Scintillator high-gain avalanche rushing photoconductor active-matrix flat panel imager: zero-spatial frequency x-ray imaging properties of the solid-state SHARP sensor structure.

PURPOSE: The authors are investigating the feasibility of a new type of solid-state x-ray imaging sensor with programmable avalanche gain: scintillator high-gain avalanche rushing photoconductor active matrix flat panel imager (SHARP-AMFPI). The purpose of the present work is to investigate the inherent x-ray detection properties of SHARP and demonstrate its wide dynamic range through programmable gain. METHODS: A distributed resistive layer (DRL) was developed to maintain stable avalanche gain operation in a solid-state HARP. The signal and noise properties of the HARP-DRL for optical photon detection were investigated as a function of avalanche gain both theoretically and experimentally, and the results were compared with HARP tube (with electron beam readout) used in previous investigations of zero spatial frequency performance of SHARP. For this new investigation, a solid-state SHARP x-ray image sensor was formed by direct optical coupling of the HARP-DRL with a structured cesium iodide (CsI) scintillator. The x-ray sensitivity of this sensor was measured as a function of avalanche gain and the results were compared with the sensitivity of HARP-DRL measured optically. The dynamic range of HARP-DRL with variable avalanche gain was investigated for the entire exposure range encountered in radiography∕fluoroscopy (R∕F) applications. RESULTS: The signal from HARP-DRL as a function of electric field showed stable avalanche gain, and the noise associated with the avalanche process agrees well with theory and previous measurements from a HARP tube. This result indicates that when coupled with CsI for x-ray detection, the additional noise associated with avalanche gain in HARP-DRL is negligible. The x-ray sensitivity measurements using the SHARP sensor produced identical avalanche gain dependence on electric field as the optical measurements with HARP-DRL. Adjusting the avalanche multiplication gain in HARP-DRL enabled a very wide dynamic range which encompassed all clinically relevant medical x-ray exposures. CONCLUSIONS: This work demonstrates that the HARP-DRL sensor enables the practical implementation of a SHARP solid-state x-ray sensor capable of quantum noise limited operation throughout the entire range of clinically relevant x-ray exposures. This is an important step toward the realization of a SHARP-AMFPI x-ray flat-panel imager.

A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis.

The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.

A solid-state amorphous selenium avalanche technology for low photon flux imaging applications.

PURPOSE: The feasibility of a practical solid-state technology for low photon flux imaging applications was investigated. The technology is based on an amorphous selenium photoreceptor with a voltage-controlled avalanche multiplication gain. If this photoreceptor can provide sufficient internal gain, it will be useful for an extensive range of diagnostic imaging systems. METHODS: The avalanche photoreceptor under investigation is referred to as HARP-DRL. This is a novel concept in which a high-gain avalanche rushing photoconductor (HARP) is integrated with a distributed resistance layer (DRL) and sandwiched between two electrodes. The avalanche gain and leakage current characteristics of this photoreceptor were measured. RESULTS: HARP-DRL has been found to sustain very high electric field strengths without electrical breakdown. It has shown avalanche multiplication gains as high as 10(4) and a very low leakage current (< or = 20 pA/mm2). CONCLUSIONS: This is the first experimental demonstration of a solid-state amorphous photoreceptor which provides sufficient internal avalanche gain for photon counting and photon starved imaging applications.

Trends and Correlates of Mean Lung Dose in Patients Receiving Breast Radiotherapy in a Single Institution from 2014 to 2018.

