Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer.

BACKGROUND: Plasma circulating tumour-specific microRNAs (miRNAs) are promising biomarkers of tumour presence and recurrence, especially for diseases whose best chance of successful treatment requires early diagnosis and timely surgery of an already malignant but not yet invasive tumour, such as colorectal cancer (CRC). METHODS: Expression levels of miRNAs previously found to be differently expressed in tumour vs normal colon tissues were investigated by quantitative real-time PCR in plasma from CRC patients and from healthy donors and confirmed in independent case control series. The validated miRNAs were also measured after surgery. Analyses were repeated on the subsets of haemolysis-free samples. RESULTS: We identified four miRNAs differently expressed between the compared groups, two (miR-21 and miR-378) of which were validated. miR-378 expression decreased in non-relapsed patients 4-6 months after surgery and miR-378 ability to discriminate CRC patients from healthy individuals was not influenced by haemolysis levels of plasma samples. CONCLUSION: The miRNA analysis on plasma samples represents a useful non-invasive tool to assess CRC presence as well as tumour-free status at follow-up. Plasma levels of miR-378 could be used to discriminate CRC patients from healthy individuals, irrespective of the level of haemoglobin of plasma samples.

Differences between in vivo and in vitro activation of cancer patient lymphocytes by recombinant interleukin 2: possible role for lymphokine-activated killer cell infusion in the in vivo-induced activation.

In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.

Comparative effects of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor after high-dose cyclophosphamide cancer therapy.

PURPOSE: We compared hematologic and clinical effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) after treatment with high-dose cyclophosphamide (HD-CTX, 7 g/m2), given as the first phase of a high-dose sequential chemotherapy program that includes a myeloablative therapy with mobilized progenitor cell autografting. PATIENTS AND METHODS: Forty-nine consecutive patients with non-Hodgkin's lymphoma, Hodgkin's disease, or poor-prognosis breast cancer received GM-CSF (n = 27) or G-CSF (n = 22) after HD-CTX in two consecutive, nonrandomized studies. Cytokines were administered in continuous intravenous (i.v.) infusion for 14 to 15 days at a median dose of 5.5 and 10 micrograms/kg/d, respectively, starting 24 hours after HD-CTX. RESULTS: Neutrophil recovery was faster with G-CSF administration (11.5 v 13.2 days; P = .01), whereas platelet counts recovered more rapidly with GM-CSF (13.7 v 16.6 days; P = .01). Prophylactic platelet transfusions were administered more frequently to patients treated with G-CSF than with GM-CSF (66% v 22% of the patients; P = .02). No clinically significant difference was observed between the two groups concerning days of absolute neutropenia or neutropenic fever. Both cytokines reduced the time to eligibility for subsequent chemotherapy administration compared with historical controls not given cytokine (14 to 16 v 20 days). Both cytokines increased circulation of hematopoietic progenitors. Most side effects were World Health Organization (WHO) median grade 1 to 2, were more frequent during GM-CSF than during G-CSF treatment, and were reversible by simple supportive measures and/or by dose reduction or suspension of the cytokine. Permanent suspension of cytokine administration was never required in either group. CONCLUSION: GM-CSF or G-CSF administration after HD-CTX reduces hematologic toxicity of high-dose chemotherapy and induces circulation of large amounts of hematopoietic progenitors suitable for autografting in cancer patients.

Lack of T-cell-mediated recognition of the fusion region of the pml/RAR-alpha hybrid protein by lymphocytes of acute promyelocytic leukemia patients.

