Longitudinal Cell-Free DNA Analysis in Patients with Small Cell Lung Cancer Reveals Dynamic Insights into Treatment Efficacy and Disease Relapse  Karinna Almodovar, PhD, Wade T. Iams, MD, Catherine B. Meador, MD, PhD, Zhiguo Zhao, MS, Sally York, MD, PhD, Leora Horn, MD, Yingjun Yan, MS, Jennifer Hernandez, BS, Heidi Chen, PhD, Yu Shyr, PhD, Lee P. Lim, PhD, Christopher K. Raymond, PhD, Christine M. Lovly, MD, PhD  Journal of Thoracic Oncology  Volume 13, Issue 1, Pages 112-123 (January 2018) DOI: 10.1016/j.jtho.2017.09.1951 Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 Mutational analysis in plasma cell-free DNA (cfDNA) from 27 patients using next-generation sequencing. (A) Study overview. (B) Summary of mutations identified by individual patient at any time point. Patients are separated by stage of disease at diagnosis (L [limited stage] versus E [extensive stage]). Alterations are color-coded per the figure legend below the image. The mutation frequencies for each gene are plotted on the right panel. TP53, tumor protein p53 gene; RB1, retinoblastoma 1 gene; PTEN, phosphatase and tensin homolog gene; NOTCH1, notch 1 gene; NOTCH2, notch 2 gene; NOTCH3, notch 3 gene; NOTCH4, notch 4 gene; MYC, v-myc avian myelocytomatosis viral oncogene homolog gene; MYCL1, v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog gene; MYCN, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog gene; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene; KIT, KIT proto-oncogene receptor tyrosine kinase gene; FGFR1, fibroblast growth factor receptor 1 gene. Journal of Thoracic Oncology 2018 13, 112-123DOI: (10.1016/j.jtho.2017.09.1951) Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Cell-free DNA detection precedes clinical or radiographic disease progression. (A) The time line for the clinical course of patient VSC-8 from diagnosis until date of death is shown. The light blue bar represents the treatment time frame, and the red dots indicate blood collection time points. Radiographic images were acquired at the time of diagnosis, on day 144, and on day 256. The red arrow in the first CT scan shows the right pleural–based primary tumor, which resolved on further imaging. Bone marrow biopsy was performed on day 289 and assessed for common SCLC markers, including CD56 (hematoxylin and eosin; original magnification, ×200 and CD56 stain; original magnification, ×200). (B) Percent mutant allelic frequency and copy number alterations for patient VSC-8 are shown. The light blue box indicates treatment time frame. A plus or minus symbol indicates presence/absence of the copy number alteration listed, and an asterisk indicates a stop. SNV, single-nucleotide variant; TP53, tumor protein p53 gene; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene; RB1, retinoblastoma 1 gene; amp, amplification; del, deletion. Journal of Thoracic Oncology 2018 13, 112-123DOI: (10.1016/j.jtho.2017.09.1951) Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 Cell-free DNA detection can clarify mixed response on imaging. (A) The time line for the clinical course of patient VSC-10 from diagnosis until last follow-up date with radiographic images is shown. The colored bars represent the active treatment time frames, including radiotherapy (RT) (as indicated by the gray bar). The purple bar indicates prophylactic cranial irradiation (PCI), and the blue shaded bar represents concurrent chemoradiation (chemorad) with weekly carboplatin and paclitaxel. The red dots indicate blood collection time points. Radiographic images were obtained at time of progression after first-line therapy (day 223) and on days 326, 417, and 494. The red arrow at day 223 indicates initially multifocal pulmonary parenchymal disease which resolved after second-line paclitaxel (day 326). The subsequent image at day 417 shows a 0.9-cm retroperitoneal lymph node of uncertain etiology. On the day 494 images, the retroperitoneal lymph node enlarged to 3.0 x 2.7 cm. (B) Percent mutant allelic frequencies and copy number alterations for patient VSC-10 are shown. The light red and blue boxes indicate treatment time periods corresponding to labeling on the time line above. The dotted line indicates the time of radiologic recurrence. A plus or minus symbol indicates presence/absence of the copy number alteration listed. CT, computed tomography; TP53, tumor protein p53 gene; NOTCH3, notch 3 gene; del, deletion. Journal of Thoracic Oncology 2018 13, 112-123DOI: (10.1016/j.jtho.2017.09.1951) Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Cell-free DNA changes correspond to remission. (A) The time line for the clinical course of patient VSC-9 from diagnosis until last follow-up date with radiographic images is shown. The colored bars represent the treatment time frames. The gray bar indicates radiation therapy (RT) and the purple bar indicates prophylactic cranial irradiation (PCI). Radiographic images were obtained at time of diagnosis, on day 93, and at last follow-up date (day 539). The red arrow shows primary mediastinal disease, which resolved on subsequent imaging. (B) Copy number alterations for patient VSC-9 are shown. The blue box indicates the time period during which cisplatin plus etoposide was administered, and the purple box indicates PCI treatment. A plus or minus symbol indicates presence/absence of the copy number alteration listed. CT, computed tomography; MYCL1, v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog gene; amp, amplification. Journal of Thoracic Oncology 2018 13, 112-123DOI: (10.1016/j.jtho.2017.09.1951) Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

Figure 5 Cell-free DNA sequencing enables early identification of treatment refractory disease. (A) The time line for the clinical course of patient VSC-14 from diagnosis until date of death is shown with radiographic images from diagnosis and on days 49, 111, and 152, demonstrating slow progression in intrathoracic disease despite all therapy. The red arrow indicates primary mediastinal disease. The color bars represent treatment time frames. The blue bar represents one cycle of carboplatin and etoposide treatment, the light yellow box represents nivolumab treatment, and the pink box indicates paclitaxel treatment. (B) Percent mutant allelic frequencies and copy number alterations for patient VSC-14 are shown. The light blue, yellow, and pink boxes indicate treatment periods corresponding to labeling on the time line above. The dotted line indicates the time of radiologic recurrence. A plus or minus symbol indicates presence/absence of the copy number alteration listed, and the asterisk indicates stop. SNV, single-nucleotide variant; TP53, tumor protein p53 gene; RB1, retinoblastoma 1 gene; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene; KIT, KIT proto-oncogene receptor tyrosine kinase gene; amp, amplification; del, deletion; NOTCH1, notch1 gene. Journal of Thoracic Oncology 2018 13, 112-123DOI: (10.1016/j.jtho.2017.09.1951) Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions