Circulating mutational portrait of cancer: manifestation of aggressive clonal events in both early and late stages

Abstract

Background: Solid tumors residing in tissues and organs leave footprints in circulation through circulating tumor cells (CTCs) and circulating tumor DNAs (ctDNA). Characterization of the ctDNA portraits and comparison with tumor DNA mutational portraits may reveal clinically actionable information on solid tumors that is traditionally achieved through more invasive approaches.

Methods: We isolated ctDNAs from plasma of patients of 103 lung cancer and 74 other solid tumors of different tissue origins. Deep sequencing using the Guardant360 test was performed to identify mutations in 73 clinically actionable genes, and the results were associated with clinical characteristics of the patient. The mutation profiles of 37 lung cancer cases with paired ctDNA and tumor genomic DNA sequencing were used to evaluate clonal representation of tumor in circulation. Five lung cancer cases with longitudinal ctDNA sampling were monitored for cancer progression or response to treatments.

Results: Mutations in TP53, EGFR, and KRAS genes are most prevalent in our cohort. Mutation rates of ctDNA are similar in early (I and II) and late stage (III and IV) cancers. Mutation in DNA repair genes BRCA1, BRCA2, and ATM are found in 18.1% (32/177) of cases. Patients with higher mutation rates had significantly higher mortality rates. Lung cancer of never smokers exhibited significantly higher ctDNA mutation rates as well as higher EGFR and ERBB2 mutations than ever smokers. Comparative analysis of ctDNA and tumor DNA mutation data from the same patients showed that key driver mutations could be detected in plasma even when they were present at a minor clonal population in the tumor. Mutations of key genes found in the tumor tissue could remain in circulation even after frontline radiotherapy and chemotherapy suggesting these mutations represented resistance mechanisms. Longitudinal sampling of five lung cancer cases showed distinct changes in ctDNA mutation portraits that are consistent with cancer progression or response to EGFR drug treatment.

Conclusions: This study demonstrates that ctDNA mutation rates in the key tumor-associated genes are clinical parameters relevant to smoking status and mortality. Mutations in ctDNA may serve as an early detection tool for cancer. This study quantitatively confirms the hypothesis that ctDNAs in circulation is the result of dissemination of aggressive tumor clones and survival of resistant clones. This study supports the use of ctDNA profiling as a less-invasive approach to monitor cancer progression and selection of appropriate drugs during cancer evolution.

Keywords: Clonality; Liquid biopsy; Lung cancer; Mutation rate; Non-invasive.

Fig. 1

Global landscape of ctDNA mutations. a Global ctDNA mutational landscape of all patients for the top 30 genes having the largest fraction of mutations. Top and left bar charts show the number of mutations and percent of mutated samples, respectively. Lower part of panel A summarizes clinical information from each patient. b ctDNA mutational landscape of patients’ stage known for the top 30 genes having the largest fraction of mutations. Two patients’ stages are unknown

Fig. 2

DNA damage repair (DDR) and chromatin remodeling gene mutations are associated with increased mutation number and may sensitize tumor to PARP inhibitor. a Patients with higher DDR and chromatin remodeling gene mutations numbers (n = 42) compared with patients with lower mutation numbers (n = 135). The black dotted line marks the median of the distribution. *** (p < 0.001), Mann-Whitney-Wilcoxon rank sum. b Composite image of axial contrast-enhanced computed tomography (CT) slicing through the liver demonstrates a progressive decrease in size of two hepatic metastases prior to treatment (left), 3 months (middle), and 5 months (right) after the initiation of olaparib therapy

Fig. 3

Higher mutation numbers in the ctDNA is associated with decreased survival. Higher mutation numbers in ctDNA is associated with poor survival. n defines the number of mutations, and survival plots are separated by mutation numbers: n = 1, 2, 3, 4, 5, and 6 mutations. Blue lines indicate more than n mutations, and the pink lines indicate equal to or less than n mutations. P values were derived using the log-rank test

Fig. 4

Gene mutations in lung carcinoma are associated with smoking status. a Mutational landscapes of lung cancers showing 30 of the most frequently mutated genes. Top and left bar charts show the number of mutations and percent of mutated samples, respectively. b EGFR and ERBB2 gene mutations concentrate mainly in never smokers

Fig. 5

TP53, PDGFBA, BRAF, ERBB2, CTNNB1, EGFR, and ARID1A mutations present in minor clones in the primary tumors are detectable in plasma ctDNA. The X axis represents allele variant fractions. Each circle represents one gene mutation present in tumor tissues as examined by Foundation1 test (F). The cases presented manifest heterogeneity and multiple clonal characteristics. Mutations also found in Guardant360 test (G) are indicated by red circle. The order of the two tests and whether the patient was treated (Tx1 for yes and Tx0 for no) is shown in the right panel

Fig. 6

Monitoring of lung cancer progression and response to therapy through longitudinal plasma ctDNA sequencing. a Compared with ctDNA mutations in five patients at two different time points, top shows the patients’ number and relative time point. Four patients demonstrated EGFR mutations. Mutation burden of patients 2 and 3 decreased after treatment. b Serial imaging at the time of plasma ctDNA testing indicated partial response for patient 3, minor response for patient 2, and progressive disease for patients 1, 4, and 5