461P Real-time PCR-based assessment of RAS/BRAF mutations in the plasma of metastatic colorectal cancer (mCRC) patients: A single institution experience

IntroductionDetection of abnormalities in the KRAS, NRAS and BRAF genes is extremely important for proper qualification of colorectal cancer (CRC) patients for therapy with anti-EGFR monoclonal antibodies. However, data about prevalence of mutations in these genes, in different localizations of CRC tumors, is limited.Material and methodsWe examined the frequency of mutations in the KRAS, NRAS and BRAF genes in 500 Caucasian CRC patients (200 women and 300 men, median age – 66 years). DNA was isolated from formalin- fixed, paraffin embeded (FFPE) tissues using Qiagen QIAmp DNA FFPE-kit. Analysis of mutations was carried out using KRAS/BRAF, NRAS and BRAF Mutation Analysis Kit for Real-Time PCR (EntroGen) using the Cobas 480 real-time PCR apparatus (Roche Diagnostics)ResultsKRAS mutations have been detected in 190 patients (38%), NRAS mutations in 20 patients (4%), whereas BRAF mutations in 24 patients (4,8%). There were no associations between age of CRC patients and frequency of KRAS, NRAS and BRAF genes mutations. These mutations were significantly more often diagnosed in women (55.5%) than in man (41%, p<0.005). Tumors of rectum and sigmoideum were the most often observed in both groups of CRC patients – with and without KRAS, NRAS and BRAF genes mutations. However, transverse colon, ascending colon and cecum cancers were the most often affected by mutationsConclusionsOur study showed that the occurrence of mutations in the KRAS, NRAS and BRAF genes is not accidental and depends on the location of CRC tumors.

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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

Journal of Clinical Medicine ◽

10.3390/jcm10010087 ◽

2020 ◽

Vol 10 (1) ◽

pp. 87

Author(s):

Vincenzo Formica ◽

Jessica Lucchetti ◽

Elena Doldo ◽

Silvia Riondino ◽

Cristina Morelli ◽

...

Keyword(s):

Colorectal Cancer ◽

Regression Analysis ◽

Metastatic Colorectal Cancer ◽

Real Time ◽

Real Time Pcr ◽

Mutational Analysis ◽

Multivariate Logistic Regression Analysis ◽

Maximum Diameter ◽

Wild Type ◽

Cox Regression Analysis

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.

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A Critical Review of Second-generation anti-EGFR Monoclonal Antibodies in Metastatic Colorectal Cancer

Current Drug Targets ◽

10.2174/1389450121666200727121011 ◽

2020 ◽

Vol 21 ◽

Author(s):

Daniel Sur ◽

Andrei Havasi ◽

Alecsandra Gorzo ◽

Claudia Burz

Keyword(s):

Colorectal Cancer ◽

Drug Resistance ◽

Clinical Trials ◽

Monoclonal Antibodies ◽

Metastatic Colorectal Cancer ◽

Second Generation ◽

Current Data ◽

Braf Mutations ◽

Durable Response ◽

Ongoing Research

Background: Anti-EGFR monoclonal antibodies (mAbs) have become a relevant solution for the treatment of patients with metastatic colorectal cancer. Current anti-EGFR monoclonal antibodies face a series of problems, including resistance and non-durable response, and RAS and BRAF mutations serve as exclusion criteria for treatment with anti-EGFR mAbs. Advances in molecular tumor profiling and information on subsequent pathways responsible for disease progression and drug resistance helped develop a new generation of anti-EGFR mAbs. These second-generation mAbs have been developed to overcome existing resistance mechanisms and to limit common side effects. For the moment, existing literature suggests that these novel anti-EGFR mAbs are far from finding their way to clinical practice soon. Objective: In this review, we summarize and evaluate current data regarding ongoing research and completed clinical trials for different second-generation anti-EGFR monoclonal antibodies. Conclusion: Anti-EGFR mAbs exhibit efficacy in advanced colorectal cancer, but second-generation mAbs failed to prove their benefit in the treatment of metastatic colorectal cancer. Understanding the biological basis of primary and acquired drug resistance could allow scientists to design better clinical trials and develop improved second-generation mAbs.

