CT perfusion in solid-body tumours. Part I: Technical issues.

Functional imaging is becoming increasingly important in both research and clinical diagnostic radiology. Perfusion computed tomography (CTP) is a readily available and widely used tool that allows an objective measurement of tissue perfusion through the mathematical analysis of data obtained from repeated scans performed after administration of contrast agent. Recently, CTP has been increasingly used in the oncological field, being studied as a potential marker of neoplastic angiogenesis, which is one of the main targets of new tumour therapies. The aim of this paper was to provide the theoretical background and practical guidance for accurately performing CTP and interpreting results of examinations in solid-body tumours. CTP could be a valid tool for functional imaging of tumours if the acquisition technique is robust, if image and data analysis is accurate and if interpretation of results is adequately inserted within a clinical context.

Gefitinib inhibits the ability of human bone marrow stromal cells to induce osteoclast differentiation: implications for the pathogenesis and treatment of bone metastasis.

Significant relief of bone pain in patients with bone metastases was observed in a clinical trial of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in breast cancer. Osteoclast activation and differentiation are regulated by bone marrow stromal cells (BMSC), a heterogeneous cell compartment that comprehends undifferentiated mesenchymal stem cells (MSC) and their specialized progeny. In this regard, we found that human primary BMSCs express immunoreactive EGFR. Expression of EGFR mRNA and protein was also demonstrated in two human, continuous MSC-like cell lines, HDS-1 and HDS-2 cells. Treatment of HDS cells with EGF produced a significant increase in the levels of activated EGFR which was not observed in the presence of gefitinib. A significant reduction in the basal levels of activation of the EGFR and of Akt was observed in HDS cells following treatment with gefitinib. Treatment of HDS cells with gefitinib produced a significant reduction in the levels of secreted macrophage colony-stimulating factor (M-CSF) and cell-associated receptor activator of NF-kappaB ligand (RANKL) in both cell lines, as assessed by using specific ELISA and Western blotting techniques. Finally, the ability to sustain the differentiation of pre-osteoclasts of conditioned medium from gefitinib-treated HDS cells was reduced by approximately 45% as compared with untreated HDS cells. These data have demonstrated for the first time that the EGFR regulates the ability of BMSCs to induce osteoclast differentiation and strongly support clinical trials of gefitinib in breast cancer patients with bone disease.

Effect of the combined treatment with 5-fluorouracil, gamma-interferon or folinic acid on carcinoembryonic antigen expression in colon cancer cells.

5-Fluorouracil (5-FU) and human recombinant gamma-interferon (gamma-IFN) were found to increase the expression of carcinoembryonic antigen (CEA) in human cancer cells in vitro. In the present study, the antimetabolite was associated with gamma-IFN or folinic acid (FA), a biochemical modulator of cellular metabolism of 5-FU, able to increase its antineoplastic activity. Treatment of two human colon cancer cell lines (HT-29 and WiDr) with 5-FU + gamma-IFN resulted in an increase of CEA expression higher than that obtainable with both agents alone, although no synergistic effects were obtained. This was demonstrated in terms of: (a) mRNA transcripts (HT-29); (b) cytoplasm and membrane CEA protein levels detected by Western blot analysis (HT-29); and (c) plasma membrane reactivity determined by flow cytometry analysis (HT-29 and WiDr). Moreover, 5-FU + gamma-IFN increased HLA class I molecules in the HT-29 cell membrane over that obtainable with gamma-IFN alone. In contrast, both agents did not induce the expression of the costimulatory molecule B7-1. Treatment with FA enhanced the antitumor effect of 5-FU but not its ability to augment CEA expression. This suggests that the FA-sensitive biochemical mechanism of action of 5-FU is not involved in its effect on CEA expression. In vivo studies showed, for the first time, that 5-FU, alone or combined with gamma-IFN, increases the amount of CEA protein over controls, either in cancer cells or in peripheral blood of nude mice bearing HT-29 cells. These results could be of potential diagnostic and/or therapeutic value when CEA protein is the target of humoral or cell-mediated immunity.

Growth factor-dependent activation of alphavbeta3 integrin in normal epithelial cells: implications for tumor invasion.

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, alphavbeta3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, alphavbeta3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. alphavbeta3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for alphavbeta3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted alphavbeta3 enrichment at FCs and impaired adhesion. Accordingly, activation of alphavbeta3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.

In vitro cultures of pathological thyroid cells: a powerful diagnostic technique.

