Health-related quality of life and inflammatory markers in malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a highly aggressive and symptomatic disease. We examined the relationship between health-related quality of life (HRQoL) and inflammatory markers, and the prognostic role of HRQoL in MPM patients. METHODS: MPM patients from two parallel phase II studies (thalidomide alone or thalidomide with chemotherapy) were included. HRQoL was assessed at baseline using the modified Lung Cancer Symptom Scale (LCSS). Baseline inflammatory markers and cytokines were measured. Spearman correlation was used to examine the relationship between inflammatory markers and HRQoL measures. The prognostic value of the HRQoL domains was examined using Cox proportional hazard model. RESULTS: Sixty-three patients were included: median age 61 years (range 44-79); 82% male; 77% Eastern Cooperative Oncology Group (ECOG) performance status 0-1; 44% epithelial histology subtype. Baseline systemic symptoms of anorexia and fatigue, the summation symptoms of overall symptomatic distress, interference with normal activity and global QoL and the aggregate score of total LCSS score were all associated with elevated neutrophil-to-lymphocyte ratio, C-reactive protein and vascular endothelial growth factor levels at baseline (rho ≥ 0.25; p < 0.05). Baseline anorexia, fatigue, cough, dyspnoea, pain, overall symptomatic distress, interference with normal activity, global QoL and total LCSS score were all significantly related to survival (p < 0.05) after adjusting for established prognostic factors (age, gender, histological subtype and performance status) and treatment effect. CONCLUSIONS: In conclusion, HRQoL seems to relate to a patient's systemic inflammatory status and is associated with survival in MPM patients.

Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer.

Tyrosine kinase inhibitors (TKIs) are the first-line therapy for non-small-cell lung cancers (NSCLC) that harbour sensitising mutations within the epidermal growth factor receptor (EGFR). However, resistance remains a key issue, with tumour relapse likely to occur. We have previously identified that cell division cycle-associated protein 3 (CDCA3) is elevated in adenocarcinoma (LUAD) and correlates with sensitivity to platinum-based chemotherapy. Herein, we explored whether CDCA3 levels were associated with EGFR mutant LUAD and TKI response. We demonstrate that in a small-cohort tissue microarray and in vitro LUAD cell line panel, CDCA3 protein levels are elevated in EGFR mutant NSCLC as a result of increased protein stability downstream of receptor tyrosine kinase signalling. Here, CDCA3 protein levels correlated with TKI potency, whereby CDCA3high EGFR mutant NSCLC cells were most sensitive. Consistently, ectopic overexpression or inhibition of casein kinase 2 using CX-4945, which pharmacologically prevents CDCA3 degradation, upregulated CDCA3 levels and the response of T790M(+) H1975 cells and two models of acquired resistance to TKIs. Accordingly, it is possible that strategies to upregulate CDCA3 levels, particularly in CDCA3low tumours or upon the emergence of therapy resistance, might improve the response to EGFR TKIs and benefit patients.

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.

Comparison of radiobiological parameters for 90Y radionuclide therapy (RNT) and external beam radiotherapy (EBRT) in vitro.

BACKGROUND: Dose rate variation is a critical factor affecting radionuclide therapy (RNT) efficacy. Relatively few studies to date have investigated the dose rate effect in RNT. Therefore, the aim of this study was to benchmark 90Y RNT (at different dose rates) against external beam radiotherapy (EBRT) in vitro and compare cell kill responses between the two irradiation processes. RESULTS: Three human colorectal carcinoma (CRC) cell lines (HT29, HCT116, SW48) were exposed to 90Y doses in the ranges 1-10.4 and 6.2-62.3 Gy with initial dose rates of 0.013-0.13 Gy/hr (low dose rate, LDR) and 0.077-0.77 Gy/hr (high dose rate, HDR), respectively. Results were compared to a 6-MV photon beam doses in the range from 1-9 Gy with constant dose rate of 277 Gy/hr. The cell survival parameters from the linear quadratic (LQ) model were determined. Additionally, Monte Carlo simulations were performed to calculate the average dose, dose rate and the number of hits in the cell nucleus. For the HT29 cell line, which was the most radioresistant, the α/ß ratio was found to be ≈ 31 for HDR-90Y and ≈ 3.5 for EBRT. LDR-90Y resulting in insignificant cell death compared to HDR-90Y and EBRT. Simulation results also showed for LDR-90Y, for doses ≲ 3 Gy, the average number of hits per cell nucleus is ≲ 2 indicating insufficiently delivered lethal dose. For 90Y doses [Formula: see text] 3 Gy the number of hits per nucleus decreases rapidly and falls below ≈ 2 after ≈ 5 days of incubation time. Therefore, our results demonstrate that LDR-90Y is radiobiologically less effective than EBRT. However, HDR-90Y at ≈ 56 Gy was found to be radiobiologically as effective as acute ≈ 8 Gy EBRT. CONCLUSION: These results demonstrate that the efficacy of RNT is dependent on the initial dose rate at which radiation is delivered. Therefore, for a relatively long half-life radionuclide such as 90Y, a higher initial activity is required to achieve an outcome as effective as EBRT.

