High expression of the phosphoinositide 3-kinase p11γ isoform can predict poor prognosis of non-small cell lung cancer.

The protein p110γ is an isoform of the catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI3Ks are involved in the regulation of cell survival, growth, proliferation, and migration and have been implicated in the oncogenesis of various cancers. In this study, p110γ expression in non-small cell lung cancer (NSCLC) and its association with clinicopathological factors and patient survival were evaluated. A total of 230 NSCLC tumors were immunohistochemically stained for p110γ. Of these, 174 (75.7%) and 56 (24.3%) were placed in the low and high expression groups, respectively. The positive rate of p110γ was significantly higher in adenocarcinoma than in squamous cell carcinoma (p⟨0.001). Advanced stage NSCLCs showed higher p110γ expression than those at an early stage (p=0.002). Irrespective of the histological tumor type, the patients with high p110γ expression had significantly worse overall survival than those with low p110γ expression (p=0.004). p110γ expression was an independent poor prognostic factor in the multivariate analysis. Our results suggest that p110γ may be involved in the development and progression of NSCLC, and that p110γ has promising potential as a prognostic factor or novel therapeutic target for NSCLC.

A Novel T Cell-Engaging Bispecific Antibody for Treating Mesothelin-Positive Solid Tumors.

As mesothelin is overexpressed in various types of cancer, it is an attractive target for therapeutic antibodies. T-cell bispecific antibodies bind to target cells and engage T cells via binding to CD3, resulting in target cell killing by T-cell activation. However, the affinity of the CD3-binding arm may influence CD3-mediated plasma clearance or antibody trapping in T-cell-containing tissues. This may then affect the biodistribution of bispecific antibodies. In this study, we used scFab and knob-into-hole technologies to construct novel IgG-based 1 + 1 MG1122-A and 2 + 1 MG1122-B bispecific antibodies against mesothelin and CD3ε. MG1122-B was designed to be bivalent to mesothelin and monovalent to CD3ε, using a 2 + 1 head-to-tail format. Activities of the two antibodies were evaluated in mesothelin-positive tumor cells in vitro and xenograft models in vivo. Although both antibodies exhibited target cell killing efficacy and produced regression of xenograft tumors with CD8+ T-cell infiltration, the antitumor efficacy of MG1122-B was significantly higher. MG1122-B may improve tumor targeting because of its bivalency for tumor antigen. It may also reduce systemic toxicity by limiting the activation of circulating T cells. Thus, MG1122-B may be useful for treating mesothelin-positive solid tumors.

Utility of Human Papillomavirus Testing for Cervical Cancer Screening in Korea.

(1) Background: Cervical cancer is one of the most common cancers in Korean women. This study was performed to discover the utility of HPV (Human Papillomavirus) testing in screening of cervical lesions and to provide the prevalence of HPV and the genotype distribution in a single center of Korea. (2) Methods: A total of 15,141 women who underwent both HPV testing and cervical cytology were enrolled in this retrospective medical record review study. (3) Results: HPV testing showed higher sensitivity than cytology for the detection of histological high-grade squamous lesions. Furthermore, the sensitivity and specificity of HPV testing varied depending on the method used. The BD Onclarity™ HPV assay had higher sensitivity (90%) than the MyHPV CHIP™ kit (all types of HPV: 82%; high-risk HPV: 76%) for high-grade squamous lesions. A combination of MyHPV CHIP™ and cytology detected 90.9% (30/33) of histological high-grade squamous lesions. A combination of BD Onclarity™ HPV assay and cytology detected 96.55% (84/87) of histological high-grade squamous lesions. In addition, HPV prevalence and genotype distribution were different depending on the HPV testing method used. (4) Conclusion: HPV testing showed higher sensitivity than cytology, but the sensitivity and specificity of HPV testing had variation depending on the method used.

Differential expression of HSP90 isoforms and their correlations with clinicopathologic factors in patients with colorectal cancer.

