Clinical Practice Use of Liquid Biopsy to Identify RAS/BRAF Mutations in Patients with Metastatic Colorectal Cancer (mCRC): A Single Institution Experience

Tumor heterogeneity represents a possible cause of error in detecting predictive genetic alterations on tumor tissue and can be overcome by testing alterations in circulating tumor DNA (ctDNA) using liquid biopsy. We assessed 72 consecutive patients with a diagnosis of metastatic colorectal cancer (mCRC) using Idylla™ Biocartis, a fully automated platform that evaluates the most frequent mutations of KRAS, NRAS and BRAF genes. We correlated the results of liquid biopsy and standard tissue-based next generation sequencing (NGS) analyses to patient clinical features. The overall agreement was 81.94%. Concordance was 85.71% and 96.15% in treatment-naïve patients and in the patient subgroup with liver metastases, respectively. In liver metastases positive, treatment-naïve patients, sensitivity, specificity and positive predictive value (PPV) were 92.31%, 100% and 100%, respectively. Circulating mutational fraction (CMF) was significantly higher in patients with liver metastases and high carcinoembryonic antigen (CEA) levels. In a subgroup of patients pre-treated with anti-Epidermal Growth Factor Receptor (EGFR) agents, emerging KRAS mutations were evidenced in 33% of cases. Testing RAS/BRAF mutations on plasma using the Idylla™ Biocartis platform is feasible and reliable in mCRC patients in clinical practice.

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Liquid biopsy-driven anti-EGFR rechallenge in patients with metastatic colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.3577 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 3577-3577

Author(s):

Stefano Mariani ◽

Marco Puzzoni ◽

Nicole Liscia ◽

Valentino Impera ◽

Andrea Pretta ◽

...

Keyword(s):

Colorectal Cancer ◽

Clinical Practice ◽

Metastatic Colorectal Cancer ◽

Liquid Biopsy ◽

Selection Criteria ◽

Wild Type ◽

Braf Mutations ◽

Previous Response ◽

Clinical Variables ◽

Ct Dna

3577 Background: The rechallenge with EGFR inhibitors represents an emerging strategy for anti-EGFR pre-treated patients with RAS wild type colorectal cancer (CRC). Unfortunately definitive selection criteria for anti-EGFR rechallenge in this setting are lacking. Very recently RAS wild type status on circulating tumor DNA (ct-DNA) at the time of rechallenge along with already known clinical criteria emerged as a potential watershed for this strategy. In the present study we explored liquid biopsy-driven anti-EGFR rechallenge strategy in the clinical practice for patients with metastatic colorectal cancer. Methods: Ct-DNA from RAS and BRAF wild type metastatic CRC patients previously treated with an anti-EGFR containing therapy was analyzed for RAS/BRAF mutations with the aim to evaluate the rechallenge strategy with anti-EGFR. The ct-DNA was analyzed for RAS-BRAF mutations using pyro-sequencing (PyroMark Q24 MDx Workstation) and nucleotide sequencing (Genetic Analyzer ABI3130) assays. Real-time PCR (Idylla) and droplet digital PCR (QX200 System) were performed to confirm the RAS-BRAF mutation status. Several clinical variables including previous response to anti EGFR containing therapy, tumor sidedness and anti-EGFR free interval were evaluated in relation to outcome. Tumor response evaluation was performed according to RECIST 1.1. Differences between categorical variables were evaluated using the Fisher’s exact test. Survival probability over time was estimated by the Kaplan–Meier method. Significant differences in the probability of survival between the strata were evaluated by log-rank test. Results: Twenty patients were included in the study. All patients were tested for RAS-BRAF mutations in ct-DNA. Fourteen patients (70%) showed a RAS-BRAF WT molecular profile, six patients (30%) showed a KRAS mutation. All the patients with ct-DNA RAS-BRAF WT profile underwent rechallenge with anti-EGFR. In details 11 patients (78.6%) underwent irinotecan+ cetuximab treatment, whereas 3 patients (21.4%) underwent panitumumab monotherapy. As for the outcome results to the rechallenge strategy, the median OS was 7 months (95% CI 5.0 to 13.0), the median PFS was 3 months (95% CI 2.0 to 6.0), the ORR was 27.3% with a DCR of 54.5%. Among the clinical variables evaluated as putative predictive/prognostic factors, previous response to anti-EGFR treatment was related to a not statistically significant improved OS (12 months vs 5 months HR:0.19 p: 0.06) and to a statistically significant improved ORR (75% vs 0% p:0.03). Conclusions: The rechallenge strategy with anti-EGFR confirmed to be feasible in clinical practice. The clinical outcome resulted consistent with the literature data. In addition to the molecular selection through the analysis of ct-DNA for RAS, previous response to anti EGFR treatment is confirmed as a prospective selection criteria for this therapeutic option.

