anti-EGFR Monoclonal Antibody Therapy Research Articles

Novel Approach for Detection of BRAF and KRAS Mutations Using ARMS (amplification refractory mutation system) Primers for Colon Cancer Therapeutic Response Prediction

Abstract Introduction/Objective KRAS and BRAF V600E mutations are confirmed predictive biomarkers for anti-EGFR monoclonal antibody therapy response and prognosis for colorectal cancer and often considered mutually exclusive with few exceptions (0.05% of mCRC cases). We have developed a multiplex PCR assay utilizing ARMS (amplification refractory mutation system) Primers and Probes for screening and further testing of BRAF mutation status in a 2 step process. Methods/Case Report The Multiplex PCR Assay or BRAF/KRAS mutation was designed as a 5 color assay. Primers and Probes consisted of Wild Type Kras and wild and mutant (V600E/V600K) type BRAF primers and probes. For specimen, Positive and negative control standards made of engineered positive control plasmids for wild type KRAS & BRAF and mutant type V600E & V600K BRAF as well as internal control of bet actin and 2 Patient samples with known BRAF V600E mutant status were run. Mutant type V600E & V600K BRAF were mixed with wild type V600 controls in different ratios as well. Results were interpreted as BRAF wild KRAS wild/ BRAF wild KRAS mutant/ BRAF V600E KRAS wild/BRAF V600K KRAS wild type and NGS was recommended in cases of concurrent BRAF and KRAS mutations. Results (if a Case Study enter NA) Positive/negative/internal control standards for wild type KRAS & BRAF and mutant type V600E & V600K BRAF showed expected results in both CFX 96 and ABI 7500 runs in triplicates. Graphs were able to determine separate Ct values and sigmoidal amplification plots that readily differentiated KRAS wild/BRAF wild/ BRAF V600E types. Mutant type V600E & V600K BRAF that were mixed with wild type V600 controls in different ratios all showed expected results and were able to differentiate between wild and mutant types with accuracy. Also, the 2 patient samples with known (Sanger sequencing-diagnosed) V600E mutation status showed positive Ct values for KRAs wild and V600E probes and negative CT values for V600 wild/V600K probes. No invalid runs or unexpected control results were observed. Conclusion The ARMS BRAF/KRAS multiplex test is a efficient, accurate 2 step assay. In the patients and positive and negative controls prepared, it was able to reliably diagnose KRAS and BRAF mutation status in a matter of 4 hours running time. Given the fact that this is a cost effective assay that can utilize most commonly used Real time PCR machines this test may be clinically implemented if larger clinical trials utilizing tissue samples from a larger number of colon cancer patients are conducted.

Novel potential mechanisms of acquired resistance to anti-EGFR monoclonal antibody (mAb) therapy detected in liquid biopsies (LBx) from patients (pts) with advanced colorectal cancer (CRC).

