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Cancer patients often suffer from cancer symptoms, treatment complications and concomitant diseases and are, therefore, often treated with several drugs in addition to anticancer drugs. Whether such drugs, here denoted as ‘concomitant drugs’, have anticancer effects or interact at the tumor cell level with the anticancer drugs is not very well known. The cytotoxic effects of nine concomitant drugs and their interactions with five anti-cancer drugs commonly used for the treatment of colorectal cancer were screened over broad ranges of drug concentrations in vitroin the human colon cancer cell line HCT116wt. Seven additional tyrosine kinase inhibitors were included to further evaluate key findings as were primary cultures of tumor cells from patients with colorectal cancer. Cytotoxic effects were evaluated using the fluorometric microculture cytotoxicity assay (FMCA) and interaction analysis was based on Bliss independent interaction analysis. Simvastatin and loperamide, included here as an opioid agonists, were found to have cytotoxic effects on their own at reasonably low concentrations whereas betamethasone, enalapril, ibuprofen, metformin, metoclopramide, metoprolol and paracetamol were inactive also at very high concentrations. Drug interactions ranged from antagonistic to synergistic over the concentrations tested with a more homogenous pattern of synergy between simvastatin and protein kinase inhibitors in HCT116wt cells. Commonly used concomitant drugs are mostly neither expected to have anticancer effects nor to interact significantly with anticancer drugs frequently used for the treatment of colorectal cancer.

Background. Little is known about the in vitro activity and cross-resistance patterns of topoisomerase II (topo II) inhibitors in primary cultures of tumor cells from patients with different diagnoses. Methods. The in vitro activity of the topo II inhibitors daunorubicin, doxorubicin, idarubicin, epirubicin, amsacrine, mitoxantrone, and etoposide was determined using the fluorometric microculture cytotoxicity assay. Four hundred seventy-six samples from patients with various diagnoses were tested with continuous drug exposure. Results. Hematologic samples were more sensitive than solid tumors to all tested drugs. Etoposide was the most active drug in solid tumors followed by epirubicin, with amsacrine the least active. The anthracyclines dox orubicin, daunorubicin, and epirubicin showed similar in vitro activity. Conversely, idarubicin showed low to moderate cross-resistance with all tested topo II inhibitors. Mitoxantrone and amsacrine showed high in vitro activity and displayed cross-resistance to the anthracyclines in the hematologic neoplasms. In the solid tumor group, mitoxantrone and amsacrine had lower activity and weak correlations with the anthracyclines. Etoposide had intermediate to weak correlations with the other drugs. Conclusions. There is a marked difference in tumortype specificity and cross resistance patterns among the different topo II inhibitors tested. This may indicate that minor differences in topo II interaction and/or other mechanisms of drug action may substantially determine the cytotoxic activity of topo II-targeted drugs. Cancer 1994; 74:2857-62.

Background

Introduction

Paclitaxel pharmacokinetics are nonlinear with saturable metabolism and saturable distribution to the tissues. The saturable distribution has in previous pharmacokinetic modeling been described as a saturable transport process, whereas the present study was undertaken to investigate alternative explanations. Using a sparse sampling scheme (on average 3.3 samples per profile), 101 plasma concentration-time profiles in 22 female patients with metastatic cancer of the breast or ovary were monitored. It was found that the observed data could be equally well described by saturable tissue binding as well as by capacity-limited tissue transport. The data were better described by a model where equilibrium was achieved with drug in the central rather than in the peripheral compartment. Models where the binding was assumed to be an instantaneous or a noninstantaneous process were tried, but the data did not allow resolution between these two possibilities. The value at which the saturable transport was half-maximal was 0.55 μM. The Kdvalues of the binding models were 0.06 to 0.12 μM. These are close to the values reported as a threshold for drug toxicity of paclitaxel, suggesting a possible connection between the binding sites involved in the pharmacokinetics and the mechanism responsible for the toxicity. For all models, a saturable elimination of paclitaxel was included using the Michaelis-Menten model.Kmfor the elimination ranged in the different models from 2.5 to 5.6 μM.

Current treatment strategies for chemotherapy of cancer patients were developed to benefit groups of patients with similar clinical characteristics. In practice, response is very heterogeneous between individual patients within these groups. Precision medicine can be viewed as the development toward a more fine-grained treatment stratification than what is currently in use. Cell-based drug sensitivity testing is one of several options for individualized cancer treatment available today, although it has not yet reached widespread clinical use. We present an up-to-date literature meta-analysis on the predictive value of ex vivo chemosensitivity assays for individualized cancer chemotherapy and discuss their current clinical value and possible future developments.

