Concurrent or sequential letrozole with adjuvant breast radiotherapy: final results of the CO-HO-RT phase II randomized trial.

BACKGROUND: We present here final clinical results of the COHORT trial and both translational sub-studies aiming at identifying patients at risk of radiation-induced subcutaneous fibrosis (RISF): (i) radiation-induced lymphocyte apoptosis (RILA) and (ii) candidates of certain single-nucleotide polymorphisms (SNPs). PATIENTS AND METHODS: Post-menopausal patients with stage I-II breast cancer (n = 150) were enrolled and assigned to either concurrent (arm A) or sequential radiotherapy (RT)-letrozole (arm B). Among them, 121 were eligible for RILA and SNP assays. Grade ≥2 RISF were the primary end point. Secondary end points were lung and heart events and carcinologic outcome. RILA was performed to predict differences in RISF between individuals. A genome-wide association study was performed to identify SNPs associated with RILA and RISF. Analyses were done by intention to treat. RESULTS: After a median follow-up of 74 months, 5 patients developed a grade ≥2 RISF. No significant difference was observed between arms A and B. Neither grade ≥2 lung nor symptomatic cardiac toxicity was observed. Median RILA value of the five patients who had grade ≥2 RISF was significantly lower compared with those who developed grade ≤1 RISF (6.9% versus 13%, P = 0.02). Two SNPs were identified as being significantly associated with RILA: rs1182531 (P = 4.2 × 10(-9)) and rs1182532 (P = 3.6 × 10(-8)); both located within the PHACTR3 gene on chromosome 20q13.33. CONCLUSIONS: With long-term follow-up, letrozole can safely be delivered concomitantly with adjuvant breast RT. Translational sub-studies showed that high RILA values were correlated with patients who did not develop RISF. REGISTERED CLINICAL TRIAL: NCT00208273.

Age dependence of T-lymphocyte apoptosis induced by high-energy proton exposure.

Peripheral blood samples from three donors of different ages were exposed to 300 kVp x-rays or 138 MeV protons (0.2, and 9 Gy dose). After 48 h incubation, CD4 and CD8 T-lymphocytes were labelled with specific monoclonal antibodies and cellular DNA was stained by propidium iodine. Radiation-induced apoptosis was followed by flow cytometry and the data were processed by LYSIS II software. The data analysis revealed an age-dependent sensitivity to radiation-induced apoptosis by 300 kVp x-rays and 138 MeV protons, for both CD4 and CD8 T-lymphocytes. Radiation-induced apoptosis was about 4 times greater in CD4 lymphocytes from the youngest donor than the oldest donor and was about 2 times greater in CD8 T-lymphocytes, both after x-ray and proton exposures. RBE values for CD4 T-lymphocytes ranged from 0.9 to 1.4 and for CD8-positive cells from 0.7 to 0.9. It is concluded that radiation-induced apoptosis of CD4 and CD8 T-lymphocytes, which is already exploited to predict patient response in conventional radiotherapy, may also be used to predict response in proton treatment planning.

[76Br]Bromodeoxyuridine PET in tumor-bearing animals.

5-bromodeoxyuridine (BUdR) provides in vitro measures of tumor cell proliferation. We used positron emission tomography to study tissue and plasma kinetics of [76Br]BUdR in tumor-bearing animals. In order to account for the slow washout of the major plasma metabolite, [76Br]bromide, a mathematical correction for the distribution volume of [76Br]bromide was applied. However, following correction specific tumor tracer retention was low or even zero and did not correlate with independent measures of proliferation. The kinetic characteristics of [76Br]BUdR make this tracer unsuitable for proliferation imaging.

Wortmannin selectively enhances radiation-induced apoptosis in proliferative but not quiescent cells.

PURPOSE: To examine whether wortmannin enhances radiation-induced apoptosis in human lymphoid cells. METHODS AND MATERIALS: Different concentrations of wortmannin (0-40 micrOM) were added to TK6 lymphoblastoid cell and whole blood cell cultures 15 min before irradiation (0-6-Gy X-rays). After irradiation, medium was changed and cells were left to incubate for 48 h. In blood samples, CD4, CD8, and CD20 lymphocytes were labeled using FITC-conjugated antibodies. All cell types were fixed in a diethyleneglycol-formaldehyde solution. DNA was stained with propidium iodide. Apoptosis was quantified using flow cytometry and confirmed using fluorescence microscopy. RESULTS: Wortmannin significantly enhances radiation-induced apoptosis in lymphoblastoid cells. Compared to the controls, wortmannin treatment only slightly enhanced radiation-induced apoptosis in quiescent T-lymphocytes and had no effect in quiescent B-lymphocytes. CONCLUSION: Wortmannin enhances radiation-induced apoptosis in a cell-type dependent manner. If the selective effect of wortmannin on proliferative tissues also exists in nonlymphoid tissues, it should enhance the therapeutic ratio of treatments for tumors located in poorly proliferative healthy tissues. Further studies are needed to compare the effects of wortmannin in human tumor cells and various normal cells including proliferative and quiescent cells.

