KRAS/NRAS/BRAF genotyping in patients with mestastatic colorectal cancer.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 718-718 ◽  
Author(s):  
Nuria Rodriguez Salas ◽  
Jesus Miranda ◽  
Lorena Ostios ◽  
Mario Muñoz ◽  
Alberto Borobia ◽  
...  
Keyword(s):  
Kras Mutation ◽  
Logistic Model ◽  
Mutational Analysis ◽  
Array Analysis ◽  
Nras Mutation ◽  
Braf Mutations ◽  
Kras Mutations ◽  
Exon 2 ◽  
World Development ◽  
Codon 61

718 Background: mCRC is the 2º cause of death in the world. Development of targeted therapies has increased the survival. The efficacy of these drugs (such Bevacizumab and monoclonal antibodies against EGFR) depends on the use of genetic biomarkers such as KRAS and NRAS. The BRAF status acts as prognostic factor. The objective of this study was to perform a mutational analysis of KRAS/NRAS/BRAF in a cohort of 326 Spanish patients, and correlate the findings with clinical factors. Methods: We analyzed KRAS by chip-array analysis (Infiniti System, USA), NRAS using pyrosequencing on a Pyromark Q96MD instrument (Qiagen, USA) and BRAF mutations (in 80 of these samples) by means of Infiniti System (AutoGenomics, USA). Results: KRAS mutations 129/326 patients had KRAS mutation (31.2% in codon 12 and 6.7% in codon 13). The median age was 68.9, 55% male, 66.4% had left-sided tumor, 83% histologically grade 2 tumor. The pattern of spread was liver (51.1%), lung (33.3%) and peritoneum (11.6%). There were 6 types of mutation in codon 12 (p.Gly12Asp, p.Gly12Cys, p.Gly12Val, p.Gly12Arg, p.Gly12Ser, p.Gly12Ala) and 1 type in codon 13 (p.Gly13Asp). We apply a logistic model (age&sex corrected) to location, and we found association between KRAS wild type and left location, OR 1.73 [CI:1.03-2.9] p = 0.035 NRAS mutations: 13 patients had NRAS mutation (3.98%), 8 male, 92% had left-sided, 46% liver metastasis and 53.9% lung metastasis. Type of NRAS mutation: 1.6% in exon 2 (1.2% in codon 12 and 0.4% in codon 13) and 1.8% in exon 3 (0.2% in codon 59 and 1.6% in codon 61). In exon 2 we found 2 types of changes, 1 in codon 12 (p.Gly12Asp) and 1 in codon 13 (p.Gly13Arg). In exon 3 we found 5 types of changes, 1 in codon 59 (p.Ala59Thr) and 4 in codon 61 (p.Gln61Glu; p.Gln61Leu; p.Gln61Arg; p.Gln61His). BRAF mutation: 9/80 patients harboured mutations, all of them at the residue V600E. Conclusions: In our cohort 39.5% had mutations in KRAS, 3.98% in NRAS and 11% in BRAF. KRAS mutation is associated to right-sided location.

Comparing KRAS mutation testing by pyrosequencing versus allele specific primary extension methods in colorectal cancer specimens.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e14522-e14522
Author(s):  
Keeran Ravin Sampat ◽  
Nirali Patel ◽  
Allison Mary Deal ◽  
Karen E Weck ◽  
Bert H. O'Neil
Keyword(s):  
Kras Mutation ◽  
Mutational Analysis ◽  
Clinical Variable ◽  
Metastatic Colon Cancer ◽  
Kras Mutations ◽  
Mutation Status ◽  
Allele Specific ◽  
Kras Codon ◽  
Kras Mutation Status ◽  
Codon 61

