KRAS Analysis Research Articles

3D cultivation of non-small-cell lung cancer cell lines using four different methods

PurposeThe aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples.MethodsFour NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex®-T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning® Elplasia® plates. Western blot was used to verify antigen protein expression. Hematoxylin-eosin staining was used to evaluate the cell morphology in the 2D and 3D cultures. Mutational analysis of KRAS and EGFR by PCR on extracted DNA from 3D tumoroids generated from cells with known mutations (A549; KRAS G12S mutation, NCI-H1975; EGFR L858R/T790M mutations).ResultsWe successfully established 3D cultures from A549, NCI-H1975, HCC-95, and HCC-1588 with all four used cultivation methods. The adenocarcinomas (A549, NCI-H1975) maintained their original IHC features in the tumoroids, while the squamous cell carcinomas (HCC-95, HCC-1588) lost their unique markers in the cultures. PCR analysis confirmed persistent genetic changes where expected.ConclusionThe establishment of tumoroids from lung cancer cell lines is feasible with various methodologies, which is promising for future tumoroid growth from clinical lung cancer samples. However, analysis of relevant markers is a prerequisite and may need to be validated for each model and cell type.

Abstract IA-08: Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer

Abstract KRAS inhibitors targeting G12C mutation have been approved by FDA for the treatment of non-small cell lung cancers and have shown promising clinical responses in pancreatic ductal adenocarcinoma (PDAC). RAS inhibitors targeting additional KRAS mutations or multiple RAS isoforms are under clinical development. Although targeting KRAS exhibits anti-tumor effect in PDAC, majority of tumors inevitably develop resistant disease, as is the case for most targeted therapies. In this new era of targeting KRAS driven cancer, understanding mechanisms driving this resistance to RAS targeted therapies and further devising combination strategies to overcome it have become a necessary, though challenging, task to achieve long term disease control. To tackle this problem, we employed various in vitro and in vivo preclinical model systems to explore the development of resistance to both genetic and pharmacological KRAS blockade. Our previous work identified the membrane proteoglycan Syndecan 1 (SDC1) as a KRAS downstream surrogate whose cell surface localization is tightly controlled by oncogenic KRAS signaling and is critical for KRAS-driven tumor development. Here, our analysis of KRAS driven genetically engineered PDAC mouse model and human pancreatic or colorectal cancer (CRC) cells with KRASG12C mutation discovered that, while cell surface SDC1 is reduced upon acute genetic or pharmacological inhibition of oncogenic KRAS, plasma membrane SDC1 is recovered in tumor cells that become resistant to KRAS inhibition (KRAS-bypass cells). Surface localization of SDC1 leads to the activation of multiple receptor tyrosine kinases (RTKs) and macropinocytosis, resulting in the resistance to KRAS blockade. We further identified that the recovery of plasma membrane SDC1 in KRAS-bypass cells is driven by YAP1 oncogene, which induces the downregulation of ARF6 GAPs, leading to the activation of ARF6, a small GTPase required for SDC1 membrane recycling. To therapeutically targeting SDC1, we have successfully developed a monoclonal antibody (22B mAb), which exhibits remarkable sensitivity and specificity for human SDC1. Our results indicate that 22B mAb exerts substantial inhibition of PDAC cell growth in vitro and desirable tumor inhibitory effects in vivo in murine PDAC tumors and human PDAC cell-derived xenograft tumors. Importantly, our 22B mAb synergizes with KRAS inhibitors, chemotherapy, or immunotherapy, resulting in significantly enhanced therapeutic effects. Taken together, our study identified SDC1 as a functional driver for the bypass of KRAS-dependence and therapeutically targeting SDC1 with a novel mAb will overcome the acquired resistance to KRAS targeted therapy. As such, our research yields important new insights in the fields of cancer biology regarding mechanisms of resistance employed by human cancers, and identifies new actionable therapeutic targets and approaches to overcome resistance to KRAS inhibition. Citation Format: Mitsunobu Takeda, Zecheng Yang, Madeline S Theardy, Alexey Sorokin, Oluwadara Coker, Shuaitong Chen, Long T Vien, Laura Bover, Haoqiang Ying, Scott Kopetz, Wantong Yao. Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-08.

Phase II trial of BXCL701 and pembrolizumab in patients with metastatic pancreatic ductal adenocarcinoma (EXPEL-PANC): Preliminary findings.