Sources of variation in patient response to radiation treatment.

PURPOSE: To investigate sources of variation in radiosensitivity displayed by cancer patients and blood donors using the leukocyte apoptosis assay. METHODS AND MATERIALS: Probes were obtained from 105 healthy blood donors, 48 cancer patients displaying normal sensitivity to radiotherapy, 12 cancer patients displaying hypersensitivity to radiotherapy, 12 Ataxia telangiectasia blood donors, and 4 additional individuals with genetic diseases of potentially modified radiosensitivity; 2 neurofibromatosis (NF) donors, a Nijmegen breakage syndrome (NBS) donor, and an Immunodeficiency, Chromosome fragility, Facial anomaly syndrome (ICF) donor. Heparinized blood was diluted in medium, irradiated, and left to incubate for 48 h. CD4 and CD8 T-lymphocyte DNA was stained with propidium iodide and the cells were analyzed by flow cytometry. RESULTS: Radiation-induced apoptosis depended on age and cell type. Cohorts of hypersensitive cancer patients, NBS and AT donors displayed compromised apoptotic response. An asymmetric apoptotic response of T-lymphocytes was observed in an ICF donor and a cryptic hypersensitivity donor. Two NF donors displayed no abnormal sensitivity to radiotherapy but compromised apoptotic T-cell response to X-rays. CONCLUSION: Our studies reveal 4 physiologic sources of variation in radiation response-2 are genetic: cryptic hypersensitivity and hereditary disease, and 2 are epigenetic: cell type and donor age. They emphasize the important role of proteins involved in the recognition and repair of DNA double-strand breaks in determining the response of individuals to radiotherapy.

High levels of delayed radiation-induced apoptosis observed in lymphoblastoid cell lines from ataxia-telangiectasia patients.

PURPOSE: Cells from ataxia-telangiectasia (A-T) patients are extremely sensitive to radiation but display decreased apoptosis, as measured during the first 3 days following radiation. To explain this apparent contradiction, we examined apoptosis in normal and A-T cells at late time points following radiation, under the assumption that radiation-induced apoptosis is delayed in the A-T cells. METHODS AND MATERIALS: Blood cells and lymphoblastoid cell lines from A-T patients, as well as healthy donors, were irradiated with X-rays. Apoptosis was measured at different time points (up to 7 and 30 days for lymphocytes and lymphoblastoid cells, respectively) using a flow cytometric method based on the reduction of intracellular DNA content (sub-G1 population). RESULTS: Compared to normal cells, CD4 and CD8 A-T lymphocytes displayed constantly reduced levels of radiation-induced apoptosis for up to 7 days after treatment. A-T lymphoblastoid cells, however, displayed a delayed and prolonged apoptosis. CONCLUSION: A-T lymphoblastoid cells show high levels of delayed radiation-induced apoptosis, which may contribute to the high cellular radiosensitivity displayed by the A-T phenotype. ATM (the gene mutated in A-T) plays different roles in the apoptotic response to ionizing radiation in quiescent lymphocytes and proliferative lymphoblastoid cells.

Distinct apoptotic phenotypes induced by radiation and ceramide in both p53-wild-type and p53-mutated lymphoblastoid cells.

The tumour suppressor gene p53 and the intracellular signalling molecule ceramide have both been shown to play crucial roles in the induction of apoptosis by ionising radiation. In this study we examined whether p53 and ceramide are involved in independent signal pathways, inducing different types of apoptosis. TK6 (p53wt/wt) and WTK1 (p53mut/mut) lymphoblastoid cells were treated with ionising radiation or N-acetyl-D-sphingosine (C2-ceramide). Flow cytometry and fluorescence microscopy studies were performed to characterise the time kinetics and morphological features of induced apoptosis. Ceramide- and radiation-induced apoptotic cells display characteristic differences in morphology and DNA staining and ceramide-induced apoptosis is expressed much faster than radiation-induced apoptosis. Radiation-induced apoptosis is p53-dependent and ceramide-induced apoptosis is p53-independent. The p53 pathway and the ceramide pathway are two independent signal pathways leading to distinct types of apoptosis. Since p53 is very often dysfunctional in tumour cells, modifying the ceramide pathway is a promising strategy to increase tumour sensitivity to radiation and other anticancer agents.

