KRAS Mutation Testing Research Articles

KRAS Mutation in Pediatric Intracranial Germ Cell Tumors.

Intracranial germ cell tumors (IGCTs) are rare, highly curable neoplasms. KRAS is a gene in the KIT/RAS signaling pathway, and KRAS mutations have been reported in patients diagnosed with IGCTs. To describe the clinicopathologic and molecular features of KRAS mutation and the treatment outcome of children diagnosed with IGCTs. Patients diagnosed with IGCTs at the Department of Pediatrics, King Chulalongkorn Memorial Hospital from 2007 to 2016 were retrospectively reviewed. DNA was extracted from formalin-fixed, paraffin-embedded tissue and used for molecular study. Mutations in codons 12, 13, and 61 of the KRAS gene were detected using the cobas® KRAS mutation test and pyrosequencing. Eighteen patients were diagnosed with IGCTs (11 males and 7 females): nine with germinomas and nine with non-germinomatous GCTs (NGGCTs). The age range of the patients was 5-14 years (median 10.5 years). Elevated markers were revealed in approximately 25% of the patients. Four patients (two with germinomas and two with NGGCTs) had leptomeningeal involvement. All patients underwent tumor biopsy and received neoadjuvant chemotherapy. Radiotherapy was administered in 16 patients, and craniospinal radiation was administered only in patients with leptomeningeal metastasis. With a median follow-up of 26 months, overall survival was 88.9% in the patients with germinomas and 37% in the patients with NGGCTs. Mutation of the KRAS gene was detected using pyrosequencing in one patient. The mutation located at codon 61, with frequency 38.3% units, nucleotide substitution CAA > CTA, and amino acid substitution, was Q61L. The patient carrying the mutant gene was diagnosed with germinoma with cerebrospinal fluid metastasis and eventually died from treatment-related toxicity. Our study revealed the treatment outcomes of IGCTs in Thai children. The metastatic germinoma patient with KRAS codon 61 mutation had a poor outcome, supporting that Q61L has a clinical correlation with IGCTs.

Liquid biopsy for KRAS, NRAS and BRAF mutation testing in advanced colorectal cancer patients: the Argentinean experience.

Objective: To analyze the frequency of KRAS, NRAS and BRAF hotspot mutations in circulating tumor DNA (ctDNA) from patients with metastatic colorectal cancer (mCRC). Methods: Observational, descriptive and retrospective study in mCRC patients with available ctDNA-based genotype of KRAS, NRAS and BRAF. Results: The frequencies of plasma mutations for KRAS, NRAS and BRAF were 34% (±7), 4% (±3) and 4% (±3), respectively. Median overall survival of plasma-tested RAS/BRAF-mutated patients was 26.6months (95% CI: 14.4-not estimable [NE]), while RAS/BRAF wild-type patients did not reach the median survival during follow-up. Median progression-free survival for RAS/BRAF wild-type and RAS/BRAF-mutated patients was 12 (95% CI: 7-NE) and 4months (95% CI: 4-NE), respectively. Conclusion: Our work supports the utility of KRAS, NRAS and BRAF analysis in liquid biopsy from mCRC patients.

Analysis of KRAS, NRAS, and BRAF Mutations, Microsatellite Instability, and Relevant Prognosis Effects in Patients With Early Colorectal Cancer: A Cohort Study in East Asia.

BackgroundEarly colorectal cancer (ECRC) refers to any size of colorectal cancer (CRC) whose depth of invasion is limited to the mucosa and submucosa. About 10% of patients with ECRC die from cancer after surgery. KRAS, NRAS, and BRAF mutations and microsatellite instability (MSI) are considered diagnostic and prognostic markers in CRC. However, their characteristics in ECRC and whether postoperative chemotherapy based on them will benefit ECRC patients or not remain unknown.Patients and MethodsPatients with ECRC and 298 patients with advanced colorectal cancer (ACRC) were collected in our hospital from January 2013 to December 2015. The Amplification Refractory Mutation System (ARMS)-PCR was used to perform the KRAS, NRAS, and BRAF mutant tests.ResultsIn ECRC patients, 43 cases of KRAS mutation were found, accounting for 69.35%. Interestingly, among KRAS mutations, there were 10 KRAS multi-site mutation patients (16.13% in 62 ECRC patients). Moreover, the NRAS mutation rate was 3.23% but no BRAF mutation was found and only 1 case of MSI-High was detected. KRAS mutation was only related to the depth of tumor invasion whereas KRAS multi-site mutations were related to mucus components and tumor size. As far as NRAS is concerned, mutations were associated with elevated CEA, mucus components, and the depth of tumor invasion. Notably, compared with 2.35% KRAS multi-site mutation in ACRC, the rate of KRAS multi-site mutation in ECRC was much higher. Furthermore, Cox regression analysis revealed that KRAS mutation could be an independent prognostic factor of ECRC in patients who have undergone endoscopic resection or surgery.ConclusionPatients with ECRC might benefit from KRAS mutation testing but not from postoperative chemotherapy.