LBA4132 Background: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options and is thought to be a “cold” tumor that does not respond to immunotherapy, due to a tumor microenvironment (TME) consisting of a desmoplastic stroma and poor T cell infiltrate. BXCL701 is an oral synthetic dipeptide that competitively inhibits dipeptidyl peptidases DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). BXCL701 exerts antitumor activity via inhibition of DPP8/9, which is associated with induction of proinflammatory cytokines, as well as inhibition of FAP, which disrupts tumor-stromal interactions. Preclinical xenograft models demonstrate synergy between BXCL701 and PD-1 blockade, reducing tumor growth and promoting an increase in intratumoral CD4+ and CD8+ T cells, macrophages and NK cells, with induction of host-protective immunity. Methods: This is a phase II trial of BXCL701 in patients with metastatic PDAC (mPDAC) following progression on 1 line of treatment for advanced disease and amenable to serial biopsies. BXCL701 is administered at 0.2 mg PO BID days 1-7 and 0.3 mg BID days 8-14 during cycle 1 (21 days) followed by 0.3 mg BID days 1-14 every 21 days in all other cycles, given with pembrolizumab 200 mg IV every 21 days (all cycles). The primary objective is to determine the 18-week progression-free survival rate (PFS18weeks). We estimate that historical 2nd-line PFS18weeks is 30% or less; using a Simon’s two-stage (minimax) design, a type I error rate of 0.05 and power of 80% if the true rate is 50%, we will need 19 patients in stage 1 and 20 in stage 2 (39 total). There is a safety lead-in phase of 6 patients. We plan to enroll 43 patients to account for a predicted 10% drop out of unevaluable patients. Correlative pharmacodynamic studies include imaging mass cytometry to examine 36 markers of the PDAC TME in tissue biopsies, as well as blood-based analyses of KRAS circulating tumor DNA, circulating markers of fibrosis, and IL-6. Enrollment began in Q3 2023 (NCT05558982). Results: Six patients have enrolled, 3 women and 3 men, median age 57.5 (range 37-80). One patient was progression-free at 18 weeks and 1 patient had stable disease (SD) at 9 weeks, not yet evaluable for the 18-week landmark. Objective response rate is 16% and disease control rate is 50% (RECIST: 1 partial response, -41%, and 2 patients with SD, -18% and 0%). Three patients had significant reductions in CA19-9 from baseline (-100%, -73%, and -97%). Median PFS and overall survival have not been reached (NR, 95% CI 1.45 months-NR and 0.92 months-NR, respectively). There have been no serious treatment-related safety events. The safety lead-in will complete after the next patient completes the 6-week safety window (1 patient was unevaluable). Conclusions: BXCL701 plus pembrolizumab is well-tolerated and shows early signs of potential clinical activity in patients with mPDAC refractory to chemotherapy. Clinical trial information: NCT05558982 .

A New Perspective on the Effectiveness of FDG PET/CT in Predicting KRAS Mutation in Colon Cancer Cases.

Aim: The main aim of this study was to evaluate the effectiveness of 18F-fluorodeoxyglucose (18FDG) positron emission tomography/computerized tomography (PET/CT) parameters in predicting the Kristen rat sarcoma viral oncogene(KRAS) mutation status of patients with colon cancer. Materials and Methods: Between April 2013 and December 2020, 79 patients who were diagnosed with colon cancer by colonoscopy underwent staging 18FDG PET/CT with this diagnosis and met all the inclusion criteria were included in this study. Clinical and prognostic features and also imaging (18FDG PET/CT and magnetic resonance imaging) reports of the patients were collected and analyzed retrospectively. Results: KRAS mutation was seen in 32 of patients (40.5%). No significant difference was observed between KRAS mutant and wild-type patients in terms of clinical features (tumor location, findings regarding metastasis, T stage, and tumor differentiation grade in patients who underwent surgery) and overall survival. Progression-free survival was significantly shorter in KRAS mutant patients (p = 0.018). Primary tumor standardized uptake value (SUVmean) was significantly higher in KRAS mutant cases in the whole group (p = 0.024) and in patients in whom KRAS analysis was performed only in the primary lesion (p = 0.036). The cutoff value for predicting KRAS mutation status was 7.01 g/mL (area under the curve [AUC]: 0.650, confidence interval [CI] 95%, 0.56-0.74). Conclusions: When colon and rectal cancer cases were evaluated separately, the primary tumor SUVmean value was significantly higher in KRAS mutant colon cancer cases. However, its effectiveness in predicting KRAS mutation status was low, similar to other parameters in the literature.

Primary Rosai-Dorfman disease of the central nervous system: A clinical, histological, and molecular appraisal.

