KRAS Exon Research Articles

Clinical Outcomes in Patients With Colon Cancer With Microsatellite Instability of Sporadic or Familial Origin Treated With Adjuvant FOLFOX With or Without Cetuximab: A Pooled Analysis of the PETACC8 and N0147 Trials.

The microsatellite instability (MSI) or deficient mismatch repair (dMMR) phenotype is usually regarded as a single biologic entity, given the absence of comparative analyses regarding prognosis and response to chemotherapy between sporadic and familial dMMR cancers. Patients with stage III colon cancers were randomly assigned to FOLFOX (leucovorin, fluorouracil, and oxaliplatin) with or without cetuximab in 2 large adjuvant phase III trials (N = 5,577). Among patients with MSI and KRAS exon 2 wild-type (WT) tumors, the prognostic and predictive impacts of sporadic versus familial dMMR cancers and BRAF V600E mutational status were determined. Multivariable Cox proportional hazards models were used to assess disease-free survival (DFS) by treatment arm, adjusting for age, sex, tumor grade, Eastern Cooperative Oncology Group performance status, pT/pN stage, and primary tumor location. Among patients with MSI status with complete data for dMMR mechanism analysis (n = 354), 255 (72%) had sporadic (BRAF mutation and/or MLH1 methylation) and 99 (28%) had familial tumors (BRAF WT and unmethylated MLH1 or loss of MSH2/MSH6/PMS2 protein expression). A large proportion of dMMR sporadic tumors were mutated for BRAF (n = 200). In patients treated with FOLFOX, DFS did not differ statistically by dMMR mechanism, whereas in patients treated with FOLFOX plus cetuximab, those with sporadic tumors had worse DFS than those with familial cancers (multivariable hazard ratio, 2.69; 95% CI, 1.02 to 7.08; P = .04). Considering the predictive utility, the interaction between treatment and dMMR mechanism was significant (P = .03). Furthermore, a nonsignificant trend toward a deleterious effect of adding cetuximab to FOLFOX was observed in patients with BRAF-mutant but not BRAF WT tumors. The addition of cetuximab to adjuvant FOLFOX was associated with shorter DFS in patients with sporadic dMMR colon cancer. Additional studies are needed to validate these results in metastatic disease.

RAS/RAF mutations and their associations with epigenetic alterations for distinct pathways in Vietnamese colorectal cancer

KRAS, NRAS, and BRAF are potential tumor-driven genes that are involved in the RAS/RAF/MAPK signaling pathway. RAS/RAF mutations importantly contribute to colorectal tumorigenesis since they remain the activated status of downstream pathways without regulation of the upstream EGFR signal. However, it has not been unclear how epigenetic alterations involved in colorectal tumorigenesis mediated by KRAS, NRAS, or BRAF mutations. Therefore, in this study, we investigated the frequency and distribution of KRAS/NRAS/BRAF mutations in Vietnamese colorectal cancer (CRC) and explored the relationship between genetic and epigenetic abnormalities in 156 tumors of CRC. Somatic mutations of KRAS (exon 2, codon 12/13; exon 3, codon 61), NRAS (exon 2, codon 12/13; exon 3, codon 61), and BRAF (exon 15, codon 600) was determined by Cobas® KRAS Mutation Test, Therascreen NRAS Pyro Kit and Cobas® 4800 BRAF V600 Mutation Test, respectively. Methylation status of BRCA1, MLH1, MGMT, p16, RASSF1A, and APC was detected by methylation-specific PCR. Distribution of each abnormality in clinicopathological features was also analyzed. Results showed the mutation rates of KRAS, NRAS, and BRAF were 41.0 %, 9.6 %, 8.3 % respectively, while the methylation rates of BRCA1, MLH1, MGMT, p16, RASSF1A, and APC were 16.7 %, 16.7 %, 32.7 %, 30.1 %, 30.1 %, and 37.2 % respectively. The distribution of KRAS mutation was mutually exclusive against that of NRAS (p < 0.001) and BRAF (p < 0.001) mutations in CRC. RAS/RAF mutations were more common in adenocarcinoma subtype (p = 0.020), whereas RASSF1A methylation was more frequent in mucinous adenocarcinoma subtype (p = 0.007). In addition, the frequency of having KRAS mutations was significantly higher in MGMT (p = 0.035) or RASSF1A (p = 0.043) methylated cases than in those without methylation. BRAF mutations were positively associated with MLH1 hypermethylation (p = 0.028) but were inversely associated with APC hypermethylation (p = 0.032). Overall, our results show specific interactions of genetic and epigenetic alterations and suggest the presence of independent oncogenic pathways in tumorigenesis of CRC.

Survival impact on regorafenib (REG) and trifluridine/tipiracil hydrochloride (FTD/TPI) for patients with metastatic colorectal cancer: Single institutional experience.

