Well-established Tumor Research Articles

Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency

The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm−/− mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm−/− mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.

RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

ObjectivesRING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well‐established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair.Materials and methodsIn this article, wild‐type, knockout and shRNA‐depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT‐PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR‐GFP, EJ5‐GFP and LacI‐LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull‐down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair.ResultsWe found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro‐apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over‐activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60.ConclusionsTaken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60.

SPARC-p53: The double agents of cancer.

Cancer is a complex disease with high incidence and mortality rates. The important role played by the tumor microenvironment in regulating oncogenesis, tumor growth, and metastasis is by now well accepted in the scientific community. SPARC is known to participate in tumor-stromal interactions and impact cancer growth in ambiguous ways, which either enhance or suppress cancer aggressiveness, in a context-dependent manner. p53 transcription factor, a well-established tumor suppressor, has been reported to promote tumor growth in certain situations, such as hypoxia, thus displaying a duality in its action. Although both proteins are being tested in clinical trials, the synergistic relation between them is yet to be explored in clinical practice. In this review, we address the controversial roles of SPARC and p53 as double agents in cancer, briefly summarizing the interaction found between these two molecules and its importance in cancer.

Self-Adjuvanting Peptide Vaccines Against Cervical Cancer

Background Cervical cancer is a common cause of cancer-related deaths in women worldwide, with a fatality rate second only to breast cancer. Human papillomaviruses (HPVs) are the main causative agents of cervical cancer, and are therefore obvious targets for vaccine development. Although two prophylactic HPV vaccines have been commercialized, therapeutic vaccines against HPVs have not been developed yet. Current vaccine technologies emphasize the power of small particles in targeting immune cells, and particles of 20-50 nm have been reported to induce optimal immune responses against a variety of pathogens and cancers. Methods We synthesized new nanoparticle-based vaccines against cervical cancer by using antigenic 8Qmin peptide epitope derived from HPV-16 E7 protein, a hydrophilic poly-(L-glutamic acid) (PGA) linker, and an 8-arm poly (tert-butyl acrylate) dendrimer-based delivery system (D8). Results Four different peptides containing 8Qmin and PGA of different lengths were successfully synthesized with high yield and purity. These were then conjugated to alkyne-functionalized D8 by copper-catalyzed alkyne-azide cycloaddition “click” reaction. The conjugates self-assembled into nanoparticles, with decreased particle size corresponding to a greater number of Glu units. The four vaccine candidates were tested in C57 black 6 (C57BL/6) mice bearing well-established (7-day-old) tumors to examine their therapeutic effects. Conclusion Interestingly, only one conjugate delayed tumor growth, and montanide adjuvanted antigen, used as a positive control, failed to demonstrate any therapeutic effect.

N6-Isopentenyladenosine Inhibits Colorectal Cancer and Improves Sensitivity to 5-Fluorouracil-Targeting FBXW7 Tumor Suppressor.

N6-isopentenyladenosine has been shown to exert potent in vitro antitumor activity on different human cancers, including colorectal cancer. Although some potential biochemical targets have been identified, its precise mechanism of action remains unclear. We found that N6-isopentenyladenosine affects colorectal cancer proliferation in in vitro models carrying different mutational status of FBXW7 and TP53 genes, and in HCT116 xenografts in SCID mice, by increasing the expression of the well-established tumor suppressor FBXW7, a component of the SCF-E3 ubiquitin ligase complex that promotes degradation of various oncoproteins and transcription factors, such as c-Myc, SREBP and Mcl1. Corroborating our previous studies, we identified for the first time the FBXW7/SREBP/FDPS axis as a target of the compound. Pull down of ubiquitinated proteins, immunoprecipitation and luciferase assays, reveal that through the increase of FBXW7/c-Myc binding, N6-isopentenyladenosine induces the ubiquitination of c-Myc, inhibiting its transcriptional activity. Moreover, in FBXW7- and TP53-wild type cells, N6-isopentenyladenosine strongly synergizes with 5-Fluorouracil to inhibit colon cancer growth in vitro. Our results provide novel insights into the molecular mechanism of N6-isopentenyladenosine, revealing its multi-targeting antitumor action, in vitro and in vivo. Restoring of FBXW7 tumor-suppressor represents a valid therapeutic tool, enabling N6-isopentenyladenosine as optimizable compound for patient-personalized therapies in colorectal cancer.