Rosai-Dorfman disease (RDD) is characterized by clonal proliferation of S-100 positive histiocytes and variable emperipolesis. It commonly affects cervical lymph nodes. Central nervous system (CNS) involvement is extremely rare. We attempted to evaluate the Cyclin D1 expression and frequency of KRAS and BRAF mutations in the RDD involving the CNS. All patients with histopathologically diagnosed RDD involving CNS were recruited from 2011 to 2022. All cases were subjected to immunohistochemistry for CD68, CD163, S100, CD1a, GFAP, CD207, EMA, ALK, BRAFV600E, IgG4, IgG, and CyclinD1. The real-time polymerase chain reaction (RT-PCR) for hotspot mutation analysis of KRAS (exons 2, 3, and 4) and BRAF (V600E) was conducted on formalin-fixed paraffin-embedded tissue using a commercial kit (EntroGen). A total of seven cases were included. The median age was 31 years, with six men and one woman. It showed spinal cord (n = 4) and intracranial (n = 3) involvement. Histologically, all cases showed histiocyte-rich inflammation with evidence of emperipolesis. These histiocytes were positive for S100, CD68, CD163, and Cyclin D1, whereas negative for CD1a, CD207, and EMA. BRAF V600E was expressed in a single case. None of the control cases (demyelination and infarction) with histiocytic infiltrate showed Cyclin D1 expression. Four RDD cases showed increased IgG4-positive plasma cells (>10/HPF) and IgG4/IgG ratio (>40%). BRAF V600E mutation was detected in one case (14.28%), while none showed KRAS mutation. RDD involving CNS is extremely rare and diagnostically challenging. Nuclear Cyclin D1 expression along with S-100 positivity in the tumor cells is a strong diagnostic clue. BRAF and KRAS mutations are rare in CNS RDD.

Abstract 5692: Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers

Abstract Aberrant expression of mutant KRAS and miRNA-21 (miR-21) are commonly attribute to malignancy and tumorigenic progression of pancreatic cancers. However, few studies have explored the molecular mechanism linking these two prognostic factors. In this study, we explored the underlying molecular bridge between KRAS and miR-21 regulation in pancreatic cancer. Functional integrative analysis of KRAS, an essential oncogenic driver in pancreatic cancer progression, and together with miR-21 revealed XRN1, a 5′-3′ exoribonuclease, as RNA-linked top-scored molecule with a strong survival correlation and malignancy in pancreatic cancer patients. Depletion of XRN1 led to severe reduction in mature miR-21 expression, while unaltered precursor miR-21 levels; indicating that the XRN1 is critical in miR-21 maturation at post-Drosha step in conjunction with Dicer. Moreover, miR-21 sensor-based experiment also confirmed the critical role of XRN1 on miR-21 biogenesis. Depletion experiments showed that XRN1 positively regulates proliferation and self-renewal capacity of pancreatic cancer cells via its 5′-3′ exoribonuclease activity and concomitantly elevated the expression of PTEN and PDCD4 which are the key miR-21 cellular downstream targets. shRNA-mediated XRN1 depletion also yielded smaller tumor volume and longer survival in pancreatic cancer xenografts and syngeneic model demonstrating the therapeutic potential of targeting XRN1. Subsequent silencing of KRAS reduced XRN1 expression in pancreatic cancer cells, suggesting that KRAS plays a key role in this XRN1-miR-21 axis. Furthermore, drug-resistant pancreatic cells showed elevated expression of both KRAS and XRN1-miR-21 indicating that this axis is also associated with drug sensitivity. In sum, these results suggest that a novel KRAS-XRN1 posttranscriptional regulatory axis plays a pivotal role in tumor progression by mediating miR-21-directed signaling, and thus may suggest a novel targeting strategy for pancreatic cancer therapeutics. Significance: This study identifies molecular regulatory interplay between KRAS and XRN1-miR-21 signaling in pancreatic cancers, elucidates how KRAS-XRN1 modulates tumorigenesis via miR-21, and demonstrates that targeting this signaling can contribute to therapeutic strategies for pancreatic cancers. [Y. Seo and S. E. Oh contributed equally to this work.] Citation Format: Yoona Seo, Seung Eon Oh, Jong Heon Kim. Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5692.