AIMS: Higher mean lung dose (MLD) in breast cancer patients has been associated with pneumonitis, pulmonary fibrosis and secondary lung cancer primaries. This study examined MLD in a single institution from 2014 to 18 to assess trends in median MLD (Gy) over time and factors associated with higher MLD to determine best practices for limiting lung toxicity. MATERIALS AND METHODS: General linear regressions were analysed to determine significant change in median MLD over time in patients receiving conventional or hypofractionated schedules for whole breast/chest wall (WB) radiotherapy with or without sequential boost or simultaneous integrated boost, WB tangential radiotherapy only and WB locoregional radiotherapy. Univariate and multivariable linear regression analysed identified factors associated with MLD. RESULTS: In total, 3894 patients were included in the analysis. The total median MLD across all years was 6.8 Gy in patients treated with conventional fractionation and 3.4 Gy in patients treated with hypofractionation. A significant increase in MLD was observed between 2014 and 2018 in patients receiving conventional or hypofractionation, conventional WB treatment with locoregional radiotherapy, conventional WB radiotherapy with simultaneous integrated boost and hypofractionated WB radiotherapy with sequential boost. Increased MLD was significantly correlated with lower lung volume and larger treatment volume due to locoregional radiotherapy, inclusion of a boost, chest wall treatment and reverse decubitus or supine positioning (P < 0.0001). CONCLUSION: A significant increase in MLD was observed over the years in patients receiving conventional and hypofractionated radiotherapy. Techniques such as prone positioning should be considered to lower MLD, particularly for patients with predisposing pulmonary risk.

Results of 15 Gy HDR-BT boost plus EBRT in intermediate-risk prostate cancer: Analysis of over 500 patients.

PURPOSE/OBJECTIVE: To report biochemical control associated with single fraction 15 Gy high-dose-rate brachytherapy (HDR-BT) boost followed by external beam radiation (EBRT) in patients with intermediate-risk prostate cancer. MATERIALS AND METHODS: A retrospective chart review of all patients with intermediate-risk disease treated with a real-time ultrasound-based 15 Gy HDR-BT boost followed by EBRT between 2009 and 2016 at a single quaternary cancer center was performed. Freedom from biochemical failure (FFBF), cumulative incidence of androgen deprivation therapy use for biochemical or clinical failure post-treatment (CI of ADT) and metastasis-free survival (MFS) outcomes were measured. RESULTS: 518 patients met the inclusion criteria for this study. Median age at HDR-BT was 67 years (IQR 61-72). 506 (98%) had complete pathologic information available. Of these, 146 (28%) had favorable (FIR) and 360 (69%) had unfavorable (UIR) intermediate-risk disease. 83 (16%) received short course hormones with EBRT + HDR. Median overall follow-up was 5.2 years. FFBF was 91 (88-94)% at 5 years. Five-year FFBF was 94 (89-99)% and 89 (85-94)% in FIR and UIR patients, respectively (p = 0.045). CI of ADT was 4 (2-6)% at 5 years. Five-year CI of ADT was 1 (0-3)% and 5 (2-8)% in FIR and UIR patients, respectively (p = 0.085). MFS was 97 (95-98)% at 5 years. Five-year MFS was 100 (N/A-100)% and 95 (92-98)% in FIR and UIR patients, respectively (p = 0.020). CONCLUSION: In this large cohort of intermediate-risk prostate cancer patients, 15 Gy HDR-BT boost plus EBRT results in durable biochemical control and low rates of ADT use for biochemical failure.

Direct-conversion flat-panel imager with avalanche gain: feasibility investigation for HARP-AMFPI.

The authors are investigating the concept of a direct-conversion flat-panel imager with avalanche gain for low-dose x-ray imaging. It consists of an amorphous selenium (a-Se) photoconductor partitioned into a thick drift region for x-ray-to-charge conversion and a relatively thin region called high-gain avalanche rushing photoconductor (HARP) in which the charge undergoes avalanche multiplication. An active matrix of thin film transistors is used to read out the electronic image. The authors call the proposed imager HARP active matrix flat panel imager (HARP-AMFPI). The key advantages of HARP-AMFPI are its high spatial resolution, owing to the direct-conversion a-Se layer, and its programmable avalanche gain, which can be enabled during low dose fluoroscopy to overcome electronic noise and disabled during high dose radiography to prevent saturation of the detector elements. This article investigates key design considerations for HARP-AMFPI. The effects of electronic noise on the imaging performance of HARP-AMFPI were modeled theoretically and system parameters were optimized for radiography and fluoroscopy. The following imager properties were determined as a function of avalanche gain: (1) the spatial frequency dependent detective quantum efficiency; (2) fill factor; (3) dynamic range and linearity; and (4) gain nonuniformities resulting from electric field strength nonuniformities. The authors results showed that avalanche gains of 5 and 20 enable x-ray quantum noise limited performance throughout the entire exposure range in radiography and fluoroscopy, respectively. It was shown that HARP-AMFPI can provide the required gain while maintaining a 100% effective fill factor and a piecewise dynamic range over five orders of magnitude (10(-7)-10(-2) R/frame). The authors have also shown that imaging performance is not significantly affected by the following: electric field strength nonuniformities, avalanche noise for x-ray energies above 1 keV and direct interaction of x rays in the gain region. Thus, HARP-AMFPI is a promising flat-panel imager structure that enables high-resolution fully quantum noise limited x-ray imaging over a wide exposure range.

