Point Mutation In Exon Research Articles

Ovarian Microcystic Stromal Tumor with Significant Nestin Expression: A Unique Case.

Ovary microcystic stromal tumor (MCST) is an extremely rare subtype of sex cord-stromal neoplasm, and only 57 cases have been reported. We herein report a unique case of ovarian MCST with positive nestin expression in a 39-year-old Chinese woman. The tumor showed microcystic stromal histological structures and characteristically expressed the CD10, WT-1, and Ki67 proteins. A molecular analysis identified a point mutation (c.110C > T) in exon 3 of the CTNNB1 gene. To our knowledge, no report has described a case of ovarian MCST with positive staining for nestin protein. Our study provides new insights into the tumor biology of ovarian MCST.

Structural Perspectives in the Development of Novel EGFR Inhibitors for the Treatment of NSCLC.

Non-small cell Lung cancer (NSCLC) is the most common type of lung cancer, which is caused by high consumption of tobacco and smoking. It is an epithelial lung cancer that affects about 2.2 million people across the globe, according to International Agency for Research on Cancer (IARC). Non-small cell lung cancer is a malignant tumor caused by EGFR mutation that occurs in the in-frame deletion of exon 19 and L858R point mutation in exon 21. Presently, clinically available inhibitors of EGFR (including erlotinib, lapatinib, gefitinib, selumetinib, etc.) are not specific and responsible for undesirable adverse effects. Moreover, to solve this problem search for newer EGFR inhibitors is the utmost need for the treatment and/or management of increasing lung cancer burden. The discovery of therapeutic agents that inhibit the specific target in tumorous cells, such as EGFR, is one of the successful strategies in treating many cancer therapies, including lung cancer. The exhaustive literature survey (2018-2023) has shown the importance of medicinally privileged pyrimidine derivatives together, fused and/or clubbed with other heterocyclic rings to design and develop novel EGFR inhibitors. Pyrimidine derivatives substituted with phenylamine, indole, pyrrole, piperazine, pyrazole, thiophene, pyridine and quinazoline derivatives substituted with phenylamine, pyrimidine, morpholine, pyrrole, dioxane, acrylamide, indole, pyridine, furan, pyrimidine, pyrazole etc. are privileged heterocyclic rings shown promising activity by inhibiting EGFR and TKIs. The present review summarizes the structure-activity relationship (SAR) and enzyme inhibitory activity, including IC50 values, percentage inhibition, and kinetic studies of potential compounds from various literature. The review also includes various aspects of molecular docking studies with compounds under clinical trials and patents filed on pyrimidine-based EGFR inhibitors in treating non-small cell lung cancer. The present review may benefit the medicinal chemist for developing novel compounds such as EGFR inhibitors.

The Comparison of Real-Time PCR and Mutation-Specific Immunohistochemistry in EGFR Mutation Analysis of Non-Small Cell Lung Carcinomas

Objective: This study aims to identify activating mutations in the epidermal growth factor receptor (EGFR) gene in patients with non-small cell lung cancer (NSCLC) and to evaluate their correlation with responses to EGFR-tyrosine kinase inhibitors (TKI) treatment. This study aims to identify activating mutations in the epidermal growth factor receptor (EGFR) gene in patients with non-small cell lung cancer (NSCLC) and to evaluate their correlation with responses to EGFR-tyrosine kinase inhibitors (TKI) treatment. We conducted a comparative analysis of Real-Time PCR and immunohistochemistry to detect EGFR mutation status in non-small cell lung cancer patients, focusing on the sensitivity, specificity, and predictive values of immunohistochemistry. Material and Method: We evaluated 788 non-small cell lung cancer samples which were analyzed for EGFR mutation status by RT-PCR. We detected 126 EGFR mutated cases among these patients. We evaluated mutation-specific EGFR immunohistochemistry directed towards the exon 19 deletions (15 bp E746-A750) and exon 21 point mutation (L858R) to the 47 EGFR mutated patients histologic material and cell blocks of cytologic specimens. Results: 32 of the 47 cases (68%) had exon 19 deletion, 14 of them (30%) had point mutation in exon 21, and one of them (2%) showed exon 18 mutation. EGFR exon 19 (15 bp E746-A750 deletion) antibody showed a sensitivity of 100%, specificity of 40%, negative predictive value of 100%, and positive predictive value of 78%. The sensitivity of the exon 21 (L858R point mutation) antibody was 93%, specificity was 91%, negative predictive value was 97% and positive predictive value was 82%. Conclusion: Our investigation indicates that mutation-specific EGFR immunohistochemistry has demonstrated a notable sensitivity and specificity for exon 21. However, while sensitive, the exon 19 (15 bp E746-A750 deletion) antibody lacked specificity. While positive immunohistochemical staining may suggest the presence of an EGFR mutation, making the patient potentially eligible for TKI treatment, it should not be the sole determinant. If immunohistochemistry results are negative, it is essential to resort to molecular tests to ensure accurate diagnosis and appropriate therapeutic guidance. With evolving diagnostic landscapes, it is crucial to harness both IHC and molecular techniques judiciously for optimal patient care.