Abstract C096: Targeting Syndecan1 to overcome acquired resistant to KRAS inhibitor in gastrointestinal cancer

Abstract Development of treatment resistance is a major impediment to effective cancer therapy. KRASG12C inhibitors have been recently approved by FDA and specific inhibitors targeting additional KRAS mutations are under clinical and preclinical development. These KRAS oncogene-specific inhibitors (KRASi) represent a promising new targeted therapy for KRAS-driven cancer types. However, as is the case for most targeted therapies, majority of tumors that initially respond to KRASG12C inhibitor quickly develop resistant disease. Unfortunately, the underlying mechanisms of KRASi resistance remain to be fully elucidated. We employed various in vitro and in vivo preclinical model systems to explore the development of KRASi resistance. Our study demonstrated that the YAP1- Syndecan 1 (SDC1) axis as the major driver for the bypass of KRAS-dependence and targeting YAP1-SDC1 will overcome the acquired resistance to KRAS targeted therapy. We previously identified the membrane proteoglycan SDC1 as a KRAS downstream surrogate whose cell surface localization is tightly controlled by oncogenic KRAS signaling and is critical for KRAS-driven tumor development. Here, our analysis of KRAS driven genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) and human pancreatic or colorectal cancer (CRC) cells with KRASG12C mutation discovered that, while cell surface SDC1 is reduced upon acute genetic or pharmacological of oncogenic KRAS, plasma membrane SDC1 is recovered in tumor cells that become resistant to KRAS inhibition (KRAS-bypass cells). Surface localization of SDC1 leads to the activation of multiple receptor tyrosine kinases (RTKs), resulting in the resistance to KRAS blockade. We further identified that the recovery of plasma membrane SDC1 in KRAS-bypass cells is driven by YAP1 oncogene, which mediates the transcription suppression of ARF6 GAPs, leading to the activation of ARF6, a small GTPase required for SDC1 membrane recycling. Genetic depletion of YAP1 or SDC1, as well as pharmacological inhibition of YAP1-SDC1 axis abolishes the tumorigenic activity of KRAS-bypass tumor cells and sensitizes KRAS-driven tumors to KRASi. Moreover, YAP1 and surface SDC1 are accumulated in CRC PDX models with acquired resistance to KRASG12C-inhibitor. Importantly, pharmacological targeting YAP1 significantly suppresses surface SDC1 in KRASi-resistant PDXs and sensitizes the tumors to KRASG12C-inhibitor. Taken together, our research identifies new actionable therapeutic targets and approaches to overcome resistance to KRAS inhibition, thus implicating the therapeutic potential to greatly improve the clinical outcome of patients with KRAS mutated cancers. Citation Format: Mitsunobu Takeda, Alexey Sorokin, Madelaine Theardy, Oluwadara Coker, Scott Kopetz, Haoqiang Ying, Giulio Draetta, Ronald A. DePinho, Wantong Yao. Targeting Syndecan1 to overcome acquired resistant to KRAS inhibitor in gastrointestinal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C096.

Crystallographic Studies of KRAS in Complex with Small Molecules and RAS-Binding Proteins.

RAS proteins play a vital role in regulating downstream signaling and essential cellular processes, positioning them as key players in normal cellular physiology and disease development. Among the various isoforms of RAS, KRAS stands out as one of the most frequently mutated genes in human cancer. The prevalence of RAS mutations in cancer often involves single amino acid substitutions at codons 12, 13, or 61. These mutations disrupt the RAS protein's inherent ability to transition between its active and inactive states, resulting in a constant activation signal and driving uncontrolled cell growth. Crystallization and structural analysis of KRAS with inhibitors and RAS-binding proteins play a pivotal role in unraveling the structural and mechanistic details of KRAS function, aiding in drug discovery efforts, and advancing our understanding of KRAS-driven diseases. Here, we present our experimental methodology for crystallizing KRAS in the presence of covalent or non-covalent small molecules and proteins acting as effectors or regulators of RAS. We detail the techniques for successful crystallization and the subsequent optimization of crystallization conditions. The resulting crystals and their structures will provide valuable insights into the key interactions between KRAS and its partner proteins or potential inhibitors, offering a foundation for developing targeted therapies that are more potent and selective against KRAS-driven cancers.

A Narrow NGS Panel in the Analysis of KRAS p.G12C Mutation in Longitudinal Series of NSCLC Patients: A Pilot Study

A Narrow NGS Panel in the Analysis of KRAS p.G12C Mutation in Longitudinal Series of NSCLC Patients: A Pilot Study

Mutation Analysis of KRAS and BRAF in Iranian Colorectal Cancer patients: A Novel Variant in Exon 15 of BRAF.

