Successful cetuximab rechallenge in metastatic colorectal cancer: A case report.

BACKGROUND: Metastatic colorectal cancer (mCRC) treatment has been evolving and increasingly driven by tumor biology and gene expression analysis. Rechallenge with epidermal growth factor receptor (EGFR) inhibitors (anti-EGFR) represents a promising strategy for patients with RAS wild-type (RAS-wt) mCRC and circulating tumor DNA has emerged as a potential selection strategy. Herein, we report the case of a RAS-wt mCRC patient who had a successful response to cetuximab rechallenge. CASE SUMMARY: Our patient was diagnosed with stage IV RAS-wt, microsatellite-stable rectosigmoid junction adenocarcinoma. He was started on first-line treatment with FOLFIRI and cetuximab and achieved partial response, allowing for a left hepatectomy (R0), followed by post-operative chemotherapy and an anterior resection; progression-free survival (PFS) of 16 months was obtained. Due to hepatic and nodal relapse, second-line treatment with FOLFOX and bevacizumab was started with partial response; metastasectomy was performed (R0), achieving a PFS of 11 months. After a 15 months anti-EGFR-free interval, FOLFIRI and cetuximab were reintroduced upon disease progression, again with partial response and a PFS of 16 months. Following extensive hepatic relapse, cetuximab was reintroduced and a marked clinical and analytical improvement was seen, after only one cycle. RAS-wt status was confirmed on circulating tumor DNA. The patient's overall survival exceeded 5 years. CONCLUSION: Our case provides real-world data to support cetuximab rechallenge in later lines of RAS-wt mCRC treatment.

Survival outcomes of neoadjuvant versus adjuvant therapy in patients with T1c, node-negative, human epidermal growth factor receptor 2-positive breast cancer: A Surveillance, Epidemiology, and End Results population-based study.

BACKGROUND: Persistent debates exist regarding the superiority of neoadjuvant therapy (NAT) over adjuvant therapy (AT) for patients with T1c, node-negative, human epidermal growth factor receptor 2-positive (HER2+) breast cancer, and relevant guidelines for these patients are lacking. METHODS: Data on patients with T1cN0M0-stage HER2+ breast cancer who received chemotherapy and surgery were extracted from 2010 to 2020 from the Surveillance, Epidemiology, and End Results database. Propensity score matching (PSM) was used to create well-balanced cohorts for the NAT and AT groups. Kaplan-Meier (KM) analysis and Cox proportional hazards models were used to assess the differences between NAT and AT in terms of overall survival (OS) and breast cancer-specific survival (BCSS). Additionally, logistic regression models were used to explore factors associated with response to NAT. RESULTS: After PSM, 2140 patient pairs were successfully matched, which achieved a balanced distribution between the NAT and AT groups. KM curves revealed similar OS and BCSS between patients receiving NAT and those undergoing AT. A multivariate Cox model identified achieving pathological complete response (pCR) after NAT, compared with AT, as a protective prognostic factor for OS (hazard ratio, 0.52; 95% CI, 0.35-0.77; p < .001) and BCSS (hazard ratio, 0.60; 95% CI, 0.37-0.98; p = .041). A logistic regression model revealed that White race and hormone receptor-negative status independently predicted pCR. CONCLUSIONS: For patients with T1cN0M0-stage HER2+ breast cancer, NAT demonstrated comparable OS and BCSS to AT. Patients who achieved pCR after NAT exhibited significantly better survival outcomes compared with those who received AT.

KEAP1-NRF2 pathway as a novel therapeutic target for EGFR-mutant non-small cell lung cancer.

