Rozelle Harvie

High blood neutrophil-to-lymphocyte ratio is an indicator of poor prognosis in malignant mesothelioma patients undergoing systemic therapy

Purpose: Asbestos-induced chronic inflammation is implicated in the pathogenesis of malignant mesothelioma (MM). We have investigated blood neutrophil-to-lymphocyte ratio (NLR), an index of systemic inflammation, as a prognostic factor in MM patients. Experimental Design: Patients with MM who had systemic therapy at participating institutes were studied. Potential prognostic factors such as age, gender, performance status, histologic subtype, and baseline laboratory parameters, including NLR, were analyzed. Overall survival from commencement of therapy was determined by the Kaplan–Meier method. Multivariate analyses using Cox Regression model were performed with significant factors (P ≤ 0.05) to determine their independent effect. Results: A total of 173 MM patients undergoing systemic therapy including 119 patients receiving first-line therapy and 54 patients receiving second- or third-line therapy were included in this retrospective evaluation. Forty-two percent of patients had an elevated NLR at baseline. The following variables were predictive of survival: female gender (P = 0.044), epithelioid histologic subtype (P < 0.001), baseline white blood cell count less than 8.3 × 109/L (P = 0.008), baseline platelet count 400 × 109/L or less (P = 0.05), and NLR of 5 or less (P < 0.001). After multivariate analysis, histologic epithelioid subtype [hazard ratio (HR) = 2.0; 95% confidence interval (CI) = 1.3–2.9; P = 0.001], and NLR less than 5 (HR = 2.7; 95% CI = 1.8–3.9; P < 0.001) remained independent predictors. The 1-year survival rate was 60% versus 26%, whereas the 2-year survival rate was 34% versus 10% for NLR less than 5 and 5 or greater, respectively. In the separate analyses of chemotherapy-naive and previously treated patient groups, NLR was an independent predictor of survival in both groups. Conclusion: Our results indicate that NLR is an independent predictor of survival for patients with MM undergoing systemic therapy. Clin Cancer Res; 16(23); 5805–13. ©2010 AACR.

DRUG RESISTANCE MECHANISMS AND MRP EXPRESSION IN RESPONSE TO EPIRUBICIN TREATMENT IN A HUMAN LEUKAEMIA CELL LINE

A drug resistant series of sublines were developed by treating the human leukaemia CCRF-CEM cell line with 16-1000 ng/ml of the anthracycline, epirubicin. The sublines developed resistance in two stages, neither involving detectable levels of P-glycoprotein. Treatment with up to 50 ng/ml epirubicin produced sublines with cross resistance limited to the anthracyclines and etoposide. Treatment with 100-1000 ng/ml epirubicin produced sublines with increased expression of the mrp gene, increased resistance to the anthracyclines and etoposide, additional cross resistance to vincristine and colchicine, decreased drug accumulation and reversal of resistance by verapamil and by buthionine sulphoximine (BSO; an inhibitor of glutathione synthesis). Our results indicate an interaction between MRP and glutathione metabolism as a mechanism for multidrug resistance.

Fractionated irradiation of H69 small-cell lung cancer cells causes stable radiation and drug resistance with increased MRP1, MRP2, and topoisomerase IIα expression

PURPOSE After standard treatment with chemotherapy and radiotherapy, small-cell lung cancer (SCLC) often develops resistance to both treatments. Our aims were to establish if fractionated radiation treatment alone would induce radiation and drug resistance in the H69 SCLC cell line, and to determine the mechanisms of resistance. METHODS AND MATERIALS H69 SCLC cells were treated with fractionated X-rays to an accumulated dose of 37.5 Gy over 8 months to produce the H69/R38 subline. Drug and radiation resistance was determined using the MTT (3,-4,5 dimethylthiazol-2,5 diphenyltetrazolium bromide) cell viability assay. Protein expression was analyzed by Western blot. RESULTS The H69/R38 subline was resistant to radiation (2.0 +/- 0.2-fold, p < 0.0001), cisplatin (14 +/- 7-fold, p < 0.001), daunorubicin (6 +/- 3-fold, p < 0.05), and navelbine (1.7 +/- 0.15-fold, p < 0.02). This was associated with increased expression of the multidrug resistance-associated proteins, MRP1 and MRP2, and topoisomerase IIalpha and decreased expression of glutathione-S-transferase pi (GSTpi) and bcl-2 and decreased cisplatin accumulation. Treatment with 4 Gy of X-rays produced a 66% decrease in MRP2 in the H69 cells with no change in the H69/R38 cells. This treatment also caused a 5-fold increase in topoisomerase IIalpha in the H69/R38 cells compared with a 1.5-fold increase in the H69 cells. CONCLUSIONS Fractionated radiation alone can lead to the development of stable radiation and drug resistance and an altered response to radiation in SCLC cells.

