Toxicity and efficacy of lomustine and bevacizumab in recurrent glioblastoma patients.

The combination of lomustine and bevacizumab is a commonly used salvage treatment for recurrent glioblastoma (GBM). We investigated the toxicity and efficacy of lomustine plus bevacizumab (lom-bev) in a community-based patient cohort and made a comparison to another frequently used combination therapy consisting of irinotecan plus bevacizumab (iri-bev). Seventy patients with recurrent GBM were treated with lomustine 90 mg/m2 every 6 weeks and bevacizumab 10 mg/kg every 2 weeks. Toxicity was registered and compared to the toxicity observed in 219 recurrent GBM patients who had previously been treated with irinotecan 125 mg/m2 and bevacizumab 10 mg/kg every 2 weeks. The response rate was 37.1% for lom-bev and 30.1% for iri-bev. Median progression-free survival (PFS) was 23 weeks for lom-bev and 21 weeks for iri-bev (p = 0.9). Overall survival (OS) was 37 weeks for lom-bev and 32 weeks for iri-bev (p = 0.5). Lom-bev caused a significantly higher frequency of thrombocytopenia (11.4% grade 3-4) compared to iri-bev (3.5% grade 3-4). Iri-bev patients had more gastrointestinal toxicity with regard to nausea, vomiting, diarrhea, constipation and stomatitis. Within the limitations of the study lom-bev is a well-tolerated treatment for recurrent GBM, although hematological toxicity may be a dose limiting factor. No significant differences between lom-bev and iri-bev were observed with regard to PFS or OS. The differences in toxicity profiles between lom-bev and iri-bev could guide treatment decision in recurrent GBM therapy as efficacy is equal and no predictive factors for efficacy exist.

Prognostic value of O-6-methylguanine-DNA methyltransferase (MGMT) protein expression in glioblastoma excluding nontumour cells from the analysis.

AIMS: It is important to predict response to treatment with temozolomide (TMZ) in glioblastoma (GBM) patients. Both MGMT protein expression and MGMT promoter methylation status have been reported to predict the response to TMZ. We investigated the prognostic value of quantified MGMT protein levels in tumour cells and the prognostic importance of combining information of MGMT protein level and MGMT promoter methylation status. METHODS: MGMT protein expression was quantified in tumour cells in 171 GBMs from the population-based Region of Southern Denmark (RSD)-cohort using a double immunofluorescence approach. Pyrosequencing was performed in 157 patients. For validation we used GBM-patients from a Nordic Study (NS) investigating the effect of radiotherapy and different TMZ schedules. RESULTS: When divided at the median, patients with low expression of MGMT protein (AF-low) had the best prognosis (HR = 1.5, P = 0.01). Similar results were observed in the subgroup of patients receiving the Stupp regimen (HR = 2.0, P = 0.001). In the NS-cohort a trend towards superior survival (HR = 1.6, P = 0.08) was seen in patients with AF-low. Including MGMT promoter methylation status, we found for both cohorts that patients with methylated MGMT promoter and AF-low had the best outcome; median OS 23.1 and 20.0 months, respectively. CONCLUSION: Our data indicate that MGMT protein expression in tumour cells has an independent prognostic significance. Exclusion of nontumour cells contributed to a more exact analysis of tumour-specific MGMT protein expression. This should be incorporated in future studies evaluating MGMT status before potential integration into clinical practice.

A comprehensive profile of recurrent glioblastoma.

In spite of relentless efforts to devise new treatment strategies, primary glioblastomas invariably recur as aggressive, therapy-resistant relapses and patients rapidly succumb to these tumors. Many therapeutic agents are first tested in clinical trials involving recurrent glioblastomas. Remarkably, however, fundamental knowledge on the biology of recurrent glioblastoma is just slowly emerging. Here, we review current knowledge on recurrent glioblastoma and ask whether and how therapies change intra-tumor heterogeneity, molecular traits and growth pattern of glioblastoma, and to which extent this information can be exploited for therapeutic decision-making. We conclude that the ability to characterize and predict therapy-induced changes in recurrent glioblastoma will determine, whether, one day, glioblastoma can be contained in a state of chronic disease.

