Frequency Of KRAS Mutations Research Articles

A Duplex CRISPR-Cas9 Ribonucleoprotein Nanomedicine for Colorectal Cancer Gene Therapy.

Based on the high frequency of concurrent adenomatous polyposis coli (APC) and KRAS mutations and their strong cooperative interaction in human colorectal cancer (CRC) promotion, we herein develop a CRISPR-Cas9-based genome-editing nanomedicine to target both APC and KRAS mutations for the treatment of CRC. To this end, a hyaluronic acid (HA)-decorated phenylboronic dendrimer (HAPD) was designed for the targeted delivery of Cas9 ribonucleoprotein (RNP), by which both APC and KRAS genetic mutations harboring in CRC cells can be synergistically disrupted. Systemic administration of Cas9 RNP targeting APC and KRAS enabled by HAPD significantly inhibits tumor growth on xenografted and orthotopic CRC mouse models and also greatly prevents CRC-induced liver metastasis and lung metastasis. Thus, this duplex genome-editing system provides a promising gene therapy strategy for the treatment of human CRC and can be extended to other types of cancers with activated Wnt/β-catenin and RAS/extracellular signal-regulated kinase (ERK) pathways.

OAB-032: Targeting GCK in RAS-mutant multiple myeloma offer a promising therapeutic approach

<h3>Background</h3> Next generation sequencing revealed frequent mutations of NRAS, KRAS or BRAF in up to 50% of newly diagnosed multiple myeloma (MM) patients. <h3>Methods</h3> Specific K- or N-RAS knockdowns led to strong decrease in MM cell viability if harbored the respective oncogenic isoform, but minimal effects were observed if only the wild-type isoform was present, presenting RAS signaling as the key driving oncogenes in the mutation bearing patients. Unfortunately, no inhibitor for RAS mutation is available for clinical use in MM. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK) is an upstream activator in the MAPK pathway. <h3>Results</h3> Our data showed that GCK (MAP4K2) knockdown in MM cells induced MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. Moreover, GCK silencing only led to significantly decreased GCK mRNA, however, did not affect IKZF1 expressions at mRNA level. We next tested the effects of GCK inhibitor TL4-12 on MM. Given the fact that RASMut MM cells have higher GCK expression level and are more sensitive to GCK knockdown, we compared mutated RAS MM cells with wild-type RAS MM cells. TL4-12 significantly inhibited the growth of mutated RAS MM cells, with IC50 5-10 fold lower compared to wild-type RAS MM cell lines. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Our data showed that GCK knockdown also downregulates IKZF1 protein level, indicating that IKZF1 is under the regulation of GCK. Concomitant treatment with proteasome inhibitor PS-341 blocked TL4-12 induced IKZF1 downregulation, suggesting that inhibition of GCK induces IKZF1 protein degradation. However, since IMiDs-resistant RPMI-8226 and JJN3 MM cells are sensitive to GCK inhibition, we hypothesized that the IKZF1 degradation mechanism induced by GCK inhibition is different from IMiDs and independent of CRBN. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells and examined the response to IMiDs and GCK inhibitor. CRBN silencing in H929 cells resulted in lenalidomide resistance. In contrast, CRBN silencing failed to rescue H929 MM from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. <h3>Conclusions</h3> Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibition downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance.

Mucinous Cystic Lesions of the Pancreas an EUS-FNA-based Study with ClinicoPathological Follow Up

Background: Mucinous cystic lesions of pancreas harbor a pre-malignant potential thus necessitating their distinction from the non-mucinous ones. To make this distinction, EUS-FNA cytology along with cyst fluid CEA and amylase levels are utilized in addition to endoscopic and radiological findings. Evaluation of K-ras mutations has emerged as a useful adjunct for the evaluation of mucinous cystic neoplasms of the pancreas. Aim: We aimed to study mucinous cystic lesions of the pancreas diagnosed on EUS-FNA cytology, in conjunction with cyst fluid CEA and amylase levels and the frequency of K-ras mutation in a cohort of patients seen at the largest cancer hospital in our country. Materials and Methods: After approval from the institutional review board, all the cases of mucinous cystic lesions of pancreas evaluated between July 2005 and August 2019 were reviewed. Patient data, including age, gender, endoscopic and radiological findings, cytological and/or histological diagnosis, cyst fluid CEA, and amylase levels were collected. Results: Twenty-three patients enrolled in the study demonstrated an equal gender distribution with a mean age of 67.4 years. The sensitivity of EUS-FNA for mucinous cystic lesions of the pancreas was 84.6%. Cyst fluid CEA levels were elevated in some MCNs but not IPMNs resulting in a sensitivity of 37.5%. The specificity of cyst fluid amylase was 90%. K-ras mutation was found to have a sensitivity and specificity of 50% and 100% respectively, for mucinous lesions of the pancreas. Conclusion: EUS-FNA is a useful technique for evaluation of pancreatic cystic lesions, especially since cytological diagnosis can be augmented by cyst fluid CEA and amylase levels. K-ras analysis can add further to the diagnostic utility of EUS-FNA

