Wild-type KRAS Research Articles

The KRAS-Mutant Consensus Molecular Subtype 3 Reveals an Immunosuppressive Tumor Microenvironment in Colorectal Cancer

Colorectal cancers (CRC) with KRAS mutations (KRASmut) are frequently included in consensus molecular subtype 3 (CMS3) with profound metabolic deregulation. We explored the transcriptomic impact of KRASmut, focusing on the tumor microenvironment (TME) and pathways beyond metabolic deregulation. The status of KRASmut in patients with CRC was investigated and overall survival (OS) was compared with wild-type KRAS (KRASwt). Next, we identified CMS, and further investigated differentially expressed genes (DEG) of KRASmut and distinctive pathways. Lastly, we used spatially resolved gene expression profiling to define the effect of KRASmut in the TME regions of CMS3-classified CRC tissues. CRC patients with KRASmut were mainly enriched in CMS3. Their specific enrichments of immune gene signatures in immunosuppressive TME were associated with worse OS. Activation of TGFβ signaling by KRASmut was related to reduced pro-inflammatory and cytokine gene signatures, leading to suppression of immune infiltration. Digital spatial profiling in TME regions of KRASmut CMS3-classified tissues suggested up-regulated genes, CD40, CTLA4, ARG1, STAT3, IDO, and CD274, that could be characteristic of immune suppression in TME. This study may help to depict the complex transcriptomic profile of KRASmut in immunosuppressive TME. Future studies and clinical trials in CRC patients with KRASmut should consider these transcriptional landscapes.

SOX plus bevacizumab versus SOX plus cetuximab as initial treatment of recurrent advanced colorectal cancer with wild-type KRAS (MCSGO-1107 study): A phase II randomized study.

136 Background: Although clinical outcomes of chemotherapy in patients with locally advanced or metastatic colorectal cancer (CRC) have improved over the last decade, the standard-of-care chemotherapy regimens for patients with unresectable RAS wild-type CRC remain to be discussed. The aim of this study was to compare S-1 and oxaliplatin (SOX) + bevacizumab (B-mab) with SOX + cetuximab (C-mab) in patients with previously untreated, unresectable, locally advanced, or metastatic CRC with wild-type KRAS. Methods: This phase II, randomized, open-label, multicenter study compared the efficacy and safety of SOX+B-mab with SOX+C-mab in patients with previously untreated, unresectable, locally advanced, or metastatic CRC with wild-type KRAS. Forty-five patients were enrolled in this study between February 2012 and October 2016. Results: The overall response rate (ORR) for the SOX+B-mab group was 59.1%, whereas that for the SOX+C-mab group was 43.5% (p = 0.29). The disease control rate (DCR) for the SOX+B-mab group was 90.9%, whereas that of the SOX+C-mab group was 91.3% (p = 0.96). For all patients, median OS were 25.3 months (95% CI: 16.5–39.4 months) in the SOX+B-mab group and 15.5 months (95% CI: 7.30–30.4 months, p = 0.167) in the SOX+C-mab group. Median PFS were 11.7 months (95% CI: 7.37–18.2 months) in the SOX+B-mab group and 5.5 months (95% CI: 3.36–10.1 months, p = 0.077) in the SOX+C-mab group. In the SOX+B-mab group, OS and PFS were not significantly different with and without early tumor shrinkage (ETS). However, in the SOX+C-mab group, patients with ETS had significantly better OS (30.4 months [95% CI: 8.0–44.3 months, p = 0.032]) and PFS (12.0 months [95% CI: 5.1–19.7 months, p = 0.003]) than those without ETS. Conclusions: The safety and efficacy of SOX+B-mab and SOX+C-mab as initial treatment for unresectable CRC with wild-type KRAS were almost the same. ETS was more correlated with PFS in the SOX+C-mab group than in the SOX+B-mab group, suggesting consideration of treatment strategy based on ETS may improve patient prognosis, especially in patients receiving the SOX+C-mab regimen. Clinical trial information: UMIN000006706 .

Emergence of RAS mutations in RAS wild type metastatic colorectal cancer patients receiving cetuximab as first-line treatment: A prospective study.

