Brian Woodward

Direct detection of early-stage cancers using circulating tumor DNA

Noninvasive liquid biopsy analysis of circulating tumor DNA permits direct detection of early-stage cancers. Finding smaller needles in haystacks The detection and analysis of cell-free DNA in patients’ blood are becoming increasingly accepted in oncology. However, this approach has generally been applied for the monitoring of patients with existing tumors. It has not been useful for early diagnosis of cancer because of insufficient sensitivity to detect really small tumors that only shed minute quantities of DNA into the blood, as well as difficulties with identifying cancer-associated genetic changes without knowing what mutations are present in the primary tumor. A method developed by Phallen et al., called targeted error correction sequencing, addresses both of these limitations and demonstrates the feasibility of detecting circulating cell-free DNA from many early tumors, suggesting its potential use for cancer screening. Early detection and intervention are likely to be the most effective means for reducing morbidity and mortality of human cancer. However, development of methods for noninvasive detection of early-stage tumors has remained a challenge. We have developed an approach called targeted error correction sequencing (TEC-Seq) that allows ultrasensitive direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing. We have used this approach to examine 58 cancer-related genes encompassing 81 kb. Analysis of plasma from 44 healthy individuals identified genomic changes related to clonal hematopoiesis in 16% of asymptomatic individuals but no alterations in driver genes related to solid cancers. Evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of 71, 59, 59, and 68%, respectively, of patients with stage I or II disease. Analyses of mutations in the circulation revealed high concordance with alterations in the tumors of these patients. In patients with resectable colorectal cancers, higher amounts of preoperative circulating tumor DNA were associated with disease recurrence and decreased overall survival. These analyses provide a broadly applicable approach for noninvasive detection of early-stage tumors that may be useful for screening and management of patients with cancer.

Monitoring Daily Dynamics of Early Tumor Response to Targeted Therapy by Detecting Circulating Tumor DNA in Urine

Purpose: Noninvasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine. Experimental Design: We tested the hypothesis that dynamic changes in EGFR activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non–small cell lung cancer (NSCLC) patients receiving osimertinib, a second-line third-generation anti-EGFR tyrosine kinase inhibitor. Results: Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pretreatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1, suggesting apoptosis with an overall decrease in fragment numbers from baselines to day 7 preceding radiographic response assessed at 6 to 12 weeks. Conclusions: These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development. The modeling of tumor lysis through the day-to-day kinetics of ctDNA released into the blood and then into the urine is demonstrated in this proof-of-concept study in lung cancer patients receiving anti-EGFR tyrosine kinase inhibitors. This strategy may determine the specific clonal populations of cells which undergo apoptosis within the first week of therapy. This has important implications for developing combinational strategies to address inter- and intralesional heterogeneity and characterizing residual disease after initial drug exposure. Clin Cancer Res; 23(16); 4716–23. ©2017 AACR.

Strategies to Overcome Bypass Mechanisms Mediating Clinical Resistance to EGFR Tyrosine Kinase Inhibition in Lung Cancer

The vast majority of patients with metastatic lung cancers who initially benefit from EGFR-targeted therapies eventually develop resistance. An increasing understanding of the number and complexity of resistance mechanisms highlights the challenge of treating tumors resistant to EGFR inhibitors. Resistance mechanisms include new, second-site mutations within EGFR (e.g., T790M and C797S), upregulation of MET kinase, upregulation of insulin growth factor receptor (IGFR), HER2 amplification, increased expression of AXL, BIM modulation, NF-κB activation, histologic switch to small-cell cancer, epithelial-to-mesenchymal transition, PDL1 expression with subsequent immune tolerance, and release of cytokines such as TGFβ and IL6. Herein, we review the growing body of knowledge regarding EGFR bypass pathways, and the development of new drugs and combination treatment strategies to overcome resistance. Mol Cancer Ther; 16(2); 265–72. ©2017 AACR.

Circulating Tumor DNA for Mutation Detection and Identification of Mechanisms of Resistance in Non-Small Cell Lung Cancer

Targeted therapies have changed the treatment landscape of non-small cell lung cancer over the past decade. Analyses of cell free circulating tumor DNA (ctDNA) provide a non-invasive and robust approach for cancer diagnosis and prognosis, real-time monitoring of treatment response, and the identification of appropriate therapeutic targets based on the detection of tumor genetic aberrations. Recent improvements in the sensitivity, specificity, and feasibility of ctDNA detection assays allow the possibility for implementation into clinical practice. This review will focus on key studies using ctDNA analysis in early lung cancer detection, prediction of treatment response, monitoring minimal residual disease and disease relapse, and the identification of resistance mechanisms. We explore how ctDNA can be used as a surrogate for tissue biopsy and an integral biomarker in the clinical management of patients with non-small cell lung cancer.