BACKGROUND: Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor erythroid-2-related factor 2 (NRF2) pathway is a major regulator protecting cells from oxidative and metabolic stress. Studies have revealed that this pathway is involved in mediating resistance to cytotoxic chemotherapy and immunotherapy, however, its implications in oncogene-addicted tumors are largely unknown. This study aimed to elucidate whether this pathway could be a potential therapeutic target for epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer. METHODS: We measured the baseline expression of NRF2 using EGFR-mutant parental cells and acquired gefitinib resistant cells. We investigated whether NRF2 inhibition affected cell death in vitro and tumor growth in vivo using a xenograft mouse model, and compared the transcriptional changes before and after NRF2 inhibition. RESULTS: Baseline NRF2 expression was enhanced in PC9 and PC9 with gefitinib resistance (PC9/GR) cells than in other cell lines, with a more prominent expression in PC9/GR. The NRF2 inhibitor induced NRF2 downregulation and cell death in a dose-dependent manner. Co-treatment with an NRF2 inhibitor enhanced osimertinib-induced cell death in vitro, and potentiated tumor growth inhibition in a PC9/GR xenograft model. Finally, RNA sequencing revealed that NRF2 inhibition resulted in the altered expression of multiple genes involved in various signaling pathways. CONCLUSION: We identified that NRF2 inhibition enhanced cell death and inhibited tumor growth in TKI-resistant lung cancer with EGFR-mutation. Thus, NRF2 modulation may be a novel therapeutic strategy to overcome the resistance to EGFR-tyrosine kinase inhibitors.

Are 19del and L858R really different disease entities?

Clinicians have recognized the similarities and differences between the two subtypes of common epidermal growth factor receptor (EGFR) mutations, but actual treatment strategies have not yet changed. The L858R mutation can be understood by considering the pharmacological conformational plasticity of the receptor protein and the presence of other co-occurring mutations, whether subtypes of EGFR or non-EGFR mutations and differences in downstream signaling pathways. As long as we know that molecular differences lead to biological differences, it is a challenge for all of us that our treatment strategies must change.

[Box: see text].

Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics.

The epidermal growth factor (EGF) receptor (EGFR) is activated by the binding of one of seven EGF-like ligands to its ectodomain. Ligand binding results in EGFR dimerization and stabilization of the active receptor conformation subsequently leading to activation of downstream signaling. Aberrant activation of EGFR contributes to cancer progression through EGFR overexpression/amplification, modulation of its positive and negative regulators, and/or activating mutations within EGFR. EGFR targeted therapeutic antibodies prevent dimerization and interaction with endogenous ligands by binding the ectodomain of EGFR. However, these antibodies have had limited success in the clinic, partially due to EGFR ectodomain resistance mutations, and are only applicable to a subset of patients with EGFR-driven cancers. These limitations suggest that alternative EGFR targeted biologics need to be explored for EGFR-driven cancer therapy. To this end, we analyze the EGFR interfaces of known inhibitory biologics with determined structures in the context of endogenous ligands, using the Rosetta macromolecular modeling software to highlight the most important interactions on a per-residue basis. We use this analysis to identify the structural determinants of EGFR targeted biologics. We suggest that commonly observed binding motifs serve as the basis for rational design of new EGFR targeted biologics, such as peptides, antibodies, and nanobodies.

Genomic profiling of colorectal cancer in large-scale Chinese patients: amplification and somatic mutations in ERBB2.

