human epidermal growth factor

Human Epidermal Growth Factor (hEGF) is a protein molecule with potent mitogenic activity, increasing the rate of wound and ulcer healing in different tissues of the human body. In recent years, the Center for Genetic Engineering and Biotechnology (CIGB) has carried out ...

Background: Erlotinib is a Human Epidermal Growth Factor Receptor Type 1/tyrosine kinase (EGFR) inhibitor which is used for non-small-cell lung cancer treatment. Despite that erlotinib is considered to have a favorable safety profile, adverse events such as interstitial lung disease (ILD) were reported in pivotal studies. The authors report the first histologically confirmed case of fatal ILD associated with erlotinib therapy. The medical record of a patient who developed fatal ILD after receiving erlotinib treatment was reviewed to identify the cause of death and other factors potentially contributive to this adverse outcome. A 55-year-old smoker with no evidence of pre-existing interstitial disease developed bilateral ILD and respiratory failure which could be explained only as a toxicity of erlotinib. He had a history of stage IV left upper lobe squamous-cell carcinoma for which he had received three successive regimens of chemotherapy (ifosfamide plus gemcitabine, docetaxel, mitomycin plus navelbine), followed five months later by erlotinib. At initiation of erlotinib treatment there were no radiological signs suggestive of ILD disease or apparent clinical signs of respiratory distress. While the patient completed two months with erlotinib therapy he developed bilateral interstitial infiltrates; despite discontinuation of erlotinib he was admitted with respiratory failure two weeks later. Diagnostic work up for other causes of pneumonitis including infectious diseases, congestive cardiac failure and pulmonary infraction was negative. Empiric treatment with oxygene, corticosteroids and later with cyclophosphamide was ineffective and the patient progressively deteriorated and died. The clinical and post-mortem examination findings are presented and the possible association relationship between erlotinib induced ILD and previous chemotherapy is discussed. Physicians should be alert to the fact that erlotinib related ILD, although infrequent, is potential fatal. The association between selective EGFR-inhibitors and ILD should be further investigated.

Learning Objectives After completing this course, the reader will be able to: Assess the predictive biomarkers currently used in breast cancer, including, for example, ER, HER-2, and TOP2A.Discuss the predictive capability of biomarkers in relation to the mechanisms of action of the corresponding treatment.Discuss the link between the targeted therapies currently used in breast cancer and the predictive biomarkers.Evaluate the role of TOP2A testing in relation to treatment with anthracyclines. CME Access and take the CME test online and receive 1 AMA PRA Category 1 Credit™ at CME.TheOncologist.com

Using 3-mercaptopropyltrimethoxysilane (MPS)-coated (PbMg1∕3Nb2∕3O3)0.63–(PbTiO3)0.37 (PMN-PT)/tin and lead zirconate titanate/glass piezoelectric microcantilever sensors (PEMSs) with single-chain variable fragment (scFv) immobilized on the MPS surface, we have demonstrated real-time, label-free detection of human epidermal growth factor receptor 2 (Her2) in a background of 1mg∕ml bovine serum albumin. Coupled with a scFv with a KD of 3.4×10−8M, the MPS-insulated PMN-PT/tin PEMS 560μm long and 720μm wide exhibited a Her2 concentration sensitivity of 5ng∕ml in a background of 1mg∕ml BSA.

Background and Objective: Determination of HER2 gene is crucial in breast carcinoma management and prognosis, as HER2 alterations are linked to a shorter disease-free period, overall survival and resistance to tamoxifen anti-estrogen therapy and other chemotherapy regimens, regardless of the nodal or hormone receptor status. This study aimed to estimate HER2 gene status of infiltrative mammary cancer cases with immunohistochemically equivocal (2+) score using Silver DNA in Situ Hybridization (SISH) technique and to investigate its association with clinicopathological variables. Methods: The study included 52 formalin-fixed paraffin embedded tissue blocks from female patients with invasive breast carcinoma with score of 2+ (equivocal) HER2 immunohistochemistry. All cases were studied by silver DNA in situ hybridization technique (SISH) for the determination of the amplified HER2 DNA. Results: The SISH technique showed that HER2 gene was not amplified in 33 cases out of 52 (63.5%); while the rest of 19 cases (36.5%) revealed amplified gene status. According to age, HER2 gene status reported non-significant difference in the age groups between cases with amplified and non-amplified gene status (P=0.173). There was a significant negative association between positive Estrogen (ER) and Progesterone (PR) status and HER2 gene amplification (P= 0.002 and 0.017, respectively). Conclusion: More than half of breast carcinoma cases with equivocal HER2 immunoreactivity showed non-amplified gene status; this needs to be considered by oncologists in their management planning of breast cancer. Amplified HER2 gene is significantly associated with negative ER and PR status that affects patients' management protocols and future outcome of the disease.

Optical probes that yield high target-to-background ratios are necessary to detect microfoci of cancer that would otherwise escape detection with white light imaging. Target-specific activation of the optical signal at tumor foci is one mechanism by which high target and low background signal can be achieved. Here, we describe a two-step activation process in which the tumors are first pretargeted with a nonfluorescent biotinylated monoclonal antibody [cetuximab (Erbitux) targeting human epidermal growth factor receptor type 1 (HER1)]. Following this, a second agent, neutravidin-BODIPY-FL fluorescent conjugate, is given and binds to the previously targeted antibody, resulting in an f10-fold amplification of the optical fluorescence signal, leading to high tumorto-background ratios. Spectral fluorescence imaging was done in a mouse model of peritoneal metastasis using a HER1-overexpressing cell line (A431) after pretargeting with biotinylated cetuximab and 3 h after administration of neutravidin-conjugated BODIPY-FL. Both aggregated tumors as well as small cancer implants were clearly visualized in vivo. For lesions f0.8 mm or greater in diameter, the spectral fluorescence imaging had a sensitivity of 96% (178 of 185) and a specificity of 98% (188 of 191). This two-step activation paradigm (pretargeting followed by neutravidinbiotin binding with an attached activatable fluorophore) could be useful in tumor-specific molecular imaging of various targets to guide surgical resections.