Heat shock protein 90 (HSP90), a molecular chaperone, plays critical roles in cellular protection against various stressful stimuli and in the regulation of cellular growth and apoptosis. HSP90 has four human isoforms; HSP90α, HSP90ß, glucose related protein 94 (GRP94), and tumor necrosis factor (TNF) receptor-associated protein 1 (TRAP1). We evaluated the differential expression of these HSP90 isoforms in colorectal cancer (CRC) and correlated their expression levels with clinicopathological factors and patient survival rates. We performed immunohistochemical staining for HSP90α, HSP90ß, GRP94, and TRAP1 in 129 CRC tumor samples and found that HSP90α expression was significantly associated with advanced pT stage (P = 0.011) and shorter recurrence-free survival (RFS) (P = 0.010), whereas GRP94 expression was correlated with low grade (P = 0.029) and better RFS (P < 0.001). HSP90ß and TRAP1 had no prognostic impact, although HSP90ß expression was positively correlated with tumor size (P = 0.008). Based on our results, HSP90α and GRP94 are potential prognostic biomarkers of CRC. In addition, the differences in expression and functional activities among four HSP90 isoforms imply that isoform selectivity should be seriously considered when HSP90 inhibitors are studied or adopted for the treatment of CRC.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes.

Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Associations of organochlorine pesticides and polychlorinated biphenyls with total, cardiovascular, and cancer mortality in elders with differing fat mass.

BACKGROUND: We investigated if certain persistent organic pollutants (POPs), namely polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides, predicted total, cardiovascular disease (CVD), and cancer mortality among the elderly, with the hypothesis that associations differ by the amount of fat mass. METHODS: We studied serum concentrations of 11 PCBs in 633 elders (age≥70 years) and of 5 OC pesticides in 675 elders within the National Health and Nutrition Examination Survey (NHANES) 1999-2004. Mean follow-up was 4.1-years. RESULTS: Neither PCBs nor OC pesticides were associated with total mortality when fat mass was not considered in analyses. However, associations of PCBs and OC pesticides with total mortality depended on fat mass (Pinteraction<0.01 and 0.06, respectively). PCBs associated inversely with total mortality for high fat mass, but not for lower fat mass. On the contrary, OC pesticides associated positively with total mortality for low fat mass and this association weakened at higher fat mass. The interaction was also observed with CVD mortality. In elders with low fat mass, higher PCBs associated with 2-3 fold higher risk of CVD mortality, while this association was absent in elders with more fat mass (Pinteraction=0.03). The positive association between OC pesticides and CVD mortality was also observed only among elderly with low fat mass (Pinteraction=0.03). CONCLUSIONS: The possibility of interaction between POPs and the amount of fat mass on risk of mortality from chronic diseases is clinically important in modern societies with an obesity epidemic and requires confirmation in other studies with larger sample size.

Persistent organic pollutants and promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase gene.

Promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) DNA repair gene is important during carcinogenesis. We explored whether organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs), were associated with hypermethylation of the MGMT gene promoter in peripheral leukocytes among 368 Koreans without cancer. Hypermethylation decreased as OCPs increased (Ptrend = 0.02), while PCB concentrations showed an inverted U-shaped association (Pquadratic < 0.01). The prevalence of MGMT promoter hypermethylation was highest within the 2nd quintile of the PCB summary score (28.4%), while it was only 2.7% in the upper 10% score. Chronic exposure to these chemicals may affect methylation of the MGMT promoter, with possibly non-monotonic dose response relationships.

Associations between organochlorine pesticides and cognition in U.S. elders: National Health and Nutrition Examination Survey 1999-2002.