Objectives: Human epidermal growth factor receptor 2 (HER2)-targeted therapies have demonstrated potential benefits for metastatic colorectal cancer (mCRC) patients with HER2 amplification, but are not satisfactory in cases of HER2 mutant CRCs. Methods: Consequently, further elucidation of amplifications and somatic mutations in erythroblastic oncogene B-2 (ERBB2) is imperative. Comprehensive genomic profiling was conducted on 2454 Chinese CRC cases to evaluate genomic alterations in 733 cancer-related genes, tumor mutational burden, microsatellite instability, and programmed death ligand 1 (PD-L1) expression. Results: Among 2454 CRC patients, 85 cases (3.46%) exhibited ERBB2 amplification, and 55 cases (2.24%) carried ERBB2 mutation. p.R678Q (28%), p.V8421 (24%), and p.S310F/Y (12%) were the most prevalent of the 16 detected mutation sites. In comparison to the ERBB2 altered (alt) group, KRAS/BRAF mutations were more prevalent in ERBB2 wild-type (wt) samples (ERBB2wt vs. ERBB2alt, KRAS: 50.9% vs. 25.6%, p < 0.05; BRAF: 8.5% vs. 2.3%, p < 0.05). 32.7% (18/55) of CRCs with ERBB2 mutation exhibited microsatellite instability high (MSI-H), while no cases with HER2 amplification displayed MSI-H. Mutant genes varied between ERBB2 copy number variation (CNV) and ERBB2 single nucleotide variant (SNV); TP53 alterations tended to co-occur with ERBB2 amplification (92.3%) as opposed to ERBB2 mutation (58.3%). KRAS and PIK3CA alterations were more prevalent in ERBB2 SNV cases (KRAS/PIK3CA: 45.8%/31.2%) compared to ERBB2 amplification cases (KRAS/PIK3CA: 14.1%/7.7%). Conclusion: Our study delineates the landscape of HER2 alterations in a large-scale cohort of CRC patients from China. These findings enhance our understanding of the molecular features of Chinese CRC patients and offer valuable implications for further investigation.

Can negative axillary ultrasound reliably predict pathologically negative axillary lymph node status in breast cancer patients with cT ≤3 cm, cN0, and HER2-positive?-a retrospective, single-institution study.

Background: Breast cancer (BC) is the leading cancer in women globally, with human epidermal growth factor receptor 2 (HER2)-positive subtype accounting for 15-20% of cases and exhibiting aggressive behavior. The standard of care for operable BC has evolved to include neoadjuvant systemic therapy, which can guide treatment decisions and improve outcomes, particularly in HER2+ BC. This study aims to investigate whether axillary ultrasound has a good negative predictive value (NPV) for early HER2 BC patients and to identify clinicopathological factors that can impact the axillary lymph node metastasis. Methods: This retrospective, single-center study evaluated the medical records of 135 patients with HER2+ BC, cT ≤3 cm, and clinically negative axillary lymph nodes from 2018 to 2020. The study aimed to determine the NPV of axillary ultrasound for pathologically negative axillary lymph node status and to identify factors associated with axillary lymph node metastasis. Results: The NPV of axillary ultrasound was 78.5%, increasing to 89.6% and 93.3% when considering 0-1 and 0-2 metastatic lymph nodes, respectively. Lymphovascular invasion (LVI) was significantly associated with axillary lymph node metastasis, with a 2.2-fold increased risk. Conclusions: Axillary ultrasound shows good predictive value for axillary lymph node negativity in HER2+ BC patients with small tumors. However, the presence of LVI increases the risk of metastasis, suggesting a need for neoadjuvant chemotherapy. These findings contribute to personalized treatment strategies for early HER2+ BC, emphasizing the role of axillary ultrasound in clinical decision-making.

Omission of axillary surgery in cN0, postmenopausal ER-positive/HER2-negative breast cancer patients undergoing breast-conserving treatment.

Background: Previous clinical trials have diminished the significance of lymph node (LN) metastasis and axillary surgery in breast cancer, particularly in cN0, postmenopausal estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative patients undergoing breast-conserving treatment (BCT). We assessed the replacement of axillary surgery with preoperative imaging modalities by analyzing the proportion of high nodal burden (HNB) patients with ≥3 LN metastases in these patients. Methods: We retrospectively identified 333 cN0, postmenopausal ER-positive/HER2-negative breast cancer patients who underwent BCT in two hospitals between January 2003 and December 2017. The proportion of LN metastasis patients and the number of metastatic LN were investigated. Risk factors of LN metastasis were analyzed and recurrence-free survival (RFS) was compared. Results: Axillary surgery confirmed LN metastasis in 81 (24.3%) of the cN0 patients. The clinical tumor size (cT) and age were factors associated with LN metastasis [cT: odds ratio (OR), 2.92, 95% confidence interval (CI): 1.69-5.05, P<0.001; age: OR, 0.33, 95% CI: 0.11-0.99, P=0.048]. However, HNB patients with ≥3 LN metastases were 15 (4.5%) of all the patients. There was statistically significant difference in the incidence of HNB between patients with cT1 tumors (3.6%) and those with cT2 tumors (7.4%) (P<0.001). Conclusions: In cN0, postmenopausal ER-positive/HER2-negative patients who underwent BCT, patients with cT1 tumors had lower rate of LN metastasis, and there were fewer instances of HNB. Therefore, in these patients, omission of axillary surgery including SLNB can be carefully considered.