Immunoaffinity purification of human O6-alkylguanine-DNA alkyltransferase using newly developed monoclonal antibodies.

As well as repairing mutagenic lesions induced by simple methylating agents, O6-alkylguanine-DNA alkyltransferase repairs precursors of cytotoxic interstrand cross-links induced by chloroethylating anticancer drugs. Moreover, levels of the transferase correlate with cellular resistance to these agents. Thus far, the human transferase has not been highly purified. In our quest to obtain the homogeneous protein we have produced four stable cloned hybridomas that secrete monoclonal antibodies against the alkyltransferase from human lymphoblasts. The specificity of these monoclonals was established by immunoblot analysis and immunoprecipitation. All these antibodies recognized the alkyltransferase only after its denaturation by sodium dodecyl sulfate. One of them, designated 19.2, was used in immunoaffinity chromatography to obtain the pure protein.

Production and characterization of antipeptide antibodies against human O6-methylguanine-DNA methyltransferase.

Four synthetic peptides from the sequence of human O6-methylguanine-DNA methyltransferase (MGMT), three corresponding to different hydrophilic regions and one corresponding to the sequence containing the alkyl acceptor residue cysteine 145, were used to immunize rabbits. The antibody against Peptide III (residues 171-184) was highly specific, and MGMT protein could be detected on Western blots of soluble protein extracts containing as little as 1 fmol of active MGMT. Antibodies against all of the peptides were able to immunoprecipitate denatured MGMT, while only the antibody against Peptide III was able to react with active enzyme. The antibody against Peptide III did not cross-react with methyltransferase from mice. The use of synthetic peptides has led to the production of a highly sensitive, specific antibody that recognizes native and denatured human MGMT. This antibody should prove useful in studies involving the detection, purification, and characterization of this enzyme.

Expression of O6-methylguanine-DNA methyltransferase in six human medulloblastoma cell lines.

Six well characterized human medulloblastoma cell lines (D283 Med, Daoy, D341 Med, D384 Med, D425 Med, and D458 Med) were examined for the expression of O6-methylguanine-DNA methyltransferase (MGMT) by activity and Western and Northern blot analysis. High levels of MGMT activity were present in D283 Med, Daoy, D341 Med, and D384 Med (1.36, 0.80, 1.68, and 1.62 pmol/mg of protein, respectively), but negligible MGMT activity was detected in D425 Med and D458 Med (0.06 and 0.05 pmol/mg of protein, respectively), which were derived separately at different times from the same patient. The presence of MGMT protein and its transcript was demonstrated in D283 Med, Daoy, D341 Med, and D384 Med, but both the protein and the mRNA were undetectable in D425 Med and D458 Med. Nevertheless, all six cell lines contained an apparently unaltered MGMT gene, as determined by Southern blot analysis. The absence of MGMT activity in D425 Med and D458 Med is likely due to the absence of the protein, resulting from a lack of transcription of the MGMT gene. The varying levels of expression of MGMT in medulloblastoma cells found in this study should provide a molecular basis for drug design and selection in chemotherapy of this tumor.

Postoperative radiation for lung cancer metastatic to the brain.

