Frequency Of KRAS Mutations Research Articles

Multi-omics-driven discovery of invasive patterns and treatment strategies in CA19-9 positive intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with a poor prognosis, predominantly CA19-9 positive. High CA19-9 levels correlate with increased aggressiveness and worse outcomes. This study employs multi-omics analysis to reveal molecular features and identify therapeutic targets of CA19-9 positive ICC, aiming to support individualized treatment. Data from seven clinical cohorts, two whole-exome sequencing cohorts, six RNA sequencing/microarray cohorts, one proteomic cohort, 20 single-cell RNA sequencing samples, and one spatial transcriptome sample were analyzed. Key findings were validated on tissue microarrays from 52 ICC samples. CA19-9 positive ICC exhibited poorer OS (median 24.1 v.s. 51.5months) and RFS (median 11.7 v.s. 28.2months) compared to negative group (all P < 0.05). Genomic analysis revealed a higher KRAS mutation frequency in the positive group and a greater prevalence of IDH1/2 mutations in the negative group (all P < 0.05). Transcriptomic analysis indicated upregulated glycolysis pathways in CA19-9 positive ICC. Single-cell analysis identified specific glycolysis-related cell subclusters associated with poor prognosis, including Epi_SLC2A1, CAF_VEGFA, and Mph_SPP1. Higher hypoxia in the CA19-9 positive group led to metabolic reprogramming and promoted these cells' formation. These cells formed interactive communities promoting epithelial-mesenchymal transition (EMT) and angiogenesis. Drug sensitivity analysis identified six potential therapeutic drugs. This study systematically elucidated the clinical, genomic, transcriptomic, and immune features of CA19-9 positive ICC. It reveals glycolysis-associated cellular communities and their cancer-promoting mechanisms, enhancing our understanding of ICC and laying the groundwork for individualized therapeutic strategies.

Elevated serum direct bilirubin is predictive of a poor prognosis for primary myelodysplastic syndrome.

The aim of this study was to assess the prognostic significance of serum direct bilirubin (DBIL) for patients newly diagnosed with myelodysplastic syndromes (MDS). The clinical, laboratory, and follow-up data of MDS patients were collected, and the associations of DBIL levels with overall survival (OS) and leukemia-free survival (LFS) were analyzed. In total, 262 MDS patients were assigned to the high DBIL level group or the normal DBIL level group in the retrospective study. High DBIL was associated with older age, reduced hemoglobin, higher levels of β2-microglobin, lactate dehydrogenase, and serum ferritin, along with the number of co-mutations (> 1) and a higher frequency of ASXL1, KIT, and KRAS mutations. Multivariate analyses found that high DBIL level was an independent adverse predictor for OS (p = 0.002, hazard ratio = 2.723, 95%CI = 1.442-5.143) but not for LFS (p = 0.057, hazard ratio = 1.678, 95%CI = 0.986-2.857). A novel nomogram based on DBIL, sex, age, β2-microglobulin, lactate dehydrogenase, the Revised International Prognostic Scoring System (IPSS-R) was constructed, which demonstrated superior accuracy compared with the IPSS-R (C-index, 0.790 vs. 0.731, respectively). An elevated DBIL level was identified as an independent adverse prognostic factor for MDS patients. An individualized prediction model was established and validated to improve prediction of OS and LFS.

Oncogenic KRAS mutations modulate BAX-mediated cell death

Kirsten rat sarcoma viral oncogene homolog (KRAS) belongs to the GTPase RAS superfamily, which regulates several cell-signaling pathways involved in the control of important cellular functions, including apoptosis. Oncogenic mutations in KRAS are considered the most common gain-of-function mutations, affecting 30–50 % of colorectal cancer (CRC) patients. While RAS proteins usually play an anti-apoptotic role, little is known about the involvement of KRAS mutations in apoptosis regulation. Here, we aimed to elucidate the role of mutated human KRAS in the regulation of BAX, a key pro-apoptotic member of the Bcl-2 family. For this purpose, we took advantage of the simpler yeast model Saccharomyces cerevisiae, using cells deficient in the main yeast RAS isoform (ras2Δ) co-expressing wild-type KRAS (KRASWT) or the most frequent KRAS mutations found in CRC - KRASG12D, KRASG12V or KRASG13D, along with human BAX. We show that, in comparison with KRASWT, KRAS mutants confer resistance to BAX-induced death and cytochrome c (cyt c) release. The modulation of BAX by KRAS isoforms seems to result from a direct interaction between these proteins, as they co-localize at the mitochondria and there is evidence they may physically interact. We further show that acetic acid significantly increased cell death in cells expressing BAX and co-expressing oncogenic KRAS mutants, but not KRASWT. This suggests a potential mechanism explaining the increased sensitivity of CRC cells harboring a KRAS-activated pathway to acetate. These findings contribute to a clearer understanding of how KRAS regulate BAX function, a relevant aspect in tumor progression.

Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity.

To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRAS G12D mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible KRAS variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of KRAS mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRAS G12D second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity. These studies define the landscape of single amino acid alterations that modulate the function of KRAS, providing a resource for the clinical interpretation of KRAS variants and elucidating mechanisms of oncogenic KRAS inactivation for therapeutic exploitation.

Integrative multi-omics analysis reveals the role of tumor-associated endothelial cells and their signature in prognosis of intrahepatic cholangiocarcinoma

This study aims to investigate the interplay between tumor-associated endothelial cells (TECs) and immune cells within the tumor microenvironment (TME) and its impact on tumor prognosis. We conducted single-cell RNA sequencing (scRNA-seq) of tumor, normal, and lymph node tissues obtained from intrahepatic cholangiocarcinoma (ICC) patients to reveal the role of TECs in tumor angiogenesis and their significant heterogeneity. Meanwhile, we identified genes highly expressed in TECs and constructed TEC signatures (TEC.Sig). Next, we calculated TEC scores of samples based on TEC.Sig. Patients with higher TEC scores exhibited a higher frequency of KRAS mutations, which was associated with increased infiltration of neutrophils and immature dendritic cells (iDCs), and decreased numbers of natural killer (NK), CD4 + T, and CD8 + T effector memory (Tem) cells, indicating an inflammation-dominated immunosuppressive phenotype. In contrast, BAP1 mutations and CXCL12 overexpression showed a contrasting trend. Spatial transcriptomics analysis and histological experiments further confirmed that TECs interacted with various tumor-killing immune cells through the CXCL12/CXCR4 axis. Multiple tumor immunotherapy datasets confirmed that the TEC.Sig could predict patient responses to immunotherapy. The TEC score is a promising and reliable biomarker for predicting genetic mutations and prognosis in ICC patients. Enhancing the regulation of the CXCL12/CXCR4 signaling pathway may represent a potential novel therapeutic target for ICC treatment.

Associations of KRAS Mutations and Clinical Characteristics of Colorectal Cancer Patients in Indonesia.

Colorectal cancer (CRC) remains a major burden worldwide, ranking third and second in incidence and mortality respectively. Detection of biomarkers including KRAS mutations can help predict prognosis and response to therapy in CRC, thus this study evaluated the frequency of KRAS mutations among Indonesian patients and their associations with clinicopathologic characteristics. Fifty-three CRC samples were collected from January to September 2022 in the Department of Surgery, Dr. Hasan Sadikin General Hospital, Bandung, Indonesia. KRAS mutations were analyzed using PCR followed by Sanger sequencing. Associations between KRAS mutations were evaluated using binary logistic regression analysis. KRAS mutations were detected in 52.8% of patients (n=28), of which 3.6% were p.Gly12Ser (n=1), 32.1% were p.Gly12Asp (n=9), 7.1% were p.Gly13Asp (n=2), 3.6% were p.Gln61His (n=1), 3.6% were p.Asp126His (n=1), and 3.6% were p.Lys169Glu (n=1). The p.Asp73= polymorphism was detected in 57.1% of the samples (n=16). KRAS mutation status did not differ significantly between the groups based on the age of onset, sex, tumor location, tumor histology, stage, and family history. KRAS mutations are present in high frequency in this cohort of CRC patients in a tertiary hospital in West Java, Indonesia. However, KRAS mutation status is not associated with the age of onset, sex, tumor location, tumor histology, stage, and family history.

