) were determined using Cox proportional hazards analysis. Results There were 49 IBEs recorded. The biologic signature classified 41% of women into a low risk group. Patients in the elevated risk group had a significantly increased risk of 10-year IBE compared to those in the low risk group (Table 1). The HR for elevated vs. low risk group was 5.0 [2.2-11], p&amp;lt;0.001, in a multivariate analysis of risk group and RT. Patients in the elevated risk group treated with BCS and RT had an 18% apparent risk difference in 10-year IBE. Patients in the low risk group had similar low 10-year risks of IBE, when treated with BCS, with or without RT. The low risk women had somewhat increased prevalence of low grade tumors (58% vs. 41%). Women with low grade and small tumors (up to 10mm) were classified into both risk groups (54% low vs. 38% elevated risk). Table 1:10-year Risks of Local Recurrence by Risk GroupBCS without RTBCS plus RTN10-Yr local IBE Risk, 95%CIn10-Yr local IBE Risk, 95%CIBaseline4028%, [11% – 31%]13122% [12% – 24%]Low Risk Group196% [0%-14%]516% [0%-12%]Elevated Risk Group2149% [20%-68%]8031% [21% - 41%] Discussion A biologic risk signature identified early breast cancer patients with low and elevated 10-year IBE risks for women treated with BCS with or without RT and no chemotherapy. Approximately 40% of women were classified into a low risk group with a 0.5% IBE risk per year. Women in the elevated risk group had 3% to 5% IBE risks per year depending on treatment. Treatment for women in this observational study was neither randomized nor strictly rules based. With further prospective validation, the biologic signature identified herein may provide a tool enabling improved management for women diagnosed with early breast cancer. Citation Format: Bremer T, Savala J, Leesman G, Wärnberg F, Sund M, Wadsten C, Whitworth PW. A biologic signature to predict ipsilateral breast event risk at 10 years for early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-57.

Risk stratification in earl- stage luminal breast cancer patients treated with and without RT.

568 Background: The goal was to develop and validate a biologic signature for 10-year ipsilateral invasive breast event (IBE) risk in luminal Stage 1 breast cancer (BC) patients treated surgically and either with or without radiation therapy (RT). Methods: This cohort was from Uppsala University and Västerås Hospitals diagnosed with Stage 1 BC and treated surgically between 1987 and 2004. Treatment was neither randomized nor strictly rules based, including adjuvant RT, Hormone Therapy (HT), and Chemotherapy (CT). Biomarkers (HER2, PR, Ki67, COX2, p16/INK4A, FOXA1 and SIAH2) were assessed on tissue microarrays in PreludeDx’s CLIA lab by board-certified pathologists. Risk groups were calculated using biomarkers and clinical factors age and size. A multivariate Cox proportional hazards analysis was used to determine hazard ratio for biologic signature. 10-year IBE risk was assessed using Kaplan-Meier survival analysis. Results: There were 423 luminal cases with biomarker data having 54 IBEs, and a median follow-up of 11.8 years. There were 372 patients treated with BCS and 51 with Mastectomy, and 325 received RT, 169 received HT, and 47 received CT. In a multivariate analysis, the biologic signature (HR = 1.6, p = 0.019) and RT (HR = 0.51, p = 0.027) were associated with IBE risk adjusting for other treatments (HT and CT) and Luminal A status (p = 0.37). For patients over 50 yrs of age with luminal A disease and treated without CT (n = 205), an elevated biologic signature identified a subset of patients with a 15% (+/- 14%) 10-year IBE risk without RT (n = 38) compared to a 4% (+/-6%) IBE risk with RT (n = 72), while patients with a low biologic signature had a 10-year IBE risk of 4% (+/- 4%) without RT (n = 26) and 3% (+/-5%) IBE risk with RT (n = 69). Conclusions: With further prospective validation, the biologic signature identified herein may provide a tool enabling improved management for women diagnosed with early luminal BC.

Early Detection Research Network: Accomplishments and Outlook.