Current treatment strategies for chemotherapy of cancer patients were developed to benefit groups of patients with similar clinical characteristics. In practice, response is very heterogeneous between individual patients within these groups. Precision medicine can be viewed as the development toward a more fine-grained treatment stratification than what is currently in use. Cell-based drug sensitivity testing is one of several options for individualized cancer treatment available today, although it has not yet reached widespread clinical use. We present an up-to-date literature meta-analysis on the predictive value of ex vivo chemosensitivity assays for individualized cancer chemotherapy and discuss their current clinical value and possible future developments.

Although medical cancer treatment has improved during the past decades, it is difficult to choose between several first-line treatments supposed to be equally active in the diagnostic group. It is even more difficult to select a treatment after the standard protocols have failed. Any guidance for selection of the most effective treatment is valuable at these critical stages. We describe the principles and procedures for ex vivo assessment of drug activity in tumor cells from patients as a basis for tailored cancer treatment. Patient tumor cells are assayed for cytotoxicity with a panel of drugs. Acoustic drug dispensing provides great flexibility in the selection of drugs for testing; currently, up to 80 compounds and/or combinations thereof may be tested for each patient. Drug response predictions are obtained by classification using an empirical model based on historical responses for the diagnosis. The laboratory workflow is supported by an integrated system that enables rapid analysis and automatic generation of the clinical referral response.

Background: The selection of cancer drugs for an individual patient is still based mostly on cancer type and stage. Predictive tests are needed to make individualized and more efficient pharmacological cancer treatment possible. Objective: To provide an overview of available, possible future development and principles for development of predictive tests for individualized selection of cancer drugs. Methods: Overview of published data. Results/conclusion: Despite increased knowledge in cancer biology, only limited progress has been made in the development and use of predictive tests. However, rapid progress in this field will be possible using already available and emerging technologies, but requires a paradigm shift in principles for the development and use of cancer drugs. Assessment of drug activity in intact tumor cells and tumor cell gene expression signatures are considered to have greatest potential for the development of versatile predictive tests.

The squamous cell carcinoma HeLa cell line and an epithelial cell line hTERT-RPE with a nonmalignant phenotype were interrogated for HeLa cell selectivity in response to 1267 annotated compounds representing 56 pharmacological classes. Selective cytotoxic activity was observed for 14 of these compounds dominated by cyclic adenosine monophosphate (cAMP) selective phosphodiesterase (PDE) inhibitors, which tended to span a representation of the chemical descriptor space of the library. The PDE inhibitors induced delayed cell death with features compatible with classical apoptosis. The PDE inhibitors were largely inactive when tested against a cell line panel consisting of hematological and nonsquamous epithelial phenotypes. In a genome-wide DNA microarray analysis, PDE3A and PDE2A were found to be significantly increased in HeLa cells compared to the other cell lines. The pathway analysis software PathwayAssist was subsequently used to extract a list of proteins and small molecules retrieved from Medline abstracts associated with the hit compounds. The resulting list consisted of major parts of the cAMP–protein kinase A pathway linking to ERK, P38, and AKT. This molecular network may provide a basis for further exploitation of novel candidate targets for the treatment of squamous cell carcinoma.

To help identify the role of modelling and simulation in the development of anti-cancer agents, their main advantages and the obstacles to their rational use, an expert meeting was organized by COST B15. This manuscript presents a synthesis of views expressed at that meeting and indicates future directions. The manuscript also shows some examples where modelling and simulation have proven to be of relevant value in the drug development process for anti-cancer agents.