Imaging brain tumor proliferative activity with [124I]iododeoxyuridine.

Iododeoxyuridine (IUdR) uptake and retention was imaged by positron emission tomography (PET) at 0-48 min and 24 h after administration of 28.0-64.4 MBq (0.76-1.74 mCi) of [124I]IUdR in 20 patients with brain tumors, including meningiomas and gliomas. The PET images were directly compared with gadolinium contrast-enhanced or T2-weighted magnetic resonance images. Estimates for IUdR-DNA incorporation in tumor tissue (Ki) required pharmacokinetic modeling and fitting of the 0-48 min dynamically acquired data to correct the 24-h image data for residual, nonincorporated radioactivity that did not clear from the tissue during the 24-h period after IUdR injection. Standard uptake values (SUVs) and tumor:brain activity ratios (Tm:Br) were also calculated from the 24-h image data. The Ki, SUV, and Tm/Br values were related to tumor type and grade, tumor labeling index, and survival after the PET scan. The plasma half-life of [124I]IUdR was short (2-3 min), and the arterial plasma input function was similar between patients (48 +/- 12 SUV*min). Plasma clearance of the major radiolabeled metabolite ([124I]iodide) varied somewhat between patients and was markedly prolonged in one patient with renal insufficiency. It was apparent from our analysis that a sizable fraction (15-93%) of residual nonincorporated radioactivity (largely [124I]iodide) remained in the tumors after the 24-h washout period, and this fraction varied between the different tumor groups. Because the SUV and Tm:Br ratio values reflect both IUdR-DNA incorporated and exchangeable nonincorporated radioactivity, any residual nonincorporated radioactivity will amplify their values and distort their significance and interpretation. This was particularly apparent in the meningioma and glioblastoma multiforme groups of tumors. Mean tumor Ki values ranged between 0.5 +/- 0.9 (meningiomas) and 3.9 +/- 2.3 microl/min/g (peak value for glioblastoma multiforme, GBM). Comparable SUV and Tm:Br values at 24 h ranged from 0.13 +/- 0.03 to 0.29 +/- 0.19 and from 2.0 +/- 0.6 to 6.1 +/- 1.5 for meningiomas and peak GBMs, respectively. Thus, the range of values was much greater for Ki (approximately 8-fold) compared with that for SUV (approximately 2.2-fold) and Tm:Br (approximately 3-fold). The expected relationships between Ki, SUV, and Tm:Br and other measures of tumor proliferation (tumor type and grade, labeling index, and patient survival) were observed. However, greater image specificity and significance of the SUV and Tm:Br values would be obtained by achieving greater washout and clearance of the exchangeable fraction of residual (background) radioactivity in the tumors, i.e., by increased hydration and urinary clearance and possibly by imaging later than 24 h after [124I]IUdR administration.

The transfer of bacteria to, and survival on, dental records.

The purpose of this study was to ascertain whether viable bacteria could be transferred from gloves to laboratory cards in dental records and, if so, to determine whether bacteria could survive on the paper. The thumbs and forefingers of two types of glove (Biogel D and Microtouch) were inoculated with Streptococcus sanguis and left for various periods of time. A sterile dental laboratory card was then held with the gloves and the number of bacteria surviving on the card determined after various periods of incubation of the card at room temperature. Bacteria were transferred to the laboratory cards and remained viable for up to 72 h. More viable bacteria were transferred from the Microtouch gloves than from the Biogel D gloves, and this was attributable, in part, to the latter gloves exerting a bactericidal effect. The results demonstrated that bacteria can be transferred from gloves to laboratory cards and that these organisms can remain viable for long periods of time. Dental records may, therefore, represent a possible route of cross-infection.

Altered apoptotic profiles in irradiated patients with increased toxicity.

