Cytokine/Chemokine Release Patterns and Transcriptomic Profiles of LPS/IFNγ-Activated Human Macrophages Differentiated with Heat-Killed Mycobacterium obuense, M-CSF, or GM-CSF.

Macrophages (Mφs) are instrumental regulators of the immune response whereby they acquire diverse functional phenotypes following their exposure to microenvironmental cues that govern their differentiation from monocytes and their activation. The complexity and diversity of the mycobacterial cell wall have empowered mycobacteria with potent immunomodulatory capacities. A heat-killed (HK) whole-cell preparation of Mycobacterium obuense (M. obuense) has shown promise as an adjunctive immunotherapeutic agent for the treatment of cancer. Moreover, HK M. obuense has been shown to trigger the differentiation of human monocytes into a monocyte-derived macrophage (MDM) type named Mob-MDM. However, the transcriptomic profile and functional properties of Mob-MDMs remain undefined during an activation state. Here, we characterized cytokine/chemokine release patterns and transcriptomic profiles of lipopolysaccharide (LPS)/interferon γ (IFNγ)-activated human MDMs that were differentiated with HK M. obuense (Mob-MDM(LPS/IFNγ)), macrophage colony-stimulating factor M-MDM(LPS/IFNγ)), or granulocyte/macrophage colony-stimulating factor (GM-MDM(LPS/IFNγ)). Mob-MDM(LPS/IFNγ) demonstrated a unique cytokine/chemokine release pattern (interleukin (IL)-10low, IL-12/23p40low, IL-23p19/p40low, chemokine (C-x-C) motif ligand (CXCL)9low) that was distinct from those of M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ). Furthermore, M-MDM(LPS/IFNγ) maintained IL-10 production at significantly higher levels compared to GM-MDM(LPS/IFNγ) and Mob-MDM(LPS/IFNγ) despite being activated with M1-Mφ-activating stimuli. Comparative RNA sequencing analysis pointed to a distinct transcriptome profile for Mob-MDM(LPS/IFNγ) relative to both M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ) that comprised 417 transcripts. Functional gene-set enrichment analysis revealed significant overrepresentation of signaling pathways and biological processes that were uniquely related to Mob-MDM(LPS/IFNγ). Our findings lay a foundation for the potential integration of HK M. obuense in specific cell-based immunotherapeutic modalities such as adoptive transfer of Mφs (Mob-MDM(LPS/IFNγ)) for cancer treatment.

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation.

Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression, HER-2/HER-3 signalling and sensitivity to pan HER blockers.

BACKGROUND: The human epidermal growth factor receptor (EGFR) is an important target for cancer treatment. Currently, only the EGFR antibodies cetuximab and panitumumab are approved for the treatment of patients with colorectal cancer. However, a major clinical challenge is a short-term response owing to development of acquired resistance during the course of the treatment. METHODS: In this study, we investigated the molecular mechanisms underlying development of acquired resistance in DiFi colorectal cancer cells to the anti-EGFR mAb ICR62 (termed DiFi62) and to the small molecule tyrosine kinase inhibitor (TKI) gefitinib (termed DiFiG) using a range of techniques. RESULTS: Compared with the findings from parental DiFi and DiFiG cells, development of acquired resistance to anti-EGFR mAb ICR62 in DiFi62 cells was accompanied by an increase in cell surface EGFR and increased phosphorylation of HER-2 and HER-3. Interestingly, DiFi62 cells also acquired resistance to treatment with anti-EGFR mAbs cetuximab and ICR61, which bind to other distinct epitopes on the extracellular domain of EGFR, but these cells remained equally sensitive as the parental cells to treatment with pan-HER inhibitors such as afatinib. CONCLUSIONS: Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed.

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

Synergistic Effects of Neratinib in Combination With Palbociclib or Miransertib in Brain Cancer Cells.

