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Background: Three different strategies to manage transient hypocalcemia after total thyroidectomy were compared to evaluate cost-effectiveness. The reliability of total serum calcium (TSCa), ionized calcium (ICa), and intact parathyroid hormone (iPTH) were investigated to achieve this goal., Methods: A multicenter, prospective randomized study was carried out with 169 patients. The strategies were "preventive" (oral calcium + vitamin D supplementation), "reactive" (therapy in hypocalcemia), and "predictive" (therapy if iPTH <10 pg/mL)., Results: TSCa had higher accuracy in identifying patients who developed hypocalcemia-related symptoms than ICa (84.6% vs 50.0%). TSCa 24 h after surgery showed 24.8% of patients with hypocalcemia, whereas TSCa 48 h after surgery identified a further 10.6% with hypocalcemia (only in the "reactive" and "predictive" groups). iPTH showed low sensitivity as a predictor of hypocalcemia. Between the 3 groups, there was no significant difference in hospitalization time or number of symptomatic hypocalcemic patients. Interestingly, the cost-per-patient was significantly different among the groups., Conclusions: None of the discussed strategies allowed for early discharge of patients without any risk of transient hypocalcemia. The "preventive" strategy was the most cost-effective, despite overtreatment., (© 2019 Wiley Periodicals, Inc.)

In recent years, the poorly remarkable goals achieved in terms of patients' important outcomes for ovarian cancer have fueled our interest toward the study of its metabolic roots. Within this research pipeline, we assessed the association between the expression of the glucose transporter GLUT1, as expressed at the tumor tissue level, and circulating pre-surgical levels of fasting glucose in a case series including data from 40 patients with high FIGO stage serous ovarian cancer. Patients who provided data to the current analysis were randomly selected from a larger cohort. To our purposes, the procedures related to serum and tissue collection, storage and biomarker assessment were highly standardized and centralized at the institutional laboratories. The GLUT1 antibody SPM498 SPRING (REF. E13810) was used at a 1:500 dilution in 2 µm slides. Staining for GLUT1 was observed at the cell membrane level in all the cases assessed, but strong staining was described in 29 (72.5%) of them. The agreement between the two independent reviewers was 100%. Strong GLUT1 staining was inversely associated with circulating levels of fasting glucose, with a particularly striking difference for patients in the lowest fasting glucose tertile (p = 0.044). These results support the biological plausibility of the association of interest. If confirmed in larger studies, our findings may help clarify the potentials of biomarkers related to energy metabolism in terms of prognosis definition, treatment assignment, and outcome interpretation for patients with high FIGO stage serous ovarian cancer., (© 2017 Wiley Periodicals, Inc.)

Background: Over the last twenty years, the efforts of the scientific community devoted to the comprehension and treatment of ovarian cancer have remained poorly remunerative, with the case-fatality ratio of this disease remaining disappointedly high. Limited knowledge of the basic principles regulating ovarian carcinogenesis and factors impacting the course of disease may significantly impair our ability to intervene in early stages and lessen our expectations in terms of treatment outcomes. In the present study, we sought to assess whether metabolic factors and anthropometric indicators, i.e., pre-treatment fasting glucose and body mass index, are associated with renown cancer related prognostic factors such as tumour stage and grade at diagnosis., Materials and Methods: Study participants were 147 women diagnosed with epithelial ovarian cancer and treated with platinum based regimens and/or surgery at the Regina Elena National Cancer Institute of Rome, Italy. Glucose levels were assessed at the institutional laboratories on venous blood collected in overnight fasting conditions and prior to any therapeutic procedure. Stage was coded according to the FIGO staging system based on the results of the diagnostic workup, while tumour grade was locally assessed by an expert pathologist. Participants' characteristics were descriptively analyzed for the overall study population and in a subgroup of 70 patients for whom data on body mass index (BMI) were available. FIGO stage and grade were compared by categories of pre-treatment fasting glucose defined upon the median value, i.e., 89 mg/dl. The association of interest was tested in regression models including BMI., Results: For the overall study population, patients in the lowest category of fasting glucose were significantly more likely to exhibit a FIGO stage III-IV at diagnosis compared with their counterpart in the highest glucose category (81.3 vs 66.7%, p: 0.021). Subgroup analysis in 70 patients with BMI data confirmed this association (81.5 vs 55.8, p: 0.049), which remained significant when tested in regression models including BMI (OR: 0.28 95% CI 0.086-0.89, p: 0.031). No relevant evidence emerged when testing the association between fasting glucose and tumour grade., Conclusions: In patients diagnosed with epithelial ovarian cancer, pre-treatment glucose levels appear to be inversely associated with FIGO stage. Further studies are warranted to eventually confirm and correctly interpret the implications of this novel finding.