38 Background: Metastatic colorectal cancers (mCRC) with wild-type KRAS are often sensitive to anti-EGFR, but occasionally develop resistance within treatment. A major limitation of treatment that selectively target kinase signaling pathways is the emergence of secondary resistance. However, interlink between emergence of KRAS mutations and secondary resistance to anti-EGFR remains to be further elucidated. In this study, we investigate evolutional changes of KRAS mutations through liquid biopsy in mCRC patients under cetuximab-treatment. Methods: Patients enrolled to receive cetuximab plus FOLFIRI or mFOLFOX6 as first-line treatment. The treatment was anticipated to be continued until disease-progression, intolerable toxic effects, or withdrawal of consent. Blood samples from patients were collected before and every three months during the cetuximab-treatment, and were required at disease-progression. These samples were evaluated for KRAS mutation using the MassARRAY platform. The primary endpoint was the percentage of patients with KRAS mutation detected during treatment. The clinical outcomes of these patients with emergence of mutant KRAS were further analyzed. Results: 108 mCRC cetuximab-treated patients, 95 patients combined with FOLFIRI and 13 patients with mFOLFOX6, were enrolled between January 2018 and June 2020. The median follow-up time was 26.5 months [interquartile range (IQR), 17.0-37.0 months]. The median duration of cetuximab-treatment was 9.0 months (IQR, 5.0-12.0 months). The percentage of detected KRAS mutations during cetuximab-treatment was 9.26%. Median emergent time of KRAS mutation from initial treatment was 5.0 months (IQR, 2.0-7.5 months). The median duration from KRAS mutation to disease-progression was 3.0 months (IQR, 2.0-6.5 months). The median progression-free survival (PFS) and overall survival (OS) of emergent RAS mutations and RAS wild-type were 8.0 versus 19.0 months ( P = 0.002) and 20.0 versus not-reach months ( P = 0.027), respectively. Conclusions: The identified KRAS mutations as a predictor of early resistance during cetuximab therapy, indicating that emergence of KRAS-mutant clones can be detected 3 months prior to radiographic progression. Emergent RAS mutations were associated with less favorable patient outcomes. Clinical trial information: NCT03401957 .

Plasma central carbon metabolite changes associated with KRAS mutation and circulating tumor DNA (ctDNA) status in colorectal cancer (CRC).

181 Background: KRAS mutations have been widely characterized as markers of poor prognosis in CRC. In stage IV CRC, KRAS mutations are predictive of benefit to anti-EGFR therapy. ctDNA has increasingly been recognized as a prognostic biomarker in CRC as well. We evaluated the association between plasma metabolites and KRAS mutation or ctDNA status in a longitudinal, observational cohort of patients with stage I-IV CRC. Methods: This was a retrospective analysis of prospectively collected blood samples from a single-institute cohort of patients with stage I-IV CRC. All blood samples were collected at pre-chemotherapy baseline. A modified Epi proColon 2.0 CE (Epigenomics AG) assay was used for plasma ctDNA testing on the methylated SEPTIN9 gene (mSEPT9). ctDNA positivity was defined as a mSEPT9 percentage of methylation reference (PMR) value greater than zero. Up to 150 metabolites of central carbon metabolism were analyzed by mass spectrometry and high-performance liquid chromatography. Analytes were compared by relative area under the curve (AUC) and differences evaluated by ANOVA. The mean AUC was used in patients with metabolites measured from > 1 timepoint of collection. Patients were stratified by ctDNA status (positive or negative) and KRAS mutant (MT) or wildtype (WT) status. Results: A total of 32 patients were included with median age 65 years (range 20-90). The majority were female (53%) and had stage IV disease (78%). Of 25 patients with stage IV CRC, 88% had pre-chemotherapy blood samples collected in the first-line setting. Most patients were KRAS MT (44%) compared to KRAS WT (37%) or unknown KRAS status (19%). The most common KRAS MT subtypes were G12D (29%), G12V (29%), G13D (21%), and G12C (14%). The mean overall survival in this cohort was 27.4 months while the mean mSEPT9 PMR value was 2553.6. When stratified by ctDNA status, ctDNA positivity was associated with decreased levels of essential amino acids (lysine, methionine, threonine) and the non-essential amino acid arginine (all p < 0.05). Compared to KRAS WT tumors, KRAS MT tumors were associated with increased levels of proline, phenylalanine, and intermediates of glycolysis (lactate), MTA cycle (SAM, 5-Methioadenosine), and O-GlcNAcylation (GlcNac, all p < 0.05). Conclusions: We are the first to demonstrate the feasibility of associating central carbon metabolites with ctDNA and KRAS mutation status. As ctDNA positivity and KRAS MT status have evolving prognostic potential in CRC, associated metabolic signatures may identify metabolic pathways for novel biomarker development. Our findings also show that KRAS MT CRC appears to be enriched in intermediates of glycolytic, methyl donor, and O-GlcNAcylation pathways.

A phase 1b/2, open-label study of amivantamab monotherapy or in combination with standard-of-care chemotherapy in participants with advanced or metastatic colorectal cancer.