Background: Aberrant expression and activation of epidermal growth factor receptor (EGFR) resulted in approval of several forms of EGFR inhibitors in the treatment of patients with a wide range of epithelial cancers. However, no EGFR inhibitor has yet been approved for the treatment of patients with brain cancer, indicating that targeting EGFR alone may not be sufficient in some patients. Methods: In this study, we investigated the role of all members of the EGFR family, other growth factor receptors, cell-cycle proteins, and downstream cell signaling pathways (e.g., mitogen-activated protein kinase (MAPK), serine/threonine protein kinase (AKT), signal transducer and activator of transcription (STAT3), Src, Abelson murine leukemia viral oncogene homolog (Abl)) on the growth of a panel of human brain cancer cell lines (HBCCLs). We examined the growth response of HBCCLs to treatment with 17 targeted agents compared to two cytotoxic drugs. Results: Of the targeted agents, the irreversible pan-human epidermal growth factor receptor (HER) inhibitors neratinib and afatinib were more effective than erlotinib and lapatinib at inhibiting the growth of all HBCCLs, and the cyclin-dependent kinase (CDK)1/2/5/9 inhibitor dinaciclib was the most potent targeted agent. We found that treatment with Src/Abl/c-kit inhibitor dasatinib, signal transducer and activator of transcription (STAT3) inhibitor stattic, Abl/platelet-derived growth factor receptor (PDGFR)α/vascular endothelial growth factor (VEGFR)2/fibroblast growth factor receptor (FGFR)1 inhibitor ponatinib, and the tropomyosin receptor kinase (TRK)/ROS proto-oncogene 1 receptor tyrosine kinase (ROS)/anaplastic lymphoma kinase (ALK) inhibitor entrectinib, also inhibited the growth of all HBCCLs. Interestingly, these agents were more effective in inhibiting growth of HBCCLs when proliferating at a slower rate. In addition to inhibiting the proliferation of HBCCLs, treatment with neratinib, dinaciclib, dasatinib, stattic and trametinib inhibited the migration of brain tumor cell line A172. Conclusions: Notably, we found that treatment with neratinib in combination with palbociclib (CDK4/6 inhibitor), or miransertib (AKT1/2/3 inhibitor) resulted in synergistic growth inhibition of all HBCCLs. Our results support that repurposing drugs like neratinib in combination with the palbociclib or miransertib may be of therapeutic potential in brain cancer and warrants further investigations.

The Combination of Afatinib With Dasatinib or Miransertib Results in Synergistic Growth Inhibition of Stomach Cancer Cells.

Background: Of various human epidermal growth factor receptor (HER) inhibitors, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan (T-Dxd) has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients, with tumor heterogeneity being one contributing factor. Methods: We investigated the effect of various types of targeted agents on growth in vitro and migration of a panel of human stomach cancer cells (HSCCLs) and the impact of cell proliferation rate on the anti-tumor activities of these agents. We also investigated the association between the cell surface expression of the HER family members, hepatocyte growth factor receptor (c-Met), anaplastic lymphoma kinase (ALK)7 and cancer stem cell markers CD44 and CD133, and the response to the targeted agents. Results: Of the 18 agents examined, the cyclin dependent kinase (CDK) 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at 50% inhibitory concentration (IC50) values between 9 nM to 23 nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor (TKI) lapatinib and the EGFR-specific TKI erlotinib in inhibiting the growth of HSCCLs. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs, with the IC50 values ranging from 2 nM to 7 µM. Many of these agents were more effective in inhibiting the growth of HSCCLs when they were proliferating at a slower rate. Treatment with neratinib, afatinib, dinaciclib, dasatinib, stattic, miransertib and paclitaxel significantly inhibited migration of stomach cancer cells. Interestingly, treatment with a combination of afatinib and dasatinib or afatinib and miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. Conclusions: These results suggest that treatment with a combination of these agents may be of therapeutic value in stomach cancer and warrants further investigations.

Treatment with a combination of the ErbB (HER) family blocker afatinib and the IGF-IR inhibitor, NVP-AEW541 induces synergistic growth inhibition of human pancreatic cancer cells.