Structure-Based Analysis of Cefaclor Pharmacokinetic Diversity According to Human Peptide Transporter-1 Genetic Polymorphism.

Cefaclor is a substrate of human-peptide-transporter-1 (PEPT1), and the impact of inter-individual pharmacokinetic variation due to genetic polymorphisms of solute-carrier-family-15-member-1 (SLC15A1) has been a topic of great debate. The main objective of this study was to analyze and interpret cefaclor pharmacokinetic variations according to genetic polymorphisms in SLC15A1 exons 5 and 16. The previous cefaclor bioequivalence results were integrated with additional SLC15A1 exons 5 and 16 genotyping results. An analysis of the structure-based functional impact of SLC15A1 exons 5 and 16 genetic polymorphisms was recently performed using a PEPT1 molecular modeling approach. In cefaclor pharmacokinetic analysis results according to SLC15A1 exons 5 and 16 genetic polymorphisms, no significant differences were identified between genotype groups. Furthermore, in the population pharmacokinetic modeling, genetic polymorphisms in SLC15A1 exons 5 and 16 were not established as effective covariates. PEPT1 molecular modeling results also confirmed that SLC15A1 exons 5 and 16 genetic polymorphisms did not have a significant effect on substrate interaction with cefaclor and did not have a major effect in terms of structural stability. This was determined by comprehensively considering the insignificant change in energy values related to cefaclor docking due to point mutations in SLC15A1 exons 5 and 16, the structural change in conformations confirmed to be less than 0.05 Å, and the relative stabilization of molecular dynamic simulation energy values. As a result, molecular structure-based analysis recently suggested that SLC15A1 exons 5 and 16 genetic polymorphisms of PEPT1 were limited to being the main focus in interpreting the pharmacokinetic diversity of cefaclor.

A First Reported Combination of Exon 18 Missense (G719X) Mutation and Exon 20 Insertion Mutation (Exon20ins) Of Epithelial Growth Factor Receptor in A Patient with Non-Small Cell Lung Carcinoma

The majority of non-small cell lung cancer (NSCLC) associated with epidermal growth factor receptor (EGFR) mutations involve exon 19 deletion mutations and exon 21-point mutation L858R (arginine for leucine substitution at amino acid 858). G719X point mutation (substitutions of the glycine at position 719 to other residues) in exon 18 and insertion in exon 20 are less commonly detected. However, the combination of the latter two mutations has never been reported. We report the first case of metastatic NSCLC with EGFR mutation harboring both exon 18 missense point mutation (G719X) and exon 20 insertion (exon20Ins).

A case of adrenomyeloneuropathy caused by a novel point mutation in the ABCD1 gene and functional verification.

Adrenoleukodystrophy is a rare neurogenetic disease, and adrenomyeloneuropathy is the most common phenotype in adults. The clinical data of a patient with adrenoleukodystrophy and spinal-peripheral neuropathy caused by a novel point mutation in exon 4 of the ABCD1 gene (c.1256T > G (p.Val419Gly)) were retrospectively analyzed. Furthermore, we constructed wild-type and mutant vectors of the ABCD1 (NM0000334) gene to validate the effect of this mutation on the expression of the ABCD1 gene and protein and to explore the mechanism of X-linked adrenoleukodystrophy occurrence and development to identify therapeutic targets for clinical treatment.