Endometrial cancers (ECs) are the most common gynecological malignancies. Based on morphology and pathogenesis, ECs are segregated into type 1 and 2 ECs. Types 1 ECs are those tumors that are estrogendriven, whereas type 2 ECs are more aggressive and are independent of hormonal status. In the proposed study, we evaluated the clinicopathological parameters of type 2 ECs and its comparison with type 1 ECs. We retrospectively analyzed seven-year data from archives of pathology, Liaquat National Hospital, from January 2013 to December 2019. All patients underwent radical surgeries for diagnosed EC on endometrial biopsy. All specimens were of total abdominal hysterectomy with bilateral salpingo-oophorectomy, omentectomy, and peritoneal sampling, along with pelvic lymphadenectomy. Records regarding tumor type, grade, depth of myometrial invasion, and ovarian, omental, nodal, and parametrial involvement were assessed. A total of 129 cases of ECs were included in the study. The mean age of the patients was 57.6 ± 9.3 years. Majority of the cases were type 1 ECs (82.2%). The most common histological type of EC was endometrioid (82.2%) followed by serous carcinoma (10.1%). Most of the tumors were grade 1 (42.6%) and the International Federation of Gynecology and Obstetrics (FIGO) stage I (72.8%). Nodal metastases were present in eight cases (6.2%) and adnexal involvement was present in 12 cases (9.3%). We found a significant association of the type of EC with lymphovascular invasion, nodal metastasis, and adnexal involvement, whereas no significant association of EC type was seen with other clinicopathological characteristics. Type 1 EC was the most frequent subtype of EC in our study. On the other hand, type 2 EC was significantly associated with nodal metastasis, lymphovascular invasion, and adnexal involvement, signifying the poor prognostic significance of this group of EC.

Background: Today, more than 70% of patients with primary node-negative breast cancer are cured by local therapy alone. Many patients receive overtreatment by adjuvant chemotherapy due to inadequate risk assessment. So far, few clinical trials have prospectively evaluated tumor biology based prognostic factors. Risk assessment by a biological algorithm including invasion factors urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) will assess up to 35-55% of node-negative patients as low-risk and thus avoid chemotherapy. In contrast, a clinical-pathological algorithm will only classify 20-40% of patients as lowrisk. High-risk node-negative patients should receive chemotherapy. Anthracycline-based regimens are accepted as a standard, the additional benefit of taxanes remains an open question. Methods/Design: The international NNBC3 ("Node Negative Breast Cancer 3-Europe") trial compares biological risk assessment (UP) using invasion factors uPA/PAI-1 with a clinical-pathological algorithm (CP). In this trial, the type of risk assessment (CP or UP) was chosen upfront by each center for its patients. Fresh frozen tissue was obtained to determine uPA/PAI-1 using an enzyme-linked immunosorbent assay (ELISA). Patients assessed as high-risk were stratified by human epidermal growth factor receptor 2 (HER2) status and then randomised to receive anthracycline-containing chemotherapy 5-Fluorouracil (F)/Epirubicin (E)/Cyclophosphymide (C) or an anthracyclinetaxane sequence (FE 100 C*6 versus FE 100 C*3 followed by Docetaxel 100 *3). Discussion: In this trial, 4,149 node-negative patients with operable breast cancer from 153 centers in Germany and France were included since 2002. Measurement of uPA/PAI-1 by ELISA was performed with standardised central quality assurance for 2,497 patients (60%) from 56 "UP"-centers. The NNBC 3-Europe trial showed that inclusion of patients into a clinical phase III trial is feasible based on biological testing of fresh frozen tumor material. In addition, 2,661 patients were classified as high-risk and thus received chemotherapy. As adjuvant chemotherapy, 1,334 high-risk patients received FE 100 C-Docetaxel 100 , and 1,327 received French FE 100 C. No unexpected toxicities were observed. Chemotherapy efficacy and comparison of UP with CP will be evaluated after longer follow-up. Trial Registration: clinical Trials.gov NCT01222052.

Synthetic CRF 1-41 in a dose of 1 mcglkg was administered i.v. Plasma cortisol (CO) and ACTH were determined before and at 30, 60, 90 & 120 min. Fourteen children ( 1 2 ~, 2F 12t4.8 yrs) were investigated including 2 normal controls, 6 with IGHD and 6 with MPHD. According to the peak plasma CO during ITT the subjects

Triple negative breast cancer (TNBC) is an aggressive clinical phenotype characterized by lack of expression (or minimal expression) of estrogen receptor (ER) and progesterone receptor (PR) as well as an absence of human epidermal growth factor receptor-2 (HER2) overexpression. It shows substantial overlap with basal-type and BRCA1-related breast cancers, both of which also have aggressive clinical courses. However, this overlap is not complete, and the expression of ER, PR, and HER2 has been noted in basal-like tumors. TNBC also includes the normal-like subtype, and not all patients with TNBC harbor BRCA1 mutations. Because of its expression profile, TNBC is not amenable to treatment with hormone therapy or the anti-HER2 monoclonal antibody trastuzumab, and systemic treatment options are currently limited to cytotoxic chemotherapy. Overall survival, whether in early-stage or advanced disease, is poor compared with that in patients who have other phenotypes. A number of targeted approaches to TNBC are undergoing clinical evaluation, including the use of agents with poly(ADP-ribose) polymerase inhibitory properties such as iniparib (the United States Adopted Name for the investigational agent BSI-201), olaparib (AZD2281), and veliparib (ABT-888), antiangiogenic agents such as bevacizumab and sunitinib, and epidermal growth factor receptor blockers such as cetuximab and erlotinib. Encouraging results with some of these agents have been reported, thereby offering the promise for improved outcomes in patients with TNBC. The clinical characteristics of TNBC and clinical experience to date with novel targeted agents under development for this aggressive phenotype is reviewed.

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virusspecific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.