A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer

KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods. This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively. KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method. These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.

Genomic and evolutionary classification of lung cancer in never smokers.

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.

Recent updates in thoracic SMARCA4-deficient undifferentiated tumor.

Germline inactivating mutations in SMARCA4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4) gene encoding for BRG1 (Brahma related gene-1) are the molecular drivers in small cell carcinoma of ovary, hypercalcemic type (SCCOHT) and in malignant rhabdoid tumors (MRT) that occur in the context of rhabdoid tumor predisposition syndrome-type 2. Somatic SMARCA4 mutations and/or loss of BRG1 have been identified in a variety of adult-onset epithelial and mesenchymal neoplasms. Among thoracic tumors, these include subsets of smoking-related non-small cell lung carcinoma (NSCLC) and a relatively rare, newly recognised tumor entity: thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Less than 100 cases of SMARCA4-UT have been reported to date. They present as large compressive and infiltrative mediastinal, lung and/or pleural masses in middle-aged male smokers. They are undifferentiated tumors composed of sheets of small/epithelioid and/or rhabdoid tumor cells variably expressing epithelial markers and consistently showing loss of BRG1 and the closely related protein, Brahma (BRM). Frequent expression of stem cell markers (SOX2, CD34, SALL4) is noted. Despite gene expression profiles similar to MRTs and SCCOHT, they show striking genomic overlap with SMARCA4-mutant NSCLC with frequent TP53, STK11, KEAP1, and KRAS mutations, high tumor mutation burden (TMB), and presence of smoking related molecular signatures in tumor cells. SMARCA4-UT show uniformly poor survival and are irresponsive to conventional therapies. Immunotherapy responses are variable but promising, although PDL1 expression appears to be of poor predictive value. Drugs exploiting genetic and epigenetic mechanisms of SMARCA4 antagonism hold promise for future targeted therapies.

The Prognostic Effect of KRAS Mutations in Non-Small Cell Lung Carcinoma Revisited: A Norwegian Multicentre Study.

Simple SummaryIn this multicentre study of 1117 patients with stage I–IV non-squamous non-small cell lung carcinoma (NSCLC), we investigated associations between KRAS and clinical characteristics and survival. We investigated survival among the following groups of patients: those with no KRAS mutations (wild type) versus those with mutated tumours, those with KRAS wild type versus KRAS G12C versus KRAS non-G12C mutated tumours and among patients with different KRAS mutation subtypes. We also grouped KRAS mutated patients according to mutation preference for the Raf, PI3K/Akt and RalGDS/Ral intracellular signalling pathways and investigated whether there were differences in survival according to their preference for these pathways. We found that the proportion of KRAS mutated patients and frequency of KRAS mutation subtypes in our study is consistent with other studies of non-Asian patients with NSCLC. In multivariable analyses, we found no significant differences in the time to disease progression or overall survival within any of the analysed groups.Background: due to emerging therapeutics targeting KRAS G12C and previous reports with conflicting results regarding the prognostic impact of KRAS and KRAS G12C in non-small cell lung cancer (NSCLC), we aimed to investigate the frequency of KRAS mutations and their associations with clinical characteristics and outcome. Since mutation subtypes have different preferences for downstream pathways, we also aimed to investigate whether there were differences in outcome according to mutation preference for the Raf, PI3K/Akt, or RalGDS/Ral pathways. Methods: retrospectively, clinicopathological data from 1233 stage I–IV non-squamous NSCLC patients with known KRAS status were reviewed. KRAS’ associations with clinical characteristics were analysed. Progression free survival (PFS) and overall survival (OS) were assessed for the following groups: KRAS wild type (wt) versus mutated, KRAS wt versus KRAS G12C versus KRAS non-G12C, among KRAS mutation subtypes and among mutation subtypes grouped according to preference for downstream pathways. Results: a total of 1117 patients were included; 38% had KRAS mutated tumours, 17% had G12C. Among KRAS mutated, G12C was the most frequent mutation in former/current smokers (45%) and G12D in never smokers (46%). There were no significant differences in survival according to KRAS status, G12C status, among KRAS mutation subtypes or mutation preference for downstream pathways. Conclusion: KRAS status or KRAS mutation subtype did not have any significant influence on PFS or OS.