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RPS24c Isoform Facilitates Tumor Angiogenesis Via Promoting the Stability of MVIH in Colorectal Cancer

Current Molecular Medicine ◽

10.2174/1566524019666191203123943 ◽

2020 ◽

Vol 20 (5) ◽

pp. 388-395 ◽

Cited By ~ 1

Author(s):

Yue Wang ◽

Youjun Wu ◽

Kun Xiao ◽

Yingjie Zhao ◽

Gang Lv ◽

...

Keyword(s):

Colorectal Cancer ◽

Ribosomal Protein ◽

Real Time ◽

Tumor Angiogenesis ◽

Real Time Pcr ◽

Molecular Mechanisms ◽

Migration And Invasion ◽

Binding Interaction ◽

Tubule Formation ◽

The Stability

Background: Colorectal cancer (CRC) is the second leading cause of death worldwide, and distant metastasis is responsible for the poor prognosis in patients with advanced-stage CRC. RPS24 (ribosomal protein S24) as a ribosomal protein, multiple transcript variant encoding different isoforms have been found for this gene. Our previous studies have demonstrated that RPS24 is overexpressed in CRC. However, the mechanisms underlying the role of RPS24 in tumor development have not been fully defined. Methods: Expression of RPS24 isoforms and lncRNA MVIH in CRC tissues and cell lines were quantified by real-time PCR or western blotting assay. Endothelial tube formation assay was performed to determine the effect of RPS24 on tumor angiogenesis. The cell viability of HUVEC was determined by MTT assay, and the migration and invasion ability of HUVEC were detected by transwell assay. PGK1 secretion was tested with a specific ELISA kit. Results: Here, we found that RPS24c isoform was a major contributor to tumor angiogenesis, a vital process in tumor growth and metastasis. Real-time PCR revealed that RPS24c isoform was highly expressed in CRC tissues, while other isoforms are present in both normal and CRC tissues with no statistical difference. Moreover the change of RPS24 protein level is mainly due to the fluctuation of RPS24c. Furthermore, we observed that silencing RPS24c could decrease angiogenesis by inhibiting tubule formation, HUVEC cell proliferation and migration. Additionally, we investigated the molecular mechanisms and demonstrated that RPS24c mRNA interacted with lncRNA MVIH, the binding-interaction enhanced the stability of each other, thereby activated angiogenesis by inhibiting the secretion of PGK1. Conclusion: RPS24c facilitates tumor angiogenesis via the RPS24c/MVIH/PGK1 pathway in CRC. RPS24c inhibition may be a novel option for anti-vascular treatment in CRC.

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Evidence-Based Second-Line Treatment in RAS Wild-Type/Mutated Metastatic Colorectal Cancer in the Precision Medicine Era

International Journal of Molecular Sciences ◽

10.3390/ijms22147717 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7717

Author(s):

Guido Giordano ◽

Pietro Parcesepe ◽

Giuseppina Bruno ◽

Annamaria Piscazzi ◽

Vincenzo Lizzi ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Checkpoint Inhibitors ◽