The novel alkylating dipeptide melphalanyl-p-L-fluorophenylalanine ethyl ester (J1) was evaluated for acute toxicity and antitumor activity in mice, with melphalan as a reference. To determine a safe and tolerable dose for efficacy studies the acute toxicity following intravenous injection in the tail vein was monitored using a 14-day schedule with up to four doses. The highest tested dose, 25 μmoles/kg, was considered close to this level, with minor effects on body weight gain but significant effects on hematological parameters. Melphalan and J1 appeared equitoxic with no statistically significant differences. Subsequently a mouse hollow fiber model was employed with subcutaneous implantation of fibers containing human tumor cells. Three different human tumor cell lines as well as two samples of primary human tumor cells (ovarian carcinoma and chronic lymphatic leukemia) were used as tumor models. At the dose level tested there was a marked and statistically significant decrease in both T-cell leukemia CCRF-CEM and small cell lung cancer NCI-H69 tumor cell growth and viability in response to J1 as compared with both placebo and melphalan treated groups. In primary ovarian carcinoma cells only J1 treatment resulted in significant tumor regression (net cell kill). In summary the results indicate that, despite an expected short half time in the blood circulation, the promising in vitrodata from the previous studies of J1 seems translatable into the in vivosituation. At equal doses of alkylating units J1, compared to melphalan, was more active in the mouse hollow-fiber model, but showed similar general toxicity.

The t(12;21) (p13;q22) translocation resulting in ETV6/RUNX1 (previously named TEL/AML1) gene fusion is present in about 25% of children with precursor B-lineage acute lymphoblastic leukemia (B-ALL). We successfully tested 275 precursor B-ALL samples from children aged 1 to 17 years to determine the relation between t(12;21) and in vitro cellular drug resistance, measured by the fluorometric microculture cytotoxicity assay (FMCA). Samples from 83 patients (30%) were positive for t(12;21). The ETV6/RUNX1+samples were significantly more sensitive than ETV6/RUNX1–samples to doxorubicin, etoposide, amsacrine, and dexamethasone, whereas the opposite was true for cytarabine. After matching for unevenly distributed patient characteristics, that is, excluding patients with high hyperdiploidy (> 51 chromosomes), t(9; 22), t(1;19), or 11q23 rearrangement, the ETV6/RUNX1+samples remained significantly more sensitive to doxorubicin (P= .001) and etoposide (P= .001). For the other drugs tested (amsacrine, cytarabine, dexamethasone, prednisolone, vincristine, 6-thioguanine, and 4-hydroperoxy-cyclophosphamide), no significant difference in cellular drug sensitivity was found. In conclusion, we found that the presence of the t(12;21) translocation in childhood precursor B-ALL is associated with a high tumor cell sensitivity to doxorubicin and etoposide. High throughput techniques should now be used to elucidate the cellular mechanisms by which ETV6/RUNX1 gene fusion is linked to increased sensitivity to these drugs.

Abstract: The feasibility of tumor sampling followed by ex vivo assessment of drug sensitivity, using the short-term fluorometric microculture cytotoxicty assay (FMCA), for selection of chemotherapy was investigated prospectively in patients with advanced cancer not amenable to standard treatment. Taxol (175 mg/m2 every 3 wk) was given to patients with tumor samples being low drug resistant (LDR) to Taxol ex vivo, to patients with no LDR drug, and if other drugs were unsuitable. The remaining patients received the most optimal drug(s) based on the FMCA results. Gastrointestinal cancer was dominating among the 61 eligible patients. Tumor sampling was safely performed in 75% by ultrasound-guided core biopsy. Eighty-two percent of the patients had Taxol. Five patients (8%) had a partial remission and 18 (30%) had stable disease. Tumor response was poorly predicted, probably because the Taxol excipient Cremophor EL is cytotoxic exclusively ex vivo. However, patients with tumor cells being LDR to at least one drug ex vivo lived significantly longer than those with no such drug.

Background:CHS 828 is a novel cyanoguanidine with cytotoxic properties which was recently shown to induce an early increase in extracellular acidification. This could hypothetically be exploited for combination with drugs interfering with, or being dependent on, pH for their effect. Methods:The isobole method and the additive model were used to evaluate the combinations CHS 828-amiloride and CHS 828-mitomycin C (MMC) in the lymphoma cell line U-937 GTB and in primary cultures of tumour cells from patients. Results:Amiloride, which blocks the Na+/H+ antiport, shifted the CHS 828 dose-response curve to the left in a synergistic manner according to the additive interaction model. MMC is a bioreductive drug with enhanced cytotoxicity at acidic pH. A lowering of pH induced by CHS 828 would theoretically create a favourable environment for bioreduction of MMC. The interaction between these drugs was mainly classified as additive by both methods, but was synergistic at the highest effect level tested. In addition, there were sub-additive to synergistic interactions between CHS 838 and MMC in 76% of the haematological samples tested. Conclusions:Circumstantial evidence indicated that the mechanisms for the interactions could be pH independent. Thus, the interaction between CHS 828 and amiloride was synergistic while the interaction between CHS 828 and MMC in tumour cells was at least additive.