The human epidermal growth factor receptor 2 (HER2) is a validated therapeutic target in breast cancer. Heterodimerization of HER2 with other HER family members results in enhanced tyrosine phosphorylation and activation of signal transduction pathways. HER2 overexpression increases the translation of fatty acid synthase (FASN), and FASN overexpression markedly increases HER2 signaling, which results in enhanced cell growth. However, the molecular mechanism and regulation of HER2 and FASN interaction are not well defined. Lapatinib is a small-molecule tyrosine kinase inhibitor that blocks phosphorylation of the epidermal growth factor receptor and HER2 in breast cancer cells, resulting in apoptosis. We hypothesized that FASN is directly phosphorylated by HER2, resulting in enhanced signaling and tumor progression in breast cancer cells. Methods: Using mass spectrometry, we identified FASN as one of the proteins that is dephosphorylated by lapatinib in SKBR3 breast cancer cells. Immunofluorescence, immunoprecipitation, Western blotting, a kinase assay, a FASN enzymatic activity assay, an invasion assay, a cell viability assay and zymography were used to determine the role of FASN phosphorylation in invasion of SKBR3 and BT474 cells. The FASN inhibitor C75 and small interfering RNA were used to downregulate FASN expression and/or activity. Results: Our data demonstrated that FASN is phosphorylated when it is in complex with HER2. FASN phosphorylation was induced by heregulin in HER2-overexpressing SKBR3 and BT474 breast cancer cells. Heregulininduced FASN phosphorylation resulted in increased FASN enzymatic activity, which was inhibited by lapatinib. The FASN inhibitor C75 suppressed FASN activity by directly inhibiting HER2 and FASN phosphorylation. Blocking FASN phosphorylation and activity by lapatinib or C75 suppressed the activity of matrix metallopeptidase 9 and inhibited invasion of SKBR3 and BT474 cells. Conclusions: FASN phosphorylation by HER2 plays an important role in breast cancer progression and may be a novel therapeutic target in HER2-overexpressing breast cancer cells.

EGFR [EGF (epidermal growth factor) receptor] overexpression correlates with poor prognosis and bad outcomes in different tumours. However, evidence for EGFR contribution in melanoma immunobiology is limited. We have expressed the full‐length human EGFR gene in a murine melanoma cell line. EGFR protein expression in stably trnasfected B16 cells in culture was defined by immunoblotting, immunohistochemistry and FACS. Additionally, transfected cells became sensitive to the lysis induced with an anti‐EGFR monoclonal antibody in the presence of complement. Exogenous human EGF addition induced cell proliferation, validating the transfected receptor functionality. Thus we have developed a system to express a functional EGFR in order to evaluate the potential contribution of EGFR expression in melanoma biology and its resulting relevance as a target for immunointerventions in nonepithelial tumours.

Purpose Pertuzumab, a human epidermal growth factor receptor 2 (HER2) –targeted monoclonal antibody, potently inhibits HER2 dimerization and HER-mediated signaling pathways. Pertuzumab and the approved HER2-targeted monoclonal antibody trastuzumab have complementary mechanisms of action and result in enhanced antitumor activity when combined. This phase II trial assessed the efficacy and safety profile of the combination in patients with HER2-positive breast cancer whose disease had progressed during prior trastuzumab-based therapy. Patients and Methods This was a multicenter, open-label, single-arm, Simon two-stage study. Patients with advanced HER2-positive breast cancer in whom disease progression had occurred during prior trastuzumab-based therapy received trastuzumab weekly (4 mg/kg loading dose, then 2 mg/kg every week) or every 3 weeks (8 mg/kg loading dose, then 6 mg/kg every 3 weeks) and pertuzumab every 3 weeks (840 mg loading dose, then 420 mg every 3 weeks). Treatment co...

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal β-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild-type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1-Dimensional 1 H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal β-sheet of hEGF are important in receptor recognition.

There is an ever-growing interest in plant molecular farming as a system for producing valuable recombinant pharmaceutical molecules, such as single-chain variable fragments, on an industrial/agricultural scale and it appears that it is going to become a reality. Human epidermal growth factor receptor-2 (HER2) is an oncogene involved in abnormal cell growth in breast cancer and is considered for the development of new cancer therapies. We describe here the cloning and expression of a scFv-␣HER2 that has been produced in Escherichia coli and in plants using both stable and transient systems in tobacco and Nicotiana benthamiana. Single-chain antibodies (ScFvs) extracted and purified from E. coli and plant tissues were tested for functionality and specificity by flow cytometry analysis on several cell lines and showed positive results when used on breast cancer slides coming from human frozen tissues. As a result, scFv-␣HER2 represents a good opportunity for application and use in diagnosis and therapy.

BACKGROUND: Circulating tumour cells (CTCs) offer a non-invasive approach to obtain and characterise metastatic tumour cells, but their usefulness has been limited by low CTC yields from conventional isolation methods. METHODS: To improve CTC yields and facilitate their molecular characterisation we compared the Food and Drug Administrationapproved CellSearch Epithelial Kit (CEK) to a simplified CTC capture method, CellSearch Profile Kit (CPK), on paired blood samples from patients with metastatic breast (n ¼ 75) and lung (n ¼ 71) cancer. Molecular markers including Human Epidermal growth factor Receptor 2 (HER2) were evaluated on CTCs by fluorescence in situ hybridisation (FISH) and compared to patients' primary and metastatic cancer. RESULTS: The median cell count from patients with breast cancer using the CPK was 117 vs 4 for CEK (Po0.0001). Lung cancer samples were similar; CPK: 145 cells vs CEK:4 cells (Po0.0001). Recovered CTCs were relatively pure (60 -70%) and were evaluable by FISH and immunofluorescence. A total of 10 of 30 (33%) breast cancer patients with HER2-negative primary and metastatic tissue had HER2-amplified CTCs. CONCLUSION: The CPK method provides a high yield of relatively pure CTCs, facilitating their molecular characterisation. Circulating tumour cells obtained using CPK technology demonstrate that significant discordance exists between HER2 amplification of a patient's CTCs and that of the primary and metastatic tumour.

Background: Today, more than 70% of patients with primary node-negative breast cancer are cured by local therapy alone. Many patients receive overtreatment by adjuvant chemotherapy due to inadequate risk assessment. So far, few clinical trials have prospectively evaluated tumor biology based prognostic factors. Risk assessment by a biological algorithm including invasion factors urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) will assess up to 35-55% of node-negative patients as low-risk and thus avoid chemotherapy. In contrast, a clinical-pathological algorithm will only classify 20-40% of patients as lowrisk. High-risk node-negative patients should receive chemotherapy. Anthracycline-based regimens are accepted as a standard, the additional benefit of taxanes remains an open question.