199 Background: While several studies have explored the evolving clonal profile of mCRC under stress of anti-EGFR using circulating tumor DNA (ctDNA), heterogeneity and small cohorts limit our understanding of acquired genomic alterations (GAs) associated with EGFR resistance. Methods: CRC LBx were profiled with FoundationOne Liquid CDx, which reports GAs in 324 cancer-related genes. Foundation Medicine’s (FMI) ctDNA tumor fraction (TF) is a composite algorithm prioritizing aneuploidy at higher levels and variant allele frequency (VAF) of canonical alterations at lower levels. Variants were considered clonal at VAF/TF ≥25%, subclonal at <25%. Among 4,546 non-MSI-H LBx, 2,776 (61%) had TF ≥1% and were used in this analysis. A subset of pts had treatment data in the US-based de-identified Flatiron Health-FM real-world clinico-genomic database (CGDB), originating from ~280 US cancer clinics (01/2011–03/2023). Results: 1,349 (49%) LBx had a clonal RAS GA, 107 (3.9%) had a clonal BRAF V600E, and 1,320 (48%) had no clonal RAS pathway driver GA. GAs in EGFR and MAP2K1 were significantly (FDR<0.0001) more common in LBx with no clonal RAS GA (162, 11%; 105, 7.4%) than with one (27, 2.0%; 24, 1.8%), consistent with prior reports of these GAs appearing in the setting of acquired resistance to anti-EGFR mAb. The 362 LBx with GAs in EGFR, MAP2K1, or subclonal RAS GAs (indicators of anti-EGFR mAb exposure based on a validated exposure signature) were compared to the 1349 LBx with a clonal RAS mutation. Post anti-EGFR LBx were significantly enriched in BRAF, TP53, FGFR3, PTPN11, ERRFI1, and MLH1 GAs (FDR<0.05) and all but TP53 had median VAF/TF <25%. The majority of clonal BRAF GAs were V600E (14/19, 74%) while the majority of subclonal BRAF GAs were Class 2 (36/61, 59%). Other subclonal GAs in receptor tyrosine kinases and RAS pathway components detected in these LBx included: NF1 (26), ROS1 (9), ALK (7), MET (7), RAF1 (7), FGFR2 (6), HRAS (6), MAP2K2 (6), and RET (4). 18/362 (4.4%) of the LBx had ≥2 of these resistance GAs (up to 5 per LBx). In CGDB, 14/25 (56%) LBx collected at progression had potential resistance GAs (median 2, maximum 10). LBx collected at progression (N = 25) had higher prevalence of GA in EGFR (28% vs. 2.3%, p=0.0003), KRAS (28% vs. 0%, p<0.0001), NRAS (20% vs. 0%, p=0.0002) and MAP2K1 (20% vs. 2.3%, p=0.008) than LBx collected before therapy start (N = 87). Conclusions: This study reports on the largest set of LBx from pts likely exposed to anti-EGFR published to date. LBx can provide a more comprehensive and quantitative picture of acquired resistance to anti-EGFR in CRC than tissue biopsy, including some potential novel mechanisms identified in this study. Further investigation into whether detection of resistance can inform clinical decisions about timing of anti-EGFR mAb use and rechallenge is warranted.

Prognostic and predictive biomarkers for anti-EGFR monoclonal antibody therapy in RAS wild-type metastatic colorectal cancer: a systematic review and meta-analysis

BackgroundRAS mutations affect prognosis in patients with metastatic colorectal cancer (mCRC) and have been identified as strong negative predictive markers for anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy, but many tumors containing wild-type RAS genes still do not respond to these therapies. Some additional biomarkers may have prognostic or predictive roles, but conclusions remain controversial.MethodsWe performed a meta-analysis and systematic review of randomized controlled trials comparing anti-EGFR mAb therapy with alternative therapy that investigated the prognostic and predictive impact of additional biomarkers in RAS wild-type (wt) mCRC patients. Hazard ratios (HRs) and 95% confidence intervals (CIs) for progression-free survival (PFS) and overall survival (OS) and odds ratios (ORs) for objective response rate (ORR) were calculated. The prognostic value of biomarkers was investigated by separately pooling HR and OR for different treatment groups in an individual study. The predictive value was assessed by pooling study interactions between treatment effects and biomarker subgroups.ResultsThirty publications reporting on eighteen trials were selected, including a total of 13,507 patients. In prognostic analysis, BRAF mutations were associated with poorer PFS [HRs = 3.76 (2.47–5.73) and 2.69 (1.82–3.98)] and OS [HRs = 2.66 (1.95–3.65) and 2.45 (1.55–3.88)] in both the experimental and control arms; low miR-31-3p expression appeared to have longer PFS and OS. In terms of predictive effect, a lack of response to anti-EGFR therapy was observed in patients with BRAF mutant tumors (Pinteraction < 0.01 for PFS). Patients with tumors with any mutation in the KRAS/NRAS/BRAF/PIK3CA gene also showed similar results compared with all wild-type tumors (Pinteraction for PFS, OS, and ORR were < 0.01, < 0.01 and 0.01, respectively). While low miR-31-3p expression could predict PFS (Pinteraction = 0.01) and OS (Pinteraction = 0.04) benefit. The prognostic and predictive value regarding PIK3CA mutations, PTEN mutations or deletions, EGFR, EREG/AREG, HER2, HER3, and HER4 expression remains uncertain.ConclusionsIn RAS wt mCRC patients receiving EGFR-targeted therapy, BRAF mutation is a powerful prognostic and therapy-predictive biomarker, with no effect found for PIK3CA mutation, PTEN mutation or deletion, but the combined biomarker KRAS/NRAS/BRAF/PIK3CA mutations predict resistance to anti-EGFR therapy. Low miR-31-3p expression may have positive prognostic and therapy predictive effects. Evidence on the prognostic and predictive roles of EGFR and its ligands, and HER2/3/4 is insufficient.