Abstract 5403: Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations

Abstract Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations Background Currently, most patients with mCRC are treated with a combination of a cytotoxic and a biologic agent. Several factors should be put into account which influencing the treatment strategy for patients with mCRC. including tumor resectability, prior systemic therapy; tumor location (left or right), and molecular characteristics such as mutation in gene Kirsten rat sarcoma viral oncogene homolog (KRAS). It has been demonstrated to be a tightly associated with poor response to anti-EGFR treatment, and even poor response to immune checkpoint inhibitors for patients with colorectal cancer. Therefore, KRAS mutational status testing has been highlighted in recent years. Methods This study queried the data from Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 2,294 CRC patients which underwent a targeted next-generation sequencing assay performed by 3DMed Clinical Laboratory Inc., a College of American Pathologists (CAP) certified and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory of 3D Medicines Inc. between January, 2017 and June, 2020 in China, to obtain a comprehensive molecular profile of KRAS mutations. Co-occurring genomic alterations, tumor mutational burden (TMB), microsatellite instability (MSI) status and PD-L1 expression (Dako 22C3 or VentanaSP263, TPS score) were analyzed. High TMB was defined as TMB ≥10 muts/Mb. Postoperative follow-up data for 27 patients were collected and analyzed. Results KRAS mutation account for 48.7% Chinese CRC patients. In addition, proportions of KRAS mutation in different exons were counted. KRAS mutation in CRC mainly occurred in exon2 (84.0%), exon3 (6.1%), exon4 (7.4%), and exon5 (0.7%). KRAS mutation in exon2, the main mutations were G12D (35.4%), G12V (20.8%), G13D (21.9%), G12C (5.9%) ad G12S (5.8%). while in exon3 and exon4, the main mutations were Q61H (50.7%) and A146T (73.2%), respectively. The proportion of MSI-H in CRC patients with and without KRAS mutation were very close. While it seemed that there were more MSI-H patients in KRAS exon3 and exon4 mutation. In addition, PD-L1 expression was detected in 1220 tumor samples by means of IHC. Among these available tumor samples, level of PD-L1expression were similar among patients without KRAS mutation, KRAS mutation in different exons, but it reveled that there were very small part of patient with strong positive PD-L1 level (TPS≥50). In addition, different prognosis were found among different mutations of KRAS exon2, in detail, worse PFS was showed in CRC patients with KRAS mutation in exon2 G12D than G12V and G13D. Conclusions CRC patients with KRAS mutations in different exons and different site have different genetic and prognostic characteristics, these patients should be managed differently in clinical practice. Citation Format: Zhengqi Wen, Hushan Zhang, Ruize Zhou, Xihong Liu, Wenliang Li. Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5403.

Tumor profiling of KRAS, BRAF, and NRAS gene mutations in patients with colorectal cancer: A Lebanese major center cohort study

