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Background and Objectives The monoclonal antibody MIB-1 is an immunohistochemical marker reacting most strongly with cells in late S phase, G2, and M portions of the cell cycle. This antibody, reactive in formalin-fixed, paraffin-embedded tissue, allows the quantitation of a proliferation index (PI) in both current clinical cases and archival material using a computerized image analyzer (CIA). Methods Since many laboratories make use of automated immunohistochemistry (AIH), this study was performed to explore the technical feasibility of using AIH (Ventana ES 320) in combination with CIA (CAS 200) to evaluate MIB-1 PI as a prognostic marker as assessed by overall survival in 50 archival (formalin-fixed, paraffin-embedded), advanced stage primary ovarian carcinomas. Results Exploratory methods confirmed that 15% was a cutpoint that could dichotomize these 50 patients into two prognostic groups based on overall survival. The median survival of patients whose carcinoma had a high MIB-1 expression (⩾15%) was 16 months compared with 30 months in the patients whose tumors demonstrated low MIB-1 expression (

The quantitation of estrogen and progesterone receptors (ER and PgR) has become the standard of care in the evaluation of patients with primary breast carcinoma. It has been demonstrated that ER and PgR detected by immunohistochemical methods in formalin-fixed paraffin-embedded tissue can be quantified by computerized image analysis. In this study, ER and PgR levels were determined by using an automated immunochemistry stainer (Ventana ES 320) and an image analyzer (CAS 200) in a series of 236 patients with stage I/II carcinoma of the breast. The degree of correlation of the ER and PgR levels determined by the dextran-coated charcoal method (DCC) with image analysis quantitation was high (r=0.75). The agreement between both methods was 77% for ER and 73% for PgR. Hormone receptor levels were correlated with prognosis as determined by overall survival. An ER level of 30 fmol/mg as determined by image analysis was established to stratify the patient population most effectively into favorable and unfavorable prognostic groups (P=0.003). An ER level of 20 fmol/mg for prognostic stratification reached statistical significance (P=0.03). The DCC method was not able to stratify the patients into prognostic groups at the traditionally accepted cutpoint of 10 fmol/mg (P=0.52). We conclude that when used in combination, automated immunohistochemistry and quantitative image analysis offer a favorable alternative to the DCC method in assessment of ER and PgR status in human mammary carcinoma. In addition, quantitative immunocytochemistry techniques may prove superior to the DCC method in specimens in which there is limited tumor volume (including fine-needle aspirates), stroma-rich tumors, and early-stage lesions including intraductal carcinoma.

Traditionally, estrogen and progesterone receptor levels have been determined by biochemical ligandbinding assays, but more recently immunohistochemical techniques have become available. They have gained popularity due to their low cost, smaller sample size requirements, and direct visualization capability of reaction location. Several antibody clones are commercially available and antibodies directed against the estrogen receptor (ER) are supplied by Ventana Medical Systems (Tucson, AZ), Abbott Laboratories (Abbott Park, IL), and lmmunotech Westbrook, ME). Antibodies directed against the progesterone receptor (PgR) are supplied by Ventana Medical Systems (Tucson, AZ), lmmunotech (Westbrook, ME), and Becton-DickinsonKelI Analysis Systems (San Jose, CA). Computer-assisted image analysis using the CAS ZOOTM (Becton-DickinsonKIS, San Jose, CA) allows quantitation of immunohistochemically determined receptor levels. Correlation of quantitated immunohistochemical ER levels with values determined by ligand-binding assay revealed the Ventana antibody to most closely predict the ligand-binding results (wk = .667). The Ventana anti-progesterone antibody quantitation most closely correlated with the ligand-binding results (wk = 435) for determination of PgR. Progesterone receptor level as determined by any of the tested methods did not stratify patients into favorable and unfavorable prognostic groups. Estrogen receptor level as determined by the Ventana antibody was the most predictive of patient outcome but this relationship did not reach statistical significance (p = .09). Most discrepancies between the ligand-binding assay and the immunohistochemical assays were associated with one of three factors: (a) low volume of neoplastic cells present due either to small sample size or high stromal content, (b) premenopausal status with circulating endogenous estrogens potentially occupying receptor sites, (c) presence of benign breast epithelium resulting in a false-positive ligand-binding assay.

