Immunohistochemical expression of CA 125 in endometrial adenocarcinoma: correlation of antigen expression with metastatic potential.

Immunohistochemical localization of CA 125 using murine monoclonal antibody OC 125 was performed on fresh frozen tissue from 44 endometrial adenocarcinomas and 26 benign endometria. Immunohistochemical evaluation incorporated both intensity and distribution of staining (CA 125 HSCORE). Thirty-seven cancers (84%) and 23 benign endometria (88%) expressed immunohistochemically detectable CA 125. Staining was confined to epithelial cells and was present both on the cell membrane and in the cytoplasm. Among the 44 endometrial cancers, CA 125 HSCORE did not correlate with histological grade, depth of myometrial invasion or estrogen/progesterone receptor levels. Following surgical staging, 13 patients (30%) were found to have extrauterine metastatic disease. The median CA 125 HSCORE of patients with metastatic disease (2.25) was significantly higher than that of patients with disease confined to the uterus (0.6) (P less than 0.001). In addition, high CA 125 HSCORE also correlated with the presence of lymph node metastasis (P less than 0.001). The results of this study suggest that high CA 125 expression by endometrial adenocarcinomas is associated with increased metastatic potential.

DNA image cytometry of keratoacanthoma and squamous cell carcinoma.

The distinction between the keratoacanthoma (KA) and the squamous cell carcinoma (SCC) can sometimes be difficult on the basis of histologic and clinical criteria. The possible diagnostic significance of DNA ploidy initiated the present study evaluating the DNA ploidy in paraffin-embedded tissue sections of 7 KA and 15 SCC, and fresh frozen tissue touch preparations of 15 of the same cases using the CAS 200 Image Analyzer. In paraffin-embedded tissue sections the main peak DNA index was based on normal epidermis, and ranged from 1.03 to 1.59 in KA and from 1.47-2.71 in SCC. The DNA Index (DI) discriminated KA from SCC in 17 of 22 cases (p less than 0.0007). The highest DNA content of single nuclei ranged from 9.0-18.0 picograms (pg) (DI 2.9-6.03) in KA and 14.8-38.6 pg (DI 4.0-11.03) in SCC. The highest DNA content discriminated KA from SCC in 16 of 22 cases (p less than 0.003). In fresh frozen tissue touch preparations from 15 of the same lesions, there was considerable overlap in DNA indices of KA (0.534-1.39) and SCC (0.464-1.41). Abnormal DNA peaks seen in histograms from three SCC in paraffin-embedded tissue sections were lost in the touch preparation histograms, probably due to inadequate sampling. Therefore, image analysis of paraffin-embedded tissue sections is better able to distinguish KA from SCC than touch preparations.

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.

DNA ploidy and proliferation index of soft tissue sarcomas determined by image cytometry of fresh frozen tissue.

The DNA content of Feulgen-stained monolayer imprint preparations from 30 fresh frozen soft tissue and bone sarcomas was analyzed using image cytometry. DNA aneuploidy showed a significant association with increasing tumor grade. Of the grade III tumors, 83.3% (15 of 18) were aneuploid; of the grade I and II sarcomas, 33.3% (4 of 12) were aneuploid (P less than 0.00861). The proliferation index (PI) was determined by the percentage of tumor nuclear area staining with the monoclonal antibody Ki-67. The PI was significantly associated with ploidy and tumor grade. Ki-67 PI was elevated (greater than 2.0%) in 73.7% (14 of 19) of the aneuploid tumors, compared with 30% (3 of 10) of the euploid tumors (P less than 0.04597). Ki-67 PI was greater than 2.0% in 77.8% (14 of 18) of the grade III tumors, compared with 27.2% (3 of 11) of the grade I and II tumors (P less than 0.01773). These findings suggest that DNA ploidy and Ki-67 PI as determined by image analysis may be a useful supplement to tumor grading. The literature examining previous studies of sarcoma DNA ploidy is reviewed.

Determination of proliferation index in advanced ovarian cancer using quantitative image analysis.

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.

Necrotizing sialometaplasia: a sheep in wolf's clothing. Healing as a diagnostic test.

Necrotizing sialometaplasia is a benign self-limited intraoral lesion that is easily confused both clinically and histologically with squamous cell carcinoma. It presents as a painless ulceration, frequently on the hard palate, that histologically shows necrosis, inflammation, squamous metaplasia, and granulation tissue. It is thought to be due to infarction of minor salivary glands and heals spontaneously in six to 12 weeks. A brief period of observation for evidence of healing can be an important diagnostic clue in distinguishing this entity from cancer, thus saving the patient unnecessary surgery or radiation therapy.