Background: Piperacillin, unlike other antibiotics, rarely causes immediate allergic reactions. Only two cases related to occupational exposure are reported in the literature., Objectives: Adoption of new methods for diagnosis of occupational allergy to drugs., Methods: An atopic nurse, aged 30 years, was referred to our hospital for an allergic work-related reaction to piperacillin. The patient had suffered two successive episodes with immediate cutaneous reaction, angioedema and dyspnoea after preparing piperacillin. Almost four years previously she had suffered from similar symptoms after taking amoxicillin. She was submitted to a clinical examination and a routine allergic test, performing also specific IgE (Phadia Pharmacia ImmunoCap) and BAT (Basophil Activation Test) for Beta-lactam antibiotics., Results: A positive response to piperacillin was observed in our case using BAT a new non-invasive and safe method, that proved useful for diagnosis of allergy. Moreover, we observed a change from an allergic reaction for therapeutic use of amoxicillin to a work-related adverse reaction to another beta-lactam, piperacillin., Conclusions: In previous clinical cases cutaneous and specific challenge tests were performed for diagnosis. At present, availability of an in vitro test, such as BAT may provide new diagnostic opportunities, and a useful tool for studying clinical cases other than, in perspective, monitoring exposed workers. Preventive measures were taken in the workplace to lower the risk of sensitization and allergic response. The nurse was transferred to a well controlled job.

Although vitamin D insufficiency is related to inferior prognosis in some cancers, limited data exist in hematologic malignancies. We evaluated the relationship between 25(OH)D serum levels and time to first treatment (TFT), a disease-specific end point, in 130 previously untreated Binet stage A chronic lymphocytic leukemia (CLL) patients. Measurement of 25(OH)D was performed by means of a direct, competitive chemiluminescence immunoassay using the DiaSorin LIAISON 25(OH)D TOTAL assay (DiaSorin, Inc., Stillwater, Minnesota). Overall, 41 patients (31.5%) had severe vitamin D insufficiency (<10 ng/mL), 66 (50.7%) had mild to moderate insufficiency (10-24 ng/mL), and 23 (17.6%) had 25(OH)D levels within the optimal range (25-80 ng/mL), with no relationship with between the season of sample collection and 25(OH)D level (P=0.188). A patient stratification according to these 3 groups led to significant difference in terms of TFT, with vitamin D insufficient patients having the shortest TFT (P=0.02). With respect to continuous 25(OH)D levels and clinical outcome, TFT was shorter as 25(OH)D decreased until a value of 13.5 ng/mL at which point the association of 25(OH)D and TFT remained constant. As a matter of fact, the 25(OH)D value of 13.5 ng/mL identified two patients subsets with different TFT risk (HR=1.91; 95% CI=1.06-3.44; P=0.03). In multivariate analysis the variable entering the model at a significant level were mutational status of IgVH (P<0.0001), serum thymidine kinase (P=0.02) and absolute lymphocyte count (P=0.03). Thus confirming the Mayo clinic experience, our data provide further evidence that 25(OH)D levels may be an important host factor influencing TFT of Binet stage A patients. Whether normalizing vitamin D levels may delay disease-progression of patients with early disease will require testing in future trials., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia (CLL) [i.e. mutational status of the immunoglobulin heavy chain variable region (IgVH), ZAP-70 and CD38 expression] and serum levels of B cell-activating factor (BAFF of the TNF family) by evaluating the impact of these variables on the time to first treatment (TFT) in a series of 169 previously untreated CLL patients in Binet stage A. Higher levels of BAFF were more frequently associated with female gender (P=0.02), younger age (P=0.01), Rai stage 0 (P=0.002), higher platelet count (P=0.005), mutated IgVH disease (P=0.002), higher occurrence of normal cytogenetic profile or presence of 13q deletion (P=0.02), low ZAP-70- (P=0.003), and CD38-expression (P=0.02). Maximally selected log-rank statistic plot identified a serum BAFF concentration of 0.313 ng/mL as the best cut-off (P<0.0001). This threshold recognized two subsets of patients with different TFT (P<0.0001). Because in multivariate analysis soluble BAFF [Hazard ratio (HR), 8.23; confidence Interval (CI) 95%,3.0-22.6, P<0.0001] and mutational status of IgVH (HR=2.60; CI 95% 1.10-6.14, P=0.03) maintained the discriminating power their combined effect on clinical outcome was assessed. When three groups were considered: (1) low-risk (n=93), patients with concordant IgVH(mut) and higher soluble BAFF; (2) intermediate-risk (n=50), patients with IgVH(mut) and low BAFF levels or IgVH(unmut) and soluble higher BAFF;(3) high-risk (n=26), patients with concordant IgVH (unmut) and low soluble BAFF, the 2-yr TFTs were, respectively, 95%, 85%, and 41% (P<0.0001). In conclusion, our results indicate that in early B-cell CLL, the biological profile including among other parameters soluble BAFF may provide a useful insight into the complex interrelationship of prognostic variables., (© 2010 John Wiley & Sons A/S.)