Insertion of a nuclear factor kappa B DNA nuclear-targeting sequence potentiates suicide gene therapy efficacy in lung cancer cell lines.

Lung cancer currently causes the majority of cancer-related deaths worldwide and new treatments are in high demand. Gene therapy could be a promising treatment but currently lacks sufficient efficiency for clinical use, primarily due to limited cellular and nuclear DNA delivery. In the present study, we investigated whether it was possible to exploit the endogenous nuclear-shuttling activity by the nuclear factor kappa B (NFκB) system, which is highly prominent in many cancers as well as lung cancer. We observed that insertion of a DNA nuclear-targeting sequence (DTS) recognized by NFκB could improve plasmid nuclear delivery and enhance the therapeutic effect of a validated transcriptionally cancer-targeted suicide gene therapy system. A clear correlation between the number of inserted NFκB-binding sites and the therapeutic effect of the suicide system was observed in both small cell lung cancer (SCLC) and non-SCLC cell lines. The effect was observed to be due to elevated nuclear translocation of the suicide gene-encoding plasmids. The results show that a significant improvement of gene therapeutic efficiency can be obtained by increasing the intracellular trafficking of therapeutic DNA. This is to our knowledge the first time a DTS strategy has been implemented for suicide gene therapy.

Needs for everyday life support for brain tumour patients' relatives: systematic literature review.

The purpose of this paper is to undertake a review of the everyday lives and the need for support felt by relatives of adults with malignant cerebral glioma. Through electronic literature searches we identified studies with qualitative, quantitative or mixed method designs. Fourteen studies were identified. They indicated that a relative often assumes the caregiver's role, taking over responsibility for the patient's illness and survival, and that the relative is often overwhelmingly exhausted by this task. The ever-changing circumstances left the relatives fearful, anxious and apprehensive. The relatives lacked information about how to provide day-to-day care and how to manage psychoses and neuropsychiatric problems at home. Likewise, they needed help from the professionals to talk with each other about potentially reduced life expectancy. Most relatives appeared to value specialist nurse support highly, and they found support groups helpful. Relatively few studies were identified, and extant research was found to be diverse in purpose, study design and study population. The majority of the studies focused only on the parts of the relatives' everyday lives in which they were taking care of and supporting the patient. Further research focusing on the impact of the illness on different part of relative's everyday life is needed.

Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas.

Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low-oxygen culture conditions and in clinical specimens of both low- and high-grade tumors. The observed global checkpoint signaling, in contrast to only focal areas of overabundant p53 (indicative of p53 mutation) in grade II astrocytomas, are consistent with DDR activation being an early event in gliomagenesis, initially limiting cell proliferation (low Ki-67 index) and selecting for mutations of p53 and likely other genes that allow escape (higher Ki-67 index) from the checkpoint and facilitate tumor progression. Overall, these results support the potential role of the DDR machinery as a barrier to gliomagenesis and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.

A chimeric fusion of the hASH1 and EZH2 promoters mediates high and specific reporter and suicide gene expression and cytotoxicity in small cell lung cancer cells.

