A report on the impact of rapid prenatal exome sequencing on the clinical management of 52 ongoing pregnancies: a retrospective review.

OBJECTIVE: Studies have shown that prenatal exome sequencing (PES) improves diagnostic yield in cases of fetal structural malformation. We have retrospectively analysed PES cases from two of the largest fetal medicine centres in the UK to determine the impact of results on management of a pregnancy. DESIGN: A retrospective review of clinical case notes. SETTING: Two tertiary fetal medicine centres. POPULATION: Pregnancies with fetal structural abnormalities referred to clinical genetics via a multidisciplinary team. METHODS: We retrospectively reviewed the notes of all patients who had undergone PES. DNA samples were obtained via chorionic villus sampling or amniocentesis. Variants were filtered using patient-specific panels and interpreted using American College of Medical Genetics guidelines. RESULTS: A molecular diagnosis was made in 42% (18/43) ongoing pregnancies; of this group, there was a significant management implication in 44% (8/18). A positive result contributed to the decision to terminate a pregnancy in 16% (7/43) of cases. A negative result had a significant impact on management in two cases by affirming the decision to continue pregnancy. CONCLUSIONS: We demonstrate that the results of PES can inform pregnancy management. Challenges include variant interpretation with limited phenotype information. These results emphasise the importance of the MDT and collecting phenotype and variant data. As this testing is soon to be widely available, we should look to move beyond diagnostic yield as a measure of the value of PES. TWEETABLE ABSTRACT: Prenatal exome sequencing can aid decision-making in pregnancy management; review ahead of routine implementation in NHS.

Long-term outcomes of real world 'watch and wait' data for rectal cancer after neoadjuvant chemoradiotherapy.

AIM: A 'watch and wait' (W&W) strategy after neoadjuvant long-course chemoradiotherapy (NACRT) remains controversial. Whilst encouraging short-term data exist, the strategy will be judged on long-term data. We present long-term, real-world UK data from a single National Health Service trust. METHODS: An analysis was performed of a prospectively maintained W&W database over 9 years between 2010 and 2018. Outcome measures include incidence and time to regrowth and overall and disease-free survival. RESULTS: We diagnosed 563 rectal cancers in 9 years. In all, 283 patients underwent rectal resection (50.3%). NACRT was used in 155 patients for margin-threatened tumours on staging MRI. Forty-nine patients (31.6%) experienced either a 'near complete' or a complete clinical response (cCR) at their 10 weeks post-NACRT assessment (MRI and endoscopy). The median age was 69 years (range 44-83), and the male to female ratio was 32:17. The median follow-up was 38 months (range 12-96). The median tumour distance from the anal verge was 7 cm (1-15 cm). Twenty-two patients had a cCR on initial assessment and 27 patients had a 'near' cCR. Of those 27 who experienced a 'near' cCR, 17 (63%) progressed to cCR on repeat assessment and 10 (37%) did not. Of these 10 patients, seven underwent standard surgical resection and three were unfit for surgery. R0 for the seven with delayed resection was 100%. Of 39 patients (22 cCR and 17 'near' cCR who progressed to cCR) (25.2% of those receiving NACRT), six patients experienced local regrowth (15.4%). The median time to local regrowth was 29 months (15-60 months). One of these six patients underwent salvage abdominoperineal resection, one was advised to have contact radiotherapy and four opted against surgery and also had contact radiotherapy. The overall survival was 100% at 2 years and 90% at 5 years. Disease-free survival was 90.47% at 2 years and 74.8% at 5 years. CONCLUSION: A W&W treatment strategy was employed safely in this patient cohort with acceptable rates of local regrowth and survival.

Recommendation to include hydroxyethyl (meth)acrylate in the British baseline patch test series.