Line Treatment ◽

Second Line ◽

Wild Type ◽

First Line ◽

Braf Mutations ◽

Mutational Status ◽

First Line Treatment

Target-oriented agents improve metastatic colorectal cancer (mCRC) survival in combination with chemotherapy. However, the majority of patients experience disease progression after first-line treatment and are eligible for second-line approaches. In such a context, antiangiogenic and anti-Epidermal Growth Factor Receptor (EGFR) agents as well as immune checkpoint inhibitors have been approved as second-line options, and RAS and BRAF mutations and microsatellite status represent the molecular drivers that guide therapeutic choices. Patients harboring K- and N-RAS mutations are not eligible for anti-EGFR treatments, and bevacizumab is the only antiangiogenic agent that improves survival in combination with chemotherapy in first-line, regardless of RAS mutational status. Thus, the choice of an appropriate therapy after the progression to a bevacizumab or an EGFR-based first-line treatment should be evaluated according to the patient and disease characteristics and treatment aims. The continuation of bevacizumab beyond progression or its substitution with another anti-angiogenic agents has been shown to increase survival, whereas anti-EGFR monoclonals represent an option in RAS wild-type patients. In addition, specific molecular subgroups, such as BRAF-mutated and Microsatellite Instability-High (MSI-H) mCRCs represent aggressive malignancies that are poorly responsive to standard therapies and deserve targeted approaches. This review provides a critical overview about the state of the art in mCRC second-line treatment and discusses sequential strategies according to key molecular biomarkers.

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P-129 Concomitant RAS and BRAF mutations: Impact on overall survival and progression free survival in metastatic colorectal cancer patients

Annals of Oncology ◽

10.1016/j.annonc.2021.05.184 ◽

2021 ◽

Vol 32 ◽

pp. S142-S143

Author(s):

A. Pretta ◽

M. Persano ◽

G. Pinna ◽

C. Donisi ◽

E. Cimbro ◽

...

Keyword(s):

Colorectal Cancer ◽

Overall Survival ◽

Metastatic Colorectal Cancer ◽

Cancer Patients ◽

Progression Free Survival ◽

Braf Mutations ◽

Free Survival ◽

Colorectal Cancer Patients

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Atypical, Non-V600 BRAF Mutations as a Potential Mechanism of Resistance to EGFR Inhibition in Metastatic Colorectal Cancer

JCO Precision Oncology ◽

10.1200/po.19.00102 ◽

2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Benny Johnson ◽

Jonathan M. Loree ◽

Alexandre A. Jacome ◽

Shehara Mendis ◽

Muddassir Syed ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Stable Disease ◽

Kinase Activity ◽

Class Ii ◽

Activity Level ◽

Class Iii ◽

Braf Mutations ◽

Egfr Inhibition ◽

Mechanism Of Resistance

PURPOSE Atypical, non-V600 BRAF ( aBRAF) mutations represent a rare molecular subtype of metastatic colorectal cancer (mCRC). Preclinical data are used to categorize aBRAF mutations into class II (intermediate to high levels of kinase activity, RAS independent) and III (low kinase activity level, RAS dependent). The clinical impact of these mutations on anti-EGFR treatment efficacy is unknown. PATIENTS AND METHODS Data from 2,084 patients with mCRC at a single institution and from an external cohort of 5,257 circulating tumor DNA (ctDNA) samples were retrospectively analyzed. Overall survival (OS) was calculated using Kaplan-Meier and log-rank tests. Statistical tests were two-sided. RESULTS BRAF mutations were harbored by 257 patients, including 36 with aBRAF mutations: 22 class III, 10 class II, four unclassified. For patients with aBRAF mCRC, median OS was 36.1 months, without a difference between classes, and median OS was 21.0 months for patients with BRAFV600E mCRC. In contrast to right-sided predominance of tumors with BRAFV600E mutation, 53% of patients with aBRAF mCRC had left-sided primary tumors. Concurrent RAS mutations were noted in 33% of patients with aBRAF mCRC, and 67% of patients had microsatellite stable disease. Among patients with aBRAF RAS wild-type mCRC who received anti-EGFR antibodies (monotherapy, n = 1; combination therapy, n = 10), no responses to anti-EGFR therapy were reported, and six patients (four with class III aBRAF mutations, one with class II, and one unclassified) achieved stable disease as best response. Median time receiving therapy was 4 months (range, 1 to 16). In the ctDNA cohort, there was an increased prevalence of aBRAF mutations and subclonal aBRAF mutations ( P < .001 for both) among predicted anti-EGFR exposed compared with nonexposed patients. CONCLUSION Efficacy of anti-EGFR therapy is limited in class II and III aBRAF mCRC. Detection of aBRAF mutations in ctDNA after EGFR inhibition may represent a novel mechanism of resistance.

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