Ivona Baricevic

High-sensitivity human papilloma virus genotyping reveals near universal positivity in anal squamous cell carcinoma: Different implications for vaccine prevention and prognosis

BACKGROUND Characterisation of human papilloma virus (HPV) infection in anal squamous cell carcinoma (ASCC) may have dual importance: first, aetiological; second, prognostic, informing outcome after chemo-radiotherapy (CRT). We undertook HPV genotyping, and allelic characterisations, to evaluate the aetiological role of HPV while simultaneously evaluating the impact of HPV genotyping on relapse-free (RFS) and overall survival (OS). METHOD Dual-primer HPV genotyping (subtypes 6, 11, 16, 18, 31, 33, 45, 52, 58) and DNA sequencing of HPV 16 positive tumours were analysed in 151 consecutively referred ASCCs, previously characterised by immunohistochemistry for p16 expression. In 110 patients treated with CRT, factors influencing RFS and OS were evaluated using univariate and multivariate models. RESULTS HPV positivity was observed in 95%. HPV 16 accounted for 89%; of these, 64% harboured the T350G E6 variant. HPV 16 positivity was significantly correlated with improved 5-year RFS (62% versus 40%; p = 0.027) and OS (59% versus 38%; p = 0.019). p16 expression was also significantly correlated with improved 5-year RFS (positive versus negative: 65% versus 16%; p < 0.0001) and OS (63% versus 13%; p < 0.0001). In multivariable models that included HPV 16 status, p16 status, sex, and age, p16 expression remained an independent prognostic factor for RFS (p < 0.0001) and OS (p = 0.002). CONCLUSION In ASCC, near-universal HPV detection rates were demonstrated, higher than generally reported in the literature, and supporting the development of multivalent HPV vaccinations for prevention. By contrast, p16 negatively, but not HPV 16 genotype, is an independent adverse prognosticator after chemo-radiotherapy in patients with ASCC.

A framework for the in vitro evaluation of cancer-relevant molecular characteristics and mitogenic potency of insulin analogues.

Summary Clinically prescribed insulin analogues are putatively linked with increased cancer risk. We developed a framework for the mandated regulatory in vitro evaluation of cancer-relevant bioassays for comparisons of insulin analogues, and showed that the cell-specific IGF-IR/IR ratio is crucial for interpretation.

Alterations of insulin-like growth factor binding protein 3 (IGFBP-3) glycosylation in patients with breast tumours

OBJECTIVES Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator of development and progression of breast cancer as it regulates the amount of free, physiologically active IGF-I and IGF-II. Changes in the glycosylation pattern within IGFBP-3 may affect its interaction with ligands. The aim of this study was to investigate whether such changes occur during disease progression. DESIGN AND METHODS IGFBP-3 in serum samples from healthy women and from women with breast tumours was characterised in terms of its concentration (IRMA), glycosylation moiety (lectin-affinity chromatography) and distribution of molecular species (immunoblotting). RESULTS In patients with benign tumours the concentration and carbohydrate content of IGFBP-3 was unaltered compared to healthy women. In patients with malignant tumours in most cases these two parameters were unchanged, but there were women whose concentration of IGFBP-3 was reduced and its structure was altered. In non-surviving cancer patients the concentration of IGFBP-3 was significantly reduced and these molecules contained a greater amount of biantennary complex type N-glycans having more mannose, fucose, bisecting GlcNAc and terminal sialic acid residues. CONCLUSION Our results showed that breast cancer progression causes alterations of IGFBP-3 glycosylation. The extent of changes increases with breast cancer severity.

Chronic insulin exposure does not cause insulin resistance but is associated with chemo-resistance in colon cancer cells.

