Effect of Human Epidermal Growth Factor on Cell Growth and Its Receptor in Human Gastric Carcinoma Cell Lines

The effect of human epidermal growth factor (hEGF) on the growthof various histological types of six human gastric carcinomacell lines was examined. The cell lines had relatively highaffinity EGF receptors (dissociation constant Kd = 10^–9to 10^–10 M). One gastric cancer cell line, MKN-74 (welldifferentiated adenocarcinoma) showed no response to hEGF, incell growth, DNA synthesis or ¹²⁵I-hEGF cell binding. Therewere no apparent correlations between histological type andcell growth, DNA synthesis or number of EGF receptors in thesecells. The number of EGF receptors and the Kd value of the gastriccarcinoma cell lines varied with their internal and externalenvironments. hEGF concentrations corresponding to maximum stimulationin DNA synthesis varied between cell lines. The results suggestsome gastric carcinoma cells to have EGF receptors and theirgrowth seemingly to be stimulated by EGF in vitro. There are,however, no obvious correlations between the effect of hEGFon the growth of human gastric carcinoma cell lines or theirhistological type.     

Antitumor Effect of PSK at a Distant Site: Inductions of Interleukin-8-like Factor and Macrophage Chemotactic Factor in Murine Tumor

The antitumor effect of PSK, a Coriolus preparation, at a distant site was analyzed with the use of a double grafted tumor system in which male BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10⁶ cells) and the left (2 × 10⁵ cells) flanks and were then injected with PSK in the right tumor on the third day thereafter. The antitumor effect of intratumoral administration of PSK in the right tumor on days 3, 4 and 5 was compared with the effect of surgical resection of the right tumor on day 5. Three out of 8 mice given PSK intratumorally became tumor-free whereas no mouse tumor-free in the left flank was found among the surgically resected mice. As regards sinecomitant immunity, tumor inoculation into the right flank followed by intratumoral administration of PSK on days 3 and 5 and surgical excision of the primary tumor on day 6 resulted in complete rejection of a tumor challenge in the left flank on day 21. The combination of presurgical intratumoral injections of PSK (more than 2 times) and postoperative oral administration of PSK appeared to be most effective in eradicating secondary tumors. Isolated TILs (tumor-infiltrating lymphocytes), obtained from the right tumor (treated with PSK) and the left tumor on day 10 in the double grafted tumor system were cultured in RPMI1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor (NCF) and macrophage chemotactic factor (MCF) activities were detected in the culture media from PSK-treated TILs that had been cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated TILs. NCF and MCF activities were also detected in the culture supernatant from PSK-treated tumor tissue on day 6. PSK-induced NCF in the murine tumor was neutralized by treatment with anti-human IL-8 IgG, and might be murine IL-8-like factor. Therefore, neutrophil and macrophage infiltrations of tumors following intratumoral injections of PSK are probably mediated by inductions of IL-8-like factor and MCF.     

EGF、EGF－R在卵巢浆液性良、恶性及交界性肿瘤中的表达

应用免疫组织化学ＡＢＣ方法，检测５２例卵巢浆液性良、恶性及交界性肿瘤中ＥＧＦ、ＥＧＦ－Ｒ的表达。结果显示：ＥＧＦ、ＥＧＦ－Ｒ在卵巢浆液性囊腺瘤中分别为５０％、３７％，交界性病变中分别为６２．５％、１００％。其中ＥＧＦ－Ｒ检测良恶性及交界性病变阳性表达率有显著性差异，Ｐ＜０．０５，提示ＥＧＦ、ＥＧＦ－Ｒ在卵巢发生发展中起不同调节作用，有利于病理的诊断和临床估计预后。     

Detection of Epidermal Growth Factor Receptor Gene Amplification in Human Squamous Cell Carcinomas Using Fluorescence in situ Hybridization

The gene for epidermal growth factor receptor (EGFR) is associated with development of certain human cancers. In this study, we employed the improved fluorescence in situ hybridization technique to detect EGFR gene amplification in cell lines and tissue sections from human squamous cell carcinomas. We detected multiple distinct signals as arrayed amplicons on metaphase chromosomes and interphase nuclei of tumor cells. Our results provide a basis for rapid and quantitative DMA diagnosis of the EGFR gene amplification in individual cells of tumor specimens.     