Mitogen-Activated Protein Kinase (MAPK) pathway and its downstream signaling pathways, play an important role in intracellular signaling. Mutations in KRAS (activating mutation) and BRAF proto-oncogenes are identified as key finding of colorectal cancer. The aim of this study was to examine mutation analysis of KRAS and BRAF in Iranian Colorectal cancer patients. We used fifty archived formalin fixed paraffin-embedded (FFPE) blocks of Iranian colorectal cancer patients. DNA was extracted from FFPE blocks for PCR assay. The quality of PCR products was determined using horizontal electrophoresis. Then, sequencing and analysis of the sequencing results were performed to investigate variation status in the sequences. KRAS exons and BRAF genes exon 15 in 50 CRC patients were analyzed, among the 19 mutant KRAS samples, 18 (36%) patients had a single base substitution (synonymous mutation) in exon 5, p. Arg161Arg (c.483G>A) and 1 (2%) patient in exon 2 (codon 12), p. Gly12Cys (c.34G>T). Also, we observed two mutations p. Val600Glu (c.1799 T>A) and p. Ser616Thr (c.1846T>A) in exon 15 of BRAF gene. We found a novel variant in BRAF gene. The p. Ser616Thr (c.1846T>A) mutation was not previously reported and we conclude that other new mutations can be identified in KRAS and BRAF which may lead to colorectal cancer.

Comparative bioinformatic analysis of KRAS, STK11 and KEAP1 (co-)mutations in non-small cell lung cancer with a special focus on KRAS G12C

ObjectivesMutations in STK11 (STK11MUT) and KEAP1 (KEAP1MUT) occur frequently in non-small cell lung cancer (NSCLC) and are often co-mutated with KRAS. Several studies linked the co-occurrence of KRASMUT + STK11MUT, as well as KRASMUT + KEAP1MUT to reduced response to immune checkpoint inhibitors (ICI) and even a negative impact on survival. Data focusing STK11 + KEAP1 co-mutations or the triple mutation (KRAS + STK11 + KEAP1) are scarce. The recent availability of KRAS-G12C inhibitors increases the clinical relevance of those co-mutations in KRAS-mutated NSCLC. Materials and methodsWe present a comprehensive bioinformatic analysis encompassing six datasets retrieved from cBioPortal. ResultsIndependent of the treatment, triple mutations and STK11MUT + KEAP1MUT were significantly associated with a reduced overall survival (OS). Across treatments, OS of patients with a KRAS G12C triple mutation was significantly reduced compared to patients with KRAS G12C-only. Under ICI-therapy, there was no significant difference in OS between patients harboring the KRAS G12C-only and patients with the KRAS G12C triple mutation, but a significant difference between patients harboring KRAS non-G12C and KRAS non-G12C triple mutations. Triple mutated primary tumors showed a significantly increased frequency of distant metastases to bone and adrenal glands compared to KRAS-only mutated tumors. Additionally, our drug response analysis in cancer cell lines harboring the triple mutations revealed the WNT pathway inhibitor XAV-939 as a potential future drug candidate for this mutational situation. ConclusionThe triple mutation status may serve as a negative prognostic and predictive factor across treatments compared to KRASMUT-only. KRAS G12C generally seems to be a negative predictive marker for ICI-therapy.

Mesonephric-like adenocarcinoma of the female genital tract: possible role of KRAS-targeted treatment—detailed molecular analysis of a case series and review of the literature for targetable somatic KRAS-mutations

PurposeMesonephric-like adenocarcinomas (MLA) of the female genital tract represent a rare and relatively recently described neoplasm exhibiting characteristic morphologic and immunohistochemical findings commonly associated with a KRAS-mutation. Most cases display an aggressive clinical behavior, but knowledge about treatment approaches is limited, especially for targeting KRAS.MethodsWe report a series of eight cases with a detailed molecular analysis for KRAS. These cases as well as the data of previously published cases with detailed information regarding KRAS-mutational events were reviewed for a potential targeted approach and its prognostic impact.ResultsBoth the uterine and ovarian MLA harbor a somatic KRAS-mutation in about 85% of the reported cases, affecting the hotspot codons 12 and 13. 15.7% of the endometrial and 15.6% of ovarian MLA are wild type for KRAS. A p.G12A-alteration was seen in 5.6% (5/89) of the endometrial and in 6.2% (2/32) of the ovarian tumors, for p.G12C in 7.9% and 6.2%, for p.G12D in 32.6% and 34.5% and for p.G12V in 36% and 37.5%, respectively. Very limited data are available regarding the prognostic impact of different mutational sites within the KRAS-gene without significant prognostic impact.ConclusionBecause of a specific p.G12C-KRAS somatic mutation, only the minority of MLA (7.9% with uterine and 6.2% with ovarian primary) are potentially targetable by sotarasib in that rare but aggressive subtype of adenocarcinoma of the female genital tract. Until now, the different location of a somatic KRAS-mutation is of no prognostic impact.