BACKGROUND: Aberrant expression and activation of the IGF-IR have been reported in a variety of human cancers and have been associated with resistance to HER targeted therapy. In this study, we investigated the effect of simultaneous targeting of IGF-IR and HER (erbB) family, with NVP-AEW541 and afatinib, on proliferation of pancreatic cancer cells. METHODS: The sensitivity of a panel of human pancreatic cancer cell lines to treatment with NVP-AEW541 used alone or in combination with afatinib, anti-EGFR antibody ICR62, and cytotoxic agents was determined using the Sulforhodamine B colorimetric assay. Growth factor receptor expression, cell-cycle distribution and cell signalling were determined using flow cytometry and western blot analysis. RESULTS: All pancreatic cancer cell lines were found to be IGF-IR positive and NVP-AEW541 treatment inhibited the growth of the pancreatic cancer cell lines with IC50 values ranging from 342 nM (FA6) to 2.73 µM (PT45). Interestingly, of the various combinations examined, treatment with a combination of NVP-AEW541 and afatinib was superior in inducing synergistic growth inhibition of the majority of pancreatic cancer cells. CONCLUSION: Our results indicate that co-targeting of the erbB (HER) family and IGF-IR, with a combination of afatinib and NVP-AEW541, is superior to treatment with a single agent and encourages further investigation in vivo on their therapeutic potential in IGF-IR and HER positive pancreatic cancers.

Therapeutic application of monoclonal antibodies in cancer: advances and challenges.

INTRODUCTION: Monoclonal antibody (mAb)-based products are highly specific for a particular antigen. This characteristic feature of the molecules makes them an ideal tool for many applications including cancer diagnosis and therapy. SOURCES OF DATA: We performed comprehensive searches of PubMed, Medline and the Food and Drug Administration website using keywords such as 'therapeutic antibodies' and 'anti-cancer antibodies'. AREAS OF AGREEMENT: Treatment of cancer patients with antibodies when used alone or in combination with chemotherapy and radiotherapy, or conjugated to drugs or radioisotopes, prolongs overall survival in cancer patients. Currently, there are 14 mAb-based drugs that have been approved for the treatment of cancer patients. AREAS OF CONTROVERSY: The response of cancer patients to antibody therapy can be of short duration. Therapeutic antibodies are expensive and may have side effects. There are no reliable predictive biomarkers for sensitivity or resistance to certain therapeutic antibodies. FUTURE FOCUS: There should be additional studies to discover novel therapeutic targets, to develop more effective antibody-based drugs with fewer side effects, to identify more reliable predictive biomarker(s) for response to therapy with antibody-based drugs and to develop alternative strategies (e.g. transgenic plants, transgenic farm animals) for production of large quantities and more affordable batches of therapeutic antibodies. AREAS TIMELY FOR DEVELOPING RESEARCH: A better understanding of cancer biology, the hallmarks of human cancers and the immune system would lead to identification of additional cell surface biomarkers. These in turn would facilitate the development of novel and biosimilar antibody-based drugs and their routine use as 'magic bullets' for the targeted therapy of human cancers.

Description of PTPRG genetic variants identified in a cohort of Chronic Myeloid Leukemia patients and their ability to influence response to Tyrosine kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) have remarkably transformed Ph+ chronic myeloid leukemia (CML) management; however, TKI resistance remains a major clinical challenge. Mutations in BCR-ABL1 are well studied but fail to explain 20-40% of resistant cases, suggesting the activation of alternative, BCR-ABL1-independent pathways. Protein Tyrosine Phosphatase Receptor Gamma (PTPRG), a tumor suppressor, was found to be well expressed in CML patients responsive to TKIs and remained at low level in resistant patients. In this study, we aimed to identify genetic variants in PTPRG that could potentially modulate TKIs response in CML patients. DNA was extracted from peripheral blood samples collected from two CML cohorts (Qatar and Italy) and targeted exome sequencing was performed. Among 31 CML patients, six were TKI-responders and 25 were TKI-non-responsive. Sequencing identified ten variants, seven were annotated and three were novel SNPs (c.1602_1603insC, c.85+14412delC, and c.2289-129delA). Among them, five variants were identified in 15 resistant cases. Of these, one novel exon variant (c.1602_1603insC), c.841-29C>T (rs199917960) and c.1378-224A>G (rs2063204) were found to be significantly different between the resistant cases compared to responders. Our findings suggest that PTPRG variants may act as an indirect resistance mechanism of BCR-ABL1 to affect TKI treatment.

Therapeutic Application of Monoclonal Antibodies in Pancreatic Cancer: Advances, Challenges and Future Opportunities.