In 2000, the National Cancer Institute established the Early Detection Research Network (EDRN) to discover, develop, and validate biomarkers to detect cancers early when they are most treatable. The EDRN′s mission recently expanded to include finding and validating biomarkers that can determine which neoplasias are likely to progress to aggressive disease and to develop and validate imaging methods to better detect and characterize early-stage cancers. EDRN investigators have made significant progress toward achieving these goals, including participation in the discovery and/or validation of 6 diagnostic tests that have been approved by the Food and Drug Administration and 12 tests that are in use in CLIA laboratories. This progress is because of the quality research conducted by the EDRN investigators, the integrated structure of the EDRN that fosters a collaborative teamwork approach, and the ability of the EDRN to collaboratively validate biomarkers and imaging methods developed by non-EDRN investigators. The network includes >30 institutions, >150 investigators, and >200 associate members (i.e., non-EDRN investigators). The development of new biomarkers for early detection of cancer is a lengthy process that begins with the discovery of promising candidate biomarkers, rigorous validation, and implementation in the clinic. Biomarkers that successfully meet clinical significance thresholds can be practice changing, and the ultimate level of success is to …

Abstract 1034: Genotyping of patient-derived xenograft tumors using short tandem repeats: Challenges and observations in ensuring concordance across passages for effective preclinical studies

Abstract Patient derived xenograft (PDX) tumor models represent the closest step to in-human trials for cancer drug research and are therefore an invaluable resource. Implantation of a cancerous tissue from a patient's primary tumor directly into an immune-deficient mouse, generating a xenograft tumor potentially mimics the tumor microenvironment in the patient and allows for the evaluation of therapies specific to a particular tumor. One of the significant challenges associated with PDX tumors is the potential loss of the human component or the take-over by mouse tissue with every passage into a new mouse model. Short tandem repeats (STRs) are short intronic sequences of DNA that are highly polymorphic and contain a pattern of nucleotides repeated in tandem. STRs can be found in distinct regions of both prokaryotes and eukaryotes organisms, with each organism containing a unique combination of the STR allelic frequencies that allow for individuals to be unambiguously identified. STR analysis is routinely used to genotype PDX tumors comparative to the original resected human cancerous tissue, ensuring concordance with original tissue and to identify mouse tissue contamination with progressive passages. The Jackson Laboratory for Genomic Medicine's CLIA laboratory has evaluated over 170 PDX tumor samples from multiple mouse model passages across 105 diverse PDX mouse models. Of those samples analyzed, genomic DNA for 33 had been extracted from fresh frozen tissue and for 143 extracted from Formalin Fixed Paraffin embedded tissue (FFPE). The current study presents the challenges and discrepancies observed in our evaluation relative to number of passages for each tumor, sample type (FFPE vs fresh frozen) - reflective of tumor heterogeneity and also potential encroachment by mouse tissue. We also present results of a cross institutional comparative STR analysis study in 123 samples wherein discordance observed in certain cases were a result of differing sample types, suggestive that tumor heterogeneity can affect the STR results and could be a leading cause for cross institutional discordant genotyping results. Citation Format: Bridgette Sisson, Andrew Hesse, Melissa Soucy, Daniel Bergeron, Shelbi Burns, Kevin Kelly, Emily Jocoy, Margaret Bundy, Honey V. Reddi. Genotyping of patient-derived xenograft tumors using short tandem repeats: Challenges and observations in ensuring concordance across passages for effective preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1034.

Abstract GS5-08: A validation of DCIS biological risk profile in a randomised study for radiation therapy with 20 year follow-up (SweDCIS)