BACKGROUND: (Meth)acrylates are potent sensitizers and a common cause of allergic contact dermatitis (ACD). The frequency of (meth)acrylate ACD has increased with soaring demand for acrylic nails. A preliminary audit has suggested a significant rate of positive patch tests to (meth)acrylates using aimed testing in patients providing a clear history of exposure. To date, (meth)acrylates have not been routinely tested in the baseline patch test series in the U.K. and Europe. OBJECTIVES: To determine whether inclusion of 2-hydroxyethyl methacrylate (2-HEMA) 2% in petrolatum (pet.) in the baseline series detects cases of treatable (meth)acrylate ACD. METHODS: During 2016-2017, 15 U.K. dermatology centres included 2-HEMA in the extended baseline patch test series. Patients with a history of (meth)acrylate exposure, or who tested positive to 2-HEMA, were selectively tested with a short series of eight (meth)acrylate allergens. RESULTS: In total 5920 patients were consecutively patch tested with the baseline series, of whom 669 were also tested with the (meth)acrylate series. Overall, 102 of 5920 (1·7%) tested positive to 2-HEMA and 140 (2·4%) to at least one (meth)acrylate. Had 2-HEMA been excluded from the baseline series, (meth)acrylate allergy would have been missed in 36 of 5920 (0·6% of all patients). The top (meth)acrylates eliciting a positive reaction were 2-HEMA (n = 102, 1·7%), 2-hydroxypropyl methacrylate (n = 61, 1·0%) and 2-hydroxyethyl acrylate (n = 57, 1·0%). CONCLUSIONS: We recommend that 2-HEMA 2% pet. be added to the British baseline patch test series. We also suggest a standardized short (meth)acrylate series, which is likely to detect most cases of (meth)acrylate allergy.

Risk and Quality in Brachytherapy From a Technical Perspective.

AIMS: To provide an overview of the history of incidents in brachytherapy and to describe the pillars in place to ensure that medical physicists deliver high-quality brachytherapy. MATERIALS AND METHODS: A review of the literature was carried out to identify reported incidents in brachytherapy, together with an evaluation of the structures and processes in place to ensure that medical physicists deliver high-quality brachytherapy. In particular, the role of education and training, the use of process and technical quality assurance and the role of international guidelines are discussed. RESULTS: There are many human factors in brachytherapy procedures that introduce additional risks into the process. Most of the reported incidents in the literature are related to human factors. Brachytherapy-related education and training initiatives are in place at the societal and departmental level for medical physicists. Additionally, medical physicists have developed process and technical quality assurance procedures, together with international guidelines and protocols. Education and training initiatives, together with quality assurance procedures and international guidelines may reduce the risk of human factors in brachytherapy. CONCLUSION: Through application of the three pillars (education and training; process control and technical quality assurance; international guidelines), medical physicists will continue to minimise risk and deliver high-quality brachytherapy treatments.

Tools to analyse and display variations in anatomical delineation.

Variations in anatomical delineation, principally due to a combination of inter-observer contributions and image-specificity, remain one of the most significant impediments to geometrically-accurate radiotherapy. Quantification of spatial variability of the delineated contours comprising a structure can be made with a variety of metrics, and the availability of software tools to apply such metrics to data collected during inter-observer or repeat-imaging studies would allow their validation. A suite of such tools have been developed which use an Extensible Markup Language format for the exchange of delineated 3D structures with radiotherapy planning or review systems. These tools provide basic operations for manipulating and operating on individual structures and related structure sets, and for deriving statistics on spatial variations of contours that can be mapped onto the surface of a reference structure. Use of these tools on a sample dataset is demonstrated together with import and display of results in the SWAN treatment plan review system.

A comparison of in-house and shared RapidPlan models for prostate radiation therapy planning.

Knowledge-based planning (KBP) can increase plan quality, consistency and efficiency. In this study, we assess the success of a using a publicly available KBP model compared with developing an in-house model for prostate cancer radiotherapy using a single, commercially available treatment planning system based on the ability of the model to achieve the centre's planning goals. Two radiation oncology centres each created a prostate cancer KBP model using the Eclipse RapidPlan software. These two models and a third publicly-available, shared model were tested at three centres in a retrospective planning study. The publicly-available model achieved lower rectum doses than the other two models. However, the planning-target-volume (PTV) doses did not meet the local planning goals and the model could not be adjusted to correct this. As a result, the plans most likely to satisfy local planning goals and requirements were created using an in-house model. For centres without an existing in-house model, a model created by another centre with similar planning goals was found to be preferred. Variations in local planning practices including contouring, treatment technique and planning goals can influence the relative performance of KBP. The value of publicly available KBP models could be enhanced through standardisation of planning goals and contouring guidelines, providing information related to the planning goals used to create the model and increased flexibility to allow local adaptation of the KBP model.

Interfraction patient motion and implant displacement in prostate high dose rate brachytherapy.

