Model-based feasibility assessment and evaluation of prostate hyperthermia with a commercial MR-guided endorectal HIFU ablation array.

PURPOSE: Feasibility of targeted and volumetric hyperthermia (40-45 °C) delivery to the prostate with a commercial MR-guided endorectal ultrasound phased array system, designed specifically for thermal ablation and approved for ablation trials (ExAblate 2100, Insightec Ltd.), was assessed through computer simulations and tissue-equivalent phantom experiments with the intention of fast clinical translation for targeted hyperthermia in conjunction with radiotherapy and chemotherapy. METHODS: The simulations included a 3D finite element method based biothermal model, and acoustic field calculations for the ExAblate ERUS phased array (2.3 MHz, 2.3 × 4.0 cm(2), ∼1000 channels) using the rectangular radiator method. Array beamforming strategies were investigated to deliver protracted, continuous-wave hyperthermia to focal prostate cancer targets identified from representative patient cases. Constraints on power densities, sonication durations and switching speeds imposed by ExAblate hardware and software were incorporated in the models. Preliminary experiments included beamformed sonications in tissue mimicking phantoms under MR temperature monitoring at 3 T (GE Discovery MR750W). RESULTS: Acoustic intensities considered during simulation were limited to ensure mild hyperthermia (Tmax < 45 °C) and fail-safe operation of the ExAblate array (spatial and time averaged acoustic intensity ISATA < 3.4 W/cm(2)). Tissue volumes with therapeutic temperature levels (T > 41 °C) were estimated. Numerical simulations indicated that T > 41 °C was calculated in 13-23 cm(3) volumes for sonications with planar or diverging beam patterns at 0.9-1.2 W/cm(2), in 4.5-5.8 cm(3) volumes for simultaneous multipoint focus beam patterns at ∼0.7 W/cm(2), and in ∼6.0 cm(3) for curvilinear (cylindrical) beam patterns at 0.75 W/cm(2). Focused heating patterns may be practical for treating focal disease in a single posterior quadrant of the prostate and diffused heating patterns may be useful for heating quadrants, hemigland volumes or even bilateral targets. Treatable volumes may be limited by pubic bone heating. Therapeutic temperatures were estimated for a range of physiological parameters, sonication duty cycles and rectal cooling. Hyperthermia specific phasing patterns were implemented on the ExAblate prostate array and continuous-wave sonications (∼0.88 W/cm(2), 15 min) were performed in tissue-mimicking material with real-time MR-based temperature imaging (PRFS imaging at 3.0 T). Shapes of heating patterns observed during experiments were consistent with simulations. CONCLUSIONS: The ExAblate 2100, designed specifically for thermal ablation, can be controlled for delivering continuous hyperthermia in prostate while working within operational constraints.

Components of a hyperthermia clinic: recommendations for staffing, equipment, and treatment monitoring.

Like other technically sophisticated medical endeavours, a hyperthermia clinic relies on skilled staffing. Physicians, physicists and technologists perform multiple tasks to ensure properly functioning equipment, appropriate patient selection, and to plan and administer this treatment. This paper reviews the competencies and tasks that are used in a hyperthermia clinic.

American College of Radiology Appropriateness Criteria(®)--locally advanced (high-risk) prostate cancer.

The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances where evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment. This review focuses on locally advanced prostate cancer and the evidence for treatment outcomes, both toxicity and efficacy, across the three major treatment modalities of external beam radiotherapy, brachytherapy and surgery. Only data that could pass contemporary quality metrics were used to form this report. This body of literature suffers from an absence of trials prospectively comparing therapies for efficacy and a lack of long-term prospective comparisons of toxicity. Upon review of these data, the authors concluded that there are several acceptable methods for the treatment of locally advanced prostate cancer that is highly dependent of the patient's clinical (both prostate cancer-specific and comorbidity-specific) parameters at diagnosis.

Catheter-based ultrasound hyperthermia with HDR brachytherapy for treatment of locally advanced cancer of the prostate and cervix.

