Extended RAS Mutational Status Analysis in Circulating Tumor DNA From Patients With Advanced Colorectal Cancer in Daily Clinical Practice. The Franco-British Institute Experience and Recommendations

Background : The development of liquid biopsies could help clinicians to shorten treatment decision and allow longitudinal molecular monitoring. We report here our experience of using the Biocartis IdyllaTM test for patients with advanced colorectal cancer in daily clinical practice. Patients and Methods: This program prospectively enrolled patients with advanced colorectalcancer at the Department of Medical Oncology of the Franco-British Institute, Levallois-Perret,France.Theprimary objective was to evaluate the feasibilty of the IdyllaTM testing in daily clinical practice. Results: From November 17, 2017 to June 12,2018, 50 patients were tested. The median time to circulating tumor (ct) extended mutational status was 4.8 hours (95% CI:4.7-5.1). Among 37 (74.0%) patients with paired plasma and tumor samples available, the overall agreement rate was 73.0%. In patients with previously untreated metastatic liver disease (n=9;18%), the overall agreement rate between paired plasma and tumor samples was 100.0%, and the weighted K coefficient was 1.00 (95% CI: 1.00-1.00). Conclusion : Extended RAS mutational status analysis in circulating tumor DNA from patients with advanced colorectal cancer is feasible and greatly helpful for daily clinical practice. We recommend restricting ctDNA testing with IdyllaTM to patients with previously untreated liver metastatic disease.


Introduction
Systemic therapy is the standard care for patients with unresectable metastatic colorectal cancer (CRC). Cetuximab and panitumumab are IgG monoclonal antibodies (MoAb), which competitively inhibit the binding of epidermal growth factor (EGF) and other ligands to EGF receptor (EGFR) and downregulate EGFR, resulting in inhibition of cell survival, growth, proliferation, and transformation. Tumor molecular testing for Kirsten rat sarcoma viral oncogene homolog (KRAS) and Neuroblastoma RAS viral oncogene homolog (NRAS) tumor genes is mandatory in order to select MCRC patients who may benefit from anti-EGFR MoAb therapy [1]. In 2014, the mean time to obtain the RAS genotyping test report in France was 25 days, and for 25% of the patients it was more than one month [2]. The murine sarcoma viral (v-Raf) oncogene homolog B (BRAF) is a human gene that encodes BRAF, a protein involved in sending signals inside cells, which are involved in directing cell growth. The BRAF V600E mutation is found in10% of patients with colorectal adenocarcinoma [3] and BRAF mutant tumors are more frequently right-sided, mucinouswith peritoneal spreadand microsatellite instability [4].
The extracellular domain (ECD) of EGFR p.S492 mutation has been described as an acquired resistance to cetuximab [5]. The  [6][7][8][9][10][11][12][13]. We report here our experience of using the Biocartis Idylla TM test for daily clinical practice in order to improve care in patients with advanced CRC.

Study Design and Population
This program prospectively enrolled patients with advanced CRC at the Department of Medical Oncology of the Franco-British Institute, Levallois-Perret, France. Clinical research study nurse was responsible for this program. A signed and dated informed consent form from each patient was required before enrollment.

Methods
The blood sampling was done in the out patient cancer unit, close to the translational research laboratory. EDTA tubes were centrifuged at 1500g for 10 minutes within 4 hours after blood samples collection, the plasma was recovered, and a second centrifugation was performed at 16000g for one minute. Circulating tumor DNA (ctDNA) was extracted and amplified from 1ml of blood plasma. The Idylla™ ctKRAS Mutation Assay is a molecular assay for the qualitative detection of 21 mutations in codons 12, 13, 59, 61,117and 146 of the KRAS gene. The Idylla™ ctNRAS-BRAF-EGFR S492R Mutation Assay is a molecular assay for the semiquantitative detection of 18 mutations in codons 12, 13, 59, 61, 117, and 146 of the NRAS gene, five mutations in codon 600 of the BRAF gene and two mutations in codon 492 of the EGFR gene. Biocartis Idylla™ platform is a CE-marked IVD in Europe.

Data Collection
Individual patient-level clinical data and tumor mutational RAS and BRAF assessment were collected in DxCare software (©2013 Medasys S.A., Clamart, France). Circulating tumor DNA information was collected using the Idylla TM Explore, a web-based application that allows analyzing data by visualization of PCR curves from the Idylla TM tests results, Cq values per target, and direct access to console results report.

Statistical Analysis
The primary objective was to evaluate the feasibility of the Idylla TM testing in daily clinical practice. Secondary objectives were: a) To evaluate the accuracy of RAS and BRAF status between paired plasma and tumor samples b) To compare the time to molecular mutational diagnosis between tumor and plasma techniques in patients with initially unknown tumor status c) To evaluate the incidence of neomutations in patients with wild-type CRC treated with anti-EGFR MoAb using a serial tracking of KRAS, NRAS, BRAF, and EFGR mutations d) To evaluate the incidence of mutation loss in patients with mutated RAS or BRAF CRC.
The feasibility of the Idylla TM testing was calculated using the time from blood sample collection to the Idylla TM processing and results. The accuracy was calculated using the interrate agreement kappa(K) coefficient [14] with its 95% circulating tumor). The strength of agreement was interpreted according to K value as follows: poor (<0.

Patient Characteristics
From November 17, 2017 to June 12,2018, 50 patients with advanced CRC had their tumor genotyped by the Idylla TM test. This testing was proposed to 30 (60.0%) patients with unknown or partially known RAS/BRAF tumor status and to 20 (40.0%) patients with known status for the RAS and BRAF tumor genes (Table1) and ( Figure 1). The mean age was 67.9 years (range: 29.7-93.4). The IdyllaTM test was done in 33(66.0%) patients before starting a new treatment sequence and in 17 (34.0%) patients with ongoing treatment sequence.