In previous studies, it was shown that the fusion region of the pml/RAR-alpha protein, expressed by acute promyelocytic leukemia (APL) cells, can be specifically recognized in vitro by donor (D. E. ) CD4 T cells in a HLA class II DR11-restricted fashion. We present here the results on the recognition of several pml/RAR-alpha peptides by APL patients expressing HLA DR11. The in vitro immunization of peripheral blood lymphocytes from four patients in remission (S. R., F. R., M. M., P. G.) with BCR1/25, a 25-mer pml/RAR-alpha, did not elicit either a polyclonal or a clonal immune response specific to the peptide. We then generated new donor anti-pml/RAR-alpha CD4(+) T-cell clones. These clones were tested for their recognition of BCR1/25. One clone (C3/5, CD3(+), CD4(+), CD8(-)) was selected for further analysis. Clone C3/5 showed specific proliferation, cytotoxicity, and cytokine (tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor) production when challenged with autologous lymphoblastic cell lines pulsed with peptide BCR1/25. C3/5 cells developed specific proliferation and cytotoxicity when challenged with peptide-pulsed lymphoblastic cell lines and peripheral blood lymphocytes from the four DR11(+) APL patients. APL blasts, available only from patients F. R. and P. G., were not lysed by C3/5 and were unable to present peptide BCR1/25. Incubation of APL cells with IFN-gamma failed to induce HLA class II molecules and recognition by the C3/5 clone. Since APL cells do not express HLA class II molecules, we tested in two donors (D. E. and C. H. R.) and in patients S. R. and P. G. whether the use of 9-mer peptides (BCR1/9) would generate a CD8/HLA class I-restricted response. No peptide-specific T-cell line or clone could be generated from both donors and patients. These findings are discussed in relation to possible therapeutic approaches to the immunotherapy of APL.

Differential recognition of a BCR/ABL peptide by lymphocytes from normal donors and chronic myeloid leukemia patients.

The BCR/ABL oncogenic fusion protein transforms normal bone marrow stem cells into neoplastic cells. It has been shown that peptides derived from the junctional region of this oncogenic fusion protein can be recognized by human T-lymphocytes obtained from normal donors. In this study, we investigated the immunogenicity in patients with chronic myeloid leukemia (CML) of a 17 mer b3/a2 Bcr/abl peptide (B/A1), which was shown to induce proliferative responses in lymphocytes from normal donors. A total of 56 CML patients in chronic phase were studied. Twenty-two patients were studied at diagnosis without any treatment (group I). Fourteen patients were receiving IFN (group II), 14 patients were being treated with hydroxyurea (group III), and 6 patients were on different regimens (group IV). Patients were initially assessed for general immunological competence using both in vivo and in vitro assays. Patients were also selected for the expression of HLA-DR0401, the HLA specificity known to present peptide B/A1 to CD4 lymphocytes. With the exception of the six patients in group IV, the results of all these assays (in vitro phytohemagglutinin/tetanus toxoid responses, in vivo skin reaction to ubiquitous antigens) in CML patients did not significantly differ from those obtained in normal donors, thus excluding the presence of generalized immunosuppression. Eight patients with HLA-DR0401 and a b3/a2 type of fusion were identified and further studied. In these eight patients dendritic cells were obtained from adherent peripheral blood mononuclear cells and used to stimulate CD4 lymphocytes. No patient developed a specific response to the bcr/abl peptide, although patients' lymphocytes proliferated in response to a promiscuous tetanus toxoid peptide in all but one case. In contrast, response to the bcr/abl peptide was observed in seven of eight HLA-DR0401 healthy donors tested. These data suggest that immunocompetent, HLA-DR0401+ CML patients are unable to respond to peptide B/A1, at difference from healthy donors. The implication of these results for the immunotherapy of CML is discussed.

Massive ex vivo generation of functional dendritic cells from mobilized CD34+ blood progenitors for anticancer therapy.

We report that blood cell autografts, collected by single leukapheresis in cancer patients (n = 11) at the time of mobilization of hematopoietic progenitors into peripheral blood following anticancer therapy with high-dose cyclophosphamide (HD-CTX) plus interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF/filgrastim), comprise 1.98 +/- 0.39 x 10(5)/kg (mean +/- SE) CD34+ progenitors of dendritic cells (DCs). This number corresponds to 140-fold more progenitors than in a control autograft collected in the steady state. DCs derived from mobilized CD34+ cells, morphologically and immunophenotypically undistinguishable from skin Langerhans cells and DCs from bone marrow and cord blood CD34+ cells, are shown to be powerful stimulators of allogeneic T cell proliferation in primary MLR and of autologous HLA-DR-restricted CD4+ T cell proliferation in response to presentation of xenogenic antigens. We show that the GM-CSF-plus-TNF-alpha-dependent ex vivo generation of DCs from mobilized CD34+ cells is 2.5-fold enhanced by flk-2/flt-3 ligand or c-kit ligand (stem cell factor) and five-fold enhanced by a combination of these growth factors. In addition, the optimal serum for the generation of DCs is autologous HD-CTX recovery-phase serum rather than fetal calf serum (FCS) or steady-state human serum, which are clinically inadequate and ineffective, respectively. In practice, the stimulation of CD34+ cells in a blood cell autograft (15.75 +/- 2.46 x 10(6)/kg) provided by the above four growth factors should permit ex vivo generation of approximately 40 x 10(9) DCs in an adult patient. These new findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.