Background: Breast cancers that overexpress the human epidermal growth factor receptor 2 (HER2) are eligible for effective biologically targeted therapies, such as trastuzumab. However, accurately determining HER2 overexpression, especially in immunohistochemically equivocal cases, remains a challenge. Manual analysis of HER2 expression is dependent on the assessment of membrane staining as well as comparisons with positive controls. In spite of the strides that have been made to standardize the assessment process, intra-and inter-observer discrepancies in scoring is not uncommon. In this manuscript we describe a pathologist assisted, computer-based continuous scoring approach for increasing the precision and reproducibility of assessing imaged breast tissue specimens. Methods: Computer-assisted analysis on HER2 IHC is compared with manual scoring and fluorescence in situ hybridization results on a test set of 99 digitally imaged breast cancer cases enriched with equivocally scored (2+) cases. Image features are generated based on the staining profile of the positive control tissue and pixels delineated by a newly developed Membrane Isolation Algorithm. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis. Results: A computer-aided diagnostic approach has been developed using a membrane isolation algorithm and quantitative use of positive immunostaining controls. By incorporating internal positive controls into feature analysis a greater Area Under the Curve (AUC) in ROC analysis was achieved than feature analysis without positive controls. Evaluation of HER2 immunostaining that utilized membrane pixels, controls, and percent area stained showed significantly greater AUC than

Introduction: Monoclonal antibodies labeled mainly with 99m Tc are being widely used as imaging agents in nuclear medicine. Recently PR81 was introduced as a new murine anti-MUC1 MAb against human breast carcinoma. This antibody reacts with the tandem repeat of 20-mer peptide of protein-8 M-1. Due to high specific reactivity, this antibody was shown to have high diagnostic potential in nuclear medicine. As the first step towards the use of an antibody for imaging purposes it has to be labeled with a radioisotope. In this study we have investigated a method for optimum labeling of this MAb with 99m Tc. Materials and Methods: The Ab reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and reduced Ab was labeled with 99mTc via stannous tartrate as a transchelator. The labeling efficiency was determined by ITLC. The integrity of reduced MAb was checked by means of SDS-PAGE and gel filtration chromatography (FPLC) on Superose 12 HR 10/30 (purity>99%). The amount of radiocolloids were measured by cellulose nitrate electrophoresis. In vitro stability of labeled product was checked in time intervals over 24 hrs by ITLC. Radioimmunoassay was used to test immunoreactivity of the labeled MAb. Biodistribution of the radiolabeled MAb was studied in normal mice at 4 and 24 hrs post-injection. Results: invitro less than %2 radiocolloids were found. There was no Ab fragmentation due to reduction procedure. Both labeled and unlabeled MAbs were able to compete for binding to the MUC1. Biodistribution studies in normal mice showed that there was no significant accumulation in any organ. Conclusion: Because of no significant loss of immunoreactivity of MAb due to labeling procedure, high in vitro stability and no accumulation in vital organs, 99m-Tc-PR81 may be a promising candidate for radioimmunoscintigraphy studies of breast cancer.

The mouse hepatitis coronavirus (MHV) infects murine cells by binding of its spike (S) protein to murine CEACAM1a. The N-terminal part of this cellular receptor (soR) is sufficient for S binding and for subsequent induction of the conformational changes required for virus-cell membrane fusion. Here we analyzed whether these characteristics can be used to redirect MHV to human cancer cells. To this end, the soR domain was coupled to single-chain monoclonal antibody 425, which is directed against the human epidermal growth factor receptor (EGFR), resulting in a bispecific adapter protein (soR-425). The soR and soR-425 proteins, both produced with the vaccinia virus system, were able to neutralize MHV infection of murine LR7 cells. However, only soR-425 was able to target MHV to human EGFR-expressing cancer cells. Interestingly, the targeted infections induced syncytium formation. Furthermore, the soR-425-mediated infections were blocked by heptad repeat-mimicking peptides, indicating ...

To explore the potential of using non-human coronaviruses for cancer therapy, we first established their ability to kill human tumor cells. We found that the feline infectious peritonitis virus (FIPV) and a felinized murine hepatitis virus (fMHV), both normally incapable of infecting human cells, could rapidly and effectively kill human cancer cells artificially expressing the feline coronavirus receptor aminopeptidase N. Also 3-D multilayer tumor spheroids established from such cells were effectively eradicated. Next, we investigated whether FIPV and fMHV could be targeted to human cancer cells by constructing a bispecific single-chain antibody directed on the one hand against the feline coronavirus spike protein-responsible for receptor binding and subsequent cell entry through virus-cell membrane fusion-and on the other hand against the human epidermal growth factor receptor (EGFR). The targeting antibody mediated specific infection of EGFR-expressing human cancer cells by both coronaviruses. Furthermore, in the presence of the targeting antibody, infected cancer cells formed syncytia typical of productive coronavirus infection. By their potent cytotoxicity, the selective targeting of non-human coronaviruses to human cancer cells provides a rationale for further investigations into the use of these viruses as anticancer agents.

Campylobacter jejuni is a leading cause of acute bacterial enteritis in humans. Poultry serves as a major reservoir of C. jejuni and is thought to act as a principal vehicle of transmission to humans. Epidermal growth factor (EGF) is a small amino acid peptide that exerts a broad range of activities on the intestinal epithelium. The aims of this study were to determine the effect of EGF on C. jejuni intestinal colonization in newly hatched chicks and to characterize its effects on C. jejuni-induced intestinal epithelial barrier disruption. White Leghorn chicks were treated with EGF daily, starting 1 day prior to C. jejuni infection, and were compared to control and C. jejuni-infected, EGF-treated chicks. Infected chicks shed C. jejuni in their feces throughout the study period. C. jejuni colonized the small intestine and cecum, disseminated to extraintestinal organs, and caused jejunal villus atrophy. EGF reduced jejunal colonization and dissemination of C. jejuni to the liver and spleen. In EGF-treated C. jejuni-infected chicks, villus height was not significantly different from that in untreated C. jejuni-infected chicks or controls. In vitro, C. jejuni attached to and invaded intestinal epithelial cells, disrupted tight junctional claudin-4, and increased transepithelial permeability. C. jejuni also promoted the translocation of noninvasive Escherichia coli C25. These C. jejuni-induced epithelial abnormalities were abolished by pretreatment with EGF, and the effect was dependent upon activation of the EGF receptor. These findings highlight EGF's ability to alter colonization of C. jejuni in the intestinal tract and to protect against pathogen-induced barrier defects. Campylobacter was first recognized as a human pathogen in the early 1970s and has since emerged as a leading cause of food-borne bacterial enteritis in the developed world (12, 22). Campylobacter-associated human enteritis ("campylobacteriosis") is characterized by fever, abdominal pain, intestinal inflammation, and diarrhea. While most cases of campylobacteriosis are self-limiting, infection is implicated as a risk factor for postinfectious complications, such as Guillain-Barré syndrome and bacteremia (38, 50). Moreover, Campylobacter is thought to be one of the most common causative microorganisms of postinfectious irritable bowel syndrome (44). Campylobacter jejuni infection is strongly associated with the handling and consumption of poultry meat. Several studies have shown that poultry serotypes of C. jejuni are associated with human infection (1, 26). Furthermore, the incidence of human campylobacteriosis has been shown to coincide with C. jejuni contamination of poultry carcasses (18, 57). Poultry is generally colonized by C. jejuni at approximately 2 weeks of age (13, 24). Despite extensive intestinal colonization, chickens usually do not show overt signs of illness. Nonetheless, weight reduction (30, 52), hepatitis (2, 37), diarrhea, and mortality (8, 52, 54) have been reported for infected poultry. Moreover, Campylobacter can disseminate to various internal organs, such