Abstract Background: Women diagnosed with ductal carcinoma in situ (DCIS) and their physicians need tools that assess individualized risk and predict treatment benefit. A DCIS biologic signature was previously validated in an observational study at Kaiser Permanente NW. We evaluated the results of the signature for predictive utility in a national randomized clinical trial (SweDCIS) by assessing the 10-year benefit of adjuvant radiotherapy (RT) on ipsilateral breast event (IBE) and invasive breast cancer (IBC) risks. Methods: The signature was validated in a prospective-retrospective study in women from the SweDCIS trial (n=1046) performed by the Swedish Breast Cancer Group. Women were treated with breast conserving surgery (BCS) between 1987-1999 and randomized to RT or no RT. A central pathology review of paraffin embedded tissue blocks (n=873) was performed at Uppsala University (UU). Freshly cut slides were provided to PreludeDx for biomarker testing. Extended follow-up of SweDCIS was published in 2014. A panel of biomarkers (HER2, PR, Ki67, COX2, p16/INK4A, FOXA1 and SIAH2) were assayed and scored in PreludeDx's CLIA lab by board-certified pathologists. Continuous Decision Scores (DS) were calculated with the biologic signature using the biomarker and clinical factors (age, size, margin, and palpability) blinded to patient outcome. The DS results were provided to the Uppsala Regional Cancer Center for analysis. A predefined and co-developed statistical analysis plan was executed. Absolute 10-year RT benefit was assessed using Kaplan-Meier survival analysis. Hazard ratios (HR) were determined using Cox proportional hazards analysis and the interaction of the DS and RT benefit was assessed. Results: Complete biomarker and clinical information was available in 584 women. In women with clear margins (n=506), 78 IBEs, including 31 IBCs, were recorded within 10 years of diagnosis. The multivariate analysis of DS (0-10 unit scale) and the RT interaction was significant for risk of IBC (p=0.048) and IBE (p&amp;lt;0.001) at 10 years. The DS defined an elevated risk group (&amp;gt;3) for which there was pronounced 10-year benefit of RT (p=0.01) with an absolute risk reduction of 9% for IBC (Table 1). The corresponding low risk group (≤3), which included 48% of all patients, demonstrated no significant RT benefit (p=0.70) with an absolute risk reduction of 1%. The continuous DS variable was correlated with IBE risk, HR 1.49/per 5 units 95%CI[1.02,2.18] (p=0.038), in addition to the RT benefit for IBE in low (p=0.04) and elevated (p&amp;lt;0.001) risk groups. Table 1. 10-year RT benefit in women from the SweDCIS trial.DS Risk GroupsIBC eventsIn Situ or IBC eventsnAbsolute RT-benefitHR [95%CI] Absolute RT-benefit HR [95%CI]Low Risk Group (DS≤3)2431%0.83 [0.32, 2.16]9%0.48 [0.24-0.97]Elevated Risk Group (DS&amp;gt;3)2639%0.24 [0.08, 0.73]17%0.31 [0.17-0.59] Discussion: Evaluation of the SweDCIS trial validated prognostic and RT predictive utility of the biologic signature. Women diagnosed with DCIS and treated with BCS±RT were stratified into clinically relevant low and elevated risk groups (≤3 vs &amp;gt;3). Women in the elevated risk group had twice the treatment benefit for IBC from RT compared to prior randomized trials, while the low risk group had no benefit from RT. Citation Format: Wärnberg F, Garmo H, Folkvaljon Y, Holmberg L, Karlsson P, Sandelin K, Linke S, Lyle S, Simin K, Leesman G, Barry T, Savala J, Whitworth P, Bremer T. A validation of DCIS biological risk profile in a randomised study for radiation therapy with 20 year follow-up (SweDCIS) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS5-08.

Abstract LB-B03: Exosome-based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients

Abstract About 60% of non-small cell lung cancer (NSCLC) patients develop resistance to targeted epidermal growth factor receptor (EGFR) inhibitor therapy through the EGFR T790M mutation. Although these patients respond well to third generation tyrosine kinase inhibitors like osimertinib, obtaining tissue biopsies for mutation profiling poses risks and is often not feasible. Detecting the mutation in a liquid biopsy from plasma has emerged as a viable alternative to biopsy, however existing liquid biopsy tests using cell-free DNA (cfDNA) often suffer from low sensitivity due to the low copy number of mutant molecules in circulation. As an example, the Food and Drug Adminstration (FDA) approved companion diagnostic for T790M mutation status in plasma (cobas® EGFR mutation Test version 2) only has a 58% sensitivity at a specificity of 80%. In this study we aimed to achieve a higher performance by combining the mutated alleles on cfDNA as well as exosomal RNA and DNA (exoNA) . ExoNA and cfDNA were extracted from the plasma of NSCLC patients with biopsy-confirmed T790M-positive (N = 102) or T790M-negative (N = 108) samples. The T790M mutation status in plasma exoNA and cfDNA was determined using an analytically validated allele-specific qPCR assay in a CLIA laboratory. The training cohort consisted of 105 samples (51 positives, 54 negatives). The validation cohort consisted of 105 samples (51 positives, 54 negatives), where ~40% of the patients had intrathoracic disease(M0/M1a). Detection of the T790M mutation in exoNA and cfDNA achieved 92% sensitivity and 89% specificity when compared to tumor biopsy results in the validation cohort. We also observed a very high sensitivity (88%) in patients with intrathoracic disease (M0/M1a), patients for whom detection by liquid biopsy has been proven particularly challenging. The combination of exoNA and cfDNA for EGFR T790M detection has a higher sensitivity and specificity compared to historical cohorts using ctDNA alone. This is especially important as it could aid giving the right treatment to patients where biopsy is not available or avoid unnecessary tumor biopsies for T790M mutation testing. Citation Format: Elena Castellanos, Dominik G. Grimm, Vasisht Tadigotla, Stefan Bentink, James Hurley, John Healy, Patricia L. Neal, Mia Sher, Dan Baughman, Chris Karlovich, Mitch Raponi, Anne Krug, Mikkel Noerholm, Luis E. Raez, Johan Skog. Exosome-based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B03.

Abstract 4954: Clinical validation of a cell-free DNA liquid biopsy approach for noninvasive molecular profiling

Abstract Clinical molecular profiling of advanced cancers enables the identification of actionable genomic alterations to guide therapeutic decisions. Although profiling of tissue samples is considered the gold standard, specimens may be unavailable or unsuitable for testing due to limited tumor purity or specimen quality. Further, genetically informed treatment decisions are increasingly necessary after disease progression and re-biopsy in this setting may not be feasible. Circulating tumor DNA (ctDNA) approaches for identification of genetic alterations in cancer patients may be more informative as the alterations reflect the current status of the tumor. ctDNA is representative of multiple tumor sites within a patient and may aid in the detection of alterations throughout the course of therapy. However, the fraction of ctDNA obtained from a blood sample is often very low (&amp;lt;1.0%) and difficult to detect. Additionally, many methods to evaluate ctDNA interrogate single hotspot or a few mutations. The next generation of ctDNA assays must identify clinically actionable genetic alterations and novel biomarkers with high precision and accuracy. To address these issues, we have developed and validated PlasmaSelect64, a ctDNA approach to comprehensively detect genetic alterations at low allele frequencies in the circulation of cancer patients. Utilizing digital genomic approaches, we demonstrated robust sensitivity and specificity in our CLIA laboratory in 64 well-established cancer genes that were identified based on clinical actionability. In addition to the evaluation of exons in 58 genes that are frequently mutated in cancer for sequence mutations, we performed a comprehensive genomic analysis of translocations in 18 genes and copy number analyses in 19 genes. We have also developed a novel approach for identification of microsatellite instability (MSI) using these error correction methodologies. To evaluate the PlasmaSelect64 approach, we developed and optimized the pre-analytical conditions for sample collection and processing with K2EDTA and Streck blood collection tubes. Analytical validation studies were performed with clinical samples and contrived cell-line mixtures containing known alterations determined by orthogonal methods. We robustly identified sequence mutations at 0.50% mutant allele frequency (MAF) with a limit of detection of 0.05% MAF, corresponding to a per-base specificity of 99.9997% and a sensitivity of 99.4%. For detection of focal amplifications and translocations, analytical method validation studies demonstrated sensitivity of 97.2% and 94.4% and specificity of &amp;gt;99% at MAFs of ≥20% and ≥0.50%, respectively. PlasmaSelect64 provides a non-invasive platform to enable detection of clinically relevant genetic alterations across a large number of genomic regions to aid in the therapeutic management of cancer patients. Citation Format: Monica Nesselbush, Samuel Angiuoli, Luis A. Diaz, Andrew Georgiadis, Shannon Glynn, Siân Jones, Laurel Keefer, Peter LoVerso, Derek Murphy, Sonya Parpart-Li, David Riley, Naomi Sengamalay, Manish Shukla, John Simmons, Snehal Talati, Rebecca Steinberg, Laura Tucker, Victor E. Velculescu, Ellen Verner, Angela Villarta, Mark Sausen. Clinical validation of a cell-free DNA liquid biopsy approach for noninvasive molecular profiling [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 4954. doi:10.1158/1538-7445.AM2017-4954