Several clinicopathologic variables influence prognosis in breast cancer, including stage, histologic grade, nodal status, and tumor size. Multiple studies have shown an independent value of proliferation index as a prognostic variable for the stratification into favorable and unfavorable groups. The monoclonal antibody MIB-1 reacts with the same antigen site, not epitope, as recognized by the Ki-67 antibody. Like Ki-67, MIB-1 reacts with cells in the late G1, S, M and G2 phases of the cell cycle, but MIB-1 has the advantage of reacting with formalin-fixed, paraffin-embedded material. The authors investigated the feasibility of using image analysis to quantitate the MIB-1 antibody staining (proliferation index [PI]) and predict survival in a series of 230 patients with stage I and stage II breast cancer. In a univariate Cox regression model, larger values of MIB-1 were related to shorter survival times (p 11 %, respectively. Higher clinical stage was associated with higher MIB-1 values and shorter survival (p = 0.01, and p = 0.003, respectively). Tumor size (p = 0.02) and nodal status (p = 0.05) were also associated with higher values of MIB-1. After adjusting for age, clinical stage, nodal status, and tumor size in a multivariate analysis, MIB-1 retained its prognostic significance (p < 0.0001) when considered as either a continuous or categorical variable. There were no significant associations between MIB-1 determined proliferation index and age (p = 0.54), histologic grade (p = 0.69), nuclear grade (p = 0.06) or the presence of vascular invasion (p =.66). There is a strong statistical relationship between cell proliferative activity, as determined by MIB-1 expression, and survival in early stage breast cancer.

It has been shown that the monoclonal antibody Ki-67 reacts with a nuclear antigen that is expressed only by proliferating cells. The feasibility of using image analysis to quantitate Ki-67 staining (proliferation index [PI]) of epithelial ovarian cancers was investigated. The PI was determined in 50 advanced-stage primary ovarian cancers. Frozen sections were immunostained with the Ki-67 monoclonal antibody, and the PI was calculated using static image analysis. Among 35 stage III ovarian carcinomas, the median PI was 8.9%, compared with 17.7% in 15 stage IV cancers (P = 0.06). There was no relationship between PI and histologic grade. The median survival time of 32 patients whose cancers had a high Ki-67 expression (> or = 7.5%) was 16.8 months, which differed significantly (P < 0.01) from the median survival of 31.5 months observed in patients whose tumors demonstrated low Ki-67 expression (< 7.5%). Quantitative image analysis of Ki-67-stained fresh-frozen ovarian cancers may provide useful prognostic information. Further studies are warranted to investigate the relationship between Ki-67 expression and other known clinicopathologic and genetic features of ovarian cancer.

To investigate epidermal growth factor receptor (EGFr) presence in the prostate, monoclonal antibody (clone EGFR1) immunohistochemical examination of radical prostatectomy specimens was performed (n = 37). All prostatic specimens contained benign prostatic hyperplasia (BPH) and/or dysplasia (prostatic intraepithelial neoplasia or PIN), as well as prostatic carcinoma (CaP). Areas of dysplasia were further categorized as to the basal cell layer and the luminal cell area. BPH, PIN, and CaP tissues in each specimen were analyzed by a single observer and graded on a scale from 0-4+. Fifteen samples were also analyzed for EGFr content utilizing a Cell Analysis Systems (CAS 200) image cytometer. EGFr immunoreactivity of BPH basal cells was significantly higher than EGFr immunoreactivity in areas of CaP (p < 0.001). EGFr staining of BPH basal cells was also significantly higher than that seen in PIN luminal cells (p < 0.001). Immunoreactivity of EGFr in PIN basal cells was significantly higher than in PIN luminal cells (p < 0.001). EGFr staining of basal cells in BPH tissues was higher than that seen in the PIN basal cell layer but the difference was not statistically significant (p = 0.06). The amount of staining present in PIN luminal cells was also significantly greater than in CaP tissues (p = 0.002). Quantitative image analysis utilizing the CAS 200 image cytometer was performed on BPH and CaP areas exclusively. EGFr immunoreactivity in basal cells of the BPH tissues was significantly greater than that seen in CaP tissues (p < 0.001). The decreased EGFr immunoreactivity in CaP may reflect a differentiating role for EGFr in normal tissues. Loss of EGFr influence may be associated with an increased proliferative state in PIN and CaP. Destruction or alteration of the epidermal grwoth factor receptor by a protease, such as prostatic specific antigen, may also explain our findings. At the present time the meaning of the different amounts of EGFr in the various types of prostate tissues is unknown.