Prospective Evaluation of Radiotherapy-Induced Immunologic and Genetic Effects in Colorectal Cancer Oligo-Metastatic Patients with Lung-Limited Disease: The PRELUDE-1 Study.

Simple SummaryThe management of advanced colorectal cancer (CRC) has been greatly improved with integrated strategies including stereotactic radiation therapy (SRT). It is a safe and effective option, particularly in oligo-metastatic (om) CRC patients. Interestingly, it has been demonstrated that SRT can induce regression of tumors in non-irradiated regions (“abscopal effect”) through stimulation of anti-tumor immune effects (“radiation-induced immunity”). We have recently shown that lung-limited omCRC is characterized by regression of tumor clones bearing specific key driver gene mutations. The aim of the PRELUDE-1 study is to assess the genetic and immunologic evolutions on tumor cancer/host cells induced by SRT in lung-limited omCRC through liquid biopsies and Next Generation Sequencing of tumor exome, HLA repertoire assessment, peripheral immune cells, and cytokine dynamics characterizations. An important secondary objective is the first prospective evaluation of the abscopal effect. The PRELUDE-1 results will help to identify subsets of patients more prone to show the abscopal effect. The PRELUDE-1 trial was registered into the clinicaltrials.gov registry on 22 April 2021, with identifier NCT04854213.Background: in recent years, the management of advanced colorectal cancer (CRC) has been greatly improved with integrated strategies including stereotactic radiation therapy (SRT). The administration of SRT has been demonstrated, particularly in oligo-metastatic (om) CRC, to be a safe and effective option. Interestingly, it has been demonstrated that SRT can induce regression of tumors in non-irradiated regions (“abscopal effect”) through stimulation of anti-tumor immune effects (“radiation-induced immunity”). We have recently shown that lung-limited omCRC is characterized by regression of tumor clones bearing specific key driver gene mutations. Aims: to assess the genetic evolution on tumor cancer cells induced by SRT in lung-limited omCRC. Secondary objectives included descriptions of the abscopal effect, responses’ duration, toxicity, and progression-free survival. A translational research will be performed to evaluate tumor genetic evolution (through liquid biopsies and Next Generation Sequencing), HLA class I repertoire, peripheral immune cells, and cytokine dynamics. Methods: PRELUDE-1 is a prospective translational study. SRT will be administered only to the largest nodule (with a maximum diameter ≤ 25 mm) in omCRC with two or three radiologically evident lesions. The sample size is based on the innovative hypothesis that radiation-induced immunity could induce regression of tumor clones bearing KRAS oncogene mutations. According to the binomial test, considering the frequency of KRAS mutations and assuming a probability of mutant KRAS→wild type KRAS of p0 = 0.0077, with α = 0.05 and 1-β = 0.60, the final sample size is 25 patients.

Uterine lavage for the detection of ovarian cancer using an expanded gene panel

Uterine lavage for the detection of ovarian cancer using an expanded gene panel

KRAS mutations in patients with colorectal cancer in Libya.

Large prospective clinical trials have demonstrated that colorectal cancers (CRCs) with wild-type KRAS respond favorably to anti-epidermal growth factor receptor treatment, thus making mutational analysis obligatory prior to treatment. In our study, frozen CRC tissues from Libyan patients were analyzed for KRAS and HRAS mutations in codons 12/13 by direct sequencing and the correlations with clinical and pathological parameters were investigated. A total of 34 CRC cases, comprising 19 men and 15 women (age range, 24-87 years), were subjected to systematic analysis for RAS mutations. Although HRAS mutations were not detected in any of the patients in the study group, KRAS codon 12/13 mutations were present in 38.2% (13/34) of the patients. The frequent types of codon 12 mutations were glycine to aspartate (G12D, 46.1%); glycine to valine (G12V, 30.8%) and glycine to cysteine (G12C, 15.4%), while the codon 13 mutations were glycine to aspartate (G13D, 7.7%). G→A mutations occurred in 53.8% (7/13) of the patients, while G→T mutations occurred in 46.2% (6/13) of the patients. Mutations occurred at the first base of codon 12 or 13 in 2/13 (15.4%) and at the second base in 11/13 (84.6%) patients. There was no significant association between clinicopathological characteristics and KRAS mutation status, except the site of the tumors harboring KRAS mutations, which was as follows: The frequency was higher among tumors located in the left colon (8/13, 61.5%) compared to other sites (P=0.027). KRAS mutations were correlated with advanced age, with 10/13 being aged >50 years and affected 8/15 female patients (53%) compared with 5/19 male patients (26%). The highest frequency of KRAS mutations was observed in highly differentiated CRCs (8/13).