43 Background: Regorafenib (REG) and trifluridine/tipiracil hydrochloride (FTD/TPI) demonstrated overall survival (OS) benefit in patients (pts) with metastatic colorectal cancer (mCRC) in the CORRECT and RECOURSE phase III trials. In Japan, REG and FTD/TPI have been approved in 2013 and 2014, respectively. However, little is known about survival impact on these agents in the real-world setting. Therefore, the aim of this retrospective study was to evaluate the effects of REG and FTD/TPI in pts with mCRC. Methods: We collected medical records from consecutive 1142 pts who had been initiated with first-line chemotherapy for mCRC from 2008 to 2016 at National Cancer Center Hospital East. The survival outcomes were compared between pts from 2008 to 2011 (cohort A) and those from 2012 to 2016 (cohort B). This study excluded pts who have not been refractory or intolerant to standard chemotherapy including fluoropyrimidine, oxaliplatin, irinotecan, and anti-EGFR antibody if KRAS exon 2/ RAS wild-type tumors. Results: A total of 590 pts were analyzed (cohort A; N = 236 and cohort B; N = 354). More patients received at least one of REG or FTD/TPI in cohort B (16.1% vs. 59.9%, p &lt; 0.001). The time from initiation to end of standard chemotherapy was comparable between the two cohort (20.0 vs. 17.5 months, p = 0.266). With a median follow-up period of 34.9 months, salvage-line OS (sOS) after standard chemotherapy was significantly longer in cohort B (4.8 vs. 6.6 months, p = 0.001), while there was only a favorable trend in cohort B in terms of OS from start of first-line treatment (27.3 vs. 28.5 months, p = 0.516). In cohort B, pts who sequentially received both of REG and FTD/TPI showed longest sOS (median, both of REG and FTD/TPI; 11.3 months, either REG or FTD/TPI; 7.0 months, neither REG nor FTD/TPI; 3.1 months). Conclusions: Our study showed that REG and FTD/TPI led to prolongation of sOS in the real-world setting, indicating the importance of strategies which make all active agents available to pts with mCRC.

Analysis of early tumor shrinkage (ETS) and depth of response (DpR) in metastatic colorectal cancer (mCRC) patients (pts) treated with second-line FOLFIRI plus panitumumab (Pani) or bevacizumab (Bev): Results from a randomized phase II WJOG6210G trial.

201 Background: The WJOG 6210G trial demonstrated a similar efficacy between Pani and Bev arms at 2nd-line setting in pts with KRAS exon2 wild-type mCRC (Shitara K, et al, Cancer Sci 2016). The exploratory analyses of clinical trials evaluating 1st-line chemotherapy for mCRC have shown significant associations of ETS and DpR with prognosis; however, there is few data in 2nd-line treatment. We investigated whether ETS and DpR are associated with clinical outcomes in mCRC pts treated with 2nd-line treatment using the data of WJOG 6210G trial. Methods: ETS was defined as tumor reduction of 20% at week 8 compared to baseline in the sum of longest diameters of target lesions according to the RECIST criteria. DpR was defined as the percentage of maximum tumor shrinkage of target lesions. Fisher’s exact test and t-test were used to compare ETS and DpR between Pani and Bev arms. A Cox regression model was used to evaluate associations between ETS and progression-free survival (PFS), and overall survival (OS). The association of DpR with PFS and OS was analyzed using Spearman’s rank correlation coefficient. Results: Eighty-seven pts were evaluable for ETS and DpR (Pani, n = 41; Bev, n = 46) among 121 pts enrolled in the WJOG6210G. Pts in the Pani arm had a higher ETS rate (39.0 % vs. 8.7 %, p &lt; 0.001) and greater DpR [median: 23.7 % (range: -49.0 to 100) vs. 0 % (range: -40.7 to 61.5), p &lt; 0.001] than pts in the Bev arm. Pts with ETS achieved longer PFS [median: 10.5 vs. 5.4 m, HR: 0.40 (95% CI: 0.20-0.80), p = 0.009] and OS [median: 22.3 vs. 14.5 m, HR: 0.49 (95% CI: 0.23-0.98), p = 0.044] than pts without ETS in the Pani arm. Similarly, the Bev arm pts with ETS had longer PFS [median: 17.1 vs. 5.6 m, HR: 0.078 (95% CI: 0.004-0.38), p = 0.0002] and OS [median: 30.9 vs. 13.2 m, HR: 0.35 (95% CI: 0.083-0.99), p = 0.048]. There were moderately linear correlations of DpR with PFS and OS in both Pani (PFS: r = 0.75, p &lt; 0.001; OS: r = 0.60, p &lt; 0.001) and Bev arms (PFS: r = 0.68, p &lt; 0.001; OS: r = 0.44, p = 0.002). Conclusions: ETS and DpR were significantly associated with PFS and OS in mCRC pts treated with 2nd-line FOLFIRI in combination with Pani or Bev. Clinical trial information: UMIN000031621.