Fresh human tumour cells from patients with ovarian cancer and chronic lymphocytic leukaemia were cultured in semipermeable hollow fibres. The fibres were implanted on immunocompetent rats, which were treated with the cyanoguanidine N-(4-chlorophenoxyhexyl)-N′-cyano-N″-4-pyridylguanidine (CHS 828). CHS 828 showed high antitumour activity against all eight tumour samples; the fibres from control animals had a mean net growth of 73% while CHS 828 treatment induced a 37% mean reduction of cell density, without observable haematological toxicity. The results show a feasibility of using tumour cells directly from patients in the hollow-fibre rat model.

The fluorometric microculture cytotoxicity assay (FMCA), a short-term in vitro assay based on the concept of total tumor cell kill, was used for testing the cytotoxic drug sensitivity of tumor cells from patients with ovarian carcinoma. A total of 125 fresh specimens was obtained, 98 (78%) of which were analyzed successfully. Data from 45 patients were available for clinical correlations. The FMCA appeared to yield clinically relevant cytotoxic drug sensitivity data for ovarian carcinoma as indicated by a comparison with tumor samples obtained from patients with non-Hodgkin's lymphoma or kidney carcinoma. Considering the most active single agent in vitro actually given in vivo, and using the median drug activity among all ovarian carcinoma samples as a cut-off, the sensitivity of the assay and its specificity were 75 and 52%, respectively. Cross-resistance in vitro was frequently observed between standard drugs but not between standard drugs and Taxol. Ten percent of the specimens showed an extreme resistance for at least 4 of 6 of the drugs investigated. Int. J. Cancer 72:1008–1012, 1997. © 1997 Wiley-Liss, Inc.

The immunohistochemical expression of the 170-kDa permeability glycoprotein (P-gp) was investigated in 41 primary untreated breast carcinomas, using the monoclonal antibodies C219 and MRK16. DNA ploidy by flow cytometry and estrogen (ER) and progesterone receptor (PgR) contents were also determined. P-gp expression, as revealed by C219 or MRK16, was observed in 6 (14%) of the investigated cancers. P-gp expression had a tendency to occur in non-diploid, high-grade tumors as well as in patients with lymph node negative disease. However, except for lymph node status, these associations were not statistically significant. No positive statistical relationships were observed between other prognostic parameters (age, tumor size, and receptor status) and P-gp expression. Considering the great heterogeneity observed in previous studies and the low expression of P-gp observed hereby, the utility of P-gp immunostaining as a guide for therapy planning in patients with breast cancer remains uncertain.

The Ca 2+receptor mechanism of the parathyroid cell was studied using La 3+as a probe. La 3+was found to bind to the cell surface without further penetration. Measurements of 45Ca fluxes and the cytoplasmic Ca 2+concentration (Ca i2+) revealed a stimulatory component in the action of La 3+on Ca 2+permeability resulting in a rise in Ca i2+These effects mimicked those obtained when raising the extracellular Ca 2+concentration from 0.5 to 3.0 mM, but the actions of La 3+and Ca 2+were not additive. The results suggest the existence of a novel Ca 2+permeability physiologically activated by binding of Ca 2+to an external receptor.

Monolayer culture of bovine parathyroid cells for 24 hours resulted in a right-shift of the dose-effect relationships for Ca2+-inhibition of parathyroid hormone (PTH) release and the dependence of the cytoplasmic Ca2+ concentration (Ca2+) on extracellular Ca2+ as well as in a less suppressible hormone release. After 4 days of culture, hormone secretion was almost non-suppressible and Cai2+ increased poorly in response to a rise in extracelluiar Ca2+. Ionomycin, a Ca2+ ionophore, raised Cai2+, but there was only a small inhibition of PTH release and the correlation between Cai2+ and secretion was weak. A deteriorated Cai2+ regulation and a decreased inhibitory action of cytoplasmic Ca2+ on PTH release were also found in ceils from human parathyroid adenomas. Functional dedifferentiation of the parathyroid cell thus results from both defective regulation and action of cytoplasmic Ca2+.