Clinical characteristics of patients in their forties who underwent surgical resection for colorectal cancer in Korea

BACKGROUNDThe proportion of young patients with colorectal cancer (CRC), especially in their 40s, is increasing worldwide. AIMTo confirm the clinical characteristics of such patients, we planned a study comparing them to patients in their 30s and 50s.METHODSPatients undergoing primary resection for CRC, patients in their 30s, 40s and 50s were included in the study. Patient and tumor characteristics, and perioperative and oncologic outcomes were compared. RESULTSMost clinical characteristics of 451 (10.5%) patients in their 40s were more similar to those of patients in their 30s than those in their 50s. On pathology data, there were more metastatic lesions (30s vs 40s vs 50s; 17.5% vs 21.1% vs 14.9%, P = 0.012) in patients in their 40s. There was a trend toward less frequent K-ras mutations among patients in their 40s (48.5% vs 33.3% vs 44.5%, P = 0.064). The proportion of patients receiving postoperative chemotherapy was also significantly greater among patients in their 40s (58.3% vs 63.9% vs 56.3%, P = 0.032). Five-year overall survival (OS) and disease-free survival (DFS) did not differ between the three groups (5-year OS, 92.2% vs 89.8% vs 92.2%, P = 0.804; 5-year total DFS, 98.6% vs 95.7% vs 96.1%, P = 0.754; 5-year local DFS, 98.6% vs 94.3% vs 94.9%, P = 0.579; 5-year systemic DFS, 86.4% vs 87.9 % vs 86.4%, P = 0.908).CONCLUSIONPatients with CRC in their 40s showed significantly more numerous metastatic lesions. The oncologic outcome of stage 1-3 patients in their 40s was not inferior compared to that of those in their 30s and 50s.

Abstract 762: Clinical characteristics and prognostic value of the KRASmutation in Chinese colorectal cancer patients

Abstract Background: The KRAS mutation are high-frequency somatic mutations found in the colorectal cancer (CRC) patients both from Western and Asian countries, but except for exon 2 of KRAS, other prevalence and prognostic value have not been adequately assessed in Asian patients. The aim of this study was to determine the mutation frequencies of whole exon mutation of KRAS in Chinese CRC patients and to investigate their impact on prognosis. Methods: 7189 tumor tissue samples (iCohort) were subjected to next-generation sequencing (NGS) for detection the KRAS mutation. 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 affliated Hospital of Soochow University (sCohort) and used ddPCR to detect KRAS mutations. Results: In the iCohort, 2706 patients (37.6%) were confirmed harboring KRAS mutation. The most frequent mutations in KRAS were G12D (32.19%), G12V (17.96%) and G13D (17.59%). In the sCohort, 51 CRC patients (35.17%) had KRAS mutations, among which KRAS G12D (64.71%), G13D (29.41%) and G14D (3.92%) were high-frequency mutations. The KRAS mutation was associated with shorter median overall survival (OS) than wild-type tumors (mOS, 69 vs 55 months; HR, 1.80; 95% Cl 1.22-2.64; P=0.0003). In 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 predictor of shorter OS. Among the common KRAS mutations, G12D was significantly associated with shorter OS (HR, 2.17; 95% Cl 1.31-3.58; P &amp;lt; 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. Our finding that KRAS G12D mutation is associated with inferior survival and a biomarker of poor prognosis in Chinese patients. Our data emphasizes the importance of molecular features in CRC patients, which could potentially be improved by G12D-specific related inhibitors. Citation Format: Ye Yuan, Wei Wang, Jiemin Zhao, Junling Zhang, Shiqing Chen, Chunti Shen, Changping Wu, Wenwei Hu. Clinical characteristics and prognostic value of the KRASmutation in Chinese colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 762.

