Edge effects in 3D dosimetry: characterisation and correction of the non-uniform dose response of PRESAGE®.

Previous work has shown that PRESAGE® can be used successfully to perform 3D dosimetric measurements of complex radiotherapy treatments. However, measurements near the sample edges are known to be difficult to achieve. This is an issue when the doses at air-material interfaces are of interest, for example when investigating the electron return effect (ERE) present in treatments delivered by magnetic resonance (MR)-linac systems. To study this effect, a set of 3.5 cm-diameter cylindrical PRESAGE® samples was uniformly irradiated with multiple dose fractions, using either a conventional linac or an MR-linac. The samples were imaged between fractions using an optical-CT, to read out the corresponding accumulated doses. A calibration between TPS-predicted dose and optical-CT pixel value was determined for individual dosimeters as a function of radial distance from the axis of rotation. This data was used to develop a correction that was applied to four additional samples of PRESAGE® of the same formulation, irradiated with 3D-CRT and IMRT treatment plans, to recover significantly improved 3D measurements of dose. An alternative strategy was also tested, in which the outer surface of the sample was physically removed prior to irradiation. Results show that for the formulation studied here, PRESAGE® samples have a central region that responds uniformly and an edge region of 6-7 mm where there is gradual increase in dosimeter response, rising to an over-response of 24%-36% at the outer boundary. This non-uniform dose response increases in both extent and magnitude over time. Both mitigation strategies investigated were successful. In our four exemplar studies, we show how discrepancies at edges are reduced from 13%-37% of the maximum dose to between 2 and 8%. Quantitative analysis shows that the 3D gamma passing rates rise from 90.4, 69.3, 63.7 and 43.6% to 97.3, 99.9, 96.7 and 98.9% respectively.

Cognitive functioning in humans with mutations of the PAX6 gene.

Fourteen patients with PAX6 gene mutations and previously identified MRI abnormalities were administered tests of cognitive functioning. No deficits were found. A subgroup with agenesis of the anterior commissure performed significantly more poorly on measures of working memory than those without this abnormality, suggesting the anterior commissure may play a role in cognitive processing in addition to an earlier identified role in sensory development and processing.

A study of eleven cutaneous malignant melanomas in adults with small-cell morphology: emphasis on diagnostic difficulties and unusual features.

AIMS: To describe the clinicopathological and immunohistochemical features of cutaneous malignant melanomas with a pure or mixed small-cell pattern in 11 adult patients, and to discuss the diagnostic difficulties encountered. METHODS AND RESULTS: Haematoxylin and eosin-stained sections of each case of cutaneous small-cell malignant melanoma, together with locally recurrent skin lesions and, where available, metastatic deposits, were re-examined. Available immunohistochemical sections were evaluated. Clinical follow-up data were obtained in each case. One patient presented with metastatic disease, the others presented with cutaneous lesions. Suggested initial diagnoses included malignant melanoma, non-Hodgkin's lymphoma, Merkel cell carcinoma and sarcoma. All the tumours were in the vertical growth phase. Nine had a junctional component, often inconspicuous. The lesions showed either a pure small-cell pattern or a mixed pattern with more conventional areas. In one case, there was colonization of a basal cell carcinoma by invasive malignant melanoma. Variable retention of small-cell morphology in local recurrences and metastases was observed, although in some cases more typically pleomorphic cells were present. In the cases tested, there was strong immunostaining for S100 protein and NKI-C3, and variable immunostaining for HMB45 and Melan-A. Non-melanocytic markers were negative. CONCLUSIONS: The possibility of a small-cell malignant melanoma should be considered in the assessment of cutaneous and non-cutaneous small-cell neoplasms. The correct diagnosis requires careful evaluation for junctional activity, melanin production and the use of a panel of melanocytic markers.

Mammalian homologues of the Drosophila eye specification genes.

The Drosophila compound eye is specified by the simultaneous and interdependent activity of transcriptional regulatory genes from four families: PAX6 (eyeless, twin of eyeless, eyegone), EYA (eyes absent), SIX (sine oculis, Optix) and DACH (dachshund). Mammals have homologues of all these genes, and many of them are expressed in the embryonic or adult eye, but the functional relationships between them are currently much less clear than in Drosophila. Nevertheless, mutations in the mammalian genes highlight their requirement both within and outside the eye in embryos and adults, and emphasize that they can be deployed in many different contexts.

Evidence of nerve sheath differentiation and high grade morphology in sclerosing epithelioid fibrosarcoma.

Sclerosing epithelioid fibrosarcoma is a recently described sarcoma in which ultrastructural evidence of fibroblastic differentiation forms part of the diagnostic criteria. This report describes a further case of this tumour, which showed evidence of both fibroblastic and perineurial differentiation by immunohistochemistry and electron microscopy, and which had areas of high grade morphology. The tumour metastasised and the patient died of disease 12 months after presentation. The relevance of these findings to diagnosis and differentiation in these tumours is discussed.

PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans.

PAX6 is widely expressed in the central nervous system. Heterozygous PAX6 mutations in human aniridia cause defects that would seem to be confined to the eye. Magnetic resonance imaging (MRI) and smell testing reveal the absence or hypoplasia of the anterior commissure and reduced olfaction in a large proportion of aniridia cases, which shows that PAX6 haploinsuffiency causes more widespread human neuro developmental anomalies.

Value and accuracy of cytology in addition to histology in the diagnosis of lung cancer at flexible bronchoscopy.

There is still disagreement as to the value and reliability of wash and brush cytology, in comparison with histology, for the diagnosis of malignancy at flexible bronchoscopy. The present study compares the yield and concordance of findings from the two modalities for visible tumours at flexible bronchoscopy. A single-centre study of 514 consecutive flexible bronchoscopy procedures, in which a lesion suspicious of cancer was seen and bronchial wash cytology, brush cytology and forceps biopsy samples were taken. All equivocal or suspicious results were taken as negative. An overall yield of 89.3% was achieved using a combination of all three tests. This was greater for endobronchial than submucosal (95% vs. 86%) tumours. Cytology alone diagnosed 17.7% of cases. Use of all three modalities allowed tumours to be differentiated between small and non-small cell types in all but 5/459 positive cases (98.9%). There were only 3/313 cases in which there was a difference in cell type (small cell vs. non-small cell) between the two modalities. We conclude that wash and brush cytology are valuable tools, in addition to forceps biopsy, at flexible bronchoscopy. All three tests should be performed routinely in cases of suspected malignancy.

A study of breast cancers detected in the incident round of the UK NHS Breast Screening Programme: the importance of early detection and treatment of ductal carcinoma in situ.

One hundred and seventy eight cancers detected on incident round screening in the UK National Health Service Breast Screening Programme were reviewed. Critical review of the immediately preceding screening films (from 3 years previously) found abnormalities at the site of the subsequently detected cancer in 93 cases (52%). Forty-eight of these (27% of the total) had microcalcification as the sole abnormality. All of these 48 women had invasive ductal carcinoma and/or ductal carcinoma in situ (DCIS) (including four cases in which DCIS was associated with another type of primary invasive breast cancer). The finding of microcalcification on the previous mammograms at the site of a subsequently detected cancer was a strong predictor for the presence of DCIS (with or without associated invasive disease) (P<0.0001). Of the women with invasive ductal carcinoma, those with microcalcification on previous films were significantly more likely to have intermediate or high grade (grade 2 or 3) tumours than those women without microcalcification on previous films (P=0.0015). Previous films were also read blind by two independent experienced breast radiologists. Cancers were correctly identified by one or both readers in 39 cases. However, 35 of the remaining 139 cases showed microcalcification which was not detected or considered significant by the readers. If only these 139 'true negative' screens are analysed, similar associations are seen between microcalcification on previous films and subsequent finding of DCIS (P=0.03) and between microcalcification on previous films and high grade invasive ductal carcinomas (P=0.015). These findings provide support for the hypothesis that microcalcification seen on previous screening films at the site of a subsequently detected invasive ductal carcinoma represents ductal carcinoma in situ. In this series, 19 of 82 women (23%) with invasive ductal carcinoma in the 'true negative' screen group had microcalcification suggestive of DCIS on mammograms taken, on average, 3 years previously. Significant microcalcification is often overlooked using current detection criteria. Early detection and treatment of DCIS is essential in order to prevent the development of aggressive invasive disease. Revision of the NHSBSP targets for DCIS detection is recommended.

Prenatal diagnosis of aniridia.

OBJECTIVE: To use molecular genetic techniques to prenatally screen for aniridia. DESIGN: Case report. METHODS: DNA was extracted from cultured fibroblasts obtained through amniocentesis. Two mutation detection methods, Ava1 restriction digestion and single-strand conformational polymorphism electrophoresis, were used to screen the PAX6 gene. MAIN OUTCOME MEASURES: The results from the amniocentesis sample were compared with DNA obtained from the affected father, firstborn infant, and unaffected mother to determine whether the fetus carried the PAX6 mutation. RESULTS: DNA from the fetus demonstrated the same banding pattern as the affected father and firstborn infant. CONCLUSIONS: The fetus carried the mutated PAX6 allele and was predicted to develop aniridia. This was later confirmed when the child was born. This case report illustrates an important use of genetic mutation screening in the clinical setting.

Anal intraepithelial neoplasia in an inflammatory cloacogenic polyp.

A rare case of anal intraepithelial neoplasia arising in an inflammatory cloacogenic polyp is reported. While the occurrence of neoplasia complicating benign anal conditions is recognised, this case re-emphasises the need for careful histological examination of all perianal lesions.

Mammalian and Drosophila dachshund genes are related to the Ski proto-oncogene and are expressed in eye and limb.