Transcriptionally targeted gene therapy is a promising experimental modality for treatment of systemic malignancies such as small cell lung cancer (SCLC). We have identified the human achaete-scute homolog 1 (hASH1) and enhancer of zeste homolog 2 (EZH2) genes as highly upregulated in SCLC compared to a panel of representative normal tissues. Here, we evaluate the use of regulatory regions from the hASH1- and EZH2-promoter regions alone and in combination for suicide gene therapy of SCLC. Two hASH1-promoter regions comprising 0.3 and 0.7 kb immediately upstream of (and including) the transcription start site were tested. Both constructs induced reporter gene activity (up to sevenfold SV40-promoter activity) in all tested classic (hASH1 positive) SCLC and in two hASH1-negative SCLC cell lines, whereas gene activity was low or absent (<4% of SV40 activity) in one hASH1-negative SCLC and in all control cell lines tested. To evaluate its therapeutic potential, the 0.7 kb hASH1 proximal-promoter region was evaluated for cytotoxicity in a suicide gene assay. The construct induced SCLC cytotoxicity at levels equivalent to those observed with the SV40 promoter, while control cells remained unaffected by the treatment. Analogously, a 1.1 kb EZH2-promoter region was evaluated by reporter and suicide gene assays. The EZH2 promoter potently induced reporter gene activity in SCLC (up to 25-fold of SV40 activity) while moderate reporter activity (up to 12% of SV40 activity), was detected in the control cells. However, in the suicide gene assay both control and SCLC cells demonstrated sensitivity indicating lack of promoter specificity. Finally, we fused the 0.7 kb hASH1 promoter to the EZH2 promoter generating a chimeric hASH1EZH2 regulatory construct. The chimeric promoter demonstrated increased activity in SCLC cells compared to the hASH1 promoter alone while retaining specificity in control cells. The hASH1EZH2 promoter thus constitutes a promising transcriptional regulator for SCLC gene therapy.

A prospective PET study of patients with glioblastoma multiforme.

OBJECTIVE: To study the post-surgical metabolic and structural cerebral changes in patients with glioblastoma multiforme (GBM). MATERIALS AND METHODS: We examined ten patients prospectively with newly diagnosed GBM. All patients were primarily treated with surgery, followed by chemotherapy (carmustine, cisplatine and etoposide) and radiotherapy. Positron emission tomography (PET) was used to measure tumor- and cerebral metabolism. CT or MRI was used to estimate tumor volume by measurements of tumor area. RESULTS: Tumor metabolism was not increased during chemotherapy (P = 0.71), but increased during radiotherapy (P = 0.01). CT/MRI showed similar results with no increase in tumor area during chemotherapy (P = 0.33) but increase during radiotherapy (P = 0.002). During the entire study, tumor metabolism and area increased evenly (P = 0.01). CONCLUSIONS: Our study did not show a gain of PET compared with structural imaging in the prospective evaluation of GBM. We found a difference in metabolic increase and tumor growth between the two treatment regimens, although this finding has limited relevance due to the design of the study.

The insulinoma-associated 1: a novel promoter for targeted cancer gene therapy for small-cell lung cancer.

The insulinoma-associated 1 (INSM1) gene is expressed exclusively during early embryonal development, but has been found re-expressed at high levels in neuroendocrine tumors. The regulatory region of the INSM1 gene is therefore a potential candidate for regulating expression of a therapeutic gene in transcriptionally targeted cancer gene therapy against neuroendocrine tumors. We analyzed expression of a reporter gene from a 1.7 kb region of the INSM1 promoter in a large number of small-cell lung cancer (SCLC) cell lines. This INSM1 promoter region showed very high levels of expression in most of the SCLC cell lines and expression was absent in cell lines of non-neuroendocrine origin. Inclusion of the general transcriptional enhancer from SV40 compromised the specificity of the promoter and did not enhance transcription in most of the SCLC cell lines. For comparison, the region of the gastrin releasing peptide (GRP) previously suggested for SCLC gene therapy was analyzed in a similar manner. High expression was observed for a number of cell lines, but unlike for the INSM1 promoter, reporter gene expression from the GRP promoter did not correlate to the relative GRP mRNA levels, demonstrating that this region may not contain all necessary regulatory elements. Expression of the suicide gene herpes simplex virus thymidine kinase (HSV-TK) from the INSM1 promoter in combination with treatment with the prodrug ganciclovir (GCV) caused a significant increase in GCV sensitivity specifically in INSM1-expressing cell lines. The INSM1 promoter is therefore a potential novel tool for transcriptionally targeted gene therapy for neuroendocrine tumors.