Insulin resistance is an adaptive process in insulin-sensitive tissues characterised by reduced insulin receptor (IR) and insulin-receptor substrate (IRS)-1 expression, increased IRS-1 serine phosphorylation and attenuated downstream signalling. We tested whether this molecular phenotype prevails in cancer cells after long-term exposure to insulin. We characterised expression of IR-related molecules, IRS-1 phosphorylation and downstream signalling in a panel of 5 colon cancer cell lines at different insulin exposures: 15 min (100 nM), approximating to acute stimulation; 48 h (100 nM), used to demonstrate adaptive changes; and 12 weeks (20 nM; chronic insulin exposure, CIE), approximating to chronic hyperinsulinaemia. To assess clinical relevance, we determined IC50 values (increased indicating chemo-resistance) in the CIE-treated cells using oxaliplatin, SN38 (irinotecan) and 5-fluorouracil (5-FU). All colon cancer cell lines (HCT 116, HT-29, C32, CaCo2, LoVo) were sensitive to 15 min insulin exposure with increased phosphorylation of Akt, PRAS40 and p70-S6K. At 48 h, there was incomplete or absent features of insulin resistance. In CIE-treated cells, there was reduced IR expression, incomplete IRS-1 adaptation, lack of signalling pathway attenuation and contra-adaptive increases in IRS-1 tyrosine phosphorylation in several cell types. In CIE cells, there were multiple examples of increased IC50 values (2- to 100-fold) following 24-h treatment with oxaliplatin and SN38, but not with 5-FU. We concluded that CIE in colon cancer cells does not completely induce an insulin resistance molecular phenotype but is associated with chemo-resistance. Adaptive changes seen in insulin-sensitive non-neoplastic cells in response to long-term insulin may not extrapolate to neoplastic cells.

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of alpha-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.

The effect of laparoscopy on the IGF system in patients diagnosed with acute cholecystitis

Objective  IGFs and IGF binding proteins in the circulation are subject to modulation by a number of catabolic states including inflammation, trauma and surgery. We sought to determine the impact of laparoscopy on the IGF system in patients diagnosed with acute cholecystitis.

The insulin-like growth factor system in the circulation of patients with viral infections

Abstract The insulin-like growth factor (IGF) system was examined in the circulation of patients with viral infections (herpes simplex virus, HSV; cytomegalovirus, CMV; rotavirus, RV and adenovirus, AV). The serum concentrations of IGF-I, IGF-II and cortisol were measured by radioimmunoassay, while IGF-binding proteins (IGFBPs) were characterised by ligand-affinity blotting. Although both IGF-I and IGF-II concentrations were significantly lower in patients with viral infections (p < 0.05) than in healthy persons, the IGF-II/IGF-I ratio was increased (p < 0.05). No correlation between the concentration of IGF-I and IGF-II and the intensity of the antibody response to infection was observed. Ligand-affinity blotting demonstrated decreased amounts of IGFBP-3 (patients with HSV, CMV, AV and some patients with RV), increased IGFBP-2 (some patients with HSV and RV) and IGFBP-1 (patients with RV). Serum cortisol was significantly elevated (p < 0.05) in patients infected with HSV, CMV and RV. The alterations observed can be interpreted as induction of the hypothalamic-pituitary-adrenal axis and suppression of the growth hormone (GH)/IGF axis under the influence of viral infection.

Insulin-like growth factors and their binding proteins in the circulation of patients with echinococcosis, trichinellosis and toxoplasmosis

BACKGROUND Insulin-like growth factors (IGFs) are polypeptide hormones that play anabolic roles in cellular growth and metabolism. Their activity is regulated by binding proteins (IGFBPs) and degradation mechanisms. The liver is regarded as the main source of circulating forms and the levels change in various disease states. The aim of the study was to explore the effects of parasitic infections on the circulating IGFs and IGFBPs. METHODS Peptide concentrations in sera of patients with echinococcosis, trichinellosis and toxoplasmosis were measured by radioimmunoassay, while IGFBP patterns were characterised by ligand-affinity blotting and gel chromatography. RESULTS IGF-I levels were reduced in trichinellosis (p=0.016), with the increased relative amounts of IGFBP-1, -2 and -4. The significantly reduced levels of IGF-I (p<0.001), IGF-II (p=0.017) and IGFBP-3 and the increased presence of IGFBP-2 were found in patients with echinococcosis. CONCLUSIONS In subjects with trichinellosis probably the combined effects of inadequate nutrition and the immunological response occurred. A possible explanation for the changes found in patients with echinococcosis is that the liver is a frequent target organ for Echinococcus granulosus. Since liver cysts were present in these patients, it can be expected that hepatic injury may affect liver metabolism leading to altered IGF/IGFBP profiles.

Association of elevated IGFBP-1 with increased IGF-II concentration in patients with carcinoma of the liver.