Cell-Free DNA from Ascites and Pleural Effusions: Molecular Insights into Genomic Aberrations and Disease Biology

Collection of cell-free DNA (cfDNA) from the blood of individuals with cancer has permitted noninvasive tumor genome analysis. Detection and characterization of cfDNA in ascites and pleural effusions have not yet been reported. Herein, we analyzed cfDNA in the ascites and pleural effusions from six individuals with metastatic cancer. In all cases, cfDNA copy number variations (CNV) were discovered within the effusate. One individual had a relevant alteration with a high copy amplification in EGFR in a never smoker with lung cancer, who showed only MDM2 and CDK4 amplification in a prior tissue biopsy. Another subject with metastatic breast cancer had cytology-positive ascites and an activating PIK3CA mutation identified in the tissue, blood, and ascites collectively. This individual had tumor regression after the administration of the mTOR inhibitor everolimus and had evidence of chromotripsis from chromosomal rearrangements noted in the cell-free ascitic fluid. These results indicate that cfDNA from ascites and pleural effusions may provide additional information not detected with tumor and plasma cell-free DNA molecular characterization, and a context for important insights into tumor biology and clonal dynamic change within primary tumor and metastatic deposits. Mol Cancer Ther; 16(5); 948–55. ©2017 AACR.

New Targets in Non–Small Cell Lung Cancer

With the implementation of genomic technologies into clinical practice, we have examples of the predictive benefit of targeted therapy for oncogene-addicted cancer and identified molecular dependencies in non-small cell lung cancer. The clinical success of tyrosine kinase inhibitors against epidermal growth factor receptor and anaplastic lymphoma kinase activation has shifted treatment emphasize the separation of subsets of lung cancer and genotype-directed therapy. Advances have validated oncogenic driver genes and led to the development of targeted agents. This review highlights treatment options, including clinical trials for ROS1 rearrangement, RET fusions, NTRK1 fusions, MET exon skipping, BRAF mutations, and KRAS mutations.

Novel Treatment Strategies for Brain Metastases in Non-small-cell Lung Cancer.

Opinion statementBrain metastases are common in patients with non-small cell lung cancer (NSCLC), and due to associated poor prognosis, this field is an important area of need for the development of innovative medical therapies. Therapies including local approaches through surgical intervention and/or radiation and evolving systemic therapies have led to improvements in the treatment of brain metastases in patients with lung cancer. Strategies that consider applying advanced radiation techniques to minimize toxicity, intervening early with effective systemic therapies to spare radiation/surgery, testing radiosensitization combinations, and developing drug penetrant molecules have and will continue to define new practice patterns. We believe that in carefully considered asymptomatic patients, first-line systemic therapy may be considered before radiation therapy and small-molecule targeted therapy may provide an opportunity to defer radiation therapy for recurrence or progression of disease. The next several years in oncology drug development will see the reporting on of brain penetrant molecules in oncogene-defined non-small cell lung cancer. Ongoing studies will evaluate immunotherapies in patients with brain metastases with associated endpoints. We hope that continued drug development and carefully designed clinical trials may afford an opportunity to improve the lives of patients with brain metastases.

Abstract 30: Quantitative monitoring of EGFR mutations in urinary circulating tumor DNA enables non-invasive pharmacodynamic assessment of anti-EGFR drug response