NOS3 and CTH gene mutations as new molecular markers for detection of lung adenocarcinoma.

Gene mutations can contribute to lung adenocarcinoma (LUAD) development, metastasis, and therapy. This study aims to identify mutations in the endothelial nitric oxide synthase (eNOS or NOS3) and cystathionine γ-lyase (CSE or CTH) genes that are connected to LUAD symptoms. Two gene polymorphisms were identified using Sanger sequencing in 31 LUAD patients' formalin-fixed paraffin-embedded (FFPE) tissues. Epidermal growth factor receptor (EGFR) mutation and programmed death-ligand 1 (PD-L1) expression were examined in 110 LUAD patients using real-time polymerase chain reaction and immunohistochemistry. Mutations in the selected genes were retrieved from the gnomAD database for all cancer types and the Mutagene and COSMIC databases for LUAD patients. The GeneMANIA prediction server was used to predict the interaction between the studied genes. Poorly and moderately differentiated tumours predominated, with pT3 N2 Mx being the most prevalent stage. Polymorphism data showed 189 NOS3 gene mutations and 34 CTH gene mutations. In 110 LUAD patients, 14 (12.73%) were PD-L1 positive and expressed 50% or more protein. Eight (7.27%) samples included EGFR mutations, including two deletions and two point mutations in exon 19, four point mutations in exon 21. In gnomAD, 4012 NOS3 mutations and 1214 CTH mutations are present. In the Mutagene and COSMIC databases, the NOS3 gene had 295 and 93 mutations, whereas the CTH gene had 61 and 36. According to the GeneMANIA prediction server, 10 genes are related to NOS3, eight with CTH, 15 with EGFR, and 5 with PD-L1. This study is the first to identify several previously unknown mutations in LUAD patients' NOS3 and CTH genes, with potential therapeutic implications.

Microcystic Stromal Tumor of ovary; Report of a rare entity

Abstract Introduction/Objective Microcystic stromal tumor (MCST) of the ovary is an extremely rare subtype of sex cord- stromal neoplasm first described by Irving and Young in 2009. MSCT is characterized by morphology with microcyst structure, solid cellular areas, and hyalinized fibrous stroma. The most reported tumors were stage I with good prognosis. Methods/Case Report We report a rare case of a microcystic stromal tumor of right ovary in a postmenopausal woman, measuring 25 cm in greatest dimension. The present patient underwent right ovarian tumor resection. Gross examination showed that the tumor was solid and cystic. Immunohistochemically, the tumor expressed CD10, WT1, cyclin D1 and vimentin, and nuclear immunoreactivity for β-catenin but negative for α-inhibin, calretinin, CK AE1/AE3, PLAP, SALL-4, CK7, P53, EMA, CD99, AFP, desmin, CgA, E-cadherin, and melanA. Next Generation Sequencing (NGS) showed rare, pathogenic, gain of function mutation CTNNB1 pT41I. The patient is free of disease at 1-year follow up. Results (if a Case Study enter NA) NA Conclusion MCST is a rare stromal tumor of the ovary. These tumors are considered benign neoplasms because almost all cases display unilateral, localized lesions and have benign outcomes, except for one recurrent case with familial adenomatous polyposis and another initial metastatic case with a CTNNB1 mutation (Zhang Y et al. Hum Pathol 2018, 78:171). MCST is histologically characterized by a microcystic growth pattern, solid cellular areas, and intervening hyalinized stroma, accompanied by immunohistochemical evidence of sex cord-stromal differentiation. Accumulation of nuclear -catenin was reported to be relevant to the pathogenesis of MCST (Maeda D et al. Am J Surg Pathol 2011, 35:1429). More specifically, Maeda et al. observed two cases of ovarian MCST that exhibited a CTNNB1 point mutation in exon 3 and strong nuclear immunoreactivity for -catenin. Other cases exhibited alterations of APC, which is associated with familial adenomatous polyposis (Lee SH et al. Genes Chromosomes Cancer 2015, 54: 353). MCST pathogenesis is linked to -catenin stabilization involving -catenin/Wnt signaling. In our case we found a rare pathogenic, gain of function CTNNB1 T41I c.122C>T mutation which maps within a Gsk3b phosphorylation site of the Ctnnb1 protein (UniProt.org). T41I is predicted to confer a gain of function to Ctnnb1 as demonstrated by nuclear accumulation of Ctnnb1 (Garcia-Rostan G et al. Cancer Res 1999, 59:1811). Our case further expands the histopathologic and molecular spectrum of this rare entity.