TPS279 Background: Amivantamab, a fully human EGFR and MET bispecific antibody, has shown clinical activity against tumors with primary activating EGFR mutations, EGFR resistance mutations, or MET pathway activation. Amivantamab has demonstrated activity in both EGFR- and MET-driven non-small cell lung cancer, with preclinical evidence demonstrating its ability to recruit immune effector cells. While two anti-EGFR antibodies are incorporated as part of the standard of care (SoC) for metastatic colorectal cancer (mCRC) patients, MET is highly expressed or amplified in subsets of mCRC and additionally plays a role in mediating resistance to anti-EGFR therapies; therefore, amivantamab may provide benefit in this setting. Methods: This open-label, multicenter, global Ph1b/2 study will assess the safety and anti-tumor activity of amivantamab as a monotherapy and characterize the safety and tolerability of amivantamab in addition to SoC chemotherapy in KRAS, NRAS, BRAF, and EGFR ectodomain wild type participants with advanced or metastatic CRC. The Ph2 amivantamab monotherapy Cohorts A and B will assess the anti-tumor activity in participants with left-sided CRC who have progressed on or after SoC fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy and an anti-VEGF treatment, without (Cohort A) or with (Cohort B) prior exposure to anti-EGFR treatment. The Ph2 amivantamab monotherapy Cohort C will assess the antitumor activity in participants with right-sided CRC who have progressed on or after SoC fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy and an anti-VEGF treatment, with or without an anti-EGFR treatment. The Ph1b dose confirmation cohorts (Ph1b-D and Ph1b-E) will assess the safety and confirm the recommended Ph2 combination dose (RP2CD) of amivantamab in addition to SoC chemotherapy regimens (mFOLFOX6 or FOLFIRI). Upon confirmation of the RP2CD, the Ph2 Cohorts D and E, which are distinct cohorts from Ph1b-D or Ph1b-E, will further characterize the safety, tolerability, and preliminary anti-tumor activity of amivantamab in addition to SoC mFOLFOX6 or FOLFIRI in mCRC patients who have progressed after front-line therapy. The primary objectives are to assess the anti-tumor activity of amivantamab as a monotherapy and characterize the safety of amivantamab when added to SoC chemotherapy in participants with mCRC (Ph2 cohorts), as well as to assess the RP2CD of amivantamab when added to SoC chemotherapy (Ph1b). The key secondary objectives are to characterize the safety of amivantamab as a monotherapy and to assess the anti-tumor activity of amivantamab when added to SoC chemotherapy in participants with mCRC. This study is currently enrolling (NCT05379595) as of August 2022 in 12 countries, with goal enrollment of 225 participants. Clinical trial information: NCT05379595 .

Biomarker analysis to predict response in patients with metastatic mismatch repair proficient colorectal cancer treated with regorafenib and nivolumab.

228 Background: We previously conducted a phase I/Ib study with regorafenib and nivolumab in patients with refractory metastatic mismatch repair proficient (pMMR) colorectal cancer (CRC). This study aimed to investigate the biomarkers that predict the treatment response. Methods: Out of the 51 patients who received regorafenib and nivolumab, 22 archival pretreatment tumor samples were subjected to the Xerna TME Panel, a machine learning-based RNA-sequencing biomarker assay and were classified into one of four TME biomarker subtypes: Angiogenesis (A), Immune Active (IA), Immune Desert (ID), or Immune Suppressed (IS). Potential predictive biomarkers including the TME subtypes, KRAS (wild type vs mutant), PD-L1 (negative vs. positive, samples with > 1% tumor cells for PD-L1 were considered positive), CD8 expression (low vs. high), and Treg cells (low vs. high) in tumor microenvironment were evaluated for correlation with overall survival (OS), progression free survival (PFS) and disease control rate (DCR, defined as complete response + partial response + stable disease). Results: Among the 22 patients, 16 (72.7%) had liver metastasis and 15 (68.2%) had lung metastasis. KRAS mutation was found in 16 (68.2%) patients. 11/21 (52.4%) were positive for PD-L1. 12 (54.5%) had high CD8 expression, whereas 9/21 (42.9%) had high Treg cells in tumor microenvironment. Ten (45.5%) patients were classified as biomarker-positive (IA + IS subtypes) and 12 (54.5%) were biomarker-negative (A + ID) based on Xerna TME panel. Two (9.1%) patients achieved partial response, 12 (54.5%) had stable disease, and five (22.7%) developed progressive disease. The median PFS was 5.6 months and median OS was 13.1 months. No significant correlation was observed between RAS mutation (p = 0.664, p = 0.609), PD-L1 expression (p = 0.287, p = 0.173), CD8 (p = 0.152, p = 0.456) and PFS or OS. Low Treg was found to be associated with prolonged PFS (median: 9.8 vs. 1.9 months, p = 0.011) but not OS (p = 0.280). Similarly, only low Treg level was related with DCR (83.3% vs. 33.3%, p = 0.032). While not reaching statistical significance, Xerna TME biomarker-positive patients showed trends for higher median PFS (7.9 months vs. 4.1 months, p = 0.254), median OS (15.75 months vs. 11.9 months, p = 0.378), and higher DCR (70% vs. 58%, p = 0.675) compared to biomarker-negative patients. Additionally, the two patients with partial responses were Xerna TME biomarker-positive. Conclusions: Our study demonstrated that low Treg in tumor microenvironment is correlated with better prognosis in patients with refractory metastatic pMMR CRC who were treated with regorafenib plus nivolumab. Xerna TME panel analysis of these patients also showed trends for predictive clinical benefit. Prospective and larger cohort studies are needed to better define predictive biomarkers for this combination in the future.