Pancreatic cancer remains as one of the most aggressive cancer types. In the absence of reliable biomarkers for its early detection and more effective therapeutic interventions, pancreatic cancer is projected to become the second leading cause of cancer death in the Western world in the next decade. Therefore, it is essential to discover novel therapeutic targets and to develop more effective and pancreatic cancer-specific therapeutic agents. To date, 45 monoclonal antibodies (mAbs) have been approved for the treatment of patients with a wide range of cancers; however, none has yet been approved for pancreatic cancer. In this comprehensive review, we discuss the FDA approved anticancer mAb-based drugs, the results of preclinical studies and clinical trials with mAbs in pancreatic cancer and the factors contributing to the poor response to antibody therapy (e.g. tumour heterogeneity, desmoplastic stroma). MAb technology is an excellent tool for studying the complex biology of pancreatic cancer, to discover novel therapeutic targets and to develop various forms of antibody-based therapeutic agents and companion diagnostic tests for the selection of patients who are more likely to benefit from such therapy. These should result in the approval and routine use of antibody-based agents for the treatment of pancreatic cancer patients in the future.

HER2 Expression Is Predictive of Survival in Cetuximab Treated Patients with RAS Wild Type Metastatic Colorectal Cancer.

The overexpressed HER2 is an important target for treatment with monoclonal antibody (mAb) trastuzumab, only in patients with breast and gastric cancers, and is an emerging therapeutic biomarker in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) mAbs cetuximab and panitumumab. In this study, we investigated the relative expression and predictive value of all human epidermal growth factor receptor (HER) family members in 144 cetuximab-treated patients with wild type RAS mCRC. The relative expression of EGFR and HER2 have also been examined in 21-paired primary tumours and their metastatic sites by immunohistochemistry. Of the 144 cases examined, 25%, 97%, 79%, 48%, and 10% were positive for EGFR, HER2, HER3, and HER4 and all four HER family members, respectively. The expression of EGFR was an indicator of poorer overall survival and the membranous expression of HER2 and HER3 3+ intensity was associated with a shorter progression free survival (PFS). In contrast, the cytoplasmic expression of HER2 was associated with better PFS. In 48% and 71% of the cases, there were discordance in the expression of EGFR or one or more HER family members in paired primary and related metastatic tumours, respectively. Our results implicate the importance of a large prospective investigation of the expression level and predictive value of not only the therapeutic target (i.e., EGFR protein) but also HER2 and other HER family members as therapeutic targets, or for response to therapy with anti-EGFR mAbs and other forms of HER inhibitors, in both the primary tumours and metastatic sites in mCRC.

Predictive value of tyrosine phosphatase receptor gamma for the response to treatment tyrosine kinase inhibitors in chronic myeloid leukemia patients.

Protein tyrosine phosphatase receptor gamma (PTPRG) is a member of the receptor-like family protein tyrosine phosphatases and acts as a tumor suppressor gene in different neoplasms. Recent studies reported the down-regulation of PTPRG expression levels in Chronic Myeloid Leukemia disease (CML). In addition, the BCR-ABL1 transcript level is currently a key predictive biomarker of CML response to treatment with Tyrosine Kinase Inhibitors (TKIs). The aim of this study was to employ flow cytometry to monitor the changes in the expression level of PTPRG in the white blood cells (WBCs) of CML patients at the time of diagnosis and following treatment with TKIs. WBCs from peripheral blood of 21 CML patients were extracted at diagnosis and during follow up along with seven healthy individuals. The PTPRG expression level was determined at protein and mRNA levels by both flow cytometry with monoclonal antibody (TPγ B9-2) and RT-qPCR, and BCR-ABL1 transcript by RT-qPCR, respectively. PTPRG expression was found to be lower in the neutrophils and monocytes of CML patients at time of diagnosis compared to healthy individuals. Treatment with TKIs nilotinib and Imatinib Mesylate restored the expression of PTPRG in the WBCs of CML patients to levels observed in healthy controls. Moreover, restoration levels were greatest in optimal responders and occurred earlier with nilotinib compared to imatinib. Our results support the measurement of PTPRG expression level in the WBCs of CML patients by flow cytometry as a monitoring tool for the response to treatment with TKIs in CML patients.

Periadventitial delivery of anti-EGF receptor antibody inhibits neointimal macrophage accumulation after angioplasty in a hypercholesterolaemic rabbit.