Bovine parathyroid cells were used to study parathyroid hormone (PTH) release and the cytoplasmic Ca 2+concentration (Ca 2+1). When the extracellular Ca 2+concentration was decreased from 3.0 to 0.5 mM, perifused cells reacted with rapid stimulation of PTH release. However, a further reductionof extracellular Ca 2+to < 10 nM resulted in prompt inhibition. Both effects were readily reversible. Using the intracellular Ca 2+indicator quin-2 also as a buffer for calcium it was possible to control Ca 2+1within the 20–600 nM range. PTH release was found to increase with Ca 2+up to 200 nM but was gradually suppressed above this concentration.

In vitro tumor models could support the process of development of new cytotoxic drugs and selection of suitable drugs for the individual patient. We investigated whether the testing of tumor cells from patients with kidney or urinary bladder carcinoma by fluorometric microculture cytotoxicity assay (FMCA) could provide clinically relevant data for these tumor types.

Primary cultures of cells from patients with chronic lymphocytic leukemia (CLL) and ovarian carcinoma (Ovca) were compared with the renal carcinoma ACHN and the lymphocytic CCRF-CEM human tumor cell lines in response to 63 toxic or nontoxic compounds. The experiments were conducted at 1, 10, and 100 μ/ml in 96-well microtiter plates for an assay time of 72 h. The plates were analyzed by the fully automated Dynatech Immuno Assay System (DIAS) using Alamar blue as a fluorometric/colorimetric indicator of metabolic activity. Drug response data were reported as the area under the tumor cell survival-concentration curve (AUC). Noncytotoxic compounds were classified as inactive by all cell systems. According to the AUC, CCRF-CEM and CLL cells were the most sensitive, followed by ACHN, and then Ovca. Many of the clinically active drugs were detected by all cell systems. However, the sensitivity pattern differed considerably between the cell types as judged from correlation analysis, and a higher proportion of clinically inactive drugs were scored as active by the cell lines compared with the primary cultures. Ovca showed the highest ratio of clinically solid tumor active/clinically inactive agents followed by CLL. The results indicate that primary cultures of human tumor cells may be a useful and valuable model for anticancer drug screening.

Bovine parathyroid cells were used to study parathyroid hormone (PTH) release and the cytoplasmic Ca2+concentration (Ca2+1). When the extracellular Ca2+concentration was decreased from 3.0 to 0.5 mM, perifused cells reacted with rapid stimulation of PTH release. However, a further reductionof extracellular Ca2+to < 10 nM resulted in prompt inhibition. Both effects were readily reversible. Using the intracellular Ca2+indicator quin‐2 also as a buffer for calcium it was possible to control Ca2+1within the 20–600 nM range. PTH release was found to increase with Ca2+up to 200 nM but was gradually suppressed above this concentration.

Effects of the protein kinase C activating phorbol ester 12-O-tetradecanoyl phorbol 13-acetate and the inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) on parathyroid hormone (PTH) release were studied in normal bovine and pathological human parathyroid cells. An increase of extracellular Ca2+from 0.5 to 3.0 mmol/l inhibited PTH release by 60% in the bovine cells with half maximal effect (ED50) at 1.31 mmol/l. This inhibition reached less than 50% in the cells from patients with primary and uremic hyperparathyroidism, and the ED50values were 1.49 and 1.42 mmol/l, respectively. The phorbol ester (0.1 μmol/l) made secretion insensitive to changes of extracellular Ca2+, an action counteracted by H-7 (50 μmol/l) in the bovine cells, whereas H-7 alone had no effects. The phorbol ester and H-7 had opposite actions on regulation of PTH release also from cells from patients with hyperparathyroidism. However, in pathological cells H-7 alone improved Ca2+inhibition of secretion by stimulating release in low Ca2+concentrations and decreasing the ED50values. The magnitude of changes in ED50values by H-7 increased with the severity of the secretory disturbance of the pathological cells. The results indicate that increased protein kinase C activity may be a factor of importance in the pathophysiology of hyperparathyroidism.