In order to get a correct predictivity from molecular and functional markers in neoplastic disease, cell cultures, correspondent to in vivo existing populations, should be available. The difficulty, as yet, to correlate in vivo conditions with in vitro molecular and functional markers, represents a hurdle for a better prognosis in several neoplastic diseases. We tackled the problem establishing cultures from surgical samples of human thyroid glands bearing various pathologies (pathological diagnosis were obtained for all samples). Cells were frozen after 2 passages, and molecular markers (thyroglobulin, TPO, TTF-1 and PAX-8) and functional parameters (TSH-dependent cAMP production and thymidine incorporation) were investigated after thawing. The "in vitro profile" (functional parameters and molecular markers) was found to correlate with the pathological diagnosis and the degree of differentiation of the starting specimens. The data presented suggest that our culture technique allows in vitro growth of cell populations that may be used to perform functional assays and may make the molecular characterization of pathological samples easier. These findings could be especially useful to better define prognosis and also help to develop innovative therapies.

Dacarbazine-induced immunogenicity of a murine leukemia is attenuated in cells transfected with mutated K-ras gene.

Strong immunogenicity is induced by antitumor triazene compounds in tumor cells through a mutagenic mechanism. A highly immunogenic <> clone, isolated from a dacarbazine-treated L5178Y leukemia of DBA/2 mice, was transfected with K-ras mutated at codon 12 (i.e. ras(m12)). This transfected clone presents at least 2 mutations, one concerning K-ras gene, and the other affecting an unrelated gene, responsible for the generation of a highly immunogenic, MHC class I restricted non-self peptide. The results indicate that cells of <> clone transfected with ras(m12) were less immunogenic than cells of the same origin transfected with the vector alone. Moreover, ras(m12)-transfected cells showed lower levels of H-2K(d) gene expression with respect to those detectable in control cells. In addition, in vivo and in vitro sensitization against <> clone carrying mutated ras did not result in a strong cytotoxic T lymphocyte response against ras(m12)-transfected non immunogenic L5178y target cells. These preliminary results suggest that K-ras mutation could down-regulate the level of tumor immunogenicity, possibly acquired through a mutagenic process affecting other unrelated genes.

Drug-induced changes of carcinoembryonic antigen expression in human cancer cells: effect of 5-fluorouracil.

Previous studies showed that 5-fluorouracil (5-FU) is capable of enhancing the membrane reactivity of the human colon carcinoma cell line HT-29 with a monoclonal antibody (COL-1) directed against carcinoembryonic antigen (CEA). In the present study, we show that short-term exposure (i.e., 1 hr) of cancer cells to 5-FU mediates a marked increase of CEA expression, that is concentration-dependent and lasts up to day 5 after treatment. This phenomenon is the result of the drug-mediated enhancement of the CEA expression, but not of the selection of the CEA-positive cells operated by the antimetabolite. This is supported by the finding that the increase of the CEA expression detected by cytofluorimetric analysis is observed not only in the parental HT-29 line, but also in its C22.20 subclone, endowed with a low basal level of CEA and with chemosensitivity to 5-FU lower than that of the parental cell line. Moreover, increase of CEA expression occurs not only in the plasma membrane, but also in the cytosolic cellular compartment, as indicated by the results of Western blot analysis. Northern blot analysis of total RNA extracted from 5-FU-treated HT-29 or C22.20 cells shows an increase in the steady-state levels of CEA and CEA-related transcripts (e.g., biliary glycoprotein). Moreover 5-FU-mediated augmentation of the CEA transcript appears to be attributable mainly to enhanced transcription rather than to increased mRNA stability. It is concluded that induction of enhanced CEA protein expression in cancer cells treated with 5-FU could be of clinical interest for the development of immunochemotherapeutic protocols based on CEA protein as the target molecule.

Macrophage colony-stimulating factor enhancement of antibody-dependent cellular cytotoxicity against human colon carcinoma cells.