Analytical Validation of Relative Average Telomere Length Measurement in a Clinical Laboratory Environment.

Average telomere length in whole blood has become a biomarker of aging, disease, and mortality risk across a broad range of clinical conditions. The most common method of telomere length measurement for large patient sample sets is based on quantitative PCR (qPCR). For laboratory-developed tests to be performed on clinical samples, they must undergo a rigorous analytical validation, currently regulated under CLIA. Whole blood samples from 40 donors were used in the analytical validation of methods for relative average telomere length (rATL) measurement. Three technical replicate DNA samples were extracted from each whole blood sample and placed in three independent wells on a sample plate. Each of these sample plates was assayed 12 times during the validation process. The study was conducted over a 20-day period, once in the morning and once in the evening, using 3 different operators. Our process of rATL measurement beginning with DNA extraction followed by qPCR-based assay resulted in repeatability and reproducibility CV of <5% and amplification efficiencies near 100%. The validated assay was used to establish a reference interval derived from 2 cohorts of individuals: (a) San Francisco Bay area (n = 504) and (b) a US cross-sectional, demographic population (n = 357). We present advances in the establishment of a highly reproducible analytically validated process for determining rATLs in a CLIA laboratory environment.

Liquid biopsy in the clinic: A prospective study of plasma circulating tumor DNA (ctDNA) next generation sequencing (NGS) in patients with advanced non-small cell lung cancers to match targeted therapy.

11536 Background: Liquid biopsy for plasma ctDNA NGS is a rapidly evolving science. Plasma ctDNA assays are commercially available and are increasingly adopted in the community with a paucity of evidence-based guidance. We set out to study the optimal timing and utility of plasma ctDNA NGS in clinic. Methods: Pts with advanced NSCLC who were driver unknown, defined as not having prior tissue NGS or clinical concern for tumor heterogeneity that may affect treatment decisions, were eligible. Peripheral blood was collected in a Streck tube (10mL), DNA extracted, and subjected to a bias-corrected hybrid-capture 21 gene targeted NGS assay in a CLIA lab with unique reads at 3000x and sensitive detection at variant allele frequency above 0.1% (ResolutionBio Bellevue, WA). Pts also had concurrent tissue NGS via MSK IMPACT. Clinical endpoints included detection of oncogenic drivers in plasma, ability to match pts to targeted therapy, concordance and turnaround time of plasma and tissue NGS. Results: Forty-one pts were prospectively accrued. Plasma ctDNA detected an oncogenic driver in 39% (16/41) of pts, of whom 17% (7/41) were matched to targeted therapy; including pts matched to clinical trials for HER2 exon 20 insertionYVMA, BRAF L597Q and MET exon14. Mean turnaround time for plasma was 7 days (4-12) and 28 days (20-43) for tissue. Plasma ctDNA was detected in 56% (23/41) of pts; detection was 40% (8/20) if blood was drawn on active therapy and 71% (15/21) if drawn off therapy, either at diagnosis or progression (Odds ratio 0.28, 95% CI 0.06 - 1.16; p = 0.06). All pts had concurrent tissue NGS; of the 10 samples resulted, there was 100% driver concordance between tissue and plasma in pts drawn off therapy. Conclusions: In pts who were driver unknown or who had clinical concern for tumor heterogeneity, plasma ctDNA NGS identified a variety of oncogenic drivers with a short turnaround time and matched them to targeted therapy. Plasma ctDNA detection was more frequent at diagnosis of metastatic disease or at progression. A positive finding of an oncogenic driver in plasma is highly specific, but a negative finding may still require tissue biopsy.