Table 2.
Correlation of p16 and pRb status in a series of breast carcinomas
| Patient ID no. | ER/PR* | DNA index/ploidy* | Proliferation index (%)* | Cyclin E† | p16† | pRb† |
|---|---|---|---|---|---|---|
| KK005 | −/− | 1.18/Aneuploid | 12.2 (H) | +++ | ++++ | + |
| KK017 | −/− | 1.72/Aneuploid | 1.5 (L) | ++++++ | ± | + |
| KK020 | −/− | 1.73/Aneuploid | 14.1 (H) | +++++ | − | − |
| KK036 | +/− | 1.84/Tetraploid | 3.3 (L) | ++ | ± | + |
| KK061 | −/− | ND | ND | ++++ | ± | − |
| KK070 | +/+ | ND | ND | + | ± | − |
| KK076 | −/− | 2.08/Tetraploid | 12.5 (H) | +++ | ± | − |
| KK086 | −/− | 1.50/Aneuploid | 36.0 (H) | +++++ | ++ | − |
| KK147 | ND | ND | ND | +++++ | +++ | + |
| KK173 | +/− | 1.91/Tetraploid | 30.2 (H) | +++++++ | ++++ | + |
| KK190 | −/− | 2.09/Tetraploid | 31.8 (H) | +++++++ | +++ | − |
| KK322 | +/− | 2.70/Aneuploid | 30.0 (H) | +++ | − | + |
| KK369 | ND | ND | 40.0 (H) | +++++++ | ++++ | + |
| KK399 | −/− | ND | ND | ++++ | ++++ | − |
| KK400 | +/− | ND | ND | ++++ | ± | − |
| KK407 | −/− | 1.89/Tetraploid | 18.0 (H) | ++++ | − | − |
| KK428 | −/− | 1.75/Aneuploid | 27.0 (H) | ++++ | − | − |
| KK429 | −/− | 1.71/Aneuploid | 28.0 (H) | +++++ | − | − |
| KK457 | ND | ND | ND | ++++++ | − | + |
| KK458 | −/− | 1.96/Tetraploid | 11.3 (H) | + | − | + |
Quantitation of immunohistochemical staining by image analysis was performed on sections stained with either the monoclonal antibody to estrogen receptor H222 (ER-ICA kit, Abbott), monoclonal antibody to progesterone receptor mPRI (Cell Analysis Systems, Lombard, IL), or monoclonal antibody to Ki67 (Dako) as described (45, 46). Ki67 staining determined growth fraction of the tumor. Values indicate percentage of positive staining: 1.0–7.0% is indicative of low (L) proliferation index, 7.1–11.9 is indicative of moderate (M) proliferation index, and >12.0% is indicative of high (H) proliferation index. For each case, the DNA ploidy was determined by quantitation of the DNA Feulgen stain by computerized microdensitometry as described (47). ND, not determined.
Cyclin E, p16, and pRb levels were measured using Western blot analysis with HE12 monoclonal antibody to cyclin E (Santa Cruz Biotechnology) as described (31, 32), monoclonal antibodies to p16, and pRb as described in text. Levels of cyclin E in tumor tissue samples were correlated with 76N normal (+) and MDA-MB-157 (++++++) tumor cell lines. For example, cyclin E in MDA-MB-157 cell line is 64-fold (i.e., ++++++) overexpressed compared with 76N cell line (i.e., +) (31). Any tumor tissue overexpressing cyclin E more than MDA-MB-157 received seven +s (i.e., +++++++). p16 levels also were correlated with MDA-MB-157 (++++) cell line. Equal protein loading was monitored by reprobing blots with actin, and all blots were analyzed by densitometry using AGFA scanner and IP Lab Gel software.