e14522 Background: KRAS mutation status is an important clinical variable for targeting treatment in metastatic colon cancer. Many community practices send out mutational analysis to laboratories which utilize allele specific primer extension (ASPE) to identify mutations in only codons 12 or 13 of the KRAS gene. Our institutional molecular pathology laboratory performs pyrosequencing (PS) of KRAS codons 12, 13, and 61 which can potentially identify more KRAS mutations. We undertook this study to determine whether there were analytical differences in the results for specific mutations between these techniques. Methods: We obtained 77 sets of paraffin slides from a local practice from patients with metastatic adenocarcinoma of the colon whose KRAS mutation status had previously been analyzed by ASPE. After macrodissection for tumor enrichment, DNA was extracted from unstained paraffin slides and analyzed for KRAS codon 12, 13, and 61 mutations using the PyroMark KRAS v2.0 test. The results of KRAS testing by PS were then compared to those by ASPE. Exact 95% confidence intervals are reported. Results: None of the patients with KRAS codon 12 or 13 mutations called by ASPE (0/77, 0% [0%-4.7%]) changed to wild-type (WT) upon retesting by PS methodology. However, six patients had a discordant result between their ASPE-based testing and PS-based testing (6/77, 7.8% [2.9%-16.2%]). Two patients had differences in the amino acid (AA) at codon 12 (2/77, 2.6% [0.3%-9.1%]). Three patients were found to have a codon 61 mutation (3/77, 3.9% [0.8%-11.0%]), including two patients who were called KRAS WT by ASPE (2/30, 7% [0.8%-22%]). Finally, one patient with a call of a codon 13 mutation by ASPE changed to WT by PS (1/77, 1.3% [0.0% - 7.0%]). Conclusions: Almost 8% of patients who had KRAS testing by PS had a discrepancy in results compared with commercial ASPE testing alone. Although ASPE appears to be very accurate in detecting KRAS mutations, it missed KRAS codon 61 mutations in about 4% of the samples tested. This may have some clinical impact as we learn more about this subgroup of patients in the future.

scholarly journals IMPACT OF KRAS MUTATIONS IN CLINICAL FEATURES IN COLORECTAL CANCER

2020 ◽  
Vol 33 (3) ◽  
Author(s):  
Renato Morato ZANATTO ◽  
Gianni SANTOS ◽  
Júnea Caris OLIVEIRA ◽  
Eduardo Marcucci PRACUCHO ◽  
Adauto José Ferreira NUNES ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Overall Survival ◽  
Kras Mutation ◽  
Direct Sequencing ◽  
Colorectal Carcinogenesis ◽  
Kras Mutations ◽  
Primary Tumor Site ◽  
Exon 2 ◽  
Metastatic Site ◽  
Colorectal Cancer Patients

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.

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

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1514 ◽  
Author(s):  
Ghimessy ◽  
Gellert ◽  
Schlegl ◽  
Hegedus ◽  
Raso ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Kras Mutation ◽  
Antiangiogenic Therapy ◽  
Hazard Ratio ◽  
Wild Type ◽  
First Line ◽  
Kras Mutations ◽  
Exon 2 ◽  
Mutational Status ◽  
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.

Smoking history and frequency of somatic KRAS mutations in adenocarcinoma of the lung

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7573-7573
Author(s):  
V. A. Miller ◽  
G. J. Riely ◽  
M. G. Kris ◽  
D. Rosenbaum ◽  
J. Marks ◽  
...  
Keyword(s):  
Cigarette Smoking ◽  
Mutational Analysis ◽  
Smoking History ◽  
Egfr Mutations ◽  
Molecular Testing ◽  
Kras Mutations ◽  
Exon 2 ◽  
Significant Difference ◽  
Never Smokers ◽  
Lung Adenocarcinomas