A phase 2 study of HLX07 as monotherapy or combination therapy in patients with locally advanced, unresectable, or metastatic esophageal squamous cell carcinoma.

4029 Background: Esophageal cancer is one of the most common cancers worldwide with esophageal squamous cell carcinoma (ESCC) being the predominant histological subtype. Most patients are diagnosed at the advanced stage, and the prognosis remains poor. About 40–84% of ESCC cases showed overexpression of EGFR which was related to shorter overall survival and disease-free survival, indicating that treatment targeting EGFR could be a new strategy. This study aimed to evaluate the efficacy and safety of HLX07, a novel recombinant humanized anti-EGFR monoclonal antibody (mAb), as monotherapy or combination therapy in patients with locally advanced, unresectable/metastatic ESCC. Methods: In this open-label, multicenter phase 2 study, patients aged 18–75 years with histologically or cytologically confirmed locally advanced, unresectable/metastatic ESCC or esophageal adenosquamous carcinoma were enrolled. Patients with no prior systemic antitumor therapy were assigned to group A and given HLX07 1000 mg plus serplulimab 200 mg (anti-PD-1 mAb) and chemotherapy (5-FU 2400 mg/m2 + cisplatin 50 mg/m2), Q2W IV. Patients who had failed first-line immuno-chemotherapy combination or at least two lines of other systemic antitumor therapy were assigned to group B and given HLX07 monotherapy (1000 mg Q2W IV). The primary endpoints were objective response rate (ORR) and progression-free survival (PFS) assessed by an independent radiological review committee and investigators per RECIST v1.1. Secondary endpoints included other efficacy measures, safety, pharmacokinetics, immunogenicity, and biomarker explorations. Results: As of February 4, 2023, 49 patients were enrolled in group A (n=30) and group B (n=19), with a median age of 64.5 and 59.0 years, respectively. 26 (86.7%) patients in group A and all patients in group B were male. The median follow-up duration was 2.9 months, and the preliminary efficacy was presented. Among the 42 efficacy evaluable patients (29 in group A and 13 in group B), investigator-assessed ORRs were 55.2% (95% CI 35.7–73.6%) and 23.1% (95% CI 5.0–53.8%) in the respective groups. Investigator-assessed median PFSs were not reached in group A and 1.5 months (95% CI 1.2–not evaluable) in group B. 15 (50.0%) patients in group A and 5 (26.3%) in group B had grade ≥3 treatment-emergent adverse events (AEs). AEs of special interest were observed in 16 (53.3%) and 11 (57.9%) patients, respectively, most commonly rash (43.3% vs 47.4%) and hypomagnesemia (33.3% vs 36.8%). No drug-related death was reported. Conclusions: The encouraging antitumor activity and manageable safety profile support further development of HLX07 as a new treatment option for patients with advanced ESCC, both in first-line and late-line settings. Clinical trial information: NCT05221658 .

Prognostic role of TP53 variants in the phase III study of FOLFIRI/cetuximab versus FOLFIRI/cetuximab followed by cetuximab (Cet) alone in first-line therapy of patients with RAS and BRAF wild-type (wt) metastatic colorectal cancer (mCRC) (ERMES study).