Abstract LB108: Validation of computationally predicted synthetic lethal interactions for KRAS

Abstract Despite dedicated research in precision medicine, identification of therapeutic strategies for oncogenic drivers remains a major challenge as many mutations are difficult to target directly. Synthetic lethality-based target prediction becomes a powerful approach for drug discovery. We previously developed a computational platform, Mining Synthetic Lethals (MiSL), that can output a list of possible synthetic lethal partners of a specific gene when fed with primary human tumor genomic and transcriptomic data. The principal logic of the MiSL algorithm is based on the Boolean implication that the SL partners of a mutation will never be deleted in the presence of the mutation in the primary tumors. High frequency of KRAS mutation in lung and colon cancers makes it an important therapeutic target. While drugs directly targeting KRAS are yet to be fully developed, we applied the principle of synthetic lethality using the MiSL platform to predict potential KRAS specific therapeutic targets. We identified 556 putative SL partners of KRAS mutations combining lung and colon cancers. To efficiently test all predictions, we used customized shRNA and CRISPR lentiviral libraries to validate all the predicted SL interactions for KRAS in colon cancer cell lines HT29 (KRAS WT) and SW620 (KRAS G12V mutant). Eight shRNAs and four sgRNAs were designed for each gene in the shRNA and CRISPR libraries. The shRNAs and sgRNAs expressed at baseline and at the end of each screen were analyzed by barcode sequencing. Synthetic lethal interactions with KRAS were determined by identifying individual sh/sgRNAs whose mean values at the end relative to the start of the screen were significantly reduced in KRAS Mut cell lines compared to WT. We also compared the results of the in vitro to an in vivo screen of the focused libraries in human tumors xenografted into mice using the same cell lines. We compared the top 50 SL candidates from each screen as ranked by MAGeCK (Model-based Genome-wide Analysis of CRISPR/Cas9 KO) and found that only 19 were shared between the in vitro and in vivo screens. Further validation of several top SL candidates coming from both in vitro and in vivo screens was performed by testing individual shRNAs/sgRNAs for growth inhibition in KRAS Mut (SW620) vs. KRAS WT (HT29) cell lines in vitro and in vivo. Individual in vitro CRISPR/Cas9 knock out of two of the candidate SL partners reduced cell viability by 50% and 57% respectively in KRAS mutant cells compared to KRAS WT cells. Furthermore, induced individual KD or KO of four SL partners resulted in inhibition of tumor growth by 84%, 68%, 95% and 60%, respectively. In summary, MiSL has shown to be a powerful platform for discovering novel targets for specified cancer types. This study has identified and validated several genes as promising targets of KRAS dependent colon cancer for further therapeutic investigation. Citation Format: He Gong, Claire Repellin, Puja Patel, Merrill Knapp, Mingze He, Jun Li, Lisa Wu, Xiaohe Liu, Subarna Sinha, Lidia Sambucetti. Validation of computationally predicted synthetic lethal interactions for KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB108.

KRAS AND BRAF mutation rates and survival outcomes in colorectal cancer in an ethnically-diverse cohort

Background: KRAS-mutant colorectal carcinoma (CRC) is resistant to anti-EGFR antibodies. Several studies compare the KRAS mutation rates between Caucasians and African Americans, but few studies have compared other ethnicities. Aims: To compare the KRAS and BRAF mutation rates, as well as overall survival rates, in CRC between ethnic groups. Additionally, we report on correlations between clinicopathological variables relevant to CRC. Methods: Clinicopathological data from 284 ethnically-diverse patients with CRC were retrospectively analysed at Liverpool Hospital (2014–2016). KRAS and BRAF mutations were detected using real-time PCR kits (from Qiagen). Ethnicity was determined using both country-of-birth and surname. Survival outcomes were available for 191 patients. Overall survival was defined as date-of-diagnosis to death from any cause. Patients still alive were censored at 50-months post-diagnosis. Results and conclusions: KRAS mutation frequency differed significantly between Caucasians and Asians (36.1% vs 56.4%, p=0.018), and Asians and South-Americans (56.4% vs 16.7%, p=0.016), but not between other groups (Middle-Easterners 45.7%). BRAF-V600E mutation frequency differed significantly between Caucasians and Middle-Easterners (16% vs 0%, p=0.029), but not between other groups (Asian 8.6%, South-American 16.7%). The 50-month survival rate did not differ between ethnicities (Caucasians 44%, Middle-Easterners 52%, Asians 58%, South-Americans 50%). Of patients who died, the median survival time was 28 months. Overall survival did not significantly differ between patients with wild-type and mutant KRAS. Tumours with BRAF-V600E mutations were more likely to display microsatellite instability (MSI) than tumours with wild-type BRAF (45.5% vs 8.3%, p<0.001). Tumours displaying MSI occurred more frequently in the right colon (p=0.007). KRAS mutations were more frequent in circumferential tumours (p=0.04), and tumours without vascular invasion (p=0.004).