7573 Background: Somatic mutations in the epidermal growth factor receptor (EGFR) gene are more common in patients with adenocarcinoma, especially those who smoked < 15 pack years (py). KRAS mutations are found in ∼25% of lung adenocarcinomas, most commonly in codons 12 and 13 of exon 2 (∼85%) and have been associated with poor prognosis in resected disease [Winton NEJM 2005] and resistance to EGFR tyrosine kinase inhibitors [Pao PLoS Med 2005]. KRAS mutations are uncommon in non-small cell lung cancer histologies other than adenocarcinoma. We sought to determine the association between quantitative measures of cigarette smoking and presence of KRAS mutations in lung adenocarcinomas. Methods: Standard direct sequencing techniques were used to identify KRAS codon 12 and 13 mutations in lung adenocarcinoma specimens from surgical resections between 2001 and 2006 and tumor specimens sent for KRAS molecular analysis in 2006. Surgical specimens were obtained from an institutional tumor bank. Detailed smoking history (age at first cigarette, packs per day, years smoked, years since quitting smoking) was obtained from the medical record and a patient-completed smoking questionnaire. Results: KRAS mutational analysis was performed on 408 lung adenocarcinomas from 242 women and 166 men. Median age was 68 (range 33–89). KRAS mutations were present in 19% (78/408, 95% CI 15 to 23%). The frequency of KRAS mutation was not associated with age or gender. The presence of KRAS mutations was not related to smoking history with 15% (9/61) of never smokers having KRAS mutations compared with 19% (51/275) of former smokers. When compared with never smokers, there was no significant difference in frequency of KRAS mutations for tumors from patients with 1–5 py (5%, p=0.44), 6- 10 py (12%, p=0.99), 11–15 py (25%, p=0.45), 16–25 py (16%, p=0.99), 26–50 py (25%, p=0.129), 51–75 py (20%, p=0.48), >75 py (20%, p=0.47) history of cigarette smoking. Conclusions: While the incidence of EGFR mutations has a strong inverse relationship with the amount of cigarettes smoked, allowing the selective molecular testing for EGFR mutations, the frequency of KRAS mutations cannot be predicted by age, gender, or smoking history. KRAS mutational analysis of all adenocarcinomas is required to reliably identify patients with KRAS mutations. No significant financial relationships to disclose.

A phase II trial of gemcitabine, irinotecan, and panitumumab in advanced cholangiocarcinoma, with correlative analysis of EGFR, KRAS, and BRAF: An interim report.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 4111-4111
Author(s):  
Davendra Sohal ◽  
Ursina R. Teitelbaum ◽  
Takeshi Uehara ◽  
Kristine Mykulowycz ◽  
Christopher D. Watt ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Growth Factor Receptor ◽  
Progression Free Survival ◽  
Tumor Stage ◽  
Primary Objective ◽  
Response To Therapy ◽  
Braf V600e ◽  
Braf Mutations ◽  
Kras Mutations ◽  
Tissue Specimens

4111 Background: Cholangiocarcinoma is an aggressive neoplasm. Current chemotherapy approaches achieve modest results. The epidermal growth factor receptor (EGFR) pathway appears to be associated with tumor stage, prognosis and response to therapy. This trial was designed to evaluate the tolerability and efficacy of the combination of panitumumab, a monoclonal anti-EGFR antibody, with gemcitabine and irinotecan, in patients with advanced cholangiocarcinoma. Molecular analysis of EGFR pathway genes was planned as well. Methods: Patients with advanced (unresectable or metastatic) cholangiocarcinoma, ECOG PS 0-2, and adequate liver, kidney and bone marrow function were treated with panitumumab (9 mg/kg) on day 1, and gemcitabine (1000 mg/m2) and irinotecan (100 mg/m2) on days 1 and 8 of a 21-day cycle. Tissue specimens were collected at diagnosis for correlative molecular analyses. Primary objective is to evaluate the 5-month progression-free survival (PFS) rate. Secondary objectives include overall response rate (ORR), overall survival (OS) and toxicity of the combination. Mutational analysis of EGFR (del 19; 858), KRAS (codons 12, 13) and BRAF (V600E) was done on samples with adequate material for testing. Results: There have been 26 (of planned 42) patients recruited to the study. A median of 6 (0-30) cycles were administered. There were no treatment related deaths. The most common gr 3 or higher toxicities were neutropenia (10 pts, 38%), thrombocytopenia (10 pts, 38%), skin rash (10 pts, 38%) and diarrhea (3 pts, 12%). During the study, there were 3 CR, 6 PR, 10 SD (disease control rate of 90%), and 2 PD (by RECIST) in 21 evaluable pts. Two pts went on to have surgical resection. Median OS is 12.7 months. Of 13 testable samples, no EGFR or BRAF mutations were identified; however, there were 7 KRAS mutations. Retrospective analysis showed no difference in OS by KRAS mutation status. Conclusions: Interim evaluation of this ongoing study showed encouraging tolerability and efficacy of this regimen. Several patients have KRAS mutations; there appears to be no association with response, however. The pre-specified efficacy criteria to continue enrollment were met.