Localization of Acidic and Basic Fibroblast Growth Factor mRNA in Human Brain Tumors

Acidic and basic fibroblast growth factors (aFGF and bFGF) are closely related peptide mitogens acting on both mesoderm- and neuroectoderm-derived cells, including fibroblasts, endothelial cells and glial cells. In order to identify the expression of mRNAs for these growth factors, in situ hybridization using human aFGF and bFGF RNA probes was performed in 24 human brain tumors. The mRNAs for aFGF and bFGF were expressed in the cells of various tumors (1/1 and 1/1 astrocytoma, 2/2 and 2/2 anaplastic astrocytomas, 6/6 and 6/6 glioblastomas, 4/4 and 4/4 meningiomas, 3/3 and 3/3 schwannomas, 1/2 and 1/2 pituitary adenomas, 4/4 and 4/4 metastatic carcinomas, 0/1 and 0/1 hemangioblastoma, 0/1 and 0/1 craniopharyngioma) and were also detected in endothelial cells and surrounding neuronal cells of brain tumors. These results suggest the possibilities that aFGF and bFGF contribute to the uncontrolled growth of tumor cells and the proliferation of endothelial cells in autocrine and paracrine manners, and that the expression of mRNAs for these growth factors in the surrounding neuronal cells results in enhancement of tumor growth.     

C－erbB－2，人表皮生长因子及其受体在大肠癌中表达的意义

用ＡＢＣ免疫组化法测定２００例大肠癌组织中Ｃ－ｅｒｂＢ－２，人表皮生长因子（ｈＥＧＦ）及其受体（ＥＧＦＲ）。结果发现：１）Ｃ－ｅｒｂＢ－２，ｈＥＧＦ，ＥＧＦＲ在２００例大肠癌中阳性表达分别为３６％、４４％、４７％，三者共同阳性为１６．５％。２）ｈＥＧＦ，ＥＧＦＲ在大肠癌ＤｕｋｅｓＣ、Ｄ期，肿瘤＞２ｃｍ、低分化腺癌，有深度浸润和淋巴结转移者阳性率显著高于其它各型（Ｐ＜０．０１）。３）Ｃ－ｅｒｂＢ－２，ｈＥＧＦ和ＥＧＦＲ阳性病例存活率明显低于这些阴性病例（Ｐ＜０．０１）。结果表明，Ｃ－ｅｒｂＢ－２，ｈＥＧＦ和ＥＧＦＲ在大肠癌的侵袭性生长中起重要作用，ｈＥＧＦ和ＥＧＦＲ可作为大肠癌患者高度恶性的生物学指标。     

Feasibility Study of Docetaxel and Cyclophosphamide Six-Cycle Therapy as Adjuvant Chemotherapy for Japanese Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer Patients

Background: We compared treatment completion rates and safety of docetaxel and cyclophosphamide sixcycletherapy (TC6) with docetaxel followed by 5FU, epirubicin and cyclophosphamide (T-FEC) therapy inJapanese patients with human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Materialsand Methods: We administered TC6 q3w or T-FEC q3w to HER2-negative breast cancer patients. The primaryendpoint of this trial was toxicity. As second endpoints, the treatment completion rate and relative dose intensitywere evaluated. Results: The TC6 and T-FEC group consisted of 22 and 21 patients, respectively. Concerninghematological toxicity, grade 3 or higher adverse reactions included neutropenia and febrile neutropenia. Asnon-hematological adverse events, exanthema and peripheral neuropathy were frequently reported in theTC6 group, whereas more patients of the T-FEC group reported nausea and vomiting. In TC6, the treatmentcompletion rate was 86.4% and the relative dose intensity of docetaxel was 93.2%. In T-FEC, the values were95.2% and 98.9%, respectively. Conclusions: These results suggest that TC6 is tolerable in Japanese, and thatthis regimen can also be performed in outpatient clinics. However, with the TC6 regimen, the compliance wasslightly lower than with the T-FEC regimen, and supportive therapy needs to be managed appropriately.     