New stochastic devices for simultaneous analysis of mismatch repair proteins and KRAS in biological samples

Two stochastic microsensors based on the immobilization of inulins: inutec and frutafit in a paste obtained from a graphene decorated with nitrogen (10.8 %), and boron (2.6 %), and paraffin oil (IR purity) were designed, characterized and validated for the molecular recognition and analysis of mismatch repair proteins like MLH1, MSH2, MSH6, PMS2, and of KRAS in biological samples such as: whole blood, urine, saliva, and tumoral tissues. The sensor based on inutec exhibited the largest working concentration range (10 decades of concentration) and the lowest limit of determination (0.32fg mL−1) when used for the assay of MLH1, and the wider working concentration range (8 decades of concentration), and the lowest limit of determination (2.30fg mL−1) when used for the assay of MSH6. The sensor based on frutafit exhibited the largest working concentration range (10 decades of concentration) when used for the determination of KRAS, and the wider working concentration range (5 decades of concentration), and the lowest limit of determination (1.00fg mL−1) when used for the assay of PMS2. No influence of the modifier (inutec and frutafit) was recorded on the linear concentration range (10 decades of concentration), and on the limit of determination (1.00 fg mL−1) for the assay of MSH2. The recoveries of MLH1, MSH2, MSH6, PMS2, and of KRAS in whole blood, urine, saliva, and tumoral tissues were higher than 98.00 with RSD (%) values lower than 0.10 %.

Evaluation of the Association Between CRNDE Plasma Expression Level, KRAS, NRAS, and BRAF Variants in Patients with advanced CRC

Colorectal cancer is one of the most common types of cancer. CRNDE is a novel-defined lncRNA. We evaluated the possibility of using CRDNE circulating lncRNA as a noninvasive biomarker. Also, we examined the effect of circulating CRNDE lncRNA on the pathogenesis of CRC and its association with the KRAS, NRAS, and BRAF somatic variants commonly observed in CRC. In this study, we enrolled plasma and FFPE tissue samples of 50 advanced CRC patients and plasma samples of 31 individuals in the control group of similar ages. Then, we performed plasma extraction, total RNA isolation, cDNA synthesis, and circulating lncRNA expression analysis, respectively. Also, we carried out KRAS, NRAS, and BRAF somatic variant analysis from FFPE tissues. Our results showed that the expression level of CRNDE (p= 0.002) were significantly upregulated in the CRC when it was compared to with the control group. The calculated area under the curve of the receiver operating characteristic was 0.70. We found a statistically significant difference between the KRAS somatic variants and the circulating CRNDE lncRNA (p= 0.031). This study demonstrated that CRNDE circulating lncRNAs may be used as a potential non-invasive biomarker in CRC. In our study, it was determined that there is a significant relationship between the frequently observed KRAS somatic variants and CRNDE in advanced-stage CRC cases. To the best of our knowledge, this is the first study to show the association of CRNDE circulating lncRNA with advanced CRC. Moreover, it is the first study to show a relationship between the KRAS somatic variants between circulating CRNDE lncRNA in advanced CRC. Keywords: CRNDE, Circulating lncRNA, KRAS, CRC, RT-PCR

A Critical Review of the Prognostic and Predictive Implications of KRAS and STK11 Mutations and Co-Mutations in Metastatic Non-Small Lung Cancer.

The Kirsten rat sarcoma viral oncogene homolog (KRAS) and serine/threonine kinase 11 (STK11) co-mutations are associated with the diverse phenotypic and heterogeneous oncogenic subtypes in non-small cell lung cancer (NSCLC). Due to extensive mixed evidence, there needs to be a review of the recent KRAS and STK11 mutation literature to better understand the potential clinical applications of these genomic biomarkers in the current treatment landscape. This critical review highlights the clinical studies that have elucidated the potential prognostic and predictive implications of KRAS mutations, STK11 mutations, or KRAS/STK11 co-mutations when treating metastatic NSCLC across various types of treatments (e.g., immune checkpoint inhibitors [ICIs]). Overall, KRAS mutations are associated with poor prognoses and have been determined to be a valid but weak prognostic biomarker among patients diagnosed with NSCLC. KRAS mutations in NSCLC have shown mixed results as a predictive clinical biomarker for immune checkpoint inhibitor treatment. Overall, the studies in this review demonstrate that STK11 mutations are prognostic and show mixed results as predictive biomarkers for ICI therapy. However, KRAS/STK11 co-mutations may predict primary resistance to ICI. Prospective KRAS/STK11-biomarker-driven randomized trials are needed to assess the predictive effect of various treatments on the outcomes for patients with metastatic NSCLC, as the majority of the published KRAS analyses are retrospective and hypothesis-generating in nature.