BackgroundIn the era of precision medicine, treatment schemes for advanced Colorectal (CRC) disease include monoclonal antibodies which block the epidermal growth factor receptor (EGFR) implicated in tumor proliferation, invasion, migration and neovascularization. Resistance to these agents has been correlated with activating downstream mutations in KRAS, BRAF and NRAS genes, among others, leading to constitutive activation of the EGFR axis bypassing EGFR blockade. The assessment of tumor RASandBRAFmutational status has thus become standard clinical practice. While multiple investigations reported roughly mutations rates of 40% in KRAS, 7% in NRAS and 5–15 % in BRAF, numbers vary across different populations with limited data specifically from the Middle East. MethodsThis is a retrospective observational Laboratory information system (LIS) chart review of all the patients with pathologically confirmed colorectal carcinoma (CRC) or metastatic CRC who underwent KRAS, NRAS and/or BRAF mutational analysis testing at the Molecular Diagnostics Laboratory of the American University of Beirut Medical Center (AUBMC) from January 2012 to December 2018, inclusive. Data retrieved included the results of mutation testing performed for KRAS, NRAS and BRAF genes, the age, gender, and tumor location for each patient. Analysis of the mutations was performed using polymerase chain reaction (PCR) hybridization StripAssay® (ViennaLab, Vienna, Austria). Results130 (47.6%) out of 273 histologically confirmed CRC cases, had positive KRAS mutations, namely in codons 12 (82%), 13 (17%), 146 (1.5%), 117 (0.75%), or 61 (0.75%). Two patients had two concomitant mutations: 12 + 12 (different mutations) and 12 + 146. Of 203 CRC cases tested for NRAS mutations, 16 (7.8%) were found to be positive for a mutation in codon 12 (37.5%), 61 (37.5%), or 13 (12.5%). Two patients had two concomitant mutations: 12 + 13 and 59 + 61. Of 172 CRC cases tested for BRAF mutations, 2 (1.2 %) were positive for the V600E -. ConclusionThis retrospective study is the first to report the frequencies of KRAS, NRAS and BRAF gene mutations in a Lebanese CRC cohort diagnosed and managed at a tertiary care center. The frequencies of the studied somatic gene mutations were similar to previously reported cohorts in other populations however the rate of BRAF mutation was lower in this cohort than expected.

Significance of KRAS mutation testing in biliary brushing cytology specimens: A 10‐year retrospective review

BACKGROUNDBiliary strictures can be caused by benign and malignant conditions. A biliary duct brushing diagnosis can be challenging because of low cellularity and overlapping morphology among different entities, leading to a variable reported sensitivity. This study aimed to assess the value of KRAS mutation testing in adding cytological diagnosis of biliary duct brushings.METHODSWith institutional review board approval, biliary duct brushing cytology specimens were collected from 269 patients with extrahepatic biliary stenosis between August 2011 and July 2021. The results of cytology and KRAS mutational analyses were evaluated in view of corresponding cytology examination and histopathological/clinical follow‐up.RESULTSKRAS mutations were identified in 50 of 269 biliary stricture brushing cases (19%). Among the cases with available follow‐up, 72% (34 of 47) of biliary brushings had confirmed malignancy when there were KRAS mutations. The overall specificity and sensitivity of KRAS mutation testing was 92% and 36%, respectively. KRAS mutation was significantly more enriched in pancreatic duct adenocarcinoma than in cholangiocarcinoma (66% vs 5%, P < .001). The absolute risk of malignancy was 3%, 28%, and 71%, respectively, in negative, atypical, and suspicious cytological diagnostic categories and the risks increased to 14%, 68%, and 95% in corresponding categories with KRAS mutation.CONCLUSIONSOur results suggested that KRAS mutational analysis can be considered supplementary to cytology diagnosis of biliary duct brushing for patients with extrahepatic biliary stenosis in clinical practice.;

Molecular Signatures of KRAS-Mutated Lung Adenocarcinoma: Analysis of Concomitant EGFR, ALK, STK11, and PD-L1 Status.