Modern approaches to the treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease. The cause of the disease is the presence of mutations in the SMN1 gene, which leads to a decrease in the expression of the SMN protein. Decreased functional activity of the SMN protein contributes to the degeneration of motor neurons. Understanding the molecular genetic cause of spinal muscular atrophy made it possible to develop and start using Nusinersen, Onasemnogene Abeparvovec and Risdiplam drugs in clinical practice for the treatment of this disease. The main approaches to the treatment of SMA are either modifying the splicing of the SMN2 gene, which, due to a point mutation in exon 7, is unable to express the full-length SMN protein, or viral delivery of a functional copy of the SMN1 gene.
 Approved drugs differ in peculiarities of use, which are associated, in particular, with the route of administration, adverse reactions and restrictions of use, including restrictions associated with the socio-economic burden of patients with SMA.
 The development and use of domestic products for the treatment of SMA with a comparable clinical effect and at a lower price would reduce the cost of drug therapy for severe forms of the disease.

Overview on Therapeutic Options in Uncommon EGFR Mutant Non-Small Cell Lung Cancer (NSCLC): New Lights for an Unmet Medical Need.

The majority of epidermal growth factor receptor (EGFR) mutations (85-90%) are exon 19 deletions and L858R point mutations of exon 21, characterized by high sensitivity to EGFR-tyrosine kinase inhibitors (TKIs). Less is known about uncommon mutations (10-15% of EGFR mutations). The predominant mutation types in this category include exon 18 point mutations, exon 21 L861X, exon 20 insertions, and exon 20 S768I. This group shows a heterogeneous prevalence, partly due to different testing methods and to the presence of compound mutations, which in some cases can lead to shorter overall survival and different sensitivity to different TKIs compared to simple mutations. Additionally, EGFR-TKI sensitivity may also vary depending on the specific mutation and the tertiary structure of the protein. The best strategy remains uncertain, and the data of EGFR-TKIs efficacy are based on few prospective and some retrospective series. Newer investigational agents are still under study, and there are no other approved specific treatments targeting uncommon EGFR mutations. Defining the best treatment option for this patient population remains an unmet medical need. The objective of this review is to evaluate existing data on the outcomes, epidemiology, and clinical characteristics of lung cancer patients with rare EGFR mutations, with a focus on intracranial activity and response to immunotherapy.

Characterization of flo4-6, a Novel cyOsPPDKB Allele Conferring Floury Endosperm Characteristics Suitable for Dry-Milled Rice Flour Production

Rice cultivars with floury endosperm provide a useful raw material for producing dry-milled rice flour, helping to enhance the processed rice food industry. To expand the genetic resources for breeding floury endosperm rice cultivars, we developed Samkwang(SA)-flo3 (SK-flo3), a floury endosperm mutant line derived from the chemical mutagenesis of Samkwang, an elite Korean japonica rice cultivar. Compared with Samkwang, SK-flo3 showed significantly lower grain hardness, which is suitable for producing dry-milled flour without the soaking and drying processes required in regular wet rice milling. The dry-milled flour of SK-flo3 exhibited excellent physicochemical properties with less damaged starch and finer flour particles relative to Samkwang. Genetic analyses revealed a G-to-A point mutation in exon 7 of cyOsPPDKB, substituting glycine with aspartic acid as a causative mutation for the floury endosperm of SK-flo3. We named this allele flo4-6 and developed a molecular marker to efficiently transfer it to commercial rice cultivars. Our results provide useful genetic resources and information for developing specialty rice cultivars for high-quality rice flour production with reduced milling costs.