Clinical Features, Molecular Alterations and Prognosis of Colorectal Adenocarcinoma With Mucinous Component in Chinese Patients.

Mucinous adenocarcinoma (MAC) is conventionally diagnosed by WHO definition when the extracellular mucin is >50% of the tumor area, while tumors with <50% mucin are designated as having a mucinous component. The study is aimed at analyzing the clinicopathologic characteristics, mutation spectrum, and prognosis of colorectal adenocarcinoma with mucinous component (CAWMC). Mutation analyses for exon 2 to 4 of KRAS gene and exon 15 of BRAF gene were performed by Sanger sequencing. Expression of DNA mismatch repairs and P53 proteins were evaluated by immunohistochemistry. Density of tumor-infiltrating lymphocyte (TIL) status was scored. We also evaluated the percentage of glands producing mucin and the morphology of the different tumor cell types in mucin pools. We retrospectively analyzed the prognosis of 43 patients with stage II/III. The overall frequencies of KRAS and BRAF mutations were 36% and 8%, respectively. Patients with MAC exhibiting high levels of mucin were related to the increase of tumor diameter (P=0.038) but were not associated with any of the other clinicopathologic parameters. The proportion or variable morphology of mucinous component did not stratify progression-free survival in stage II/III cases. TIL was the most significant predictor of progression-free survival among stage II/III CAWMC. It is interesting to note that signet ring cell carcinoma does not portend a worse prognosis for patients with high TIL levels. Combining use the grade of TIL status with the WHO grade of the entire tumor can help identify patients with a high risk of recurrence more accurately.

Mutational landscape with a focus on KRAS mutations in non-small lung cancer in Taiwan.

e20513 Background: Lung cancer is the leading cause of cancer death in Taiwan. The poor prognosis of lung cancer patients with advanced disease and recent development of precision medicine motivates the investigation of molecular targets. The purpose of the study is to estimate the prevalence of KRAS gene mutations and 15 other gene targets among non-small cell lung cancer (NSCLC) patients in Taiwan. Methods: This retrospective study included 933 NSCLC patients diagnosed between January 1992 and July 2018 at six medical centers in Taiwan, including Taichung Veteran’s General Hospital, Chung San Medical University Hospital, China Medical University Hospital, Kaohsiung Veteran’s General Hospital, Tri-Service General Hospital and National Taiwan University Hospital. Patients ≥18 years, had primary histologically confirmed NSCLC diagnosis at time before any treatment, and availability of archival tumor tissue with &gt;30% tumor cellularity. Mutational Analysis: The Formalin-Fixed Paraffin-Embedded (FFPE) tumor blocks were analyzed for the prevalence of somatic mutations. The entire coding sequence of 16 genes was analyzed by next generation sequencing of macrodissected DNA from FFPE recut slides at a depth of greater than 300x and interrogated for somatic pathogenic variants. RNA was not available; hence this limits sensitivity to detect fusions. Results: The mean age at diagnosis was 62 years, 498/933 (53%) were women, 411/843 (49%) were smokers or former smokers, and 829/933 (89%) of adenocarcinoma histopathology. Stage was available for 723 patients with a distribution of 328/723 (45%) Stage I, 120/723 (17%) Stage II, 210/723 (29%) Stage III, and 65/723 (9%) Stage IV. EGFR mutations were identified in 327/933 (35.1%) and TP53 mutations in 179/933 (19.2%). KRAS mutations were identified in 72/933 (7.7%) patients, with KRAS G12C mutations observed in 23/933 (2.5%), KRAS G12D in 16/933 (1.7%), and KRAS G12V in 14/933 (1.5%) (Table). In addition, co-occurring mutations with KRAS were observed and are highlighted, including 14 TP53, 4 PIK3CA, 2 EGFR, 2 CTNNB1, 1 STK11, 1 BRAF and 1 PTEN. Conclusions: EGFR and TP53 were the most frequent mutations identified among all stage NSCLC in Taiwanese populations, followed by KRAS. KRAS G12C and KRAS G12D, the two most frequent KRAS mutations, were identified in 32% and 22% of the KRAS-mutant tumors. Although the prevalence of any KRAS mutation or KRAS G12C mutation is lower than typically seen in Western countries, it is similar to observations among Asian populations. These findings broaden the understanding of the mutational landscape of NSCLC patients in Taiwan to inform development of new therapeutic approaches. [Table: see text]