PURPOSE: Although resection of single brain metastases and postoperative whole-brain radiation therapy (WBRT) improves survival, compared with treatment using WBRT alone, the value of postoperative WBRT after resection of brain metastases is controversial. We analyzed the largest reported series of lung cancer patients with resected brain metastases to evaluate the impact of postoperative WBRT. MATERIALS AND METHODS: Between 1974 and 1989, 185 patients with non-small-cell lung cancer (NSCLC) underwent resection of brain metastases. Patients who had received preoperative WBRT (23%, 42 of 185) were excluded. The remaining patients were divided into group A (no WBRT; n = 32), group B (patients received WBRT and were prognostically matched to group A; n = 32), and group C (all other WBRT patients; n = 79). Most patients received postoperative doses of 30 Gy in 10 fractions. Higher doses were used in 16% of group B and 18% of group C patients. RESULTS: Overall 5-year survival rates were as follows: group A, 12%; B, 8%; C, 16%. Overall brain failures occurred in 38% of patients in group A, 47% in group B, and 42% in group C. The use of WBRT (group A v groups B plus C) had no apparent impact on survival or on overall brain failure rates. In particular, no improvement in either of these parameters could be demonstrated when group B was compared with group A. Focal failure (defined as failure within the brain adjacent to the site of the resected brain metastases) occurred as follows: group A, 34% (11 of 32); groups B plus C, 23% (25 of 111) (P = .07). WBRT significantly reduced focal failure for patients with adenocarcinoma (group A, 33% [eight of 24]; groups B plus C, 14% [11 of 79]; P = .05). Nonfocal failure (anatomically distinct from the resected metastasis) occurred in 9% of patients in group A (three of 32), 21% in groups B plus C (23 of 111) (P = .07). CONCLUSION: Long-term survival is possible when NSCLC brain metastases are resected. Postoperative WBRT as used in this series only had an impact on the focal control of brain metastases and this effect was of borderline significance. The lack of conclusive benefit supports the need for ongoing randomized trials to test the value of adjuvant postoperative WBRT. Brain failures were relatively common in all three groups of patients, which suggests that doses greater than 30 Gy need to be studied.

A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

In Vivo MR Imaging of Fibrin in a Neuroblastoma Tumor Model by Means of a Targeting Gd-Containing Peptide.

PURPOSE: A magnetic resonance imaging contrast agent based on a tetrameric Gd-DTPA-like system linked to a fibrin-targeting peptide (Gd-F) has been designed for in vivo tumor characterization. PROCEDURES: Gd-F was synthesized following Fmoc-SPPS strategy. Binding was measured using soluble fibrin DD(E) fragment and a dried fibrin assay. Contrast efficiency was tested on human and mouse clots and in vivo on Neuro2A tumor model. An anti-thrombotic drug was used to evaluate Gd-F sensitivity for changes in fibrin availability at the tumor site. RESULTS: The high relaxivity of Gd-F (42 mM(-1) s(-1), per molecule) yielded a strong signal enhancement in human and murine clots. High contrast was also measured in vivo in Neuro2A tumors, with a persistent enhancement in tumor rim and stroma. Upon treatment with an anti-thrombotic drug, the contrast uptake was significantly reduced in the tumor area confirming the specificity of the probe. CONCLUSIONS: Gd-F resulted to be an efficient probe for tumor delineation and for monitoring fibrin deposits during tumor progression and anti-thrombotic therapy.

External radiation of brain metastases from renal carcinoma: a retrospective study of 119 patients from the M. D. Anderson Cancer Center.

PURPOSE: Approximately 10% of patients with metastatic renal cell carcinoma are diagnosed with brain metastases. Most of these patients receive palliative radiotherapy and die of progressive brain metastatic disease. This retrospective study examines the M. D. Anderson Cancer Center experience with such patients who received only whole brain radiation therapy (WBRT). METHODS AND MATERIALS: Records of 200 patients with brain metastases from renal carcinoma who were treated at M. D. Anderson Cancer Center between 1976 and 1993 were reviewed. Of these patients, 119 received WBRT only and constitute the basis of this study. Different prognostic factors were analyzed. RESULTS: Overall median survival time from diagnosis of the brain metastases was 4.4 months. Multiple brain tumors were treated in 70 patients (58.8%) who had a survival of 3.0 months compared with 4.4 months for patients having a single brain metastasis (p = 0.043). Among 117 patients the causes of death were neurologic in 90 (76%), systemic cancer in 19 (16%), and unknown in 9 (8%). Survival rates at 6 months, 1 year, and 2 years, were 33.6, 16.8, and 5.9%, respectively. Patients in whom brain metastases were diagnosed synchronously with a renal primary (n = 24) had a median survival time of 3.4 months compared with 3.2 months for those 95 who were diagnosed metachronously (p < 0.79, NS). In the Cox multivariate analysis of 13 possible prognostic factors, only a single brain metastasis (p = 0.0329), lack of distant metastases at the time of diagnosis (p = 0.0056), and tumor diameter < or = 2 cm (p < 0.0016) were statistically significant. CONCLUSION: These unsatisfactory results with WBRT suggest that more aggressive approaches, such as surgery or radiosurgery should be applied whenever possible.