We have isolated mammalian homologues of the Drosophila dachshund gene. Two domains of high conservation, one of which contains an alpha-helical, coiled-coil motif, show similarity to the Ski family of genes. We therefore propose that Dachshund belongs to a superfamily including these genes. Mouse Dachshund (Dach) is expressed in the eye and limb, structures affected by the Drosophila loss-of-function mutant, and rib primordia, CNS and genital eminence. Pax6 and Dach show overlapping but non-identical expression patterns. Dach expression is unaffected in smalleye mouse brain, indicating that Pax6 is not directly activating Dach. In Drosophila eye development dachshund is a component of an interacting network of proteins. Genes homologous to many of these exist in mammals; Dach joins this expanding group.

Combined SSCP/heteroduplex analysis in the screening for PAX6 mutations.

We demonstrate the use of combined SSCP and heteroduplex analysis in the detection of PAX6 mutations using non-radioactive silver staining. A panel of aniridia patients was screened by this approach and we show that a greater number of mutations was detected than would have been found by running each technique alone. Six previously unreported aniridia mutations in PAX6 are also described..

Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.

Mutation or deletion of the PAX6 gene underlies many cases of aniridia. Three lines of evidence now converge to implicate PAX6 more widely in anterior segment malformations including Peters' anomaly. First, a child with Peters' anomaly is deleted for one copy of PAX6. Second, affected members of a family with dominantly inherited anterior segment malformations, including Peters' anomaly are heterozygous for an R26G mutation in the PAX6 paired box. Third, a proportion of Sey/+ Smalleye mice, heterozygous for a nonsense mutation in murine Pax-6, have an ocular phenotype resembling Peters' anomaly. We therefore propose that a variety of anterior segment anomalies may be associated with PAX6 mutations.

PAX6 mutations in aniridia.

Aniridia is a congenital malformation of the eye, chiefly characterised by iris hypoplasia, which can cause blindness. The PAX6 gene was isolated as a candidate aniridia gene by positional cloning from the smallest region of overlap of aniridia-associated deletions. Subsequently PAX6 intragenic mutations were demonstrated in Smalleye, a mouse mutant which is an animal model for aniridia, and six human aniridia patients. In this paper we describe four additional PAX6 point mutations in aniridia patients, both sporadic and familial. These mutations highlight regions of the gene which are essential for normal PAX6 function. In addition, the frequency at which we have found PAX6 mutations suggests that lesions in PAX6 will account for most cases of aniridia.

Identification and preliminary characterization of a protein motif related to the zinc finger.

We have identified a protein motif, related to the zinc finger, which defines a newly discovered family of proteins. The motif was found in the sequence of the human RING1 gene, which is proximal to the major histocompatibility complex region on chromosome six. We propose naming this motif the "RING finger" and it is found in 27 proteins, all of which have putative DNA binding functions. We have synthesized a peptide corresponding to the RING1 motif and examined a number of properties, including metal and DNA binding. We provide evidence to support the suggestion that the RING finger motif is the DNA binding domain of this newly defined family of proteins.

Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization.

Fluorescence in situ hybridization (FISH) with biotin-labeled probes mapping to 11p13 has been used for the molecular analysis of deletions of the WAGR (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) locus. We have detected a submicroscopic 11p13 deletion in a child with inherited aniridia who subsequently presented with Wilms tumor in a horseshoe kidney, only revealed at surgery. The mother, who has aniridia, was also found to carry a deletion including both the aniridia candidate gene (AN2) and the Wilms tumor predisposition gene (WT1). This is therefore a rare case of an inherited WAGR deletion. Wilms tumor has so far only been associated with sporadic de novo aniridia cases. We have shown that a cosmid probe for a candidate aniridia gene, homologous to the mouse Pax-6 gene, is deleted in cell lines from aniridia patients with previously characterized deletions at 11p13, while another cosmid marker mapping between two aniridia-associated translocation breakpoints (and hence a second candidate marker) is present on both chromosomes. These results support the Pax-6 homologue as a strong candidate for the AN2 gene. FISH with cosmid probes has proved to be a fast and reliable technique for the molecular analysis of deletions. It can be used with limited amounts of material and has strong potential for clinical applications.

Molecular genetics of the Pax gene family.

Three members of the Pax gene family are now known to be responsible for the established mouse developmental phenotypes Splotch, Small eye and undulated; two of these genes are implicated in the human congenital diseases Waardenburg's syndrome and aniridia. The mouse mutants will act as model systems for these human disorders and, in addition, will provide insights into the processes of vertebrate development.

The human BDNF gene maps between FSHB and HVBS1 at the boundary of 11p13-p14.

To map in detail the human gene for brain derived neurotrophic factor (BDNF) we have used a PCR-based assay to amplify the gene from somatic cell hybrids containing human chromosome 11 with deletion or translocation breakpoints in the WAGR region. The BDNF gene maps between the FSHB and HVBS1 loci, an interval of approximately 4 Mb at the boundary of 11p13 and 11p14.