Expanded KRAS and NRAS assays increase detection of mutations that predict for resistance to therapy in colorectal cancer patients.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 519-519
Author(s):  
Garrett Larson ◽  
Anna Israyelyan ◽  
Heinz-Josef Lenz ◽  
Stephanie H. Astrow
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
Targeted Therapies ◽  
Kinase Inhibitor ◽  
Meta Analysis ◽  
Screening Strategy ◽  
Nras Mutation ◽  
Braf Mutations ◽  
Exon 2 ◽  
Exon 4

519 Background: The selection of targeted therapies is guided by the analysis of somatic mutations. The identification of RAS activating mutations can be used to examine tyrosine kinase inhibitor therapeutic eligibility and prognosis. Beyond known mutations in RAS exon 2 (codons 12 and 13), the identification of additional mutations in RAS exons 3 and 4 (codons 61, 117 and 146) also predict for resistance to EGFR therapy in colorectal cancer (CRC). Meta-analysis supports screening for these additional mutations in any screening strategy prior to administration of EGFR mAb therapy in metastatic CRC patients (Sorich, MJ, et al., Ann Oncol, Aug 12, 2014). Methods: We expanded our existing allele-specific KRAS and sequencing-based NRAS assays to include codons 61, 117, and 146 and analytically validated these assays to CAP/CLIA standards. DNA, from microdissected colon tumor tissue that was wild-type for RAS exon 2, was tested for exon 3 and 4 mutations and included over 15 additional mutations. Results: Forty-two (9.1%) samples were identified as bearing either an exon 3 or 4 KRAS mutation amongst 461 colon cancer specimens. Exon 4 codon 146 mutations were more prevalent than three of the commonly screened exon 2 mutations: G12A, G12R, and G12S. Five (1.8%) of samples were identified as carrying exon 3 or 4 NRAS mutations amongst 272 colon cancer specimens. This included a single codon 146 mutation in exon 4. As is seen with exon 2, RAS mutations at exons 3 and 4 were mutually exclusive of activating BRAF mutations with ~10% of patients harboring V600E. The collection of additional data studying KRAS and NRAS mutation status is currently ongoing. Conclusions: The KRAS expanded coverage contributed an additional 5.5% to overall burden of specimens bearing mutations. The NRAS expanded coverage contributed an additional 1.8% to the mutational burden. These analyses in clinical cohorts support the observations made in a trial population (Douillard JV, et al. NEJM 369:1023-34, 2013). The expanded RAS coverage identifies additional patients unlikely to respond to EGFR-targeted therapies that would otherwise have been assessed as “no mutation detected” in using assays restricted to RAS exon 2.

scholarly journals Frequency and Spectrum of KRAS Mutations in Moroccan Patients with Lung Adenocarcinoma