Background:KRAS mutations are the most common oncogenic driver mutations of non-small cell lung cancer (NSCLC) in the Western world. Mutations of the KRAS gene are most prevalent in the patient population of current and former cigarette smokers. With the recent pivotal approval of a targeted inhibitor therapy for patients with KRAS p.G12C mutated and pretreated NSCLC, analysis of the heterogeneity of KRAS mutations and concomitant molecular alterations in patients with these tumors at all clinical stages is indicated.Methods:In this retrospective analysis, patient pathology records were reviewed for all cases receiving a pathologic diagnosis of NSCLC within our hospital system. All data were collected with IRB approval. Cases of indeterminate tumor type favoring a non-lung primary, as well as non-adenocarcinoma NSCLC (eg, squamous) were excluded from the cohort. In this hospital system, molecular testing for KRAS mutations is part of a molecular biomarker panel that is reflex ordered at initial diagnosis by the pathologist and may be performed as a single gene test or as a solid organ cancer hotspot panel by next generation sequencing. For each patient, KRAS mutational status and specific KRAS mutations, if present, were collated. Additional information assessed for this study included patient demographics (age, gender, and smoking history), tumor staging if available, PD-L1 expression levels by immunohistochemistry (IHC), and the presence of other genetic alterations (EGFR, ALK, and STK11).Results:Between January 1, 2017 and January 1, 2019, there were 276 patients diagnosed with NSCLC of all stages who had KRAS mutational analysis performed in our hospital system and who met the criteria for inclusion into the study cohort. A KRAS driver mutation was detected in 29% of these patients. The most frequently identified KRAS mutation was p.G12C (38%), followed by p.G12D (21%) and p.G12V (13%). KRAS-mutated lung adenocarcinoma was significantly associated with current or former patient smoking status in this cohort (29/202 (14%) smokers and 1/74 (1%) non-smokers; P = .0006). PD-L1 expression of at least 1% by IHC was present in 43% of KRAS-mutated lung adenocarcinomas and 45% of non-KRAS-mutated adenocarcinomas. In this study, KRAS mutations were not found to co-occur with gene alterations in EGFR, ALK, or STK11. In 48% of cases, at least one genetic alteration (KRAS, ALK, EGFR, or STK11) was identified.Conclusions:In this study cohort, KRAS-mutated lung adenocarcinoma demonstrated significant mutational heterogeneity, which is consistent with previously published studies. KRAS mutational status was also significantly associated with a current or former smoking history. Notably, p.G12C was the most frequently identified KRAS mutation in this cohort, with a frequency of 38%. This finding is particularly relevant given the recent approval of a KRAS p.G12C-specific targeted inhibitor therapy and the continued development of additional KRAS targeted therapies that may prove effective in treating NSCLC. These findings also highlight the necessity of considering molecular testing for KRAS mutations in patients with NSCLC and a smoking history, as this population most frequently harbors KRAS mutations and may benefit from these emerging targeted therapies.

Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples.

The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.

P24.01 Turnaround Time and Variant Prevalence of a Blood-based KRAS Test in Patients With NSCLC

Mutations in KRAS have historically been a harbinger of poor prognosis in patients with non-small cell lung cancer (NSCLC). These patients may have better outcomes using novel KRAS-targeting therapies currently in development, particularly if rapid turnaround, blood-based, KRAS mutation testing can identify treatment-eligible patients and help expedite time-to-treatment. A commercially available blood-based genomic test identifies the three most common KRAS mutations in NSCLC, G12C, G12V and G12D (KRAS G12C/V/D).

Mutations in the KRAS gene as a predictive biomarker of therapeutic response in patients with colorectal cancer

Abstract Introduction: Despite the important role of general KRAS mutational status in the selection of an adequate therapeutic protocol in patients with colorectal cancer (CRC), studies that focus on its specific mutations and their significance on progression of disease are scarce. This study aimed to determine the significance of specific KRAS mutations in response to standard chemotherapy protocols with oxaliplatin-based (FOLFOX 4, OXFL) in the first-line and irinotecan-based chemotherapy (FOLFIRI, IFL) in the second-line therapy, and to evaluate the correlation between these mutations and clinicopathological characteristics of CRC patients. Methods: Genomic DNA was extracted from the FFPE tumour tissue sections while the KRAS mutation test was performed by using PCR methods. Results: Prevalence of KRAS gene mutations in CRC patients was 45%. Mutated KRAS was more frequent in later stages of tumor infiltrations (P =0.0017), on the right side of the colon (P= 0.0044), and in patients who developed metastases in the first 6 months after CRC diagnosis than in patients who developed metastases after 24 months (P=0.0083). In a group of patients with a poor therapeutic response to standard chemotherapy the most frequent mutations in KRAS gene were G12D and G12V (63.88%), while in a group of patients with a good response to therapeutic protocols the most prevalent mutation was G12A (66.66%). Conclusion: Our results indicate that there was a significant difference in biological behaviour between tumours harboring different mutations in KRAS gene. Overall, mutation G12A could be a novel prognostic biomarker for CRC patients treated with standard chemotherapy.

