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Cloud P Paweletz
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ES25 - Liquid Biopsy (ID 27)
- Event: WCLC 2019
- Type: Educational Session
- Track: Treatment of Early Stage/Localized Disease
- Presentations: 1
- Now Available
- Moderators:Max Diehn, Lucia Milla Collado
- Coordinates: 9/10/2019, 14:30 - 16:00, Colorado Springs (1994)
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ES25.03 - Leveraging the Quantitative Nature of Cfdna Genotyping for Lung Cancer Care (Now Available) (ID 3289)
14:30 - 16:00 | Presenting Author(s): Cloud P Paweletz
- Abstract
- Presentation
Abstract
Nearly 70 years after the discovery of free-floating DNA in the blood, also known as cell free DNA (cfDNA) (1), plasma genotyping of cfDNA is transforming cancer care with promise in non-invasive genotyping, early diagnosis, and disease prognostication by detection of minimal residual disease (MRD). At present the most widespread use of cfDNA in lung cancer is detection of mutations in EGFR and KRAS or re-arrangements in ALKand ROS1 in the metastatic setting. In fact, the only FDA approved liquid biopsy test among all cancers is Roche’s Cobas plasma EGFR mutation test for non-small cell lung cancer. (NSCLC) (2), while many diagnostic companies have developed PCR or next generation sequencing (NGS) laboratory developed tests that are commonly reimbursed by payors in house. Next-generation sequencing (NGS) permits broader inquiries, allowing assessment of the mutation status of thousands to millions of bases.
The use of cfDNA in early cancers is confounded by the fact that early detection demands ultra-sensitive assays of low abundant biological markers. As proof of concept Bettegowda et al. performed digital PCR on cfDNA of 640 cancer patients of varying cancer type and stage. Intriguingly they found that rates of tumor DNA differed by tissue of origin, and 47% of stage I cancers and 55% of stage II cancers had detectable circulating tumor DNA (ctDNA) (3). An observation that as we and others have confirmed is a main source for false negatives (4). At present, NGS efforts are focused either on targeted approaches using either barcoded targeted amplicon (TAmSeq) or hybrid capture approaches (CAPP-Seq) covering 10Mb to 50Mb at reported sensitivities of 0.01% to 0.50% for fit for purpose build NGS assays. However, pushing assay sensitivity increases false positives.
Recently, we and other have found that false positives are reported in many commercial assays and are routinely attributed to ‘tumor heterogeneity’ . These can be attributed to DNA shed from normal cells, including germline variants or non-cancerous somatic variants from clonal hematopoiesis (CH) (5-8). The latter is particularly challenging because CH can involve cancer-associated genes (e.g. TP53, JAK2, KRAS). To limit false positives and to investigate the common link between cancer-related mutations within the blood and underlying malignancies broad sequencing of cfDNA should also detect other cancer-related mutations, such as inactivating mutations in tumor suppressors and include assaying of the patients blood cells to filterout germlines. Thus, deep and broad sequencing could provide sensitivity needed to detect low levels of cfDNA alterations in early stage patients. Indeed, Abbosh et al. perform multi region whole-exome sequencing of early-stage NSCLC tumors to show an abundance of clonal mutations in these tumors (9). Abbosh and colleagues provide an intriguing solution to this issue by requiring the detection of two or more SNVs for the determination of the presence of cancer.In this presentation we present factors affecting ultras sensitive assays with particular emphasis on interpretation of commercial tests and future use of cfDNA assays in early cancers.
1. Mandel P and Metais P. Les acides nucleiques du plasma sanguin chez l’homme [in French]. C R Seances Soc Biol Fil 1948;142:241-243.
2.Center for Drug Evaluation and Research. Approved Drugs - cobas EGFR Mutation Test v2 [Internet]. U S Food and Drug Administration Home Page. Center for Drug Evaluation and Research; Available from: https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm504540.htm
3.Bettegowda C, Sausen M, Leary R, Kinde I, Agrawal N, Bartlett B, et al. Detection of Circulating Tumor DNA in Early and Late Stage Human Malignancies. Science Translational Medicine 2014;16:224ra24.
4. Oxnard GR, Thress KS, Alden RS, et al: Association Between Plasma Genotyping and Outcomes of Treatment With Osimertinib (AZD9291) in Advanced Non–Small-Cell Lung Cancer. Journal of Clinical Oncology 34:3375–3382, 2016
5.Hu Y, Ulrich BC, Supplee J, et al: False-Positive Plasma Genotyping Due to Clonal Hematopoiesis. Clin Cancer Res 24:4437-4443, 2018
6.Hu Y, Alden RS, Odegaard JI, et al: Discrimination of Germline EGFR T790M Mutations in Plasma Cell-Free DNA Allows Study of Prevalence Across 31,414 Cancer Patients. Clin Cancer Res 23:7351-7359, 2017
7.Slavin TP, Banks KC, Chudova D, et al: Identification of Incidental Germline Mutations in Patients with Advanced Solid Tumors Who Underwent Cell-Free Circulating Tumor DNA Sequencing. Journal of Clinical Oncology 36:3459-3465, 2018
8.Oxnard GR, Tara M, Earl H, et al. Genome-wide sequencing for early stage lung cancer detection from plasma cell-free DNA (cfDNA): The Circulating Cancer Genome Atlas (CCGA) study. Journal of Clinical Oncology : LBA8501-LBA8501, 2018
9.Abbosh C, Birkbak NJ, Wilson GA, Jamal-Hanjani M, Constantin T, Salari R, et al. Phylogenetic ctDNA Analysis Depicts Early Stage Lung Cancer Evolution. Nature 2017;545:446-451.