PURPOSE: To quantify movement of prostate cancer patients undergoing treatment, using an in-house developed motion sensor in order to determine a relationship between patient movement and high dose rate (HDR) brachytherapy implant displacement. METHODS: An electronic motion sensor was developed based on a three axis accelerometer. HDR brachytherapy treatment for prostate is delivered at this institution in two fractions 24 h apart and 22 patients were monitored for movement over the interval between fractions. The motion sensors functioned as inclinometers, monitoring inclination of both thighs, and the inclination and roll of the abdomen. The implanted HDR brachytherapy catheter set was assessed for displacement relative to fiducial markers in the prostate. Angle measurements and angle differences over a 2 s time base were binned, and the standard deviations of the resulting frequency distributions used as a metric for patient motion in each monitored axis. These parameters were correlated to measured catheter displacement using regression modeling. RESULTS: The mean implant displacement was 12.6 mm in the caudal direction. A mean of 19.95 h data was recorded for the patient cohort. Patients generally moved through a limited range of angles with a mean of the exception of two patients who spent in excess of 2 h lying on their side. When tested for a relationship between movement in any of the four monitored axes and the implant displacement, none was significant. CONCLUSIONS: It is not likely that patient movement influences HDR prostate implant displacement. There may be benefits to patient comfort if nursing protocols were relaxed to allow patients greater freedom to move while the implant is in situ.

Quantitative MRI: Defining repeatability, reproducibility and accuracy for prostate cancer imaging biomarker development.

INTRODUCTION: Quantitative MRI (qMRI) parameters have been increasingly used to develop predictive models to accurately monitor treatment response in prostate cancer after radiotherapy. To reliably detect changes in signal due to treatment response, predictive models require qMRI parameters with high repeatability and reproducibility. The purpose of this study was to measure qMRI parameter uncertainties in both commercial and in-house developed phantoms to guide the development of robust predictive models for monitoring treatment response. MATERIALS AND METHODS: ADC, T1, and R2* values were acquired across three 3 T scanners with a prostate-specific qMRI protocol using the NIST/ISMRM system phantom, RSNA/NIST diffusion phantom, and an in-house phantom. A B1 field map was acquired to correct for flip angle inhomogeneity in T1 maps. All sequences were repeated in each scan to assess within-session repeatability. Weekly scans were acquired on one scanner for three months with the in-house phantom. Between-session repeatability was measured with test-retest scans 6-months apart on all scanners with all phantoms. Accuracy, defined as percentage deviation from reference value for ADC and T1, was evaluated using the system and diffusion phantoms. Repeatability and reproducibility coefficients of variation (%CV) were calculated for all qMRI parameters on all phantoms. RESULTS: Overall, repeatability CV of ADC was <2.40%, reproducibility CV was <3.98%, and accuracy ranged between -8.0% to 2.7% across all scanners. Applying B1 correction on T1 measurements significantly improved the repeatability and reproducibility (p<0.05) but increased error in accuracy (p<0.001). Repeatability and reproducibility of R2* was <4.5% and <7.3% respectively in the system phantom across all scanners. CONCLUSION: Repeatability, reproducibility, and accuracy in qMRI parameters from a prostate-specific protocol was estimated using both commercial and in-house phantoms. Results from this work will be used to identify robust qMRI parameters for use in the development of predictive models to longitudinally monitor treatment response for prostate cancer in current and future clinical trials.

Standard versus hypofractionated intensity-modulated radiotherapy for prostate cancer: assessing the impact on dose modulation and normal tissue effects when using patient-specific cancer biology.

Hypofractionation of prostate cancer radiotherapy achieves tumour control at lower total radiation doses, however, increased rectal and bladder toxicities have been observed. To realise the radiobiological advantage of hypofractionation whilst minimising harm, the potential reduction in dose to organs at risk was investigated for biofocused radiotherapy. Patient-specific tumour location and cell density information were derived from multiparametric imaging. Uniform-dose plans and biologically-optimised plans were generated for a standard schedule (78 Gy/39 fractions) and hypofractionated schedules (60 Gy/20 fractions and 36.25 Gy/5 fractions). Results showed that biologically-optimised plans yielded statistically lower doses to the rectum and bladder compared to isoeffective uniform-dose plans for all fractionation schedules. A reduction in the number of fractions increased the target dose modulation required to achieve equal tumour control. On average, biologically-optimised, moderately-hypofractionated plans demonstrated 15.3% (p-value: <0.01) and 23.8% (p-value: 0.02) reduction in rectal and bladder dose compared with standard fractionation. The tissue-sparing effect was more pronounced in extreme hypofractionation with mean reduction in rectal and bladder dose of 43.3% (p-value: < 0.01) and 41.8% (p-value: 0.02), respectively. This study suggests that the ability to utilise patient-specific tumour biology information will provide greater incentive to employ hypofractionation in the treatment of localised prostate cancer with radiotherapy. However, to exploit the radiobiological advantages given by hypofractionation, greater attention to geometric accuracy is required due to increased sensitivity to treatment uncertainties.

