MicroRNA expression profiling of sputum for the detection of early and locally advanced non-small-cell lung cancer: a prospective case-control study.

BACKGROUND: Non-small-cell lung cancer (nsclc) is associated with very poor overall survival because 70% of patients present with locally advanced or metastatic disease at the time of diagnosis. Micrornas (mirnas) are a class of short, noncoding rna molecules whose presence in samples of biologic fluids such as sputum has demonstrated promise as a potential means of detecting nsclc. We investigated the stage-specific nsclc detection potential of an efficient panel of 3 mirnas (mir-21, mir-210, mir-372) using a single sputum sample. METHODS: A single spontaneously expectorated sputum sample was prospectively collected from 21 early nsclc (≤stage ii) patients, 22 advanced nsclc (≥stage iii) patients, and 10 control subjects. Mirna expression profiles were determined by quantitative real-time polymerase chain reaction and were analyzed by unsupervised hierarchical cluster analysis. RESULTS: Mean tumour size (±95% confidence interval) in the early and advanced nsclc patients was 3.3 cm ± 0.9 cm and 4.8 cm ± 0.7 cm respectively. Adenocarcinoma constituted 61.9% of the early and 45.5% of the advanced nsclc cases respectively. In comparing the early nsclc group with the control group, the mirna panel yielded a diagnostic sensitivity of 67% and a specificity of 90.0%. For the advanced nsclc group, the mirna panel detected nsclc with a sensitivity and specificity of 64% and 100% respectively. CONCLUSIONS: A sputum mir-21, mir-210, and mir-372 expression profile might provide a sensitive and highly specific means for detecting nsclc. Sputum mirna analysis demonstrates promise as a potential complementary screening tool.

Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells, thus providing a therapeutic potential. In this study, we examined a large panel of human malignant glioma cell lines and primary cultures of normal human astrocytes for their sensitivity to TRAIL. Of 13 glioma cell lines, 3 were sensitive (80-100% death), 4 were partially resistant (30-79% death), and 6 were resistant (< 30% death). Normal astrocytes were also resistant. TRAIL-induced cell death was characterized by activation of caspase-8 and -3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation. Decoy receptor (DcR1 and DcR2) expression was limited in the glioma cell lines and did not correlate with TRAIL sensitivity. Both sensitive and resistant cell lines expressed TRAIL death receptor (DR5), adapter protein Fas-associated death domain (FADD), and caspase-8; but resistant cell lines expressed 2-fold higher levels of the apoptosis inhibitor phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). In contrast, cellular FADD-like IL-1beta-converting enzyme-like inhibitory protein (cFLIP) expression was similar in sensitive and resistant cells. Transfection of sense PED/PEA-15 cDNA in sensitive cells resulted in cell resistance, whereas transfection of antisense in resistant cells rendered them sensitive. Inhibition of protein kinase C (PKC) activity restored TRAIL sensitivity in resistant cells, suggesting that PED/ PEA-15 function might be dependent on PKC-mediated phosphorylation. In summary, TRAIL induces apoptosis in > 50% of glioma cell lines, and this killing occurs through activation of the DR pathway. This caspase-8-induced apoptotic cascade is regulated by intracellular PED/PEA-15, but not by cFLIP or decoy receptors. This pathway may be exploitable for glioma and possibly for other cancer therapies.

Held-breath self-gating technique for radiotherapy of non-small-cell lung cancer: a feasibility study.

PURPOSE: To examine the feasibility of a held-breath self-gating (HBSG) technique in the radiotherapy of lung cancer. MATERIAL AND METHODS: Sixteen consecutive eligible and consenting patients undergoing radiotherapy for non-small-cell lung cancer were accrued for this study. The patients underwent a standardized training session and were then asked to breath hold at four points in the breathing cycle (maximal and end tidal, inspiration and expiration) while under fluoroscopic visualization. Plain films and video imaging with digital image analysis were used to record and measure the movement of the diaphragm, a tumor surrogate, in the superior-inferior axis. These measurements were obtained during and between multiple separate breath holds within one session and between breath holds in sessions held at least one day apart. RESULTS: Maximal inspiration and expiration tend to provide the best positional reliability, and the standard deviation of diaphragmatic position ranged from 0.13 to 2.57 mm, with an average of 0.97 mm. During a single breath hold, the diaphragmatic movement averaged 2.62 mm with a standard deviation of 1.28 mm. The day-to-day variation of diaphragmatic positions was less than 5 mm. The held-breath self-gating technique resulted in a reduction of diaphragmatic movement by an average of 11.9 mm when compared to that seen during tidal breathing. CONCLUSION: In the radiotherapeutic management of non-small-cell lung cancer, this HBSG technique offers a simple method that provides superior immobilization of the diaphragm compared to tidal breathing. Clinical implementation of this technique should allow for a reduction of treatment margins, thus sparing more normal tissues and facilitating dose escalation.

