Regional chemotherapy directed by individual chemosensitivity testing in vitro: a prospective decision-aiding trial.

A prospective decision-aiding trial was performed to select drugs for regional chemotherapy of various liver tumors (n = 36) by individual drug testing. The drugs were chosen for hepatic artery infusion according to the individual chemosensitivity of tumor biopsies in the human tumor colony-forming assay (HTCA). In vitro HTCA sensitivity correlated with complete response (CR) + partial response (PR) + no change (NC) 93% of the time and with CR + PR 55% of the time. The test sensitivity was 90%, and the specificity was 67% for CR + PR + NC versus progressive disease (PD), whereas the sensitivity and specificity were 89% and 28%, respectively, for CR + PR versus NC + PD. The overall predictive accuracy of the test was 86% for CR + PR + NC versus PD and 58% for CR + PR versus NC + PD. Overall, 83% of this heterogenous patient group with various tumors achieved CR + PR + NC and a 50% clinical response (CR + PR). In vitro-sensitive patients showed a significantly lower intrahepatic progression rate (7% PD) than in vitro-resistant patients (57%; P < 0.05). These results indicate that the HTCA could identify active drugs for individualized hepatic artery infusion, and patients may profit from the use of in vitro-sensitive drugs.

Flow-cytometric determination of peptide-class I complex formation. Identification of p53 peptides that bind to HLA-A2.

A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.

Thymidylate synthase is a predictor for response and resistance in hepatic artery infusion chemotherapy.

The value of intratumoral thymidylate synthase (TS) quantitation as a predictive parameter for hepatic artery infusion (HAI) chemotherapy in patients with colorectal liver metastases was investigated. Relative TS mRNA levels were determined in 29 tumor samples using a quantitative RT-PCR amplification method. The median level of expression was 3.0 x 10(-3) (no units) and varied considerably among the tumors over a range of 135-fold. Patients with low TS levels were 4.1-fold more likely to respond (P < 0.03) compared to patients with high TS levels. Our results indicate that TS quantitation is a valuable predictive marker for tumor response to HAI therapy.

Mutational inactivation of the p53 gene in the human erythroid leukemic K562 cell line.

The K562 human chronic myelogenous leukemia (CML) cell line has attained widespread use as a model for studying hematologic malignancy and erythroid differentiation. Sequencing of the p53 gene in the K562 cell line demonstrated a mutation in exon 5 characterized by a single base insertion (cytosine) between codons 135 and 136. This frameshift mutation leads to an N-terminal truncated protein of 147 amino acids. Only the mutated sequence was present suggesting that the normal allele has been lost. Reverse transcription PCR (RT-PCR) detected a p53 transcript but Western blotting and immunohistochemical staining of cells failed to detect p53 protein. The identification of an inactivation mutation of p53 in the K562 cell line further supports the argument that p53 mutations play a role in myeloid blast transformation of CML.

Palliative and adjuvant regional chemotherapy in pancreatic cancer.

To improve the dismal prognosis of patients (pts) with pancreatic cancer we treated 32 patients with non-resectable (UICC III, 17 pts; UICC IV, 15 pts--group 1) and 20 patients with resected (UICC I, 1 pt; UICC II, 3 pts; UICC III, 16 pts--group 2) pancreatic cancer with palliative (group I) and adjuvant post-operative (group II) coeliac axis intra-arterial cyclic infusions (CAI). CAI consisted of mitoxantrone 10 mg/m2 on day 1, folinic acid 170 mg/m2 and 5-FU 600 mg/m2 during days 2-4, and cis-platinum 60 mg/m2 on day 5 for up to 11 (group I) or six (group II) cycles. In a total of 211 cycles toxicities at the level of WHO III occurred in 0-6% and of WHO IV in 0%. The median survival times, compared with institutional historical controls (treated vs controls), were 12 vs 4.8 months in UICC III (P < 0.006) and 4 vs 2.9 months in UICC IV (P < 0.05) group I pts, and 21 vs 9.3 months in group II (P < 0.0003). Hepatic disease progression appeared to be suppressed with CAI, which also appears to be effective for palliative and adjuvant treatment in non-resectable and resected pancreatic cancer.

In vitro and in vivo cytotoxicity of alkyl lysophospholipid ET-18-OCH3 and thioether lipid BM 41.440.

Screening for cytotoxicity in the clonogenic assay in human tumor xenografts and L1210 mouse leukemia revealed comparable dose-dependent effects of the alkyl lysophospholipid ET-18-OCH3 and the thioether lipid BM 41.440. The efficacy in human tumors only was marginal at low doses. In vivo tests of both agents were carried out in nude mice bearing two of the tumors that proved most sensitive in vitro and in mice inoculated with L1210 leukemia. Only small effects on the growth of the human tumors and no effects on L1210 leukemia were observed. In view of clinical rules for definition of remission, no convincing antitumor effects were obtained.