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KRAS, NRAS, BRAF, HER2 and microsatellite instability in metastatic colorectal cancer – practical implications for the clinician

Radiology and Oncology ◽

10.2478/raon-2019-0033 ◽

2019 ◽

Vol 53 (3) ◽

pp. 265-274 ◽

Cited By ~ 11

Author(s):

Vlad-Adrian Afrăsânie ◽

Mihai Vasile Marinca ◽

Teodora Alexa-Stratulat ◽

Bogdan Gafton ◽

Marius Păduraru ◽

...

Keyword(s):

Colorectal Cancer ◽

Clinical Practice ◽

Microsatellite Instability ◽

Metastatic Colorectal Cancer ◽

Growth Factor Receptor ◽

Genetic Alterations ◽

Braf V600e ◽

Pulmonary Metastasectomy ◽

Ras Gene ◽

The Right

Abstract Background Colorectal cancer is a successful model of genetic biomarker development in oncology. Currently, several predictive or prognostic genetic alterations have been identified and are used in clinical practice. The RAS gene family, which includes KRAS and NRAS act as predictors for anti-epithelial growth factor receptor treatment (anti-EGFR), and it has been suggested that NRAS mutations also play a role in prognosis: patients harboring NRAS alterations have a significantly shorter survival compared to those with wild type tumours. BRAF V600E mutations are rare and occur mostly in tumors located in the ascending colon in elderly female patients. BRAF is instrumental in establishing prognosis: survival is shorter by 10–16 months in BRAF-mutant patients, and BRAF may be a negative prognostic factor for patients who undergo hepatic or pulmonary metastasectomy. Moreover, this mutation is used as a negative predictive factor for anti-EGFR therapies. Two new biomarkers have recently been added to the metastatic colorectal cancer panel: HER2 and microsatellite instability. While HER2 is still being investigated in different prospective studies in order to validate its prognostic role, microsatellite instability already guides clinical decisions in substituted with advanced colorectal cancer. Conclusions There are current evidences that support using above mentioned genetic biomarkers to better identify the right medicine that is supposed to be used in the right patient. This approach contributes to a more individualized patient-oriented treatment in daily clinical practice.

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KRAS and BRAF Mutations as Prognostic and Predictive Biomarkers for Standard Chemotherapy Response in Metastatic Colorectal Cancer: A Single Institutional Study

Cells ◽

10.3390/cells9010219 ◽

2020 ◽

Vol 9 (1) ◽

pp. 219 ◽

Cited By ~ 3

Author(s):

Nuria Garcia-Carbonero ◽

Javier Martinez-Useros ◽

Weiyao Li ◽

Alberto Orta ◽

Nuria Perez ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Predictive Biomarkers ◽

Braf V600e ◽

Chemotherapy Response ◽

First Line ◽

Standard Chemotherapy ◽

Braf Mutations ◽

Kras Mutations ◽

Line Standard

KRAS mutation is a confirmed predictive biomarker for anti-EGFR monoclonal antibody therapy response for metastatic colorectal cancer. However, its prognosis impact and the predictive potential for first-line standard chemotherapy remains unclear. On the other hand, V600E mutation is the most frequent and studied mutation in the BRAF gene, and it has been associated with a poor outcome of patients and a low response to anti-EGFR treatment. Thus, the aim of this study is to evaluate the role of KRAS and BRAF mutations as prognosis factors and predictive biomarkers for 1st line standard chemotherapy in metastatic colorectal cancer. KRAS mutations and BRAF V600E mutations exhibited a poor outcome (p = 0.021 and p < 0.0001, respectively). Cox multivariate analysis showed that the presence of liver metastasis (HR = 1.595; 95% CI: 1.086–2.343; p = 0.017), KRAS mutation (HR = 1.643; 95% CI: 1.110–2.431; p = 0.013) and BRAF V600E mutation (HR = 5.861; 95% CI: 2.531–13.570; p < 0.0001) were statistically significant co-variables for progression-free survival. Interestingly, patients with KRAS mutations were associated with a poor response to first line standard chemotherapy (p = 0.008). In contrast, the BRAF V600E mutation did not have any impact on the first line standard chemotherapy response (p = 0.540). Therefore, in the present study, we provide new insight on the role of KRAS and BRAF, not only as prognosis biomarkers, but also as first line standard chemotherapy response biomarkers in metastatic colorectal cancer.