3544 Background: The ERMES study explored the optimal intensity of anti-EGFR-based first line therapy for RAS/BRAF wt mCRC once achieved disease control. Although the study did not demonstrate non-inferiority of maintenance with Cet alone versus standard treatment, preliminary results suggested that a strategy of de-escalation treatment with only Cet might be effective in selected patients. Conflicting results have been previously reported for the predictive and prognostic role of TP53 variants in mCRC patients receiving anti-EGFR monoclonal antibody therapy. Here we describe the role of TP53 mutations within the ERMES study. Methods: Patients with untreated RAS/BRAF wt mCRC were randomly assigned (1:1) to receive either FOLFIRI/Cet until PD/toxicity (arm A) or FOLFIRI/Cet for 8 cycles followed by Cet alone (arm B). Tumor tissue samples were tested using the Oncomine Solid Tumor DNA kit covering hot spot mutations in 22 genes, including KRAS, NRAS, BRAF and TP53. Variant calling was performed using the Variant Caller Ion Torrent suite 5.16 software. The prognostic value of TP53 variants was assessed in the intention-to-treat (ITT) patients’ population with available sequencing data. Results: Tumor tissue specimens were available for 418/593 (70.5%) patients of the ITT population. Testing failed in 29 cases, whereas KRAS/NRAS/BRAF V600E variants were found in 36/389 (9.2%) patients, who were enrolled on the basis of local testing. Progression free survival (PFS) data were not available for 4 cases. Therefore, the final population in analysis was 349 cases. TP53 variants were detected in 165/349 (47.3%) patients, of which 86/165 (52.1%) in arm A and 79/165 (47.9%) in arm B. The median PFS (mPFS) of TP53 wild type patients in the overall population was 9.4 months versus 10.4 months in the TP53 mutant subgroup (HR 0.8439; 95%CI, 0.673-1.058; p = 0.142). In arm A, the mPFS of TP53 wild type patients was 10.8 months versus 10.7 months in TP53 mutant patients, with an HR 1.053 (95%CI, 0.7582-1.462; p = 0.76). The mPFS of TP53 wild type patients in arm B was 8.3 months versus 10.2 months in the TP53 mutant cohort, with an HR 0.7 (95%CI, 0.5087-0.9657; p = 0.0298). No significant difference was observed for the overall response rate (ORR) in both arms between TP53 mutant and wild type patients. Conclusions: These preliminary data suggest a possible prognostic role of TP53 variants in RAS/BRAF wild type mCRC patients receiving first line FOLFIRI/cetuximab followed by Cet alone. Comprehensive genomic profiling is ongoing to identify genomic signatures associated with sensitivity to Cet.

Long-term outcomes of nasopharyngeal carcinoma treated with helical tomotherapy using simultaneous integrated boost technique: A 10-year result.

To evaluate the long-term survival and treatment-related toxicities of helical tomotherapy (HT) in nasopharyngeal carcinoma (NPC) patients. One hundred and ninety newly diagnosed non-metastatic NPC patients treated with HT from September 2007 to August 2012 were analyzed retrospectively. The dose at D95 prescribed was 70-74Gy, 60-62.7Gy and 52-56Gy delivered in 33 fractions to the primary gross tumor volume (pGTVnx) and positive lymph nodes (pGTVnd), the high risk planning target volume (PTV1), and the low risk planning target volume (PTV2), respectively, using simultaneous integrated boost technique. The statistical analyses were performed and late toxicities were evaluated and scored according to the Common Terminology Criteria for Adverse Events (version 3.0). The median follow-up time was 145 months. The 10-year local relapse-free survival (LRFS), nodal relapse-free survival (NRFS), distant metastasis-free survival (DMFS) and overall survival (OS) were 94%, 95%, 86%, and 77.8%; respectively. Fifty (26.3%) patients had treatment-related failures at the last follow-up visit. Distant metastasis, occurred in 25 patients, was the major failure pattern. Multivariate analysis showed that age and T stage were independent predictors of DMFS and OS, Concomitant chemotherapy improved overall survival, but anti-EGFR monoclonal antibody therapy failed. The most common late toxicities were mainly graded as 1 or 2. Helical tomotherapy with simultaneous integrated boost technique offered excellent long-term outcomes for NPC patients, with mild late treatment-related toxicities. Age and clinical stage were independent predictors of DMFS and OS. And, concurrent chemotherapy means better OS. Further prospective study is needed to confirm the superiority of this technology and to evaluate the roles of anti-EGFR monoclonal antibody treatment.