A Rare Mutation Associated with Cerebral Arteriopathy with Subcortical Infarct and Leukoencephalopathy (CADASIL) (P2-5.002)

<h3>Objective:</h3> To report a rare cause of CADASIL with a point mutation in exon 5 of NOTCH 3 gene causing a non-cysteine change and its implication. <h3>Background:</h3> CADASIL is a rare genetic non-amyloid arteriopathy primarily affecting small brain vessels. Its prevalence in population is 2 to 5/100,000, but may be underestimated and underdiagnosed. Although there is over 200 different NOTCH-3 mutations described worldwide, 95% of them are missense mutation in the exons 2–24, which leads to a gain or loss of a cysteine residue in one of 34 epidermal growth factor-like repeat (EGFr) domains of the NOTCH3 protein. Non-cysteine mutations are rare and their role is not well determined. <h3>Design/Methods:</h3> Case report <h3>Results:</h3> 58-year-old man with a three-year progressive cognitive decline, history of headaches, fatigue, and generalized weakness was admitted to the neurology wards for worsening weakness and increased need for assistance in daily life. Neurological exam was significant for mild left hemiparesis. MoCA score was 19/30. Brain MRI showed one subacute and numerous chronic lacunar ischemic strokes including in the corpus callosum, also seen on previous outpatient MRIs. These changes had been attributed to demyelinating disease by the outpatient physician. An extensive workup including autoimmune was unrevealing. Classical leukoencephalopathy was not present, but hyperintensities in the temporal lobe raised concern for CADASIL. DNA sequencing revealed a non-cysteine mutation with c.754 G&gt;A transition in exon 5 of NOTCH 3 gene resulting in a substitution of Valine to Methionine (V252M). This is the second case of this mutation reported in the literature. Non-cysteine mutation may affect NOTCH-3 signaling output and possibly lead to a milder leukoencephalopathy and extensive involvement of corpus callosum with lacunar infarctions resembling demyelinating disease. <h3>Conclusions:</h3> CADASIL with a point mutation in exon 5 of NOTCH 3 gene causing a non-cysteine change may present with milder disease and not classical MRI changes. <b>Disclosure:</b> Dr. Delpirou Nouh has nothing to disclose. Dr. Mahmoud has nothing to disclose. Juliane Chainakul has nothing to disclose. Robert Hamilton has nothing to disclose. Dr. Sidorov has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Allergan. Dr. Sidorov has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for BioHaven .

Abstract 3630: TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignancy of the skeleton, and inordinately affects young adults. Despite improved outcomes with current multimodal care, the prognosis for patients with metastatic disease is grim. Peripheral neural regulation of cancer growth has long been suggested by clinical observations in pancreatic and prostate cancers. Prior reports suggest peripheral nerve sprouting in osteosarcoma, but the functional roles of sensory nerves in osteosarcoma are unknown. Here, the role of TrkA+ sensory nerves in OS disease progression was assayed. Methods: All experiments were conducted under IACUC approval at Johns Hopkins University. TrkAF592A mice which are homozygous for a phenylalanine-to-alanine point mutation in exon 12 of Ntrk1 gene were crossbred with NOD scid mice (TrkAF592A/Scid). TrkA inhibition was performed by 1NMPP1 treatment. In vivo tumor growth was examined using a periosteal xenograft model. 1 million human 143B-luc OS cells were injected. Analysis was performed using serial measurements of tumor size and IVIS imaging. Nerve distribution and macrophage polarization within the tumor microenvironment was documented using immunostaining for Beta III Tubulin (TUBB3), CD86, CD206 and F4/80. Results: Inhibition of TrkA led to a significant reduction in Tubb3+ nerves (88.4%) within and around the OS xenografts. In addition, tumors within TrkAF592A/Scid animals demonstrated slower growth, including a 51.5% reduction in tumor size and 60.0% reduction in IVIS luciferase activity at 4 wks. Histologic examination showed a reduction in macrophage infiltration and reprogramming of macrophages from an M2-like to M1-like phenotype in tumors with chemical denervation. Remarkably, this was associated with a reduction in Ki67 proliferative index (80.1%) and CD31 immunofluorescent staining (60.8%) within the tumors. Overall survival of mice with TrkA inhibition was significantly prolonged (p=0.0003). Retrograde tracing identified the sensory nerve fibers innervating the periosteum originated from L3-L5 lumbar DRGs and RNA seq identified OS-derived neurotrophins and axon guidance molecules. Discussion: Disruption of TrkA+ nerves significantly mitigates OS disease progression in a mouse xenograft model, including a significant improved overall survival. These findings are associated with a reduction of macrophage infiltration and macrophage re-polarization. Ongoing studies are examining the molecular mechanisms that mediate nerve-to-macrophage interaction in peripheral nerves and DRG neurons using a combination of sequencing approaches. Significance/Clinical Relevance: Sensory neurons positively regulate osteosarcoma growth and disease progression. Use of chemical or microsurgical approaches to denervate tumors may represent a future adjunctive therapy to both improve pain and prognosis in high grade bone sarcomas. Citation Format: Qizhi Qin, Sowmya Ramesh Ramesh, Thomas L. Clemens, Aaron W. James. TrkA+ sensory neurons modulate macrophage phenotype in osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3630.