Fulvestrant is a new oestrogen receptor (ER) antagonist that is licensed for the treatment of postmenopausal women with advanced breast cancer progressing following antioestrogen treatment and may also be effective in those progressing after non-steroidal aromatase inhibitors. The use of fulvestrant in a Compassionate Use Programme (CUP) in a 'real-life' setting has permitted its activity and tolerability profile in patients with different disease characteristics to be observed. Here, we present five case reports of fulvestrant use in postmenopausal women with advanced breast cancer progressing after prior endocrine therapy. Clinical experience from the CUP supports the published clinical trial data and suggests that fulvestrant is a valuable new treatment for postmenopausal women with advanced breast cancer, including those with visceral metastases and human epidermal growth factor receptor 2-positive disease.

Current treatment options for ovarian cancer, which is one of the most widespread gynecological malignancies, are limited, mainly because patients with advanced-stage disease often develop resistance to chemotherapeutics. In breast cancer cells, several studies suggest that overexpression of the human epidermal growth factor receptor-2 (HER-2) leads to increased resistance against certain, but not all cytotoxic drugs. In ovarian carcinoma, conflicting data on the correlation of HER-2 expression and tumor cell sensitivity exist. In this paper, we explore the role of HER-2 expression and signaling levels pertaining to paclitaxel (Taxol) chemoresistance by applying three different and independent strategies in SKOV-3 ovarian carcinoma cells. Firstly, we show that treatment with the HER-2 inhibitory antibody trastuzumab (Herceptin), which is well established in tumor therapy, results in markedly increased, rather than decreased, cellular paclitaxel resistance. Next, we present two newly developed low molecular weight inhibitors of HER-2 tyrosine kinase activity, D-69491 and D-70166. With both drugs, the decrease in cellular paclitaxel sensitivity upon HER-2 inhibition is confirmed. Finally, for more detailed analysis we stably downregulate HER-2 expression by ribozyme-targeting. Using clonal ribozyme-transfected SKOV-3 cells with different residual HER-2 levels, we establish a 'HER-2 gene dose effect' of paclitaxel cytotoxicity. We show that this effect is due to differential induction of apoptosis and differential cell cycle inhibition by paclitaxel. Finally, paclitaxel-or HER-2-mediated alterations in the phosphorylation of MAP kinases p42/44, Stress-activated protein kinase/Jun-terminal kinase (SAPK/JNK), and p38, and effects on the activation of caspase-3, caspase-7, and bcl-2 are discussed. We conclude that paclitaxel cytotoxicity in SKOV-3 cells is 'HER-2 dose-dependent' and identify cell proliferation as one underlying cellular event of this effect.

Purpose To correlate both primary lesion 18 F-fluorodeoxyglucose (FDG) maximum standardized uptake value (SUVmax) and diffusion-weighted imaging (DWI) apparent diffusion coefficient (ADC) with clinicopathological prognostic factors and compare the prognostic value of these indexes in breast cancer. Methods The study population consisted of 44 patients with 44 breast cancers visible on both preoperative FDG PET/CT and DWI images. The breast cancers included 9 ductal carcinoma in situ (DCIS) and 35 invasive ductal carcinomas (IDC). The relationships between both SUVmax and ADC and clinicopathological prognostic factors were evaluated by univariate and multivariate regression analysis and the degree of correlation was determined by Spearman's rank test. The patients were divided into a better prognosis group (n=24) and a worse prognosis group (n=20) based upon invasiveness (DCIS or IDC) and upon their prognostic group (good, moderate or poor) determined from the modified Nottingham prognostic index. Their prognostic values were examined by receiver operating characteristic analysis. Results Both SUVmax and ADC were significantly associated (p<0.05) with histological grade (independently), nodal status and vascular invasion. Significant associations were also noted between SUVmax and tumour size (independently), oestrogen receptor status and human epidermal growth factor receptor-2 status, and between ADC and invasiveness. SUVmax and ADC were negatively correlated (ρ=-0.486, p=0.001) and positively and negatively associated with increasing of histological grade, respectively. The threshold values for predicting a worse prognosis were ≥4.2 for SUVmax (with a sensitivity, specificity and accuracy of 80%, 75% and 77%, respectively) and ≤0.98 for ADC (with a sensitivity, specificity and accuracy of 90%, 67% and 77%, respectively). Conclusion SUVmax and ADC correlated with several of pathological prognostic factors and both indexes may have the same potential for predicting the prognosis of breast cancer.