Recombinant adenoviral vector-lipofectAMINE complex for gene transduction into human T lymphocytes.

We have evaluated, as a vector for gene transfer into human T lymphocytes, a recombinant adenovirus (rAd-MFG-AP) carrying a modified, membrane-exposed, alkaline phosphatase (AP) as reporter gene. CD3+ cells were selected from the buffy coat of healthy donors by the immunomagnetic technique. The positive cell population, comprising 96+/-2% CD3+ cells, was cultured with clinical-grade cytokine(s) for 3-7 days prior to rAd-MFG-AP transduction and the transgene expression was evaluated 48 hr later by indirect immunofluorescence flow cytometry assay with an anti-alkaline phosphatase antibody. The best efficiency of transduction was achieved on incubation of CD3+ cells with IL-2 plus either IL-12 (AP+ cells, 12+/-3%) or IL-7 (AP+ cells, 11+/-3%). To increase further the efficiency of transduction, we have combined LipofectAMINE and rAd-MFG-AP with the aim to enhance the uptake of viral particles into the target cells. The percentage of CD3+ cells transduced by rAd-MFG-AP-LipofectAMINE complex was 24+/-4% (range, 20-35%) after incubation with IL-2 plus IL-7 and 22+/-4% (range, 18-32%) after incubation with II-2 plus IL-12. Forty-eight hours after the incubation with rAd-MFG-AP, the transduced T lymphocytes were subjected to fluorescence-activated cell sorting and fractionated into AP+ and AP- cell subpopulations. The AP+ cell fraction, comprising 96.8% of AP+ cells, was evaluated by FACScan analysis for T lymphocyte surface antigens. The immunophenotyping of the transduced T lymphocytes has shown that there was not a particular subtype of T lymphocytes more susceptible to rAd-MFG-AP transduction. In addition, the transgene expression did not modify T lymphocyte functions, as demonstrated by results obtained by cytotoxicity assay before and after rAd-MFG-AP-LipofectAMINE complex transduction. In conclusion, human T lymphocytes can be efficiently transduced, under clinically applicable conditions, by adenovirus-LipofectAMINE complex after 7 days of culture with IL-2 and IL-12 or IL-7.

Large-scale collection of circulating haematopoietic progenitors in cancer patients treated with high-dose cyclophosphamide and recombinant human GM-CSF.

Circulating haematopoietic progenitors from 36 cancer patients were collected by continuous-flow leukapheresis during the phase of rapid haematopoietic recovery after pancytopenia induced by high-dose cyclophosphamide and then cryopreserved for autologous transplantation. 20 of the patients also received intravenous infusion of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) for 7, 10 or 14 days after cyclophosphamide. 106 leukapheresis procedures were done for 2-5 consecutive days. Leukapheresis was started significantly earlier in patients receiving rhGM-CSF. In these patients, yields of peripheral blood elements (leucocytes, mononuclear cells, haematopoietic progenitors and platelets) were significantly higher than in controls treated with cyclophosphamide only. In particular, the mean number of granulocyte-monocyte colony-forming cells was 43.88 X 10(4) vs. 6.16 X 10(4) per kg patient body weight per leukapheresis. Side-effects of leukapheresis were limited to central venous catheter occlusion and fever in 4% and 2% of all procedures, respectively.

Curability of advanced Burkitt's lymphoma in children by intensive short-term chemotherapy.