Monocyte recruitment and their differentiation into macrophages are both early events in native and accelerated atherosclerosis that follows angioplasty. We have investigated the putative functional role of the epidermal growth factor receptor (EGFR) present on rabbit monocytes/macrophages. The impact of periadventitial delivery of an EGFR-specific, blocking monoclonal antibody (ICR62, which inhibits EGF-binding to its receptor) was investigated in a rabbit model of accelerated atherosclerosis induced by a combination of carotid injury and 4 weeks of a 2% cholesterol-diet. Two weeks after the initiation of the diet, a balloon-catheter angioplasty of the left common carotid artery was performed and a collar placed around the injured carotid artery immediately, for the delivery of ICR62 antibody, isotype-matched antibody or saline control. Monocyte/macrophage accumulation, cell proliferation and neointimal thickening were determined 2 weeks after the delivery of the antibodies. The function of the EGFR on rabbit monocytes was also investigated in vitro, using chemotaxis assays. Treatment with ICR62 was associated with a significant reduction in macrophage accumulation and neointimal thickening and a 76% reduction in neointimal area of the vessel wall compared with controls. In vitro ICR62 inhibited macrophage and smooth muscle cell migration towards EGFR ligands including EGF and HB-EGF. These findings suggest that EGFR ligation may be important in the development of early atherosclerotic lesions following balloon-catheter angioplasty, and periadventitial delivery may provide a feasible approach for administration of the inhibitors of EGFR-binding such as ICR62.

Synergistic activity of agents targeting growth factor receptors, CDKs and downstream signaling molecules in a panel of pancreatic cancer cell lines and the identification of antagonistic combinations: Implications for future clinical trials in pancreatic cancer.

Pancreatic cancer is one of the most aggressive, heterogeneous and fatal type of human cancers for which more effective therapeutic agents are urgently needed. Here, we investigated the sensitivity of a panel of seven human pancreatic cancer cell lines (HPCCLs) to treatment with various tyrosine kinase inhibitors (TKIs), cyclin­dependent kinase (CDK) inhibitors, an inhibitor of STAT3 stattic, and a cytotoxic agent gemcitabine both as single agents and in combination. The membranous expression of various receptors and the effect of selected agents on cell cycle distribution, cell signaling pathways and migration was determined using flow cytometry, western blot analysis and scratch wound healing assays, respectively. While the expression of both HER­3 and HER­4 was low or negative, the expression of EGFR and HER2 was high or intermediate in all HPCCLs. Of all the agents examined, the CDK1/2/5/9 inhibitor, dinacicilib, was the most potent agent which inhibited the proliferation of all seven HPCCLs with IC50 values of ≤10 nM, followed by SRC targeting TKI dasatinib (IC50 of ≤258 nM), gemcitabine (IC50 of ≤330 nM), stattic (IC50 of ≤2 µM) and the irreversible pan­HER TKI afatinib (IC50 of ≤2.95 µM). Treatment with afatinib and dasatinib inhibited the ligand­induced phosphorylation of EGFR and SRC respectively. Statistically significant associations were found between HER2 expression and response to treatment with the ALK/IGF­IR/InsR inhibitor ceritinib and fibroblast growth factor receptor (FGFR)1/2/3 inhibitor AZD4547, HER3 and IGF­IR expression and their response to treatment with TKIs targeting HER family members (erlotinib and afatinib), and c­MET and ALK7 expression and their response to treatment with stattic. Interestingly, treatment with a combination of afatinib with dasatinib and gemcitabine with dasatinib resulted in synergistic tumor growth inhibition in all HPCCLs examined. In contrast, the combination of afatinib with dinaciclib was found to be antagonistic. Finally, the treatment with afatinib, dasatinib and dinaciclib strongly inhibited the migration of all HPCCLs examined. In conclusion, the CDK1/2/5/9 inhibitor dinaciclib, irreversible pan­HER TKI afatinib and SRC targeting TKI dasatinib were most effective at inhibiting the proliferation and migration of HPCCLs and the combination of afatinib with dasatinib and gemcitabine with dasatinib led to synergistic tumor growth inhibition in all HPCCLs examined. Our results support further investigation on the therapeutic potential of these combinations in future clinical trials in pancreatic cancer.

Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer.

Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens and the development of targeting agents. Here, we report the development of two novel mAbs against CFPAC-1 human pancreatic cancer cells. Using ELISA, flow cytometry, immunoprecipitation, mass spectrometry, Western blot and immunohistochemistry, we found that the target antigens recognised by the two novel mAbs KU44.22B and KU44.13A, are integrin α3 and CD26 respectively, with high levels of expression in human pancreatic and other cancer cell lines and human pancreatic cancer tissue microarrays. Treatment with naked anti-CD26 mAb KU44.13A did not have any effect on the growth and migration of cancer cells nor did it induce receptor downregulation. In contrast, treatment with anti-integrin α3 mAb KU44.22B inhibited growth in vitro of Capan-2 cells, increased migration of BxPC-3 and CFPAC-1 cells and induced antibody internalisation. Both novel mAbs are capable of detecting their target antigens by immunohistochemistry but not by Western blot. These antibodies are excellent tools for studying the role of integrin α3 and CD26 in the complex biology of pancreatic cancer, their prognostic and predictive values and the therapeutic potential of their humanised and/or conjugated versions in patients whose tumours overexpress integrin α3 or CD26.

Aberrant DNA methylation of PTPRG as one possible mechanism of its under-expression in CML patients in the State of Qatar.

BACKGROUND: Several studies showed that aberrant DNA methylation is involved in leukemia and cancer pathogenesis. Protein tyrosine phosphatase receptor gamma (PTPRG) expression is a natural inhibitory mechanism that is downregulated in chronic myeloid leukemia (CML) disease. The mechanism behind its downregulation has not been fully elucidated yet. AIM: This study aimed to investigate the CpG methylation status at the PTPRG locus in CML patients. METHODS: Peripheral blood samples from CML patients at time of diagnosis [no tyrosine kinase inhibitors (TKIs)] (n = 13), failure to (TKIs) treatment (n = 13) and healthy controls (n = 6) were collected. DNA was extracted and treated with bisulfite treatment, followed by PCR, sequencing of 25 CpG sites in the promoter region and 26 CpG sites in intron-1 region of PTPRG. The bisulfite sequencing technique was employed as a high-resolution method. RESULTS: CML groups (new diagnosed and failed treatment) showed significantly higher methylation levels in the promoter and intron-1 regions of PTPRG compared to the healthy group. There were also significant differences in methylation levels of CpG sites in the promoter and intron-1 regions amongst the groups. CONCLUSION: Aberrant methylation of PTPRG is potentially one of the possible mechanisms of PTPRG downregulation detected in CML.

Epidermal growth factor receptor inhibitors in cancer treatment: advances, challenges and opportunities.

Aberrant expression of the epidermal growth factor receptor (EGFR) system has been reported in a wide range of epithelial cancers. In some studies, this has also been associated with a poor prognosis and resistance to the conventional forms of therapies. These discoveries have led to the strategic development of several kinds of EGFR inhibitors, five of which have gained US Food and Drug Administration approval for the treatment of patients with non-small-cell lung cancer (gefitinib and erlotinib), metastatic colorectal cancer (cetuximab and panitumumab), head and neck (cetuximab), pancreatic cancer (erlotinib) and breast (lapatinib) cancer. Despite these advances and recent studies on the predictive value of activating EGFR mutation and KRAS mutations with response in non-small-cell lung cancer and colon cancer patients, there is currently no reliable predictive marker for response to therapy with the anti-EGFR monoclonal antibodies cetuximab and panitumumab or the small molecule EGFR tyrosine kinase inhibitors gefitinib and erlotinib. In particular, there has been no clear association between the expression of EGFR, determined by the US Food and Drug Administration-approved EGFR PharmDX kit, and response to the EGFR inhibitors. Here, we discuss some of the controversial data and explanatory factors as well as future studies for the establishment of more reliable markers for response to therapy with EGFR inhibitors. Such investigations should lead to the selection of a more specific subpopulation of cancer patients who benefit from therapy with EGFR inhibitors, but equally to spare those who will receive no benefit or a detrimental effect from such biological agents.

Co-expression and prognostic significance of putative CSC markers CD44, CD133, wild-type EGFR and EGFRvIII in metastatic colorectal cancer.

The presence of colorectal cancer stem cells (CSCs) have been associated with tumour initiation and resistance to therapy. This study investigated the co-expression and prognostic significance of the CSCs biomarkers CD44 and CD133 with wild-type EGFR (wtEGFR) and EGFRvIII in colorectal cancer (CRC). The expression of these biomarkers were determined in tumours from 70 patients with metastatic CRC by immunohistochemistry, and in a panel of human CRC cell lines, and their variants with acquired-resistance to EGFR inhibitors, by flow cytometry. The expression of CD44, CD133, wtEGFR and EGFRvIII were present in 17%, 23%, 26% and 13% of cases and the co-expression of CD44/CD133 with wtEGFR and EGFRvIII were present in 9% and 3% of the cases respectively. Only co-expression of CSCs/EGFRvIII (P = 0.037), and amphiregulin (P = 0.017) were associated with worse overall survival. Interestingly, disease-free survival was improved in BTC expressing patients (P = 0.025). In vitro CD133 expression and its co-expression with CD44 were associated with primary-resistance to irinotecan and acquired-resistance to anti-EGFR inhibitors respectively. Our results suggest co-expression of CSCs and EGFRvIII could be potential biomarkers of worse overall survival and resistance to therapy in patients with mCRC and warrants further validation in a larger cohort.

Co-targeting the EGFR and IGF-IR with anti-EGFR monoclonal antibody ICR62 and the IGF-IR tyrosine kinase inhibitor NVP-AEW541 in colorectal cancer cells.

The aberrant expression of the epidermal growth factor receptor (EGFR) has been reported in a wide range of epithelial tumours. In some studies, co-expression of insulin-like growth factor receptor-I (IGF-IR) have been associated with resistance to the EGFR inhibitors. Here, we investigated the sensitivity of a panel of human colorectal tumour cell lines, including two newly established lines Colo2 and Colo13, to treatment with anti-EGFR mAb ICR62 and IGF-IR tyrosine kinase inhibitor NVP-AEW541 alone and in combination. We also determined the association between the expression levels of EGFR and IGF-IR with their responses to ICR62 and/or NVP-AEW541. In contrast to DiFi cells, which contained high levels of EGFR but lower level of IGF-IR, the remaining 11 colorectal tumour cells expressed low levels of both EGFR and IGF-IR and such cells were relatively resistant to ICR62 or NVP-AEW-541 when used alone. Interestingly, compared to the results with the single agent, the effect of combination of NVP-AEW541 and ICR62 was found to be additive on inhibiting the growth of Colo13, CCL235, CCL244 cells but antagonistic in other (CCL218) cells. While overexpression of the EGFR seems to be associated with response to ICR62, no clear correlation was found between the expression levels of EGFR and IGF-IR, or the levels of phosphorylated EGFR and response to treatment with NVP-AEW541, in single or combination setting with ICR62. Our results suggest that combining EGFR and IGF-IR inhibitors may enhance antitumour response in a fraction of colorectal cancer cells and warrants further study in colorectal cancer.

Responses of human colorectal tumor cells to treatment with the anti-epidermal growth factor receptor monoclonal antibody ICR62 used alone and in combination with the EGFR tyrosine kinase inhibitor gefitinib.

The anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab has been approved for the treatment of patients with metastatic colorectal cancer. However, there is currently no reliable marker for response to therapy with the EGFR inhibitors. In this study, we investigated the sensitivity of 10 human colorectal tumor cell lines (DiFi, CCL218, CCL221, CCL225, CCL227, CCL228, CCL231, CCL235, CCL244, and HCT-116) to treatment with our anti-EGFR monoclonal antibody, ICR62, and/or the EGFR tyrosine kinase inhibitor, gefitinib. Of the cells examined, only DiFi contained high levels of constitutively active EGFR and were highly sensitive to treatment with both ICR62 (IC(50) = 0.52 nmol/L) and gefitinib (IC(50) = 27.5 nmol/L). In contrast, the growth of other tumor cell lines, which contained low levels of the EGFR, HER-2, and pAkt but comparable or even higher basal levels of phosphorylated mitogen-activated protein kinase (pMAPK), were relatively resistant to treatment with both inhibitors. Both ICR62 and gefitinib induced EGFR down-regulation, reduced the basal levels of pEGFR at five known tyrosine residues, pMAPK, and pAkt, and increased the sub-G(1) population in DiFi cells. However, treatment with a combination of ICR62 and gefitinib neither sensitized colorectal tumor cells that were insensitive to treatment with the single agent nor enhanced the growth-inhibitory effect of the single agent in DiFi cells. These results indicate that basal levels of pMAPK and pAkt are not good indicators of response to the EGFR inhibitors in colorectal cancer cells and dual targeting of the EGFR by a combination of ICR62 and gefitinib is not superior to treatment with a single agent.