Antibody-dependent cellular cytotoxicity (ADCC) has been suggested to be an important defense mechanism against tumors. The effects of recombinant human macrophage colony-stimulating factor (rhM-CSF) on ADCC activity of human monocytes were investigated. Human peripheral monocytes were pre-incubated for 72 h with rhM-CSF at various concentrations (50, 100, 200, 400 U/ml) and then used as effector cells in a 24-h 111-Indium release assay. Human carcinoma cell lines LS-174T, CBS, and KLE were used as targets to react with anti-carcinoma monoclonal antibodies (mAbs: murine D612, murine CC49, and chimeric CC49). A significant increase in ADCC activity was observed after monocytes were incubated in 100-400 U/ml of human rhM-CSF. Variation in ADCC activity of monocytes among donors was observed. The enhancement of ADCC activity was blocked by the addition of a neutralizing antibody to rhM-CSF. Less D612 mAb was required for the rhM-CSF-treated monocytes to mediate an equivalent level of ADCC activity as compared to the untreated monocytes. Because of the low levels of rhM-CSF required in these studies to enhance ADCC, treatment of monocytes alone with comparable levels of rhM-CSF did not enhance antibody-independent cytotoxicity. Moreover, it is demonstrated here that recombinant human interleukin-4 (rhIL-4) and rhM-CSF can have a synergistic effect of monocyte-mediated ADCC on human tumor cells. These results thus indicate that rhM-CSF augments ADCC of human peripheral blood monocytes using mAbs to human carcinomas, suggesting a potential role for rhM-CSF in cancer immunotherapy.

Enhanced interleukin-2 production in human tumor-infiltrating lymphocytes engineered by 3'-truncated interleukin-2 gene.

Tumor-infiltrating lymphocytes (TILs), T lymphocytes associated with solid tumors that can be grown with interleukin (IL)-2 in vitro, preferentially accumulate at tumor sites after adoptive transfer. Therefore, TILs can be considered for use as cellular vehicles in gene therapy. We transduced melanoma TILs with the IL-2 gene and clarified functional characteristics of the TIL transductants. TILs transduced with 3'-end-truncated IL-2 gene (480 bp) produced high amounts of IL-2 detected in supernatants when compared to TILs transduced with the native IL-2 gene containing 3'-end adenine-thymidine (AT)-rich sequences (650 bp). The level of IL-2 in supernatants was higher with the addition of anti-Tac antibody (Ab) to block the consumption of IL-2 by the TILs. These TILs could proliferate autonomously in the absence of exogenous IL-2, and the proliferation of TILs could be completely blocked by anti-IL-2 Ab or anti-IL-2 receptor Ab. Thus TILs transduced with IL-2 gene can proliferate through the autocrine loop. However, the expression of IL-2 from TILs transduced with the IL-2 gene was downregulated after 2 to 3 weeks of G418 selection. Our study indicates the feasibility of transduction and expression of a truncated 480-bp IL-2 gene into TILs and the possibility of employing adoptive immunotherapy protocols using TILs modified with this IL-2 gene.

Differential effects of recombinant interferon-alpha and 5-fluorouracil against colon cancer cells or against peripheral blood mononuclear cells.

Comparative studies on the suppressive effects of recombinant interferon-alpha (IFN-alpha), 5-fluorouracil (5-FU), or IFN-alpha + 5-FU have been performed in vitro on colon carcinoma cells (HT-29 cell line) and PHA-stimulated mononuclear cells (MNC) of peripheral blood obtained from healthy donors. IFN-alpha was used at 500 U/ml against HT-29 cells and at 1000 U/ml against MNC on day 1 of culture; 5-FU was used at 250 microM against HT-29 and at 1400 microM against MNC on day 2 of culture. The results show that: (a) IFN-alpha inhibited MNC and HT-29 cells by 13.4% and 32.9%, respectively; (b) 5-FU inhibited MNC and HT-29 cells by 54.7% and 87.0%, respectively; (c) IFN-alpha + 5-FU resulted in a stronger inhibition of HT-29 cells (i.e., 96.1%). In contrast, that combination was significantly less suppressive than 5-FU alone when MNC were used as targets (i.e., 35.9% inhibition). Natural cell-mediated cytotoxic activity relative to 10(6) MNC was not markedly altered by all agents alone or in combination. Moreover, treatment with IFN-alpha, 5-FU or IFN-alpha + 5-FU resulted in a marked increase in the number of HT-29 cells positive for the CEA surface antigen. These data seem to provide further rational support of the clinical use of IFN-alpha + 5-FU in colorectal cancer, based on the differential toxicity of this drug combination on tumor versus normal immunocompetent cells.

In vitro tumour cell growth inhibition: a comparative study between allosensitized cytotoxic T lymphocytes and lymphokine activated killer cells.