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).

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

Changes in gene expression associated with stable drug and radiation resistance in small cell lung cancer cells are similar to those caused by a single X-ray dose.

Small cell lung cancer (SCLC) initially responds well to chemotherapy and fractionated radiotherapy, but resistance to these treatments eventually develops in the vast majority of cases. To understand how resistance develops in the H69 SCLC cell line, we compared the changes in gene expression associated with 37.5 Gy fractionated X-ray treatment that produced the stable radiation- and drug-resistant H69/R38 cell subline to the changes associated with a single 4- or 8-Gy X-ray treatment. Gene expression was determined by suppression subtractive hybridization combined with Northern blot analysis and two-dimensional (2D) protein electrophoresis. Stable radiation and drug resistance was associated with coordinate changes in the expression of genes of the cytoskeleton, protein synthesis, cell cycle, redox/stress and metabolic pathways. The pattern of these changes was remarkably similar to the changes seen 24 h after a single X-ray treatment of the H69 cells but differed from the changes in expression associated with a single X-ray treatment of the resistant H69/ R38 cells. Stable radiation and drug resistance may be caused by the constitutive expression of those genes transiently expressed by sensitive cells in response to a single X-ray dose. The repeated treatments received during fractionated irradiation may promote the change from a transient to a constitutive pattern of gene expression.

Cellular models of drug- and radiation-resistant small cell lung cancer.

BACKGROUND: The H69-EPR, H69-CP, H69-VP and H69/R38 resistant sublines of the classic small cell lung cancer (SCLC) line have proven useful in studies of resistance and its circumvention with paclitaxel. MATERIALS AND METHODS: The suppressor/oncogene profile of these sublines determined by Western and Northern blot was compared to the variant H82 SCLC cell profile. Two-dimensional electrophoresis/mass spectrometry was used to determine the effect of paclitaxel on protein expression. RESULTS: The H69-EPR and H69-CP resistant sublines were similar to the variant H82 cells for bcl-2, p21waf1, p53, N-myc and c-myc expression while the H69-VP subline retained the classic H69 pattern. A 1-h treatment with 10 ng/ml paclitaxel substantially reversed the resistance except for the H69/R38 subline and tended to reverse the resistance-associated changes in protein expression in the H69-EPR subline. CONCLUSION: Although some resistant sublines express a variant pattern of suppressor/oncogenes with low bcl-2, resistance is substantially reversed by paclitaxel treatment.

Establishing a panel of chemo-resistant mesothelioma models for investigating chemo-resistance and identifying new treatments for mesothelioma.

Mesothelioma is inherently chemo-resistant with only 50% of patients responding to the standard of care treatments, and consequently it has a very grim prognosis. The aim of this study was to establish a panel of chemo-resistant mesothelioma models with clinically relevant levels of resistance as tools for investigating chemo-resistance and identifying new treatments for mesothelioma. Chemo-resistant cell lines were established in vitro and characterized in vivo using syngeneic Fischer rats. Tumors derived from all chemo-resistant cell lines were immunohistochemically classified as mesothelioma. Homozygous deletion of p16(INK4A)/p14(ARF) and increased expression of several ATP-binding cassette transporters were demonstrated, consistent with findings in human mesothelioma. Further, the acquisition of chemo-resistance in vitro resulted in changes to tumor morphology and overall survival. In conclusion, these models display many features corresponding with the human disease, and provide the first series of matched parental and chemo-resistant models for in vitro and in vivo mesothelioma studies.

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.

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.