Obesity-Associated Breast Cancer: Analysis of Risk Factors and Current Clinical Evaluation.

Several studies show that a significantly stronger association is obvious between increased body mass index (BMI) and higher breast cancer incidence. Additionally, obese and postmenopausal women are at higher risk of all-cause and breast cancer-specific mortality compared with non-obese women with breast cancer. In this context, increased levels of estrogens, excessive aromatization activity of the adipose tissue, overexpression of pro-inflammatory cytokines, insulin resistance, adipocyte-derived adipokines, hypercholesterolemia, and excessive oxidative stress contribute to the development of breast cancer in obese women. Genetic evaluation is an integral part of diagnosis and treatment for patients with breast cancer. Despite trimodality therapy, the four-year cumulative incidence of regional recurrence is significantly higher. Axillary lymph nodes as well as primary lesions have diagnostic, prognostic, and therapeutic significance for the management of breast cancer. In clinical setting, because of the obese population primary lesions and enlarged lymph nodes could be less palpable, the diagnosis may be challenging due to misinterpretation of physical findings. Thereby, a nomogram has been created as the "Breast Imaging Reporting and Data System" (BI-RADS) to increase agreement and decision-making consistency between mammography and ultrasonography (USG) experts. Additionally, the "breast density classification system," "artificial intelligence risk scores," ligand-targeted receptor probes," "digital breast tomosynthesis," "diffusion-weighted imaging," "18F-fluoro-2-deoxy-D-glucose positron emission tomography," and "dynamic contrast-enhanced magnetic resonance imaging (MRI)" are important techniques for the earlier detection of breast cancers and to reduce false-positive results. A high concordance between estrogen receptor (ER) and progesterone receptor (PR) status evaluated in preoperative percutaneous core needle biopsy and surgical specimens is demonstrated. Breast cancer surgery has become increasingly conservative; however, mastectomy may be combined with any axillary procedures, such as sentinel lymph node biopsy (SLNB) and/or axillary lymph node dissection whenever is required. As a rule, SLNB-guided axillary dissection in breast cancer patients who have clinically axillary lymph node-positive to node-negative conversion following neoadjuvant chemotherapy is recommended, because lymphedema is the most debilitating complication after any axillary surgery. There is no clear consensus on the optimal treatment of occult breast cancer, which is much discussed today. Similarly, the current trend in metastatic breast cancer is that the main palliative treatment option is systemic therapy.

Osimertinib after definitive chemoradiotherapy in unresectable stage III epidermal growth factor receptor-mutated non-small-cell lung cancer: analyses of central nervous system efficacy and distant progression from the phase III LAURA study.