A clinical treatment delivery platform has been developed and is being evaluated in a clinical pilot study for providing 3D controlled hyperthermia with catheter-based ultrasound applicators in conjunction with high dose rate (HDR) brachytherapy. Catheter-based ultrasound applicators are capable of 3D spatial control of heating in both angle and length of the devices, with enhanced radial penetration of heating compared to other hyperthermia technologies. Interstitial and endocavity ultrasound devices have been developed specifically for applying hyperthermia within HDR brachytherapy implants during radiation therapy in the treatment of cervix and prostate. A pilot study of the combination of catheter based ultrasound with HDR brachytherapy for locally advanced prostate and cervical cancer has been initiated, and preliminary results of the performance and heating distributions are reported herein. The treatment delivery platform consists of a 32 channel RF amplifier and a 48 channel thermocouple monitoring system. Controlling software can monitor and regulate frequency and power to each transducer section as required during the procedure. Interstitial applicators consist of multiple transducer sections of 2-4 cm length × 180 deg and 3-4 cm × 360 deg. heating patterns to be inserted in specific placed 13g implant catheters. The endocavity device, designed to be inserted within a 6 mm OD plastic tandem catheter within the cervix, consists of 2-3 transducers × dual 180 or 360 deg sectors. 3D temperature based treatment planning and optimization is dovetailed to the HDR optimization based planning to best configure and position the applicators within the catheters, and to determine optimal base power levels to each transducer section. To date we have treated eight cervix implants and six prostate implants. 100 % of treatments achieved a goal of >60 min duration, with therapeutic temperatures achieved in all cases. Thermal dosimetry within the hyperthermia target volume (HTV) and clinical target volume (CTV) are reported. Catheter-based ultrasound hyperthermia with HDR appears feasible with therapeutic temperature coverage of the target volume within the prostate or cervix while sparing surrounding more sensitive regions. (NIHR01CA122276).

Dosimetric equivalence of nonstandard HDR brachytherapy catheter patterns.

PURPOSE: To determine whether alternative high dose rate prostate brachytherapy catheter patterns can result in similar or improved dose distributions while providing better access and reducing trauma. MATERIALS AND METHODS: Standard prostate cancer high dose rate brachytherapy uses a regular grid of parallel needle positions to guide the catheter insertion. This geometry does not easily allow the physician to avoid piercing the critical structures near the penile bulb nor does it provide position flexibility in the case of pubic arch interference. This study used CT datasets with 3 mm slice spacing from ten previously treated patients and digitized new catheters following three hypothetical catheter patterns: conical, bi-conical, and fireworks. The conical patterns were used to accommodate a robotic delivery using a single entry point. The bi-conical and fireworks patterns were specifically designed to avoid the critical structures near the penile bulb. For each catheter distribution, a plan was optimized with the inverse planning algorithm, IPSA, and compared with the plan used for treatment. Irrelevant of catheter geometry, a plan must fulfill the RTOG-0321 dose criteria for target dose coverage (V100(Prostate) > 90%) and organ-at-risk dose sparing (V75(Bladder) < 1 cc, V75(Rectum) < 1 cc, V125(Urethra) << 1cc). RESULTS: The three nonstandard catheter patterns used 16 nonparallel, straight divergent catheters, with entry points in the perineum. Thirty plans from ten patients with prostate sizes ranging from 26 to 89 cc were optimized. All nonstandard patterns fulfilled the RTOG criteria when the clinical plan did. In some cases, the dose distribution was improved by better sparing the organs-at-risk. CONCLUSION: Alternative catheter patterns can provide the physician with additional ways to treat patients previously considered unsuited for brachytherapy treatment (pubic arch interference) and facilitate robotic guidance of catheter insertion. In addition, alternative catheter patterns may decrease toxicity by avoidance of the critical structures near the penile bulb while still fulfilling the RTOG criteria.

Evaluation of ultrasound-based prostate localization for image-guided radiotherapy.

To evaluate the use of the ultrasound-based BAT system for daily prostate alignment. Prostate alignments using the BAT system were compared with alignments using radiographic images of implanted radiopaque markers. The latter alignments were used as a reference. The difference between the BAT and marker alignments represents the displacements that would remain if the alignments were done using ultrasonography. The inter-user variability of the contour alignment process was assessed. On the basis of the marker alignments, the initial displacement of the prostate in the AP, superoinferior, and lateral direction was -0.9 +/- 3.9, 0.1 +/- 3.9, and 0.2 +/- 3.4 mm respectively. The directed differences between the BAT and marker alignments in the respective directions were 0.2 +/- 3.7, 2.7 +/- 3.9, and 1.6 +/- 3.1 mm. The occurrence of displacements >/=5 mm was reduced by a factor of two in the AP direction after the BAT system was used. Among eight users, the average range of couch shifts due to contour alignment variability was 7, 7, and 5 mm in the antero-posterior (AP), superoinferior, and lateral direction, respectively. In our study, the BAT alignments were systematically different from the marker alignments in the superoinferior, and lateral directions. The remaining random variability of the prostate position after the ultrasound-based alignment was similar to the initial variability. However, the occurrence of displacements >/=5 mm was reduced in the AP direction. The inter-user variation of the contour alignment process was significant.

Prostate volume change after radioactive seed implantation: possible benefit of improved dose volume histogram with perioperative steroid.

PURPOSE: To evaluate the changes in prostate volume associated with radioactive seed implantation and identify factors that influence prostate swelling. METHODS AND MATERIALS: Between June 1997 and August 1999, 161 patients implanted for prostate carcinoma at the University of California, San Francisco, had prostate volume measurements taken at 4 time points (preplan, preimplant, postimplant, postimplant dosimetry). Patient records were reviewed for treatment with perioperative steroids, hormone therapy (nHT), and external beam radiotherapy (EBRT). One and 2-way analysis of variance (ANOVA) methods were used to test differences in mean effects among patient subsets. RESULTS: A mean 20% volume increase was noted immediately postimplant overall (p < 0.0001), and even with EBRT and/or HT. Steroids were associated with a mean volume decrease of 19.9%, by 3-4 weeks post-procedure (p < 0.0001). Without steroids, only a 3.8% mean change was seen (p = ns). Steroid use resulted in a significant increase in mean dose-volume histogram (DVH) (p = 0.001); however, this benefit was only observed among patients who did not receive steroid. A consistently high DVH occurred with steroid use. CONCLUSION: A significant decrease in prostate volume and improved DVH are associated with steroid use. The diminished benefit of steroid use and higher mean DVH achieved in later years suggests the existence of a significant "learning curve" for brachytherapy procedures.

Mismatch cleavage detects pathogenic microorganisms.

When a DNA probe hybridizes a DNA target and generates a G/A mismatch in the probe-target DNA heteroduplex, the mismatch enzyme, mutY, will cut the A base at the site of the mismatch. This specific cleavage at the mismatched A on the known probe will reveal the complementary DNA sequences of the targets. This study shows mismatch cleavage assays identify the complementary sequence of cryptic plasmid target in the extract of chlamydia infected cells in culture. In addition, the specific cleavage at a single base permits differentiation of two sequences with one base difference. This was shown in differentiating the subtypes of human immunodeficiency virus (HIV) type 1. The addition of amines in the assays increases the sensitivity by freeing the target for recycling. The combined assay system of high sensitivity and demonstrated specificity allows further evaluation for direct identification of pathogenic microorganisms in patient samples.

Normal tissue dosimetric comparison between HDR prostate implant boost and conformal external beam radiotherapy boost: potential for dose escalation.

PURPOSE: To compare the dose and volume of bladder and rectum treated using high-dose-rate (HDR) prostate implant boost versus conformal external beam radiotherapy boost, and to use the dose-volume information to perform a critical volume tolerance (CVT) analysis and then estimate the potential for further dose escalation using HDR brachytherapy boost. METHODS AND MATERIALS: Using CT scan data collected before and after patients underwent HDR prostate implant, a 7-field conformal prostate-only external beam treatment plan and HDR brachytherapy treatment plan were constructed for each patient. Doses to the normal structures were calculated. Dose-volume histograms (DVH) were plotted for comparison of the two techniques. Wilcoxon signed rank test was performed at four dose levels to compare the dose to normal structures between the two treatment techniques. The acute and late effects of HDR brachytherapy were calculated based on the linear-quadratic (LQ) model. CVT analyses were performed to calculate the potential dose gain (PDG) using HDR brachytherapy boost. RESULTS: The volume of bladder and rectum receiving high dose was significantly less from implant boost. On the average, 0.19 cc of the bladder received 100% of the brachytherapy prescription dose, compared with 5.1 cc of the bladder receiving 100% of the prescription dose in the 7-field conformal external beam radiotherapy boost. Similarly, 0.25 cc of the rectum received 100% of the dose with the implant boost, as compared to 2.9 cc in the conformal external beam treatment. The implant also delivered higher doses inside the prostate volume. On average, 47% of the prostate received > or =150% of the prescription dose. The CVT analysis revealed a range of PDG using the HDR brachytherapy boost which depended on the following variables: critical volume (CV), critical volume tolerance dose (CVTD), number of HDR fractions (N), and the dose of external beam radiotherapy (XRT) delivered with brachytherapy boost. The PDG varied from -3.45% to 10.53% for tumor with an alpha-beta ratio of 10 and 7.14% to 64.6% for tumor with an alpha-beta ratio of 1.5 based on the parameters used for calculation in this study. CONCLUSIONS: HDR brachytherapy can provide better sparing of rectum and bladder while delivering a higher dose to the prostate. Even with the increased late effects of high dose per fraction, there is still a potential for dose escalation beyond external radiotherapy limits using HDR brachytherapy.

Mismatch cleavage detects base deletion in cystic fibrosis gene.

The delta F508 is the most common defect in the cystic fibrosis (CF) gene; it involves in a 3-base deletion in codon 508 and results in the loss of a phenylalanine residue at amino acid position 508. Our previous results have shown the mismatch enzyme cleavage at the mismatch of a DNA duplex in identifying a specific DNA sequence or a point mutation. The assay is simple and reliable. By manipulating the melting temperature (Tm) for the hybrids of the DNA targets and the deoxynucleotide probes, the mismatch cleavage assays are able to detect the most common defective CF gene, delta F508. The assays with a delta F508 and a normal wild-type probe can differentiate the three genotypes, i.e., delta F508/delta F508, delta F508/normal and normal/normal. Furthermore, the addition of ammonium acetate amplifier to the assay for recycling the target DNA can increase the sensitivity to a level that is sufficient to detect the mutated target in a few micrograms of genomic DNA without the aid of PCR amplification. The detection of the base deletion, the amplification of sensitivity and the differentiation among the genotypes of normal, carrier delta F508 and mutant delta F508 suggest the useful application of mismatch cleavage in genetic diagnosis at the DNA level.

A modified design of microwave tissue coagulator monopolar antenna for applications in laparoscopic hepatic surgery.

Massive hemorrhage is of primary concern during hepatic surgery. In this study, we not only modified the monopolar antenna of the microwave tissue coagulator but also investigated its feasible applications in laparoscopic hepatic surgery. In addition, the conventional antenna of the microwave tissue coagulator was used as a model. A catheter was attached to the antenna. The network analyzer was used to analyze the impedance of the novel monopolar antenna. Moreover, we designed and constructed a novel monopolar antenna for the microwave tissue coagulator. The antenna is gas-leak-proof, having sufficient stiffness and length and retaining similar microwave emission strength, pattern, and shielding property as the conventional antenna. In vitro tests in porcine liver confirm the coagulation and hemostatic effects of the novel type laparoscopic monopolar antenna. Results presented herein confirm the effectiveness of a laparoscopic monopolar antenna for a microwave tissue coagulator with sufficient coagulation effects.

Re-examining the role of prostate-specific antigen density in predicting outcome for clinically localized prostate cancer.

OBJECTIVES: To evaluate the prognostic significance of prostate-specific antigen density (PSAD) in clinically localized prostate cancer and determine whether this index is independent of or superior to prostate-specific antigen (PSA) in predicting outcome of patients treated with external beam radiotherapy. METHODS: Between January 1989 and December 1993, 175 evaluable patients with clinically localized prostate cancer received definitive radiotherapy using computed tomography (CT)-guided conformal techniques. PSAD was defined as the ratio of the pretreatment serum PSA to the prostate volume measured from CT treatment planning scans by one investigator. All PSA values were determined using the Hybritech assay. Biochemical failure was defined as two consecutive elevations in PSA separated by at least 3 months and a final PSA value greater than 1 ng/mL. RESULTS: Multivariate analysis including PSA and Gleason score revealed both to be statistically significant predictors of biochemical disease-free survival (P = 0.048 and P < 0.001, respectively). PSAD did not achieve significance on regression analysis. A direct multivariate analysis including PSA and PSAD required dichotomization in order to reduce high correlation. This analysis demonstrated a relative risk (RR) for failure of 1.27 (NS) for high PSA versus low PSA compared with a RR of 1.20 (NS) for high PSAD versus low PSAD. A regression model containing all three variables indicated only the Gleason score as significant in predicting biochemical failure. CONCLUSIONS: These data do not suggest that PSAD is either an independent prognostic factor or a stronger discriminant of outcome than PSA in patients with clinically localized prostate cancer treated with definitive external beam radiotherapy. Larger patient numbers with longer follow-up data, use of a clinical end point, or an analysis restricted to the appropriate subgroup may demonstrate the utility of PSAD in the future.

Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme.

The Escherichia coli MutY gene was cloned into a modified pET-11 plasmid which was then transfected into an E.coli HMS174 host for overproduction of the MutY mismatch repair (MR) enzyme. Approximately 30-50% of the total cellular protein in the transformed HMS174 cells was isopropyl-beta-D-thiogalactoside-induced MutY protein, as estimated from the staining intensity on an SDS-PAGE gel following electrophoresis. The MutY protein was purified to near homogeneity by cellulose phosphate ion-exchange chromatography followed by gel filtration chromatography. The purified MutY protein had enzyme activities which cleaved the A of a G/A mismatch at the 3' end of the first phosphodiester bond and then the 5' end of the second phosphodiester bond of the A. It also cut the A of a C/A mismatch, but to a much lesser extent, and the activity was DNA sequence-dependent. The reliability of the assay in determining the site and nature of a DNA mutation was examined in human tumor DNA samples with known or unknown p53 mutations. In the assay, polymerase chain reaction-amplified DNA fragments from normal and mutated p53 genes were mixed, denatured and annealed to generate mismatches of G/A or C/A for cleavage by the MutY MR enzyme. The assay results revealed the site and nature of known G:C<-->T:A mutations. In addition, a previously unknown G:C to T:A mutation, which was misread in the sequencing analysis of a tumor DNA preparation, was identified by this assay.

Increased susceptibility to staphylococcal enterotoxin B intoxication in mice primed with actinomycin D.

Mice (BALB/cJ, C3H/HeN, and C3H/HeJ) primed with actinomycin D became highly susceptible to lethal intoxication with staphylococcal enterotoxin B (SEB). The mice underwent toxicosis and toxic shock and died. Actinomycin D-primed C3H/HeN and C3H/HeJ mice showed equal sensitivity to SEB, suggesting that bacterial lipopolysaccharide derived from gram-negative bacteria in the gut may not be an important cofactor in intoxication. In a time course study of the illness, prominent pathological changes characterized by blood congestion and thickening of alveolar septa were seen in the lung, while blood congestion, inflammation, epithelial cell flattening, and villous blunting were seen in the small intestine. In lymphoid tissues, such as the spleen, congestion, inflammation, and lymphoid cell depletion were the major reactions. The pathological features of the mice had many similarities to those of rhesus monkeys intoxicated with intravenous SEB. The actinomycin D-primed C3H/HeJ mice are thus an ideal mouse model for studying SEB toxicosis and toxic shock.

Detection of DNA point mutations with DNA mismatch repair enzymes.

We have developed a simple and reliable procedure to screen gene mutations using DNA mismatch repair (MR) specific mut Y enzyme of Escherichia coli and thymidine DNA glycosylase from HeLa cells. The mut Y enzyme cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches. Previously, we showed the determination of G:C-->T:A mutations in the N-ras gene in two human tumor samples with mut Y G/A MR enzyme. As low as 1-2% mutant DNAs in a sample of mutant and wild-type DNA can be detected with a synthetic DNA to create G/A mispairing for the assay. In this paper, we simplify the assay, include G/T MR thymidine glycosylase from HeLa cells and evaluate the application for screening DNA point mutations of p53 and ras genes. In this method, DNA fragments amplified from normal and mutated genes by polymerase chain reaction (PCR) were mixed and annealed to create DNA mismatches for cleavage by mismatch repair enzymes. The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography. In theory, the enzymes that cut G/A or G/T mispairs will detect the mutations of G:C-->A:T, A:T-->G:C, G:C-->T:A and T:A-->G:C. Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes. The reliability of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-amplified DNA fragments. Our data showed that, following mismatch repair enzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected. In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of 32P on mutant or wild-type DNA fragments. The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indicate the advantages of the method over other techniques in testing DNA mutants.

p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines.

