Clinical Utility of Plasma KRAS, NRAS and BRAF Mutational Analysis with Real Time PCR in Metastatic Colorectal Cancer Patients-The Importance of Tissue/Plasma Discordant Cases.

Vincenzo Formica,Silvia Riondino,Nicola Renzi,Fiorella Guadagni,Emanuela Dell’Aquila,Elena Doldo,Daniele Nitti,Mario Roselli,Jessica Lucchetti,Patrizia Ferroni,Cristina Morelli,Augusto Orlandi,Antonella Nardecchia,Renato Argirò,Giampiero Palmieri

doi:10.3390/jcm10010087

Abstract

Background: Tumor tissue (T) mutational analysis represents the standard for metastatic colorectal cancer (mCRC); however, circulating tumor DNA (ctDNA) detected by liquid biopsy in plasma (PL) can better represent tumor heterogeneity. Methods: mCRC patients undergoing standard first-line chemotherapy with known T-KRAS/NRAS/BRAF status were enrolled in the present prospective study. PL mutations were assessed within 2 weeks before chemotherapy start with real time PCR and correlated with T status and Progression free survival (PFS). Clinical and biochemical variables including also total number of tumor lesions (TNL) and the sum of maximum diameter (SMD) of all lesions were assessed as potential predictors of T/PL discordance. RESULTS: Among 45 enrolled patients, all BRAF mutations were concordant between T and PL and there were 20% of patients RAS discordant: 9% wild type in T and mutated in PL and 11% mutated in T and wild type in PL. T mutations were significantly associated to median PFS (mPFS of 4.5, 8.3 and 22.9 months for T-BRAF mutated, T-RAS mutated, and T-wild type patients, respectively, p for trend 0.00014). PL mutations further refined prognosis: RAS wild type in T and mutated in PL had significantly shorter PFS than concordant RAS wild type in T and PL: mPFS 9.6 vs. 23.3 months, respectively, p = 0.02. Patients RAS mutated in T and wild type in PL had longer PFS than concordant RAS mutated in T and PL: 24.4 vs. 7.8 months, respectively, p = 0.008. At a multivariate cox regression analysis for PFS, PL mutations were independent prognostic factor superior to T analysis (HR 0.13, p = 0.0008). At multivariate logistic regression analysis TNL and SMD were significant predictors of discordant cases. Conclusions: PL mutational analysis allows a better prognostication than T analysis alone and could help in mCRC treatment management.

Highlights

Since 1948, when cell-free nucleic acids were first detected in human plasma [1], the interest in the molecular characteristics of the plasma cell-free DNA of cancer patients has increasingly grown in the scientific community.Cell-free DNA is fragmented DNA found in the compartment of non-cellular molecules of the blood
Background: Tumor tissue (T) mutational analysis represents the standard for metastatic colorectal cancer; circulating tumor DNA detected by liquid biopsy in plasma (PL) can better represent tumor heterogeneity
Patients were treated with one of the following standard first-line regimens based on TRAS/BRAF mutational analysis: RAS/BRAF wild type (WT) patients received panitumumabFOLFOX (panitumumab, 6 mg/kg intravenous (IV) infusion on day 1 before FOLFOX chemotherapy regimen-oxaliplatin 85 mg/m2 IV on day 1, leucovorin, 200 mg/m2 on day 1 and 5-fluorouracil 400 mg/m2 IV bolus followed by 1200 mg/m2 IV infusion over 22 h on days 1 and 2, every 14 days); mutated patients received bevacizumab-FOLFOX (bevacizumab 5 mg/kg IV on day 1 and FOLFOX chemotherapy regimen -oxaliplatin 85 mg/m2 intravenous (IV) infusion on day 1, leucovorin, 200 mg/m2 on day 1 and 5-fluorouracil 400 mg/m2 IV bolus followed by 1200 mg/m2 IV infusion over 22 h on days 1 and 2, every 14 days)

Summary

Introduction

Since 1948, when cell-free nucleic acids were first detected in human plasma [1], the interest in the molecular characteristics of the plasma cell-free DNA of cancer patients has increasingly grown in the scientific community. The turnaround time of tumor tissue analysis is usually longer than that of ctDNA analysis, making the latter more attractive from a technical standpoint This would be especially useful for metastatic colorectal cancer (mCRC) patients who need rapid molecular profiling in order to receive the optimal targeted agent in a timely fashion [7]. In the present prospective study, we assessed the clinical utility of PL liquid biopsy for KRAS, NRAS and BRAF mutation testing in mCRC using qualitative Real Time PCR (Easy PGX), in consecutive patients with known T mutational status prior to starting a standard first-line chemotherapy. As RAS/BRAF mutations are both predictive and prognostic in mCRC patients treated with optimal first-line therapy [17,18], progression free survival (PFS) was chosen as a surrogate for the assessment of test usefulness. Written informed consent was obtained from all patients and the study procedures were performed in accordance with the Declaration of Helsinki and adhered to the international Good Clinical Practice guidelines

Molecular Analysis Cell-Free DNA Extraction and Amplification

Statistical Analysis

Patients’ Characteristics

Survival Analysis

Predictors of Discordant Cases

Conclusions