ISRN Oncology ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-4 ◽  
Author(s):  
Ibrahim Elghissassi ◽  
Hanane Inrhaoun ◽  
Anwar Boukir ◽  
Fouad Kettani ◽  
Lamia Gamra ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Kras Mutation ◽  
Mutation Frequency ◽  
Asian Population ◽  
Wild Type ◽  
North African ◽  
Kras Mutations ◽  
Exon 2 ◽  
Lung Adenocarcinomas ◽  
Moroccan Patients

Background. In lung adenocarcinoma, the frequency of KRAS mutations is ethnicity dependent with a higher proportion in African Americans and white Caucasians than in Asians. The prevalence of these mutations among North Africans patients is unknown. The objective of this study was to report the frequency and spectrum of KRAS mutations in a group of Moroccan lung adenocarcinoma patients. Methods. Tumor specimens from 117 Moroccan patients with lung adenocarcinoma were selected to determine frequency and spectrum of KRAS mutations. KRAS mutations in codons 12 and 13 of exon 2 were analyzed using conventional DNA sequencing. Results. The overall frequency of the KRAS mutations was 9% (11/117). In the population with KRAS mutations, there was a trend towards more male (P=0.06) and more smokers (P=0.08) compared to patients with wild type KRAS. KRAS mutations were located at codon 12 in 10 out of 11 patients (91%). The G12C mutation was the most frequent KRAS mutation (73%). Conclusion. This is the first study to date examining the frequency and spectrum of KRAS mutations in lung adenocarcinomas in North African and Arab populations. KRAS mutation frequency in Moroccan patients was comparable with the frequency observed in East-Asian population. KRAS mutations are more likely observed in males and smokers and to be transversions. Further studies, in larger numbers of patients, are needed to confirm these findings.

Does multiple mutational analysis of the EGFR pathway have a prognostic role in advanced colorectal cancer (CRC)?

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 3612-3612
Author(s):  
Luisa Foltran ◽  
Giuseppe Aprile ◽  
Giovanna De Maglio ◽  
Federica Edith Pisa ◽  
Mariaelena Casagrande ◽  
...  
Keyword(s):  
Braf Mutation ◽  
Advanced Colorectal Cancer ◽  
Mutational Analysis ◽  
Wild Type ◽  
Prognostic Role ◽  
Braf Mutations ◽  
Kras Mutations ◽  
Pik3ca Mutations ◽  
Negative Prognostic Factor ◽  
Impact On Survival

3612 Background: BRAF mutation is widely recognised as a strong negative prognostic factor in advanced CRC, while the prognostic value of KRAS mutations in codons 12-13 remains controversial. Exploring mutations in other downstream components of the EGFR pathway may have an impact on survival. Methods: A consecutive cohort of 201 metastatic CRC patients treated with systemic chemotherapy were analysed for KRAS (12-13-61-146), BRAF, PIK3CA and NRAS genotypes by pyrosequencing on PyroMarkTMQ96 ID instrument (Qiagen, Germany) with commercially available kits Anti-EGFR MoAb response (Diatech Pharmacogenetics, Italy). Accurate microdissection guaranteed more than 70% of cancer cells for each sample. For the purpose of the survival analysis 4 categories were created: (1) KRAS mutated (codons 12-13 only); (2) BRAF mutated; (3) any of KRAS codons 61-146, PIK3CA or NRAS mutations; (4) all-wild type. Log-rank and Cox proportional tests were applied for statistical analysis. Results: KRAS mutations were present in 96 (47.8%) patients: 86 (42.8%) were in codons 12-13. BRAF mutations were found in 11 (5.5%) samples while PIK3CA and NRAS in 33 (16.4%) and 7 (3.5%), respectively. All mutations were mutually exclusive except for 24 (11.9%) patients with concomitant KRAS/PIK3CA mutations. Median survivals for different categories are shown. Patients harbouring BRAF mutation had the worst outcome (p=0.0006). Mutations of any codon of KRAS (12-13-61-146) also negatively impacted on survival (p=0.026), while the all wild-type (KRAS/BRAF/PIK3CA/NRAS) patients had the longest survival (p=0.002). Conclusions: This study suggests the usefulness for early molecular profiling for advanced CRC patients. Mutational analysis of all EGFR pathway components may identify different prognostic subgroups. This information may drive treatment selection in clinical practice and stratification in clinical trials. [Table: see text]