The treatment programme (regimen I) we designed in 1982 for advanced Burkitt's lymphoma was modified in 1986 as regimen IIA and IIB for patients presenting without or with bone marrow (BM) and/or nervous system involvement, respectively. Following a 5-week course of cytoreductive chemotherapy, including vincristine (VCR), cyclophosphamide (CPM), doxorubicin (DXR), high-dose methotrexate (HDMTX) and intrathecal methotrexate and cytarabine (ARAC), high-dose ARAC and cisplatin were given as a 4-day continuous infusion. Regimen I continued with an additional 3-week course including VCR, CPM, DXR and HDMTX, which was omitted in regimen IIA. In regimen IIB the initial cytoreductive chemotherapy was complemented by adding etoposide and increasing HDMTX doses, and by modifying the high-dose ARAC administration modality and was followed, once the bone marrow had recovered, by ifosfamide that concluded the programme. A total of 44 children (22 in regimen I and 22 in regimens IIA and IIB) were treated, with an overall response rate of 98%. 4 patients died as a result of treatment related complications. Survival, progression-free and event-free survival rates were 73, 70 and 63%, respectively, for regimen I, and 82, 90 and 82%, respectively, for regimen IIA and IIB. A short chemotherapeutic regimen, using alternating phase-specific and non-specific agents, is able to cure the majority of patients with advanced Burkitt's lymphoma.

Diversity of effects of two antitumor anthracycline analogs on the pathway of activation of PKC in intact human platelets.

Two antitumor antibiotics doxorubicin and daunorubicin were tested for their ability to influence the activation of protein kinase C in human platelets. Daunorubicin was found to inhibit the phosphorylation of the 40 K PKC substrate induced by thrombin and 12-O-tetradecanoyl-phorbol-13-acetate as well as the phosphorylation of the 20 K protein induced by thrombin. The serotonin release associated to these phosphorylative events was also inhibited by daunorubicin. In contrast the effects of doxorubicin, though inhibitory on the release reaction, were always stimulatory of the phosphorylations. Doxorubicin alone was able to induce the phosphorylation of both 40 K and 20 K phosphoproteins in a concentration-dependent manner. Whereas the stimulation by doxorubicin was not influenced by pretreatment with dibutyryl-cyclic-AMP which inhibits the effects of thrombin, this effect was inhibited by daunorubicin, neomycin and stimulated by the diacylglycerol-kinase inhibitor R 59 022. It is proposed that doxorubicin activates the protein kinase C by causing the breakdown of phosphoinositides.

Large-scale enrichment of mobilized CD34+ peripheral blood hematopoietic progenitors by removal of nylon wool-adherent mature cells.

With the aim of facilitating the ex vivo manipulation of peripheral blood hematopoietic progenitors (CPCs = circulating progenitor cells) collected by leukapheresis, we removed polymorphonuclear cells and monocytes that naturally adhere to nylon wool fibers. Leukapheresed cells harvested at the time of hematopoietic recovery after cancer therapy with high-dose cyclophosphamide plus hematopoietic growth factors were incubated with nylon wool fibers for 1 h at 37 degrees C. Evaluation of the cells non-adherent to the nylon wool in all experiments (n = 14) showed that the median recovery of nucleated cells and CPCs detected as CD34+ cells, CFU-GM and BFU-E was 16.4% (range 4.8%-34.0%), 60.0% (range 30.8-80.8%), 60.9% (range 33.4-74.5%) and 65.5% (range 30.8-69.2%), respectively. Therefore exposure to the nylon wool determined a selective removal of mature cells and a complementary enrichment of CPCs. The wide range of results depended on the significantly different cell compositions of the unmanipulated leukaphereses. The latter from patients receiving rhG-CSF (n = 10) comprised a median of 88.5% (range 77.8-93.8%) and 11.5% (range 6.2-22.2%) polymorphonuclear and mononuclear cells, respectively. In contrast, leukaphereses from patients receiving rhGM-CSF or PIXY321 (n = 4) comprised a median of 71.1% (range 55.4-85.0%) and 28.9% (range 15.0-44.6%) polymorphonuclear and mononuclear cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Benefits of blood cell transplant cryopreservation with oxypolygelatine (Gelifundol) plasma substitute.