There is limited evidence about whether background exposure to organochlorine pesticides is related to impairment of cognitive function in general populations. This study was performed to investigate cross-sectional associations between serum concentrations of organochlorine pesticides and cognitive function, a predictor of dementia, among U.S. elders without overt dementia. Study subjects were 644 elders aged 60-85, participating in the National Health and Nutrition Examination Survey 1999-2002. We selected 6 organochlorine pesticides (p,p'-dichlorodiphenyltrichloroethane (DDT), p,p'-dichlorodiphenyldichloroethylene (DDE), trans-nonachlor, oxychlordane, heptachlor epoxide, and ß-hexachlorocyclohexane) which were commonly detected in current general population. Cognitive function was assessed with the Digit-Symbol Substitution Test. All 6 compounds showed statistically significant or marginally significant inverse associations with cognitive score after adjusting for covariates including education levels. The strongest association was observed with p,p'-DDT. With the outcome of low cognitive score defined as <25th percentile, elders in the highest quartile of p,p'-DDT, p,p'-DDE, and ß-hexachlorocyclohexane had 2 to 3 times higher risks than those in the lowest quartile. In particular, when their concentrations were further divided with the cutoff points of 90th and 95th percentiles, p,p'-DDT in the highest 5th percentile showed 6.5 (95% confidence interval: 2.6-16.3) times higher risk of low cognitive score. On the other hand, non-persistent pesticides like organophosphates or pyrethroid showed little association with this cognitive score. The potential role of background exposure to organochlorine pesticides in the development of dementia should be explored in future prospective studies and in-vitro/in-vivo experimental studies.

Paradoxical Associations of Insulin Resistance With Total and Cardiovascular Mortality in Humans.

BACKGROUND: Insulin resistance is linked to many human chronic diseases. Paradoxically, however, impaired insulin signaling contributes to longevity in various organisms and is suggested as an adaptive mechanism against external stressors, including obesity. We formulated a novel hypothesis that insulin resistance can be beneficial in obese humans, insofar as it does not cause glucose dysmetabolism. METHODS: N = 5,241 participants aged ≥40 with normal fasting glucose were combined across the 1988-1994 and 1999-2004 National Health and Nutrition Examination Survey datasets. Mean follow-up period was 6.6 years. Insulin resistance was measured with homeostasis model assessment (HOMA-IR). Outcomes were all causes (n = 724), cardiovascular diseases (CVD, n = 316), and cancer mortality (n = 190). RESULTS: Supporting the hypothesis, obese persons with high HOMA-IR showed a decreased risk of total and CVD mortality compared to those with the lowest HOMA-IR. Adjusted hazard ratios were 1.0, 0.8, 0.4, and 0.4 (p(trend) = .02) for all death and 1.0, 0.6, 0.2, and 0.2 (p(trend) < .01) for CVD death. On the other hand, lean persons with high HOMA-IR showed about twice the total and CVD mortality compared to persons with the lowest HOMA-IR (p(trend) < .01, respectively). CONCLUSIONS: Insulin resistance in obese individuals may begin as an adaptive mechanism and can be beneficial if it is not associated with glucose dysmetabolism. In contrast, insulin resistance in lean individuals associated with higher risk of total and CVD mortality. Insulin resistance may be multifaceted and conventional approaches to regard insulin resistance itself as a pathological condition may be reconsidered in this light.

Enhancement of the tumor penetration of monoclonal antibody by fusion of a neuropilin-targeting peptide improves the antitumor efficacy.

The limited localization and penetration of monoclonal antibodies (mAb) into solid tumors restricts their antitumor efficacy. Here, we describe a solid tumor-targeting antibody with enhanced tumor penetration activity. We designed a 22-residue peptide (A22p), which was extracted from the C-terminal basic region of semaphorin 3A (Sema3A) but modified to have higher affinity with neuropilin receptors (NRP), and genetically fused it to the C-terminus of Fc of human immunoglobulin G1 via a 15-residue (G4S)3 linker, generating Fc-A22p, for the bivalent binding to NRPs. In contrast to Fc or the monovalent A22p peptide alone, Fc-A22p homed to tumor vessels and induced vascular permeability through VE-cadherin downregulation and penetrated tumor tissues by interacting with NRPs in mice bearing human tumor xenografts. We extended the Fc-A22p platform by generating mAb-A22p antibodies of two clinically approved solid tumor-targeting mAbs, the anti-EGF receptor mAb cetuximab (erbitux), and the anti-Her2 mAb trastuzumab (herceptin). The mAb-A22p antibodies retained the intrinsic antigen binding, natural Fc-like biophysical properties, and productivity in mammalian cell cultures, comparable with those of the parent mAbs. In mouse xenograft tumor models, the mAb-A22p antibodies more efficiently homed to tumor vessels and spread into the extravascular tumor parenchyma, which significantly enhanced antitumor efficacy compared with the parent mAbs. Our results suggest that mAb-A22p is a superior format for solid tumor-targeting antibodies due to its enhanced tumor tissue penetration and greater antitumor efficacy compared with conventional mAbs.