Evolution of Sub-Clones with KRAS Mutations In Pediatric Patients with MLL-AF4 Rearrangements

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2454-2454
Author(s):  
Silvia Bresolin ◽  
Emanuela Giarin ◽  
Luca Trentin ◽  
Lueder Hinrich Meyer ◽  
Giuseppe Basso ◽  
...  
Keyword(s):  
Mouse Model ◽  
Kras Mutation ◽  
Lymphoblastic Leukemia ◽  
Mutation Screening ◽  
Selective Advantage ◽  
Human Leukemia ◽  
Kras Mutations ◽  
Specific Patient ◽  
Exon 2 ◽  
Ras Genes

Abstract Abstract 2454 Mixed Lineage Leukemia (MLL) gene rearrangements are common genetic translocations occurring in human leukemia. Numerous MLL-partner genes have been identified so far; of these MLL-AF4 was found to characterize the largest subgroup of fusion proteins in pediatric acute lymphoblastic leukemia (ALL). A recent study reported that mutations in kras accelerate leukemo-lymphogenesis in a MLL-AF4 transgenic mouse model. Activating mutations in RAS genes, frequently occuring in codons 12, 13 and 61, prevent the hydrolysis of RAS-GTP and result in the constitutive activation of the RAS proteins enhancing cell survival and proliferation. Mutations in RAS genes were found in several types of human cancers, including leukemia and ALL with MLL rearrangements. We analyzed the presence of KRAS mutations in pediatric MLL-AF4 rearranged ALL samples at diagnosis and relapse with the aim to establish the frequency of KRAS mutations in MLL-AF4 leukemia and to tentatively associate RAS mutations to the leukemogenic process. In this study 40 ALL patients were included (23 infant and 17 non infant); of these, 14 pairs at diagnosis and relapse, 22 at diagnosis only and 4 samples at relapse only. The study was approved by the institutional ethical committee and informed consent was obtained in accordance with the Declaration of Helsinki. Mutation screening was performed for exon 2 and exon 3 of KRAS by 454 technology (Roche Applied Science) with GS Junior Sequencing Instruments; to recognize each specific patient, primer pairs included a 10-base molecular identifier barcode sequence (MID). We generated amplicon ultra-deep sequencing of the 2 amplicons with a median coverage per amplicon of 1800 reads per sample. Twenty patients (55,6%) out of 36 with t(4;11) at diagnosis harbored KRAS mutations (range of penetrance of 0,4–29,7% for mutated sequences). Interestingly, in some patients more than one clone with different mutations at difference penetrance in KRAS codons were present. Of the 18 relapse samples 4 (22%) harbor KRAS mutations (penetrance of 1.3–38% of reads). Sanger sequencing was used to validate the presence of mutations in patients with more of 20% of penetrance identified with 454 technology. To evaluate the role of KRAS mutations in the leukemogenic process we analyzed matched paired diagnostic and relapse samples; in patients with mutations at diagnosis, 3 distinct situations were identified in the relapsed samples, pointing different changes between diagnosis and relapse: (1) presence of KRAS mutations in a specific codon at diagnosis and disappearance of this clone at relapse (6 of 9 cases, 67%); (2) decrease of the penetrance of a mutation between diagnosis and relapse and appearance of a new different mutation at relapse (2 of 9 cases, 22%); (3) clonal evolution of the same mutation and increase of the percentage of the mutated sequence from diagnosis to relapse (1 of 9 cases, 11%). Apparently, patients with MLL-AF4 rearrangements at diagnosis and relapse presented with subclones and during the relapse phase previous clones disappeared while new clones appeared and only in one case we can assume a selective advantage of the clone with the KRAS mutation. To further explore the propagation of KRAS mutated subclones in MLL-AF4, we transplanted 2 patients at diagnosis into a NOD/SCID xenograft mouse model. Xenografted samples obtained from leukemia bearing mice transplanted further onto subsequent recipients in serial passages. KRAS mutations were analyzed for each of the passages as mentioned above. One of the diagnostic samples used for transplantation harbored KRAS G12S mutation. The human cells of this patient isolated after the first passage in the mouse carried the same mutation of the diagnosis but with a very low penetration, suggesting a minority clone that decreased further in the second passage. No trace of mutations was found in the third passage. The other diagnostic sample for which serial transplanted samples were analyzed had no KRAS mutation at diagnosis and no new mutations appeared during further passages in the mice. The disappearance of the mutated KRAS subclone during passages in the recipient mice seems to reflect the negative selection for mutated clones observed in patients between diagnosis and relapse. Overall, these data showed that KRAS mutations are frequently occurring in MLL-AF4 leukemias at diagnosis however these mutations do not seem to add a selective advantage to the blast cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clinical characteristics and prognostic value of the KRAS mutation in Chinese colorectal cancer patients