We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia (CLL) [i.e. mutational status of the immunoglobulin heavy chain variable region (IgV(H)), ZAP-70- and CD38-expression] and serum levels of CD26 (dipeptidyl peptidase IV, DPP IV) by evaluating the impact of these variables on the time to first treatment (TFT) in a series of 69 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA we found that with exception of a borderline significance for ZAP-70 (P = 0.07) and CD38 (P = 0.08), circulating levels of CD26 did not correlate with either Rai substages (P = 0.520) or other biomarker [beta2-microglobulin (P = 0.933), LDH (P = 0.101), mutational status of IgV(H) (P = 0.320)]. Maximally selected log-rank statistic plots identified a CD26 serum concentration of 371 ng/mL as the best cut-off. This threshold allowed the identification of two subsets of patients with CD26 serum levels higher and lower that 371 ng/mL respectively, whose clinical outcome was different with respect to TFT (i.e. 46% and 71% at 5 yr respectively; P = 0.005). Along with higher serum levels of CD26, the univariate Cox proportional hazard model identified absence of mutation in IgV(H) (P < 0.0001) as predictor of shorter TFT. As in multivariate analysis all these parameters maintained their discriminating power (mutational status of IgV(H,)P < 0.0001; soluble CD26, P = 0.02) their combined effect on clinical outcome was assessed. When three groups were considered: (1) Low-risk group (n = 31), patients with concordant IgVH(mut) and low level of soluble CD26; (2) intermediate risk group (n = 26), patients with discordant pattern; (3) high-risk group (n = 12), patients with concordant IgVH(unmut) and high level of soluble CD26, differences in the TFT were statistically significant, with a TFT at 5 yr of respectively 88%, 51% and 43% (P < 0.0001). Our results indicate that in early B-cell CLL biological profile including among other parameters soluble CD26 may provide a useful insight into the complex interrelationship of prognostic variables. Furthermore, CD26 along with mutational status of IgV(H) can be adequately used to predict clinical behavior of patients with low risk disease.