Treatment Protocols in the Efficacy and Safety of Anti-EGFR Medicines in Combination with Standard Therapy for Patients with Nasopharyngeal Cancer: A Meta-Analysis.

This study was conducted to compare the efficacy of standard therapy (radiotherapy/RT/CT) with that of antiepidermal growth factor receptor (anti-EGFR) monoclonal antibody (NPC) therapy in patients with advanced nasopharyngeal cancer. A meta-analysis was performed to meet the objective of this study. The English databases PubMed, Cochrane Library, and Web of Science were searched. The literature review compared anti-EGFR-targeted therapy with conventional therapy practices. The main outcome measure was overall survival (OS). Secondary goals were progression-free survival (PFS), locoregional recurrence-free survival (LRRFS), distant metastasis-free survival (DMFS), and adverse events (grade 3). The database search resulted in 11 studies, with a total of 4219 participants. It was found that combining an anti-EGFR regimen with conventional therapy did not enhance OS (hazard ratio [HR] = 1.18; 95%confidence interval [CI] = 0.51-2.40; p = 0.70) or PFS appreciably (HR = 0.95; 95%CI = 0.51-1.48; p = 0.88) in patients with nasopharyngeal carcinoma. While LRRFS increased considerably (HR = 0.70; 95%CI = 0.67-1.00; p = 0.01), the combined regimen did not improve DMFS (HR = 0.86; 95%CI = 0.61-1.12; p = 0.36). Treatment-related adverse events included haematological toxicity (RR = 0.2; 95%CI = 0.08-0.45; p = 0.01), cutaneous reactions (RR = 7.05; 95%CI = 2.15-23.09; p = 0.01), and mucositis (RR = 1.96; 95%CI = 1.58-2.09; p = 0.01). Individuals who have nasopharyngeal cancer do not have an increased chance of surviving until a local recurrence of their disease if they get normal therapy in addition to an anti-EGFR regimen. However, this combination does not enhance overall survival. On the other hand, this factor adds to an increase in the number of adverse effects.

REVERCEII (ACCRU-GI-1809): A randomized phase II study of regorafenib followed by anti-EGFR monoclonal antibody therapy versus the reverse sequencing for metastatic colorectal cancer patients previously treated with fluoropyrimidine, oxaliplatin and irinotecan.

TPS213 Background: Regorafenib (R) is an oral multikinase inhibitor that blocks several protein kinases involved in angiogenesis and oncogenesis; it has a survival benefit in refractory metastatic colorectal cancer (mCRC). The current standard (std) treatment in patients (pts) with RAS wildtype (WT) mCRC is sequential treatment with an anti-EGFR antibody (AEA) followed by R. However, R, which is orally administered once daily, may be more convenient and thus preferable for pts than AEA. REVERCE, a Japanese trial, demonstrated a significant 5.8 month (mo.) survival benefit with regorafenib administered prior to AEA compared to the std sequence. Based off these findings, the proposed phase II trial is to confirm the observed survival benefit from regorafenib sequencing prior to anti-EGFR monoclonal antibody therapy in REVERCE in a US patient population. Methods: REVERCEII is an Academic and Community Cancer Research United (ACCRU) network-led randomized phase II study of R (dose escalation from 80mg to 160mg based on tolerance) prior to AEA (R+AEA) compared to standard sequencing (AEA+R) in pts with refractory RAS WT mCRC. Patients are randomized 1:1 to receive R (Arm A) vs. AEA (with or without irinotecan per investigator choice) (Arm B). At the time of disease progression or intolerance, patients will receive sequential treatment until disease progression. Eligibility criteria include histologically confirmed mCRC, ECOG ≤ 2, acceptable organ function, and patients must have had prior fluoropyrimidine, oxaliplatin and irinotecan, and no prior AEA nor R. The primary objective is to compare the overall survival (OS), the primary endpoint, between evaluable patients (eligible, consented, started protocol treatment) who were randomized to R+AEA (arm A) and AEA+R (arm B). With 83 OS events, we have 87% power to detect an improvement in median OS from 9 months to 14.5 mo., assuming 1-sided significance level of 0.15, and exponential distribution. The total sample size is 124 patients. Secondary endpoints include progression-free survival, objective response, and adverse events. The total study duration is expected to be 3 years. Clinical trial information: NCT04117945. Clinical trial information: 04117945.