Introduction-The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets. Discussion-In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with 64 Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (Z HER2:477 ) and dimeric [(Z HER2:477 ) 2 ] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA). The resulting DOTA-Affibody conjugates were radiolabeled with 64 Cu and evaluated in nude mice bearing subcutaneous SKOV3 tumors. Biodistribution experiments showed that tumor uptake values of 64 Cu-DOTA-Z HER2:477 and 64 Cu-DOTA-(Z HER2:477 ) 2 were 6.12±1.44% and 1.46±0.50% ID/g, respectively, in nude mice (n=3 each) at 4 h postinjection. Moreover, 64 Culabeled monomer exhibited significantly higher tumor/blood ratio than that of radiolabeled dimeric counterpart at all time points examined in this study. MicroPET imaging of 64 Cu-DOTA-Z HER2:477 in SKOV3 tumor mice clearly showed good and specific tumor localization. This study demonstrates that 64 Cu-labeled Z HER2:477 is a promising targeted molecular probe for imaging HER2 receptor expression in living mice. Further work is needed to improve the excretion properties, hence dosimetry and imaging efficacy, of the radiometal-based probe.

Purpose-Two-helix scaffold proteins (~ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an 18 F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors. Methods-An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I 2 oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-18 F-fluorobenzyl aldehyde ( 18 F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, 18 F-FBO-MUT-DS, was performed at 37°C. 18 F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 μCi/μg, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted. Results-Cell uptake assays showed high and specific cell uptake (~12% applied activity at 1 h) by incubation of 18 F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K D ) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of 18 F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 μg of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p<0.05). displays excellent HER2 targeting ability and tumor PET imaging quality. The two-helix scaffold proteins are suitable for development of 18 F-based PET probes.

Introduction Although breast carcinoma (BC) is the most common malignancy affecting Jordanian females and the affected population in Jordan is younger than that in the West, no information is available on its biological characteristics. Our aims in this study are to evaluate the expression of estrogen receptor (ER) and progesterone receptor (PR) and Her-2/neu overexpression in BC in Jordan, and to compare the expression of these with other prognostic parameters for BC such as histological type, histological grade, tumor size, patients' age, and number of lymph node metastases.

Computer-aided prognosis (CAP) is a new and exciting complement to the field of computer-aided diagnosis (CAD) and involves developing and applying computerized image analysis and multi-modal data fusion algorithms to digitized patient data (e.g. imaging, tissue, genomic) for helping physicians predict disease outcome and patient survival. While a number of data channels, ranging from the macro (e.g. MRI) to the nano-scales (proteins, genes) are now being routinely acquired for disease characterization, one of the challenges in predicting patient outcome and treatment response has been in our inability to quantitatively fuse these disparate, heterogeneous data sources. At the Laboratory for Computational Imaging and Bioinformatics (LCIB) 1 at Rutgers University, our team has been developing computerized algorithms for high dimensional data and image analysis for predicting disease outcome from multiple modalities including MRI, digital pathology, and protein expression. Additionally, we have been developing novel data fusion algorithms based on non-linear dimensionality reduction methods (such as Graph Embedding) to quantitatively integrate information from multiple data sources and modalities with the overarching goal of optimizing meta-classifiers for making prognostic predictions. In this paper, we briefly describe 4 representative and ongoing CAP projects at LCIB. These projects include (1) an Image-based Risk Score (IbRiS) algorithm for predicting outcome of Estrogen receptor positive breast cancer patients based on quantitative image analysis of digitized breast cancer biopsy specimens alone, (2) segmenting and determining extent of lymphocytic infiltration (identified as a possible prognostic marker for outcome in human epidermal growth factor amplified breast cancers) from digitized histopathology, (3) distinguishing patients with different Gleason grades of prostate cancer (grade being known to be correlated to outcome) from digitized needle biopsy specimens, and (4) integrating protein expression measurements obtained from mass spectrometry with quantitative image features derived from digitized histopathology for distinguishing between prostate cancer patients at low and high risk of disease recurrence following radical prostatectomy.

Purpose Pertuzumab is a humanized monoclonal antibody inhibiting human epidermal growth factor receptor 2 (HER2) dimerization. The aim of this phase II trial was to assess the antitumor activity and safety profile of pertuzumab monotherapy in patients with HER2-negative metastatic breast cancer. The utility of biomarkers detected in paraffin-embedded tissue as predictors of response was also explored. Patients and Methods This was an international, multicenter, open-label, randomized phase II study. Patients (n = 79) with centrally confirmed HER2-negative metastatic breast cancer were randomly assigned to receive pertuzumab once every 3 weeks with a loading dose of 840 mg followed thereafter by either 420 mg (arm A) or 1,050 mg (arm B). Patients were stratified by country and prior taxane therapy. Results Of 79 patients who were randomly assigned, 78 were included in the intent-to-treat population. In arm A (n = 41), two patients had partial responses, and 18 patients (44%) experien...

Introduction Substantial preclinical evidence has indicated that inhibition of integrin linked-kinase (ILK) correlates with cytotoxic/ cytostatic cellular effects, delayed tumor growth in animal models of cancer, and inhibition of angiogenesis. Widely anticipated to represent a very promising therapeutic target in several cancer indications, it is increasingly evident that optimal therapeutic benefits obtained using ILK targeting strategies will only be achieved in combination settings. The purpose of this study was to investigate the therapeutic potential of the ILK small molecule inhibitor, QLT0267 (267), alone or in combination with chemotherapies commonly used to treat breast cancer patients. used in combination with Dt. In vivo studies focusing on low Her2-expressing breast cancer cells (LCC6) implanted orthotopically demonstrated that treatment with 267/Dt engendered improved therapeutic effects when compared with mice treated with either agent alone. Conclusions The findings indicate that the 267/Dt drug combination confers increased (synergistic) therapeutic efficacy towards human breast cancer cells that express low levels of Her2.

Protein-protein interactions (PPI) play a crucial role in many biological processes, and modulation of PPI using small molecules to target hot spots has therapeutic value. As a model system we will use PPI of human epidermal growth factor receptors (EGFRs). Among the four EGFRs, EGFR-HER2 and HER2-HER3 are well known in cancer. We have designed a small molecule that is targeted to modulate HER2-mediated signaling. Our approach is novel because the small molecule designed disrupts dimerization not only of EGFR-HER2 but also of HER2-HER3. In the present study we have shown, using surface plasmon resonance (SPR) analysis, that a peptidomimetic, compound 5, binds specifically to HER2 protein extracellular domain and disrupts the dimerization of EGFRs. To evaluate the effect of compound 5 on HER2 signaling in vitro, Western blot and PathHunter assays were used. Results indicated that compound 5 inhibits the phosphorylation of HER2 kinase domain and inhibits the heterodimerization in a dose-dependent manner. Molecular modeling methods were used to model the PPI of HER2-HER3 heterodimer.

