Patients and controls
The patient was a 35.7 year old woman with strong family history of breast cancer recruited into a clinical trial of MRI screening for young woman at high risk for breast cancer with dense breast tissue . Gadolinium enhancement images revealed a small 1 cm lesion in the upper-outer quadrant of the left breast, identified pathologically as an infiltrating ductal carcinoma. The patient underwent a modified radical mastectomy of the left breast and chose to also undergo a contralateral prophylactic total mastectomy. Blood and tissue were obtained for analysis with consent under Magee-Womens Hospital (of the University of Pittsburgh Medical Center) IRB # MWH-94-108.
Data from this hereditary breast cancer patient were compared to that from two additional patients as well as previously published controls. The first new control patient was a 20 year old women undergoing breast reduction mammoplasty. The second contemporaneous control patient was a 36 year old woman undergoing cosmetic surgery on her contralateral breast two years after successful lumpectomy to remove an apparently sporadic stage IIA breast tumor (2.5 cm, negative for estrogen and progesterone receptors, 13 lymph nodes negative). She had undergone standard radiotherapy and chemotherapy with adriamycin and cyclophosphamide. Histopathological analysis confirmed that the breast tissue from both of these control patients was free of cancer and within the acceptable histological range of normal.
Patient tissue culture and analysis
Fresh tissues from the patient were obtained within 5 hours of surgery. After pathological evaluation, excess tissue not needed for diagnosis was placed into DMEM containing 10% fetal calf serum and 3x antibiotic antimycotic (Sigma, St. Louis, MO) at 4°C. This tissue was then processed as described in Latimer et al.  and placed into culture on a diluted form of matrigel (1:1 with DMEM) in the novel MWRIα medium .
Eight samples of the principal patient's tissue were obtained for culture after bilateral mastectomy surgery. We were not able to obtain a sample of her tumor, because it was utilized entirely for clinical diagnosis. We were able to obtain 4 pieces of histologically normal non-tumor adjacent tissue at increasing 1 cm intervals from the tumor margin from her left (ipsilateral) breast. In addition, we obtained 4 similar pieces of fresh tissue from her contralateral breast. All were placed into primary explant (HME) culture.
For analysis of cell growth and in vitro differentiation, explants were cultured and imaged every second day using a digital Hamamatsu Orca camera for 30–60 days. Images were analyzed on a Macintosh G4 computer using QED imaging software (Media Cybernetics, Inc., Silver Spring, MD).
Control tissue cultures
Breast reduction mammoplasty tissues were obtained from patients ages 20–70 at Magee-Womens Hospital under the above IRB. A neighboring piece of mammoplasty tissue (from the same 0.25 cm2 sample) to that placed into primary culture was fixed and processed in paraffin. These sections were examined by a pathologist to verify the histological features and normality of the tissue. Breast tissue was processed as previously described . Tissue was rinsed three times in PBS containing antibiotics, disaggregated and placed into MWRIα medium  on a thin coat of matrigel. Peripheral blood lymphocytes (PBLs) were obtained with consent from normal healthy control subjects ages 20–50 working at Magee-Womens Hospital or students at the University of Pittsburgh. Foreskin fibroblast (FF) tissue was obtained as discarded tissue from newborn infants after circumcision and utilized between passages 7 and 10. These control populations have been previously described in greater detail [30, 46]. Breast tissue samples from the two new control patients were processed in the same manner.
Analysis of S-phase indices
Primary cultures of mammary tissue, established 10–14 days, were labeled with 3H-thymidine for a period of 2 hours followed by a chase with cold thymidine for 2 hours and then processed for autoradiography. After a 10–12 day exposure, slides were processed and analyzed by two independent, blinded scorers who evaluated the tissue samples for the percentage of cells in S phase (characterized by complete coverage of the nucleus with silver grains).