Vascular endothelial growth factor (VEGF)-D and clinical outcomes in metastatic colorectal cancer (mCRC) patients (pts) treated with second-line FOLFIRI plus bevacizumab (Bev): A biomarker study of the WJOG 6210G trial.

226 Background: The WJOG 6210G trial demonstrated a similar efficacy between FOLFIRI plus Bev and FOLFIRI plus panitumumab (Pani) at 2nd-line treatment in pts with KRAS exon2 wild-type mCRC (Shitara K, et al, Cancer Sci 2016). Recently, angiogenesis biomarker studies of clinical trials have identified VEGF-D as a potential predictor for angiogenesis inhibitors in mCRC. We evaluated whether VEGF-D level are associated with clinical outcomes in the WJOG 6210G trial. Methods: Plasma samples which were collected at pre-treatment were analyzed. VEGF-D level in plasma was measured by means of magnetic bead panel (G &amp; G SCIENCE CO., LTD.). The correlations of VEGF-D level with progression-free survival (PFS) and overall survival (OS) were evaluated by cox regression analyses. Results: Ninety-nine pts were evaluable for VEGF-D level (Bev, n = 51; Pani, n = 48) among 121 pts enrolled in the WJOG6210G. The median VEGF-D level was 441 pg/ml (range 60-1570). VEGF-D level was not affected by the period from last administration of 1st-line Bev to start of 2nd-line treatment. When the median VEGF-D level was adopted as cut-off value, pts with high VEGF-D achieved a shorter PFS and OS than pts with low VEGF-D in the Bev arm [HR: 0.58 (95%CI 0.32-1.07), p = 0.08; HR: 0.62 (95%CI 0.34-1.11), p = 0.10]. In pts with high VEGF-D, the Bev arm tended to have a shorter OS and PFS compared to Pani arm [median: 12.4 m vs. 17.5 m, HR: 1.58 (95%CI 0.87-2.88); median: 4.7 m vs. 5.5 m, HR: 1.31 (95%CI 0.72-2.40)]; whereas, comparable OS and PFS in pts with low VEGF-D [median: 18.9 m vs. 16.5 m, HR: 1.08 (95%CI 0.59-2.01); median: 5.9 m vs. 7.2 m, HR: 1.10 (95%CI 0.62-1.99)]. OS was stratified by VEGF-D level quartile, then OS of Bev arm in each quartile was compared with that of Pani arm (Table). Clinical trial information: UMIN000031621. Conclusions: Our study suggests clinically meaningful association between VEGF-D level and clinical outcomes in mCRC pts treated with 2nd-line FOLFIRI plus Bev. [Table: see text]

Biomarker Analysis in A Randomized Phase 2 Study of Panitumumab Versus Cetuximab in Colorectal Cancer (WJOG6510GTR)

Background: The randomized phase II WJOG6510G study demonstrated the non-inferiority of panitumumab to cetuximab in terms of progression-free survival (PFS) in patients with wild-type KRAS exon 2 metastatic colorectal cancer. In this study, we performed exploratory analyses of updated survival data using the KRAS exon 2 and RAS/BRAF statuses.

Relation of cetuximab-induced skin toxicity and early tumor shrinkage in metastatic colorectal cancer patients: results of the randomized phase 3 trial FIRE-3 (AIO KRK0306)

Cetuximab-induced skin toxicity (Cet-ST) is positively associated with outcome in metastatic colorectal cancer (mCRC). Besides its predictive relevance for targeted therapy, we investigated its prognostic impact with early tumor shrinkage (ETS) ≥20%, another on-treatment surrogate for clinical outcome in FIRE-3. FIRE-3 evaluated first-line FOLFIRI (folinic acid, fluorouracil and irinotecan) plus cetuximab (FOLFIRI/Cet) versus FOLFIRI plus bevacizumab (FOLFIRI/Bev) in mCRC patients with RAS-WT tumors (i.e. wild-type in KRAS and NRAS exons 2-4). Retrospective data on Cet-ST that occurred during cycles 1-3 of treatment were correlated with efficacy endpoints, including ETS. To control for guarantee-time bias, only patients who had completed three or more treatment cycles were considered. Of 199 patients treated with FOLFIRI/Cet, 181 (91.0%) completed three or more treatment cycles. A significant survival benefit of FOLFIRI/Cet over FOLFIRI/Bev was only evident in patients developing Cet-ST grade 2-3 [41.0 versus 26.6 months; hazard ratio (HR)= 0.73; 95% confidence interval (CI): 0.61-0.87; P < 0.001] compared with Cet-ST grade 0-1 (HR= 0.90; 95% CI: 0.67-1.20; P= 0.48). Regarding prognosis, Cet-ST grade 2-3 (n= 75; 41.4%), compared with Cet-ST grade 0-1 (n= 106; 58.6%), was associated with prolonged overall survival (OS; HR= 0.62; 95% CI: 0.42-0.91; P=0.01). In multivariate analysis, both Cet-ST (HR= 0.66; 95% CI: 0.50-0.87; P= 0.003) and ETS (HR= 0.55; 95% CI: 0.41-0.74; P < 0.0001) were independently prognostic for OS. Absence of both Cet-ST grade ≥2 and ETS identified a subgroup of patients with very poor prognosis (median OS 15.1 months). In FIRE-3, the addition of cetuximab to FOLFIRI was associated with superior OS compared with FOLFIRI/Bev only in patients developing Cet-ST grade ≥2. Regarding prognostic relevance, both Cet-ST and ETS were independent and early predictors of survival. The present analysis supports that a combined evaluation of on-treatment parameters such as Cet-ST and ETS may help to guide treatment of mCRC.