2021 ◽  
pp. 172460082110171
Author(s):  
Ye Yuan ◽  
Yingting Liu ◽  
Ye Wu ◽  
Junling Zhang ◽  
Chunti Shen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Overall Survival ◽  
Cancer Patients ◽  
High Frequency ◽  
Kras Mutation ◽  
Chinese Patients ◽  
Wild Type ◽  
Kras Mutations ◽  
Exon 2 ◽  
Colorectal Cancer Patients

Background: The KRAS mutations are high-frequency somatic mutations found in colorectal cancer patients from Western and Asian countries however, with the exception of exon 2 of KRAS, other prevalence and prognostic values have not been adequately assessed in Asian patients. The aim of this study was to determine the mutation frequencies of whole exon mutations of KRAS in Chinese colorectal cancer patients and to investigate their impact on prognosis. Methods: A total of 7189 tumor tissue samples (iCohort) were subjected to next-generation sequencing for detection of KRAS mutations. All pathologic or likely pathologic mutations of KRAS were considered. In addition, clinical features and prognostic dates were collected from 145 patients at The Third Affiliated Hospital of Soochow University, China (sCohort) and used droplet digital™ polymerase chain reaction to detect KRAS mutations. Results: In the iCohort, 2706 patients (37.6%) were confirmed harboring KRAS mutations. The most frequent of these mutations were G12D (32.19%), G12V (17.96%), and G13D (17.59%). In the sCohort, 51 colorectal cancer patients (35.17%) had KRAS mutations, among which KRAS G12D (64.71%), G13D (29.41%), and G14D (3.92%) were high-frequency. The KRAS mutations were associated with shorter median overall survival than wild-type tumors (69 vs. 55 months; HR 1.80; 95% Cl 1.22, 2.64; P=0.0003). In the Cox multivariate analysis, age (HR 1.562; 95% Cl 1.10, 2.22; P=0.013), tumor differentiation (HR 0.417; 95% Cl 0.19, 0.90; P=0.026), and KRAS mutation (HR 1.897; 95% Cl 0.19, 0.90; P=0.001) remained independent predictors of shorter overall survival. Among the common KRAS mutations, G12D was significantly associated with shorter overall survival (HR 2.17; 95% Cl 1.31, 3.58; P < 0.0001) compared with KRAS wild-type patients. Conclusions: Our findings indicate that KRAS genes are frequently mutated, and over 30% harbored the KRAS G12D mutation subtype. We found that the KRAS G12D mutation is associated with inferior survival and is a biomarker of poor prognosis in Chinese patients. Our data emphasize the importance of molecular features in colorectal cancer patients, which could potentially be improved by G12D-specific related inhibitors.

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