BACKGROUND: Distant metastases in non-small-cell lung cancer (NSCLC) are a poor prognostic factor that negatively impact quality of life. The central nervous system (CNS) is a common site of distant progression in epidermal growth factor receptor-mutated (EGFRm) NSCLC. Osimertinib is a third-generation EGFR-tyrosine kinase inhibitor recommended for advanced EGFRm NSCLC and as adjuvant treatment for resected EGFRm NSCLC. In LAURA (NCT03521154), osimertinib demonstrated statistically significant improvement in progression-free survival (PFS) versus placebo in unresectable stage III EGFRm NSCLC without progression during/following chemoradiotherapy (CRT). CNS efficacy and time to death or distant metastases (TTDM) analyses are reported here. PATIENTS AND METHODS: Patients without progression during/following definitive platinum-based CRT were randomised 2 : 1 to receive osimertinib (80 mg daily) or placebo until progression [by blinded independent central review (BICR)] or discontinuation. The primary endpoint was PFS by BICR. CNS PFS by neuroradiologist BICR and TTDM by BICR were secondary endpoints. RESULTS: Overall, 216 patients were randomised (143 osimertinib, 73 placebo). Median CNS PFS by neuroradiologist BICR was not reached [95% confidence interval (CI) not calculable (NC)-NC] with osimertinib versus 14.9 months (95% CI 7.4 months-NC) with placebo; hazard ratio (HR) for CNS PFS: 0.17 (95% CI 0.09-0.32). CNS PFS analysis by investigator assessment was consistent with BICR assessment. The cumulative incidence of CNS progression at 12 months was 9% (95% CI 5% to 14%) with osimertinib and 36% (95% CI 24% to 47%) with placebo. There was clinically meaningful improvement in TTDM with osimertinib versus placebo; HR for TTDM: 0.21 (95% CI 0.11-0.38). The cumulative incidence of distant metastases at 12 months was 11% (95% CI 6% to 17%) with osimertinib and 37% (95% CI 26% to 48%) with placebo. CONCLUSIONS: Osimertinib demonstrated clinically meaningful improvements in CNS PFS and TTDM versus placebo, supporting osimertinib post-CRT as the standard of care in unresectable stage III EGFRm NSCLC.

Targeted and combination immunotherapies using biologics for gastric cancer: the state-of-the-art.

INTRODUCTION: Gastricadenocarcinoma (GAC) remains a prevalent cancer worldwide and its incidence is increasingin South America. The heterogenous nature of GAC makes advances in managementchallenging. AREAS COVERED: Despitechallenges, recent therapeutic targets are individualizing treatment. Forlocalized disease with microsatellite-instability-high/deficientmismatch repair, immunotherapy is now an adopted practice. In the advancedunresectable setting, those harboring human epidermalgrowth factor receptor-2 (HER2) expression continue to be a separateentity. EXPERT OPINION: Future targets are developing. Among these includeclaudin 18.2 (CLDN18.2), fibroblast growth factor receptor 2b(FGFR2b), and trophoblast cellsurface antigen-2 (TROP-2). FDA approval of zolbetuximab's, an anti-CLDN18.2monoclonal antibody, is expected soon. Additionally, bemarituzumab, ananti-FGFR2b monoclonal antibody, has shown improvements in combination withchemotherapy in those with HER2 negative GAC with FGFR2 overexpression. Thiscombination is now being investigated in a phase 3 trial. Lastly, TROP-2 has emergedas an exciting solid tumor target and study is expected in GAC. All three ofthese therapeutic targets have seen an abundance of drug development in recentyears, and we anticipate newer targeted agents driving therapeutic decisions inGAC in the coming years.

Analysis of genomic alternations in epidermal growth factor receptor (EGFR)-T790M-mutated non-small cell lung cancer (NSCLC) patients with acquired resistance to osimertinib therapy.