Abstract 4476: BI-732, a novel fourth-generation EGFR-TKI, demonstrates promising activities against the C797S-mediated EGFR-TKI resistance

Abstract Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a standard modality of the 1st-line treatments for patients with EGFR-mutated non-small-cell lung cancer (NSCLC). The FDA approved osimertinib, a third-generation EGFR-TKI, is highly selective for EGFR-activating mutations (exon 19 deletion [del19] or L858R point mutation in exon 21 [L858R]) as well as EGFR-T790M mutation. Nevertheless, resistance inevitably emerges and leads to disease progression. Acquired EGFR-C797S mutation is the most common on-target resistance mechanism to osimertinib in first- and second-line settings, and there are currently no approved targeted therapies. We aimed to evaluate the efficacy of BI-732, a fourth-generation EGFR-TKI developed to overcome the C797S mutation. Materials and Methods: We constructed several types of Ba/F3 cells expressing EGFR mutations containing the C797S mutation as follows: EGFRdel19/C797S, EGFRL858R/C797S, EGFRdel19/T790M/C797S, and EGFRL858R/T790M/C797S. We also constructed PC9 cells harboring EGFRdel19/C797S (named PC9-DC cells) using the CRISPR/Cas9 system. Furthermore, we generated two patient-derived cells, YU-1097 with EGFRdel19/T790M/C797S and YU-1182 with EGFRL858R/C797S. The in vitro efficacy of BI-732 was tested in several cell lines harboring the EGFR-C797S mutation mentioned above. We further evaluated in vivo antitumor activity of BI-732 in subcutaneous YU-1097 xenograft model and intracranial YU-1097-tumor model.Results: BI-732 effectively inhibited the viability of Ba/F3 cells expressing EGFRdel19/C797S (IC50, 6.8 nM), EGFRL858R/C797S (IC50, 213.4 nM), EGFRdel19/T790M/C797S (IC50, 3.8nM), and EGFRL858R/T790M/C797S (IC50, 15.2 nM), concomitant with marked reduction in EGFR and downstream signal phosphorylation. Moreover, BI-732 exhibited comparable nanomolar in vitro activity to osimertinib on EGFR activating mutation while sparing wild-type EGFR activity. We further confirmed the superior antiproliferative efficacy of BI-732 in PC9-DC (IC50, 24.9 nM), YU-1182 (IC50, 73 nM) and YU-1097 cells (IC50, 2.9 nM). Oral administration of BI-732 at a concentration of 25 mg/kg as a single agent resulted in significant tumor regression with 183.2% tumor growth inhibition (TGI) in the subcutaneous YU-1097 xenograft model. In a combination treatment strategy with osimertinib, BI-732 showed enhanced antitumor activity at much lower concentrations than monotherapy. BI-732 also demonstrated the ability to penetrate brain-blood barrier (BBB) in the intracranial YU-1097-tumor models.Conclusions: BI-732 is a potent, selective, and orally available fourth-generation EGFR-TKI for EGFR mutations including C797S and has efficient BBB penetration. Our findings suggest that BI-732 can be effective against EGFR mutant NSCLC, especially EGFRDel19/T790M/C797S and EGFRDel19/C797S, that have progressed with previous EGFR inhibitors. Citation Format: Eun Ji Lee, Jiyun Lee, Seung Yeon Oh, You Won Lee, Ju young Kim, Su-Jin Choi, Sewon Park, Mi Ra Yu, Jae Hwan Kim, Kyoung-Ho Pyo, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Anke Baum, Lydia Woelflingseder, Harald Engelhardt, Mark Petronczki, Flavio Solca, Mi Ran Yun, Byoung Chul Cho. BI-732, a novel fourth-generation EGFR-TKI, demonstrates promising activities against the C797S-mediated EGFR-TKI resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4476.