In a series of 84 chronic lymphocytic leukemia (CLL) patients we sought to establish whether BAFF (B-cell activating factor of the TNF family) circulating levels correlated with clinical characteristics of disease. BAFF serum levels were significantly higher in 20 healthy controls (i.e., median 695 ng/mL, range 389-1040) in comparison to the whole population of CLL patients (median 376, range 93-8914; P<0.0001). After setting a cut-off at the median value observed in healthy controls (i.e., 695 ng/mL) we found that 6 out of 15 (40%) patients with familial CLL had increased BAFF levels while the same occurred only in 5 out of 64 (7.2%) patients with sporadic CLL (P=0.0007). No significant difference in age (P=0.82), sex (P=0.97), Binet clinical stage (P=0.20), incidence of autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP) (P=0.47), mutational status of IgVH (P=1.00), CD38 (P=0.34) and ZAP-70 expression (P=0.16) could be detected between patients with sporadic and familial CLL, respectively. The only feature characterizing familial CLL patients was a higher serum BAFF level (sporadic CLL 336 ng/mL, range 93-925; familial CLL 601 ng/mL, range 138-8914; P=0.002). Our data suggest that BAFF levels are elevated in patients with familial CLL. The small cohort of patients used implies that a larger study is needed to reinforce the observation.

Angiogenesis is involved in the pathogenesis of B-cell chronic lymphocytic leukemia (CLL), and high microvascular density has been found in CLL to be associated with a poor prognosis. In this study, we assessed serum levels of adiponectin in 69 patients with Binet stage A B-CLL, and these values were retrospectively correlated with bone marrow (BM) microvessel area and serum levels of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), angiogenin, PECAM-1 (CD31), matrix metalloproteinase-9 (MMP-9), interleukin-8 (IL-8), syndecan-1, and the percentage of CD38(+) or ZAP-70(+) CLL cells. The positive correlation between serum levels of adiponectin and VEGF (P = .03) does not translate into an increase of the extent of BM angiogenesis (P = .404), FGF-2 (P = .348), angiogenin (P = .402), and CD31 (P = .248) serum concentrations. Accordingly, IL-8 (P = .175), syndecan-1 (P = .06), and MMP-9 (P = .144) circulating levels were not likely to reflect adiponectin concentration. Furthermore, patients with higher levels of adiponectin had a more favorable biological profile as defined by a lower number of both CD38(-) (r = -0.294; P = .02) and ZAP-70(+) (r = -0.285; P = .04). Finally, we evaluated the presence of adiponectin in B-CLL cells at gene expression level. RMA intensity values for adiponectin gene transcript denote a homogeneous low expression in B-CLL cells, whereas VEGF transcript was highly expressed with a degree of interpatient variability. Overall, these data seem to indicate that adiponectin could be involved as an antiangiogenic factor in B-CLL.

The correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukaemia (CLL) [i.e., mutational status of the immunoglobulin heavy chain variable region (IgV(H)), ZAP-70- and CD38-expression] and adiponectin serum concentration was evaluated in a cohort of 69 previously untreated Binet stage A CLL patients. Adiponectin levels inversely correlated with absolute peripheral blood lymphocyte count (r = -0.254; P = 0.03), CD38-positive CLL cells (r = -0.294; P = 0.04) and ZAP-70 (r = -0.285; P = 0.03). The univariate Cox proportional hazard model demonstrated that, in addition with lower serum levels of adiponectin (P = 0.01), the unmutated IgV(H) condition (P = 0.002) and ZAP-70-positivity (P = 0.02) were associated with a shorter time to first treatment (TFT). However, in multivariate analysis only ZAP-70 positivity emerged as predictor of the TFT (P = 0.008). The levels of adiponectin in CLL were evaluated in 60 patients from an independent cohort investigated by gene expression profiling. Adiponectin gene expression was invariably low suggesting a limited (if any) role of leukemic cells in the production of circulating adiponectin levels. In contrast, both adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA were highly expressed by CLL cells with a degree of inter-patient variability. Our results, although preliminary, lend support to the idea that adiponectin secretion by bone marrow adipocytes might represent a possible promising drug target in the field of hematology.