Multicentre phase I/II study of PI-88, a heparanase inhibitor in combination with docetaxel in patients with metastatic castrate-resistant prostate cancer

BACKGROUND Docetaxel (Taxotere) improve survival and prostate-specific antigen (PSA) response rates in patients with metastatic castrate-resistant prostate cancer (CRPC). We studied the combination of PI-88, an inhibitor of angiogenesis and heparanase activity, and docetaxel in chemotherapy-naive CRPC. PATIENTS AND METHODS We conducted a multicentre open-label phase I/II trial of PI-88 in combination with docetaxel. The primary end point was PSA response. Secondary end points included toxicity, radiologic response and overall survival. Doses of PI-88 were escalated to the maximum tolerated dose; whereas docetaxel was given at a fixed 75 mg/m(2) dose every three weeks RESULTS Twenty-one patients were enrolled in the dose-escalation component. A further 35 patients were randomly allocated to the study to evaluate the two schedules in phase II trial. The trial was stopped early by the Safety Data Review Board due to a higher-than-expected febrile neutropenia of 27%. In the pooled population, the PSA response (50% reduction) was 70%, median survival was 61 weeks (6-99 weeks) and 1-year survival was 71%. CONCLUSIONS The regimen of docetaxel and PI-88 is active in CRPC but associated with significant haematologic toxicity. Further evaluation of different scheduling and dosing of PI-88 and docetaxel may be warranted to optimise efficacy with a more manageable safety profile.

The potential of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide to circumvent three multidrug-resistance phenotypes in vitro.

Abstract The effectiveness of N-[2-(dimethylamino) ethyl]acridine-4-carboxamide (DACA) relative to that of amsacrine, idarubicin, daunorubicin and paclitaxel against three different forms of multidrug resistance (MDR) was determined using two sublines of the CCRF-CEM human leukaemia cell line, the P-glycoprotein-expressing CEM/VLB100 subline and the MRP-expressing CEM/E1000 subline, and two extended-MDR sublines of the HL60 human leukaemia cell line, HL60/E8 and HL60/V8. DACA was effective against P-glycoprotein-mediated MDR and MRP- mediated MDR, whereas the extended-MDR pheno- type showed only low levels of resistance (<2-fold) to DACA. In comparison, idarubicin was ineffec- tive against the MRP and extended-MDR phenotypes. Repeated exposure of the K562 human leukaemia cell line to DACA (55, 546 or 1092 nM for 3 days over 10 weeks) did not result in the development of any significant drug resistance. We conclude that DACA has the potential to treat refractory leukaemia.

Increased MRP expression is associated with resistance to radiation, anthracyclines and etoposide in cells treated with fractionated γ‐radiation

The failure of chemotherapy is often associated with the failure of radiotherapy in the treatment of cancer. To investigate this relationship, the CCRF‐CEM (CEM) human T‐cell leukaemia cell line was treated with fractionated γ‐radiation totalling 75 Gy (10 cycles of 1.5 Gy daily for 5 days). This produced the CEMRR subline which was 1.5‐fold resistant to radiation compared with the parental CEM cells. The CEMRR subline was also resistant to daunorubicin, idarubicin and etoposide but not to paclitaxel, cis‐platinum or chlorambucil. Treatment with 50 μM buthionine sulphoximine, an inhibitor of glutathione synthesis, reversed the daunorubicin resistance in the CEMRR subline. Multidrug resistance‐associated protein (MRP) mRNA was 6‐fold higher in the CEMRR subline than in the CEM cells, and there was no detectable expression of P‐glycoprotein in either the CEM cells or the CEMRR subline. Treatment of the CEM cells with 2 Gy of γ‐radiation caused an increase in MRP‐mRNA within 4 hr which, by 24 hr, was greater than 5‐fold that of the untreated CEM cells. No change in MRP mRNA was observed in the CEMRR subline with similar treatment. We conclude that MRP is involved in the immediate response to radiation and it may account for the drug resistance that often develops following radiation treatment. Int. J. Cancer 73:164–167, 1997.

The predictive role of serum VEGF in an advanced malignant mesothelioma patient cohort treated with thalidomide alone or combined with cisplatin/gemcitabine.