Molecular characterisation of pancreatic ductal adenocarcinoma with NTRK fusions and review of the literature

AimsThe majority of pancreatic ductal adenocarcinomas (PDACs) harbour oncogenic mutations in KRAS with variants in TP53, CDKN2A and SMAD4 also prevalent. The presence of oncogenic fusions including NTRK fusions are...

Adenocarcinomas with mesonephric features in gynecologic tract: a clinicopathological analysis of seven cases

Objective: To investigate and compare the histologic characteristics of adenocarcinomas with mesonephric features located in different parts of the gynecologic tract. Methods: Two cases of mesonephric adenocarcinomas (MA) of the cervix and 5 cases of mesonephric-like adenocarcinomas (MLA) of the uterus and ovary were collected in Women's Hospital, School of Medicine, Zhejiang University from January 2018 to October 2020. Hematoxylin-eosin staining, immunohistochemistry and KRAS mutation testing were performed together with review of literature. Results: MA of the cervix as well as MLA of the uterus and ovary had similar morphologic features, showing an admixture of glandular, tubular, papillary and solid growth patterns. However, both MA cases were located in cervical stroma, which demonstrated residual mesonephric ducts present at the periphery. All four uterine MLA cases extensively involved the endometrium and myometrium. The ovarian MLA case was associated with endometriosis. No residual mesonephric ducts were present in the MLA cases. Immunohistochemically, GATA3 was positive in all seven MA/MLA cases. TTF1 was expressed only in 4/5 MLA cases. ER and PR were negative and p53 was wild-type in all cases. KRAS mutation was detected in all five cases. During the 6-32 months of follow-up, one patient developed recurrence and the others were tumor-free. Conclusions: In the gynecologic tract, both MA in cervix and MLA in uterus and ovary have similar morphologic features, immunohistochemical expression and KRAS mutation. However, distinct from MA that originates from mesonephric remnant, MLA is closely related to Mullerian epithelium.

Familial Adenomatous Polyposis-associated Traditional Serrated Adenoma of the Small Intestine: A Clinicopathologic and Molecular Analysis.

Familial adenomatous polyposis (FAP) is an inherited cancer predisposition syndrome associated with numerous gastrointestinal tract adenomatous polyps, as well as gastric fundic gland polyps and pyloric gland adenomas in the upper gastrointestinal tract. While colonic FAP-associated traditional serrated adenomas (TSAs) have been reported in a few studies, small bowel FAP-associated adenomas with TSA morphology have not been characterized. This study describes the clinicopathologic and molecular findings of this type of adenoma in the small bowel of patients with FAP. We reviewed small bowel adenomas in 45 consecutive FAP patients to identify adenomas with zones showing slit-like serrations, cells with eosinophilic cytoplasm, ectopic crypt formation, and vesicular nuclei. Sporadic small bowel adenomas from 51 consecutive patients were also reviewed for adenomas with the same features. Of the 177 polyps from 45 FAP patients and 60 polyps from 51 nonsyndromic patients, 18 TSAs from 9 FAP patients (20%) and 10 TSAs from the sporadic group (19.6%) were identified. FAP patients presented at a younger age than nonsyndromic patients (median: 43 vs. 66; P=0.0048). FAP-associated TSAs were asymptomatic and smaller than sporadic TSAs (median size: 0.6 vs. 2.5 cm; P=0.00006). Immunostaining for β-catenin and testing for BRAF and KRAS mutations were performed in a subset of the cohort. Nuclear β-catenin was seen in 1 FAP-associated TSA and 3 nonsyndromic TSAs. All TSAs (FAP-associated and nonsyndromic) showed wild-type BRAF, while KRAS mutations were identified only in the nonsyndromic setting. In summary, small bowel FAP-associated and sporadic TSAs share a similar morphology, and the BRAF-serrated pathway does not contribute to their pathogenesis.