In an effort to confirm previous reports we analyzed clinico-biological implications of increased serum levels of thrombopoietin (TPO) in a series of 71 previously untreated Binet stage A B-cell chronic lymphocytic leukemia (CLL) patients. Serum levels of TPO did not correlate with peripheral blood lymphocytosis (p = 0.928), Rai substages (p = 0.516), platelet count (p = 0.572), hemoglobin level (p = 0.228), LDH (p = 0.144) and beta2-microglobulin (p = 0.520). The same applied when correlation with ZAP-70 (p = 0.562), CD38 (p = 0.258) or mutational status of IgV(H) (p = 0.0794) were sought. The risk of disease-progression according to known and putative prognostic parameters was also evaluated as time to first treatment (TFT). The univariate Cox proportional hazard model demonstrated that the absence of IgV(H) mutational status (p = 0.0005) and ZAP-70-positivity (p = 0.02) were associated with a shorter TFT. In contrast, Kaplan-Meier estimates of TFT, plotted after setting as cut-off the median value for TPO (i.e., 46 pg/mL), failed to demonstrate any statistical difference between two groups (p = 0.342). Looking for cellular source of TPO we investigated the presence of TPO at gene expression level in 60 B-CLL patients belonging to an independent series. Here we provide evidence for the presence of a low TPO gene expression transcript in B-CLL cells. In conclusion, our results indicate that in early B-cell CLL circulating level of TPO does not provide a useful insight into the complex interrelationship of prognostic variables.

We wondered whether there was any association between the extent of increased angiogenesis and IgV(H) gene mutational status, expression of CD38 and ZAP-70 in early B-cell chronic lymphocytic leukemia (CLL) patients. Circulating levels of vascular endothelial growth factor (VEGF) correlated positively with ZAP-70-expression (P=0.03), CD38- expression (P=0.03) and mutational status of IgV(H) (P=0.005). The same did not apply when correlations were sought with either bone marrow angiogenesis or serum levels of basic fibroblatic growth factor (FGF-2). Studies to determine how to integrate variables reflecting increased angiogenesis with other prognostic markers such as CD38, ZAP-70, IgV(H) status and cytogenetic abnormalities are needed to optimize risk stratification for individual patients.

The association between angiogenin and cancer progression and poor outcome in solid tumors has been documented, but its significance in leukemias has not been evaluated. Using an ELISA technique (Quantikine Human Angiogenin Immunoassay; R&D Systems), we measured serum angiogenin levels in 77 previously untreated Binet stage A B-cell chronic lymphocytic leukemia (CLL) patients. No difference in angiogenin serum levels could be found between patients (median: 295 ng/mL; range: 74-1700) and 15 age- and sex-matched healthy controls (median: 264 ng/mL; range: 29-1835) (P = NS; Mann-Whitney test). Increased angiogenin serum level was associated with higher LDH (P = 0.03) and beta2-m (P = 0.007) concentrations. However, angiogenin did not reflect the extent of bone marrow (BM) angiogenesis as evaluated by microvessel area (P = 0.611), circulating levels of vascular endothelial growth factor (VEGF) (P = 0.873) and basic fibroblastic growth factor (FGF-2) (P = 0.421). When the 25 patients with available data were stratified into the four major cytogenetic categories (normal karyotype, 13q as a sole aberration, 12q trisomy, 11q or 17p deletion) and aberrations were compared with angiogenin serum levels, no correlation was found (P = 0.651; Kruskall-Wallis test). A cut-off of angiogenin serum level corresponding to median (i.e. 330 ng/mL) or higher identified later upstaging and longer progression-free survival (PFS). The 5-yr PFS was 51.5% for patients with angiogenin levels lower than median and 85% for patients with higher values [P = 0.03; hazard ratio (HR) = 2.86; 95% CI: 1.08-6.72]. Although in multivariate analysis only Rai substages (P = 0.00001) and peripheral blood lymphocytosis (P = 0.009) retained their prognostic significance, angiogenin could be incorporated into the Rai substages thus leading to the identification of the following risk categories: (i) stage 0 (angionenin >330 ng/mL); (ii) stage 0 (angiogenin <330 ng/mL) + stage I-II (angiogenin >330 ng/mL); and (iii) stage I-II (angiogenin <330 ng/mL). The 40-month PFS were as follows: 85%, 65%, 25% (chi(2) for trend = 6.33; d.f. = 1; P = 0.01). In conclusion, serum angiogenin levels although not increased in comparison with healthy controls, may predict clinical outcome of patients with early CLL and help to refine Rai's stratification.