There is general agreement that several distinct subpopulation of lymphocytes, including major histocompatibility complex (MHC)-restricted T lymphocytes and non-restricted natural killer, or lymphokine-activated killer (LAK), cells are active in lysing neoplastic cells. In this study experiments were designed to compare the inhibitory effects of LAK cells and allosensitized cytotoxic T lymphocytes (CTL) on in vitro growth of an Epstein-Barr virus-transformed B-cell line (BSM) and of a HTLV-I producer T-cell line (MT-2). It was found that allosensitized CTL are more efficient at inducing BSM, or MT-2, cell growth inhibition than LAK cells. These results are consistent with the hypothesis that MHC-restricted T effector cells could mediate higher tumour suppressive effects than non-MHC restricted LAK cells.

Transfer of the IL-6 gene into a human colorectal carcinoma cell line and consequent enhancement of tumor antigen expression.

cDNA encoding the human IL gene (580 bp), inserted into a retroviral expression vector carrying neomycin resistance selective marker, was introduced into HT-29 human colon carcinoma cells by lipofection. Interleukin-6 activity was measured by ELISA and bioassay using B9 cells. Interleukin-6 secreted by transfected HT-29 cells was shown to be biologically active. The expression of the human tumor associated antigen CEA (carcinoembryonic antigen), HLA classes I and II, and ICAM-1 antigens in the transfected HT-29 cells were also analyzed by flow cytometry. Significant enhancement in the expression of CEA but not in the expression of HLA class I, HLA class II and ICAM-1 antigens, was observed in the transfected HT-29 cells as compared to the parental HT-29 cells. These results provide experimental evidence that enhancement of tumor antigen expression on tumor cells can be induced by IL-6 gene transfection, and suggest another potential role for the use of IL-6 gene transfer in the immunotherapy of human cancers.

A human T cell line engineered to secrete chimeric monoclonal antibody.

Both monoclonal antibodies (MAbs) and human T cells have been used in human tumor immunotherapy protocols. Tumor-infiltrating lymphocytes (TILs) and MAbs that can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) via human effector cells have shown antitumor effects in both animal models and clinical trials. One potential novel approach would be to combine these two modalities in the creation of a T cell capable of secreting antitumor immunoglobulins (Ig), in essence, creating an antitumor Ig "factory" at the tumor site. In the studies reported here, we have cloned the D612 MAb Ig genes and generated a chimeric D612 IgG1 containing the murine variable region and human constant region. D612 MAb has been shown to mediate lysis of human colon carcinomas via effector cell-mediated ADCC. We have demonstrated that following transfection, chimeric D612 can be expressed and secreted by the human T-cell line MOLT-4 at a rate of 0.25 micrograms/ml per 10(6) cells in 72 hours. The secreted Ig retained its antigen-binding properties as assayed by competition radioimmunoassay and also its ability to mediate ADCC against human tumor cells. To our knowledge, this is the first demonstration of the production of a chimeric IgG by human T cells and opens the possibility of a therapeutic approach in which TILs secrete humanized antitumor MAb capable of mediating ADCC at the tumor site.

Immuno-chemotherapy of advanced colorectal cancer with alpha-2a interferon and 5-fluorouracil. Immunopharmacological studies.

Twelve patients with metastatic colorectal cancer received alternating cycles of low immunomodulating doses of alpha-IFN + 5-Fluorouracil (5-FU) or 5-FU alone. Hematological, biochemical and physical evaluation showed that both treatment cycles were well tolerated. However, transient fever and moderate flu-like symptoms were observed following alpha-IFN administration. Treatment with 5-FU alone produced long-lasting inhibition of CD8+ T lymphocytes, but did not depress NK activity (NKA). Combined treatment with alpha-IFN produced a short-term increase of NKA and antagonized the effect of 5-FU on CD8+ cells on day 5 of the cycle. Parallel studies on in vitro models showed antiproliferative effects of 5-FU on PHA-stimulated MNC and confirmed the preferential inhibition of CD8+ cells. Pretreatment with alpha-IFN did not reverse the effect of 5-FU on CD8+ lymphocytes, but partially protected MNC from the toxic effects of the drug. This was presumably due to the cytostatic effects induced by alpha-IFN on MNC before exposure to the cycle-specific antineoplastic agent. This investigation suggests that alpha-IFN could play a positive role in immuno-chemotherapy of colorectal cancer through multiple mechanisms not entirely related to direct antitumor effects of the agent.

Role of vincristine in immunochemotherapy of leukemia in mouse or human models.