BIOM-06. CLINICAL AND PROGNOSTIC SIGNIFICANCE OF KRAS MUTATIONS FOR BRAIN METASTASES FROM COLORECTAL ADENOCARCINOMA IN THE UNITED STATES

Abstract BACKGROUND KRAS mutations in colorectal adenocarcinomas have been associated with metastatic involvement of the brain (BM); and predict a lack of benefit to EGFR-targeted antibodies. Herein we leverage national data to evaluate the prevalence and impact of KRAS mutations on colorectal BMs. METHODS Newly-diagnosed patients with stage 4 colorectal adenocarcinoma were identified from the National Cancer Database, comprising >70% of newly-diagnosed cases in the U.S. from 2010–2016. Multivariable logistic regression was used to evaluate predictors of brain involvement, including KRAS mutation status. Overall survival (OS) was estimated with Kaplan-Meier techniques, and compared by logrank test and multivariable Cox regression. RESULTS Of 86,719 patients newly presenting with stage 4 colorectal adenocarcinoma, 1.5% (n=1,318) had BMs. 39.7% of BM cases had KRAS mutation testing, in which 57.9% (n=361) of BM cases were KRAS-mutant, as opposed to only 45.6% (n=13,259) of non-BM cases (p< 0.001). Mutant KRAS persisted as independently associated with BMs (OR 1.37, 95%CI: 1.09–1.72, p=0.006) following adjustment for age at diagnosis, sex, AJCC cT and cN status, and metastatic lung, liver, or bone involvement. Overall, BM patients displayed a median OS of 5.3 months (95%CI: 4.6–6.1): 7.5 months (95%CI: 5.7–10.6) if KRAS-wildtype vs. 12.0 months (95%CI: 10.4–15.4) if KRAS-mutant (p=0.01). The improved OS associated with KRAS-mutant BMs also persisted (HR 0.63, 95%CI: 0.47–0.84, p=0.001) after adjusting for the aforementioned clinical variables, in addition to comorbidity index, chemoradiotherapy, metastasectomy, and treating hospital type. CONCLUSIONS Nationally, testing colorectal BM patients for KRAS mutation status—a predictive biomarker for EGFR-mab therapy—remains underutilized. KRAS-mutant colorectal adenocarcinomas were more likely to have metastatic brain involvement, with KRAS-wildtype BMs demonstrating significantly worse OS than KRAS-mutant BMs. Our data suggest that KRAS mutation testing is underutilized in colorectal BM patients and provides useful prognostic information.

Diagnostic value of digital droplet polymerase chain reaction and digital multiplexed detection of single-nucleotide variants in pancreatic cytology specimens collected by EUS-guided FNA

EUS-guided FNA is recommended as a first-line procedure for the histopathologic diagnosis of pancreatic cancer. Molecular analysis of EUS-FNA samples might be used as an auxiliary tool to strengthen the diagnosis. The current study aimed to evaluate the diagnostic performances of K-ras testing using droplet digital polymerase chain reaction (ddPCR) and a novel single-nucleotide variant (SNV) assay performed on pancreatic EUS-FNA samples. EUS-FNA specimens from 31 patients with pancreatic masses (22 pancreatic ductal adenocarcinomas, 7 chronic pancreatitis, and 2 pancreatic neuroendocrine tumors) were included in the study. K-ras testing was initially performed by ddPCR. In addition, mutational status was evaluated using an SNV assay by NanoString technology, using digital enumeration of unique barcoded probes to detect 97 SNVs from 24 genes of clinical significance. The overall specificity and sensitivity of cytologic examination were 100% and 63%, respectively. K-ras mutation testing was performed using ddPCR, and the sensitivity increased to 87% with specificity 90%. The SNV assay detected at least 1 variant in 90% of pancreatic ductal adenocarcinoma samples; the test was able to detect 2 K-ras codon 61 mutations in 2 cases of pancreatic ductal adenocarcinoma, which were missed by ddPCR. The overall diagnostic accuracy of the cytologic examination alone was 74%, and it increased to 91% when the results of both molecular tests were considered for the cases with negative and inconclusive results. The current study illustrated that integration of K-ras analysis with cytologic evaluation, especially in inconclusive cases, can enhance the diagnostic accuracy of EUS-FNA for pancreatic lesions.

Diameter of Superior Rectal Vein - CT Predictor of KRAS Mutation in Rectal Carcinoma.