Background and Methods: Serum levels of matrix metalloproteinase-9 (MMP-9) which agree with progression in solid and haematological tumours were correlated to the risk of disease progression in 62 patients with early (Binet stage A) B-cell chronic lymphocytic leukaemia (CLL). Sera were taken at diagnosis and tested by an enzyme-linked immunosorbent assay., Results: MMP-9 levels positively correlated with haemoglobin levels (P = 0.03) and platelet count (P = 0.03). No association was found with main clinico-haematological features representative of tumour mass, such as peripheral blood lymphocytosis, bone marrow histology, Rai substages and beta-2 microglobulin (beta-2m). A cut-off of MMP-9 levels corresponding to 33rd percentile (203 ng/mL) or higher identified earlier upstaging and shorter progression-free survival. MMP-9 was a significant prognostic marker in multivariate analysis and partially independent of Rai substages, which suggests its inclusion into such a staging system to better stratify prognostically Rai stages I and II patients., Conclusions: MMP-9 serum levels predict disease behaviour and help to refine the prognosis of stage A CLL patients.

The purpose of this review is to examine the indications and contraindications of antioxidant therapy in arterial hypertension. It has been suggested that oxidative stress plays a role in hypertension, increasing pressure values and leading to complications. Oxidative stress, at present, appears as one of several metabolic abnormalities described in essential hypertension. It is still uncertain whether this abnormality is primary or secondary; in any case, measurement of the main oxidant and antioxidant activities in plasma and blood cells may be useful in order to recognize and monitor oxidative stress. Antioxidant therapy could be useful to counteract the effects of oxidative stress on blood vessels, arterial pressure and low-density lipoproteins. Although antioxidant therapy with drugs or physiological substances in hypertensive subjects has been examined in some clinical trials, these experimental observations do not appear sufficient to draw definitive conclusions because long-term randomized, controlled studies do not exist. At present, it seems appropriate to propose some dietary recommendations which, apart from their antioxidant properties, are useful and do not have untoward effects. The safest approach to treat or to prevent oxidative stress consists of a diet that includes foods with a high antioxidant content (i.e. vitamins C and E and flavonoids). This diet consists fundamentally of fruits, vegetables and grains. Moreover it seems useful to use antihypertensive drugs that are able to decrease oxidative stress. The addition of physiologic antioxidant substances should be considered only in hypertensive subjects with a marked increase in oxidant or decrease in antioxidant factors. Indeed reactive oxygen species play a pivotal role in many physiological reactions, and their excessive inhibition could be dangerous. Further controlled, randomized long-term trials with antioxidants in hypertension are necessary to establish the efficacy and tolerability of antioxidants in the adjuvant therapy of hypertension.

We verified the variations of primary tumour steroid receptor status and proliferative activity at different times and phases (follicular vs luteal) of the menstrual cycle and their relationship with short clinical outcome in a cohort of 248 N- breast cancer patients. Steroid receptor content (ER and PgR) was evaluated by DCC assay and proliferative activity by 3H-Thymidine autoradiographic assay (TLI). Median age was 44 years, 60% of tumors were T1, and cytohistological grade was G1-2 in 54% of cases. At surgery, 57% were in the luteal phase while 43% were in the follicular phase. No significant variations were found in mean TLI or ER and PgR characteristics of the primary tumors surgically treated in different periods of the menstrual cycle; however, the ER level resulted significantly higher in 4th with respect to the 3rd week of menstrual cycle, while PgR level was higher in PgR+ cases treated during the 3rd week. The number of relapses and disease-free survival curves after 36 months median follow-up did not differ significantly for patients treated in different periods of the menstrual cycle (12% and 9% of disease relapses in luteal and follicular phases; p=n.s.). We can conclude, therefore, that TLI, ER and PgR expressions could vary significantly during menstrual cycle only in certain specific tumor subgroups.