In two groups of 11 patients with poor prognosis malignancies undergoing high-dose sequential chemotherapy, we have evaluated the cryopreservation of blood cell transplants with oxypolygelatine-containing (55% oxypolygelatine, 6% hydroxyethylstarch, 5% dimethyl sulfoxide) vs standard human serum-containing (55% human serum, 6% hydroxyethylstarch, 5% dimethyl sulfoxide) cryoprotectant mixtures. Evidence is presented demonstrating that substitution of human serum proteins with oxypolygelatine has no detrimental effect either in vitro on the post-thawing recovery of hematopoietic progenitors or in vivo on the capacity of marrow reconstituting function in patients treated with myeloablative cancer therapy and autologous blood cell transplant. Oxypolygelatine is commercially available for clinical use as a plasma expander, is 30-fold less expensive than human serum albumin, is certified free of foreign serum proteins and antibodies as well as free of pyrogen, viral, mycoplasmal and bovine spongiform encephalopathy contaminants. Because of these characteristics, oxypolygelatine permits avoidance of: (1) the use of expensive serum albumin; (2) the fastidious preparation of autologous plasma or serum, and (3) the risk of infection associated with the infusion of allogeneic serum. Because of these practical advantages, we recommend the clinical use of oxypolygelatine as a substitute for human serum proteins for the routine cryopreservation of blood cell transplants.

Elimination of bcl-2-IgH-positive follicular lymphoma cells from blood transplants with high recovery of hematopoietic progenitors by the miltenyi CD34+ cell sorting system.

Contamination of autologous blood cell transplants with cells of follicular non-Hodgkin's lymphoma (F-NHL) may contribute to relapse of the malignancy after potentially curative high doses of chemotherapy and radiotherapy. In an attempt to circumvent this limitation, we have evaluated various techniques of selection of CD34+ cells to eliminate malignant cells from blood cell transplants of five patients with F-NHL undergoing high-dose sequential therapy. The contamination of F-NHL cells was evaluated using a nested PCR assay for the detection of bcl-2-IgH rearrangement with a sensitivity of one F-NHL cell in 10(5) normal cells. In two experiments with blood cell transplant fractions of 0.5 x 10(9) nucleated cells, negative selection of CD34+ cells by removal of B cells and other mature cells that naturally adhere to nylon wool fibers decreased the number of CD19+ B cells detectable by flow cytometry but failed to eliminate bcl-2-IgH-positive F-NHL cells detectable by PCR. In contrast, positive selection of CD34+ cells by the Miltenyi MiniMACS high gradient magnetic cell sorting system in five separate experiments resulted in: (1) the elimination of F-NHL cells in four out of five cases as detected both flow cytometry and bclk-2-IgH PCR; (2) a highly purified population of hematopoietic progenitors comprising 90.8% +/- 2.3% CD34+ cells; and (3) the recovery of 77.9% +/- 3.2% CD34+ cells. These favorable results were confirmed on a large-scale with a blood cell transplant comprising 5.8 x 10(9) nucleated cells in which positive selection of CD34+ cells by the Miltenyi SuperMACS system resulted in: (1) the elimination of F-NHL cells as detected both by flow cytometry and bcl-2-IgH PCR; (2) a highly purified population of hematopoietic progenitors comprising 94.6% CD34+ cells; and (3) the recovery of 62.7% CD34+ cells. These results, attained with the newly available Super MACS system, compare favorable with previous techniques because they show the feasibility of eliminating F-NHL cells from blood cell transplants without relevant nonspecific loss of hematopoietic progenitors.

A new polymorphism (Ser362Thr) of the L-myc gene is not associated with lung adenocarcinoma risk and prognosis.

The non-coding variation in the second intron of the L-myc gene, generating an EcoRI polymorphism, is associated with lung cancer risk and prognosis. We carried out sequence analysis of the L-myc gene in lung adenocarcinoma (ADCA) patients to identify functional polymorphisms and identified a new single nucleotide polymorphism (SNP) in the third exon of the gene causing a Ser362Thr conservative amino acid change in the C-terminus of the encoded protein. This polymorphism showed significant linkage disequilibrium with the L-myc EcoRI polymorphism located at 1751 bp distance. Genotyping of the Ser362Thr SNP in 220 Italian ADCA patients and in 230 general population controls revealed a similar low frequency (0.10-0.11) of the Thr allele in both groups. The multivariate odds ratio was 0.68 (95% confidence interval (CI) 0.38-1.22). In the ADCA patients, no significant association between the Ser/Thr polymorphism and survival was observed. Thus, the present results do not support candidacy of the L-myc Ser362Thr polymorphism for the functional polymorphism of the L-myc genomic region.