Detection of Epidermal Growth Factor Receptors and E-Cadherins in the Basolateral Membrane of A431 Cells by Laser Scanning Fluorescence Microscopy

We have examined the localization of epidermal growth factor (EGF) receptors and E-cadherins in cultured A431 cells, an epidermoid carcinoma cell line, using a laser scanning fluorescence microscope. The fluorescence signals generated by monoclonal antihodies against EGF receptor and E-cadherin were localized mainly in the cell-cell contact sites, about 3 μm in width. When these areas for cell adhesion were scanned perpendicularly, fluorescence was detected from a point 4μ from the cell base for a distance of 8 μm towards the cell surface (about 16 μm from the cell base). These data suggest the subcellular localization of EGF receptors and E-cadherins in the basolateral membrane of A431 cells.     

Human Epidermal Growth Factor Receptor Family‐Targeted Therapies in the Treatment of HER2‐Overexpressing Breast Cancer

Breast cancer characterized by overexpression of human epidermal growth factor receptor 2 (HER2) has been associated with more aggressive disease progression and a poorer prognosis. Although an improved understanding of breast cancer pathogenesis and the role of HER2 signaling has resulted in significant survival improvements in the past 20 years, resistance to HER2‐targeted therapy remains a concern. A number of strategies to prevent or overcome resistance to HER2‐targeted therapy in breast cancer are being evaluated. This article provides a comprehensive review of (a) the role of HER2 signaling in breast cancer pathogenesis, (b) potential receptor and downstream therapeutic targets in breast cancer to overcome resistance to HER2‐targeted therapy, and (c) clinical trials evaluating agents targeting one or more members of the HER family and/or downstream pathways for the treatment of breast cancer, with a focus on metastatic disease.     

A Deletion Mutation within the Ligand Binding Domain Is Responsible for Activation of Epidermal Growth Factor Receptor Gene in Human Brain Tumors

Two transplantable cell lines of human glioblastoma multiforme GL-3 and GL-5 carried an amplification and overexpression of structurally altered epidermal growth factor (EGF) receptor gene: the 140 kilodalton EGF receptors in these cases exhibited a constitutively expressed tyrosine kinase activity without the ligand. Here, we isolated the abnormal EGF receptor cDNA from GL-5 cell line, and demonstrated that this cDNA bears a single large intramolecular deletion mutation 801 base pairs long within the ligand binding domain of EGF receptor. In other regions no amino acid substitution was observed. At the level of genomic DNA, this deletion appeared to start from the 1st intron and terminate in the 6th intron of the EGF receptor gene. However, in the two lines of glioblastoma, GL-3 and GL-5, the positions of the start or the end of the deletion mutation in these introns were not identical, suggesting an involvement of a unique recombination mechanism in the formation of deletion mutation. A weak but ligand-independent transforming activity was observed in the deletion-carrying EGF receptor cDNA.     

Association of Human Epidermal Growth Factor Receptor 2 with Radiotherapy Resistance in Patients with T1N0M0 Breast Cancer

Purpose

Preclinical studies have shown that human epidermal growth factor receptor 2 (HER2) status is associated with resistance to radiotherapy (RT). In this study, we evaluated the overall survival of a T1N0M0 breast cancer cohort in Korea according to the use of RT and the HER2 status.

Methods

We analyzed data collected from 11,552 patients with invasive breast cancer who were enrolled in the Korean Breast Cancer Society Registration Program between 1999 and 2007. Data on the TNM stage, estrogen receptor status, progesterone receptor status, HER2 status, operation method, and the use of RT were analyzed.

Results

The median follow-up period was 51 months. A significant improvement in overall survival after RT was observed only in the HER2(-) group. In this group, the 10-year overall survival rate was 95.5% for patients who did not receive RT and 96.3% for patients who received RT (p=0.037). In contrast, in the HER2(+) group, RT was not associated with a survival benefit (p=0.887). Multivariate analysis showed that RT was significantly associated with a reduction in mortality in the HER2(-) group (hazard ratio, 0.738; 95% confidence interval, 0.549-0.993; p=0.045).

Conclusion

We found that postoperative RT was not associated with a survival benefit in HER2(+) breast cancer patients, suggesting that HER2(+) breast cancers could be RT resistant.     