PURPOSE: A retrospective study of radiation-induced apoptosis in CD4 and CD8 T-lymphocytes, from 12 cancer patients who displayed enhanced toxicity to radiation therapy and 9 ataxia telangiectasia patients, was performed to test for altered response compared to healthy blood-donors and normal cancer patients. METHODS AND MATERIALS: Three milliliters of heparinized blood from each donor was sent via express post to the Paul Scherrer Institute (PSI) for subsequent examination. The blood was diluted 1:10 in RPMI medium, irradiated with 0-, 2-, or 9-Gy X-rays, and incubated for 48 h. CD4 and CD8 T-lymphocytes were then labeled using FITC-conjugated antibodies, erythrocytes were lysed, and the DNA stained with propidium iodide. Subsequently, cells were analyzed using a Becton Dickinson FACScan flow cytometer. Radiation-induced apoptosis was recognized in leukocytes as reduced DNA content attributed to apoptosis-associated changes in chromatin structure. Apoptosis was confirmed by light microscopy, electron microscopy, and by the use of commercially available apoptosis detection kits (in situ nick translation and Annexin V). Data from hypersensitive individuals were compared to a standard database of 105 healthy blood-donors, and a database of 48 cancer patient blood donors who displayed normal toxicity to radiation therapy. To integrate radiosensitivity results from CD4 and CD8 T-lymphocytes after 2 and 9 Gy, z-score analyses were performed. RESULTS: A cohort of 12 hypersensitive patients was evaluated; 8 showed enhanced early toxicity, 3 showed enhanced late toxicity, and 1 showed both. The cohort displayed less radiation-induced apoptosis (-1.8 sigma) than average age-matched donors. A cohort of 9 ataxia telangiectasia homozygotes displayed even less apoptosis (-3.6 sigma). CONCLUSION: The leukocyte apoptosis assay appears to be a useful predictor of individuals likely to display increased toxicity to radiation therapy; however, validation of this requires a prospective study.

Anti-neuroblastoma antibody chCE7 binds to an isoform of L1-CAM present in renal carcinoma cells.

Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti-neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 M(r) cell surface protein. The protein was partially purified by immuno-affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH(2)-terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1-CAM), and both were characterized by the NH(2)-terminal deletion of 5 amino acids, comprising exon 2 of L1-CAM. Reverse trancription-polymerase chain reaction (RT-PCR) analysis of the regions spanning exon 2 and exon 27 of L1-CAM indicated that in neuroblastoma cells both transcripts for the full-length and exon-deleted forms are present, whereas in the renal carcinoma cell lines only the exon-deleted L1-CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1-CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1-CAM mRNA levels are correlated with protein expression.

Effect of hMSH6 cDNA expression on the phenotype of mismatch repair-deficient colon cancer cell line HCT15.

Mismatch recognition in human cells is mediated primarily by a heterodimer of hMSH2 and hMSH6. Cells mutated in both alleles of the hMSH6 gene are deficient in the correction of base/base mispairs and insertion/deletion loops of one nucleotide and thus exhibit a strong mutator phenotype, evidenced by elevated mutation rates and microsatellite instability, as well as by tolerance to methylating agents. The decrease in replication fidelity associated with a loss of mismatch correction implies that with each division, these cells are likely to acquire new mutations throughout their genomes. Should such secondary mutations occur in genes linked to replication fidelity or involved in the maintenance of genomic stability, they might contribute to the observed mutator phenotype. The human colon tumour line HCT15 represents one such case. Although it carries inactivating mutations in both hMSH6 alleles, it has also been shown to contain a missense mutation in the coding sequence of the proofreading domain of the polymerase-delta gene. In an attempt to find out whether the phenotype of HCT15 cells was indeed brought about solely by the lack of hMSH6, we stably transfected them with a vector carrying the wild-type hMSH6 cDNA. Our results show that although the levels of transgenic hMSH6 were low, expression of the wild-type protein resulted in a substantial restoration of mismatch binding, mismatch repair capacity and the stability of mononucleotide repeats, as well as in the reduction of mutation rates. Although methylation tolerance of the hMSH6-expressing cells was not markedly affected, the G2 cell cycle checkpoint, absent in N-methyl-N'-nitro-N-nitrosoguanidine-treated control cells, was restored.

TP5 triggers signal transduction involving mitogen activated protein kinases in monocytes.

The pentapetide thymopentin (TP5) corresponding to the aminoacids RKDVY represents the residues 32-36 of thymopoietin (TP), which was originally isolated from bovine thymus. Both were observed to induce T-cell differentiation and maturation. Recently however it was shown, that TP represents the N-terminal 49 aa of the human thymopoietin (TMPO) isoforms TMPO alpha, beta and gamma, which are localized in the nucleus. TP5 was investigated in a variety of diseases and showed efficacy by improving the immune balance, whereby different cells increased in cell number or activity. Findings which support the assumption of multifunctional efficacy and a description of TP and TP5 modulating T cells lack any interpretation on molecular level. In the present study we investigated the binding of TP5 on white blood cells. We identified monocytes and neutrophils as TP5-binding cells by displacing fluorescein-labelled TP5 with an excess of unlabelled TP5 in competition assays. Binding of TP5 on cell surface proteins resulted in cellular signalling and we report here that TP5 triggers signal transduction involving mitogen activated protein kinases p42/p44 (MAPKs) in monocytes.

m-THPC-mediated photodynamic therapy (PDT) does not induce resistance to chemotherapy, radiotherapy or PDT on human breast cancer cells in vitro.