Adagrasib: First Approval.

Adagrasib (KRAZATI™) is an orally available, potent, irreversible, small molecule inhibitor of KRAS G12C mutant isoform being developed by Mirati Therapeutics for the treatment of solid tumours harbouring KRAS G12C oncogenic driver mutation, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Adagrasib covalently binds to the mutant cysteine in KRAS G12C and locks the mutant KRAS protein in its inactive state, thereby preventing downstream signalling without affecting wild-type KRAS protein. In December 2022, adagrasib received its first approval in the USA for the treatment of adults with KRAS G12C-mutated locally advanced or metastatic NSCLC (as determined by an FDA approved test) who have received ≥1 prior systemic therapy. It was approved under accelerated approval based on objective response rate and duration of response, and its continued approval for this indication may be contingent upon verification and description of a clinical benefit in a confirmatory trial(s). The drug is under regulatory review for NSCLC in the European Union and is in development for CRC in the US. Clinical studies of adagrasib in solid tumours, including CRC, are underway in several countries. This article summarizes the milestones in the development of adagrasib leading to this first approval for KRAS G12C-mutated locally advanced or metastatic NSCLC.

Predictive and prognostic features of metastatic colorectal cancer arising from the transverse colon: A pooled analysis of the CCTG/AGITG CO.17 and CO.20 randomized trials.

124 Background: Primary tumour location is predictive of anti-EGFR benefit and prognostic in metastatic colorectal cancer (mCRC). Transverse colon cancers are often categorized as right sided, but the optimal cut point is unclear. Canadian Cancer Trials Group (CCTG)/Australasian Gastro-Intestinal Trials Group ( AGITG) CO.17 compared Cetuximab (Cet) vs. best supportive care (BSC) in mCRC. CCTG/AGITG CO.20 studied the addition of Brivanib Alaninate to Cet in pre-treated KRAS wildtype (WT) mCRC. We investigated the predictive and prognostic features of transverse colon primary location in a pooled cohort from these trials. Methods: Data from patients with RAS WT mCRC from CO.17 and KRAS WT mCRC from CO.20 randomized to cetux were analyzed for treatment outcomes according to location - right, transverse and left. The cecum to transverse colon was considered right sided, while the splenic flexure to rectum was considered left sided. Results: 553 patients were included, 201 (36.3%) from CO.17 and 352 (63.7%) from CO.20. Primary site distribution was: 32 (5.8%) transverse, 101 (18.3%) right and 420 (75.9%) left. On multivariate analysis from 457 (82.6%) patients treated with Cet, left side was associated with superior OS (HR, 0.40; 95% CI, 0.24-0.68, p=0.0006) and PFS (HR, 0.48; 95% CI,0.29-0.79, p=0.004) compared to transverse colon. No significant difference was noted in OS (HR, 0.74; 95% CI, 0.41-1.31, p=0.30) and PFS (HR, 0.79; 95% CI, 0.46-1.36, p=0.40) between right side versus transverse colon. Sidedness was not associated with prognostic difference in OS or PFS in the 96 (17.4%) patients receiving BSC alone. Outcomes according to primary site and treatment are shown. Conclusions: Transverse mCRC has comparable prognostic and predictive features to right sided mCRC. In keeping with previous studies, left side was predictive of greater Cet benefit and presented better overall prognosis when single agent Cet was used after 5-FU, oxaliplatin and irinotecan. [Table: see text]

Impact of KRAS Mutation on Survival Outcome of Patients With Metastatic Colorectal Cancer in Jordan.