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BRAF and KRAS mutations in metastatic colorectal cancer: future perspectives for personalized therapy

Gastroenterology Report ◽

10.1093/gastro/goaa022 ◽

2020 ◽

Vol 8 (3) ◽

pp. 192-205 ◽

Cited By ~ 2

Author(s):

Zi-Nan Li ◽

Lin Zhao ◽

Li-Feng Yu ◽

Min-Jie Wei

Keyword(s):

Colorectal Cancer ◽

Clinical Trials ◽

Survival Rate ◽

Metastatic Colorectal Cancer ◽

Early Stage ◽

Unmet Need ◽

Growth Factor Receptor ◽

Kras Mutations ◽

Viral Oncogene ◽

Viral Oncogene Homolog

Abstract Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide and 30% of patients with CRC experience metastasis. Patients with metastatic colorectal cancer (mCRC) have a 5-year overall survival rate of <10%. V-raf murine sarcoma viral oncogene homolog B1 (BRAF) and V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog (KRAS) mutations are mostly studied in mCRC, as clinical trials found that first-line chemotherapy with anti-epidermal growth factor receptor agent confers limited efficacy for mCRC. Treatment decisions for early-stage mCRC do not consider BRAF or KRAS mutations, given the dramatically poor prognosis conferred by these mutations in clinical trials. Thus, it is necessary to identify patients with mCRC harboring BRAF or KRAS mutations to formulate rational therapeutic strategies to improve prognosis and survival. BRAF and KRAS mutations occur in ∼10% and ∼44% of patients with mCRC, respectively. Although the survival rate of patients with mCRC has improved in recent years, the response and prognosis of patients with the aforementioned mutations are still poor. There is a substantial unmet need for prospective personalized therapies for patients with BRAF- or KRAS-mutant mCRC. In this review, we focus on BRAF and KRAS mutations to understand the mechanisms underlying resistance and improving the response rate, outcomes, and prognosis of patients with mCRC bearing these mutations and to discuss prospective personalized therapies for BRAF- and KRAS-mutant mCRC.

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cfDNA for accurate determination of RAS and BRAF mutations using OncoBEAM liquid biopsy in metastatic colorectal cancer patients: Results of the real-world multicentric ColoBEAM study.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.3542 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 3542-3542 ◽

Cited By ~ 3

Author(s):

Alexandre Harle ◽

Celine Gavoille ◽

Olivier Bouche ◽

Meher Ben Abdelghani ◽

Jérôme Edouard Plaza ◽

...

Keyword(s):

Colorectal Cancer ◽

Liver Metastasis ◽

Metastatic Colorectal Cancer ◽

Liquid Biopsy ◽

Accurate Determination ◽

Braf V600e ◽

Molecular Testing ◽

Braf Mutations ◽

Blood Samples

3542 Background: Determination of KRAS, NRAS ( RAS) and BRAF mutations is a standard of care for the management of patients with metastatic colorectal cancer (mCRC). RAS mutations are well characterized resistance biomarkers to anti-EGFR antibodies and BRAF V600 mutations indicate poor prognosis. Tissue biopsy has traditionally been used to determine RAS and BRAF status, but liquid biopsy analysis of circulating tumor DNA (ctDNA) has demonstrated utility as a less invasive tool to expedite molecular testing results to the clinic. The ColoBEAM study reports the performance of plasma mutation testing in a real-life prospective series of 278 patients across 8 centers. Methods: Plasma derived ctDNA was prepared from 20mL blood samples prospectively collected from mCRC patients who had not received chemotherapy in the prior 15 days. ctDNA was centrally assessed using OncoBEAM and results compared to those obtained by routine analysis of tissue. Both tissue and blood samples with discrepant RAS results were blindly reassessed with OncoBEAM. Results: Of 278 patients enrolled, 202 blood samples were available for OncoBEAM testing. RAS and BRAF V600E mutations were detected in tissue in 132/202 (65.4%) and 4/198 (2.0%) patients, respectively. Analysis of the first ctDNA sample as compared to tissue DNA resulted in a kappa coefficient (κ) of 0.52 [0.41 – 0.63] and accuracy of 75.2% (65.1% sensitivity; 94.3% specificity). OncoBEAM testing of a second sample resulted (κ) of 0.66 [0.56 - 0.76] and accuracy of 83.2% (77.3% sensitivity; 94.3% specificity). Of the 4 samples with a BRAF V600E mutation in tumor tissue 2 were detected in blood. In the subgroup of patients with liver metastasis (n=136), accuracy was 88.2% (87.4% sensitivity; 90.2% specificity) for RAS and BRAF status with (κ) of 0.73 [0.61 – 0.86]. In a subgroup of chemotherapy naïve patients with liver metastasis (n=49), accuracy was 91.8% (93.3% sensitivity; 89.5% specificity) for RAS and BRAF status with (κ) of 0.83 [0.67 – 0.99]. Conclusions: The results of the ColoBEAM study confirm plasma ctDNA as a credible surrogate marker to tissue DNA for RAS and BRAF status assessment and may be incorporated as a first-line theragnostic assessment. New testing on a second sample for wild-type status demonstrated 91.8% concordance between blood and tissue. Clinical trial information: NCT02751177.