Photodynamic therapy (PDT) uses laser light to activate a photosensitizer that has been absorbed preferentially by cancer cells after systemic administration. A phototoxic reaction ensues resulting in cell death and tissue necrosis. Some cells, however, may survive PDT. This study was performed to determine if surviving human breast cancer cells (MCF-7) can become resistant to PDT, chemotherapy or radiotherapy. The MCF-7 cells were cultured under standard conditions prior to being exposed to the photosensitizer, 5,10,15,20-meta-tetra(hydroxyphenyl)chlorin (m-THPC), for 24 h and then irradiated with laser light (652 nm). Surviving cells were allowed to regrow by allowing a 2 week interval between each additional PDT. After the third and final treatment, colony formation assays were used to evaluate the sensitivity of cultured cells to ionizing radiation and PDT and the ATP cell viability assay tested in vitro chemosensitivity. Flow cytometry was used to analyze the cell cycle. No alterations in the cell cycle were observed after three cycles of PDT with m-THPC. Similar responses to chemotherapy and ionizing radiation were seen in control and treatment groups. The m-THPC-sensitized PDT did not induce resistance to subsequent cycles of PDT, chemo- or radiotherapy. Photodynamic therapy with m-THPC may represent a novel adjunctive treatment of breast cancer that may be combined with surgery, chemotherapy or ionizing radiation.

Successful treatment of invasive aspergillosis in chronic granulomatous disease by bone marrow transplantation, granulocyte colony-stimulating factor-mobilized granulocytes, and liposomal amphotericin-B.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency with complete absence or malfunction of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the phagocytic cells. Life-threatening infections especially with aspergillus are common despite optimal antimicrobial therapy. Bone marrow transplantation (BMT) is contraindicated during invasive aspergillosis in any disease setting. We report an 8-year-old patient with CGD who underwent HLA-genoidentical BMT during invasive multifocal aspergillus nidulans infection, nonresponsive to treatment with amphotericin-B and gamma-interferon. During the first 10 days post-BMT, the patient received granulocyte colony-stimulating factor (G-CSF)-mobilized, 25 Gy irradiated granulocytes from healthy volunteers plus G-CSF beginning on day 3 to prolong the viability of the transfused granulocytes. This was confirmed in vitro by apoptosis assays and in vivo by finding nitroblue tetrazolium (NBT)-positive granulocytes in peripheral blood 12 and 36 hours after the transfusions. Clinical and biological signs of infection began to disappear on day 7 post-BMT. Positron emission tomography with F18-fluorodeoxyglucose (FDG-PET) and computed tomography (CT) scans at 3 months post-BMT showed complete disappearance of infectious foci. At 2 years post-BMT, the patient is well with full immune reconstitution and no sign of aspergillus infection. Our results show that HLA-identical BMT may be successful during invasive, noncontrollable aspergillus infection, provided that supportive therapy is optimal.

Potential tumour doubling time: determination of Tpot for various canine and feline tumours.

Spontaneous tumours in dogs and cats are an excellent model for clinical human research, such as in developing proton conformation radiotherapy for humans. The kinetics of tumour cells can be used effectively to predict prognosis and response to therapy in patients with tumours. Knowledge of the kinetic parameters in these tumours is therefore important. In the present study the kinetic parameters evaluated included the labelling index (LI), relative movement (RM), mitotic index (MI), and potential doubling time (Tpot). These parameters were determined using in vivo labelling with bromodeoxyuridine, flow cytometry and histological preparation. Samples were obtained and evaluated from 72 dogs and 20 cats, presenting as patients in our clinic. Within the groups of epithelial and mesenchymal tumours from dogs and cats, the kinetic parameters LI, RM and MI were compared with Tpot. Significant correlations were observed for the comparison Tpot and LI. No correlation was found between Tpot and RM.

Programmed cellular response to ionizing radiation damage.

Three forms of radiation response were investigated to evaluate the hypothesis that cellular radiation response is the result of active molecular signaling and not simply a passive physicochemical process. The decision whether or not a cell should respond to radiation-induced damage either by induction of rescue systems, e.g. mobilization of repair proteins, or induction of suicide mechanisms, e.g. programmed cell death, appears to be the expression of intricate cellular biochemistry. A cell must recognize damage in its genetic material and then activate the appropriate responses. Cell type is important; the response of a fibroblast to radiation damage is both quantitatively and qualitatively different from that of a lymphocyte. The programmed component of radiation response is significant in radiation oncology and predicted to create unique opportunities for enhanced treatment success.