IMPACT OF KRAS MUTATIONS IN CLINICAL FEATURES IN COLORECTAL CANCER.

ABSTRACT Background: KRAS mutations are important events in colorectal carcinogenesis, as well as negative predictors of response to EGFR inhibitors treatment. Aim: To investigate the association of clinical-pathological features with KRAS mutations in colorectal cancer patients treated. Methods: Data from 69 patients with colorectal cancer either metastatic at diagnosis or later, were retrospectively analyzed. The direct sequencing and pyrosequencing techniques were related to KRAS exon 2. The mutation diagnosis and its type were determined. Results: KRAS mutation was identified in 43.4% of patients. The most common was c.35G>T (p.G12V), c.35G>A (p.G12D) and c.38G>A (p.G13D). No correlation was found between KRAS mutation and age (p=0.646) or gender (p=0.815). However, mutated group had higher CEA levels at admission (p=0.048) and codon 13 mutation was associated with involvement of more than one metastatic site in disease progression (p=0.029). Although there was no association between primary tumor site and mutation diagnosis (p=0.568), primary colon was associated with worse overall survival (p=0.009). Conclusion: The KRAS mutation was identified in almost half of patients. Mutated KRAS group had higher levels of CEA at admission and the mutation at codon 13 was associated with involvement of more than one metastatic site in the course of the disease. Colon disease was associated with the worst overall survival.

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.

Results of Liquid Biopsy Studies by Next Generation Sequencing in Patients with Advanced Stage Non-small Cell Lung Cancer: Single Center Experience from Turkey.

Several studies demonstrated the utility of plasma-based cell-free circulating tumor DNA (ccfDNA) in determination of mutations in non-small cell lung cancer (NSCLC). We aimed to report our results of next generation sequencing (NGS) using liquid biopsy in patients with NSCLC. Patients with advanced stage NSCLC were enrolled and their genomic profiling results were recorded. Next generation sequencing targeted panel includes 19 hot-spot genes. The plasma was separated from the peripheral blood sample and ccfDNAs were isolated for NGS. We performed genomic profiling in 100 patients (20 females and 80 males) with a median age of 59.3 (range 26-79). A second liquid biopsy was performed in eight patients who developed progressive disease after the first treatment. The study population had adenocarcinoma (AC) (n = 73), squamous cell carcinoma (SCC) (n = 14), or NSCLC-NOS (not otherwise specified) (n = 13). In the SCC group, three of 14 patients had variants on EGFR and MET genes. In the AC and NSCLC-NOS groups, 39 out of 86 patients (45.3%) had variants. The most common one was in the EGFR gene (n = 27, 31.4%) including seven mutations related to drug resistance and two were polymorphisms. Three patients had both driver and resistance mutations (EGFR T790M, n = 2; KRAS exon 2 G12S and MET exon 14 E1012K, n = 1). Fifteen patients (17.4%) had an activating EGFR mutation and eight patients (9.3%) had variants in the KRAS gene. We reported our results regarding genomic profiling related to treatment using liquid biopsy in patients with NSCLC. Advantages of this method are the non invasiveness and reproducibility.