BACKGROUND AND OBJECTIVES: Genomic alterations after resistance to osimertinib therapy in advanced T790M-mutated non-small cell lung cancer (NSCLC) are complex and poorly understood. In this study, we aimed to detect these genomic alternations via comprehensive next-generation sequencing (NGS) of tissue and liquid biopsies. PATIENTS AND METHODS: From September 2020 to June 2021, 31 stage IIIB/IV T790M-mutated NSCLC patients who exhibited progressive disease after osimertinib therapy and provided written informed consent were recruited. Liquid and tissue biopsy samples for NGS testing were collected from 31 and 18 patients, respectively. Eighteen study patients had paired NGS data from tissue and liquid biopsies. RESULTS: With respect to the T790M mutation status, the preservation and loss rates were 33% and 67%, respectively, in both liquid and tissue biopsy samples. Five patients (16.1%) had the C797S mutation (4 liquid samples and 1 tissue sample). Two (6.5%) had MET mutations, 3 (9.7%) had BRAF-V600E mutations, and 1 (3.2%) had a KRAS-G12C mutation. Among the 18 patients who underwent tissue rebiopsies, those with preserved T790M mutation had significantly longer progression-free survival (PFS) with osimertinib therapy than those with T790M mutation loss (10.8 vs. 5.0 months, P = 0.045). Among all patients, those with T790M mutation loss in liquid biopsy samples had longer PFS after osimertinib therapy (10.8 vs. 7.5 months, P = 0.209) and postprogression survival (17.7 vs. 9.6 months, P = 0.132) than those with preserved T790M mutation based on liquid biopsies. CONCLUSIONS: NGS using either tissue or liquid biopsy samples from advanced T790M-mutated NSCLC patients with acquired resistance to osimertinib therapy can detect various genomic alternations. Future studies focusing on subsequent tailored therapies on the basis of NGS results are warranted.

Major pathologic response and long-term clinical benefit in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer after neoadjuvant chemotherapy.

BACKGROUND: The majority of HR+/HER2-breast cancer patients can also achieve long-term survival despite not attaining pCR, indicating limited prognostic value of pCR in this population. This study aimed to identify novel pathologic end points for predicting long-term outcomes in HR+/HER2-breast cancer after neoadjuvant chemotherapy. METHODS: We analyzed HR+/HER2-breast cancer patients with stage II-III tumors who underwent curative surgery after neoadjuvant chemotherapy from three hospitals. Major pathologic response (MPR), defined as the presence of Miller-Payne grades 3-5 and positive lymph node ratio of ≤10 %, was used as a pathological evaluation indicator. We assessed the association between MPR and event-free survival (EFS) and performed Multivariable Cox regression to identify independent factors associated with EFS. RESULTS: From January 2010 to December 2020, 386 patients were included in the final analysis. 28 patients (7.3 %) achieved pCR and 118 patients (30.6 %) achieved MPR. The median duration of follow-up was 54.4 months,5-year EFS was 87 % in the MPR group vs. 68 % in the non-MPR group. Multivariate analysis showed that low PR expression, high clinical stage, lower Miller-Payne grades and Positive lymph node ratio were independent poor prognostic factors for EFS (all P values < 0.05). The prognostic effect of MPR remained in multivariable models (hazard ratio (HR), 0.45; 95 % confidence interval (CI), 0.26-0.76; P = 0.008), In non-pCR patients, those who achieved MPR exhibited a similar EFS compared with pCR patients (HR, 2.25; 95 % CI, 0.51-9.84; P = 0.28). CONCLUSION: MPR may be a novel pathologic end point in HR+/HER2-breast cancer after neoadjuvant chemotherapy, holding greater applicability in the prognosis evaluation than pCR.

AB020. Flagellin synergistically enhances anti-tumor effect of EGFRvIII peptide in a glioblastoma-bearing mouse brain tumor model.

BACKGROUND: Glioblastoma (GBM) is the most aggressive type of brain tumor with heterogeneity and strong invasive ability. Treatment of GBM has not improved significantly despite the progress of immunotherapy and classical therapy. Epidermal growth factor receptor variant III (EGFRvIII), one of GBM-associated mutants, is regarded as an ideal therapeutic target in EGFRvIII-expressed GBM patients because it is a tumor-specific receptor expressed only in tumors. Flagellin B (FlaB) originated from Vibrio vulnificus, is known as a strong adjuvant that enhances innate and adaptive immunity in various vaccine models. This study investigated whether FlaB synergistically could enhance the anti-tumor effect of EGFRvIII peptide (PEGFRvIII). METHODS: EGFRvIII-GL261/Fluc cells were used for GBM-bearing mouse brain model. Cell-bearing mice were inoculated with phosphate-buffered saline (PBS), FlaB alone, PEGFRvIII alone, and PEGFRvIII plus FlaB. Tumor growth based on magnetic resonance imaging (MRI) and the survival rate was investigated. T cell population was examined by flow cytometry analysis. Both cleaved caspase-3 and CD8+ lymphocytes were shown by immunohistochemistry (IHC) staining. RESULTS: The PEGFRvIII plus FlaB group showed delayed tumor growth and increased survival rate when compared to other treatment groups. As evidence of apoptosis, cleaved caspase-3 expression and DNA disruption were more increased in the PEGFRvIII plus FlaB group than in other groups. In addition, the PEGFRvIII plus FlaB group showed more increased CD8+ T cells and decreased Treg cells than other treatment groups in the brain. CONCLUSIONS: FlaB can enhance the anti-tumor effect of PEGFRvIII by increasing CD8+ T cell response in a mouse brain GBM model.

Methods for production and assaying catalysis of isolated recombinant human aspartate/asparagine-ß-hydroxylase.

Aspartate/asparagine-ß-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies.

Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids.

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-ß receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFß-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

Review of In Situ Hybridization (ISH) Stain Images Using Computational Techniques.

Recent advancements in medical imaging have greatly enhanced the application of computational techniques in digital pathology, particularly for the classification of breast cancer using in situ hybridization (ISH) imaging. HER2 amplification, a key prognostic marker in 20-25% of breast cancers, can be assessed through alterations in gene copy number or protein expression. However, challenges persist due to the heterogeneity of nuclear regions and complexities in cancer biomarker detection. This review examines semi-automated and fully automated computational methods for analyzing ISH images with a focus on HER2 gene amplification. Literature from 1997 to 2023 is analyzed, emphasizing silver-enhanced in situ hybridization (SISH) and its integration with image processing and machine learning techniques. Both conventional machine learning approaches and recent advances in deep learning are compared. The review reveals that automated ISH analysis in combination with bright-field microscopy provides a cost-effective and scalable solution for routine pathology. The integration of deep learning techniques shows promise in improving accuracy over conventional methods, although there are limitations related to data variability and computational demands. Automated ISH analysis can reduce manual labor and increase diagnostic accuracy. Future research should focus on refining these computational methods, particularly in handling the complex nature of HER2 status evaluation, and integrate best practices to further enhance clinical adoption of these techniques.

Epidermal Growth Factor Receptor Emerges as a Viable Target for Reducing Tumorigenicity of MDCK Cells.

The MDCK cell line is perceived as better than the embryos of hen eggs for the production of influenza vaccines, but the tumorigenicity of these cells is concerning. Epidermal growth factor receptor (EGFR) is likely to be a crucial target that contributes to the tumorigenicity of MDCK cells. In this study, EGFR-knockdown and EGFR-overexpression cell lines were established. EGFR's influence on cell growth, migration, clonogenic ability, and flu virus susceptibility was evaluated in vitro, and its role in cell tumorigenicity was examined in nude mice. GST pull-down coupled with mass spectrometry (MS) and bioinformatics analysis identified EGFR-interacting proteins. The expression levels of these proteins, as well as those of PI3K-AKT- and MAPK-ERK-signaling-pathway-related molecules, were confirmed at both gene and protein levels. The result indicates that EGFR overexpression can enhance cell proliferation, migration, and clonal formation; EGFR knockdown could effectively curtail tumorigenesis and amplify the titers of influenza viruses in MDCK cells. An analysis of the underlying mechanism identified a total of 21 interacting proteins implicated in tumor formation, and among these, AKT1, CDK4, GNB2, and MAPK8 were confirmed at both gene and protein levels. EGFR can activate key factors of the PI3K-AKT signaling pathway, AKT and PI3K, and promote their phosphorylation levels. Consequently, we concluded that EGFR interacts with GNB2, facilitating transmembrane signal transduction, activating the PI3K-AKT signaling cascade, controlling cell cycle alterations, stimulating cell proliferation, and promoting tumorigenesis.

Synthesis and Biological Evaluation of Novel Cationic Rhenium and Technetium-99m Complexes Bearing Quinazoline Derivative for Epidermal Growth Factor Receptor Targeting.