Fractionated stereotactic radiosurgery and concurrent taxol in recurrent glioblastoma multiforme: a preliminary report.

PURPOSE: Surgery and systemic chemotherapy offer modest benefit to patients with recurrent glioblastoma multiforme. These tumors are associated with rapid growth and progressive neurological deterioration. Radiosurgery offers a rational alternative treatment, delivering intensive local therapy. A pilot protocol to treat recurrent glioblastoma was developed using fractionated stereotactic radiosurgery with concurrent intravenous (i.v.) Taxol as a radiation sensitizer. METHODS AND MATERIALS: The treatment outcome was analyzed in 14 patients with recurrent glioblastoma treated with fractionated stereotactic radiosurgery and concurrent Taxol. Median tumor volume was 15.7 cc and patients received a mean radiation dose of 6.2 Gy at 90% isodose line, 4 times weekly. The median dose of Taxol was 120 mg/m2. RESULTS: The median survival was 14.2 months, 1-year survival was 50%. CONCLUSIONS: Survival for this small group of patients was similar to or better than historical controls or patients treated with single-fraction radiosurgery alone. This data should stimulate the investigation of both fractionated radiosurgery and the development of radiation sensitizers to further enhance treatment.

Resection of brain metastases from non-small-cell lung carcinoma. Results of therapy. Memorial Sloan-Kettering Cancer Center Thoracic Surgical Staff.

The treatment of patients with a solitary brain metastasis has been evolving, with most centers recommending resection in patients with good performance status. To evaluate the results of resection of brain metastases from non-small-cell lung cancer, we reviewed our 16-year experience with 185 consecutive patients undergoing resection of brain metastases from 1974 to 1989, inclusive. There were 89 men and 96 women; ages ranged from 34 to 75 years (median 54). Sixty-five (35%) had synchronous and 120 (65%) metachronous brain metastases. Discounting the brain metastasis, 68 patients (37%) had stage I, 13 (7%) stage II, 62 (33%) stage IIIA, 30 (16%) stage IIIB, and 12 (6%) stage IV carcinoma. There was no significant difference in age, locoregional stage (TN), or histologic features in patients with synchronous versus metachronous lesions. The overall survival rates (n = 185) were as follows: 1 year, 55%; 2 years, 27%; 3 years, 18%; 5 years, 13%; and 10 years, 7% (median 14 months). There was no significant difference in survival between patients with synchronous and metachronous lesions. To evaluate the impact of locoregional stage and treatment of the primary site, we analyzed only those patients with synchronous brain metastases. Multivariate analysis demonstrated that locoregional stage had no significant effect on survival (p = 0.97), but complete resection of the primary disease significantly prolonged survival (p = 0.002). Therefore complete resection, and not stage, of the locoregional primary lesion is the primary determinant of survival in patients undergoing resection of brain metastases from non-small-cell lung cancer.

Insulin increases expression of apobec-1, the catalytic subunit of the apolipoprotein B mRNA editing complex in rat hepatocytes.

We have previously shown that chronic insulin treatment of rat hepatocytes increases the fraction of edited apolipoprotein B (apoB) mRNA from approximately 50% to as much as 90%. We have now examined the effect of insulin on apobec-1 mRNA abundance and demonstrate that increased editing of apoB mRNA following insulin treatment is accompanied by elevated apobec-1 mRNA levels in primary rat hepatocytes. Time-course measurements of the effects of insulin on apoB mRNA editing and apobec-1 mRNA abundance showed that both were elevated almost maximally within 48 hours and sustained for at least 5 days of insulin treatment.

Surgical resection of brain metastases from renal cell carcinoma in 50 patients.