Background: Acquisition of the EGFR T790M resistance mutation is a hallmark of disease progression in patients with metastatic EGFR mutant lung adenocarcinoma treated with first generation anti-EGFR inhibitors. Utilizing a single copy sensitivity mutation detection platform and implementing daily collection of urine samples, we sought to demonstrate the feasibility of detecting EGFR mutations in urinary ctDNA and understand mechanisms of resistance to targeted therapies in patients with EGFR-mutated lung adenocarcinoma. Methods: In a biomarker study of 100 patients with EGFR-mutant metastatic lung adenocarcinoma (34 patients enrolled), urine was collected at either daily or monthly time points up to 4 months prior to radiologic detection of progression on erlotinib, and at multiple time points post-progression on next line therapy. Urinary ctDNA was extracted by a method that preferentially isolates short, fragmented ctDNA. Quantitative analysis of EGFR activating mutations and T790M resistance mutation was performed using blocker technology and PCR enrichment coupled with NGS detection (MiSeq). Urine was collected daily after the initiation of second line anti-EGFR therapy in 10 patients. Early pharmacodynamic events that occur within the first hours to days of anti-EGFR therapy were further studied by quantitating ctDNA for EGFR exon 19deletions, L858R, and T790M. Results: Interim analysis was conducted on 34 patients receiving first line anti-EGFR therapy with erlotinib; twenty-two of 34 patients demonstrated radiographic progression. Analysis of longitudinal samples revealed that the EGFR T790M mutation was detected in the urine specimens of 15 out of 22 (68%) patients on erlotinib. All 10 patients who were positive for T790M mutation by tissue were also positive by urine. Urine testing identified five additional T790M-positive patients who had a high clinical suspicion of T790M progressive disease. Three of these patients were tissue negative but both plasma and urine positive for T790M. EGFR T790M was detected up to 15 weeks prior to radiolographic detection of progression on first line erlotinib. Early peaks in ctDNA on days 1-4 correlated with tumor lysis. An observed sustained decrease in mutational levels after week 1 of therapy confirms the cytostatic effect of the tyrosine kinase inhibitor (TKI). The size of the initial peaks in ctDNA for EGFR exon 19deletions, L858R, and T790M correlated with CT radiographic response after two cycles of therapy. Conclusion: We demonstrate that the T790M mutation can be successfully detected in urinary ctDNA months before progression on anti-EGFR TKIs. Urinary ctDNA testing identifies additional patients who potentially are eligible for anti-T790M treatment. Initial results from 10 patients demonstrated that kinetic changes in EGFR ctDNA mutational load after drug adminstration can be used with pharmacokinetic data to better understand dyanmic changes in tumor biology, drug bioavailability, and assist in early drug development. The clinical utility of daily kinetic monitoring of ctDNA in urine after drug adminstration is being further validated in a larger study. Citation Format: Hatim Husain, Karena Kosco, Saege Hancock, Errin Samuelsz, Shiloh Guerrero, Brian Woodward, Cecile Rose Vibat, Vlada Melnikova, Mark Erlander, Scott Lippman, Razelle Kurzrock. Quantitative monitoring of EGFR mutations in urinary circulating tumor DNA enables non-invasive pharmacodynamic assessment of anti-EGFR drug response. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 30.

Abstract 2410: Cell-free DNA derived from ascites: Detection of copy number and somatic mutations using OncoScan FFPE® Assay

Background: There is increased interest in molecular analysis of cell-free DNA isolated from body fluids for the evaluation of tumor progression. These “liquid” biopsies are usually obtained from blood. Their advantages include the fact that they can be repeatedly accessed, are less invasive, and may be sensitive to changes in tumor profile from multiple metastatic sites. Materials and Methods: Eleven ascites samples from patients with metastatic epithelial neoplasms (gastric, N = 3; pancreas, N = 3; ovarian, N = 2; breast, N = 2; and lung cancer, N = 1) were investigated. Cell-free DNA was isolated from supernatant of ascites fluid (50 ml) after centrifugation using commercially available DNA purification kits (Norgen Bioteck Corp and Qiagen), and analyzed using the OncoScan FFPE Assay kit. Results: Cell-free DNA yields ranged from 1.7 ng to 230 ng per mL of ascites fluid, indicating wide variability in DNA content. Of the 11 patients, all had detectable aberrations, including copy number alterations affecting from Conclusions. Combined copy number and somatic analysis of ascites cell-free DNA revealed clinically relevant aberrations, including ones not found in primary tumor tissue. Comparison to circulating cells in ascites, cell-free DNA in blood, as well as tumor molecular profile is ongoing. Citation Format: Hatim Husain, Sumathi Venkatapathy, German Gomez, Brian Woodward, Suzanna Lee, Lubena Khambaty, Lily Chen, Radha Duttagupta, Eric T. Fung, Razelle Kurzrock. Cell-free DNA derived from ascites: Detection of copy number and somatic mutations using OncoScan FFPE® Assay. [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 2410. doi:10.1158/1538-7445.AM2015-2410