Background Colorectal cancer (CRC) is the most prevalent cancer in males, with an incidence rate (IR) of 13.1%, and the second most prevalent cancer in females, with an IR of 8.4%, coming after breast cancer in Jordan. The present study was motivated by conflicting clinical data regarding the prognostic impact of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation in patients with metastatic colorectal cancer (mCRC). Our study aimed to investigate if KRAS mutation conferred a negative prognostic value in Jordanian patients with mCRC. Materials and methods The current study is a retrospective study that collected data from a cohort of 135 mCRC patients diagnosed between 1 January 2017 and 1 January 2022 at our Oncology Department at the Jordanian Military Cancer Center (MCAC) using our patients' electronic medical records. The last follow-up date was 1 September 2022. From the cohort, we obtained data regarding age, sex, date of diagnosis, metastatic spread, KRAS status, either mutated KRAS or wild-type KRAS, and location of the primary tumor. All patients underwent tumor tissue biopsies to determine KRAS mutational status based on quantitative polymerase chain reaction and reverse hybridization from an accredited diagnostic laboratory at Jordan University Hospital. Statistical analysis was carried out to address the associations betweenKRAS mutation and the patients-tumor characteristics and their prognosis on survival. Results KRAS mutation was found in 40.3% of the participants in the study, and 56.7% had the wild type. There was a predilection of KRAS mutation, with 67% on the right side versus 33% on the left side (p= 0.018). Kaplan-Meier survival analysis showed worse survival outcomes in KRAS mutant patients (p= 0.002). The median overall survivalin the KRAS mutant patients was 17 months (95%confidence interval (CI): 13.762-19.273) compared to 21 months (95% CI: 20.507-27.648) in patients with wild-type KRAS. Additionally, the Cox regression model identified that KRAS mutation carries a poorer prognosis on survival outcome hazard ratio (HR: 2.045, 95% CI: 1.291-3.237, p = 0.002). The test also showed statistical significance in the metastatic site (lung only). But this time, it was associated with a better survival outcome (HR: 0.383, 95% CI: 0.186-0.788, p = 0.009). Conclusion The present study shows that the presence of KRAS mutation has been found to negatively impact the prognosis and survival outcome of Jordanian patients with mCRC.

A Cost-effectiveness Analysis of Adding Cetuximab to the First-line Treatment of Metastatic Colorectal Carcinoma in Iran; Considering Genetic Screening for Precision Medicine.

This study aimed to evaluate the cost-effectiveness of cetuximab in different genetic populations of metastatic colorectal carcinoma patients, including KRAS and RAS wild types and mutants, when added to FOLFIRI treatment regimens for evidence-based disease management in Iran. A Markov decision model was designed in TreeAge software with the three states of stable, progress, and death. Clinical outcomes were extracted from published clinical studies, and costs were extracted from the Iranian local data. The primary outcome was an incremental cost-effectiveness ratio (ICER) in the simulated population. The cost-utility model from the perspective of the health system indicated that the average direct medical costs of a patient that has not been genetically screened are $56,985.27 and $20,767.74 in FOLFIRI + cetuximab and FOLFIRI regimens, respectively. However, costs per patient in the KRAS wild-type population were $21,845.52 in FOLFIRI and $78,321.22 in FOLFIRI + cetuximab. In RAS wild-type patients, FOLFIRI and FOLFIRI + cetuximab costs per patient were $23,111.62 and $84,976.39, respectively. Incremental QALYs for the above scenarios were 0.069, 0.193, and 0.285, respectively. Therefore, the ICER of add-on cetuximab in Iran compared to the treatment alternatives in the scenarios with and without KRAS screening was $520,771.55/QALY, $292,768.16/QALY, and $217,460.51/QALY. Although genetic screening in precision medicine reduces costs per outcome, according to the willingness-to-pay threshold of $4349.50 in the Iranian health system, add-on cetuximab to the FOLFIRI regimen is not a cost-effective strategy even with genetic screening and a 20% price reduction.

Whole-cell energy modeling reveals quantitative changes of predicted energy flows in RAS mutant cancer cell lines

Cellular utilization of available energy flows to drive a multitude of forms of cellular "work" is a major biological constraint. Cells steer metabolism to address changing phenotypic states but little is known as to how bioenergetics couples to the richness of processes in a cell as a whole. Here, we outline a whole-cell energy framework that is informed by proteomic analysis and an energetics-based gene ontology. We separate analysis of metabolic supply and the capacity to generate high-energy phosphates from a representation of demand that is built on the relative abundance of ATPases and GTPases that deliver cellular work. We employed mouse embryonic fibroblast cell lines that express wild-type KRAS or oncogenic mutations and with distinct phenotypes. We observe shifts between energy-requiring processes. Calibrating against Seahorse analysis, we have created a whole-cell energy budget with apparent predictive power, for instance in relation to protein synthesis.

Mutant KRAS Drives Immune Evasion by Sensitizing Cytotoxic T-Cells to Activation-Induced Cell Death in Colorectal Cancer.

The roles of oncogenic KRAS in tumor immune evasion remain poorly understood. Here, mutant KRAS is identified as a key driver of tumor immune evasion in colorectal cancer (CRC). In human CRC specimens, a significant reduction in cytotoxic CD8+ T-cell tumor infiltration is found in patients with mutant versus wild type KRAS. This phenomenon is confirmed by preclinical models of CRC, and further study showed KRAS mutant tumors exhibited poor response to anti-PD-1 and adoptive T-cell therapies. Mechanistic analysis revealed lactic acid derived from mutant KRAS-expressing tumor cells sensitized tumor-specific cytotoxic CD8+ T-cells to activation-induced cell death via NF-κB inactivation; this may underlie the inverse association between intratumoral cytotoxic CD8+ T-cells and KRAS mutation. Importantly, KRAS mutated tumor resistance to immunotherapies can be overcome by inhibiting KRAS or blocking lactic acid production. Together, this work suggests the KRAS-mediated immune program is an exploitable therapeutic approach for the treatment of patients with KRAS mutant CRC.

The efficacy of immune checkpoint inhibitors in biliary tract cancer with KRAS mutation.

With a 15% incidence, KRAS is one of the most common mutations in biliary tract cancer (BTC) and is a poor prognostic factor. Immune checkpoint inhibitors (ICIs) as salvage therapy have modest activity in BTC. There are limited data on the efficacy of ICIs according to KRAS mutation in BTC. We evaluated the efficacy of ICIs in BTC patients with or without KRAS mutations. Retrospective observational study. We conducted molecular profiling in BTC patients who received ICIs as salvage therapy. The expression of programmed death ligand 1 (PD-L1) on tumor cells was assessed using immunohistochemistry. The TruSightTM Oncology 500 assay from Illumina was used as a cancer panel. We analyzed overall survival (OS) and progression-free survival (PFS) of ICI in BTC patients according to KRAS mutation and PD-L1 expression. A total of 62 patients were included in this analysis. The median age was 68.0 years; 47 patients (75.8%) received pembrolizumab and 15 (24.2%) received nivolumab as salvage therapy. All patients received gemcitabine plus cisplatin as the frontline therapy, and 53.2% received fluoropyrimidine plus oxaliplatin (FOLFOX) before ICI. The median number of lines of prior chemotherapy was 2.5. The KRAS mutation was found in 13 patients (19.1%), and 28 patients (45.2%) showed 1% or more of tumor cells out of visible tumor cells positive for PD-L1. There was no statistical correlation between KRAS mutation and PD-L1 expression. The median OS and PFS with ICI were 5.6 [interquartile range (IQR): 3.3-8.0] and 3.8 (IQR: 3.0-4.5) months, respectively. There were no statistically significant differences in PFS with ICIs according to KRAS mutation (mutant type versus wild type) and PD-L1 expression (positive versus negative). In subgroup analysis, patients with both KRAS mutation and PD-L1 positivity had longer PFS compared with patients with KRAS mutation and PD-L1 negativity (10.1 versus 2.6 months, p = 0.047). This finding was not shown in patients with wild-type KRAS. Our analysis suggested that PD-L1 expression might be a useful biomarker for ICIs in BTC patients with KRAS mutation but not in those with wild-type KRAS.

The role of molecular testing in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is highly aggressive and has few treatment options. To personalize therapy, it is critical to delineate molecular subtypes and understand inter- and intra-tumoral heterogeneity. Germline testing for hereditary genetic abnormalities is recommended for all patients with PDA and somatic molecular testing is recommended for all patients with locally advanced or metastatic disease. KRAS mutations are present in 90% of PDA, while 10% are KRAS wild type and are potentially targetable with epidermal growth factor receptor blockade. KRASG12C inhibitors have shown activity in G12C-mutated cancers, and novel G12D and pan-RAS inhibitors are in clinical trials. DNA damage repair abnormalities, germline or somatic, occur in 5-10% of patients and are likely to benefit from DNA damaging agents and maintenance therapy with poly-ADP ribose polymerase inhibitors. Fewer than 1% of PDA harbor microsatellite instability high status and are susceptible to immune checkpoint blockade. Albeit very rare, occurring in <1% of patients with KRAS wild-type PDAs, BRAF V600E mutations, RET and NTRK fusions are targetable with cancer agnostic Food and Drug Administration-approved therapies. Genetic, epigenetic, and tumor microenvironment targets continue to be identified at an unprecedented pace, enabling PDA patients to be matched to targeted and immune therapeutics, including antibody-drug conjugates, and genetically engineered chimeric antigen receptor or T-cell receptor - T-cell therapies. In this review, we highlight clinically relevant molecular alterations and focus on targeted strategies that can improve patient outcomes through precision medicine.

The Prevalence and Prognosis of Microsatellite Instability-High/Mismatch Repair-Deficient Colorectal Adenocarcinomas in the United States.

Microsatellite instability (MSI) and DNA mismatch repair (MMR) status is an indispensable biomarker in the management of colorectal cancers. We therefore examined the epidemiology of MSI-high/MMR-deficient colorectal cancers in the United States. Adults presenting with colorectal adenocarcinoma in 2018-2019 were identified from the US National Cancer Database. Attributes associated with MSI-high/MMR-deficiency were identified using multivariable logistic regression and reported using average adjusted probabilities (%AAP) and 99.9% CIs. As a secondary aim, the survival associated with MSI/MMR status was assessed. Among 101,259 colorectal adenocarcinomas in 2018-2019, 82.0% were microsatellite stable/MMR-proficient, 3.8% MSI-low, and 14.2% MSI-high/MMR-deficient-including 16.6%, 19.9%, 12.4%, and 7.3% of stage I, II, III, and IV cancers, respectively. In locoregional cancers, MSI-high/MMR-deficiency was associated with a bimodal age distribution, female sex, right-sided colonic origin, wild-type KRAS, and a prior diagnosis of cancer (all P < .001). By race/ethnicity, colorectal adenocarcinomas were MSI-high/MMR-deficient in 16.9%AAP of non-Hispanic White (99.9% CI, 16.5 to 17.4) patients, compared with 11.3%AAP of non-Hispanic Black (99.9% CI, 10.3 to 12.4), 12.4%AAP of Asian/Pacific Islander (99.9% CI, 10.5 to 14.3), and 15.1%AAP of Hispanic (99.9% CI, 13.4 to 16.7) patients (all P < .001). Histologically, MSI-high/MMR-deficiency was associated with increasing grade, from 11.3%AAP of well-differentiated tumors (99.9% CI, 10.2 to 12.4) to 28.4%AAP of poorly differentiated cases (99.9% CI, 27.1 to 29.8; P < .001). Compared with conventional histology (15.2%AAP, 99.9% CI, 14.8 to 15.6), medullary (41.1%AAP, 99.9% CI, 33.0 to 49.3; P < .001) and mucinous (24.6%AAP, 99.9% CI, 22.8 to 26.3; P < .001) subtypes-but not signet-ring cell histology (15.5%AAP, 99.9% CI, 11.6 to 19.4; P = .79)-were more frequently MSI-high/MMR-deficient when adjusting for clinicopathologic features including grade. Our findings establish the epidemiology, features, and prognostic implications of MSI-high/MMR-deficiency among colorectal adenocarcinoma patients in the United States.

Overall survival of patients with metastatic KRAS wild type colorectal cancer patients treated with anti-EGFR third line monotherapy

Introduction. There is no evidence-based data comparing upfront chemotherapy doublets with anti-EGFR monoclonal antibody with sequential treatment utilizing anti-EGFR monotherapy as a consecutive line of treatment in patients with metastatic colorectal cancer. Here we report real-world survival data for patients with colorectal cancer (CRC) treated with anti-EGFR monoclonal antibody as 3rd line monotherapy. Material and methods. It was single center retrospective study. We collected retrospectively data of wild-type KRAS metastatic CRC patients who have failed oxaliplatin- and irinotecan based therapy and were treated with anti-EGFR monoclonal antibody as the 3. line monotherapy in 2009–2017 in Copernicus Memorial Hospital, Lodz, Poland. Last observation was recorded in February 2020. We calculated median overall survival (since commencement of palliative systemic treatment), median progression free survival and median OSIII (overall survival sine commencement of monotherapy with anti-EGFR agent). Results. 130 patients were included in the study. 40.6% were females. The median age was 63 years (range 38–83). 57% of patients were initially diagnosed with metastatic/inoperable colorectal cancer. 80 patients were treated with 3. line cetuximab, 50 — with panitumumab. At the moment of data analysis 123 deaths were recorded. OS since start of palliative systemic treatment was calculated for 120 patients and its median was 25.8 months. MPFS since start of anti-EGFR antibody was 4.3 months, mOSIII —10.7 months. Conclusions. 3rd line treatment of metastatic colorectal cancer with anti-EGFR antibodies is effective. It is good option for patients, who are not fit enough or not willing to have 1st line triplet therapy.