Voxel-level biological optimisation of prostate IMRT using patient-specific tumour location and clonogen density derived from mpMRI.

AIMS: This study aimed to develop a framework for optimising prostate intensity-modulated radiotherapy (IMRT) based on patient-specific tumour biology, derived from multiparametric MRI (mpMRI). The framework included a probabilistic treatment planning technique in the effort to yield dose distributions with an improved expected treatment outcome compared with uniform-dose planning approaches. METHODS: IMRT plans were generated for five prostate cancer patients using two inverse planning methods: uniform-dose to the planning target volume and probabilistic biological optimisation for clinical target volume tumour control probability (TCP) maximisation. Patient-specific tumour location and clonogen density information were derived from mpMRI and geometric uncertainties were incorporated in the TCP calculation. Potential reduction in dose to sensitive structures was assessed by comparing dose metrics of uniform-dose plans with biologically-optimised plans of an equivalent level of expected tumour control. RESULTS: The planning study demonstrated biological optimisation has the potential to reduce expected normal tissue toxicity without sacrificing local control by shaping the dose distribution to the spatial distribution of tumour characteristics. On average, biologically-optimised plans achieved 38.6% (p-value: < 0.01) and 51.2% (p-value: < 0.01) reduction in expected rectum and bladder equivalent uniform dose, respectively, when compared with uniform-dose planning. CONCLUSIONS: It was concluded that varying the dose distribution within the prostate to take account for each patient's clonogen distribution was feasible. Lower doses to normal structures compared to uniform-dose plans was possible whilst providing robust plans against geometric uncertainties. Further validation in a larger cohort is warranted along with considerations for adaptive therapy and limiting urethral dose.

Chloride channel myotonia: exon 8 hot-spot for dominant-negative interactions.

Myotonia congenita (MC) is the commonest genetic skeletal muscle ion channelopathy. It is caused by mutations in CLCN1 on chromosome 7q35, which alter the function of the major skeletal muscle voltage-gated chloride channel. Dominant and recessive forms of the disease exist. We have undertaken a clinical, genetic and molecular expression study based upon a large cohort of over 300 UK patients. In an initial cohort of 22 families, we sequenced the DNA of the entire coding region of CLCN1 and identified 11 novel and 11 known mutations allowing us to undertake a detailed genotype-phenotype correlation study. Generalized muscle hypertrophy, transient weakness and depressed tendon reflexes occurred more frequently in recessive than dominant MC. Mild cold exacerbation and significant muscle pain were equally common features in dominant and recessive cases. Dominant MC occurred in eight families. We noted that four newly identified dominant mutations clustered in exon 8, which codes for a highly conserved region of predicted interaction between the CLC-1 monomers. Expressed in Xenopus oocytes these mutations showed clear evidence of a dominant-negative effect. Based upon the analysis of mutations in this initial cohort as well as a review of published CLCN1 mutations, we devised an exon hierarchy analysis strategy for genetic screening. We applied this strategy to a second cohort of 303 UK cases with a suspected diagnosis of MC. In 23 individuals, we found two mutations and in 86 individuals we identified a single mutation. Interestingly, 40 of the cases with a single mutation had dominant exon 8 mutations. In total 48 individuals (from 34 families) in cohort 1 and 2 were found to harbour dominant mutations (37% of mutation positive individuals, 30% of mutation positive families). In total, we have identified 23 new disease causing mutations in MC, confirming the high degree of genetic heterogeneity associated with this disease. The DNA-based strategy we have devised achieved a genetic diagnosis in 36% of individuals referred to our centre. Based on these results, we propose that exon 8 of CLCN1 is a hot-spot for dominant mutations. Our molecular expression studies of the new exon 8 mutations indicate that this region of the chloride channel has an important role in dominant negative interactions between the two chloride channel monomers. Accurate genetic counselling in MC should be based not only upon clinical features and the inheritance pattern but also on molecular genetic analysis and ideally functional expression data.

Comparison of 192Ir air kerma calibration coefficients derived at ARPANSA using the interpolation method and at the National Physical Laboratory using a direct measurement.