Associations of organochlorine pesticides and polychlorinated biphenyls in visceral vs. subcutaneous adipose tissue with type 2 diabetes and insulin resistance.

Background exposure to organochlorine (OC) pesticides and polychlorinated biphenyls (PCBs) has been linked to type 2 diabetes. As OC pesticides and PCBs mainly accumulate in adipose tissue and there are physiological and clinical differences between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), we explored if there were associations of OC pesticides and PCBs in VAT or SAT with type 2 diabetes and insulin resistance. Participants were 50 patients with or without type 2 diabetes who underwent surgery for either cancer or benign liver or gallbladder lesions. We analyzed 14 OC pesticides and 22 PCB congeners in both VAT and SAT. Insulin resistance was estimated using homeostasis model assessment (HOMA). Although concentrations of OC pesticides and PCBs were strongly correlated between VAT and SAT, absolute concentrations differed substantially between them. In particular, concentrations of all PCBs were consistently about 5-10 times higher in VAT than SAT, but these patterns were independent of diabetes status. Some OC pesticides or PCBs, such as dichlorodiphenyltrichloroethanes (DDTs), chlordanes, and PCBs with 5 or less chlorides showed significant associations with diabetes or insulin resistance. For example, when tertiles of concentration-based summary measures were used, adjusted ORs were 1.0, 2.3, and 9.0 (P trend=0.02) for DDTs in VAT and 1.0, 2.1, and 5.7 (P trend=0.08) for PCBs with 5 or less chlorides. This study generally confirmed previous findings using serum concentrations. It would be useful to study pharmacodynamics of POPs in VAT and SAT further.

Associations of serum ferritin and transferrin % saturation with all-cause, cancer, and cardiovascular disease mortality: Third National Health and Nutrition Examination Survey follow-up study.

OBJECTIVES: Even though experimental studies have suggested that iron can be involved in generating oxidative stress, epidemiologic studies on the association of markers of body iron stores with cardiovascular disease or cancer remain controversial. This study was performed to examine the association of serum ferritin and transferrin saturation (%TS) with all-cause, cancer, and cardiovascular mortality. METHODS: The study subjects were men aged 50 years or older and postmenopausal women of the Third National Health and Nutrition Examination Survey 1988-1994. Participants were followed-up for mortality through December 31, 2006. RESULTS: Serum ferritin was not associated with all-cause, cancer, or cardiovascular mortality for either men or postmenopausal women. However, all-cause, cancer, and cardiovascular mortality were inversely associated with %TS in men. Compared with men in the lowest quintile, adjusted hazard ratios for all-cause, cancer, and cardiovascular mortality were 0.85, 0.86, 0.76, and 0.74 (p for trend < 0.01), 0.82, 0.73, 0.75, and 0.63 (p for trend < 0.01), and 0.86, 0.81, 0.72, and 0.76 (p for trend < 0.01), respectively. For postmenopausal women, inverse associations were also observed for all-cause and cardiovascular mortality, but cancer mortality showed the significantly lower mortality only in the 2nd quintile of %TS compared with that of the 1st quintile. CONCLUSIONS: Unlike speculation on the role of iron from experimental studies, %TS was inversely associated with all-cause, cancer and cardiovascular mortality in men and postmenopausal women. On the other hand, serum ferritin was not associated with all-cause, cancer, or cardiovascular mortality.

Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice.

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.

In vivo and in vitro evaluation of Cy5.5 conjugated epidermal growth factor receptor binding peptide.

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or (125)I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5≫EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various (125)I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of (125)I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.