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Phase III Trial of Cetuximab With Continuous or Intermittent Fluorouracil, Leucovorin, and Oxaliplatin (Nordic FLOX) Versus FLOX Alone in First-Line Treatment of Metastatic Colorectal Cancer: The NORDIC-VII Study

Journal of Clinical Oncology ◽

10.1200/jco.2011.38.0915 ◽

2012 ◽

Vol 30 (15) ◽

pp. 1755-1762 ◽

Cited By ~ 354

Author(s):

Kjell Magne Tveit ◽

Tormod Guren ◽

Bengt Glimelius ◽

Per Pfeiffer ◽

Halfdan Sorbye ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Phase Iii ◽

Line Treatment ◽

First Line ◽

Braf Mutations ◽

Kras Mutations ◽

Phase Iii Trial ◽

Significant Difference ◽

First Line Treatment

Purpose The NORDIC-VII multicenter phase III trial investigated the efficacy of cetuximab when added to bolus fluorouracil/folinic acid and oxaliplatin (Nordic FLOX), administered continuously or intermittently, in previously untreated metastatic colorectal cancer (mCRC). The influence of KRAS mutation status on treatment outcome was also investigated. Patients and Methods Patients were randomly assigned to receive either standard Nordic FLOX (arm A), cetuximab and FLOX (arm B), or cetuximab combined with intermittent FLOX (arm C). Primary end point was progression-free survival (PFS). Overall survival (OS), response rate, R0 resection rate, and safety were secondary end points. Results Of the 571 patients randomly assigned, 566 were evaluable in intention-to-treat (ITT) analyses. KRAS and BRAF mutation analyses were obtained in 498 (88%) and 457 patients (81%), respectively. KRAS mutations were present in 39% of the tumors; 12% of tumors had BRAF mutations. The presence of BRAF mutations was a strong negative prognostic factor. In the ITT population, median PFS was 7.9, 8.3, and 7.3 months for the three arms, respectively (not significantly different). OS was almost identical for the three groups (20.4, 19.7, 20.3 months, respectively), and confirmed response rates were 41%, 49%, and 47%, respectively. In patients with KRAS wild-type tumors, cetuximab did not provide any additional benefit compared with FLOX alone. In patients with KRAS mutations, no significant difference was detected, although a trend toward improved PFS was observed in arm B. The regimens were well tolerated. Conclusion Cetuximab did not add significant benefit to the Nordic FLOX regimen in first-line treatment of mCRC.

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P-217 Value of liquid biopsy using high-affinity plasma DNA binding magnetic beads and qualitative real-time PCR for KRAS, NRAS and BRAF mutations in metastatic colorectal cancer patients

Annals of Oncology ◽

10.1016/j.annonc.2020.04.299 ◽

2020 ◽

Vol 31 ◽

pp. S161

Author(s):

J. Lucchetti ◽

V. Formica ◽

E. Doldo ◽

C. Morelli ◽

L. Vergilii ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Cancer Patients ◽

Real Time Pcr ◽

Liquid Biopsy ◽

Magnetic Beads ◽

Plasma Dna ◽

Braf Mutations ◽

High Affinity ◽

Colorectal Cancer Patients

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Anti-Metastatic Activity of an Anti-EGFR Monoclonal Antibody against Metastatic Colorectal Cancer with KRAS p.G13D Mutation

International Journal of Molecular Sciences ◽

10.3390/ijms21176037 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6037

Author(s):

Tomokazu Ohishi ◽

Yukinari Kato ◽

Mika K. Kaneko ◽

Shun-ichi Ohba ◽

Hiroyuki Inoue ◽

...