The reference air kerma rate from 192Ir High Dose Rate (HDR) brachytherapy sources can be measured using a suitably calibrated Farmer chamber and an appropriate in-air calibration jig. When a primary standard for 192Ir gamma rays is available, a calibration coefficient for the chamber and jig combination can be determined directly. In Australia, due to the absence of such a standard, the chamber must be calibrated by interpolation of the response in 60Co and in a kilovoltage x-ray beam. Corrections for the effect of the jig, scatter and beam non-uniformity must then be measured or calculated before the reference air kerma rate can be determined. We compare the air-kerma calibration coefficient of a PTW 30010 PMMA/A1 Farmer chamber (referred to as Farmer chamber throughout this report) obtained from the 192Ir primary standard at the National Physical Laboratory in the UK with the corresponding coefficient obtained by interpolating Australian calibrations using 60Co and 250 kV x-rays and determining suitable correction factors. The resulting chamber/jig calibration coefficients differ by 0.2% which is well within the combined standard uncertainties of 1.2% and 0.6% reported by ARPANSA and NPL respectively.

Radiobiological parameters in a tumour control probability model for prostate cancer LDR brachytherapy.

To provide recommendations for the selection of radiobiological parameters for prostate cancer treatment planning. Recommendations were based on validation of the previously published values, parameter estimation and a consideration of their sensitivity within a tumour control probability (TCP) model using clinical outcomes data from low-dose-rate (LDR) brachytherapy. The proposed TCP model incorporated radiosensitivity (α) heterogeneity and a non-uniform distribution of clonogens. The clinical outcomes data included 849 prostate cancer patients treated with LDR brachytherapy at four Australian centres between 1995 and 2012. Phoenix definition of biochemical failure was used. Validation of the published values from four selected literature and parameter estimation was performed with a maximum likelihood estimation method. Each parameter was varied to evaluate the change in calculated TCP to quantify the sensitivity of the model to its radiobiological parameters. Using a previously published parameter set and a total clonogen number of 196 000 provided TCP estimates that best described the patient cohort. Fitting of all parameters with a maximum likelihood estimation was not possible. Variations in prostate TCP ranged from 0.004% to 0.67% per 1% change in each parameter. The largest variation was caused by the log-normal distribution parameters for α (mean, [Formula: see text], and standard deviation, σ α ). Based on the results using the clinical cohort data, we recommend a previously published dataset is used for future application of the TCP model with inclusion of a patient-specific, non-uniform clonogen density distribution which could be derived from multiparametric imaging. The reduction in uncertainties in these parameters will improve the confidence in using biological models for clinical radiotherapy planning.

The fragile X tremor ataxia syndrome in the differential diagnosis of multiple system atrophy: data from the EMSA Study Group.

The recent identification of fragile X-associated tremor ataxia syndrome (FXTAS) associated with premutations in the FMR1 gene and the possibility of clinical overlap with multiple system atrophy (MSA) has raised important questions, such as whether genetic testing for FXTAS should be performed routinely in MSA and whether positive cases might affect the specificity of current MSA diagnostic criteria. We genotyped 507 patients with clinically diagnosed or pathologically proven MSA for FMR1 repeat length. Among the 426 clinically diagnosed cases, we identified four patients carrying FMR1 premutations (0.94%). Within the subgroup of patients with probable MSA-C, three of 76 patients (3.95%) carried premutations. We identified no premutation carriers among 81 patients with pathologically proven MSA and only one carrier among 622 controls (0.16%). Our results suggest that, with proper application of current diagnostic criteria, FXTAS is very unlikely to be confused with MSA. However, slowly progressive disease or predominant tremor are useful red flags and should prompt the consideration of FXTAS. On the basis of our data, the EMSA Study Group does not recommend routine FMR1 genotyping in typical MSA patients.

Development of a registration framework to validate MRI with histology for prostate focal therapy.