Congenital giant axonal neuropathy.

Giant axonal neuropathy (GAN) is a distal sensorimotor neuropathy, characterized by neurofilamentous axonal swellings, with usual onset at 2 to 3 years of age. We report a case of congenital GAN with hypotonia at birth. At 7 months of age, nerve conduction studies showed almost complete lack of sensory and motor responses in the lower extremities. A sural nerve biopsy specimen disclosed absence of myelinated axons. Autopsy, following death at 15 months of age, revealed axonal swellings in peripheral nerves and distal degeneration of long spinal cord tracts. The neurofilamentous content of the axonal swellings was confirmed by Glees-Marsland staining and immunoperoxidase reaction with antibodies to neurofilaments. Axonal swellings did not stain with periodic acid-Schiff and were not seen in the cerebral cortex or brain stem, distinguishing this process from infantile neuroaxonal dystrophy. This patient illustrates congenital GAN with subsequent rapid progression.

Comparison of the effect of ischaemia and anoxia on the sarcolemma of the dog heart.

Contraction-band necrosis, a striking morphologic lesion, is common to many types of myocardial injury including the calcium paradox and ischaemic injury with reperfusion. This lesion is characterized by explosive swelling, massive calcium overload, and severe disruption of the myofibrils due to the formation of contraction bands. The studies reviewed in this paper provide evidence that in ischaemia and reperfusion, these changes are preceded by sarcolemmal injury that occurs during the period of ischaemia. Sarcolemmal injury was evaluated by electron microscopy and by measurements of inulin diffusible space (IDS) in thin slices of myocardium incubated in vitro. Reversibly injured ischaemic myocytes have ultrastructurally intact plasma membranes which are impermeable to inulin. Longer durations of ischaemia, sufficient to produce contraction-band necrosis during reperfusion, result in fragmentation of plasma membranes during the ischaemic intervals, and the IDS is markedly increased during subsequent incubation. Thus ultrastructural evidence of membrane damage is present early in ischaemia and is associated temporally with the increased IDS. The role of anoxia, per se, in inducing membrane damage was investigated in tissue slices incubated at 37 degrees C in crystalloidal media gassed with nitrogen. Anoxic slices produced lactate and lost ATP and adenine nucleotides, but cell volume and the IDS were not significantly increased for at least five hours (twice the time required for severe membrane damage to develop in total ischaemia) and the plasmalemma remained intact by electron microscopy. Thus, despite depletion of high energy phosphates, membrane damage, detectable by alterations in IDS or ultrastructure, occurs much more slowly during anoxia alone than during ischaemia. These results suggest that anoxia, per se, may not be the cause of membrane damage in ischaemia.

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 tissue 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 metastases, 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 flow cytometry studies. In 9 patients with primary tumors with a Breslow 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).

Heterogeneity of antigen expression in benign and malignant breast and ovarian epithelial cells.

Expression of II distinct antigen families has been studied in normal and malignant epithelial cells from breast and ovary by avidin-biotin immunoperoxidase staining of frozen tissue sections. Each of the antigens was expressed in a fraction of the normal and malignant breast tissues from different individuals. Among breast and ovarian cancers, a similar fraction of tumors expressed each of the determinants. An epitope on a 42-kDa glycoprotein was expressed significantly more often by ovarian carcinomas than by benign ovarian epithelium. Several determinants on 66-, 240-kDa or high-molecular-weight proteins or glycoproteins were expressed significantly more often on benign breast epithelium than on normal ovarian epithelium. Substantial heterogeneity in antigen expression was observed among different tumor specimens. Each of 14 epithelial ovarian cancers expressed a distinctive combination of antigens. Among 18 breast cancers, 16 distinct antigenic phenotypes were observed. Interestingly, comparable heterogeneity was observed in the expression of epitopes on benign breast and ovarian epithelium, compatible with the possibility that the monoclonal reagents recognized polymorphic determinants. Substantial variations in antigen expression were also observed among cells within each neoplasm. However, when antibodies against 5 different determinants were used in combination, a majority of cells could be stained intensely in all 13 breast carcinomas tested and more than 95% of tumor cells could be stained in 10 of the 13.

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.

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.

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.