Dose-volume complication analysis for visual pathway structures of patients with advanced paranasal sinus tumors.

PURPOSE: The purpose of the present work was to relate dose and volume information to complication data for visual pathway structures in patients with advanced paranasal sinus tumors. METHODS AND MATERIALS: Three-dimensional (3D) dose distributions for chiasm, optic nerve, and retina were calculated and analyzed for 20 patients with advanced paranasal sinus malignant tumors. 3D treatment planning with beam's eye view capability was used to design beam and block arrangements, striving to spare the contralateral orbit (to lessen the chance of unilateral blindness) and frequently the ipsilateral orbit (to help prevent bilateral blindness). Point doses, dose-volume histogram analysis, and normal tissue complication probability (NTCP) calculations were performed. Published tolerance doses that indicate significant risk of complications were used as guidelines for analysis of the 3D dose distributions. RESULTS: Point doses, percent volume exceeding a specified published tolerance dose, and NTCP calculations are given in detail for patients with complications versus patients without complications. Two optic nerves receiving maximum doses below the published tolerance dose sustained damage (mild vision loss). Three patients (of 13) without optic nerve sparing and/or chiasm sparing had moderate or severe vision loss. Complication data, including individual patient analysis to estimate overall risk for loss of vision, are given. CONCLUSION: 3D treatment planning techniques were used successfully to provide bilateral sparing of the globe for most patients. It was more difficult to spare the optic nerves, especially on the ipsilateral side, when prescription dose exceeded the normal tissue tolerance doses. NTCP calculations may be useful in assessing complication risk better than point dose tolerance criteria for the chiasm, optic nerve, and retina. It is important to assess the overall risk of blindness for the patient in addition to the risk for individual visual pathway structures.

Targeted radiotherapy of multicell neuroblastoma spheroids with high specific activity [125I]meta-iodobenzylguanidine.

PURPOSE: Iodine-125 induces cell death by a mechanism similar to that of high linear energy transfer (high-LET) radiation. This study investigates the cytotoxicity of high-specific-activity [125I]meta-iodobenzylguanidine (125I-mIBG) in human SK-N-MC neuroblastoma cells grown as three-dimensional multicellular spheroids. MATERIALS AND METHODS: Spheroids were incubated with high-specific-activity 125I-mIBG (6 mCi/microg, 1000 times that of the conventional specific activity used for autoradiography). Cytotoxicity was assessed by fluorescence viability markers and confocal microscopy for intact spheroids, fluorescence-activated cell sorting and clonogenic assay, and clonogenic assays for dispersed whole spheroids. Distribution of radioactive mIBG was determined by quantitative light-microscope autoradiography of spheroid cryostat sections. Dose estimation was based on temporal knowledge of the retained radioactivity inside spheroids, and of the radiolabel's emission characteristics. Findings were compared with those of spheroids treated under the same conditions with 131I-mIBG, cold mIBG, and free iodine-125. RESULTS: 125I-mIBG exerted significant cell killing. Complete spheroids were eradicated when they were treated with 500 microCi of 125I-mIBG, while those treated with 500 microCi or 1000 microCi of 131I-mIBG were not. The observed difference in cytotoxicity between treatments with 125I- and 131I-mIBG could not be accounted for by the absorbed dose of spheroid alone. The peripheral, proliferating cell layer of the spheroids remained viable at the moderate radioactivity of 100 microCi for both isotopes. Cytotoxicity induced by 125I-mIBG was quantitatively comparable by the peripheral rim thickness to that of 131I-mIBG at the dose of 100 microCi. The peripheral rim thickness decreased most significantly in the first 17 hours after initial treatment. There was no statistical decrease in the rim thickness identified afterwards for the second, third, and fourth days of incubation. CONCLUSION: The cytotoxic effect of high-specific-activity 125I-mIBG appears to be comparable to, if not more efficient than that of conventionally used 131I-mIBG at the same level of total radioactivity. 125I-mIBG may improve the therapeutic index over that of 131I-mIBG in the clinical management of metastatic neuroblastoma due to the short range of Auger electrons.