Hormone Receptor and Human Epidermal Growth Factor Receptor 2 Detection in Invasive Breast Carcinoma: A Retrospective Study of 12,467 Patients From 19 Chinese Representative Clinical Centers

IntroductionWe evaluated the current status of hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) detection in invasive breast carcinoma (IBC) in various laboratories across China.Materials and MethodsThe Breast Pathology Study Group of the Chinese Society of Pathologists collected HR and HER2 data from 12,467 IBC cases from 19 representative clinical centers in China. The data from every center were compared with the pooled data from the other centers.ResultsThe assessment of HR status showed that the overall positive estrogen receptor (ER) and progesterone receptor (PR) rates were 71.7% and 63.7% (range, 60.9%-87.9% and 43.9%-84.8%), respectively. The ER results in 3 centers and the PR results in 6 centers were outside the 99.5% confidence interval and were considered to be outliers (P < .0005). Of the 12,467 cases, 62.4% were ER⁺/PR⁺, 9.3% were ER⁺/PR⁻, 1.3% were ER⁻/PR⁺, and 27.0% were ER⁻/PR⁻. The assessment of HER2 status showed that the overall positive rate of HER2 (with a definition of immunohistochemistry [IHC] 3+ or IHC2+/ in situ hybridization-positive) was 24.7% (range, 13.7%-35.7%) in each center. Three centers were outside the 99.5% confidence interval and were considered to be outliers (P < .0005). The proportion of HER2 IHC3+ was 21.1%. The positive rates of HER2 gene amplification in IHC 0/1+, 2+, 3+ cases were 2.0%, 17.6%, and 85.9%, respectively.ConclusionsAs the largest study of HR and HER2 status in Chinese patients with IBC, the data from the present study have indicated that the overall rates of HR and HER2 were comparable to those reported in previous studies. However, the rates varied among the laboratories. Individual centers had not met the target values, and they need to improve the detection.     

Safety and Tolerability of Docetaxel,Cyclophosphamide, and Trastuzumab Compared to Standard Trastuzumab-Based Chemotherapy Regimens for Early-Stage Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer

Purpose

We evaluated the tolerability and cardiac safety of docetaxel, cyclophosphamide, and trastuzumab (TCyH) for the treatment of early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer and compared to the standard trastuzumab-based chemotherapy regimens doxorubicin with cyclophosphamide followed by paclitaxel and trastuzumab (AC-TH) and docetaxel, carboplatin, and trastuzumab (TCaH).

Methods

We retrospectively reviewed early-stage, resectable, HER2-positive breast cancer patients treated with trastuzumab-based chemotherapy at a single comprehensive cancer center between 2004 and 2011. Patient characteristics, comorbidities, relative dose intensity (RDI) of each regimen, tolerability, and cardiac toxicity were evaluated.

Results

One hundred seventy-seven patients were included in the study (AC-TH, n=114; TCaH, n=39; TCyH, n=24). TCyH was solely administered in the adjuvant setting, whereas two-thirds of the AC-TH and TCaH groups were administered postoperatively. Patients treated with TCyH tended to have a more significant underlying cardiac history, higher Charlson comorbidity index, and were of an earlier stage. All patients treated with TCyH received granulocyte colony stimulating factor primary prophylaxis. No febrile neutropenia or grade ≥3 hematologic toxicity was observed in the TCyH group as compared to the AC-TH and TCaH groups. There were no significant differences in the rates of early termination, hospitalization, dose reduction, or RDI between the regimens. The symptomatic congestive heart failure rate between AC-TH, TCaH, and TCyH groups was not significantly different (4.4% vs. 2.6% vs. 8.3%, respectively, p=0.57). There was also no significant difference in the rate of early trastuzumab termination between patients treated with each regimen.

Conclusion

TCyH is well tolerated and should be investigated as an alternative adjuvant chemotherapy option for patients who are not candidates for standard trastuzumab-containing regimens. Larger clinical trials are necessary to support the wider use of TCyH as an adjuvant regimen.     