Background Addition of taxanes to preoperative chemotherapy in breast cancer increases the proportion of patients who have a pathological complete response (pCR). However, a substantial proportion of patients do not respond, and the prognosis is particularly poor for patients with oestrogen-receptor (ER)/progesterone-receptor (PR)/human epidermal growth factor receptor 2 (HER2; ERBB2)-negative (triple-negative) disease who do not achieve a pCR. Reliable identifi cation of such patients is the fi rst step in determining who might benefi t from alternative treatment regimens in clinical trials. We previously identifi ed genes involved in mitosis or ceramide metabolism that infl uenced sensitivity to paclitaxel, with an RNA interference (RNAi) screen in three cancer cell lines, including a triple-negative breast-cancer cell line. Here, we assess these genes as a predictor of pCR to paclitaxel combination chemotherapy in triple-negative breast cancer. Methods We derived a paclitaxel response metagene based on mitotic and ceramide genes identifi ed by functional genomics studies. We used area under the curve (AUC) analysis and multivariate logistic regression to retrospectively assess the metagene in six cohorts of patients with triple-negative breast cancer treated with neoadjuvant chemotherapy; two cohorts treated with paclitaxel (n=27, 30) and four treated without paclitaxel (n=88, 28, 48, 39). Findings The metagene was associated with pCR in paclitaxel-treated cohorts (AUC 0•79 [95% CI 0•53-0•93], 0•72 [0•48-0•90]) but not in non-paclitaxel treated cohorts (0•53 [0•31-0•77], 0•59 [0•22-0•82], 0•53 [0•36-0•71], 0•64 [0•43-0•81]). In multivariate logistic regression, the metagene was associated with pCR (OR 19•92, 2•62-151•57; p=0•0039) with paclitaxel-containing chemotherapy. Interpretation The paclitaxel response metagene shows promise as a paclitaxel-specifi c predictor of pCR in patients with triple-negative breast cancer. The metagene is suitable for development into a reverse transcription-PCR assay, for which clinically relevant thresholds could be established in randomised clinical trials. These results highlight the potential for functional genomics to accelerate development of drug-specifi c predictive biomarkers without the need for training clinical trial cohorts.

Background: The cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2-positive (HER2+) and 11 HER2negative (HER2-) paraffin-embedded breast tumors. Methods: Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance t-statistics tested for differences in expression levels between HER2 + and HER2 -tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI).

Metaplastic breast carcinomas constitute a heterogeneous group of neoplasms, accounting for less than 1% of all invasive mammary carcinomas. Approximately 70-80% of metaplastic breast carcinomas overexpress the epidermal growth factor receptor (EGFR). Human epidermal growth factor receptor (HER)2 and EGFR have attracted much attention in the medical literature over the past few years owing to the fact that humanized monoclonal antibodies against HER2 and therapies directed against the extracellular ligand-binding domain or the intracellular tyrosine kinase domain of EGFR have proven successful in treating certain types of human cancer. We investigated whether HER2 and EGFR overexpression was present and evaluated gene amplification in a series of metaplastic breast carcinomas. Twenty-five metaplastic breast carcinomas were immunohistochemically analyzed using a monoclonal antibody (31G7) for EGFR and two antibodies for HER2 (Herceptest and CB11) and scored using the Herceptest scori...

Purpose To evaluate the maximum-tolerated dose (MTD), safety profile, and immunogenicity of two chimeric, B-cell epitopes derived from the human epidermal growth factor receptor (HER2) extracellular domain in a combination vaccine with a promiscuous T-cell epitope (ie, MVF) and nor-muramyl-dipeptide as adjuvant emulsified in SEPPIC ISA 720. Patients and Methods Eligible patients with metastatic and/or recurrent solid tumors received three inoculations on days 1, 22, and 43 at doses of total peptide that ranged from 0.5 to 3.0 mg. Immunogenicity was evaluated by enzyme-linked immunosorbent assay, flow cytometry, and HER2 signaling assays. Results Twenty-four patients received three inoculations at the intended dose levels, which elicited antibodies able to recognize native HER2 receptor and inhibited both the proliferation of HER2-expressing cell lines and phosphorylation of the HER2 protein. The MTD was determined to be the highest dose level of 3.0 mg of the combination vaccine. Th...

Purpose Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Noninvasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule Z HER2:342 , which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2expressing xenografts using radioiodinated Z HER2:342 . The goal of this study was to develop a method for 99m Tc labelling of Z HER2:342 using the MAG3 chelator, which was incorporated into Z HER2:342 using peptide synthesis, and evaluate the targeting properties of the labelled conjugate. Methods MAG3-Z HER2:342 was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for 99m Tc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of 99m Tc-MAG3-Z HER2:342 and 125 I-para-iodobenzoate -Z HER2:342 was compared 6 h p.i. Results Synthetic MAG3-Z HER2:342 possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with 99m Tc with an efficiency of about 75-80%. Labelled 99m Tc-MAG3-Z HER2:342 retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. 99m Tc-MAG3-Z HER2:342 showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression. Conclusion Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled sitespecific 99m Tc labelling of the Z HER2:342 Affibody molecule with preserved targeting capacity.

Although human epidermal growth factor receptor 2 (HER2) overexpression is implicated in tumor progression for a variety of cancer types, how it dysregulates signaling networks governing cell behavioral functions is poorly understood. To address this problem, we use quantitative mass spectrometry to analyze dynamic effects of HER2 overexpression on phosphotyrosine signaling in human mammary epithelial cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). Data generated from this analysis reveal that EGF stimulation of HER2-overexpressing cells activates multiple signaling pathways to stimulate migration, whereas HRG stimulation of these cells results in amplification of a specific subset of the migration signaling network. Self-organizing map analysis of the phosphoproteomic data set permitted elucidation of network modules differentially regulated in HER2-overexpressing cells in comparison with parental cells for EGF and HRG treatment. Partial least-squares regression analysis of the same data set identified quantitative combinations of signals within the networks that strongly correlate with cell proliferation and migration measured under the same battery of conditions. Combining these modeling approaches enabled association of epidermal growth factor receptor family dimerization to activation of specific phosphorylation sites, which appear to most critically regulate proliferation and/or migration.