PURPOSE: Focal therapy has been proposed as an alternative method to whole-gland treatment for prostate cancer when aiming to reduce treatment side effects. The authors recently validated a radiobiological model which takes into account tumor location and tumor characteristics including tumor cell density, Gleason score, and hypoxia in order to plan optimal dose distributions for focal therapy. The authors propose that this model can be informed using multiparametric MRI (mpMRI) and in this study present a registration framework developed to map prostate mpMRI and histology data, where histology will provide the "ground truth" data regarding tumor location and biology. The authors aim to apply this framework to a growing database to develop a prostate biological atlas which will enable MRI based planning for prostate focal therapy treatment. METHODS: Six patients scheduled for routine radical prostatectomy were used in this proof-of-concept study. Each patient underwent mpMRI scanning prior to surgery, after which the excised prostate specimen was formalin fixed and mounted in agarose gel in a custom designed sectioning box. T2-weighted MRI of the specimen in the sectioning box was acquired, after which 5 mm sections of the prostate were cut and histology sections were microtomed. A number of image processing and registration steps were used to register histology images with ex vivo MRI and deformable image registration (DIR) was applied to 3D T2w images to align the in vivo and ex vivo MRI data. Dice coefficient metrics and corresponding feature points from two independent annotators were selected in order to assess the DIR accuracy. RESULTS: Images from all six patients were registered, providing histology and in vivo MRI in the ex vivo MRI frame of reference for each patient. Results demonstrated that their DIR methodology to register in vivo and ex vivo 3D T2w MRI improved accuracy in comparison with an initial manual alignment for prostates containing features which were readily visible on MRI. The average estimated uncertainty between in vivo MRI and histology was 3.3 mm, which included an average error of 3.1 mm between in vivo and ex vivo MRI after applying DIR. The mean dice coefficient for the prostate contour between in vivo and ex vivo MRI increased from 0.83 before DIR to 0.93 after DIR. CONCLUSIONS: The authors have developed a registration framework for mapping in vivo MRI data of the prostate with histology by implementing a number of processing steps and ex vivo MRI of the prostate specimen. Validation of DIR was challenging, particularly in prostates with few or mostly linear rather than spherical shaped features. Refinement of their MR imaging protocols to improve the data quality is currently underway which may improve registration accuracy. Additional mpMRI sequences will be registered within this framework to quantify prostate tumor location and biology.

HIV counselling and testing: overemphasizing high acceptance rates a threat to confidentiality and the right not to know.

OBJECTIVE: To examine factors affecting the readiness for HIV-related voluntary confidential counselling and testing (VCT). METHODS: In a population-based HIV survey in selected urban and rural areas in Zambia, adults aged > or = 15 years were selected by stratified random cluster sampling. The participants were asked to provide a saliva sample for anonymous HIV testing (n=4812, consent rate 93.5%) and, as a part of an interview, were asked about previous HIV testing experience and if they wished to be counselled and tested for HIV. Those indicating interest (initially willing) were provided with an invitation letter to see a counsellor. In rural areas, VCT was provided by personnel brought in from outside the local community, whereas in urban areas it was provided by locally recruited staff. RESULTS: The overall HIV test rate was 6.5%, but rates appeared to be considerably biased towards higher educational groups. The proportion initially willing was 37% while 3.6% actually came for counselling and were tested (9.3% of those initially willing), of which 47% returned for the result. Actual use was four to five times higher in rural compared with urban areas. Self-perceived risk and high-risk behaviour were positively associated with initial willingness but not with actual use. CONCLUSIONS: The readiness for VCT in the general population was found to be very low. Provision factors such as concerns about confidentiality and length of time waiting for the test result contributed to the low utilization rate. Results of this study contrast sharply with reported VCT acceptance rates of 70-90% among women attending antenatal care in Zambian and in other African populations, suggesting an urgent need to evaluate testing policy and practice of antenatal VCT in particular.

PCR amplification of 40-year-old paraffin-embedded tumor-tissues - comparison of 4 different DNA extraction and purification methods.

Four different methods of DNA extraction from formalin-fixed, paraffin-embedded tissues were compared for their ability to produce DNA suitable as a template for polymerase chain reaction (PCR). Seven paraffin-embedded rhabdomyosarcoma tissue blocks from the 1950s and one from 1960 were treated with the following extraction methods: 1. Proteinase K digestion and phenol/chloroform extraction; 2. Proteinase K digestion followed by boiling to inactivate the enzyme; 3. Proteinase K digestion, addition of Chelex-100, followed by boiling; and 4. Proteinase K digestion and Prep-A-Gene purification. DNA extracted by methods 1 and 2 was degraded, but DNA of high molecular weight was recovered in every sample extracted by methods 3 and 4 - even though some degradation was observed. Extracted DNA was used as a template for PCR amplification of exon 4 of the PAX-3 gene. PCR was successful in 7 out of 8 samples prepared using methods 3 and 4, producing levels of amplified product equivalent to those obtained with control DNA obtained from fresh lymphocytes. Only very weak products were found in samples prepared by methods 1 (2 out of 8) and 2 (4 out of 8). These results indicate that chelation of polyvalent ions (Chelex-100 method) or obviating the need for boiling (Prep-A-Gene) may protect DNA during extraction.