Phase II study of gemcitabine, oxaliplatin and capecitabine in patients with KRAS exon 2 mutated biliary tract cancers

Background: Molecular markers may identify subgroups of patients with clinically distinct behavior and response to treatment. In some gastrointestinal tumors, KRAS has prognostic value and negative predictive value. This is the first prospective study to report the outcome of combination chemotherapy in biliary tract cancer patients with KRAS mutation.Methods: From 2009 to 2015, 25 patients were included from two Scandinavian centers. Main inclusion criteria were non-resectable biliary tract cancer, ECOG performance status 0–2 and tumor KRAS mutation. A bi-weekly cycle of chemotherapy was administered as gemcitabine 1000 mg/m2 and oxaliplatin 85 mg/m2 day 1, followed by 7 days of oral capecitabine 1000 mg/m2. Response evaluation was done every six treatment and the primary endpoint was the fraction with progression free survival (PFS) at 6 months. The study also included a non-preplanned analysis of circulating tumor specific DNA.Results: Chemotherapy was given for a median of 5 months (range 0–14) and among 17 patients evaluable for response, best responses were complete response (1), partial response (2), and stable disease (14). Eighteen patients had CT-verified progression, six died between evaluations and one patient is still progression-free. Median PFS was 6.8 months (95% CI 3.1–11.0) and median overall survival (OS) was 11.2 months (95% CI 6.6–14.3). The fraction with PFS at 6 months was 52% (95% CI 31–69%). Exploratory analyses found an improved survival in patients with a low level of plasma DNA.Conclusion: Pretreatment molecular characterization was feasible in BTC, but the rate of KRAS mutations was low. The study met its primary endpoint with a fraction of PFS at six months of 52%. The effect of combination chemotherapy with gemcitabine, oxaliplatin and capecitabine in this selected population was comparable to results from unselected groups with PFS and OS of 6.8 and 11.2 months, respectively. ClinicalTrials.gov NCT00779454

Abstract C13: A “mouse hospital” for preclinical testing of diagnostic and treatment modalities in pancreatic ductal adenocarcinoma (PDA)

Abstract A major challenge for cancer drug development is deciding which compounds to bring forward to clinical trials. Mouse models provide a helpful prescreening tool, but xenograft and subcutaneous models can be poorly predictive of a treatment’s efficacy against a naturally occurring tumor. Preclinical models that more accurately reproduce all the features of a tumor, including its microenvironment, may provide a more accurate readout for response to therapy. Genetically engineered mouse (GEM) models of pancreatic ductal adenocarcinoma (PDA) have been developed that exploit the fact that virtually all PDAs exhibit activating mutations in Kras (exon 12) and that over 75% have mutations in p53. These models use a pancreas-specific Cre recombinase to create mutations in Kras and p53 in the pancreatic epithelium. Tumors in these “KPC” mice recapitulate the salient clinical, histopathologic, and molecular features of the human disease. Importantly, the tumor arises in an immune-competent host. A variant of the KPC model, known as KPCY, also has a fluorescent lineage label on pancreatic epithelial cells, permitting the evaluation of a treatment’s effects on metastatic incidence or growth. Our KPC and KPCY mice are on the C57 BL/6 strain background. Alternatively, syngeneic models provide a less labor-intensive tool for evaluating the efficacy of cancer therapies in the presence of a functional immune system. The mouse hospital employs both laparotomy and a relatively new technique of ultrasound-guided injection for orthotopic implantation of a murine PDA cell line in C57 BL/6 mice. This technique is faster, less invasive, and requires less recovery time than a traditional laparotomy. The mission of the Pancreatic Cancer Mouse Hospital of the Abramson Cancer Center is to evaluate novel PDA therapies on behalf of investigators who are at the University of Pennsylvania or are collaborating with Penn investigators. Our mouse studies are designed to mimic human clinical trials, including the use of eligibility criteria and randomized treatment assignment. Ultrasound is used for screening and to measure changes in tumor in response to therapy; serial studies permit measurement of time to diagnosis and rate of tumor progression. Additional endpoints include overall survival, serum chemistries, histology, tissue collection for pharmacodynamics studies, and immune subtyping as would be performed in a human clinical trial. Citation Format: Cynthia Clendenin, Rina Sor, Deirdre McMenamin, Ben Z. Stanger, Gregory Beatty, Robert H. Vonderheide. A “mouse hospital” for preclinical testing of diagnostic and treatment modalities in pancreatic ductal adenocarcinoma (PDA) [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C13.

Clinical Impact of Circulating Tumor RAS and BRAF Mutation Dynamics in Patients With Metastatic Colorectal Cancer Treated With First-Line Chemotherapy Plus Anti-Epidermal Growth Factor Receptor Therapy.