BackgroundThe purpose of this study was to investigate the feasibility of CT parameters to predict the presence of KRAS mutations in rectal cancer patients. The relationship between the presence of a KRAS mutation and pathological findings was evaluated simultaneously.MethodsEighty-nine patients (29 females, 60 males, age 27–90, mean 59.7±12 years) with pathologically proven rectal cancer were enrolled. A KRAS mutation test was completed following surgery. Parameters evaluated on CT included the tumor location, the diameter of the superior rectal vein (SRV) and inferior mesenteric vein (IMV), the presence of calcification, ulceration, lymph node enlargement (LNE), distant metastasis, tumor shape (intraluminal polypoid mass, infiltrative mass, or bulky), circumferential extent (C0–C1/4, C1/4–C1/2, C1/2–C3/4, or C3/4–C1), enhanced pattern (homogeneous or heterogeneous), CT ratio, and the length of the tumor (LOT). Pathological findings included lymphovascular emboli, signet ring cell, peripheral fat interval infiltration, focal ulcer, lymph node metastasis, tumor pathological type, and differentiation extent. The correlations between KRAS status and CT parameters, and KRAS status and pathological findings were investigated. The accuracy of CT characteristics for predicting KRAS mutation was evaluated.ResultsA KRAS mutation was detected in 42 cases. On CT image, the diameter of the SRV was significantly increased in the KRAS mutation group compared to in the KRAS wild-type group (4.6±0.9 mm vs 4.2±0.9 mm, p=0.02), and LNE was more likely to occur in the KRAS mutation group (73.3% vs 26.7%, p=0.03). There was no significant difference between the KRAS mutation group and the KRAS wild-type group on the other CT parameters (location, IMV, calcification, ulcer, distant metastasis, tumor shape, enhanced pattern, circumferential extent, CT ratio, and LOT). In the pathological findings, a KRAS mutation was more likely to occur in the middle differentiation group (p=0.03). No significant difference was found between the KRAS mutation group and the KRAS wild-type group in the presence of lymphovascular emboli, signet ring cell, peripheral fat interval infiltration, focal ulcer, lymph node metastasis, and tumor pathological type. With the best cut-off value of 4.07 mm, the AUC of the SRV to predict a KRAS mutation was 0.63 with a sensitivity of 76.2% and a specificity of 48.9%.ConclusionIt was feasible to use the diameter of the SRV to predict a KRAS mutation in rectal cancer patients, and LNE also can be regarded as an important clue on preoperative CT images.

ALL-351: NRAS, KRAS, JAK2, CRLF2, TP53 Mutations in Adult ALL patients

Context Testing for NRAS, KRAS, JAK2, CRLF2 and TP53 mutations in adult ALL patients and studying of molecular pathways are critical for understanding the biology of ALL. Objective Assessment of mutations in NRAS, KRAS, JAK2, CRLF2, TP53 genes and analysis of signaling pathways in de novo adult ALL patients. Design NRAS, KRAS, JAK2, CRLF2, TP53 mutations were studied B-ALL (Ph+/Ph−). TP53 gene mutations were investigated in B-ALL (Ph+/Ph−) and T-ALL patients.TP53 gene was also evaluated in non-tumor cells of 5 patients to exclude germline mutations. Gene expression profiling was performed for 5 B-ALL patients. Setting Sanger sequencing of hot-spot regions NRAS, KRAS, JAK2, CRLF2 and TP53 (exons 2-10) was conducted. PanCancer Pathways Panel by NanoString was used for gene expression analysis. KEGG pathway analysis for overexpressed genes (of the 13 canonical pathways) was carried out using the DAVID tool. Patients or other participants The study included 95 B-ALL patients, m:f (39:45), median age 31 years; 64 T-ALL, m:f (44:20), median age 27 years, 27 Ph+ ALL, m:f (10:17), median age 33 years. 109 patients treated by protocol ALL-2016 ( NCT03462095 ), 50 patients – by ALL-2009 ( NCT01193933 ), 23 – by ALL-2012 (for Ph«+»ALL) and 4 – by other protocols. Median follow-up 20.8 months. Results NRAS (9.7%), KRAS (12.9%), JAK2 (3.16%) mutations was detected only in B-ALL patients. CRLF2 mutations mutation was not detected. NRAS, KRAS, JAK2 mutations didn't impact disease outcome in B-ALL patients treated by ALL-2009, ALL-2016 protocols. TP53 mutations more often were detected in B-ALL patients (11.2%) compared with T (3.1%) and Ph+ (7.4%). They were associated with poorer OS (TP53-WT – 66.9% vs 48% TP53 mut, p=0.094) and a lower incidence of CR (TP53-WT – 90.6% vs 70.4% TP53 mut, p = 0.033). In 4 out of 5 patients, TP53 mutations were detected in non-tumor cells what considers leukemia as a manifestation of Li-Fraumeni syndrome. The most significant pathways affected were pathways in cancer (p = 6.7E-33) and cell cycle (p = 3.3E-31). Conclusions TP53 mutations are associated with unfavorable prognosis. Most TP53 mutations are of hereditary origin. NRAS, KRAS, JAK2 mutations did not affect the disease outcome. Pathway analysis provides a useful tool for understanding the ALL biology, and we plan to continue it and recruit more patients. The reported study was funded by RFBR, project number 19-31-27001.