Two immunotoxins were prepared using monoclonal antibodies (mAb) directed towards two distinct epitopes of the gp185HER-2 extracellular domain, and the type I ribosome inactivating protein (RIP) plant toxin saporin 6. Cell protein synthesis inhibition assay reveals that the immunotoxins display a potent and specific cytotoxicity that is characterized by a slow rate, since the time required to inhibit incorporation of radiolabeled leucine completely ranges from 36 h to 60 h depending on the target cell line and the immunotoxin. Because this feature may hamper the immunotherapeutic use of these conjugates we analysed this further by studying the early phases of internalization of immunotoxins by immunoelectron microscopy. The results of this study have demonstrated that the distribution pattern of the immunotoxins and of the unconjugated mAb over the cell surface overlaps. Similarly the mAb and immunotoxins are internalized into the cell by two different pathways: via clathrin-coated pits or via smaller uncoated pits and vesicles. A higher degree of internalization is achieved when the two immunotoxins are used in combination. Unlike the slow kinetics of cell intoxication the process of immunotoxin endocytosis is characterized by a rapid rate of internalization (above 40% at 5 min in the SK-BR-3 cell line). Although these findings provide no clue to explain the mechanisms of the slow rate of cytotoxicity of the two immunotoxins their rapid internalization indicates that these reagents can be exploited in immunotherapeutic approaches to gp185HER-2-expressing malignancies.

Over-expression of the HER-2 oncogene correlates with poor prognosis in breast and ovarian carcinomas. Using a sensitive immunohistochemical assay, we have detected low levels of gp185HER-2 in intradermal nevi (78%) and in primary (75%) and metastatic melanomas (58%). The HER-2 gene product expressed by cultured melanoma cells had the expected molecular weight, but no levels of tyrosine phosphorylation could be detected. Consistently, we were unable to inhibit in vitro growth of melanoma cells with an anti-gp 185HER-2 MAb, in conditions in which the growth of SKBr-3 breast-carcinoma cells was severely impaired. However, immunotoxins to gp 185HER-2 were able to kill gp185HER-2-positive melanoma cells. These data indicate that low levels of gp185HER-2 are expressed by the melanocyte lineage, with no correlation with transformation or tumor progression. Nevertheless, gp185HER-2 appears a suitable target for immunotherapy of cutaneous melanoma.

The shc genes encodes three widely expressed proteins of 46, 52 and 66 kDa. Overexpression of p46shc and p52shc in NIH3T3 fibroblasts induces a tumorigenic phenotype. Shc products are phosphorylated on tyrosine by the activated epidermal growth factor receptor (EGFR) and become physically associated with EGFR via their SH2 domain. Thus Shc oncoproteins may play a role in mitogenic signal transduction. Here we report that Shc products are substrates also of the erbB-2 kinase and form complexes with the erbB-2 product in intact cells. In vitro, the bacterially expressed Shc SH2 domain is sufficient to reconstitute the high affinity Shc/erbB-2 interaction. The erbB-2 region required for Shc binding was narrowed down to the most COOH-terminal 179 residues of gp185erbB-2; within this region, phosphorylation of one or more of the erbB-2 autophosphorylation sites is required for Shc/gp185erbB-2 complex formation as well as optimal phosphorylation of Shc products by the erbB-2 kinase. Thus, Shc proteins may play a role in signal transduction by gp185erbB-2.

The oncogene HER-2/neu encodes a transmembrane glycoprotein of 185 kDa (gp185HER-2) with tyrosine-kinase activity. Gene amplification and high levels of expression of gp185HER-2 have been found to correlate with poor clinical outcome in breast and ovarian carcinomas. Employing a somatic cell hybrid fusion protocol, which yields a high frequency production of hybridomas, we have analyzed the extent of the murine immune response to the gp 185 extracellular domain. In a single fusion experiment, using as immunogen NIH 3T3 cells expressing high levels of a transfected human HER-2 gene, we have generated mAbs, mainly of IgG1 isotype, displaying high affinity (10(7)-10(10) mol/L) to gp 185. Analysis of the epitope specificity has allowed the identification of five distinct groups of spatially related epitopes, each provided with different immunodominancy, and all resistant to formalin fixation. The use of inhibitor of N-linked glycosylation tunicamicyn has demonstrated that the mAbs bind to epitopes localized in the protein core of gp185HER-2. Because recent reports have shown that gp185HER-2 has a restricted expression in normal tissues and is homogenously detectable in metastatic foci of gp 185 + primary tumors, antibodies to this macromolecule, in addition to their prognostic value, may represent reagents for in vitro and in vivo diagnostic applications, as well as for the development of therapeutic strategies.