Feasibility of the Idylla TM Testing
Seventy three samples analyses (including longitudinal testing) were performed. ctKRAS mutation assay was done in 73 samples and ctNRAS/BRAF/EGFR mutation assay was performed for 60 samples. The median time from blood sample collection to the Idylla TM processing was 34 minutes (95% CI: 29-39). The median time to ctKRAS results and to full ctKRAS/NRAS/BRAF/EGFR results were 2.8 hours (95% CI: 2.7-2.9) and 4.8 hours (95% CI: 4.7-5.1), respectively.

Time to Molecular Mutational Diagnosis: Plasma vs Tumor
In 30 (60%) patients with initially unknown tumor status, the median time to molecular profile using liquid biopsies was 4.

Accuracy of RAS and BRAF Status Between Paired Plasma and Tumor Samples
Among 37 (74.0%) patients with paired plasma and tumor samples available, 27 (73.0%) had a concordant molecular profile between plasma and tumor. Positive, negative, and overall agreement rates were 47.0%, 95.0% and 73.0%, respectively. The weighted K coefficient was 0.44 (95% CI: 0.17-0.70). Among 10 (20%) patients with discordant results between plasma and tumor, five patients had single extra-liver metastatic site (lung, n=3; peritoneum, n=2) and five patients received prior therapy for metastatic disease (Table 2). When selecting only patients with paired plasma and tumor samples with previously untreated metastatic liver disease (n=9; 18%), there were no discordant results between plasma and tumor. The positive, negative and overall agreement rates were 100.0%, and the weighted K coefficient was 1.00 (95% CI: 1.00-1.00).

Neomutations in Wild-Type Crc Patients
Of the nine (18%) patients with wild-type RAS/RAF CRC treated with anti-EGFR MoAb using a serial tracking of KRAS, NRAS, BRAF and EFGR mutations, two (22.2%) had neomutations. The first patient was treated with FOLFIRI-cetuximab as firstline therapy for right-sided unresectable metastatic CRC. A ctNRAS G12A/V neomutation was not confirmed in further liquid biopsy and was contemporary of partial response (Figure 2A). The second patient was treated with cetuximab-irinotecan as third line therapy for left sided metastatic CRC. A ctKRAS G12C neomutation was confirmed in further liquid biopsy and preceded radiologic disease progression ( Figure 2B). Of note, an EGFR S492R mutation was found in one patient with locally advanced right-sided KRAS wildtype CRC (insufficient tumor material for NRAS and BRAF analysis). This patient received prior FOLFOX first-line therapy without anti-EGFR MoAb. In this patient, panitumumab was preferred to cetuximab as second-line therapy due to the mutation of the extracellular domain of EGFR. Panitumumab was stopped after 3 cycles due to clinical and biological (CEA) progression while CTscan stabilization was observed.

Loss of Mutation in Mutant CRC Patients
Of the eight (16%) patients with previously treated (i.e., at least one prior line of therapy) mutant RAS or BRAF metastatic CRC, six patients were ctRAS/RAF wild-type and two patients were ctRAS or ctBRAF mutant.

Discussion
This is the first prospective report of using the Idylla TM ctKRAS and ctNRAS/BRAF/EGFR testing for patients with advanced CRC in daily clinical practice. Clinical research study nurse was responsible for this program. Blood sampling was located close to the translational research laboratory inorder to shorten the preanalytical time, with a median time from sample collection to the Idylla TM processing of 34 minutes, including the centrifugation process. Molecular diagnostic testing of four genes (KRAS, NRAS, BRAF and EGFR) took less than 5 hours, enabling physicians to make timely therapeutic decisions, and compare favorably with 56 days necessary to obtain a full tumor molecular evaluation.In

5/6
the unselected population, the overall agreement between plasma and tumor was 73%. Discordant resultswere observed in patients with extra-liver disease and/or in patients having received prior therapy for metastatic disease. Thus, when aiming to obtain a rapid molecular diagnosis, the Idylla TM testing should be restricted to patients with previously untreated CRC with liver metastasis, with an overall agreement rate of 100%. Serial tracking of the RAS and BRAF mutation emergence in patients with wild-type CRC treated with anti-EGFR MoAb was previously described [15].
In our study, a transient ctNRAS mutation was found in one patient treated with cetuximab-based therapy, but was not associated with disease progression, whereas a confirmed ctKRAS mutation preceded failure of cetuximab-based therapy. Liquid biopsies could help to identify patients having tumors with secondary resistance to drugs and to develop different treatment strategies using agents targeting neomutations for those patients. Serial tracking of the RAS or BRAF mutation loss may also be offered to patients with mutant RAS or BRAF CRC. It has been previously suggested that patients with metastatic CRC harboring mutated primary tumors, thus not candidate to EGFR MoAb, frequently have wild-type RAS circulating tumor cells in blood [16]. Preliminary data suggest that patients with mutant KRAS CRC can frequently (50%) switch to a prevalent wild-type KRAS disease in course of treatment with anti-angiogenic drugs [17]. This phenomenon was concomitant to disease progression. In our series, six of the eight patients with previously treated mutant RAS or BRAF metastatic CRC were ct wild-type. Those 'NeoWild-Type' patients may be eligible to anti-EGFR therapy and warrant larger prospective evaluation in this particular setting.

Conclusion
Extended RAS mutational status analysis in circulating tumor DNA from patients with advanced CRC is feasible and helpful for daily practice routine.We recommend restricting the Idylla TM ctDNA testing to patients with previously untreated liver metastatic disease.