465P Identification of actionable alterations in primary colorectal tumors using NGS-based comprehensive genomic profiling

Increasing numbers of actionable alterations have emerged in colorectal cancer. Aside from anti-EGFR monoclonal antibody therapy for patients with KRAS, NRAS, and BRAF wild-type tumors, around 60% of patients could benefit from single or combined targeted therapies. To inform potential therapy options, a next-generation sequencing (NGS)-based comprehensive genomic profiling is essential prior treatment. In this study, we aim to identify rational targeted therapy options in colorectal cancer.

P-156 A phase Ib/II study of cetuximab and pembrolizumab in metastatic colorectal cancer

Anti-EGFR monoclonal antibody (mAb) therapy can increase EGFR-specific T cells in peripheral blood (Trivedi et al Clin Cancer Res 2016). Thus, therapeutic strategies jointly targeting the EGFR-RAS-MAPK pathway as well as block critical immune checkpoints may be of benefit for patients with metastatic colorectal cancer (mCRC). We conducted a phase Ib/II study of cetuximab, a mAb targeting EGFR, with the anti-PD-1 mAb pembrolizumab in RAS wild-type (RAS-wt) mCRC; the results of the phase Ib part have been previously reported (Boland et al ASCO GI 2018).

Molecular landscape and prognostic value of HER2 low-expression on metastatic colorectal cancer.

229 Background: HER2 dual blockade showed the promise in clinical trials in patients (pts) with HER2-positive (HER2-Pos) metastatic colorectal cancer (mCRC). A phase I/II study of a novel anti-HER2 antibody-drug conjugate (HER2-ADC) showed promising activities across different cancer subtypes including HER2-Pos mCRC, and also showed a hint of activity for low-expressing (HER2-L) mCRC, regardless of RAS mutation. However, molecular landscape and prognostic value of HER2-L on mCRC are unclear. Methods: The eligibility included pts with mCRC who had undergone surgical resection of primary tumor. Using the specimen, HER2 expression was evaluated by immunohistochemistry (IHC) and FISH. Definition of HER2 positivity was as follows; HER2-Pos as IHC 3+ or IHC 2+/FISH positive: HER2-L as IHC 2+/FISH negative or IHC 1+: Negative (HER2-Neg) as IHC 0+. NGS tests were also done. Results: Between 2005 and 2015, a total of 370 pts were analyzed; 60% were male, with a median age of 64 y/o. Fifteen pts (4%) with HER2-Pos, 21 (6%) with HER2-L, and 334 (90%) with HER2-Neg were identified. Proportion of left-sided primary tumor was similar among groups ranging from 72% to 78%. No difference was observed in terms of major clinicopathological characteristics. The proportion of co-altered RAS mutations in HER2-L was significantly higher than HER2-Pos ( p = 0.037) and similar to HER2-Neg (27% in HER2-Pos vs. 62% in HER2-L vs. 56% in HER2-Neg). As of median follow-up of 24.8 months, median overall survival (mOS) in HER2-L was significantly better than that in HER2- Pos ( p = 0.029, 18.2 months in HER2-Pos vs. 33.3 in HER2-L vs. 27.9 in HER2-Neg). In 58 pts harboring RAS wild-type who received an anti-EGFR monoclonal antibody therapy, median progression-free survival (mPFS) in HER2-L tended to be better than that in HER2- Pos, with 2.2 months in HER2-Pos, 7.8 in HER2-L, and 5.5 in HER2-Neg ( p = 0.099). Conclusions: Since HER2-L mCRC had the high prevalence of co-altered RAS mutations but showed a better prognosis and might benefit more from an anti-EGFR therapy than HES2-Pos, the HER2-L mCRC seems to have a different biological behavior from HER2-Pos in terms of molecular landscape and prognostic value on mCRC.