Incidence and Management of Diarrhea With Adjuvant Pertuzumab and Trastuzumab in Patients With Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer

BackgroundThe APHINITY (BIG 4-11) study showed that pertuzumab significantly improved the rates of invasive disease-free survival among patients with human epidermal growth factor receptor 2 (HER2)-positive, operable breast cancer when added to adjuvant trastuzumab and chemotherapy. Because diarrhea was a common adverse event that could compromise treatment administration, we evaluated the incidence and management of diarrhea in the APHINITY study.Patients and MethodsThe APHINITY trial is a prospective, randomized, multicenter, multinational, double-blind, placebo-controlled trial. The eligible patients were randomly assigned to receive standard adjuvant chemotherapy and 1 year of trastuzumab combined with pertuzumab or placebo. The diarrhea incidence, severity (National Cancer Institute common terminology criteria for adverse events, version 4.0), onset, and management were analyzed.ResultsA total of 4805 patients were randomized. Diarrhea of any grade was the most common adverse event and occurred in 71% of patients in the pertuzumab arm versus 45% in the placebo arm. Diarrhea grade 3 to 4 was observed in 10% and 4% in the pertuzumab and placebo arms, respectively. The greatest incidence of diarrhea was reported during the concomitant administration of HER2-targeted therapy and taxane (61% vs. 34% of patients experienced an event with pertuzumab vs. placebo, respectively). A marked decrease was observed on chemotherapy cessation. Antidiarrheal agents were commonly used, and diarrhea rarely caused treatment dose modifications or discontinuation.ConclusionDiarrhea was a common adverse event in the APHINITY study. Most episodes were low grade and were generally manageable with common antidiarrheal agents. The incidence of diarrhea was greater with the combination of a taxane and HER2-targeted treatment and decreased once chemotherapy was stopped.     

Effect of Hormone Therapy on Long-term Outcomes of Patients with Human Epidermal Growth Factor Receptor 2- and Hormone Receptor-Positive Metastatic Breast Cancer: Real World Experience in China

Background: Among human epidermal growth factor receptor 2 (HER2)-positive breast cancer, more thanhalf are also hormone receptor (HR)-positive. Although HR is a predictive factor for the efficacy of hormonetherapy, there are still some uncertainties in regard to the effects on patients with HR-positive and HER2-positivemetastatic breast cancers due to the potential resistance to hormone therapy caused by co-expression of HRand HER2. There are no clinical trials directly comparing the efficacy of hormonal therapy with chemotherapy.Materials and Methods: To examine the real-world effect of hormone therapy on patients with HR-positiveand HER2-positive metastatic breast cancers, a cross-sectional study of a representative sample of the Chinesepopulation was conducted. The study included 113 patients who received first-line and second-line palliativetreatment between 2005 and 2010 in the Cancer Institute and Hospital, Chinese Academy of Medical Science.The effect of hormone therapy on overall survival (OS) was studied. Results: The patients who received hormonetherapy (n=51) had better overall survival in contrast to those who received chemotherapy with anti-HER2therapy (n=62) in first- or second-line treatment. The difference was of borderline statistical significance (51.8mvs 31.9m, p=0.065). In addition, the effect of hormone therapy did not differ significantly with other prognosticfactors, including age (≤50 years or >50 years), disease free survival (≥2 years or < 2 years) and site of metastasis(visceral or bone/soft tissue). On multivariate analysis, administration of hormone therapy was associated witha trend toward a favorable prognosis (p=0.148, HR=0.693, 95%CI 0.422-1.139). Age more than 50 years wasthe sole independent harmful prognostic factor (p<0.001, HR=2.797, 95%CI 1.676-4.668). Conclusions: Ourdata suggest that hormonel therapy may improve outcomes of the patients with ER-positive and HER2-positivemetastatic breast cancer.     