Determination of Factor II Codons Genotype in Southeastern Iranian Patients With Hereditary Deficiency of Factor II

Objectives: Congenital prothrombin (factor II) deficiency is an inherited rare bleeding disorder with an autosomal recessive manner. The prevalence of this disorder is about one in 2000000 people in general population, but it is more common in areas with a high rate of consanguinity. To date, there is no report on the absence of prothrombin, which is a life-threating disorder. Considering the importance of factor II in body homeostasis, this study aimed to find any possible mutation of coagulation factor II codons in patients with inherited factor II deficiency in southeastern Iran. Materials andMethods: This study was conducted on 12 patients with inherited deficiency of prothrombin. Early diagnosis was based on clinical symptoms, laboratory evaluation, and family history. Then, the function level of prothrombin was measured, the initial diagnosis of disease was confirmed, and polymerase chain reaction (PCR) analysis was performed. Finally, gene sequencing and genotyping of factor II was done. Results: Molecular analysis indicated a point mutation in exon 7 in three patients and a frameshift mutation in exon 14 due to addition of a thymine base at position 1760-1761 in one patient, both of which have been reported for the first time. Conclusions: Molecular methods performed on patients from Southeastern Iranian population in terms of coagulation factor II deficiency revealed a substitution mutation in exon 7 in three patients and a frameshift mutation in exon 14 in one patient, both of which were reported for the first time. Considering the significant difference between the clinical symptoms of the present study and previous studies, probably the type of mutations reported in this study (for the first time) caused these clinical symptoms, but statistical studies did not show any relationship between the type of mutation and the occurrence of clinical symptoms. And it needs more investigations on more patients, with a larger population.

The complexity of EGFR exon 19 deletion and L858R mutant cells as assessed by proteomics, transcriptomics, and metabolomics

Most lung adenocarcinoma-associated EGFR tyrosine kinase mutations are either an exon 19 deletion (19Del) or L858R point mutation in exon 21. Although patients whose tumors contain either of these mutations exhibit increased sensitivity to tyrosine kinase inhibitors, progression-free and overall survival appear to be longer in patients with 19Del than in those with L858R. In mutant-transfected Ba/F3 cells, 19Del and L858R were compared by multi-omics analyses including proteomics, transcriptomics, and metabolomics. Proteome analysis identified increased plastin-2, TKT, PDIA5, and ENO1 expression in L858R cells, and increased EEF1G expression in 19Del cells. RNA sequencing showed significant differences between 19Del and L858R cells in 112 genes. Metabolome analysis showed that amino acids, adenylate, guanylate, NADPH, lactic acid, pyruvic acid glucose 6-phosphate, and ribose 5-phosphate were significantly different between the two mutant cells. Because GSH was increased with L858R, we combined osimertinib with the GSH inhibitor buthionine sulfoximine in L858R cells and observed synergistic effects. The complexity of EGFR 19Del and L858R mutant cells was demonstrated by proteomics, transcriptomics, and metabolomics analyses. Therapeutic strategies for lung cancer with different EGFR mutations could be considered because of their different metabolic phenotypes.