Dosimetric evaluation of lung tumor immobilization using breath hold at deep inspiration.

PURPOSE: To examine the dosimetric benefit of self-gated radiotherapy at deep-inspiration breath hold (DIBH) in the treatment of patients with non-small-cell lung cancer (NSCLC). The relative contributions of tumor immobilization at breath hold (BH) and increased lung volume at deep inspiration (DI) in sparing high-dose lung irradiation (> or = 20 Gy) were examined. METHODS AND MATERIALS: Ten consecutive patients undergoing radiotherapy for Stage I-IIIB NSCLC who met the screening criteria were entered on this study. Patients were instructed to BH at DI without the use of external monitors or breath-holding devices (self-gating). Computed tomography (CT) scans of the thorax were performed during free breathing (FB) and DIBH. Fluoroscopy screened for reproducible tumor position throughout DIBH, and determined the maximum superior-inferior (SI) tumor motion during both FB and DIBH. Margins used to define the planning target volume (PTV) from the clinical target volume included 1 cm for setup error and organ motion, plus an additional SI margin for tumor motion, as determined from fluoroscopy. Three conformal treatment plans were then generated for each patient, one from the FB scan with FB PTV margins, a second from the DIBH scan with FB PTV margins, and a third from the DIBH scan with DIBH PTV margins. The percent of total lung volume receiving > or = 20 Gy (using a prescription dose of 70.9 Gy to isocenter) was determined for each plan. RESULTS: Self-gating at DIBH was possible for 8 of the 10 patients; 2 patients were excluded, because they were not able to perform a reproducible DIBH. For these 8 patients, the median BH time was 23 (range, 19-52) s. The mean percent of total lung volume receiving > or = 20 Gy under FB conditions (FB scan with FB PTV margins) was 12.8%. With increased lung volume alone (DIBH scan with FB PTV margins), this was reduced to 11.0%, tending toward a significant decrease in lung irradiation over FB (p = 0.086). With both increased lung volume and tumor immobilization (DIBH scan with DIBH PTV margins), the mean percent lung volume receiving > or = 20 Gy was further reduced to 8.8%, a significant decrease in lung irradiation compared to FB (p = 0.011). Furthermore, at DIBH, the additional benefit provided by tumor immobilization (i.e., using DIBH instead of FB PTV margins) was also significant (p = 0.006). The relative contributions of tumor immobilization and increased lung volume toward reducing the percent total lung volume receiving > or = 20 Gy were patient specific; however, all 8 of the patients analyzed showed a dosimetric benefit with this DIBH technique. CONCLUSION: Compared to FB conditions, at DIBH the mean reduction in percent lung volume receiving > or = 20 Gy was 14.3% with the increase in lung volume alone, 22.1% with tumor immobilization alone, and 32.5% with the combined effect. The dosimetric benefit seen at DIBH was patient specific, and due to both the increased lung volume seen at DI and the PTV margin reduction seen with tumor immobilization.

Results of primary and adjuvant CT-based 3-dimensional radiotherapy for malignant tumors of the paranasal sinuses.