RAS and BRAF mutations can be detected as a mechanism of acquired resistance in circulating tumor (ct) DNA in patients with metastatic colorectal cancer treated with anti-epidermal growth factor receptor therapy. RAS and BRAF mutational status was assessed in ctDNA in a baseline plasma sample and a serum sample collected at the time of the last available determination (named secondary extraction) from patients with KRAS exon 2 wild-type metastatic colorectal cancer treated in two first-line prospective biomarker-designed clinical trials (PULSE, ClinicalTrials.gov identifier: NCT01288339; and POSIBA, ClincialTrials.gov identifier: NCT01276379). Analysis of extended RAS and BRAF in tissue and plasma from 178 patients with KRAS exon 2 wild-type metastatic colorectal cancer showed a sensitivity of 64.1% and a specificity of 90%. The median overall survival (OS) of baseline patients with RAS and BRAF mutations in ctDNA was 22.3 months (95% CI, 15.6 to 29 months) and 8.9 months (95% CI, 6.3 to 11.4 months), respectively, which was significantly inferior to the median OS of 40.4 months (95% CI, 35.9 to 44.9 months) in two patients with wild-type disease (P < .001). Acquisition of RAS/BRAF mutations occurred in nine of 63 patients (14%) with progressive disease (PD; ie, blood draw within 1 month before PD or after PD) compared with six of 73 patients (8%) with no PD or blood extraction for ctDNA analysis before 1 month of PD (P = .47). Median OS in patients with RAS/BRAF acquisition was 23.9 months (95% CI, 19.7 to 27.9 months) compared with 40.6 months (95% CI, not reached to not reached) in patients who remained free of mutations (P = .016). Our results confirm that baseline RAS and BRAF testing in ctDNA discriminates survival. The emergence of RAS/BRAF mutations has limited relevance for the time to progression to anti-epidermal growth factor receptor therapy.

Molecular Alterations in Adult Histiocytic Neoplasms

Introduction Histiocytic neoplasms are rare disorders of the monocyte-macrophage-dendritic cell lineage that pose significant management challenges to the clinicians. Over the last five years, there has been emerging data on the role of MAPK/ERK pathway (RAS-RAF-MEK-ERK) in their pathogenesis. In this study, we report the molecular alterations among patients with histiocytic neoplasms seen at our institution. Methods We pursued target-capture next generation sequencing (NGS) on tissue specimen of patients with various histiocytic neoplasms- Erdheim-Chester disease (ECD), Langerhans cell histiocytosis (LCH), Rosai-Dorfman disease (RDD), histiocytic sarcoma (HS), and Langerhans cell sarcoma (LCS). We used three NGS platforms, Tempus® xO (1,714 genes), Tempus® xT (596 genes), or Foundation One® CDX (324 genes). All platforms used formalin-fixed paraffin-embedded tumor tissue specimens for detection of substitutions, insertion and deletion alterations, and copy number alterations, and gene rearrangements. The Tempus assays also utilized matched blood or saliva samples for germline DNA sequencing in addition to whole-transcriptome RNA sequencing. The tumor cellularity threshold for pursuing RNA sequencing was 30% so the test was not pursued specimens for lower cellularity. Results We included 31 cases with histiocytic neoplasms in this study. The disease distribution was as follows: ECD (n=6), LCH (n=7), RDD (n=12), ECD-RDD overlap (n=3), HS (n=2), and LCS (n=1). Among the 13 patients with ECD and LCH, BRAF-V600E mutations were found in 8 (62%) cases. We found several novel DNA alterations in this study in all disease groups: ECD (BRAFV471F), LCH (MAP2K1 in-frame deletion), and RDD (frameshift mutations of DNMT3A, SMARCA4, KMT2A, and CDC73 truncation). In one LCH patient without any DNA alterations, we found MAPK1 and MAP2K1 overexpression on RNA sequencing. We also had two cases with HS, both of which were found to have KRAS exon 3 mutations. One of these cases had concomitant JAK2V617F+ myelofibrosis and the tissue specimen for HS demonstrated TET2 and SRSF2 mutations as well. Our cohort included one patient with multifocal lymph node LCS that demonstrated NRAS and TP53 mutations, who did not respond to MEK-inhibitor therapy. Alterations in epigenetic regulators (TET2, DMNT3A, KMT2A, ASXL1) were found in 5 (16%), potentially suggesting clonal hematopoiesis in a subgroup of histiocytosis. Conclusions Histiocytic neoplasms demonstrate diverse somatic oncogenic alterations. In our study, we noted several novel alterations beyond the MAPK-ERK pathway in RDD that may point toward other potentially pathogenic mechanisms for these rare disorders. In cases where the DNA sequencing does not reveal pathogenic alterations, transcriptome analysis may aid in the diagnosis and management. OffLabel Disclosure: Trametinib use for Langerhans cell sarcoma