Keyword(s):

Colorectal Cancer ◽

Monoclonal Antibody ◽

Metastatic Colorectal Cancer ◽

Growth Factor Receptor ◽

High Sensitivity ◽

Kras Mutations ◽

Cytotoxicity Activities ◽

Hct 116 Cells

The now clinically-used anti-epidermal growth factor receptor (EGFR) monoclonal antibodies have demonstrated significant efficacy only in patients with metastatic colorectal cancer (mCRC), with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS). However, no effective treatments for patients with mCRC with KRAS mutated tumors have been approved yet. Therefore, a new strategy for targeting mCRC with KRAS mutated tumors is desired. In the present study, we examined the anti-tumor activities of a novel anti-EGFR monoclonal antibody, EMab-17 (mouse IgG2a, kappa), in colorectal cancer (CRC) cells with the KRAS p.G13D mutation. This antibody recognized endogenous EGRF in CRC cells with or without KRAS mutations, and showed a high sensitivity for CRC cells in flow cytometry, indicating that EMab-17 possesses a high binding affinity to the endogenous EGFR. In vitro experiments showed that EMab-17 exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against CRC cells. In vivo analysis revealed that EMab-17 inhibited the metastases of HCT-15 and HCT-116 cells in the livers of nude mouse metastatic models, unlike the anti-EGFR monoclonal antibody EMab-51 of subtype mouse IgG1. In conclusion, EMab-17 may be useful in an antibody-based therapy against mCRC with the KRAS p.G13D mutation.

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PTEN Expression and KRAS Mutations on Primary Tumors and Metastases in the Prediction of Benefit From Cetuximab Plus Irinotecan for Patients With Metastatic Colorectal Cancer

Journal of Clinical Oncology ◽

10.1200/jco.2008.20.2796 ◽

2009 ◽

Vol 27 (16) ◽

pp. 2622-2629 ◽

Cited By ~ 304

Author(s):

Fotios Loupakis ◽

Luca Pollina ◽

Irene Stasi ◽

Annamaria Ruzzo ◽

Mario Scartozzi ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Growth Factor Receptor ◽

Progression Free Survival ◽

Pten Expression ◽

Kras Mutations ◽

Primary Tumors ◽

Pten Loss ◽

Egfr Inhibition ◽

Paired Samples

PurposePTEN, AKT, and KRAS are epidermal growth factor receptor (EGFR) downstream regulators. KRAS mutations confer resistance to cetuximab. This retrospective study investigated the role of PTEN loss, AKT phosphorylation, and KRAS mutations on the activity of cetuximab plus irinotecan in patients with metastatic colorectal cancer (mCRC).Patients and MethodsA cohort of patients with irinotecan-refractory mCRC who were treated with cetuximab plus irinotecan was tested for PTEN immunoreactivity (ie, immunohistochemistry; IHC), pAKT IHC, and KRAS mutations. Analyses were performed both on primary tumors and on related metastases, and the association among IHC, mutational results, and treatment outcomes was investigated.ResultsOne-hundred two patients were eligible. Ninety-six primary tumors, 59 metastases, and 53 paired samples were available. Forty-nine primary tumors (58% of assessable samples) had a preserved PTEN expression (PTEN-positive), whereas 35 (40% of assessable samples) were pAKT-positive. Levels of concordance between primary tumors and metastases were 60%, 68%, and 95% for PTEN, pAKT, and KRAS, respectively. PTEN status on primary tumors and pAKT status both on primary tumors and on metastases did not predict response or progression-free survival (PFS). On metastases, 12 (36%) of 33 patients with PTEN-positive tumors were responders compared with one (5%) of 22 who had PTEN-negative tumors (P = .007). The median PFS of patients with PTEN-positive metastases was 4.7 months compared with 3.3 months for those with PTEN-negative metastases (hazard ratio [HR], 0.49; P = .005). Patients with PTEN-positive metastases and KRAS wild type had longer PFS compared with other patients (5.5 months v 3.8 months; HR, 0.42; P = .001).ConclusionPTEN loss in metastases may be predictive of resistance to cetuximab plus irinotecan. The combination of PTEN IHC and KRAS mutational analyses could help to identify a subgroup of patients with mCRC who have higher chances of benefiting from EGFR inhibition.

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