OBJECTIVES: Metastases are frequently diagnosed among patients with renal cell carcinoma (RCC). Of 709 patients with brain metastases (BMET) who were operated on at our institution between 1974 and 1993, 50 (7%) were of renal origin. METHODS: Medical records were reviewed retrospectively. Survival time was calculated by the Kaplan-Meier method and Cox proportional hazards model. RESULTS: There were 38 men and 12 women. The median age was 60 years. The primary RCC was resected in 47 patients. Forty patients had a metachronous diagnosis of RCC and BMET. Median interval between the diagnosis of RCC and BMET was 17 months. In all 50 patients overall median survival (MS) from diagnosis of primary RCC was 31.4 months and from craniotomy was 12.6 months. Postoperative mortality was 10% (5 patients). In patients with primary RCC in the left kidney (n=25) versus right kidney (n=25) median survival from craniotomy was longer; 21.3 versus 7.4 months (P<0.014). Twenty-three patients (46%) had intratumoral hemorrhage. Eight patients had cerebellar metastasis (MS, 3.0 months) and 9 had multiple metastases resected (MS, 7.6 months). Thirty-eight patients had both brain and pulmonary metastases, and 16 of them had pulmonary resection (MS, 18.6 versus 8.0 months; P<0.03). Twenty-two patients received whole-brain radiation therapy (WBRT) after craniotomy and 18 did not receive WBRT (MS, 13.3 versus 14.5 months; P<0.62). The 1-year, 2-year, 3-year, and 5-year survival was 51%, 24%, 22%, and 8.5% respectively. CONCLUSIONS: Only the resection of lung metastasis, supratentorial location of BMET, left-sided localization of primary RCC, and lack of neurologic deficit before craniotomy were statistically significant prognostic factors in Cox regression analysis. In the absence of effective systemic treatment, we suggest that patients with BMET from RCC be considered for operative resection for treatment and palliation.

Cytosine methylation and suppression of O6-methylguanine-DNA methyltransferase expression in human rhabdomyosarcoma cell lines and xenografts.

Human tumor cell lines that do not express O6-methylguanine-DNA methyltransferase (MGMT) in detectable quantities (Mer-) are hypersensitive to the effects of O6-guanine-alkylating agents. Because the Mer- phenotype enhances tumor response to such agents, we investigated possible mechanisms involved in regulation of MGMT expression in a panel of Mer+ and Mer- pediatric rhabdomyosarcoma xenograft and cell lines. All Mer- cell and xenograft lines lacked not only MGMT activity but also the protein and mRNA as well, suggesting that its expression is transcriptionally regulated. Transfection of Mer+ and Mer- rhabdomyosarcoma cell lines with MGMT gene promoter-CAT constructs yielded similar levels of CAT expression, indicating that Mer- cells possessed the necessary factors to support transcription. Methylation in the 5'-untranslated region of the MGMT gene was assayed by Southern analysis using methylation-sensitive restriction enzymes. Digestion with HpaII and its methylation-insensitive isoschizomer, MspI, revealed little overall correlation between methylation and MGMT expression. However, methylation in a single SmaI site at position-69 was observed in all MGMT deficient lines but not in any MGMT expressing lines. These results suggest that methylation of specific cytosines in the MGMT promoter may play a role in suppressing its expression, as well as being a potentially useful marker for the Mer- phenotype.

Identification of nitrosourea-resistant human rhabdomyosarcomas by in situ immunostaining of O6-methylguanine-DNA methyltransferase.

Cellular levels of O6-methylguanine-DNA methyltransferase (MGMT) correlate strongly with cellular resistance to carcinogenic and chemotherapeutic agents that produce adducts at the O6-position of guanine in DNA. Although biochemical and molecular assays can indicate the average MGMT content of tissues or tumors, they cannot distinguish mixed populations of cells, such as those that exist in tumor biopsy samples. We have determined MGMT at the cellular level in a panel of pediatric rhabdomyosarcoma xenografts by in situ immunostaining with a human MGMT-specific antibody employing a very sensitive procedure that involves biotin-avidin coupled horseradish peroxidase with silver-enhanced diaminobenzidine-nickel staining. Two xenograft tumor lines known to be MGMT-deficient were not stained, whereas the nuclei in three MGMT-expressing lines were clearly stained. This is the first demonstration of an in situ procedure that discriminates drug-sensitive MGMT-deficient tumors from drug-resistant MGMT expressing tumors. This procedure should prove useful, therefore, for predicting the susceptibility of tissues and tumors to O6-guanine alkylating agents.