PNS336 NETWORK META-ANALYSIS (NMA) WITH TIME-VARYING HAZARD: A COMPARATIVE ANALYSIS OF SURVIVAL OUTCOMES

In oncology, the standard approach to NMA of survival outcomes is to assume proportional hazards (PH), i.e. to assume that the hazard ratio (HR, or treatment effect) remains constant over time. This, however, may not always hold true. The objective of this analysis was to perform a NMA with alternative methodologies and compare the results to understand how the selected method might influence survival estimates. Using a Cochrane review as a basis, the studies included in the NMA assessed progression-free and overall survival (PFS/OS) in metastatic colorectal cancer patients (KRAS exon 2 wild-type) receiving first-line epidermal growth factor receptor inhibitors. Kaplan-Meier curves were digitised, individual patient-level data (IPD) reconstructed and the PH assumption tested. The NMA was conducted with the four following methods: 1) using HRs reported in the publications; 2) considering treatment effects on shape and scale of parametric survival curves; 3) fractional polynomials; 4) restricted cubic splines. Methods 2–4 required IPD. Six randomised control trials (RCTs) were included in the analyses, of which five investigated cetuximab/chemotherapy vs chemotherapy alone and one panitumumab/chemotherapy vs chemotherapy alone. The PH assumption did not hold for PFS and/or OS in all RCTs. Substantial differences were observed in hazards, over time, with each of the four methods. Comparing the four NMA methods, differences were observed in the estimated median PFS and OS: for cetuximab/chemotherapy, the standard approach yielded median PFS and OS of 10.1 and 24.2 months, respectively, with up to a 15% variation with the alternative methods. This study highlights the importance of using an appropriate approach to model selection in survival analysis, as model selection may have a substantial impact on the estimated relative treatment effects and consequently on the long-term cost-effectiveness of health technologies.

Molecular: Genetic analysis of uterine carcinosarcomas

Abstract Background Tissue precursors of uterine carcinosarcomas remain unknown, therefore targeted therapy has not been determined. Methods This research is based on the investigation of formalin‐fixed, paraffin‐embedded tissue blocks from 17 women undergoing primary surgical treatment between 1961–2010, that were retrieved from the pathoanatomical department of the Institute of Oncology named after N.N.Petrov. In the present study, molecular investigations were applied to determine the pathogenesis of uterine carcinosarcomas. We have analyzed microsatellite instability, mutations of TP53 (exons 5-9), PTEN (exons 5, 8), K-RAS (exon 1) and loss of heterozygosity of variable loci of the chromosome 17 in epithelial and mesenchymal components of uterine carcinosarcomas. DNAs were extracted by proteinase K digestion («Fisher» US) by Herrington, C.S. & McGee, J.O. Mutations of TP53, PTEN and K-RAS were defined by SSCP with the next sequence. Highly sensitive markers BAT 26 and BAT 40 were used for analysis of microsatellite instability. Results The loss of heterozygosity of variable markers of the short arm of the 17th chromosome was detected (in 9 of 17 tumours- 52,9%). All 17 tumours were microsatellite stable. K-ras mutations were detected in 2 of 17 tumours - 11,7%. One tumour contained identical mutations in both components, other one contained mutations only in the epithelial component. Mutations in the TP53 were identified in 13 of 17 tumours - 76,5%. Five (29,4%) tumours contained identical mutations in both components, 8 (47,1%) - only in the epithelial component and 4 showed wild-type TP53. PTEN mutations were detected in 11 of 17 tumours - 64,7%. Six tumours (35,3%) contained wild-type PTEN. Tumours with PTEN mutations showed worse prospects than ones with wild-type PTEN: 1-yr overall survival - 12,5% vs 60 %, р Conclusions Uterine carcinosarcomas are the microsatellite stable tumours. This type of tumours is associated with chromosomal instability of the genome. PTEN mutations are matched with poor prognosis and low overall survival. The present study of PTEN damages may be important for targeted therapy by the PARP inhibitors. Legal entity responsible for the study Russian Medical Academy for Continuous Professional Education. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Customisation of therapeutic strategy in metastatic colorectal cancer by use of liquid biopsies: Updated results of an observational study

Abstract Background Metastatic colorectal cancer (mCRC) affects 20% of patients diagnosed with colorectal malignancy and is a major cause of cancer-related death worldwide. Epidermal growth factor receptor (EGFR) blockade with monoclonal antibodies, in combination with chemotherapy, has been shown to benefit patients with RAS wild type tumours. Our project explores real-time assessment of mCRC mutational profile, by use of two methods of circulating tumour DNA (ctDNA) analysis, and its potential to better guide therapeutic decisions. Methods Baseline plasma samples were prospectively collected from 64 patients at diagnosis of mCRC and at disease progression. Analysis of KRAS and NRAS exons 2, 3 and 4 was performed with the highly sensitive BEAMing digital polymerase chain reaction (PCR) and next-generation sequencing (NGS). In a subgroup of patients, with baseline RAS mutations, mid-treatment liquid biopsies were performed, to assess the change in ctDNA RAS mutational load as response biomarker. Mutational status of matched tumour tissue, generated with a conventional PCR-based method or NGS, was prospectively recorded for all patients. Results Preliminary results of our study, presented at the EACR-ESMO Joint Conference on Liquid Biopsies, have shown detection of RAS mutations in the ctDNA from over 50% of patients, with a satisfactory plasma-tissue concordance. An update on these data, as well as on the predictive value of ctDNA RAS mutational load dynamics, will be presented. Disease progression data, in light of baseline RAS status, will also be revisited, as almost 20% of patients have progressed on first-line treatment. In addition, the concordance of BEAMing and NGS as liquid biopsy methods is to be studied in a subgroup of patients with available plasma samples. Conclusions The larger sample size and the maturity of this updated analysis further add on the value of our work, the results of which remain in line with the literature. Our study serves as a real-life example of the potential of liquid biopsies to detect RAS mutation status changes, allowing for precision-oncology therapeutic approaches in the continuum of care of metastatic colorectal cancer patients. Legal entity responsible for the study Department of Medical Oncology, University General Hospital of Ioannina. Funding Amgen, Hellenic Society of Medical Oncology (HeSMO) and Society for Study of Clonal Heterogeneity of Neoplasia (EMEKEN). Disclosure G. Pentheroudakis: Research grant / Funding (institution): Amgen. All other authors have declared no conflicts of interest.

