Columnar cell lesions of the breast include a morphologic spectrum of simple columnar cell change, columnar cell hyperplasia, columnar cell hyperplasia with atypia and ductal carcinoma in situ of micropapillary/cribriform type. Invasive carcinomas of low grade are often seen in association with this spectrum. The biologic significance of these lesions that are commonly found on breast biopsies is unknown. Three cases of formalin-fixed, paraffin-embedded breast tissues, each displaying the entire spectrum of columnar cell lesions through ductal carcinoma in situ and including foci of invasive carcinoma were microdissected at multiple sites to evaluate neoplasia progression. Minute tissue targets were microdissected (4-8/case) from unstained 4-microm thick recut paraffin sections and included non-neoplastic breast and sites of columnar cell change, hyperplasia, atypia, ductal carcinoma in situ and invasive carcinoma. Allelic imbalance for a broad panel of microsatellite markers in proximity to known tumor suppressor genes was quantitated using automated polymerase chain reaction/gel electrophoresis. Genomic loci evaluated 1p, 3p, 5q, 9p, 9q, 10q, 17p, 17q, 19q, 22q. The presence, topographic relationship and time course of mutational damage was correlated with columnar morphologic features. Detailed allelic imbalance information was obtained from each microdissection tissue target producing a detailed fingerprint of mutational damage in each case. Allelic damage was targeted predominately at 9q, 10q, 17p and 17q. Simple columnar cell change was without mutational changes and only present in one case of columnar cell hyperplasia. The remainder of the cases all show progressive accumulation of allelic damage in columnar cell changes with atypia, ductal carcinoma in situ and invasive carcinoma. The fractional mutation percentage increased progressively from columnar cell hyperplasia through invasive carcinoma. Low level of allelic imbalance was demonstrable in columnar cell lesions by the microdissection approach. A gradient of progressive mutational change could be delineated in each case manifesting allelic loss damage. Allelic loss damage appeared to preferentially target loci at 9q, 10q, 17p and 17q. The findings are consonant with the hypothesis that a select group of atypical columnar cell lesions are morphologic precursors to invasive carcinoma. Integrated molecular pathology analysis used here can help define the significance of columnar cell lesions and its role in breast cancer tumorigenesis on an individual patient basis.