Insulin-like growth factors (IGFs) are mitogens for numerous types of cells including cancer cells. The aim of this work was to analyze some of the components of the IGF system to assess which could be potential clinical biomarkers for monitoring patients diagnosed with liver cancer. Compared to healthy persons, patients with liver cancer had a lower concentration of IGF-I and a higher concentration of IGFBP-1, whereas the concentrations of IGF-II and IGFBP-3 remained unchanged. The IGF-I:IGFBP-3 ratio decreased in patients with cancer, while the IGF-II:IGFBP-1 ratio was not altered. Patients with primary carcinoma and those scheduled for surgery had lower IGF-I and higher IGF-II and IGFBP-1 concentrations than patients with secondary carcinoma and those not eligible for surgery. It may be postulated that a liver with primary cancer is induced to increase IGF-II and IGFBP-1 synthesis more than a liver involved in metastatic response. Similarly, in patients eligible for liver surgery an increase in IGF-II may reflect a gradual change in the concentration associated with a different stage of disease. As increased synthesis of certain IGFBPs is necessary to compensate decreased production of the others or increased IGF production, determination of serum IGF-II, IGFBP-1 and their ratio may aid in estimating the compensatory capacity of the liver affected by cancer.

Ligand-Binding Activity and Immunoreactivity of Insulin-Like growth Factor Binding Proteins in Patients with Colorectal Carcinoma And Postoperative Sepsis

Summary Background: Postoperative sepsis alters the growth hormone (GH)/insulin-like growth factor (IGF) axis leading to reduction in IGF-I and IGF-binding protein (IGFBP) 3 and elevation of GH-independent IGFBPs. The aim of this work was to investigate the differences in ligand binding and immunoreactivity of the circulating IGFBPs between surviving and nonsurviving septic patients that underwent colorectal surgery. Methods: Ligand binding was detected only for IGFBP-3 and IGFBP-2 and, consecutively, immunoreactivity was assayed for these IGFBPs. Results: In survivors both ligand- and immunoblotting re - vea led two IGFBP-3 isoforms (40 and 45 kDa). In non-survivors ligand-blotting hardly detected the IGFBP-3 doublet, intact immunoreactive IGFBP-3 was not detected and the major species was the 29 kDa fragment. Immunoblotting with an anti-IGFBP-2 antibody indicated an increase in the amount of intact and fragmented IGFBP-2 along the progression of illness in the non-survivors. The amount of intact IGFBP-2 in the survivors did not differ during the treatment. The amount of immunoreactive IGFBP-2 corresponded to its ligand-binding activity. Conclusions: According to the results reported here, ligandreactivity and immunoreactivity of IGFBP-3 should be taken into account as variables that affect the GH/IGF axis in critically ill patients. The degree of their variation is not directly correlated to the total amount of IGFBP-3 determined by the immunoassay, but they may be related to patients’ meta bolic potential to combat a disease. Krotok sadržaj Uvod: Fbstoperativna sepsa izaziva promene u sprezi hor- mon rasta (GHVinsulinu slični faktori rasta (IGF). Smanjuju se koncentracije IGF-I i IGF vezujućeg proteina (IGFBP) 3, a povećavaju IGFBP koji ne zavise od GH. Glj rada bio je da se ispita ligand-vazujuća aktivnost i imunoreaktivnost IGFBP molekula u cirkulaciji pacijenata operisanih od koJorektalnog karcinoma sa postoperativnom sepsom i rslićitim ishodom u smislu preživljavanja Metode: Ligand-vezujuća aktivnost detektovana je ligand blotomza IGFBP-2 i IGFBP-3, te jeza ove IGFBP odredivana i imunoreaktfcnost. Rezultati: Kod preživelih, ligand i imunoblotom utvrđeno je prisustvo dve IGFBP-3 izofomie (40 i 45 kDa). Kod umrlih, u ligand blotu IGFBP-3 dublet bio je jedva vidljiv, dok je u imunoblotu najjača traka pripadala fragmentu {29 kDa). Ukupna količina IGFBP-2 (intaktni molekuli i fragmenti) bila je znatno veća kod pacijenata koji nisu prđivet i progresivno je rasla sa približavanjem letalnog ishoda. Ligand-vezujuća aktivnost je takode bila srazmerno veća. Zaključak: Dobijeni rezultati upućuju na značaj određivanja ligand-vezujuće aktivnosti i imunoreakttvnosti IGFBP-3 kod pacijenata u kritičnom stanju. Promene IGFBP-3 aktivnosti nisu direktno odraz promene koncentracije IGFBP-3 odre- đene imunotestom, te su stoga važne u proceni metabo- ličkog potencijala pacijenta da se suprotstavi bolesti.