Paget's disease is a skin cancer characterized by characteristic (Paget) cells scattered in the epidermis. Although its prognosis is generally favorable with surgical resection, the clinical outcome turns unfavorable in cases with recurrence and metastasis. Therefore, establishment of effective therapeutic regimens is required for advanced Paget's disease. The human epidermal growth factor receptor 2 (HER2) protein, a transmembrane growth factor receptor, is frequently overexpressed in malignancies, causing activation of the phosphatidylinositol 3 kinase (PI3K) and extracellular signal-regulated kinase (ERK) signal pathways. Recently, HER2-targeting molecular therapy using trastuzumab (Herceptin; Genentech, Inc, South San Francisco, Calif) was revealed to be effective in advanced breast cancers overexpressing HER2 protein. Here, we analyzed the correlation between activation of the HER2 signal pathways and clinicopathologic parameters of 36 extramammary Paget's disease samples from 34 Japanese patients, using immunohistochemical analyses for HER2, phosphorylated HER2, phosphorylated AKT, and phosphorylated ERK proteins. We found overexpression of the HER2 protein in 19.4% (7) of the lesions, 3 of which showed HER2 amplification by chromogenic in situ hybridization. Phosphorylated HER2 protein was detected in 12 lesions (33.3%), including 2 of the 7 HER2-overexpressing lesions.

Using 3-mercaptopropyltrimethoxysilane (MPS)-coated (PbMg1∕3Nb2∕3O3)0.63–(PbTiO3)0.37 (PMN-PT)/tin and lead zirconate titanate/glass piezoelectric microcantilever sensors (PEMSs) with single-chain variable fragment (scFv) immobilized on the MPS surface, we have demonstrated real-time, label-free detection of human epidermal growth factor receptor 2 (Her2) in a background of 1mg∕ml bovine serum albumin. Coupled with a scFv with a KD of 3.4×10−8M, the MPS-insulated PMN-PT/tin PEMS 560μm long and 720μm wide exhibited a Her2 concentration sensitivity of 5ng∕ml in a background of 1mg∕ml BSA.

Introduction: Identification of gene expression-based breast cancer subtypes is considered a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene-expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and only a limited understanding exists of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and to deliver specific epigenotypes associated with particular breast cancer subtypes. Methods: By using a microarray approach, we analyzed DNA methylation in regulatory regions of 806 cancerrelated genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biologic validation by pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A, and 48 luminal B paired breast cancer/ adjacent tissues. With the all-subset selection method, we identified the most subtype-predictive methylation profiles in multivariable logistic regression analysis. Results: The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identified novel subtype-specific epigenotypes that clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. Conclusions: Our results provide evidence that well-defined DNA methylation profiles enable breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.

Pertuzumab represents a new class of targeted anticancer agents, human epidermal growth factor receptor (HER) dimerization inhibitors. The aim of this single-arm phase II clinical study was to assess the efficacy and safety of single-agent pertuzumab in castration-resistant prostate cancer (CRPC) patients who had experienced progression after prior chemotherapy. Patients and Methods Patients received pertuzumab every 3 weeks. All castration-resistant patients had experienced progression after at least one taxane-based regimen. Patients received a loading dose of 840 mg pertuzumab (cycle 1) followed by 420 mg for subsequent cycles. The primary end point was overall response and safety. A separate retrospective analysis of actual survival time versus predicted survival time for a patient population with comparable prognostic features was performed. Results Patients were enrolled (N ϭ 42) and treated (n ϭ 41). No patients had complete or partial response (as defined by Response Evaluation Criteria in Solid Tumors Group or Ն 50% decline in prostate-specific antigen). Of 30 efficacy-assessable patients, five had stable disease (SD) for at least 23 weeks; one of five had SD for 36 weeks. Pertuzumab was well tolerated; diarrhea was the most common adverse effect (61.0%, grades 1 to 3). Retrospective analysis of survival using a validated nomogram suggested that survival was prolonged with pertuzumab treatment, compared with historic controls with similar baseline prognostic features. Conclusion Pertuzumab was well tolerated and resulted in no objective responses, but several patients had SD more than 23 weeks from a heavily pretreated population. Retrospective analysis suggested prolonged median survival time with pertuzumab compared with historical controls. Thus, inhibition of HER dimerization may have clinical utility in CRPC patients.

Background. p53 overexpression has been identified as a poor prognostic marker in breast cancer. We investigate the value of p53 status within the context of stage and intrinsic subtype classification (subtype), in a group of African-American (AA) women of lower socioeconomic status (SES) with primary breast cancer. Methods. Participants were 331 consecutive AA women treated at an urban hospital (median follow-up 41 months) with known subtype [luminal A = estrogen receptor (ER)? and/or progesterone receptor (PR)?, human epidermal growth factor receptor 2 (HER2)-; luminal B = ER? and/ or PR?, HER2?; HER2? = ER-, PR-, HER2?; basal = ER-, PR-, HER2-, cytokeratin (CK)5/6?, and/or HER1?; and unclassified = negative for all five markers] and p53 (Pab1801 antibody) immunohistochemical status. Proportional hazards regression models were used to select and evaluate factors prognostic for all-cause mortality. Results. p53? status was associated with grade 3 tumors, ER/PR-status, and basal subtype. On univariate analysis, factors related to survival were stage, grade [(3/1) hazard ratio (HR) = 2.64; 95% confidence interval (CI), 1.15-6.07], subtype [(ex. basal/luminal A) HR = 2.15; 95% CI, 1.34-3.45], and p53 status [(?/-) HR = 1.77; 95% CI, 1.15-2.72]. Multivariable modeling indicated that p53? status remained a negative prognostic factor (HR = 1.63; 95% CI, 1.01-2.59) after adjustment for effects of age, stage, grade, and subtype; 5-year adjusted survival was significantly greater for p53-(66.7%) than p53? cases (54.7%). Conclusion. p53 status is an independent predictor of survival after consideration of other strong prognostic factors such as stage, tumor grade, and subtype, and thus may be useful in identifying AA women at high risk of breast cancer mortality.

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical "handle" by which immunoconjugates and multimers can be engineered.

Background: Biomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness. Methodology/Principal Findings: By creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies. Conclusions/Significance: The microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy.