Robert B. Kinney

Overexpression of HER-2/neu in endometrial cancer is associated with advanced stage disease.

Prior studies have shown that overexpression of HER-2/ neu occurs in one third of breast and ovarian cancers and that overexpression is associated with poor prognosis. We used a monoclonal antibody to assess immunohistochemically the level of HER-2/ neu expression in normal and malignant endometrium. In 24 normal endometrial samples light to moderate (1+ to 2+) staining for HER-2/ neu was seen in the glands, and there was no variation in intensity of staining during the menstrual cycle. Among 95 endometrial adenocarcinomas, nine (9%) were found to have heavier staining for HER-2/ neu than was seen in normal endometrium (3+). High expression of HER-2/ neu was found in 27% of patients with metastatic disease compared with 4% of patients with disease confined to the uterus (p neu expression also was associated with absence of estrogen receptor (p neu overexpression in endometrial cancer.

Differential Immunoreactivity of Epidermal Growth Factor Receptor in Benign, Dysplastic and Malignant Prostatic Tissues

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.

c-erbB-2 Expression in Breast Cancer Detected by Immunoblotting and Immunohistochemistry

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.

Ploidy analysis of epithelial ovarian cancers using image cytometry

We used a computerized image analysis system to determine the DNA content of 103 epithelial ovarian cancers using touch imprints of frozen tumor samples. Similar to prior studies of ploidy using flow cytometry, we found that most ovarian cancers (78%) were aneuploid while a minority (22%) were diploid. There was no relationship between ploidy and stage, histologic grade, or the ability to perform optimal cytoreductive surgery. Also, like prior studies using flow cytometry, negative second-look laparotomy and survival were somewhat more common in advanced-stage patients with diploid cancers than in those with aneuploid cancers. We conclude that ploidy of ovarian cancers can be determined using a computerized image analysis system to quantitate feulgen staining of cells in touch imprints. Ploidy is unlikely to play a role in treatment planning for patients with advanced-stage disease. Larger studies of patients with early-stage disease are needed, however, to determine whether ploidy is a more accurate means of predicting which patients are most likely to benefit from adjuvant therapy.

DNA ploidy of malignant melanoma determined by image cytometry of fresh frozen and paraffin-embedded tissue

Image analysis of DNA content was performed from single nuclei of melanoma monolayer imprints made from fresh frozen (issue of 14 patients with primary malignant melanoma and 16 patients with local recurrences at the incision site and local or distant metastases. This procedure requires fewer cells and is an advantage when the quantity of tumor available is limited, especially in thin low Breslow depth cutaneous melanomas. Image analysis allowed reproducible measurement of DNA ploidy from 100 cells. The frequency of aneuploidy was similar in primary and metastatic melanomas. Three of 3 patients with euploid primary melanomas showed no evidence of recurrences or meta‐stases, though one died of unrelated disease with short follow‐up. The 4 patients with primary melanoma who developed metastases had aneuploid primaries; two of these patients died of metastatic disease. Three of 4 patients with euploid metastatic tumors were free of disease at last follow‐up, and 1 patient died with stable disease. Nine of 12 patients with aneuploid tumors died of metastatic disease. The frequency of DNA ploidy in the present image analysis study correlated with previous (low cytometry studies. In 9 patients with primary tumors with a Bin‐slow depth greater than 0.75 mm, the. DNA content was also determined in nuclei obtained from formalin‐fixed paraffin‐embedded tissue. The frequency of aneuploidy was higher in fresh tissue (7 of 9) as compared with paraffin‐embedded tissue of the same cases (4 of 9).

Detection of a Novel Marker in the Bronchial Secretions of Patients with Non-Small Cell Lung Cancer using the 4B5 Monoclonal Antibody

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.

Overexpression of HER-2/neu in endometrial cancer is associated with advanced stage disease

Prior studies have shown that overexpression of HER-2/neu occurs in one third of breast and ovarian cancers and that overexpression is associated with poor prognosis. We used a monoclonal antibody to assess immunohistochemically the level of HER-2/neu expression in normal and malignant endometrium. In 24 normal endometrial samples light to moderate (1+ to 2+) staining for HER-2/neu was seen in the glands, and there was no variation in intensity of staining during the menstrual cycle. Among 95 endometrial adenocarcinomas, nine (9%) were found to have heavier staining for HER-2/neu than was seen in normal endometrium (3+). High expression of HER-2/neu was found in 27% of patients with metastatic disease compared with 4% of patients with disease confined to the uterus (p less than 0.005). High HER-2/neu expression also was associated with absence of estrogen receptor (p less than 0.005) and with increased mortality from cancer. Further studies are needed to determine the significance of HER-2/neu overexpression in endometrial cancer.