A double determinant radioimmunoassay Mov2-MOv8 for monitoring ovarian carcinomas: definition of the methodology.

A double-determinant radioimmunoassay for the detection of circulating antigens associated with human ovarian carcinoma was developed using two monoclonal antibodies: MOv2 and MOv8 employed respectively as catcher and tracer. The development of the method through three different procedures enabled us to detect the presence of CaMOv2-CaMOv8 carrying molecules in 14 out of 15 ascitic fluids from ovarian carcinoma patients whose tumors were found to be positive with MOv2 and MOv8 monoclonal antibodies by immunofluorescence. Moreover, 13 out of 15 ovarian carcinoma patients presented high levels of antigen in their serum (60-170 Ua/ml). Low levels of antigen were observed in the normal population, the values ranging from 30-40 Ua/ml. However, in 13 out of 100 apparently healthy women high levels of antigen were found in the serum.

Determinants of Helicobacter pylori seroprevalence among Italian blood donors.

BACKGROUND/AIM: Helicobacter pylori is a worldwide infection. It is estimated that approximately 50% of the general population is affected, but this percentage varies considerably between countries. To investigate the prevalence of H. pylori infection, a cross-sectional epidemiological study, based on the serological determination of the IgG antibodies against H. pylori, was carried out in healthy Italian blood donors by using a commercially available kit. METHODS: From March 1995 to March 1997, a total of 2598 consecutive volunteer blood donors were tested for the presence of antibodies against H. pylori. All patients answered a detailed questionnaire which collected sociodemographic characteristics, and smoking, alcohol drinking and dietary habits. Test-positive subjects with gastrointestinal symptoms underwent endoscopy, with biopsies taken for histological diagnosis. RESULTS: The global prevalence of H. pylori infection in our study was 1161/2598 (45%). It was directly correlated with age (67% in subjects aged > or = 50 years). The prevalence of H. pylori infection was higher in men (46.4%) than women (38.4%), and more frequent in patients with a low educational level, in the lower quintile of height and in the upper quintile of body mass index (BMI). No significant association with smoking and alcohol drinking was found. Inverse associations were found with elevated consumption of milk (chi-square for trend 5.49, P < 0.05), but not other examined food groups. Multivariate analysis selected sex, age, BMI and educational level as the variables independently related to H. pylori infection. CONCLUSION: This study confirms relatively high prevalence of H. pylori seropositivity among Italian healthy adults and points to sex, age, BMI and sociocultural class as persisting determinant features of H. pylori infection.

Adjuvant therapy with essential fatty acids (EFAs) for primary liver tumors: some hypotheses.

Hepatocarcinoma is responsible for approximately 1 million deaths annually. It is usually discovered at an advanced stage and, if inoperable, has a poor prognosis. New therapies combining chemotherapy, hyperthermia, radiotherapy and immunomodulators have been recently attempted with various levels of success. Once the tumor is detected at an early stage, some possibilities of cure seem to emerge either by intratumoral percutaneous injection (PEI) of alcohol or by chemoembolization and interstitial hyperthermia. When the tumor volume is more than 5 cm, these therapies are less successful and radiotherapy can be used. All the techniques described have some limits; PEI, for instance, does not achieve a complete eradication of lesions > 3 cm and a non-homogenous alcohol distribution within the tumor leads to areas of necrosis. Radiotherapy, even if effective, is limited by dose-related radiation hepatitis. Another important limiting factor is the incomplete response to therapy and tumor recurrence. Essential fatty acids, especially gamma linolenic acid (GLA) and eicosapentaenoic acid (EPA) are discussed here for their ability to control primary tumor proliferation and increase response to chemotherapy, radiotherapy and hyperthermic treatment, thanks to their effects on cellular membranes (increased lipoperoxidation and modification of tumor stroma).

HLA binding characteristics and generation of cytotoxic lymphocytes against peptides derived from oncogenic proteins.