A murine monoclonal antibody designated 4B5 was raised against the high molecular weight fraction of pooled sputum from patients with non-small cell lung cancer (NSCLC). Immunohistochemical staining indicated that 4B5 binds to histologically normal bronchial epithelium distant from tumor in 72% (39 of 54) of patients with NSCLC, but it binds to the primary cancer in only 13% (7 of 54) of the same patients. The antibody reacted less intensely with the bronchial epithelium in 16.6% (3 of 18) of autopsied patients without significant lung disease. The antigen recognized by 4B5 is a high molecular weight glycoprotein of more than 400 kilodaltons, judged by gel filtration and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and western blot analysis. Antigenic activity persisted after heating and resisted treatment with neuraminidase, but it was destroyed using protease and periodate. Multiple epitopes were present on each molecule recognized by 4B5. The determinants recognized by this antibody deserve additional study as possible markers of premalignant change in patients with NSCLC.

To investigate HER-2/neu oncoprotein immunoreactivity, monoclonal antibody TA1 immunohistochemical examination of flash-frozen radical prostatectomy specimens was performed (n = 35). All prostatic specimens contained benign prostatic hyperplasia (BPH) and/or prostatic intraepithelial neoplasia (PIN), as well as prostatic carcinoma (CaP). HER-2/neu oncoprotein immunoreactivity in BPH tissues was not significantly different than that for the PIN basal cell layer (P = 0.10) or for the PIN luminal cells (P = 0.17). There was significantly more HER-2/neu oncoprotein immunoreactivity in BPH than in areas of CaP (P < 0.001). There was no significant difference in the amount of immunoreactivity present in PIN basal cells when compared to the PIN luminal cells (P = 0.49). Both the PIN basal cells and luminal cells stained for the HER-2/neu oncoprotein to a higher degree than cells in the CaP areas (P < 0.001 in both cases). HER-2/neu oncoprotein immunoreactivity is present at a significantly higher degree in BPH and PIN than in malignant prostatic epithelium.

Immunohistochemical examination of radical prostatectomy specimens from 57 patients was performed to determine the differential expression of transforming growth factor alpha in the human prostate. In addition, epidermal growth factor receptor (EGFr) immunoreactivity was assessed in each case. Stromal versus epithelial staining was determined for each histological subtype: benign prostatic hypertrophy (BPH), prostatic intra-epithelial neoplasia (PIN), and prostatic cancer (CaP) by a single pathologist reviewer. TGFa staining was predominant in stroma while EGFr was localized to the epithelial basal cell layer. Immunoreactivity of both TGFa (P = 0.002) and EGFr (P < 0.001) revealed a significant reduction in CaP compared to BPH or PIN. Autocrine stimulation of EGFr by TGFa or other unrecognized factors may be present in CaP. Conversely, altered stromal influence of CaP via TGFa may be present. These observations could form the basis for future cancer therapeutic strategies using antagonist factors.

Image analysis of nuclear DNA content (DNA ploidy) was performed on smears of basal cell carcinoma (BCC) obtained during Mohs microscopically-controlled surgery from 51 tumors. DNA ploidy was compared with histologic growth pattern and the contour of the invading edge. There was a statistically significantly increased frequency of DNA aneuploidy in smears from BCC exhibiting partial or total diffuse (infiltrative and superficial multicentric) growth patterns (80%; 32 of 40) as compared to solely circumscribed growth patterns (0%; 0 of 11) (p < 0.001).

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.

Overexpression of the nuclear phosphoprotein p53 is one of the most frequently detected abnormalities in human cancer and appears to be associated with mutation of the p53 gene. In this study of breast cancer, p53 overexpression was detected in two (15%) of 15 pure intraductal tumors, 73 (25%) of 291 primary invasive carcinomas, 13 (50%) of 26 lymph nodes containing metastatic breast cancer, and two of four established breast cancer cell lines. Sequence analysis of selected specimens confirmed that p53 overexpression was associated with mutation of the gene, while no mutations were detected in specimens without p53 overexpression. Thus, overexpression of p53 occurs in all stages of breast cancer and is consistently associated with the production of mutant proteins. Immuno-histochemical analysis is a simple method which reliably predicts the presence of most p53 gene mutations in breast cancer specimens.