Prevalence of colorectal cancer biomarkers and their impact on clinical outcomes in Riyadh, Saudi Arabia.

ObjectivesKRAS, NRAS, and BRAF mutations are commonly present in colorectal cancer (CRC). We estimated the frequency of KRAS, NRAS, and BRAF mutations and assessed their impact on survival and other clinical variables among Saudi patients.DesignRetrospective cohort study design.SettingsOncology department of a tertiary hospital in Riyadh, Saudi Arabia. We gathered information from 2016 to 2018.ParticipantsCohort of 248 CRC patients to assess the demographic data, pathological tumour features, response to treatment modalities, disease progression, and metastasis.Statistical analysis usedCorrelation analysis using the chi-square test. Survival analysis using a Kaplan Meier method. Cox regression analysis to calculate the hazard ratios.ResultsDemographic data revealed that 84% of patients were diagnosed with CRC above the age of 50 years. Only 27% of patients presented with distant metastasis. KRAS mutations were the most prevalent (49.6%), followed by NRAS mutations (2%) and BRAF mutations (0.4%). Wild type tumours were found among 44.4% of patients. KRAS mutation showed no significant correlation with the site, type, pathological grade, and stage of the tumour.The mean survival time was shorter among patients with KRAS mutations than among patients with wild type KRAS tumours (54.46 vs. 58.02 months). Adjusted analysis showed that the survival time was significantly affected by patients’ age at diagnosis (P = 0.04). Male patients had an increased risk of mortality by 77% (hazard ratio: 1.77).ConclusionsSaudi CRC patients had a high frequency of KRAS mutations and a low frequency of BRAF mutations. The KRAS mutation status did not affect the patients’ survival.

Comprehensive Molecular and Clinicopathologic Analysis of 200 Pulmonary Invasive Mucinous Adenocarcinomas Identifies Distinct Characteristics of Molecular Subtypes.

Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically. A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping (n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS). Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion (F11R-NRG2). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS-mutant IMAs, NRG1-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (P < 0.0001). This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1-rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.

Next-Generation Sequencing Reveals High Uncommon EGFR Mutations and Tumour Mutation Burden in a Subgroup of Lung Cancer Patients.

Xuanwei County in Southwest China shows the highest incidence and mortality rate of lung cancer in China. Although studies have reported distinct clinical characteristics of patients from Xuanwei, the molecular features of these patients with non-small cell lung cancer (NSCLC) remain unclear. Here, we comprehensively characterised such cases using next-generation sequencing (NGS). Formalin-fixed, paraffin-embedded tumour samples from 146 patients from Xuanwei with NSCLC were collected for an NGS-based target panel assay; their features were compared with those of reference Chinese and The Cancer Genome Atlas (TCGA) cohorts. Uncommon EGFR mutations, defined as mutations other than L858R, exon 19del, exon 20ins, and T790M, were the predominant type of EGFR mutations in the Xuanwei cohort. Patients harbouring uncommon EGFR mutations were more likely to have a family history of cancer (p = 0.048). A higher frequency of KRAS mutations and lower frequency of rearrangement alterations were observed in the Xuanwei cohort (p < 0.001). Patients from Xuanwei showed a significantly higher tumour mutation burden than the reference Chinese and TCGA cohorts (p < 0.001). Our data indicates that patients from Xuanwei with NSCLC harbouring G719X/S768I co-mutations may benefit from treatment with EGFR-tyrosine kinase inhibitors. Our comprehensive molecular profiling revealed unique genomic features of patients from Xuanwei with NSCLC, highlighting the potential for improvement in targeted therapy and immunotherapy.

Features of Childhood Colorectal Carcinomas and Frequency of K-ras Mutations

Features of Childhood Colorectal Carcinomas and Frequency of K-ras Mutations