PURPOSE: This study reports our clinical experience supporting the normal tissue-sparing capability of 3-dimensional (3-D) treatment planning when applied to advanced neoplasms of the paranasal sinuses. METHODS AND MATERIALS: Between 1986 and 1992, computed tomography (CT)-based 3-D radiotherapy was used to treat 39 patients with advanced stage malignant tumors of the paranasal sinuses as all or part of initial treatment. Fifteen unresectable patients were treated with primary radiotherapy to a median prescribed total dose of 68.4 Gy. Twenty-four patients were treated with postoperative adjuvant radiotherapy for close margins (< 5 mm), microscopic or gross residual disease. The median prescribed total doses were 55.8 Gy, 59.4 Gy and 67.8 Gy, respectively. Globe-sparing fields were used in the primary treatment plans of 37 patients (95%). The median follow-up is 4.5 years (range, 19-86 months). RESULTS: For the unresectable patients who were treated with radiotherapy alone, the local control rate at 3 years is 32%. The actuarial overall survivals at 3 and 4 years are 32%. For the patients who received postoperative adjuvant radiotherapy, none of the five patients irradiated for close surgical margins recurred locally. Three of the 14 with microscopic residual (21%) recurred locally at 26, 63, and 74 months from the start of irradiation. Four of the five with gross residual (80%) recurred locally with a median time to recurrence of 2 years. The local control rates at 3 and 5 years for the adjuvant group are 75% and 65%, respectively. The actuarial overall survival at 3 and 5 years are 65% and 60%, respectively. None of the first sites of local disease progression were judged to have occurred outside the high-dose region. There was one case of mild osteoradionecrosis successfully treated with conservative treatment, one case of limited optic neuropathy and one case of possible radiation-induced cataract. There was no blindness related to irradiation. CONCLUSION: This study indicates that computed tomography-based 3-D radiotherapy can preserve critical structures unaffected by tumor invasion and achieve the generally expected local control rates when it is used as all or part of initial treatment for extensive malignant tumors of the paranasal sinus. The presence of gross disease was a major adverse prognostic factor in this study. Additional therapeutic maneuvers are essential to improve the local control and survival rate in patients with advanced paranasal sinus carcinomas.

Killing of EMT-6 cells by decays from isotopes incorporated on sensitizer adducts.

EMT-6 tumor cell killing by decays from 3H and 125I incorporated by adduct formation of radiolabeled sensitizers was studied in vitro. Hypoxic radiosensitizers become covalently bound to cellular molecules after metabolic reduction, and EMT-6 tumor cells can tolerate over 10(9) adducts/cell of misonidazole without loss of colony-forming ability. Cells were incubated under hypoxic conditions in the presence of [3H]misonidazole or [125I]iodoazomycinriboside for various times and the amounts of bound 3H and 125I were determined. Cells were stored as monolayers at 22 degrees C, in suspension culture at 4 degrees C, and frozen in complete medium plus 8% DMSO at -196 degrees C for various times to facilitate the accumulation of radioactive decays before plating in vitro for colony-forming assays at 37 degrees C. At 22 degrees C in monolayer culture, EMT-6 tumor cells tolerated 950 and 1720 decays/cell of 3H and 125I, respectively, without evidence of radiotoxicity. This number of decays/cell over the exposure times used represents 1.54 x 10(6) 3H/cell and 8.4 x 10(4) 125I/cell, respectively. Significant cell killing was detected after similar amounts of isotope decay when cells were held at 4 degrees C. When cells were frozen in the presence of 8% DMSO, they were more resistant to inactivation by isotope decays or by gamma rays than cells in liquid phase at 4 degrees C. These data suggest that selective hypoxic tumor cell suicide by 3H or 125I decays from bound sensitizer at 37 degrees C will be an inefficient process, at least for drugs with specific activities as tested. These data are consistent with data on cell inactivation by isotopes incorporated into cells by other procedures.

The development of target-eye-view maps for selection of coplanar or noncoplanar beams in conformal radiotherapy treatment planning.

Three-dimensional conformal radiotherapy allows the use of tightly conformed, multiple coplanar or noncoplanar beams. However, visualizing the spatial relationships between the target volume and adjacent critical structures is not always obvious or intuitive. Tools such as beam's eye view (BEV) have aided in this process and been very useful. In this study, a target-eye-view (TEV) map is developed as a functional extension of BEVs. The TEV map for a critical structure is created by checking the BEVs for all gantries and table rotations. For each possible BEV, the amount of overlap between the planning target volume (PTV) and the organ at risk (OAR) is determined. This information is presented in a Mercator spherical map, where the color tone indicates the amount of overlap between the PTV and the OAR. A composite TEV map is then created by summing the TEV grading scores for all OARs. The composite map shows beam orientations with the most overlap being light and the least overlap being dark, thus simplifying the selection of appropriate beam angles. The accuracy of the TEV maps has been confirmed separately with corresponding BEVs generated by a three-dimensional treatment planning system.

Implantation metastasis of primary central nervous system lymphoma complicating radiotherapy outcome.