Phase II trial of BXCL701 and pembrolizumab in patients with metastatic pancreatic ductal adenocarcinoma (EXPEL-PANC).

TPS4194 Background: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options and is thought to be a “cold” tumor that does not respond to immunotherapy, due to a tumor microenvironment (TME) consisting of a desmoplastic stroma and poor T cell infiltrate. BXCL701 is an oral synthetic dipeptide that competitively inhibits dipeptidyl peptidases DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). BXCL701 exerts antitumor activity via inhibition of DPP8/9, which is associated with induction of proinflammatory cytokines, as well as inhibition of FAP, which disrupts tumor-stromal interactions. Preclinical xenograft models demonstrate synergy between BXCL701 and an anti-PD-1 monoclonal antibody, reducing tumor growth and promoting an increase in intratumoral CD4+ and CD8+ T cells and NK cells. The combination of BXCL701 and pembrolizumab is already being studied in a phase II trial in patients with prostate cancer (NCT03910660) and a multi-cohort basket study (NCT04171219). Methods: This is a phase II trial of BXCL701 administered at 0.2 mg PO BID days 1-7 and 0.3 mg BID days 8-14 during cycle 1 (21 days) followed by 0.3 mg BID days 1-14 every 21 days in all subsequent cycles, given with pembrolizumab 200 mg IV every 21 days (all cycles). The primary objective is to determine the 18-week progression-free survival rate (PFS18weeks) in patients with metastatic PDAC treated in the second-line setting. We estimate that historical 2nd-line PFS18weeks is 30% or less; using a Simon’s two-stage (minimax) design, a type I error rate of 0.05 and power of 80% when the true response rate of 50%, we will need 19 patients in stage 1 and 20 in stage 2 (a total of 39). We plan to enroll 43 patients to account for a predicted 10% drop out of unevaluable patients. Eligible patients must have metastatic PDAC and disease progression or intolerance to only 1 line of therapy for metastatic disease, without exposure to prior immunotherapy. Patients must have measurable disease amenable to serial biopsies. Correlative pharmacodynamic studies include imaging mass cytometry to examine 34 markers of the PDAC TME in tissue biopsies, as well as blood-based analyses of KRAS circulating tumor DNA, circulating markers of fibrosis, and IL-6. Enrollment began in Q1 2023. Clinical trial information: NCT05558982 .

The five-year KRAS, NRAS and BRAF analysis results and treatment patterns in daily clinical practice in Slovenia in 1st line treatment of metastatic colorectal (mCRC) patients with RAS wild-type tumour (wtRAS) - a real- life data report 2013-2018.

We preformed a Phase IV non-interventional study to assess KRAS, NRAS and BRAF status in metastatic colorectal cancer (mCRC) patients suitable for 1st line treatment and to evaluate the decisions for 1st line treatment considering the treatment goals in the RAS wild type (wt) patients. The aim of our study was also to evaluate the influence of a waiting period for biomarkers analysis on the start of first-line treatment. Patients with histologically confirmed mCRC adenocarcinoma suitable for first-line treatment fulfilling all inclusion criteria were included in the study. The KRAS, NRAS and BRAF analysis was performed from tissue samples of primary tumor site or metastatic site. All included patients have given consent to participate in the study by signing the informed consent form. From April 2013 to March 2018 at the Institute of Oncology Ljubljana 650 patients were included, 637 of them were treated with first- line systemic treatment according to RAS and BRAF status. Remaining 13 patients with mCRC did not receive systemic first-line treatment. The distribution of patients with KRAS mutated and wild-type tumors, was almost equal, 48.8% and 47.9% respectively, 89 % of the patients had wt NRAS tumours and 86.1% had wt BRAF tumours. The most frequently prescribed treatment was bevacizumab-based therapy (53.1%), either in combination with doublet chemotherapy or with mono-chemotherapy. EGFR inhibitors cetuximab and panitumumab were prescribed in wt RAS mCRC patients (30.9%). The waiting period for biomarkers analysis was two weeks. Our real-world data, single centre 5-year analysis showed that the distribution between wild type and mutated type tumors of the patients with mCRC was approximately the same, as worldwide, so the Slovenian population with mCRC has the same ratio distribution of KRAS, NRAS and BRAF wild and mutated genes. We concluded that a two-week waiting period for biomarkers analysis did not influence the first line treatment decision, so it was in the accordance with the worldwide treatment guidelines based on evidence-based medicine.