Differential response to gefitinib of cells expressing normal EGFR and the mutant EGFRvIII.

Epidermal growth factor receptor (EGFR) is frequently amplified and/or mutated in a number of human tumours and abnormal signalling from this receptor is believed to contribute to the malignant phenotype seen in these tumours. Gefitinib is a small molecule inhibitor that specifically binds and inhibits the EGFR tyrosine kinase and has been shown to inhibit the growth, proliferation, survival and invasion of a range of tumour cells overexpressing EGFR. However, clinical response to gefitinib has failed to correlate with EGFR levels and activity, indicating that other molecular mechanisms such as downstream signalling and mutations could be of importance in predicting clinical response. We therefore investigated the effect of the specific EGFR inhibitor gefitinib on the phosphorylation level, signalling and growth of cells expressing the naturally occurring constitutively active EGFR variant EGFRvIII, a low nontransforming level of EGFR and a high transforming level of EGFR. Results show that levels of gefitinib sufficient to suppress EGFR phosphorylations, EGFR-mediated proliferation and EGFR-mediated anchorage-independent growth are not sufficient to inhibit these features in cells expressing EGFRvIII. Furthermore, the data indicate that long-term exposure of EGFRvIII-expressing cells to low concentrations of gefitinib (0.01-0.1 microM) result in increased phosphotyrosine load of the receptor, increased signalling to ERK and stimulation of proliferation and anchorage-independent growth, presumably by inducing EGFRvIII dimerisation. Higher concentrations of gefitinib (1-2 microM), on the other hand, significantly decreased EGFRvIII phosphotyrosine load, EGFRvIII-mediated proliferation and anchorage-independent growth. Further studies are needed to investigate the implications of these important findings in the clinical setting.

Assessment of the hypothalamo-pituitary-adrenal axis in patients treated with radiotherapy and chemotherapy for childhood brain tumor.

The impact of cranial irradiation (CIR) and chemotherapy on the hypothalamo-pituitary (HP)-adrenal (HPA) axis was assessed in a population-based follow-up study of patients treated for childhood brain tumor not directly involving the HP axis. HPA function was evaluated and compared with that in healthy controls (n = 17), measuring basal cortisol and the peak cortisol response to an insulin tolerance test (ITT) and an ACTH test(.) The cortisol cut-off level was 500 nmol/liter. The biological effective dose (BED) of radiotherapy was determined for the HP region and spine and was expressed in Gray units, as BED gives a means of expressing the biological effects of different dosage schedules in a uniform way. Seventy-three children (46 males and 27 females), less than 15 yr of age when diagnosed during 1970-1997 in the Eastern part of Denmark, were included. The median age at time of radiotherapy was 8.4 yr (range, 0.8-14.9). The median length of follow-up was 15 yr (range, 2-29). Fourteen patients (19%) had basal cortisol levels below 500 nmol/liter and did not respond with a peak cortisol above the cut-off level to either an ACTH test (30 or 60 min) or an ITT, and thus, they had insufficiency of the HPA axis. Even though a peak cortisol above 500 nmol/liter was reached in the rest of the cohort (n = 59) after either an ACTH test (30 or 60 min) or an ITT, they had significantly lower peak cortisol levels compared with controls (P = 0.0099). Thirteen patients failed the ACTH test (30 min), but passed the ACTH test (60 min), implying a risk of misinterpreting the cortisol capacity of the patient if only the ACTH test (30 min) is obtained. The basal cortisol levels and the cortisol levels in the ACTH test (30 min) and the ACTH test (60 min) were significantly lower in the patient group compared with controls. There was a significant correlation between the peak cortisol after the ITT compared with the peak cortisol after the ACTH test (30 or 60 min; r(s) = 0.56; P = 0.0006), but 48% failed the ITT, and there was discordance in 10 of 33 (30%) patients who passed the ACTH but failed the ITT, indicating the recommendation of continuous use of the ITT as the gold standard for evaluation of the HPA axis. Stepwise backward multiple linear regression analysis showed that the best-fit model to predict the peak cortisol level after an ITT included BED (P = 0.04) and length of follow-up (P = 0.06). In contrast, age at RT, chemotherapy, BED to the spine, and gender were not included in the model. In conclusion, these data suggest that CIR for a childhood brain tumor may affect the HPA axis, resulting in secondary adrenal insufficiency, whereas adjuvant chemotherapy does not seem to add to the deleterious effect of CIR. We recommend life-long surveillance of the HPA axis and performing regular ITTs.

