Using cfRNA as a tool to evaluate clinical treatment outcomes in patients with metastatic lung cancers and other tumors

Abstract

Aim: We report an exploratory analysis of cfRNA as a biomarker to monitor clinical responses in non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer (CRC). An analysis of cfRNA as a method for measuring PD-L1 expression with comparison to clinical responses was also performed in the NSCLC cohort. Methods: Blood samples were collected from 127 patients with metastatic disease that were undergoing therapy, 52 with NSCLC, 50 with breast cancer, and 25 with CRC. cfRNA was purified from fractionated plasma, and following reverse transcription (RT), total cfRNA and gene expression of PD-L1were analyzed by real-time polymerase chain reaction (qPCR) using beta-actin expression as a surrogate for relative amounts of cfDNA and cfRNA. For the concordance study of liquid biopsies and tissue biopsies, the isolated RNA was analyzed by RNAseq for the expressions of 13 genes. We had to close the study early due to a lack of follow-up during the Covid-19 pandemic. Results: We collected a total of 373 blood samples. Mean cfRNA PCR signals after RT were about 50-fold higher than those of cfDNA. cfRNA was detected in all patients, while cfDNA was detected in 88% of them. A high concordance was found for the expression levels of 13 genes between blood and solid tumor tissue. Changes in cfRNA levels followed over the course of treatments were associated with response to therapy, increasing in progressive disease (PD) and falling when a partial response (PR) occurred. The expression of PD-L1 over time in patients treated with immunotherapy decreased with PR but increased with PD. Pre-treatment levels of PD-L1 were predictive of response in patients treated with immunotherapy. Conclusion: Changes in cfRNA correlate with clinical response to the therapy. Total cfRNA may be useful in predicting clinical outcomes. PD-L1 gene expression may provide a biomarker to predict response to PD-L1 inhibition.

Keywords: NSCLC; PD-L1; cfDNA; cfRNA; liquid biopsies.

Figure 1

PCR signals of beta-actin from cfRNA (as its cDNA) and from cfDNA in identical aliquots of patients’ plasma. P < 0.001 for the difference in median signal values (Wilcoxon/Kruskal-Wallis rank sums). PCR: Polymerase chain reaction.

Figure 2

Concordance between the expressions of 13 genes in paired plasma and tissue samples a is determined by RNAseq.

Figure 3

Changes in levels of cfRNA and cfDNA during therapy. cfRNA and cfDNA were measured at initiation of therapy and at various times during the chemotherapy using PCR amplification of B-actin. Treatment efficacy was determined by CT scans. PCR: Polymerase chain reaction.

Figure 4

Changes in cfRNA levels during therapy when cfDNA is unmeasurable. cfRNA and cfDNA were measured at initiation of treatment and at various times during chemotherapy using PCR amplification of Β-actin. Treatment efficacy of chemotherapy regimens was determined by CT scans. PCR: Polymerase chain reaction.

Figure 5

Changes in total cfRNA vs. outcome in response to various therapies across different tumor types. The bars represent analyses of 154 BC, 84 CRC, 135 NSCLC patient samples. BC: Breast cancer; CRC: colorectal cancer; NSCLC: non-small cell lung cancer.

Figure 6

Association of response to chemotherapy with PD-L1 gene expression determined using cfRNA in patients’ blood draws.

Figure 7

Measurement of PD-L1 gene expressions in NSCLC patients during treatments with immunotherapy agents. Chemotherapy includes carbo-pemetrexed; targeted therapy includes erlotinib, osimertinib, and crizotinib. NSCLC: Non-small cell lung cancer.

Figure 8

Pre-treatment measurement of PD-L1 gene expressions. (A) NSCLC patients treated with immunotherapy. P = 0.003 for the difference in median values. (Wilcoxon /Kruskal Wallis Rank Sums). Immunotherapy includes pembrolizumab, nivolumab, atezolizumab. (B) Non-immunotherapy of NSCLC patients. NSCLC: Non-small cell lung cancer.