Synergistic antitumor effects between Vincristine (VCR) and allograft responses have been found in mice bearing allogeneic retrovirus-induced leukemia. In this model VCR depressed weakly allograft reactivity if given before but not after antigen administration. In a parallel human tumor model in vitro using HTLV-1 induced MT-2 leukemia, additive but not synergistic immuno-chemotherapeutic effects were obtained with allogeneic mononuclear cells (MNC) combined with VCR at 0.1 but not at 1 micrograms/ml. In this case natural immunity (NI) rather than antigen-dependent immunity (ADI) was involved in the combined effects of VCR + MNC. In the in vitro model pretreatment of effector cells with 1 or 0.1 micrograms/ml of VCR depressed natural cell-mediated cytotoxicity (NCMC). However when the drug was added to the effector + target cells during the 4 h cytotoxicity assay, 1 but not 0.1 micrograms/ml of the drug was capable of depressing NCMC function. These results would provide valuable information for developing in vitro immuno-chemotherapy studies in human tumor systems, including those characterized by the presence of tumor-associated oncogenic retroviruses, capable of depressing both NI and ADI functions.

Combined effects of host antitumor immune responses and chemotherapy. Studies with hexamethylmelamine.

The antitumor activity of hexamethylmelamine (HMM) was tested in various mouse tumor models in the presence or absence of host-vs-tumor graft responses. The drug was moderately active against Sarcoma-180 growing in different strains of non-sensitized mice. Strong protection was afforded when recipients were preimmunized with irradiated tumor cells 15 days before tumor challenge followed by HMM treatment. The drug did not show antitumor activity against two radiation-induced lymphomas of congenic mice of B10 background, inoculated into H-2 compatible hosts, or into mice incompatible for subregions of H-2. In this model HMM increased mortality of allogeneic mice presumably through impairment of host-vs-lymphoma graft resistance. In conclusion this study shows that synergistic or antagonistic effects can be obtained by combining chemotherapy with antitumor immune responses.

Combined effects of immunity and antitumor drugs against cancer. II. In vitro studies with cis-diamminedichloroplatinum in a human leukemia system.

Human K562 leukemic cells were exposed in vitro to cis-diamminedichloroplatinum (DDP) followed by addition of intact or irradiated mononuclear cells (MNC) obtained from peripheral blood of normal donors. tumor inhibition provoked by DDP was significantly enhanced by normal MNC, but not by irradiated cells at the effector: leukemic cell ratio of 2:1. In contrast MNC alone did not show appreciable effects on K562 cells. The NK activity of MNC was also inhibited by exposure to gamma rays. The combined effects of DDP + MNC do not appear to be due to increased susceptibility of DDP-pretreated K562 cells to NK-mediated cytolysis. Actually leukemic cells treated with 10 micrograms/ml of DDP and cultured for 48 h at 37 degrees C, showed decline of susceptibility to the cytotoxic effects of MNC. These studies suggest that natural immunity could be of potential value in the clinical use of DDP.

Combined effects of immunity and antitumor drugs against cancer. I. In vivo studies with cis-diamminedichloroplatinum and cyclophosphamide in mouse models.

Cis-diamminedichloroplatinum (DDP) or cyclophosphamide (Cy) were given to mice bearing L1210 or LSTRA leukemia in H-2 compatible tumor-host combinations. Little anti-tumor activity was afforded by DDP against both leukemias inoculated in entirely histocompatible recipients. However, when the drug was given to mice incompatible for minor histocompatibility loci (MMHL) with the tumor, the efficacy of the treatment was markedly augmented and a substantial number of long-term survivors was found among BALB/c mice inoculated with L1210 cells. On the other hand, no difference in survival times was found between histocompatible or allogeneic mice inoculated with both leukemias, not subjected to chemotherapy. The LSTRA model was much less susceptible to this type of DDP-mediated antineoplastic immuno-chemotherapy synergism. Moreover no synergistic effects with allograft reaction were detected with Cy in both L1210 and LSTRA models, although Cy was markedly more active than DDP against leukemic cells in histocompatible recipients.

[Dynamic electrocardiography for identification of temporally altered activity in pacemakers (author's transl)]. / Utilità dell'elettrocardiografia dinamica nello svelare malfunzioni transitorie della stimolazione endocardica permanente.

Four cases of subjects with implanted pacemakers are reported. The patients temporarily presented suspected symptoms of altered activity of the pacemakers. On the other hand usual tests demonstrated a regular activity of the pacemakers. The dynamic electrocardiography, by a regular registration during 24/48 hours and during usual working, documented; once a partial hole in the circuits of the generator, twice a partial break of the catheter, once a displacement. It is underlined the importance of dynamic electrocardiography to identify temporarily altered activity of pacemakers, undiagnosticable by disposable usual tests.