Summary: Increasing the extracellular Ca2+ concentration from 0.5 to 3.0 mM induced marked increments in cytoplasmic Ca2+ concentration (Ca2+i) and inhibition of parathyroid hormone (PTH) release of freshly isolated bovine parathyroid cells. 1,25-dihydroxycholecalciferol (1,25(OH)2D3; 0.1–100 ng/ml) did not affect (Ca2+i) and was also without acute effect on the secretion. During 4 days of monolayer culture, the parathyroid cells underwent significant increases in both number and size, and presence of 10–100 ng/ml 1,25(OH)2D3 almost completely inhibited the cell proliferation, whereas the hypertrophy was unaffected. One day of culture with 0.1–100 ng/ml 1,25(OH)2D3 was without effect on PTH release but after 4 days there was a dose-related reduction of recretion. At this time point and irrespective of the culture condition, PTH release was no longer suppressed by high extracellular Ca2+. Furthermore, Ca2+i increased little upon increments in the extracellular Ca2+ concentration as compared with freshly isolated cells. It is concluded that after prolonged exposure to 1,25(OH)2D3, PTH release is inhibited and, at high concentrations, the parathyroid cells cease to proliferate. However, 1,25(OH)2D3 does not affect the development of functional dedifferentiation of parathyroid cells during monolayer culture.

The automated fluorometric microculture cytotoxicity assay (FMCA) is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein by viable cells after a 72-hr culture period in microtiter plates. The FMCA was adopted for chemosensitivity testing of tumor cells from patients with ovarian carcinoma. Thirty-seven samples of solid tumors and malignant effusions were obtained from 35 patients at diagnosis or relapse. Tumor cells from solid samples and effusions were prepared by enzymatic digestion and centrifugation, respectively, followed by Percoll or Ficoll purification. The fluorescence was proportional to the number of cells/well and considerably higher in tumor cells than in contaminating normal cells. The effect of up to 19 cytotoxic drugs was successfully assessed in 70% of the samples and there was a good correlation between drug sensitivity data reported by the FMCA and the DiSC assay performed in parallel. The overall drug sensitivity pattern in vitrocorresponded well to the clinical experience. The effect of cisplatin varied considerably between patients and resistance was found also in cases not previously exposed to cytotoxic drugs. The FMCA is a rapid and simple method that seems to report clinically relevant cytotoxic drug sensitivity data in ovarian carcinomas. In the future, this method may contribute to optimizing chemotherapy by assisting in individualized drug selection and new drug development.

A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples. Copyright 1994, 1999 Academic Press

A 72 hours fluorometric microculture cytotoxicity assay (FMCA) was used for the study of chemotherapeutic drug resistance in tumor cell suspensions from patients with acute myelocytic leukemia (AML). A marked heterogeneity with respect to sensitivity was observed for a panel of cytotoxic drugs tested in 76 samples from 60 patients with treated or untreated AML. Primary resistance to vincristine (Vcr) and prednisolone in untreated AML was observed as well as ‘acquired’ resistance to several other antileukemic drugs. Cross resistance patterns for AML active drugs revealed significant positive relationships between anthracyclines, VP 16 and amsacrine (Amsa), whereas mitoxantrone (Mitox) was more weakly correlated. Sensitivity to cytosine arabinoside was unrelated to the anthracyclines, VP16, Amsa and Mitox butshowed a significant relationship to 6-thioguanine. Several resistance modifying agents, including the novel non-imrnunosuppressive cyclosporin A analogue PSC 833, were able to potentiate the effects of doxorubicin and Vcr at concentrations achievable in the clinic. However, the pattern of activity was heterogenous and the frequency of responsive samples was higher in relapse compared tode novocases. Individualin vitro/in vivocorrelations based on quartile distributions of ail accumulated drug sensitivity data from AML patients indicated a high specifity with respect to the identification of drug resistance. The results suggest that the FMCA may provide clinically valuable information on chemotherapeutic drug resistance in AML.

Influence of vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide, and substance P was investigated on dispersed parathyroid cells of adult cattle. At a physiological concentration of extracellular calcium, vasoactive intestinal polypeptide stimulated the parathyroid hormone release in a dose-dependent manner, whereas no effects were noted for the other peptides. The dependency of PTH secretion upon extracellular calcium was shifted to the right by vasoactive intestinal polypeptide at 10−6mol/l, with a tendency for greater effects at low (0.5 mmol/l) than high concentrations (2.0-3.0 mmol/l) of the cation. Vasoactive intestinal polypeptide significantly enhanced cAMP release of the parathyroid cells, whereas no influence was noted on cytoplasmic calcium or pH within the cells. The results suggest that vasoactive intestinal polypeptide stimulates the PTH release by interaction with cAMP production of the parathyroid cells. This effect may contribute to the development of hypercalcemia in patients with neuroendocrine tumours secreting vasoactive intestinal polypeptide.