Computed tomography-guided stereotactic biopsy is commonly used in the diagnosis of brain lesions. An uncommonly reported risk of the procedure is the potential of implantation metastasis. This phenomenon has been reported in central nervous system malignancies. Although the role of prophylactic local radiotherapy at biopsy sites is well recognized in solid tumors, it has not been reported to occur after stereotactic biopsy of a brain tumor. The authors report a case of locally progressive primary central nervous system lymphoma at an unsuspiciously underdosed biopsy site complicating radiotherapy outcome.

Dosimetry study of [I-131] and [I-125]- meta-iodobenz guanidine in a simulating model for neuroblastoma metastasis.

The physical properties of I-131 may be suboptimal for the delivery of therapeutic radiation to bone marrow metastases, which are common in the natural history of neuroblastoma. In vitro and preliminary clinical studies have implied improved efficacy of I-125 relative to I-131 in certain clinical situations, although areas of uncertainty remain regarding intratumoral dosimetry. This prompted our study using human neuroblastoma multicellular spheroids as a model of metastasis. 3D dose calculations were made using voxel-based Medical Internal Radiation Dosimetry (MIRD) and dose-point-kernel (DPK) techniques. Dose distributions for I-131 and I-125 labeled mIBG were calculated for spheroids (metastases) of various sizes from 0.01 cm to 3 cm diameter, and the relative dose delivered to the tumors was compared for the same limiting dose to the bone marrow. Based on the same data, arguments were advanced based upon the principles of tumor control probability (TCP) to emphasize the potential theoretical utility of I-125 over I-131 in specific clinical situations. I-125-mIBG can deliver a higher and more uniform dose to tumors compared to I-131 mIBG without increasing the dose to the bone marrow. Depending on the tumor size and biological half-life, the relative dose to tumors of less than 1 mm diameter can increase several-fold. TCP calculations indicate that tumor control increases with increasing administered activity, and that I-125 is more effective than I-131 for tumor diameters of 0.01 cm or less. This study suggests that I-125-mIBG is dosimetrically superior to I-131-mIBG therapy for small bone marrow metastases from neuroblastoma. It is logical to consider adding I-125-mIBG to I-131-mIBG in multi-modality therapy as these two isotopes could be complementary in terms of their cumulative dosimetry.

Effect of radiation on cytokine and cytokine receptor messenger-RNA profiles in p53 wild and mutated human glioblastoma cell lines.

OBJECTIVE: Glioblastoma cells produce cytokines with proinflammatory or immunosuppressive properties, or both, which, in addition to altered p53 gene expression, have been shown to be associated with glioblastoma resistance to radiotherapy. The reported data concerning cytokines have been isolated and sometimes discordant, and a comprehensive profile analysis of cytokines and their corresponding receptors in irradiated glioblastomas has received limited attention. The object of this study was to test the hypothesis that radiation alone in clinically relevant doses would not significantly alter expression of endogenous cytokines and their receptors in human glioblastoma celll ines with wild-type and mutant p53. DESIGN AND METHOD: Culture specimens of 4 glioblastoma cell lines of different p53 gene expression (U87, U118, U251, U373) were irradiated with cobalt 60 at a dose of 10 Gy. After 48 hours, radiosensitivity was defined through a colony formation assay, cell cycle distribution was analyzed by flow cytometry, and cytokine and cytokine receptor messenger-RNA (mRNA) profiles were defined with an RNase protection assay. Different single doses of radiation at varying time intervals after culture were applied also to wild-type p53 cell lines. RESULTS: All cell lines were relatively radioresistant at lower doses of 1 and 2 Gy. Immunosuppressive cytokine and cytokine receptor mRNA of the Th2 (IL-13Ralpha, IL-4) and Th3 family (TGF-beta1, 2 and 3, TGF-betaRI and RII) were expressed. In contrast, only 2 proinflammatory Th1 cytokine receptor genes (IFN-gammaRa and IFN-gammaRbeta), but no significant Th1 cytokine gene expression, were detected. Even though the population examined included a large fraction of reproductively dead cells, cytokine and cytokine receptor mRNA profiles were not altered significantly by irradiation in all cell lines, regardless of the p53 status. CONCLUSION: These results suggest that cobalt irradiation alone at clinically relevant doses does not significantly alter the cytokine and cytokine receptor profiles in human glioblastoma cell lines.