There is a need for new treatment strategies and prognostic markers for the management of malignant mesothelioma (MM). The activity of thalidomide/cisplatin/gemcitabine (arm A) or thalidomide alone (arm B) was investigated in two parallel phase II studies in patients with advanced MM, using 6 month progression free survival (PFS) as the principal end-point. The predictive role of pre-treatment and 8 week follow-up serum C-reactive protein (CRP), interlukin-6 (IL-6), interlukin-6 soluble receptor (sIL-6R), mesothelin (SMRP) and vascular endothelial growth factor (VEGF) was also assessed. The proportion of patients with stable disease for >6 months was similar in both studies (arm A 35%, arm B 29%) and toxicity was mainly grade I/II. In univariate analyses only pre-treatment VEGF and CRP were correlated with survival. At 8 weeks post treatment, increased survival was found with low (median) VEGF and CRP (P<0.05). Change in VEGF over the first 8 weeks of treatment was also predictive for survival (P<0.05). When pre-treatment VEGF was >median, decreasing VEGF was associated with increased survival (P<0.05). In conclusion, thalidomide alone, or in combination with cisplatin/gemcitabine, controlled disease for >6 months in ∼30% of patients. Patients with decreasing VEGF during treatment had longest survival. Pre-treatment VEGF or CRP and early change in VEGF on treatment may predict treatment benefit and should be examined in future studies.

Predicting the clonogenic survival of A549 cells after modulated x-ray irradiation using the linear quadratic model

In this study we present two prediction methods, mean dose and summed dose, for predicting the number of A549 cells that will survive after modulated x-ray irradiation. The prediction methods incorporate the dose profile from the modulated x-ray fluence map applied across the cell sample and the linear quadratic (LQ) model. We investigated the clonogenic survival of A549 cells when irradiated using two different modulated x-ray fluence maps. Differences between the measured and predicted surviving fraction were observed for modulated x-ray irradiation. When the x-ray fluence map produced a steep dose gradient across the sample, fewer cells survived in the unirradiated region than expected. When the x-ray fluence map produced a less steep dose gradient across the sample, more cells survived in the unirradiated region than expected. Regardless of the steepness of the dose gradient, more cells survived in the irradiated region than expected for the reference dose range of 1-10 Gy. The change in the cell survival for the unirradiated regions of the two different dose gradients may be an important factor to consider when predicting the number of cells that will survive at the edge of modulated x-ray fields. This investigation provides an improved method of predicting cell survival for modulated x-ray radiation treatment. It highlights the limitations of the LQ model, particularly in its ability to describe the biological response of cells irradiated under these conditions.

A preliminary investigation of cell growth after irradiation using a modulated x-ray intensity pattern.

In this study we have investigated a spatial distribution of cell growth after their irradiation using a modulated x-ray intensity pattern. An A549 human non-small cell lung cancer cell line was grown in a 6-well culture. Two of the wells were the unirradiated control wells, whilst another two wells were irradiated with a modulated x-ray intensity pattern and the third two wells were uniformly irradiated. A number of plates were incubated for various times after irradiation and stained with crystal violet. The spatial distribution of the stained cells within each well was determined by measurement of the crystal violet optical density at multiple positions in the plate using a microplate photospectrometer. The crystal violet optical density for a range of cell densities was measured for the unirradiated well and this correlated with cell viability as determined by the MTT cell viability assay. An exponential dose response curve was measured for A549 cells from the average crystal violet optical density in the uniformly irradiated well up to a dose of 30 Gy. By measuring the crystal violet optical density distribution within a well the spatial distribution of cell growth after irradiation with a modulated x-ray intensity pattern can be plotted. This method can be used for in vitro investigation into the changes in radiation response associated with treatment using intensity modulated radiation therapy (IMRT).

Serum galactosyltransferase isoenzymes as markers for solid tumours in humans

High resolution agarose isoelectric focusing was used to compare the galactosyltransferase isoenzyme patterns of serum from 9 healthy controls with those from 38 patients with either breast, lung, ovarian, stomach or colonic cancer. At least 12 peaks of enzyme activity were found in every sample, the healthy controls having major forms with isoelectric points of 4.74, 4.87, 4.96, 5.16 and 5.23. Thirty patients (79%) had elevated levels of at least one isoenzyme and 23 (61%) had at least 3 isoenzymes elevated compared to only 10 (26%) patients who had elevated total serum galactosyltransferase activity. The isoenzymes which were most often elevated in the cancer patient group had isoelectric points of 4.93, 5.16 and 4.61. One isoenzyme with an isoelectric point of 4.43 was preferentially elevated in patients with ovarian cancer. Those isoenzymes containing little or no sialic acid were rarely elevated in cancer patients. Although no cancer-associated isoenzyme was detected the quantitative differences observed in the cancer patient group were striking.