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

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.

KRAS and NRAS mutational gene profile of metastatic colorectal cancer patients in Jordan.

BackgroundA constitutively active RAS protein in the absence of stimulation of the epidermal growth factor receptor (EGFR) is the result of mutations in KRAS and NRAS genes. Mutations in the KRAS exon 2 and outside exon 2 have been found to predict the resistance to anti-EGFR monoclonal therapy. A substantial proportion of metastatic colorectal cancer cases (mCRC) exhibit RAS mutations outside KRAS exon 2, particularly in KRAS exon 3 and 4 and NRAS exons 2 and 3. No data about RAS mutations outside KRAS exon 2 are available for Jordanian patients with mCRC. We aim to study the molecular spectrum, frequency, and distribution pattern of KRAS and NRAS mutations in Jordanian patients with mCRC.MethodsA cohort of 190 Jordanian metastatic colorectal cancer patients were enrolled in the trial. We detected mutations in exon 2 of the KRAS and NRAS gene as well as mutations outside of exon 2 using the StripAssay technique. The KRAS StripAssay covered 29 mutations and 22 NRAS mutations.ResultsMutations were observed in 92 (48.42%) cases, and KRAS exon 2 mutations accounted for 76 cases (83.69%). KRAS G12D was the most common mutation, occurring in 18 cases, followed by KRAS G12A in 16 cases, and G12T in 13 cases. Mutations outside of KRAS exon 2 represented 16.3% of the mutated cases. Among those, 6 cases (6.48%) carried mutations in NRAS exon 2 and 3, and 10 cases (10.87%) in KRAS exon 3 and 4.ConclusionThe frequency of NRAS and KRAS mutations outside of exon 2 appears to be higher in Jordanian patients in comparison with patients from western countries. KRAS mutations outside of exon 2 should be tested routinely to identify patients who should not be treated with anti-EGFR antibodies.

Mutation Status and Immunohistochemical Correlation of KRAS, NRAS, and BRAF in 260 Chinese Colorectal and Gastric Cancers.

KRAS, NRAS and BRAF are kinases involved in the RAS-RAF-MAPK signaling pathway and also potential tumor-driven genes. Patients with KRAS/NRAS/BRAF mutations are resistant to anti-EGFR monoclonal antibody therapy. The main purpose of this study is to investigate the mutation status and distribution of KRAS/NRAS/BRAF in Chinese colorectal and gastric cancers, and to explore the histopathological changes and related immunohistochemical marker changes caused by these mutations. The mutation status of KRAS (exons 2, codon 12/13), NRAS (exons 2/3/4, codon 12/13/59/61/117/146) and BRAF (exons 15, codon 600) were detected by amplification refractory mutation system polymerase chain reaction (ARMS-PCR) in 86 colon cancer, 140 rectal cancer and 34 gastric cancer tissues. Then, the frequencies and distribution of KRAS/NRAS/BRAF mutations were described in detail. Furthermore, the relationship between KRAS/NRAS/BRAF mutations and the features of histopathological and related immunohistochemical markers were analyzed. The results showed that KRAS/NRAS/BRAF mutation rates in colon cancer were 44.2, 1.2, and 3.5%; in rectal cancer were 37.1, 4.3, and 0.7%; in gastric cancer were none, none and 2.9%. The mutation rate of KRAS in female (48.8%) is significantly higher than that of male (27.8%), and the mutation rate increased with the higher degree of differentiation. Additionally, the mutation rate of BRAF detected by ARMS-PCR (1.77%) was significantly lower than that by immunohistochemistry (4.11%). It also showed that the KRAS/NRAS/BRAF mutation status had a certain relationship with the expression of some immunohistochemical markers. This study provides more data support for clinical research on KRAS/NRAS/BRAF mutation in CRCs or gastric cancers.

