Methylation of the retinoic acid receptor-B2 (RARB2) P2 promoter is hypothesized to be an important mechanism for loss of RARB2 function during early mammary carcinogenesis. The frequency of RARB2 P2 methylation was tested in (a) 16 early stage breast cancers and (b) 67 random periareolar fine needle aspiration (RPFNA) samples obtained from 38 asymptomatic women who were at increased risk for breast cancer. Risk was defined as either (a) 5-year Gail risk calculation z z1.7%; (b) prior biopsy exhibiting atypical hyperplasia, lobular carcinoma in situ, or ductal carcinoma in situ; or (c) known BRCA1/2 mutation carrier. RARB2 P2 promoter methylation was assessed at two regions, M3 (À À51 to 162 bp) and M4 (104-251 bp). In early stage cancers, M4 methylation was observed in 11 of 16 (69%) cases; in RPFNA samples, methylation was present at M3 and M4 in 28 of 56 (50%) and 19 of 56 (38%) cases, respectively. RPFNAs were stratified for cytologic atypia using the Masood cytology index. The distribution of RARB2 P2 promoter methylation was reported as a function of increased cytologic abnormality. Methylation at both M3 and M4 was observed in (a) 0 of 10 (0%)

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1. The reaction of slowly growing pathogenic bacteria with specific fluorescent antibody provided a means for their rapid identification.2. Fluorescein isothiocyanate was used to label the globulins of antisera as it had a number of advantages over fluorescein isocyanate.3. Several other isothiocyanates and a few sulphonyl chlorides tested were inferior to flurescein isothiocyanate as labelling agents. Some of the compounds caused protein precipitation, and others gave a less intense or less distinctive fluorescence than did dluorescein isothiocyanate.4. The tests were performed by taking impressions of bacterial micro-colonies on glass cover-slips, drying and fixing the preparations, staining them with labelled antiserum, washing in saline and mounting in glycerol saline. The preparations were than examined microscopically under ultra-violet light.5. Micro-colonies ofBr. suis, Past. tularensisandPast. pestis, when treated with homologous labelled antiserum, fluoresced brightly under ultra-violet light and the individual bacteria were sharply defined and stained at the periphery.6. Micro-colonies of these and other bacteria stained with normal labelled serum, or heterologous labelled antiserum, showed either very dim fluorescence under ultra-violet light, with poor definition of in dividual bacteria, or no fluorescence at all.7. The technique described permitted specific identification ofBr. suis, Past. pestisandPast. tularensiswithin 20 hr. of inoculation of agar plates with material suspected of containing one of these organisms.This investigation arose out of a line of work suggested by Dr G. S. Wilson. We should like to thank Dr D. W. Henderson and Major L. H. Kent for providing facilities for the work, Mr E. O. Powell for continual encouragement and supervision of the microscopy, Dr D. W. Henderson, Dr M. C. Lancaster and Dr D. A. L. Davies for supplying antisera, and Mr T. W. Pearce for valuable technical assistance and the photographs.

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