EGFR-Mutant Non-small Cell Lung Cancer: State-of-the-Art and Future Perspectives

EGFR mutations are the first identified targetable driver alterations in advanced non-small cell lung cancer (NSCLC), for which specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) have been developed. These small molecules, administered orally, changed the natural history of patients with EGFR-mutated NSCLC, reporting impressive response and survival data. Osimertinib, a third-generation EGFR-TKI, can be considered the standard first-line therapy for the ‘common’ EGFR mutations, which include the exon 19 deletion and Leu858Arg point mutation in exon 21, accounting for 90% of cases. The ‘uncommon’ EGFR mutations, highly heterogeneous and with a low frequency, seem to be more sensitive to afatinib and osimertinib, a second-generation EGFR-TKI, excluding the EGFR exon 20 insertions mutations, for which a platinum-based regimen should be recommended while waiting for specific targeted inhibitors to reach the market. However, after an initial activity to first-line EGFR-TKI treatment, a disease progression is reported due to the presence of an intrinsic resistance or the onset of an acquired resistance. The latter can be broadly grouped into EGFR-dependent or EGFR-independent mechanisms of resistance, for which several new drugs and strategic approaches are under investigation. This review focuses on the state-of-the-art EGFR-TKIs in the treatment of metastatic NSCLC harbouring EGFR mutations, and also discusses potential future perspectives.

Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

Mutation and up-regulation of TP53 in ovine pulmonary adenocarcinoma lung cells as a model of human lung cancer.

Ovine pulmonary adenocarcinoma (OPA) is a model of human lung cancer‎ and fatal viral disease that causes neoplasia in sheep respiratory cells. ‎In the current study, 986 lung samples was inspected in the slaughterhouse, and finally twenty OPA ‎ lung organs were clinically diagnosed and five healthy lung organs were assigned as the control sample. Three SSCP patterns were detected for the affected lungs animals in comparison with the healthy lungs. In addition, sequencing results indicated three different single point mutations in exon 4 of TP53 within infected lungs, whereas no mutations were observed in exon 9 of this gene. Real-time PCR results showed up-regulation of the TP53 gene in all the infected lung cells compared to healthy cells. There was significant correlation between the mutations in exon 4 and OPAand can be used as a useful tool in determining the mechanism of lung cancer.

1000P Common and uncommon mutations in NSCLC: Differences in response to treatment with tyrosine kinase inhibitors: A single-center retrospective analysis

Uncommon EGFR mutations are a heterogeneous group, discovered in ∼10% of the population with EGFR-positive non-small cell lung cancer (NSCLC), whose sensitivity to tyrosine-kinase inhibitors is not yet widely demonstrated. We propose to retrospectively analyze how patients with various EGFR mutations respond differently to treatment with tyrosine-kinase inhibitors. 48 patients with EGFR-positive NSCLC were identified and divided into subgroups by mutation. Progression-free survival (PFS), overall survival (OS) and time to treatment failure (TTF) in those who were treated with Afatinib (Giotrif®) were evaluated. Subgroups were compared using the log-rank test and a multivariate analysis was performed. Patients with common mutations, deletion in exon 19 and L85R, had median OS of 31 months (95% CI: 22-51) and 23 months (95% CI: 6-33) and a median PFS of 16 months (95% CI: 9-33) and 10 months (95% CI: 6-22), respectively. OS and PFS, in patients with common mutations, were superior to those with an uncommon mutation with a p value of 0.003 and 0.0001. Patients with mutations in exon 18 and 21 and patients with mutations in exon 20 had the worst outcome with an OS of 6.5 months (95% CI: 4-30) a PFS of 2.5 months (95% CI: 1-4) and an OS of 3 months (95% CI: 2-4) and a PFS of 4.5 (95% CI: 4-5), respectively. Patients with these mutations also had significantly lower TTF. Multivariate analysis confirmed the results: patients with point mutations in exon 19-21 and mutations in exon 20 had the highest risk of death (HR 6.01 CI: 1.02-35.46, p value=0.001 and HR 12.81 CI: 2.46-62.19 p value=0.004).Table: 1000PMutationNo.FrequencyProgression Free Survival (months)Overall survival (months)Del191837.5%15.5 (9-33)31 (22-51)L858R1837.5%10 (6-22)23 (6-33)Complex mutations24.1%8.25 (0.5-16)8.25 (0.5-16)DelIns1948.3%11 (2-30)21.5 (2-43)Exon 18-2148.3%2.5 (1-4)6.5 (4-30)Exon 2024.1%4.5 (4-5)3 (2-4) Open table in a new tab Patients with uncommon mutations respond heterogeneously to tyrosine-kinase inhibitor treatments. Patients with deletion-insertion or complex mutations respond less than patients with common mutations but they are sensitive to treatment, whereas patients with insertion in exon 20 or mutations in exons 18 and 21 respond very poorly.