Rebalancing of actomyosin contractility enables mammary tumor formation upon loss of E-cadherin

E-cadherin (CDH1) is a master regulator of epithelial cell adherence junctions and a well-established tumor suppressor in Invasive Lobular Carcinoma (ILC). Intriguingly, somatic inactivation of E-cadherin alone in mouse mammary epithelial cells (MMECs) is insufficient to induce tumor formation. Here we show that E-cadherin loss induces extrusion of luminal MMECs to the basal lamina. Remarkably, E-cadherin-deficient MMECs can breach the basal lamina but do not disseminate into the surrounding fat pad. Basal lamina components laminin and collagen IV supported adhesion and survival of E-cadherin-deficient MMECs while collagen I, the principle component of the mammary stromal micro-environment did not. We uncovered that relaxation of actomyosin contractility mediates adhesion and survival of E-cadherin-deficient MMECs on collagen I, thereby allowing ILC development. Together, these findings unmask the direct consequences of E-cadherin inactivation in the mammary gland and identify aberrant actomyosin contractility as a critical barrier to ILC formation.

Bispecific T-Cell Engager -Armored Chimeric Antigen Receptor T Cells Overcome Antigen Escape From EGFRvIII-Targeted Therapy For Glioblastoma

Abstract INTRODUCTION Immune therapy with T cells engineered to express chimeric antigen receptors (CARs) represents a promising therapy for patients with glioblastoma (GBM). However, clinical responses have been limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR T cells directed against a single target. In clinical studies with CART-EGFRvIII, EGFRvIII-targeted T cells successfully localized to the brain tumor microenvironment, but ultimately failed to prevent disease progression with post-treatment specimens demonstrating high levels of wild-type EGFR despite reduced expression of EGFRvIII. METHODS We developed a novel bicistronic CAR construct engineered for local delivery of bispecific T-cell engagers (BiTEs) that target residual tumor. Specifically, EGFRvIII-targeted CAR T cells were engineered to secrete BiTEs against wild-type EGFR, which is frequently amplified and overexpressed in GBM. RESULTS Human T cells were efficiently transduced with the dual CART.BiTE transgene. These modified cells secreted biologically active EGFR-specific BiTEs that not only redirected CAR T cells but also recruited and activated untransduced bystander T cells against wild-type EGFR. Recapitulating clinical data, EGFRvIII CAR T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive GBM. Conversely, CART.BiTE cells cured mice even in the setting of antigen-loss, against heterogeneous and well-established intracerebral tumors in mice. Unlike CAR T cells directly targeting EGFR, which caused toxicity in human skin grafts in vivo, secreted BiTE-EGFR was both locally effective and did not result in toxicity against grafted human skin. CONCLUSION This is the first instance in which CARs and BiTEs have been combined into a single platform of immune therapy. Our results demonstrate that CARs and BiTEs can be combined strategically to mitigate antigen heterogeneity in GBM and also provide a unique T-cell-based delivery method for BiTEs to tumors in the brain.

A comparison between Nonlinear Least Squares and Maximum Likelihood estimation for the prediction of tumor growth on experimental data of human and rat origin

Several mathematical models have been developed to explain the growth of tumors and used to fit experimental or clinical data. Their predictive power – i.e. their ability to forecast the future growth on the basis of present knowledge – however, has been rarely explored. Here, we investigate whether a Hidden Markov Model (HMM) based on the well-established Gompertz tumor growth function with additive Gaussian noise could effectively be used to predict the future growth of experimental tumors. The idea behind this work is that one might achieve more accurate predictions if estimates of the unknown parameters of the HMM are used instead of those obtained by fits of the deterministic Gompertz model to the data. We use the principle of Maximum Likelihood (ML) to estimate unknown parameters related to growth dynamics and noise, and we compare its effectiveness to the classical Nonlinear Least Squares minimization approach. The analyses show that our approach can provide better growth predictions when the data contain adequate information concerning the tumors saturation phase. The forecasts could also be improved by taking into account prior knowledge about the unknown parameters when the information concerning the saturation phase was inadequate. We conclude that by using HMMs in combination with the principle of ML, one can obtain more reliable growth predictions for individual tumors.

NQO1 targeting prodrug triggers innate sensing to overcome checkpoint blockade resistance

Lack of proper innate sensing inside tumor microenvironment (TME) limits T cell-targeted immunotherapy. NAD(P)H:quinone oxidoreductase 1 (NQO1) is highly enriched in multiple tumor types and has emerged as a promising target for direct tumor-killing. Here, we demonstrate that NQO1-targeting prodrug β-lapachone triggers tumor-selective innate sensing leading to T cell-dependent tumor control. β-Lapachone is catalyzed and bioactivated by NQO1 to generate ROS in NQO1high tumor cells triggering oxidative stress and release of the damage signals for innate sensing. β-Lapachone-induced high mobility group box 1 (HMGB1) release activates the host TLR4/MyD88/type I interferon pathway and Batf3 dendritic cell-dependent cross-priming to bridge innate and adaptive immune responses against the tumor. Furthermore, targeting NQO1 is very potent to trigger innate sensing for T cell re-activation to overcome checkpoint blockade resistance in well-established tumors. Our study reveals that targeting NQO1 potently triggers innate sensing within TME that synergizes with immunotherapy to overcome adaptive resistance.