Successful treatment of an iatrogenic giant femoral artery pseudoaneurysm with percutaneous thrombin injection.

A femoral artery pseudoaneurysm (FAP) is one of the most troublesome complications following invasive procedures related to the femoral arterial access. Post-procedure FAP rarely occurs; however, its occurrence tends to increase with the more frequently antiplatelet agents, anticoagulants, and larger-sized catheter used for interventional procedures. Traditionally, surgical repair has been considered as the standard treatment modality for FAP; however, less invasive methods currently exist such as blind manual or ultrasound-guided compression repair (UGCR) as well as percutaneous thrombin injection, both of which have replaced the need for surgery. We report a case of a giant pseudoaneurysm in a femoral artery, which had developed as a complication of stenting in a patient with carotid artery stenosis and ischemic heart disease, and was subsequently successfully treated using percutaneous thrombin injection.

Characterization, biodistribution and small-animal SPECT of I-125-labeled c-Met binding peptide in mice bearing c-Met receptor tyrosine kinase-positive tumor xenografts.

c-Met is a receptor tyrosine kinase involved in tumor cell growth, invasion, metastases and angiogenesis. Overexpression of c-Met is frequently observed in several tumor types. Here, we report the in vitro cell-binding properties and biodistribution and SPECT/CT imaging in glioma (U87MG) xenograft-bearing mice of (125)I-labeled c-Met-binding peptides (cMBPs) including analogs conjugated to amino acid and aliphatic carbon linkers. In vitro assays showed that the peptide without any linker and those with GGG and 8-aminooctanoic acid linkers had low cellular internalization and that IC(50) values of peptides were 1.5 microM, 65 nM and 85.3 nM, respectively. Biodistribution studies showed the GGG-containing peptide had higher tumor uptake and a higher tumor-to-blood activity concentration ratio than other receptor-binding ligands. SPECT/CT studies with a dedicated small-animal imaging system were performed in U87MG-bearing athymic mice. Although U87MG tumor xenografts could be visualized by SPECT/micro-CT using the various (125)I labeled cMBPs, image contrast and overall quality were unremarkable.

A neutralizable epitope is induced on HGF upon its interaction with its receptor cMet.

A new conformational neutralizable epitope is created on heptocyte growth factor (HGF), when it interacts with its receptor, cMet. By immunizing rabbits with HGF-cMet complex, we successfully generated a monoclonal antibody (SFN68) that inhibits HGF-cMet interaction, and blocks the biological function mediated by HGF. To define the epitope, we screened out an epitope-mimicking peptide, KSLSRHDHIHHH, from a phage display of combinatorial peptide library. In molecular mimicry this peptide bound to cMet and inhibited HGF-cMet interaction. No humoral response was induced to this epitope-mimicking peptide when immunization was done with HGF alone.

Clonality analysis for normal and cancerous colon tissues with human androgen receptor gene polymerase chain reaction.

We assessed the feasibility of clonality analysis with human androgen receptor gene polymerase chain reaction in terms of the sensitivity and specificity for normal and cancerous colonic tissues taken from fourteen informative cases selected from 22 women with colonic adenocarcinoma. Ten crypts microdissected from each 10-microm-thick cryostat sections and whole tissues were used as samples. DNA was extracted from the samples and amplified with and without prior enzyme digestion. These products were analyzed by capillary electrophoresis for clonality. Of the whole-tissue DNA, none of the normal tissues and seven (50.0%) of the cancerous tissues showed monoclonality. Of the microdissected samples, monoclonality was found in 88.4% (107/121) of normal crypts and 95.9% (117/122) of cancerous crypts. Samples composed of crypts with short and long alleles were found in eight of the 14 normal colonic mucosae, but in none of the cancerous tissues. We concluded that the sensitivity of this method is limited for both whole-tissue DNA and microdissected-tissue DNA, because monoclonality from small samples does not always indicate monoclonality of the entire lesion. The high specificity of this method, however, allows polyclonal results in whole tissues to be confirmed by additional analysis of microdissected tissues.