Identification of CDH4 Gene Copy Number Alteration and Its Association with Clinical Profile of Colorectal Cancer Patient

Background: The genes cadherin 4 (CDH4), Kirsten rat sarcoma viral oncogene homolog (KRAS), V-Raf murine sarcoma viral oncogene homolog B (BRAF), and microsatellite instability (MSI) each play a role in the development of colorectal cancer (CRC). CDH4 gene is located in chromosome 20q13.33 and its amplification or gain is the earliest mutational event found in the majority of CRC and colon polyps. This study aimed to identify copy number alteration in the CDH4 gene and its association with the clinical profile of CRC patients, including gender, age, tumor location and differentiation, frequency of BRAF and KRAS mutations, and MSI status. Methods: The DNA was extracted from 50 tumors and adjacent normal tissues based on the manufacturer’s instructions. Detection of MSI status was carried out by pentaplex polymerase chain reaction (PCR) and PCR products were size separated by capillary electrophoresis (CE) using an ABI 310 Genetic Analyzer. Both KRAS and BRAF mutations were identified by PCR and sequencing while CDH4 copy measurement was measured using TaqManTM Copy Number Assay. Results: Our findings showed 22 (44%) samples with no changes in the copy number of CDH4 gene. Interestingly, 21 (42%) cases had an amplification of the CDH4 gene or CDH4 gene gain, and seven (14%) cases decreased in CDH4 gene expression or CDH4 gene loss. We found an association between changes in the CDH4 gene and gender (p=0.001). However, there was no association between changes in the CDH4 gene and age (p=0.979), tumor location (p=0.145) and differentiation (p=0.648), the frequency of BRAF (p=0.171) and KRAS mutations (p=0.184) and MSI status (p=0.923). Conclusions: Copy number alteration in CDH4 gene in CRC patients and this alteration is significantly associated with gender. Further studies with a larger number of samples are essential to confirm this result and to identify the cause of CDH4 copy number alteration and its biological significance.

Frequency of KRAS and BRAF mutations in colorectal carcinoma and their association with clinical-pathological characteristics in a tertiary hospital in Kenya.

Colorectal carcinoma is a leading cause of cancer morbidity and mortality globally. Its management includes the use of targeted therapy which require assessment for biomarkers to choose eligible patients. KRAS and BRAF mutations are biomarkers predictive of response to anti-EGFR therapy. This study aimed at determining the frequency of BRAF V600E and KRAS exon 2,3,4 mutations in colorectal carcinoma patients at the Aga Khan University Hospital Nairobi, Kenya. Study participants were patients who had colectomy for colorectal carcinoma. They were identified from the laboratory information system. The patients age, gender and tumor location were determined from the medical records. The histological diagnosis, pathological tumor and nodal stage were confirmed by examining hematoxylin and eosin-stained slides prepared from the colectomy specimen. DNA was extracted from the specimens using Qiagen QIAamp DNA FFPE Tissue Kit and PCR performed using EntroGen KRAS/BRAF mutation analysis kit following manufacturer's protocol. One hundred fourteen patients were enrolled. Colorectal carcinoma was significantly more common in males than females. The mean age at diagnosis was 58 years. Majority of the tumors were in the right colon, were of pathological tumor stage T3 and had nodal involvement. Forty six percent (46%) of the cases had KRAS mutations while 5.3% had BRAF V600E mutation. KRAS mutation was associated with a high pathological tumor stage and nodal involvement. Colorectal carcinoma in our patients is more common in males and tend to occur at a younger age. The patients tend to have a high tumor pathological stage and nodal involvement at diagnosis. The high frequency of KRAS exon 2,3,4 mutation and low frequency of BRAF V600E mutations is similar to what has been reported in literature.

Identification and validation of inflammatory subtypes in intrahepatic cholangiocellular carcinoma

BackgroundInflammation plays a critical role in tumor development. Inflammatory cell infiltration and inflammatory mediator synthesis cause changes in the tumor microenvironment (TME) in several cancers, especially in intrahepatic cholangiocellular carcinoma (ICC). However, methods to ascertain the inflammatory state of patients using reliable biomarkers are still being explored.MethodWe retrieved the RNA sequencing and somatic mutation analyses results and the clinical characteristics of 244 patients with ICC from published studies. We performed consensus clustering to identify the molecular subtypes associated with inflammation. We compared the prognostic patterns, clinical characteristics, somatic mutation profiles, and immune cell infiltration patterns across inflammatory subtypes. We performed quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) to confirm gene expression. We performed logistic regression analyses to construct a nomogram predicting the inflammatory status of patients with ICC.ResultsOur results confirmed that ICC can be categorized into an inflammation-high subtype (IHS) and an inflammation-low subtype (ILS). Patients from each group had distinct prognosis, clinical characteristics, and TME composition. Patients with ICC in the IHS group showed poorer prognosis owing to the immunosuppressive microenvironment and high frequency of KRAS and TP53 mutations. Cancer-associated fibroblast (CAF)-derived COLEC11 reduced myeloid inflammatory cell infiltration and attenuated inflammatory responses. The results of qRT-PCR and IHC experiments confirmed that COLEC11 expression levels were significantly reduced in tumor tissues compared to those in paracancerous tissues. Patients with ICC in the IHS group were more likely to respond to treatment with immune checkpoint inhibitors (ICIs) owing to their higher tumor mutational burden (TMB) scores, tumor neoantigen burden (TNB) scores, neoantigen counts, and immune checkpoint expression levels. Finally, we developed a nomogram to effectively predict the inflammatory status of patients with ICC based on their clinical characteristics and inflammatory gene expression levels. We evaluated the calibration, discrimination potential, and clinical utility of the nomogram.ConclusionThe inflammatory response in IHS is primarily induced by myeloid cells. COLEC11 can reduce the infiltration level of this group of cells, and myeloid inflammatory cells may be a novel target for ICC treatment. We developed a novel nomogram that could effectively predict the inflammatory state of patients with ICC, which will be useful for guiding individualized treatment plans.

Clinicopathological significance of mutation profile detected by next generation sequencing in different metastatic organs of non-small cell lung cancers

BackgroundThe primary tumor and it’s metastases show heterogeneity in molecular studies for targeted therapies in Non-Small Cell Lung Cancer(NSCLC), the leading cause of cancer-related deaths worldwide. The study aimed to identify somatic mutations in biopsies from NSCLC patients’ metastatic organs using Next-Generation Sequencing(NGS) and examine their association with clinicopathological parameters. Materials and methodsThe study included 128 NSCLC patients and, NGS was performed on tumor biopsies from different metastatic organs at Molecular Pathology laboratory of the Department of Medical Pathology in Aydın Adnan Menderes University Faculty of Medicine. The age, gender, histopathological diagnoses, metastatic organs, smoking and mutation status were all recorded, along with the analysis results of 72 genes and 4149 primers in the panel of the NGS system. Results53.9 % of the cases had a history of smoking and patients with brain metastases had a higher smoking rate(p=0.000). The most common occurrence(39.8 %) was lymph node metastasis, followed by brain(19.5 %). There was a strong correlation between mutation presence and metastasis in the liver(p=0.012), bone(p=0.002), and pleura(p=0.008). Smokers had a higher frequency of KRAS(p=0.000) and TP53(p=0.001) mutations. Brain metastases showed a statistically significant NF1 mutation(p=0.001), while the liver exhibited a significant BRAF mutation(p=0.000). NF1-TP53, PTEN-TP53 and NF1-PTEN were the most common concomitant mutations and, the brain was the most common metastatic organ in which they occurred. ConclusionOur results suggest prizing assessing detected mutations, in the prediction, follow-up and management of metastases, especially in patients with lung adenocarcinoma. The assessment also needs to consider the tumor's mutation status in metastatic organs. New therapeutic agents targeting NF1 mutations will be available in the future to treat NSCLC, especially in metastases.

HRS-4642: The next piece of the puzzle to keep KRAS in check

KRASG12D is the most frequent KRAS mutation in human cancer. In this issue, Zhou etal. describe a novel KRASG12D inhibitor, HRS-4642, that shows potent and selective anti-tumor activity across various models and synergizes with proteasome inhibitors. Responses have also been observed in patients during an ongoing phase 1 trial.

Lymph node metastasis in early invasive lung adenocarcinoma: Prediction model establishment and validation based on genomic profiling and clinicopathologic characteristics.

The presence of lymph node (LN) metastasis directly affects the treatment strategy for lung adenocarcinoma (LUAD). Next-generation sequencing (NGS) has been widely used in patients with advanced LUAD to identify targeted genes, while early detection of pathologic LN metastasis using NGS has not been assessed. Clinicopathologic features and molecular characteristics of 224 patients from Ruijin Hospital were analyzed to detect factors associated with LN metastases. Another 140 patients from Huashan Hospital were set as a test cohort. Twenty-four out of 224 patients were found to have lymph node metastases (10.7%). Pathologic LN-positive tumors showed higher mutant allele tumor heterogeneity (p < 0.05), higher tumor mutation burden (p < 0.001), as well as more frequent KEAP1 (p = 0.001), STK11 (p = 0.004), KRAS (p = 0.007), CTNNB1 (p = 0.017), TP53, and ARID2 mutations (both p = 0.02); whereas low frequency of EGFR mutation (p = 0.005). A predictive nomogram involving male sex, solid tumor morphology, higher T stage, EGFR wild-type, and TP53, STK11, CDKN2A, KEAP1, ARID2, KRAS, SDHA, SPEN, CTNNB1, DICER1 mutations showed outstanding efficiency in both the training cohort (AUC = 0.819) and the test cohort (AUC = 0.780). This study suggests that the integration of genomic profiling and clinical features identifies early-invasive LUAD patients at higher risk of LN metastasis. Improved identification of LN metastasis is beneficial for the optimization of the patient's therapy decisions.

Molecular characteristics of patients with colorectal signet-ring cell carcinoma with different ABO blood groups

BackgroundColorectal signet-ring cell carcinoma (SRCC) is a rare subtype of malignant adenocarcinoma, accounting for approximately 1 % of colorectal cancer (CRC) cases. Its biomarkers and molecular characteristics remain controversial, and there are no specific therapeutic targets or strategies for its clinical treatment. MethodsA retrospective study was conducted between January 2010 and December 2021. 1058 colorectal cancer cases from the Sun Yat-sen University Cancer Center and 489 cases from the Tumor Genome Atlas Project were included in the analysis, of which 64 were SRCC. Data extraction included patient demographics, blood types and risk factors, including clinical variables and genomics (either a 19-gene panel NGS or 1021-gene panel NGS). Univariate analyses were performed to identify factors significantly associated with overall survival. ResultsThe blood groups of 27 (42.2 %), 18 (28.1 %), 12 (18.8 %), and seven (10.9 %) patients were classified as O, A, B, and AB, respectively. We found that O was a unique blood group characterized by a low frequency of KRAS mutations, a high frequency of heterozygosity at each HLA class I locus, and a high tumor mutational burden (TMB). Patients in blood group A with high-frequency KRAS mutations and those in blood group B with anemia and metabolic abnormalities required targeted treatment. Furthermore, genetic alterations in SRCC differed from those in adenocarcinoma and mucinous adenocarcinoma. ConclusionsOur study revealed genomic changes in SRCC patients across different blood groups, which could advance the understanding and precise treatment of colorectal SRCC.

Clinicopathological and molecular features of genome-stable colorectal cancers.

Colorectal cancers (CRCs) are traditionally divided into those with either chromosomal instability (CIN) or microsatellite instability (MSI). By utilizing TCGA data, the Laird team found a subset of CRCs, namely, genome-stable CRCs (GS CRCs), which lack both CIN and MSI. Although the molecular features of GS CRCs have been described in detail, the clinicopathological features are not well defined. A total of 437 CRCs were analyzed for copy number variation (CNV) statuses in eight genes (ARID1A, EGFR, FGFR1, KDM5B, MYBL2, MYC, SALL4, and SETDB1) using droplet-digital PCR. CRCs that showed CNV in ≤ one gene and no MSI were defined as GS-like CRCs. Clinicopathological and molecular features of GS-like CRCs were compared with those of CIN-like CRCs. GS-like CRCs comprised 4.6% of CRCs and showed a predilection toward the proximal colon, lower nuclear optical density, KRAS mutation, PIK3CA mutation, and aberrant expression of KRT7. Survival analysis showed no significant difference between the three subgroups. Through our study, the GS-like subtype was found to comprise a minor proportion of CRCs and have proclivity toward a proximal bowel location, hypochromatic tumor nuclei, aberrant KRT7 expression, and a high frequency of KRAS and PIK3CA mutations.