Advancing Care for Head and Neck Cancers in a Multidisciplinary Tumour Board in the East

Managing head and neck cancers is an excellent example of the importance of teamwork, with head and neck surgeons, clinical oncologists, radiologists, pathologists and other allied health professionals specialised in this disease site working together. The reliable imaging and dedicated pretreatment work-up entailing the comprehensive anatomical description of tumour involvement by the radiologists, the expertise of surgeons in performing en-bloc gross tumour resection, the uneventful speedy postoperative rehabilitation and recovery by the speech therapists and nutritionists, as well as the dedicated treatment planning of clinical oncologists in delivering precise preoperative or postoperative (chemo)radiotherapy to maximise the therapeutic potentials are the pillars of treatment success. A multidisciplinary tumour board involving all of these key players is essential to provide the highest level of recommendation based on evidence-based medicine and to bring patients new hopes and the best chance of cure. This review illustrates the seamless collaborative teamwork within a well-established multidisciplinary tumour board in managing one of the most intractable cancers in the East, taking enlightenment and inspiration from the West.

WWOX somatic ablation in skeletal muscles alters glucose metabolism

ObjectiveWWOX, a well-established tumor suppressor, is frequently lost in cancer and plays important roles in DNA damage response and cellular metabolism. MethodsWe re-analyzed several genome-wide association studies (GWAS) using the Type 2 Diabetes Knowledge Portal website to uncover WWOX's association with metabolic syndrome (MetS). Using several engineered mouse models, we studied the effect of somatic WWOX loss on glucose homeostasis. ResultsSeveral WWOX variants were found to be strongly associated with MetS disorders. In mouse models, somatic ablation of Wwox in skeletal muscle (WwoxΔSKM) results in weight gain, glucose intolerance, and insulin resistance. Furthermore, WwoxΔSKM mice display reduced amounts of slow-twitch fibers, decreased mitochondrial quantity and activity, and lower glucose oxidation levels. Mechanistically, we found that WWOX physically interacts with the cellular energy sensor AMP-activated protein kinase (AMPK) and that its loss is associated with impaired activation of AMPK, and with significant accumulation of the hypoxia inducible factor 1 alpha (HIF1α) in SKM. ConclusionsOur studies uncover an unforeseen role of the tumor suppressor WWOX in whole-body glucose homeostasis and highlight the intimate relationship between cancer progression and metabolic disorders, particularly obesity and type-2 diabetes. Subject areasGenetics, Metabolic Syndrome, Diabetes.

Matrix metalloproteinase 2 (MMP-2) and its tissue inhibitor 2 (TIMP-2) in pancreatic cancer (PC).

ObjectivesThe incidence rate of pancreatic cancer (PC) is similar to mortality rate, thus searching specific tumor biomarkers of PC is sorely needed. Matrix metalloproteinase-2 (MMP-2) and the imbalance between MMP-2 and its tissue inhibitor (TIMP-2) play a critical role in tumor progression. We aim to assess the diagnostic and prognostic usefulness of serum MMP-2 and TIMP-2 as potential biomarkers in comparison to well-established tumor markers of PC (CA 19-9, carbohydrate antigen 19-9 and CEA, carcinoembryonic antigen).ResultsWe indicated the significant differences between serum TIMP-2 concentrations in PC patients, CP individuals and control group. The diagnostic sensitivity of TIMP-2 was the highest among all proteins tested and increased up to 96% in combined measurement with MMP-2. The area under ROC curve (AUC) for TIMP-2 was larger than for MMP-2, but lower than for classical tumor markers.MethodsPresented study comprised on 226 subjects, including 92 PC patients, 43 chronic pancreatitis (CP) patients and 91 healthy volunteers. The serum concentrations of these proteins were measured using immunological methods.ConclusionsPresented findings suggest higher usefulness of TIMP-2 than MMP-2 as potential biomarker in the diagnosis of PC patients, however more studies on large population are needed to support our results.

Single cell transcriptomic landscapes of pattern formation, proliferation and growth in Drosophila wing imaginal discs.

Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at the individual cell level remains unclear. In the past decades, studies using Drosophila wing imaginal discs as a model system have provided valuable insights into pattern formation, growth control and regeneration. Here, we provide single cell transcriptomic landscapes of pattern formation, proliferation and growth of wing imaginal discs. We found that patterning information is robustly maintained in the single cell transcriptomic data and can provide reference matrices for computationally mapping single cells into discrete spatial domains. Assignment of wing disc single cells to spatial subregions facilitates examination of patterning refinement processes. We also clustered single cells into different proliferation and growth states and evaluated the correlation between cell proliferation/growth states and spatial patterning. Furthermore, single cell transcriptomic analyses allowed us to quantitatively examine disturbances of differentiation, proliferation and growth in a well-established tumor model. We provide a database to explore these datasets at http://drosophilayanlab-virtual-wingdisc.ust.hk:3838/v2/This article has an associated 'The people behind the papers' interview.

TET2 Deficiency Sets the Stage for B-cell Lymphoma.

TET2 is a well-established tumor suppressor in the context of myeloid malignancies, but its role in lymphoma development has been less clear. In this issue of Cancer Discovery, Dominguez and colleagues report that TET2 function is critical for germinal center exit and plasma cell differentiation, and its deficiency can lead to B-cell lymphoma phenotypes.See related article by Dominguez et al., p. 1632.

Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice.

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8+ T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8+ T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8+ T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP-E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. Cancer Immunol Res; 6(11); 1301-13. ©2018 AACR.

Recent advances in the histological and molecular classification of endometrial stromal neoplasms.

This review addresses known features and recent developments in the histological, immunohistochemical, and molecular characterization of endometrial stromal neoplasms. We discuss the spectrum of these tumors, from the benign endometrial stromal nodule to low-grade endometrial stromal sarcoma to uterine undifferentiated sarcomas with a special emphasis on the expanding group of high-grade stromal sarcomas, recently added to the 2014 WHO classification, not only discussing the well-established YWHAE-FAM22 tumors but also two new groups, presenting with BCOR alterations including those with BCOR tandem internal duplications or NTRK fusions. It is likely that this high-grade category of endometrial stromal tumors will expand as increasing molecular data is available.

RKIP: A Key Regulator in Tumor Metastasis Initiation and Resistance to Apoptosis: Therapeutic Targeting and Impact.

RAF-kinase inhibitor protein (RKIP) is a well-established tumor suppressor that is frequently downregulated in a plethora of solid and hematological malignancies. RKIP exerts antimetastatic and pro-apoptotic properties in cancer cells, via modulation of signaling pathways and gene products involved in tumor survival and spread. Here we review the contribution of RKIP in the regulation of early metastatic steps such as epithelial–mesenchymal transition (EMT), migration, and invasion, as well as in tumor sensitivity to conventional therapeutics and immuno-mediated cytotoxicity. We further provide updated justification for targeting RKIP as a strategy to overcome tumor chemo/immuno-resistance and suppress metastasis, through the use of agents able to modulate RKIP expression in cancer cells.

Novel clinicopathological and molecular characterization of metanephric adenoma: a study of 28 cases

BackgroundMetanephric adenoma is a rare, benign renal neoplasm with occasional misdiagnosis. However, its molecular characterization is not fully understood.MethodsIn this study, we use the hybrid capture-based Next-Generation Sequencing to sequence a panel of 295 well-established oncogene or tumor suppressor genes in 28 cases of MA patients in China. Novel clinicopathological markers associated with the mitogen-activated protein kinase (MAPK) pathway in metanephric adenoma were detected by immunohistochemistry.ResultsIt was found that except for BRAF (22/28) mutations (c.1799 T > A, p.V600E), NF1 (6/28), NOTCH1 (5/28), SPEN (5/28), AKT2 (4/28), APC (4/28), ATRX (3/28), and ETV4 (3/28) mutations could also be detected. Meanwhile, a novel and rare gene fusion of STARD9-BRAF, CUX1-BRAF, and LOC100507389-BRAF was detected in one MA patient. In addition, although MEK phosphorylation was normally activated, the phosphorylation level of ERK was low in metanephric adenoma cases. Highly expressed p16 and DUSP6 may have contributed to these results, which maintained MA as a benign renal tumor.ConclusionsThis study provides novel molecular and pathological markers for metanephric adenoma, which could improve its diagnosis and increase the understanding of its pathologic mechanism.

Caloric restriction inhibits mammary tumorigenesis in MMTV-ErbB2 transgenic mice through the suppression of ER and ErbB2 pathways and inhibition of epithelial cell stemness in premalignant mammary tissues.