Preparation and purification of cationic solid lipid nanospheres--effects on particle size, physical stability and cell toxicity.

Cationic solid lipid nanospheres (SLN) were prepared by the microemulsion technique with polysorbate 80 (Tween 80) and butanol as surfactants. The SLN (diameter 100-500 nm, zetapotential around +15 mV) consisted mainly of stearylamine (SA) and different triglycerides. Three different purification methods, ultrafiltration, ultracentrifugation and dialysis, were investigated and compared with the cellular toxicity and physical stability of the dispersions. The cell toxicity was dependent on both the SLN composition and the purification method. Dialysis was found to easily and efficiently remove excessive surfactant determined by dynamic light scattering (DLS), leading to reduced cell toxicity and increased physical stability of the SLN on storage. The cationic SLN might constitute a promising DNA delivery system.

A population-based study of thyroid function after radiotherapy and chemotherapy for a childhood brain tumor.

The effect of craniospinal irradiation (CSI) vs. cranial irradiation (CIR) only with or without chemotherapy (CT) on the hypothalamus/pituitary (HP) thyroid axis was assessed in a population-based study of patients treated for a childhood brain tumor not directly involving the HP axis. Thyroid function was evaluated and compared with that in healthy controls (n = 27), measuring TSH, free T4, total T4, total T3, and TRH. The biological effective dose (BED) of radiotherapy, determined for the HP region and spine and expressed in grays (Gy) as BED, gives a means of expressing the biological effects of different dosage schedules in a uniform way. Seventy-one children (45 males and 26 females), less than 15 yr of age when diagnosed between 1970-1997 in the eastern part of Denmark, were included. Twenty-nine had received CSI, and 42 had received CIR only. The median age at time of radiotherapy was 8.4 yr (range, 0.8-14.9). The median length of follow-up was 12.0 yr (range, 2.0-28.0). There was no significant difference between CSI and the CIR only patients with respect to median BED to the HP region. Primary hypothyroidism was found in 24%, of whom 71% had been treated with CSI and 29% with CIR only; 73% had compensated hypothyroidism, and 27% had overt primary hypothyroidism. Central hypothyroidism was found in 6%. Free T4 and total T3 were significantly lower in the CSI and CIR only groups compared with controls. As the CIR only group had significantly higher median basal TSH levels compared with controls and as the CSI compared with the CIR only group and controls had significantly higher median basal TSH levels, we speculate that this was probably due to scattered irradiation from both cranial and spinal fields to the thyroid gland. There was a significant relation between basal TSH and time of follow-up (r(s) = -0.39; P = 0.001). Stepwise backward multiple linear regression analysis showed that the best-fit model to predict basal TSH was free T4 (P < 0.0001), the length of follow-up (P = 0.02), and total T3 (P = 0.06). In contrast, age at radiotherapy, BED to the HP region and spine, and whether the patient had been treated with CT were not included in the model. The TRH test showed significantly exaggerated and prolonged TSH responses for the CSI and CIR only groups compared with controls, indicating HP dysfunction. In conclusion, these data suggest that both CSI and CIR for childhood brain tumor may affect the HP-thyroid axis, resulting in hypothyroidism. CT had no significant influence on HP-thyroid function. We recommend prolonged surveillance of pituitary-thyroid function in long-term survivors of childhood brain tumor and institution of thyroid hormone replacement if the levels of TSH and free T4 are above and below the normal range, respectively, to ensure normal growth and metabolism.

Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line.

Previous studies have shown a correlation between expression of the EGF receptor type III mutation (EGFRvIII) and a more malignant phenotype of various cancers including: non-small-cell lung cancer, glioblastoma multiforme, prostate cancer and breast cancer. Thus, a detailed molecular genetic understanding of how the EGFRvIII contributes to the malignant phenotype is of major importance for future therapy. The GeneChip Hu6800Set developed by Affymetrix was used to identify changes in gene expression caused by the expression of EGFRvIII. The cell line selected for the study was an EGF receptor negative small-cell-lung cancer cell line, GLC3, stably transfected with the EGFRvIII gene in a Tet-On system. By comparison of mRNA levels in EGFRvIII-GLC3 with those of Tet-On-GLC3, it was found that the levels of mRNAs encoding several transcription factors (ATF-3, JunD, and c-Myb), cell adhesion molecules (CD36, CD24), signal transduction related molecules (MKP-1) and other molecules related to cancer (CD98, thymosin beta-10) were altered in the EGFRvIII transfected cell line. Northern hybridisations and Western blot analyses were used to verify selected results. The results indicate that expression of EGFRvIII alters expression of genes involved in the control of cell growth, survival and motility.

The type III epidermal growth factor receptor mutation. Biological significance and potential target for anti-cancer therapy.

Mutations in the epidermal growth factor receptor occur frequently in a number of human tumours including gliomas, non-small-cell lung carcinomas, ovarian carcinomas and prostate carcinomas. The type III epidermal growth factor receptor mutation (variously named EGFRvIII, de2-7 EGFR or AEGFR), which lacks a portion of the extracellular ligand binding domain, is the most common. Here, we review the current status with regard to the role of EGFRvIII in human cancers. A detailed discussion of the formation of EGFRvIII and its structure at the protein level are likewise included along with a discussion of its more functional roles. The design and use (preclinical and clinical) of small molecule inhibitors, antibodies, and antisense oligonucleotides against wild-type EGFR are considered in detail as these strategies can be directly adapted to target EGFRvIII. Finally, the status of EGFRvIII targeted therapy is reviewed.

Specific killing of P53 mutated tumor cell lines by a cross-reactive human HLA-A2-restricted P53-specific CTL line.

p53 is upregulated in the majority of spontaneous tumors and the HLA class I molecule HLA-A2 is expressed by approximately 50% of the caucasians. Potentially, these facts make HLA-A2-binding p53 peptides for CTL-inducing immunotherapy applicable to a broad range of cancer patients. In our study, we investigated the CTL-inducing capacity of autologous monocyte-derived dendritic cells (DC) maturated by exposure to CD40L and pulsed with a pool of 4 wild-type, HLA-A2-binding p53 peptides, and the p53-specific CD8(+) CTL lines established from healthy HLA-A2-positive donors were characterized. Reactivity to p53(65-73) and p53(187-197) peptides was obtained in the T-cell lines. Interestingly, cold target inhibition experiments demonstrated that the simultaneous recognition of the 2 peptides was the result of cross-reactivity, which was confirmed by killing experiments at the clonal CTL level. Furthermore, 4 HLA-A2(+) p53-mutated tumor cell lines were lysed by the CTL line, indicating that these peptides are endogenously processed and presented on HLA-A2 molecule. Thus, monocyte-derived DC pulsed with a pool of peptides are able to induce CTL reactivity to wild-type p53 peptides presented by several cancer cell lines. In addition, the recognition of 2 different p53 peptides by the same CTL clone suggests a promiscuous peptide recognition by the TCR involved. Taken together, these in vitro results suggest that vaccination with autologous DC pulsed with multiple p53 epitopes may induce an effective tumor-specific CTL response in vivo with the potential to eradicate p53-upregulated spontaneously occurring tumors.