AIMS AND BACKGROUND: Structurally altered proteins (derived from chromosomal translocations or gene mutations) can be considered tumor specific antigens and represent an attractive target for a T-cell mediated response. T lymphocytes recognize antigens in the form of peptides bound to HLA-molecules. MATERIALS AND METHODS: Peptides derived from oncogenic proteins were screened for the presence of HLA binding motifs; actual binding were evaluated by HLA stabilization experiments using transfectants and specific anti-HLA antibodies. Specific lymphocytes were induced by in vitro peptide sensitization and screened by thymidine uptake or cellular cytotoxic assays. RESULTS: We identified peptides derived from EWS/FLI-1 fusion protein and from mutated K-RAS protein (encompassing respectively the fusion point and the mutation at position 12) that showed binding motif for HLA-Cw*0702 and HLA-A3 respectively. The actual binding of these peptides was analysed in a stabilization assay. We detected binding for the EWS/FLI-1 peptide and for 5 RAS peptides (1 wild type and 4 mutated). The effect of temperature, beta 2-microglobulin (beta 2-m) and fetal calf serum (FCS) on the binding and the stability of the HLA/peptide complex was studied. A low temperature (26 degrees C) increased the binding both in HLA-A3 and HLA-Cw*0702, while FCS reduced it. beta 2-m increased the binding to the HLA-A3 molecule but did not influence the binding to the HLA-Cw*0702. The stability of already formed complexed was somewhat different in the HLA-A3 and HLA-Cw*0702 system: both were more stable at 26 degrees C than at 37 degrees C but while the beta 2-m and FCS did not influence the stability of the HLA-A3/peptide complex, they seemed to cause opposite effects in the HLA-Cw*0702 system (beta 2-m stabilized and FCS destabilized the complex). Finally, we were able to generate a specific CD8+ CTL line against a K-RAS mutated peptide. CONCLUSIONS: Although binding motifs and actual HLA binding can be detected in several cases, the generation of a cellular response is infrequent, confirming that HLA binding is necessary but not sufficient to obtain an in vitro response. Further optimization of culture conditions, type of Antigen Presenting Cells (APC), peptides, use of stabilizers like beta 2-m are still needed.

Local adoptive immunotherapy of advanced head and neck tumors with LAK cells and interleukin-2.

Since October 1987 a pilot phase I-II study on the effect of loco-regional injections of recombinant interleukin 2 (rIL-2) in association with LAK cells has been performed in advanced, recurrent head and neck cancer patients. Fourteen patients were treated with autologous LAKs and rIL-2 (Glaxo) given peritumorally and in the mastoid region (rIL-2 only in the latter site). LAKs (2-70 x 10(7)) + rIL-2 were injected on the first day of therapy, followed by 9 daily injections of rIL-2 only. The total daily dose of rIL-2 was escalated from 2,400 to 1.8 x 10(6) IU. Clinical evaluation was performed 30 days from the onset of therapy; 3 partial (95%, 66% and 50% reduction) and 3 minor responses were seen in the evaluated patients. All the other patients with a progressive disease after the first cycle were shifted to palliative chemotherapy. The partial responses were found in patients with a tumor burden less than 20 cm2. Cervical node metastasis did not respond to treatment. No relevant side effects occurred. These results indicate that loco-regional immunotherapy with rIL-2 and LAK cells can produce clinical responses in advanced head and neck cancer patients.

Inhibition of human melanoma growth in nude mice by autologous, alloactivated peripheral blood lymphocytes.

Peripheral blood lymphocytes of melanoma patients were stimulated in vitro by a pool of allogeneic lymphocytes and shown to be cytotoxic against autologous melanoma cells. To evaluate the in vivo antitumor activity of the cytotoxic alloactivated autologous peripheral blood lymphocytes, tumor neutralization (Winn) assay was carried out by injecting such lymphocytes admixed with autologous melanoma cells in athymic BALB/c nude mice. In 3 of 6 cases, complete inhibition of tumor growth was obtained at lymphocytes to tumor cells ratio of 10:1 and in one case also of 5:1. In all cases the appearance of tumors was delayed and the growth rate was significantly reduced in a dose-dependent fashion as compared to control mice injected with tumor cells alone. We conclude that in vitro alloactivated peripheral blood lymphocytes can inhibit and/or impair the growth of autologous melanoma cells in nude mice.