Caloric intake influences the onset of many diseases, including cancer. In particular, caloric restriction (CR) has been reported to suppress mammary tumorigenesis in various models. However, the underlying cancer preventive mechanisms have not been fully explored. To this end, we aimed to characterize the anticancer mechanisms of CR using MMTV-ErbB2 transgenic mice, a well-established spontaneous ErbB2-overexpressing mammary tumor model, by focusing on cellular and molecular changes in premalignant tissues. In MMTV-ErbB2 mice with 30% CR beginning at 8 weeks of age, mammary tumor development was dramatically inhibited, as exhibited by reduced tumor incidence and increased tumor latency. Morphogenic mammary gland analyses in 15- and 20-week-old mice indicated that CR significantly decreased mammary epithelial cell (MEC) density and proliferative index. To understand the underlying mechanisms, we analyzed the effects of CR on mammary stem/progenitor cells. Results from fluorescence-activated cell sorting analyses showed that CR modified mammary tissue hierarchy dynamics, as evidenced by decreased luminal cells (CD24highCD49flow), putative mammary reconstituting unit subpopulation (CD24highCD49fhigh) and luminal progenitor cells (CD61highCD49fhigh). Mammosphere and colony-forming cell assays demonstrated that CR significantly inhibited mammary stem cell self-renewal and progenitor cell numbers. Molecular analyses indicated that CR concurrently inhibited estrogen receptor (ER) and ErbB2 signaling. These molecular changes were accompanied by decreased mRNA levels of ER-targeted genes and epidermal growth factor receptor/ErbB2 family members and ligands, suggesting ER-ErbB2 signaling cross-talk. Collectively, our data demonstrate that CR significantly impacts ER and ErbB2 signaling, which induces profound changes in MEC reprogramming, and mammary stem/progenitor cell inhibition is a critical mechanism of CR-mediated breast cancer prevention.

Development of Specific New ELISA for Bioanalysis of Cetuximab: A Monoclonal Antibody Used for Cancer Immunotherapy

Background: Cetuximab (CET) is a monoclonal antibody that inhibits Epidermal Growth Factor Receptor (EGFR) used for immunotherapy of different types of cancer. Additionally, CET used in combination therapy, potentiates the effects of chemotherapy and radiation therapy in eradicating well-established tumors. Recently, a combination of CET and newly developed chemotherapeutic candidate drugs are being investigated for use as new-generation chemotherapy. Introduction: Due to the increasing importance of this class of biopharmaceuticals, our dynamic field of research has arisen around the establishment of an immunoassay method with a highly specific and sensitive to quantify CET at low concentration levels in human plasma. This study describes the development and full validation of a new enzyme-linked immunosorbent assay (ELISA) with high sensitivity and selectivity for bioanalysis of CET. Methods: The assay involved the non-competitive binding reaction of CET to its specific antigen (human epidermal growth factor protein; EGFR) followed by a chromogenic enzyme reaction for immune detection of the CET- EGFR complex and color development. Technically, CET was captured by EGFR antigen protein immobilized onto a 96-well assay plate. The CET- EGFR complex formed onto the plate wells was quantified, for the first time, using alkaline phosphatase enzyme labeled anti-human IgG (ALP-IgG) and para-nitrophenyl phosphate substrate (pNPP) as a chromogenic substrate for alkaline phosphatase enzyme. Results: The optimum conditions for conducting the proposed ELISA were established and its analytical performance was evaluated as per the guidelines for the validation of immunoassays for bioanalysis. The assay Limit Of Detection (LOD) was 1.5 ng/mL and the effective working dynamic range was 5- 6250 ng/mL. The accuracy and precision of the assay were proved. Conclusion: We have developed and validated a highly sensitive and selective ELISA for quantitation of CET in plasma samples. The CET- EGFR complex formed onto the plate wells were quantified, for the first time, using alkaline phosphatase enzyme labeled anti-human IgG (ALP-IgG) and paranitrophenyl phosphate substrate (pNPP) as a chromogenic substrate for alkaline phosphatase enzyme. An analyst can process a batch of ~ 200 samples, in triplicate, per day. The proposed ELISA method for CET shows acceptable precision and, accuracy and adequate sensitivity to contribute to study its pharmacokinetic (PK), Pharmacodynamic (PD), Therapeutic Drug Monitoring (TDM) and appears to be suitable for employment in all clinical laboratories. Keywords: Cancer immunotherapy, therapeutic monoclonal antibodies, cetuximab, bioanalysis, ELISA.