To explore the prognostic characteristics of colon cancer based on tertiary lymphoid structure-related genes and reveal the characteristics of tumor microenvironment and drug prediction

In order to construct a prognostic evaluation model of TLS features in COAD and better realize personalized precision medicine in COAD. Colon adenocarcinoma (COAD) is a common malignant tumor of the digestive system. At present, there is no effective prognostic marker to predict the prognosis of patients. Tertiary lymphoid structure (TLS) affects cancer progression by regulating immune microenvironment. Mining COAD biomarkers based on TLS-related genes helps to improve the prognosis of patients. In order to construct a prognostic evaluation model of TLS features in COAD and better realize personalized precision medicine in COAD. The mRNA expression data and clinical information of COAD and adjacent tissues were downloaded from the Cancer Genome Atlas database. The differentially expressed TLS-related genes of COAD relative to adjacent tissues were obtained by differential analysis. TLS gene co-expression analysis was used to mine genes highly related to TLS, and the intersection of the two was used to obtain candidate genes. Univariate, LASSO, and multivariate Cox regression analysis were performed on candidate genes to screen prognostic markers to construct a risk assessment model. The differences of immune characteristics were evaluated by ESTIMATE, ssGSEA and CIBERSORT in high and low risk groups of prognostic model. The difference of genomic mutation between groups was evaluated by tumor mutation burden score. Screening small molecule drugs through the GDSC library. Finally, a nomogram was drawn to evaluate the clinical value of the prognostic model. Seven TLS-related genes ADAM8, SLC6A1, PAXX, RIMKLB, PTH1R, CD1B, and MMP10 were screened to construct a prognostic model. Survival analysis showed that patients in the high-risk group had significantly lower overall survival rates. Immune microenvironment analysis showed that patients in the high-risk group had higher immune indicators, indicating higher immunity. The genomic mutation patterns of the high-risk and low-risk groups were significantly different, especially the KRAS mutation frequency was significantly higher in the high-risk group. Drug sensitivity analysis showed that the low-risk group was more sensitive to Erlotinib, Savolitinib and VE _ 822, which may be used as a potential drug for COAD treatment. Finally, the nomogram constructed by pathological features combined with RiskScore can accurately evaluate the prognosis of COAD patients. This study constructed and verified a TLS model that can predict COAD. More importantly, it provides a reference standard for guiding the prognosis and immunotherapy of COAD patients.

Altering the gut microbiome and TME in advanced liver cancers: A phase II study of nivolumab, tadalafil, and oral vancomycin in refractory HCC or liver-dominant metastatic CRC or PDAC.

e14642 Background: The gut-liver axis shapes the environment that promotes liver disease. In murine models of liver cancer, our group showed that altering gut microbiome with oral vancomycin can induce an antitumor effect by modifying bile acids (BA) that then recruit natural killer T cells (NKTC). However, these NKTC were met by an immunosuppressive counter-regulatory response where myeloid-derived suppressor cells (MDSC) accumulated, impairing their cytotoxicity. Tadalafil, a PDE5 inhibitor, can suppress these MDSC in the TME. The aim of this study was to examine if the immunomodulatory effects of oral vancomycin and tadalafil can enhance nivolumab activity in advanced primary liver cancer or liver metastases. Methods: This is a phase II study of adult patients (pts) with refractory hepatocellular carcinoma (HCC) or liver-dominant metastatic colorectal (mCRC) or pancreatic (mPDAC) cancers. Pts received IV nivolumab 480mg on D1, oral vancomycin 125mg every 6 hours on D1-21, and tadalafil 10mg once daily in 28d cycles until disease progression or unacceptable toxicity. Primary endpoint was best overall response (BOR) according to RECIST v1.1. Secondary endpoints included safety/tolerability and mOS. Correlative studies were performed on paired stool, blood, and tumor biopsies. Results: From 6/2019 - 7/2021, 22 heavily pretreated pts were enrolled: 6 HCC, 9 PDAC, 7 CRC. Median age was 63y (41-82), and 27% were female. Molecular testing showed MSS, TMB low to intermediate (1.57-13.35), and frequent mutations in KRAS and TP53. The BOR was SD (3pts, 18.75%), followed by PD (13pts, 81.25%); there were no PR or CR (ORR = 0%). Six pts had symptomatic deterioration prior to restaging scans. At a median follow-up of 4m (0.9-23.0m), all 22 pts had died of progressive disease with mOS of 4m. When stratified by site of origin, mOS was 9.5m (95%CI: 1.3-21.9m) in HCC and 3.6m (95%CI: 1.9-6.6m) in liver metastases (log-rank test, p=0.27). The most common grade 3-4 trAEs were electrolyte disturbances and anemia. The stool microbiome composition of Bacteroides species did not change with treatment as detected by shotgun sequencing. Plasma secondary BA, measured by the mixed meal test, decreased with treatment (p&lt;0.05, n=18, two-way ANOVA). The classical to non-classical monocytes ratio increased with treatment in flow cytometry analysis of PBMC immune cells (p&lt;0.05, n=14, one-way ANOVA). Transcriptomic analysis of treated liver tumor biopsies showed an increase in BA metabolism. Conclusions: Although well tolerated, the combination of oral vancomycin and tadalafil with nivolumab did not produce any meaningful clinical responses. Correlative studies suggested enhanced activation of BA metabolization, but TME immune profiling did not reveal substantial changes in regulatory suppressor cells or NKTC activity. Clinical trial information: NCT03785210 .