Background/Objectives: Epidermal growth factor receptor (EGFR) plays a vital role in cell proliferation and survival, with its overexpression linked to various malignancies, including non-small cell lung cancer (NSCLC). Although EGFR tyrosine kinase inhibitors (TKIs) are a key therapeutic strategy, acquired resistance and relapse remain challenges. This study aimed to synthesize and evaluate novel rhenium-based complexes incorporating EGFR TKIs to enhance anticancer efficacy, particularly in radiosensitization. Methods: We synthesized a rhenium tricarbonyl complex (Complex 2) and its 99mTc analog (Complex 2') by incorporating triphenylphosphine instead of bromine as the monodentate ligand and PF6- as the counter-ion, resulting in a positively charged compound that forms cationic structures. Cytotoxicity and EGFR inhibition were evaluated in A431 cells overexpressing EGFR using MTT assays, Western blotting, and flow cytometry. Radiosensitization was tested through MTT and clonogenic assays. The 99mTc complex's radiochemical yield, stability, and lipophilicity were also assessed. Results: Complex 2 exhibited significant cytotoxicity with an IC50 of 2.6 µM and EGFR phosphorylation inhibition with an IC50 of 130.6 nM. Both complex 1 and 2 induced G0/G1 cell cycle arrest, with Complex 2 causing apoptosis. Radiosensitization was observed at doses above 2 Gy. Complex 2' demonstrated high stability and favorable lipophilicity (LogD7.4 3.2), showing 12% cellular uptake after 30 min. Conclusions: Complexes 2 and 2' show promise as dual-function anticancer agents, offering EGFR inhibition, apoptosis induction, and radiosensitization. Their potential as radiopharmaceuticals warrants further in-depth investigation in preclinical models.

The Impact of Extensive Surgical Resection of Butterfly Glioblastomas on Outcomes in the Presence of TERT Mutation and EGFR Amplification: A Retrospective Cohort Study.

BACKGROUND AND OBJECTIVES: This study aimed to assess if extensive surgical resection enhances outcomes in wild-type Isocitrate Dehydrogenase (IDH) butterfly glioblastoma (B-GBM) patients, despite the presence of Telomerase Reverse Transcriptase (TERT) mutation and Epidermal Growth Factor Receptor (EGFR) amplification. METHODS: The study, retrospectively conducted from 2014 to 2022, involved 723 GBM patients, 41 of whom met the criteria for IDH wild-type B-GBM. Exclusion criteria comprised prior diagnoses or treatments for low-grade glial tumors. Surgeons, employing two approaches-partial and extensive surgery-categorized patients based on age, sex, tumor location, corpus callosum involvement, and genetic characteristics. The interval between initial surgery and tumor recurrence/tumor-free period (TR/TFP) and overall survival (OS) were recorded and compared between the partial and extensive resection groups, analyzing the impact of resection width on TR/TFP and OS. Preoperative assessments utilized thin-section cranial computed tomography (CT) and contrast-enhanced magnetic resonance imaging (MRI). Intraoperatively, tumor excision was guided by sodium fluorescein, and margins were delineated via neuronavigation. Genetic alterations (TERT mutations and EGFR amplifications) were correlated with surgical type, TR/TFP, and OS. Karnofsky Performance Scale (KPS) evaluations were performed pre- and post-operatively and at key intervals, comparing outcomes between surgical groups. Standard radiotherapy and chemotherapy regimens were administered to all patients. RESULTS: Extensive resection yielded significantly longer TR/TFP compared to partial resection, despite TERT gene mutation and EGFR amplification being linked to shorter TR/TFP and OS. Its impact on OS, however, was not significant. KPS scores indicated a superior quality of life after extensive resection, with sustained improvement upon recurrence. CONCLUSIONS: Extensive resection of B-GBM, even in the presence of adverse genetic alterations, may prolong TR/TFP, offering patients a period of improved comfort with minimal distress.