Miz1 promotes KRAS-driven lung tumorigenesis by repressing the protocadherin Pcdh10

Targeting KRAS-mutated non-small-cell lung cancer (NSCLC) remains clinically challenging. Here we show that loss of function of Miz1 inhibits lung tumorigenesis in a mouse model of oncogenic KRAS-driven lung cancer. In vitro, knockout or silencing of Miz1 decreases cell proliferation, clonogenicity, migration, invasion, or anchorage-independent growth in mutant (MT) KRAS murine or human NSCLC cells but has unremarkable impact on non-tumorigenic cells or wild-type (WT) KRAS human NSCLC cells. RNA-sequencing reveals Protocadherin-10 (Pcdh10) as the top upregulated gene by Miz1 knockout in MT KRAS murine lung tumor cells. Chromatin immunoprecipitation shows Miz1 binding on the Pcdh10 promoter in MT KRAS lung tumor cells but not non-tumorigenic cells. Importantly, silencing of Pcdh10 rescues cell proliferation and clonogenicity in Miz1 knockout/knockdown MT KRAS murine or human tumor cells, and rescues allograft tumor growth of Miz1 knockout tumor cells in vivo. Miz1 is upregulated in MT KRAS lung tumor tissues compared with adjacent non-involved tissues in mice. Consistent with this, Miz1 is upregulated while Pcdh10 is downregulated in human lung adenocarcinomas (LUAD) compared with normal tissues, and high Miz1 levels or low Pcdh10 levels are associated with poor survival in lung cancer patients. Furthermore, the Miz1 signature is associated with worse survival in MT but not WT KRAS LUAD, and Pcdh10 is downregulated in MT compared to WT KRAS LUAD. Taken together, our studies implicate the Miz1/Pcdh10 axis in oncogenic KRAS-driven lung tumorigenesis.

Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

BackgroundAlternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.MethodsWe recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.ResultsMass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.ConclusionProteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

Detection of circulating KRAS mutant DNA in extracellular vesicles using droplet digital PCR in patients with colon cancer.

Extracellular vesicles secreted by tumor cells contain double-stranded DNA called extracellular vesicle DNA (evDNA). EvDNA is genomic DNA that reflects cancer driver mutations. However, the significance of evDNA analysis in the diagnosis and surveillance of colon cancer remains unclear. This study aimed to investigate the clinical utility of extracellular vesicles and evDNA isolated from the plasma of colon cancer patients harboring KRAS G12D and G13D mutations. Cell-free DNA (cfDNA) and evDNA were collected from the plasma of 30 patients with colon cancer. KRAS mutation status (G12D and G13D) was detected using a droplet digital polymerase chain reaction assay (ddPCR). Sensitivity and specificity were evaluated in patients with wild-type KRAS tumors. Mutation status was correlated with carcinoembryonic antigen (CEA) levels and overall survival (OS). Thirty cfDNA and evDNA pairs showed a KRAS fractional abundance (FA) ranging from 0 to 45.26% and 0 to 83.81%, respectively. When compared with eight wild-type KRAS samples, cfDNA exhibited 70% sensitivity and 100% specificity, whereas evDNA achieved 76.67% sensitivity and 100% specificity. The concentration of evDNA was significantly lower than that of cfDNA, but it obtained a higher FA than cfDNA, while showing a positive correlation with CEA. Our findings demonstrate the feasibility of evDNA as a complementary tool to aid current methods of patient evaluation in the diagnosis and surveillance of colon cancer.

Multiplex Digital Polymerase Chain Reaction on a Droplet Array SlipChip for Analysis of KRAS Mutations in Pancreatic Cancer.

Pancreatic cancer is a terminal disease with high mortality and very poor prognosis. A sensitive and quantitative analysis of KRAS mutations in pancreatic cancer provides a tool not only to understand the biological mechanisms of pancreatic cancer but also for diagnosis and treatment monitoring. Digital polymerase chain reaction (PCR) is a promising tool for KRAS mutation analysis, but current methods generally require a complex microfluidic handling system, which can be challenging to implement in routine research and point-of-care clinical diagnostics. Here, we present a droplet-array SlipChip (da-SlipChip) for the multiplex quantification of KRAS G12D, V, R, and C mutant genes with the wild-type (WT) gene background by dual color (FAM/ROX) fluorescence detection. This da-SlipChip is a high-density microwell array of 21,696 wells of 200 pL in 4 by 5424 microwell format with simple loading and slipping operation. It does not require the same precise alignment of microfeatures on the different plates that are acquired by the traditional digital PCR SlipChip. This device can provide accurate quantification of both mutant genes and the WT KRAS gene. We collected tumor tissue, paired normal pancreatic tissue, and other normal tissues from 18 pancreatic cancer patients and analyzed the mutation profiles of KRAS G12D, V, R, and C in these samples; the results from the multiplex digital PCR on da-SlipChip agree well with those of next-generation sequencing (NGS). This da-SlipChip moves digital PCR closer to the practical point-of-care applications not only for detecting KRAS mutations in pancreatic cancer but also for other applications that require precise nucleic acid quantification with high sensitivity.