Gonadal status in male survivors following childhood brain tumors.

The effect of radiotherapy (RT) and chemotherapy (CT) on gonadal function was assessed in males treated for a childhood brain tumor not directly involving the hypothalamus/pituitary (HP) axis in a population-based study with a long follow-up time. All males <15 yr at the time of diagnosis (median: 9.0 yr, range: 0.8 to 14.9 yr) and diagnosed from January 1970 through February 1997 in the eastern part of Denmark and [gte]18 yr at the time of follow-up (median: 25.8 yr, range:18.5 to 39.3 yr) were included. Thirty males fulfilled the criteria. The median age at time of RT was 9.0 yr (range: 0.8 to 14.9 yr) and the median length of follow-up was 18 yr (range: 2.0 to 28.0 yr). The biological effective dose of RT was determined to the HP region and to the spine and expressed in gray because the biological effective dose gives a means of expressing the biological effect on normal tissue of different dosage schedules in a uniform way. Levels of serum FSH, luteinizing hormone (LH), sexual hormone-binding globulin, testosterone, and inhibin B were measured and compared with healthy age-matched male controls (n = 347), and the patients had a GnRH stimulation test performed with determination of peak FSH and LH. Patients treated with RT + CT (n = 13), compared with patients treated with RT only (n = 17), had significantly higher median peak FSH (8.33 vs. 3.79 IU/L, P = 0.03) and median peak LH (20.0 vs. 12.8 IU/L, P = 0.03), and significantly lower median inhibin B (86.0 vs. 270 pg/ml, P = 0.03), and median inhibin B/FSH ratio (12.8 vs. 107.9, P = 0.04), which indicates gonadal damage. Inhibin B and inhibin B/FSH ratio were also significantly lower in the RT + CT group, compared with controls (median: 86.0 vs. 215 pg/ml, P = 0.02), (median:12.8 vs. 67; P = 0.01), respectively. We found a significantly inverse correlation between basal FSH and inhibin B and FSH and total testicular volume (r(s) = -0.83; P < 0.0001), (r(s) = -0.67; P < 0.0001), respectively, and a significant correlation between inhibin B and total testicular volume (r(s) = 0.63; P < 0.0001). Stepwise backward multiple linear regression analysis showed the best-fit model to predict inhibin B levels included total testicular volume (P = 0.002) and CT (P = 0.09). Median basal LH in the RT-only group was significantly lower, compared with controls (3.44 vs. 2.45 IU/L; P = 0.0001) indicating secondary hypogonadism, and in both the RT + CT group and the RT-only group, levels of testosterone were significantly lower, compared with our reference population (12.8 vs. 21.9 nmol/L; P = 0.001, and 14.7 vs. 21.9 nmol/L; P = 0.0003), respectively. In conclusion these data suggest that cranial irradiation for a childhood brain tumor may affect the HP axis, and adjuvant CT can reduce inhibin B indicating primary gonadal damage. Thus, such patients may have normal or even low levels of FSH despite damage to the seminiferous epithelium, and because the fertility status by a semen analysis for psychological reasons can be difficult to obtain in this group of patients, we suggest inhibin B as the most useful direct serum marker of spermatogenesis in the follow-up of individuals who have received both cranial irradiation and gonadotoxic chemotherapy. However, because the number of patients with RT + CT and RT only are small, these data must be confirmed in further studies.

Cranial radiotherapy of childhood brain tumours: growth hormone deficiency and its relation to the biological effective dose of irradiation in a large population based study.