Thirteen human lung cancer cell lines, 7 representing small cell lung cancer (SCLC) and 6 different types of non-SCLC, were tested for sensitivity to tumour necrosis factor alpha (TNF-α) and interferon alpha and gamma (IFN-α and γ) using an automated fluorometric microculture cytotoxicity assay (FMCA). One SCLC line (H-82) was found to be sensitive to IFN-α in short-term (72 h) culture, whereas after prolonged (5 days) culture two additional SCLC cell lines responded to IFN-γ. TNF-α inhibited the growth of one large cell carcinoma cell line (H-157), whereas all SCLC lines were found to be insensitive. The combination of IFN-γ and TNF-α produced no further response compared with the single agents used alone. By continuous cultivation of the IFN-α-sensitive cell line H-82 in the presence of increasing concentrations of IFN-α, an IFN-α-resistant subline (H-82R) was established. This line displayed a high degree of resistance (>100 fold) to IFN-α and cross-resistance to IFN-γ. There was no alteration in the number of IFN binding sites, in the growth rate, the expression of selected surface markers for SCLC or the expression of multidrug resistance markers in the H-82R subline compared with the parental H-82 cell line. The results demonstrate a heterogeneous response of SCLC cell lines to IFN-α and γ and TNF-α with only a minority of the cell lines responding to these agents by growth inhibition. The IFN-α and γ H-82R subline may serve as a valuable tool in future studies on the mechanisms of IFN antitumour activity.

No relevant conflicts of interest to declare.

Background. There has been a long history of Individualized Tumor Response (ITR) testing to aid drug selection in tailored therapy for leukemia and lymphoma. Almost all the published evidence suggests that the results produced provide useful prognostic information and added value that is independent of other patient variables. Trials have shown ITR test results correlate with subsequent patient response and survival. However, results have not been so readily accessible due, in part, to multiple descriptive terms. Terminology. As a result, groups undertaking ITR with leukemias, lymphomas and solid tumors have agreed to use the term ’Individualized Tumor Response’ to describe the ’effect of anticancer treatments on whole living tumor cells freshly removed from cancer patients’. This term does not include tests with subcellular fractions, animals or cell lines. Methods. All methods measure cell death: fresh tumor is removed from the patient, treated with an anticancer regimen in vitro for 2–7 days and tumor survival assessed. ITR testing for chemotherapy and radiotherapy has recently been extended to cover newer agents (Weisenthal, 2007). Results. Multiple publications have shown assays correlate well with each other, allowing this meta-analysis. We found 1929 published ITR test results compared with patient response and analysed them to determine relative risk (Rel Risk) of clinical response after treatment with ITR-test-sensitive (ITR+) vs. ITR-test-resistant (ITR-) regimens: Comparison of ExVTR test results with clinical response rate (RR) and relative risk (Rel Risk) Diagnosis # Clinical RR RR ITR+ RR ITR− Rel Risk IVT+ Rel Risk IVT− *, 2P=0.025; **, 2P=0.002; *** 2P<0.0001; #, number of patients ALL 304 81.3 90.6 49.3 0.90** 1.65*** ANLL 621 72.0 87.9 35.8 0.82*** 2.01*** CLL 720 69.0 82.5 18.8 0.83*** 3.66*** NHL 85 54.1 72.9 11.5 0.74* 4.69*** Other 199 61.3 78.2 8.3 0.78*** 7.36*** Total 1929 70.4 84.6 28.3 0.83*** 2.48*** Overall, the response rate for patients given ITR+ drugs (84.6%) is significantly greater than for patients treated with ITR- drugs (28.3%). The relative risk of not responding was significantly reduced by treatment with an IVT+ regimen (0.83; 95% Confidence Interval 0.80–0.86) but increases to 2.48 (2.15–2.87) when treated with an IVT- regimen. Published comparisons of ITR with patient survival have all shown better survival for patients treated with ITR+ drugs compared with patients treated with ITR- drugs. A randomised controlled clinical trial of the efficacy of ITR in CLL is ongoing. Conclusions. Compared with newer methods being developed to provide tailored therapy (such as genomic analyses), ITR testing provides a functional profile of how whole tumor cells respond to toxic insult. This is far closer to what happens in the patient than the measurement of the effect on genes or proteins. Further investment in ITR testing - technologies that produce clinically significant results for tailored therapy - is warranted.