Non-clinical evaluation of NT-112, an autologous T cell product engineered to express an HLA-C*08:02-restricted TCR targeting KRAS G12D and resistant to TGF-b inhibition.

e14533 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in oncogenes such as KRAS. Activating mutations in KRAS are among the most prevalent oncogenic driver mutations in human cancers, with KRAS G12D being the most frequent KRAS mutation in colorectal carcinoma and pancreatic cancer. NT-112 is an autologous engineered T-cell product expressing an HLA-C*08:02-restricted TCR that specifically targets the KRAS G12D mutation. NT-112 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-112, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-C*08:02-restricted KRS G12D neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-112 product functionality was evaluated in both in vivo models and in vitro co-culture experiments with a panel of cell lines including those endogenously expressing the KRAS G12D mutation. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. For determination of NT-112 product safety, cross-reactivity and allo-reactivity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-112. Results: High reactivity of the NT-112 TCR against KRAS G12D and HLA-C*08:02-expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against KRAS WT, minimal cross-reactivity in Alanine and Glycine scans, and lack of T-cell activation in the absence of HLA-C*08:02. In vivo, NT-112 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (&lt;0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-112 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-112 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant KRAS-targeted cancer immunotherapy.

Young adults (YA) with non-small cell lung cancer (NSCLC): Snapshot of the oncogenic drivers and immune landscapes.

8538 Background: Approximately 5% of NSCLC occurs in patients 50 years or younger (median age:71) representing distinct clinicopathological features. Reports characterizing genomic alterations are scarce. Using a large real-world (RW) dataset, we characterize oncogenic drivers, and immune landscapes to better understand YA with NSCLC. Methods: 42,326 NSCLC specimens were analyzed using next-generation sequencing of DNA (DNA-592 panel or whole exome) or RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). YA (&lt;=50 years) were categorized into three groups (years): 18-30 (A1, n=61), 31-40 (A2, n=277) &amp; 41-50 (A3, n=1,549); vs &gt;50 (A4, n=40,439). Composition of tumor microenvironment (TME) was estimated from bulk RNA sequencing using QuanTIseq method. RW survival on Osimertinib (Osi-OS) was obtained from insurance claims and calculated from initiation of Osi treatment to last contact and was compared between ages 18-50 and &gt;50 years. Hazard ratio (HR) was calculated using the Cox proportional hazards model. Statistical significance was determined using Chi-square, Fisher’s Exact, Mann-Whitney U and log-rank tests and corrected for multiple comparisons where applicable (q&lt;0.05). Results: Consistent with previous reports, NSCLC in YA was more prevalent in females, non-smokers and was associated with adenocarcinoma histology. Among driver alterations, ALK(IHC+), ROS1, RET and NTRK1 fusion were enriched and KRAS mutations were reduced in YA. Interestingly, while the prevalence of KRASG12D from transition (non-smoker related) and EGFRE746_A750 decreased with age, KRASG12C from transversion increased with age in YA. YA were also associated with improved Osi-OS (HR: 0.79, median Osi-OS=37.4 months vs 32 months in &gt;50 years old, p=0.019). High tumor mutation burden (&gt;=10 mut/Mb), the prevalence of mutations in TP53, KMT2D, NF1, RBM10, NFE2L2 and NOTCH1 increased while GNAS decreased with age. The expression of immune checkpoint genes such as PDCD1LG2(A1/A4: 0.63, A3/A4: 0.89), LAG3(A2/A4: 0.73, A3/A4: 0.89) and IFNG (A1/A4: 0.32, A3/A4: 0.75) were reduced, while M2 macrophage (A2/A4: 1.2) and neutrophil (A2/A4: 1.2) were increased in YA (all q&lt;0.05). Conclusions: We report an increased prevalence of RET and NTRK1 fusions in YA and key differences in distributions of frequent KRAS and EGFR mutations in YA (vs A4) along with decreased expression of immune related genes in the tumor microenvironment. The implications are under active investigation. [Table: see text]