OBJECTIVE: The study was to determine the incidence of GH deficiency (GHD) following cranial radiotherapy (RT) for a childhood brain tumour in a large population based study and analyse the biological effective dose (BED) to the hypothalamus/pituitary (HP) region as a risk factor. DESIGN: BED was assessed by use of the linear-quadratic (LQ) model, which gives a means of expressing the biological effect of various treatment schedules in a uniform way. In patients aged >/= 18 years (n = 53) GH status was assessed by an insulin-tolerance test (ITT) (n = 34), however, in patients with seizure disorders (n = 19), and in 20 children aged < 18 years GH status was assessed by an arginine test. Cut-off levels for GHD, indicating GH substitution, were defined by a peak GH response of < 9 mU/l and < 15 mU/l for patients >/= 18 and < 18 years, respectively. PATIENTS: Ninety-one children aged < 15 years eligible for the study, diagnosed between 1970 and 1997 in the Eastern part of Denmark, the Faroe Islands and Greenland, with a primary brain tumour not directly involving the HP axis. 84% (n = 76) agreed to participate. Three patients were excluded due to hypothyroidism detected at time of testing. MEASUREMENTS: Serum GH and levels of serum insulin-like growth factor-I (s-IGF-I) and serum insulin-like growth factor binding protein-3 (s-IGFBP-3) were measured. BED was assessed to the HP region. RESULTS: The median age at the time of RT was 8.7 years (range: 0.8-14.9 years) and the median time of follow-up was 15 years (range: 2-28 years). Fifty-eight patients (80%) had GHD and they had received a median BED of 77.5 Gy to the HP region, whereas the median BED was 54.5 Gy for 15 patients without GHD (P = 0.002). Peak GH and BED were correlated (rs = -0.53, P < 0001). Median IGF-I SDS and IGFBP-3 SDS were -2.5 (-5.2-0.7 SDS) and -1.7 (-5.8-0.9 SDS), respectively, and IGF-I SDS was correlated to peak GH (rs = 0.45, P < 0.001). Peak GH and length of follow-up were related (rs = -0.28, P = 0.018). Stepwise backward multiple linear regression analysis showed that the best-fit model to predict the peak GH release following ITT/arginine stimulation included BED (P < 0.0001) and length of follow-up (P = 0.05). CONCLUSIONS: The data of this study suggest that the majority of long-term survivors of brain tumours develop GH deficiency following radiotherapy in childhood and that the adverse effects of radiotherapy may be directly related to the biologically effective dose. With longer follow-up fewer patients might respond normally to GH stimulation tests.

Gene delivery by an epidermal growth factor/DNA polyplex to small cell lung cancer cell lines expressing low levels of epidermal growth factor receptor.

In the present study, we wanted to determine whether efficient gene delivery using an epidermal growth factor (EGF)/DNA polyplex could be accomplished in small cell lung cancer (SCLC) cell lines expressing low EGF receptor (EGFR) levels. EGFR expression levels and transduction efficiencies with polyplexes were examined in five SCLC cell lines and two controls. EGFR expression was examined by binding assays and demonstrated low EGFR levels ranging from 3.6 to 87.4 fmol/mg protein. The SCLC cell lines exhibited high sensitivity to adenovirus infection, which was an important determinant for transduction efficiency when adenovirus was used as an endosomolytic agent. The transduction efficiencies with EGF/DNA polyplexes ranged from 41% +/- 3.5% to 73% +/- 4.6% in the EGFR-positive SCLC cell lines. In the controls lacking EGFRs, only 5% +/- 1.0% and 8% +/- 1.8% of the cells were transduced. Furthermore, the transduction efficiency could be reduced from 50% +/- 4.9% to 18% +/- 1.1% when excess EGF was added to compete with the EGF/DNA polyplexes. In the present study, receptor-targeted gene delivery to SCLC cell lines has been demonstrated for the first time. Our results indicate that even low receptor expression levels in the target cells are sufficient for efficient and specific in vitro gene delivery with EGF/DNA polyplexes.