Theoretical considerations and in vitro concentration response studies with two human colorectal carcinoma cell lines. The rational experimental base for clinical studies in regional chemotherapy.

The theoretical advantage of regional vs. systemic chemotherapy was calculated, based on pharmacokinetic considerations. The relevance of exposure time and high local concentrations of chemotherapeutic drugs for regional chemotherapy was elucidated in time dependent concentration response curves with two human cell lines. The theoretical pharmacological advantage of regional vs. systemic chemotherapy was defined by the formula Rd = (AUCi. a. 1 + AUCi. a. 2)/(AUCi. v.) and in hepatic artery infusion is for adriamycin (ADM) 5.8-.6, cis-platinum (CDDP) 8, epirubicine (EPI) 6.3, 5-fluorouracil (5-FU) 22-58, mitomycin C (MMC) 4.6, mitoxantrone (NOV) 6.3. All drugs but 5-FUDR exerted concentration response behaviour in the cell line-experiments. In the cell lines cytotoxicity depended on exposure time so that concentration chi time products at (IC50), (c chi t (IC50)), were calculated to determine an optimal in vitro exposure time. Based on these results and clinical considerations, optimal clinical exposure times could be defined for regional chemotherapy. The results may be of high relevance for e.g. hepatic artery infusion at the lower and chemoembolization or intraperitoneal instillation at the higher test concentration, respectively.

Morphological studies on the subchronic toxicity of a standardized liver- and spleen-extract.

A short-term safety study on the liver- and spleen-extract Factor AF2 has been carried out. Daily injections of 4 or 33.3 mg/kg body weight for six weeks were not followed by drug-induced changes.

Addition of interleukin 12 to low dose interleukin 2 treatment improves antitumor efficacy in vivo.

Interleukin 12 (IL-12) enhances lysis mediated by NK- and lymphokine activated killer (LAK) cells. It also causes proliferation of IL-2 stimulated T and NK cells in vitro. For these IL-2 complementing properties murine pulmonary metastases of a coloncarcinoma line were treated with IL-12 and IL-2 or with the individual agents. Results were compared to sham treated controls. IL-2 alone mediated significant tumor reduction but provoked pulmonary edema and concomittand toxicity, graded in three steps. IL-12 combined with an IL-2 dose reduced by 81% still resulted in significant antitumoral activity. Toxicity, however, was not discernable from sham treated controls. IL-12 thus appears as an attractive cytokine for combination with IL-2 in antitumor therapy. Particularly treatment of tumors, like gastrointestinal tract cancers, so far mainly resistant to cell mediated antitumor therapy, might profit from this approach.

Inhibition of nitric oxide synthesis does not improve interleukin-2-mediated antitumor effects in vivo.

Nitric oxide (NO) decreases cytotoxicity and proliferation of cytotoxic lymphocytes (CTLs) in vitro. Both can be prevented by inhibitors of the NO synthase (NOS). To elucidate whether inhibition of the IL-2-induced NOS could boost efficacy of IL-2-stimulated CTLs in vivo, we assessed lung metastases in mice injected with IL-2, the NOS inhibitor aminoguanidine (AG), their combination and the diluent. No improvement was observed for IL-2 + AG compared to IL-2 while NO production was normalized. Since NO causes one of the two major side effects of IL-2 treatment, hypotension, we further studied whether capillary leak could be attributed to NO, too. While IL-2-inducible NO was reduced to control levels by AG, pulmonary edema was unaffected. Thus a decrease in NO does not improve antitumor effects of IL-2-stimulated CTLs nor does it attenuate IL-2-associated capillary leak.

Identification of wild-type and mutant p53 peptides binding to HLA-A2 assessed by a peptide loading-deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay.

Mutations of the p53 gene are the most frequently observed genetic changes in human cancers; often leading to an overexpression of the wild-type (wt) p53 protein. Demonstrable T cell reactivity against tumor cells overexpressing wt or mutant p53-derived peptides could support the application of such epitopes in cancer immunotherapies. As the binding of peptide to MHC class I molecules is a prerequisite for antigen-specific T cell recognition, we evaluated the ability of wt and mutant p53 peptides to bind to HLA-A2.1 using two independent flow cytometry-based assay systems, the T2 major histocompatibility complex (MHC) class I peptide stabilization assay (stabilization assay) and the peptide-induced MHC class I reconstitution assay (reconstitution assay). The twenty selected wt sequences each conformed to the previously reported HLA-A2.1 peptide binding motif. Seven of the wt p53 and 2/13 mutant p53 peptides derived from the previously chosen wt peptides bound to HLA-A2.1 in both the stabilization and the reconstitution assays. An additional six wt and six mutant p53 peptides, presumably exhibiting lower affinity for HLA-A2.1, were identified only in the reconstitution assay. Those p53 peptides binding HLA-A2.1 may provide useful immunogens for the generation of HLA-A2.1-restricted cytolytic T lymphocytes in vitro and in vivo.