To investigate factors involved in progression of breast cancer, we estimated the growth fraction of malignant cell populations in various stages of mammary cancer growth. Frozen sections were immunostained with the Ki-67 monoclonal antibody and the proliferation index determined using static image analysis. Pure intraductal carcinoma, intraductal carcinoma coexisting with invasive disease, and metastatic sites coexisting with primary tumors were studied. The proliferation index of pure intraductal carcinomas (mean 4.5%, median 1.8%) was not significantly different from invasive mammary cancers (mean 5.1%, median 2.2%). The proliferation index determined for the in situ component of primary cancers (mean 3.8%, median 1.5%) was not significantly different from values obtained from the invasive component of growth (mean 4.2%, median 2.1%). Variability between in situ and invasive components for individual cases was minimal in tumors whose proliferation index was less than 3.0%; for tumors with higher proliferation indices, the differences were greater. However, there was no trend toward a decrease or increase in growth fraction for the two components of primary tumor growth. The mean proliferative index for primary tumors (mean 4.9%, median 4.0%) was not significantly different from the mean proliferative score from a matched group of metastatic sites in the same patients (mean 5.7%, median 5.5%). Comparison of individual cases uncovered differences in some tumors; again no consistent trends in either direction were noted. An increase (or decrease) in growth activity does not accompany the transition from intraductal (in situ) disease to invasive mammary cancer, nor does a change in growth fraction necessarily accompany progression of mammary cancer from the primary to regional metastatic site.

Evidence that the c-erbB-2 proto-oncogene is important in prognosis and oncogenesis in a number of human malignancies is increasing. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized to determine the amplification and protein expression of this proto-oncogene, respectively. These extraction techniques are often time consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Immunohistochemistry (IHC), on the other hand, is more often time and cost effective. In addition, IHC may offer enhanced sensitivity over extraction techniques because of the in situ nature of analysis. In data presented here, 71 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilution DNA hybridization and IHC, respectively. In 65 (92%) of 71 cases, high-level expression was associated with gene amplification, whereas moderate or low-level expression was associated with a normal diploid gene copy number. In five of the six discrepant cases, IHC predicted amplification which was not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. In 39 of the 71 total cases, Western immunoblotting was compared with IHC in the assessment of oncoprotein expression. Concordance was found in 33 (85%) of 39 cases. In four of the six discrepant cases, high levels of c-erbB-2 expression were demonstrated by IHC but not by immunoblotting. In these cases, intraductal disease and stroma-rich tumors again led to a relative paucity of neoplastic tissue within the specimens. We conclude that IHC offers a favorable alternative to either Southern analysis or Western immunoblotting in the assessment of c-erbB-2 gene copy number and expression levels of oncoprotein in human mammary carcinoma. Furthermore, IHC may prove advantageous to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

Objective: To determine the utility of measuring the tumor proliferation index as a prognostic marker in patients with non—small-cell lung cancer. Design: Immunostaining for the proliferationassociated antigen Ki-67, quantitated using computerassisted image cytometry, was used to derive the tumor proliferation index for 61 fresh-frozen, banked specimens of non—small-cell lung cancer. DNA ploidy was measured concomitantly for all specimens. A median follow-up of 38 months was achieved for survival analyses. Setting: A large southeastern United States private referral institution and affiliated hospital provided the study environment. Participants: A consecutive, convenience sample of 61 patients was enrolled based on resected tissue preservation and viability over a five-year accruement. Main Outcome Measures: Significant associations between DNA content, proliferation index, established clinicopathological parameters, and outcome were examined. Results: A significant inverse association between patient survival and tumor proliferation index was found that was independent of other established clinicopathological predictors of outcome. Patients whose tumors harbored a proliferation index of less than 3.5 survived significantly longer than patients with tumors demonstrating higher values. No association between DNA content and proliferation index was uncovered. Conclusion: Measurement of the proliferation index, as derived from quantitative Ki-67 immunostaining and analyzed by image cytometry, may provide significant complementary, if not independent, prognostic information for patients with non—small-cell lung cancer. (Arch Surg. 1993;128:1382-1390)