Association of Human Epidermal Growth Factor Receptor-2 Expression and Clinicopathological Findings in Patients with Colorectal Cancer

Background: To determine the frequency of HER-2 overexpression in colorectal cancer (CRC) patients,and to explore the relationship between clinicopathological prognostic factors and their effects on survival,based on immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) analysis. Materials andMethods: The study included 80 patients with a histologically proven diagnosis of CRC that received adjuvantFOLFOX-4 chemotherapy at our department between March 2006 and September 2010. Patient data wereanalyzed retrospectively. Results: The median follow-up period and age of the patients were 24 months and59 years, respectively. In immunohistochemical staining, 3+ staining was found in 2 patients (2.5%) while 2+was in 13 (16%) . FISH for HER-2 was performed for all of these 15 patients; samples which were 3+ showedpositivity but the ones with 2+ were negative. There was no significant correlation between HER-2 expressionand age, gender, tumor localization, histological subtype, grade, lymphovascular and perineural invasion, orpTN stage (P>0.05), even when the patients with HER-2 overexpression were analyzed separately. There wasalso no significant relationship between progression-free survival (PFS) and overall survival (OS), and HER-2expression, gender, tumor localization, obstruction-perforation, bleeding, histological type, grade, lymphovascularand perineural invasion, or pT staging (P>0.05); however, there was a significant relationship between lymphnode involvement, and PFS and OS (P<0.05). Conclusions: Evaluation of HER-2 overexpression in a morecomprehensive, multi-center, prospective trial with standardized methods will be an appropriate approach.     

Expressional Correlation of Human Epidermal Growth Factor Receptor 2, Estrogen/Progesterone Receptor and Protein 53 in Breast Cancer

Background: This study aimed to show the localization of estrogen / progesterone receptors, human epidermalgrowth factor receptor 2 (Her-2) and protein 53 (p53) by immunohistochemistry in a series of consecutive breastcancer patients. Materials and Methods: The study covered invasive breast cancers from 299 patients presentingat the Oncogenetic Clinic and Pathology Centers of Ahwaz Jondishapour University of Medical Sciences Hospitalin Iran during the time period from 2009 to 2011. The Scarff-Bloom Richardson scoring method was used. Results:Of the 299, 27% (80/299) were <40, 33% (100/299) were 41-50, and the remaining 40% (119/299) were>50 yearsold. The highest incidence of breast cancer in this study population was in the group of more than 50 year age,and the most common histological type of breast cancer was the invasive ductal carcinoma, which accountedfor 68% (203/299) of the cases. Out of possible total of 207, 6% (13/207), 41% (85/207), and 53% (109/207) werescored as grade І, ІІ, ІІІ, respectively. Conclusion: Our findings demonstrated a lack of association betweenlabeling for the markers studied and tumor size and age of the patients. We confirmed an association betweenER labeling and nuclear grade of breast cancer. The conflicting results obtained compared with the literature bebecause of differences in the immunohistochemical techniques applied in the various studies and to the scoringsystems used.     

Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice

The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α) and vascular endothelial growth factor (VEGF) but were negative for EGFR, human epidermal growth factor receptor 2 (HER2), and VEGFR. Double immunofluorescence staining revealed that tumor-associated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR), and phosphorylated VEGFR (pVEGFR). Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase) or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01); this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001). AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, and increased the level of apoptosis in both tumor-associated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.     

Mega Base Map of the Epidermal Growth Factor (EGF) Receptor Gene Flanking Regions and Structure of the Amplification Units in EGF Receptor-hyperproducing Squamous Carcinoma Cells

We have established a mega base scale physical map of the 5'- and 3'- flanking regions of the epidermal growth factor (EGF) receptor gene using CpG-recognition rare-cutting restriction enzymes and pulsed-field gel electrophoresis. In this map, a methylation-free CpG island (HTF island) is located within an 8-kilobase pair (kb) Eco RI fragment which includes exon 1 of the EGF receptor gene. From this HTF island, a 390-kb Not I fragment was identified as the longest 5'-flanking region and a 540-kb Mlu I fragment as the longest 3'-flanking region. Utilizing this map information, we have analyzed the structure of the flanking regions of amplified EGF receptor genes which are found in various squamous carcinoma cells. Among seven cell lines tested, four cell lines carrying EGF receptor genes in amounts more than 20 times that of normal cells showed amplification together with large 5'- and 3'- flanking regions. The amplified 5'-flanking regions were rearranged in different forms but were distinct in each cell line. The amplified 3'-flanking regions were at least 540 kb in size and common to all the cell lines, except that A431 had rearrangement points within 540 kb downstream of the HTF island. Thus, the size of amplification units appears to be large and different in each cell line.