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MA12 - New Frontiers from Pathology to Genomics (ID 138)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Mesothelioma
- Presentations: 1
- Now Available
- Moderators:Prasad S Adusumilli, Francisco Perez Ochoa
- Coordinates: 9/09/2019, 14:00 - 15:30, Melbourne (1991)
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MA12.06 - Patient-Derived Organotypic Tumor Spheroids (PDOTS) Facilitate Therapeutic Screening for Malignant Pleural Mesothelioma (Now Available) (ID 2561)
14:00 - 15:30 | Author(s): Cloud P Paweletz
- Abstract
- Presentation
Background
While genotype directed therapies are an essential aspect of personalized medicine in non-small cell lung cancer (NSCLC), this modality is not currently an option in mesothelioma. Instead there is a need for improved functional testing via predictive platforms that can help identify the susceptibility of patient tumors to drug therapies. Here, we demonstrate the use of a novel ex vivo functional system utilizing 3D microfluidic culture and patient-derived organotypic tumor spheroids (PDOTS) as a platform to study the tumor microenvironment and predict tumor responses to treatment in mesothelioma.
Method
We evaluated 31 mesothelioma patient specimens under an IRB approved protocol. PDOTS of mesothelioma were generated as previously described (Larios et al. AACR. 2017; Jenkins et al. Cancer Discovery. 2017). Samples were treated with standard chemotherapy (pemetrexed and cisplatin combined) as well as immunotherapy (ipilimumab and pembrolizumab combined) and live/dead quantification was conducted using dual labeling de-convolution fluorescence microscopy. Positive responses ex vivo included samples with significant cell death to control while positive in vivo responses were based on radiologic lack of tumor recurrence using the response evaluation criteria in solid tumors (RECIST, version 1.1) to assess for disease progression.
Result
We found that in treatment naïve specimens prolonged ischemic times were associated with decreased tissue viability (ischemia >25 minutes resulted in decrease of live cells from an average of 81% to 56%), lower tumor yield (< 50% tumor content), and decreased generation of spheroids (< 20 spheroids/well). Specimens with prior treatment were consistently associated with low tissue viability irrespective of ischemic times. Of the 31 specimens studied, 10 samples met viability and tumor content standards to undergo further treatment with standard chemotherapy and immunotherapy, and 5 of those samples were tracked to available patient-treatment response data. Ultimately, comparison of ex vivo and in vivo treatment responses demonstrated that 4 of 5 samples treated with standard chemotherapy had concordant responses to those of patients who received the same or similar post-operative therapy. Notably, our discordant sample exhibited large variation in standard deviations due to technical variability.
Conclusion
Here we demonstrate that analysis of ex vivo mesothelioma tissue correlates to in vivo responses. These results suggest that PDOTS can serve as a predictive platform for therapies. Further work streamlining human tissue collection and optimizing factors that affect formation of PDOTS prior to ex vivo treatment analysis should be further investigated.
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P1.01 - Advanced NSCLC (ID 158)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.01-46 - Response Assessment Using Plasma Cell-Free DNA (cfDNA) – When Is the Optimal Time to Assess Response? (ID 958)
09:45 - 18:00 | Author(s): Cloud P Paweletz
- Abstract
Background
Plasma cfDNA analysis is routine for non-invasive genotyping of advanced NSCLC, however response assessment using plasma cfDNA is not well characterized. We hypothesized that response in cfDNA would be an early process occurring well before routine imaging timepoints.
Method
We retrospectively analyzed a total of 48 baseline and serial on-treatment plasma samples collected from 16 patients enrolled across three Experimental Therapeutics Clinical Trials Network (ETCTN) phase I trials of osimertinib combinations in advanced EGFR-mutant NSCLC. For validation, we also retrospectively analyzed a total of 201 baseline and serial on-treatment samples from an institutional cohort of 67 advanced NSCLC patients receiving systemic treatment. Using droplet digital PCR (ddPCR) of key EGFR or KRAS driver mutations, plasma response was defined as any decrease in mutation concentration to below baseline levels. We compared the magnitude of initial (baseline to day 11-30) and subsequent (day 11-30 to day 36-84) plasma response. Finally, we prospectively assessed response using serial amplicon-based plasma next-generation sequencing (NGS) in a pilot cohort of 8 NSCLC patients starting systemic therapy.
Result
Of 15 ETCTN patients with any plasma response, best plasma response was seen at the initial response timepoint in 12 patients (80.0%) and ≥90% of the total plasma response was seen at the initial response timepoint in 14 patients (93.3%). In the validation cohort of 61 patients with any plasma response (Figure), best plasma response was seen at the initial response timepoint in 39 patients (63.9%) and ≥90% of the total plasma response was seen at the initial response timepoint in 52 patients (85.2%). Complete plasma responses (-100%) were seen as early as 11 days after initiating therapy. In the prospective clinical cohort, plasma NGS detected genomic alterations and enabled monitoring of changes in mutant allele fraction in all 8 patients. The median turnaround time of the assay was 8 days.
Conclusion
Plasma response is an early phenomenon, with the vast majority of plasma response seen within 30 days, and as early as 11 days. These findings suggest that early plasma cfDNA analysis may permit response assessment well before standard imaging timepoints, with potential as an early marker of drug effect. Additional investigation to understand the relationship between early plasma response, radiographic response, and durability of treatment effect is still needed.