An Ex Vivo Human Tumor Assay Shows Distinct Patterns of EGFR Trafficking in Squamous Cell Carcinoma Correlating to Therapeutic Outcomes

EGFR overexpression is associated with squamous cell carcinoma development. Altered endocytosis and polarization of receptor tyrosine kinases, including EGFR, affect migration and invasion in three-dimensional culture. These studies have been completed via genetic sequencing, cell line, or three-dimensional invitro and invivo murine models. Here, we describe an imaging method that allows exvivo examination of ligand-induced endocytosis of EGFR in non-dissociated human tumors. We analyzed sets of tumor samples from advanced cutaneous squamous cell carcinoma and head and neck squamous cell carcinoma, actinic keratosis, intraepidermal carcinoma, and cutaneous squamous cell carcinoma. We show that EGFR endocytosis is dysregulated in advanced SCC and correlates with anti-EGFR monoclonal antibody therapy outcomes. In actinic keratosis, intraepidermal carcinoma, and well-differentiated cutaneous squamous cell carcinoma, different patterns of epidermal growth factor ligand uptake and binding were observed at the leading edge of different dysplastic lesions, suggesting that these differences in EGFR endocytosis might influence the metastatic potential of dysplastic squamous epithelium. These studies in live exvivo human tumors confirm that endocytosis dysregulation is a physiological event in human tumors and has therapeutic implications.

EGFR protein expression of the metastatic colorectal cancer as a prognostic/predictive factor for anti-EGFR antibody therapy.

e15548Background: Anti-EGFR monoclonal antibody therapy is a standard treatment of advanced colorectal cancer. Unlike in case of other target therapies the target of anti-EGFR monoclonal antibodies...

Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology.

- To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. - The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. - Twenty-one guideline statements were established. - Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented.

Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology.

Purpose Molecular testing of colorectal cancers (CRCs) to improve patient care and outcomes of targeted and conventional therapies has been the center of many recent studies, including clinical trials. Evidence-based recommendations for the molecular testing of CRC tissues to guide epidermal growth factor receptor (EGFR) -targeted therapies and conventional chemotherapy regimens are warranted in clinical practice. The purpose of this guideline is to develop evidence-based recommendations to help establish standard molecular biomarker testing for CRC through a systematic review of the literature. Methods The American Society for Clinical Pathology (ASCP), College of American Pathologists (CAP), Association for Molecular Pathology (AMP), and the American Society of Clinical Oncology (ASCO) convened an Expert Panel to develop an evidence-based guideline to help establish standard molecular biomarker testing, guide targeted therapies, and advance personalized care for patients with CRC. A comprehensive literature search that included over 4,000 articles was conducted to gather data to inform this guideline. Results Twenty-one guideline statements (eight recommendations, 10 expert consensus opinions and three no recommendations) were established. Recommendations Evidence supports mutational testing for genes in the EGFR signaling pathway, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize molecular testing for predictive and prognostic molecular biomarkers involve selection of assays, type of specimens to be tested, timing of ordering of tests and turnaround time for testing results. Additional information is available at: www.asco.org/CRC-markers-guideline and www.asco.org/guidelineswiki.

Molecular Biomarkers for the Evaluation of Colorectal Cancer.

Objectives: To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. Methods: The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. Results: Twenty-one guideline statements were established. Conclusions: Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented.

Clinical translation of predictive markers for anti-EGFR monoclonal antibody therapy in metastatic colorectal cancer

Altered function of the epidermal growth factor receptor (EGFR) and associated mitogen-activated protein (MAP) kinase pathway plays a key role in disease progression in metastatic colorectal cancer (mCRC).