The extended spectrum of RAS-MAPK pathway mutations in colorectal cancer.

Current clinical guidelines recommend mutation analysis for select codons in KRAS and NRAS exons 2, 3, and 4 and BRAF V600E to guide therapy selection and prognostic stratification in advanced colorectal cancer. This study evaluates the impact of extended molecular testing on the detection of RAS-MAPK pathway mutations. Panel next-generation sequencing results of colorectal cancer specimens from 5795 individuals from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (AACR Project GENIE) were included. Mutations in RAS-MAPK pathway genes were analyzed and functionally annotated. Colorectal cancers had recurrent pathogenic pathway activating mutations in KRAS (44%), NRAS (4%), HRAS (<1%), BRAF (10%), MAP2K1 (1%), RAF1 (<1%), and PTPN11 (<1%). The proportion of colorectal cancers with pathogenic RAS pathway mutations was 37% when only KRAS codon 12 and 13 mutations were considered, 46% when also including select KRAS and NRAS exons 2, 3, and 4 mutations, 53% when including BRAF V600E mutations, and 56% when including all pathogenic mutations. Panel next-generation sequencing testing identifies additional RAS-MAPK pathway driver mutations beyond current guideline recommendations. These mutations have potential implications in treatment selection for patients with advanced colorectal cancer.

KRAS Mutations Predict Response and Outcome in Advanced Lung Adenocarcinoma Patients Receiving First-Line Bevacizumab and Platinum-Based Chemotherapy.

Bevacizumab, combined with platinum-based chemotherapy, has been widely used in the treatment of advanced-stage lung adenocarcinoma (LADC). Although KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutation is the most common genetic alteration in human LADC and its role in promoting angiogenesis has been well established, its prognostic and predictive role in the above setting remains unclear. The association between KRAS exon 2 mutational status and clinicopathological variables including progression-free survival and overall survival (PFS and OS, respectively) was retrospectively analyzed in 501 Caucasian stage IIIB-IV LADC patients receiving first-line platinum-based chemotherapy (CHT) with or without bevacizumab (BEV). EGFR (epidermal growth factor receptor)-mutant cases were excluded. Of 247 BEV/CHT and 254 CHT patients, 95 (38.5%) and 75 (29.5%) had mutations in KRAS, respectively. KRAS mutation was associated with smoking (p = 0.008) and female gender (p = 0.002) in the BEV/CHT group. We found no difference in OS between patients with KRAS-mutant versus KRAS wild-type tumors in the CHT-alone group (p = 0.6771). Notably, patients with KRAS-mutant tumors demonstrated significantly shorter PFS (p = 0.0255) and OS (p = 0.0186) in response to BEV/CHT compared to KRAS wild-type patients. KRAS mutation was an independent predictor of shorter PFS (hazard ratio, 0.597; p = 0.011) and OS (hazard ratio, 0.645; p = 0.012) in the BEV/CHT group. G12D KRAS-mutant patients receiving BEV/CHT showed significantly shorter PFS (3.7 months versus 8.27 months in the G12/13x group; p = 0.0032) and OS (7.2 months versus 16.1 months in the G12/13x group; p = 0.0144). In this single-center, retrospective study, KRAS-mutant LADC patients receiving BEV/CHT treatment exhibited inferior PFS and OS compared to those with KRAS wild-type advanced LADC. G12D mutations may define a subset of KRAS-mutant LADC patients unsuitable for antiangiogenic therapy with BEV.

Analytical validation of the Target Selector ctDNA platform featuring single copy detection sensitivity for clinically actionable EGFR, BRAF, and KRAS mutations.

BackgroundPersonalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection.MethodsThe Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days.ResultsA total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence.ConclusionsHighly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management.