Analysis of KRAS–Ligand Interaction Modes and Flexibilities Reveals the Binding Characteristics

KRAS, a common human oncogene, has been recognized as a critical drug target in treating multiple cancers. After four decades of effort, one allosteric KRAS drug (Sotorasib) has been approved, inspiring more KRAS-targeted drug research. Here, we provide the features of KRAS binding pockets and ligand-binding characteristics of KRAS complexes using a structural systems pharmacology approach. Three distinct binding sites (conserved nucleotide-binding site, shallow Switch-I/II pocket, and allosteric Switch-II/α3 pocket) are characterized. Ligand-binding features are determined based on encoded KRAS-inhibitor interaction fingerprints. Finally, the flexibility of the three distinct binding sites to accommodate different potential ligands, based on MD simulation, is discussed. Collectively, these findings are intended to facilitate rational KRAS drug design.

Mutation status of full RAS and BRAF in 169 Moroccan patients with colorectal cancer.

216 Background: Morocco has the highest cancer mortality rate in MENA Countries. Colorectal cancer (CRC) is classified as the first digestive cancer and remains a burden in Morocco. According to the data on GLOBOCAN 2020, there were 4324 new CRC patients and 2374 deaths, accounting for 7.3% and 2.8% of all cancers, respectively. Our study aimed to investigate the frequency of the full RAS ( KRAS, NRAS) and BRAF genes in CRC patients from Morocco and their possible associations with clinico-pathological features. Methods: Archived FFPE of 169 CRC patients were screened for KRAS, NRAS and BRAF mutations by Idylla technology. Results: Full RAS mutations were identified in 46.1% (42% in KRAS, 4.1% in NRAS). In KRAS gene, exon 2 mutations accounted for 84.5% (69% in codon 12, 15.5 % in codon 13). Within codon 12, KRAS G12D and G12C were more frequently detected (29.5% and 16.9%, respectively). Detection of KRAS mutations, and particularly G12D and G12C subtypes, are of large significance for CRC patients, have possible therapeutic implications. Within codon 13, the most frequently observed mutation was G13D (15.5 %). Outside exon 2, the mutation rate was 35.1% (8.4% in exon 3 and 26.7% in exon 4). Concurrent KRAS mutations were identified in 8 cases, which suggests that multiple mutations can occur in the same or different codons. In NRAS gene, the mutation rates of exon 2 and 3 were 71.4% and 57.1% respectively. G13V and Q61K were the most common mutations, accounting for 28.6 % of each. Concurrent KRAS mutations were identified in 2 cases. Of the 169 samples, mutations in the BRAF gene at V600E were detected in 3.5%. Combined mutational analysis of KRAS, NRAS and BRAF was able to identify 49.6% of patients with CRC as likely non-responders to anti-EGFR therapy. There was an association between KRAS mutations and age, which were higher in the age group>50 years old (p=0.022). Tumors in the left colon (36.61%) are more likely to harbor mutations in KRAS than the rectum (19.7%) in both sexes. The adenocarcinoma well-differentiated was the most frequent for patients with KRAS mutations (54.9%). No significant clinicopathologic correlations with NRAS and BRAF mutations were identified. Conclusions: Beside established anti-CRC treatment, better understanding of the causality of CRC can be established by combining epidemiology and genetic/epigenetic on CRC etiology in Morocco. This approach may be able to significantly reduce the burden of disease in the country. Moreover, the Moroccan government should develop policy on CRC prevention and public health programs which may serve as a feasible setting to increase public awareness on lifestyle risk factors.

Morphological and Molecular Genetic Retrospective Analysis of KRAS, NRAS, BRAF and MSI (Microsatellite Instability) Pattern in Colorectal Cancer

Gastroenterology Medicine & Research Morphological and Molecular Genetic Retrospective Analysis of KRAS, NRAS, BRAF and MSI (Microsatellite Instability) Pattern in Colorectal Cancer Flavia Vrenna, Anastasia Nocita, Giuseppina Alberto, Rosanna Patarino and Federico Tallarigo* Department of Surgical Pathology, Department of Medicine and Oncology, St John of God Hospital, Crotone, Italy *Corresponding author: Federico Tallarigo, Department of Surgical Pathology, Department of Medicine and Oncology, St John of God Hospital, Crotone, Italy Submission:December 05, 2022;Published: January 26, 2023 DOI: 10.31031/GMR.2023.07.000662 ISSN 2637-7632Volume7 Issue3