A G:C-->T:A mutational hotspot at codon 249 of the p53 tumor suppressor gene has previously been identified in hepatocellular carcinoma (HCC) of patients from Qidong, China and southern Africa in which aflatoxin B1 (AFB1) and hepatitis B virus (HBV) are known synergistic risk factors. We have examined p53 mutation patterns of HCC from geographic areas in which the risk factors vary. Nine HCC lines and four hepatoblastoma lines (HB) were examined for p53 gene mutations and the relationship with HBV infection. Five of the nine HCC lines had homozygous mutation or deletion randomly distributed in exons 6-8, whereas none of the four HB cell lines had p53 mutations. One of the four HB lines (HepG2) had an N-ras mutation at codon 61 position 2. The p53 point mutations in the three HCC cell lines from Japan resulted in the amino acid changes of cysteine for tyrosine in cell line HuH 7 at codon 220 (A:T-->G:C), alanine for glycine in cell line HLF at codon 244 (G:C-->C:G), and serine for arginine in cell line HLE at codon 249 (G:C-->C:G). In addition, the deletion of 18 base pairs from codon 264 position 3 to codon 270 position 1 has resulted in the deletion of Leu-Gly-Arg-Asn-Ser-Phe from the amino acids sequences 256-270 in the Japanese cell line HuH 4. The cell line PLC/PRF/5 that showed p53 mutation at codon 249 (G:C-->T:A) with substitution of serine for arginine was derived from a South African patient. Our results indicate that whereas the p53 gene is not mutated in the HB cell lines, the HCC cell lines frequently contain an abnormal p53 gene. In addition, p53 point mutations were not detected in the four Japanese HCC cell lines that were positive for genomic integration of HBV X-gene and surface antigen gene. The three Japanese HCC cell lines with p53 mutations did not contain HBV sequences, indicating that hepatocarcinogenesis associated with p53 mutation does not require the genomic integration of HBV sequences.

Mutagenicity of formaldehyde in Chinese hamster lung fibroblasts: synergy with ionizing radiation and N-nitroso-N-methylurea.

Cultured Chinese hamster V79 cells, a widely utilized model system in risk assessment of environmental agents, have been utilized to measure toxicity and mutagenicity of formaldehyde with or without previous exposure to either the alkylating agent N-nitroso-N-methylurea or to ionizing radiation. Each of these agents caused a dose-dependent decrease in colony forming efficiency and a parallel increase in 6-thioguanine resistant colonies. Significant mutant frequencies were induced by 0.3 up to 1 mM formaldehyde, 2 and 4 Gy of radiation and 0.2 and 0.5 mM N-nitroso-N-methylurea. Exposure of cells to ionizing radiation or N-nitroso-N-methylurea followed by submutagenic concentrations of formaldehyde potentiated both the cytotoxicity and the mutagenicity as compared with the corresponding separate effects caused by each of these agents. Taken together, these studies clearly demonstrate genotoxic effects in vitro of three recognized carcinogens, i.e. formaldehyde, N-nitroso-N-methylurea and ionizing radiation. Moreover, the synergies now demonstrated in regards to cytopathic consequences indicate interactive effects between formaldehyde and these agents, representing both a chemical and a physical carcinogen.

Detection of single DNA base mutations with mismatch repair enzymes.

A novel method for identifying DNA point mutations has been developed by using mismatch repair enzymes. The high specificity of the Escherichia coli MutY protein has permitted the development of a reliable and sensitive method for the detection and characterization of point mutations in the human genome. The MutY protein is involved in a repair pathway that can convert A/G or A/C mismatches to C/G or G/C basepairs, respectively. A/G or A/C mismatches formed by hybridization between two amplified genomic DNA samples or between specific DNA probes and target DNA are nicked at the mispaired adenine strand by MutY protein. As little as 1% of the mutant sequence can be detected by the mismatch repair enzyme cleavage (MREC) method in a mixture of normal and mutated DNAs (e.g., mutant cells are only present in 1% of the normal cell background). By using different probes, the assay also can determine the nucleotide sequence of the mutation. We have applied this method to detect single-base substitutions in human oncogenes.