Unscheduled DNA synthesis
NER was measured by autoradiography of unscheduled DNA synthesis after UV damage (UDS) [47, 48]. After a total of 10–14 days in culture, without passaging, cultures were irradiated with UV light at 254 nm at a mean fluence of 1.2 Joules/m2 for 12 seconds in the absence of culture medium, for a total dose of 14 J/m2. Each sample was represented by at least two chamber slides. One chamber of each 2-chamber slide was shielded from the UV dose to be used as an unirradiated control sample. Primary cultures had not reached confluence and were still actively growing at the time the UDS assay was performed. Control FF were plated subconfluently 2 days before the UDS assay to insure that they also were not in a quiescent state brought on by confluence. After UV exposure, all cultures were incubated in medium supplemented with 10 μCi ml [3H]methyl-thymidine (~80 Ci mmol-1) (PerkinElmer Life Sciences, Boston, MA) for 2 hours at 37°C. Labeling medium was then replaced with unlabeled chasing medium containing 10-3 M non-radioactive thymidine (Sigma) and incubated for a further 2 hours to clear radioactive label from the intracellular nucleotide pools. After incubation in the post-labeling medium, cells were fixed in 1X SSC, 33% acetic acid in ethanol, followed by 70% ethanol and finally rinsed in 4% perchloric acid over night at 4°C. All slides were dried and subsequently dipped in photographic emulsion (Kodak type NTB2) and exposed for 10 to 14 days in complete darkness at 4°C.
The length of exposure of emulsion was determined in each experiment by preparing FF "tester" slides. After 10–12 days these tester slides were developed and grain counting was performed. If the nuclei over the foreskin fibroblasts averaged 50 or more grains per nucleus, then the rest of the experimental slides were developed. If the grain count was below this level, the remaining slides were left to expose 1–3 days longer before being developed.
After photographic development of emulsion, all slides were stained with Giemsa, then examined at a total magnification of 1000X on a Zeiss Axioskop under oil emersion for grains located immediately over the nuclei of non-S phase cells . Local background grain counts were evaluated in each microscopic field over an area the same size as a representative nucleus, and this total was subtracted from the grain count of each nucleus in that field. The average number of grains per nucleus was quantified for each side of the chamber slide, both unirradiated and irradiated. The final NER value for each slide was calculated by subtracting the unirradiated mean (grains per nucleus) from the irradiated mean (grains per nucleus), after the initial subtraction of local background in each field. NER was initially expressed as a percentage of the activity of concurrently analyzed FF. Four FF slides were scored per experiment, by an average of three counters. 200 nuclei were counted per slide, for a total of 800, with an average of 61.6 grains/nucleus. Six slides were evaluated for the patient's PBL sample, two by each of three counters. An average of 195 nuclei were scored per slide (for a total of almost 1200), with an average of 7.5 grains/nucleus. Four slides were counted for the contemporaneous breast reduction control, two each by two counters. There were an average of 200 nuclei per slide and 14.1 grains/nucleus. Six slides were scored from the patient's ipsilateral breast tissue sample, two by each of three independent counters, and five slides were counted from the contralateral sample, again by three independent counters. An average of just over 100 nuclei were evaluated per slide for each sample, for a total of almost 600 nuclei for the ipsilateral sample and over 500 for the contralateral sample. As the NER capacities indicate, these samples had very similar counts; about 35 grains/nucleus for the ipsilateral sample and 28 grains/nucleus for the contralateral sample. Finally, four slides were counted from the contralateral sample of a sporadic breast cancer patient, by three counters. There were an average of 200 nuclei per slide and 29.4 grains per nucleus.
To ensure accuracy and guard against transcription errors, raw grain counts from the UDS assay were processed independently in duplicate, once using StatView (version 5.0.1, SAS Institute, Inc., Cary, NC), and once using the Data Analysis Toolpack of the Excel 2001 spreadsheet program (Microsoft Corp., Redmond, WA). The final count from slides of the same cell type within the same experiment and developed the same day were averaged together and expressed as a percentage of concurrently analyzed FF. These results were then normalized by comparison to the average for the tissue type control population .