Real-world applications of the new grading system in lung adenocarcinoma: A study of 907 patients focusing on revealing the relationship between pathologic grade and genetic status

BackgroundThe status of the lung adenocarcinoma (LUAD) grading system and the association between LUAD differentiation, driver genes, and clinicopathological features remain to be elucidated. MethodsWe included patients with invasive non-mucinous LUAD, evaluated their differentiation, and collected available clinicopathological information, gene mutations, and analyzed clinical outcomes. ResultsAmong the 907 patients with invasive non-mucinous LUAD, 321 (35.4 %) were poorly differentiated, 422 (46.5 %) were moderately differentiated, and 164 (18.1 %) were well differentiated. EGFR mutation was more common in the LUADs accompanied without CGP (complex glandular pattern) than LUADs with CGP (p < 0.001). Correlation analysis between mutations and clinical characteristics showed that EGFR gene mutation (p < 0.001), KRAS gene mutation (p < 0.05), and ALK gene rearrangement (p < 0.001) were significantly related to the degree of tumor differentiation, and the KRAS and ALK gene mutation frequencies were higher in the low-differentiation group than in the high and medium differentiation groups. The EGFR mutation frequency was higher in the well/moderately differentiated adenocarcinoma group. ConclusionsOur study adds to the evidence regarding the role of the grading system in prognosis. EGFR, KRAS, and ALK are related to the degree of tumor differentiation.

Expression patterns of HNF4α, TTF-1, and SMARCA4 in lung adenocarcinomas: impacts on clinicopathological and genetic features.

HNF4α expression and SMARCA4 loss were thought to be features of non-terminal respiratory unit (TRU)-type lung adenocarcinomas, but their relationships remained unclear. HNF4α-positive cases among 241 lung adenocarcinomas were stratified based on TTF-1 and SMARCA4 expressions, histological subtypes, and driver mutations. Immunohistochemical analysis was performed using xenograft tumors of lung adenocarcinoma cell lines with high HNF4A expression. HNF4α-positive adenocarcinomas(n = 33) were divided into two groups: the variant group(15 mucinous, 2 enteric, and 1 colloid), where SMARCA4 was retained in all cases, and the conventional non-mucinous group(6 papillary, 5 solid, and 4 acinar), where SMARCA4 was lost in 3/15 cases(20%). All variant cases were negative for TTF-1 and showed wild-type EGFR and frequent KRAS mutations(10/18, 56%). The non-mucinous group was further divided into two groups: TRU-type(n = 7), which was positive for TTF-1 and showed predominantly papillary histology(6/7, 86%) and EGFR mutations(3/7, 43%), and non-TRU-type(n = 8), which was negative for TTF-1, showed frequent loss of SMARCA4(2/8, 25%) and predominantly solid histology(4/8, 50%), and never harbored EGFR mutations. Survival analysis of 230 cases based on histological grading and HNF4α expression revealed that HNF4α-positive poorly differentiated (grade 3) adenocarcinoma showed the worst prognosis. Among 39 cell lines, A549 showed the highest level of HNF4A, immunohistochemically HNF4α expression positive and SMARCA4 lost, and exhibited non-mucinous, high-grade morphology in xenograft tumors. HNF4α-positive non-mucinous adenocarcinomas included TRU-type and non-TRU-type cases; the latter tended to exhibit the high-grade phenotype with frequent loss of SMARCA4, and A549 was a representative cell line.