Histology and its prognostic effect on KRAS-mutated colorectal carcinomas in Korea.

KRAS mutation is frequently identified in advanced colorectal carcinoma (CRC); however, its prognostic significance and the associated histological features have remained to be clarified. In the present study, the precise histological results and prognostic value of KRAS-mutated CRCs were investigated in patients from South Korea. A retrospective review of the results from KRAS mutation testing, as well as evaluation of the histology of 310 cases of CRC at various stages, were performed. Cross-tabulation and survival analysis were performed according to the KRAS status. Patients with KRAS mutation more frequently exhibited serrated and papillary architectures (P=0.009 and P=0.014, respectively). KRAS mutation was an independent unfavorable prognostic factor for overall survival (OS) according to multivariate analysis (P=0.001), whereas no association was observed with disease-free survival (DFS) (P=0.611). Of note, in the subgroup of KRAS-mutated carcinomas, the presence of a solid component on histology was associated with less favorable OS (P=0.032). Furthermore, among the wild type cases, patients with a micropapillary component had a worse OS than those who did not (P=0.018). However, no subgroup or specific histological features were associated with DFS. In summary, KRAS-mutated CRCs had a moderate association with particular histological features, and according to the KRAS mutational status, there was a certain degree of association between histology and prognosis.

Sinonasal papillomas: A single centre experience on 137 cases with emphasis on malignant transformation and EGFR/KRAS status in “carcinoma ex papilloma”

Among the three major histological subtypes of sinonasal papillomas, inverted (ISP) and oncocytic (OSP) sinonasal papillomas tend to undergo malignant transformation to carcinoma. However, criteria determining risk of recurrence and malignant progression have not been established. Recently, EGFR and KRAS mutations were detected to be characteristic for ISP and OSP, respectively. In this study, we analyzed 137 sinonasal papilloma cases (132 ISP and 5 OSP) for clinicopathological characteristics, frequency of recurrences/malignant transformation, and histological types and genetic features of carcinoma ex Schneiderian papilloma. OSP presented at a higher age than ISP (median, 75 vs. 57 years) and affected predominantly females. Overall frequency of recurrences and malignant transformation was 23.1% and 9.5%, respectively. Rates of recurrence (33.3% vs. 22.0%) and malignant transformation (33.3% vs. 8.8%) were higher in OSP compared to ISP, respectively. Carcinomas (n = 10) occurred mostly synchronously, more frequently in females and mainly associated with ISP (n = 9). Squamous cell carcinoma (SCC) was the most frequently associated malignancy. Concordant EGFR (in ISP/associated carcinoma) and KRAS (in the OSP/associated carcinoma) mutations were detected in all successfully analyzed matching papilloma/carcinoma pairs, confirming their shared clonal origin. Results of this large study are in line with recent studies showing frequent EGFR and KRAS mutations in sinonasal carcinoma ex Schneiderian papilloma. As the papilloma component might on occasion be missed on biopsy of synchronous carcinoma ex papilloma, EGFR and KRAS mutation testing represents a promising molecular surrogate for sinonasal “carcinoma ex papilloma”, at the same time offering an opportunity for targeting mutant EGFR in this rare cancer type.

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.