Loss of cyclin-dependent kinase inhibitor p27Kip1 is a novel prognostic factor in localized human prostate adenocarcinoma.

p27Kip1 is a cyclin-dependent kinase inhibitor that negatively regulates cell proliferation by mediating cell cycle arrest in G1. This study was undertaken to assess the prognostic value of p27Kip1 in localized human prostate cancer. Archival material from 113 radical prostatectomy specimens obtained between 1985 and 1993 was stained immunohistochemically for p27Kip1 protein using a commercially available antibody. Patient charts were reviewed for preoperative serum prostate-specific antigen, clinical and pathological staging, Gleason tumor grade, time to biochemical and clinical recurrence, and survival. Strong p27Kip1 staining was uniformly seen in benign prostatic epithelial components in all tumor sections. p27Kip1 staining was reduced in most prostate cancers and was variable in prostatic intraepithelial neoplasia. Decreased p27Kip1 staining (<25% of nuclei stained positive for p27Kip1) correlated with seminal vesicle involvement (P = 0.0032) and with higher Gleason grade (P = 0.0114). On univariate analysis, low p27Kip1 predicted an increased risk of treatment failure in the node-negative cohort (P = 0.0037) and in the subset who did not receive neoadjuvant hormonal therapy (P = 0.049). Low p27Kip1 expression was an independent predictor of treatment failure on multivariate analysis of lymph node negative prostate cancers following radical retropubic prostatectomy (n = 102; P = 0.047). Seminal vesicle involvement (P = 0.034) and positive surgical margins (P = 0.047) were also independent prognostic factors for disease recurrence. In patients who received preoperative neoadjuvant hormonal therapy, low p27Kip1 in the pathological specimen was an even stronger predictor of outcome than it was in the entire group (n = 23, P = 0.015).

Proliferative activity and p53 expression in adenoid cystic carcinoma of the breast.

Adenoid cystic carcinoma is a rare type of invasive breast carcinoma that has a good prognosis. We studied a series of four cases of adenoid cystic carcinoma in which we correlated the clinical and pathological features. The pathological features examined included light microscopy; electron microscopy; immunohistochemistry using antibodies to keratin, vimentin, S100 protein, actin, estrogen and progesterone receptors, and proliferation marker MiB-1, and p53 suppressor protein; image cytometric analysis for measurement of DNA ploidy; and molecular analysis using polymerase chain reaction single strand conformation polymorphism to assess point mutation of the p53 gene. All of the cases had a low nuclear grade, were negative for estrogen and progesterone receptors, and were DNA diploid. Three of the cases showed no evidence of metastases and had small primary tumors with low proliferative activity and absence of p53 protein expression. In contrast, one of the cases showed axillary lymph node metastases and in this case the primary tumor was large with a higher proliferative activity and expression of p53 protein, suggesting that these factors might play a role in the biological behavior of adenoid cystic carcinoma.

DNA ploidy of hydatidiform moles and nonmolar conceptuses: a study using flow and tissue section image cytometry.

This study was performed to investigate the role of DNA cytometry in the evaluation of molar and nonmolar pregnancies. DNA ploidy analysis was performed on paraffin-embedded tissue from 53 molar (35 complete, 18 partial) and 24 nonmolar (13 hydropic, 11 nonhydropic) conceptuses. Nuclear suspensions were analyzed by both flow and image cytometry and there was excellent correlation (96%) in the classification of DNA diploid, triploid, and tetraploid cases using these two methods. DNA ploidy analysis revealed a high proportion of tetraploid nonmolar conceptuses (42%) and complete moles (47%). The majority of partial moles were triploid (89%). Tissue sections from all cases were also studied by image cytometry to identify the cellular subpopulations (decidua, villous stromal cells, inner trophoblast, and extravillous trophoblast) with abnormal DNA content. In the triploid cases, all of the villous cell subtypes had an abnormal DNA content consistent with the development of partial moles from a triploid conceptus. In contrast, the majority of tetraploid cases showed high proliferative activity of the extravillous trophoblast whereas the other villous cell subtypes were diploid. These results suggest that tetraploid complete moles may arise from a diploid conceptus with the development of tetraploidy related to polyploidization of the hyperplastic extravillous trophoblast. Tissue section image cytometric DNA analysis can aid in our interpretation of ploidy results and our understanding of the biology of molar pregnancies.

DNA image cytometry of prostatic carcinoma: a comparison of needle core biopsy and subsequent prostatectomy specimens.

DNA ploidy has recently been identified as an objective prognostic factor in prostatic carcinoma. Although the diagnosis of prostatic carcinoma is increasingly being made with the use of needle core biopsies, the optimal method for the cytometric analysis of these specimens has yet to be determined. In addition, the degree to which the biopsy is representative of the subsequent prostatectomy specimen with respect to DNA heterogeneity has not been adequately addressed. In this study, image cytometric (ICM) DNA analysis was performed on tissue sections from 12 prostatic needle core biopsies and the results were compared with similar ICM analysis of the subsequent prostatectomy specimens. Multiple blocks (n = 48) of the prostatectomy specimens were utilized to prepare tissue sections and nuclear suspensions and each set of preparations were analyzed by ICM in a parallel comparison study. There was concordance of 0.80 in the classification of DNA diploid and aneuploid tumors by ICM analysis of tissue sections and nuclear suspensions from paraffin blocks. In all of the discordant cases, DNA aneuploid populations were identified by ICM analysis of tissue sections only. This is attributed to difficulties in obtaining a representative nuclear suspension from disaggregated paraffin-embedded prostatic tissue which often has a very desmoplastic stroma. ICM analysis of tissue sections seems to be an optimal method for DNA ploidy analysis of prostatic carcinoma and is well suited to small volume biopsy material. Determination of DNA ploidy status in prostatic biopsies was predictive of the subsequent prostatectomy specimens with a concordance of 0.92.

A comparative study of DNA quantitation in breast carcinoma with image cytometric analysis and in vitro fine-needle aspiration with flow cytometric analysis.

The DNA ploidy status of 53 fresh primary breast carcinomas was analyzed in a comparative study of flow cytometric (FCM) and image cytometric (ICM) analyses. Samples for FCM analysis were obtained with an in vitro fine-needle aspiration technique. Touch imprints from the same tumors were analyzed by three independent observers by ICM analysis. An ICM comparative study of "sequential" and "visually selected" nuclei also was performed. There was an overall concordance of 0.85 in the classification of diploid and nondiploid tumors by FCM and ICM analyses. In most discordant cases, a nondiploid population was identified by FCM analysis alone. This is attributed to the superior resolution of and sampling/preparatory method used for FCM analysis. There was an overall concordance of 0.91 in the classification of diploid and nondiploid tumors by ICM analysis. Selective analysis of atypical nuclei resulted in increased sensitivity in the detection of DNA aneuploid populations by ICM analysis.

Detection of false DNA aneuploidy and false DNA multiploidy in flow cytometric DNA analysis.

Although false DNA aneuploid peaks have previously been described in normal tissue, criteria for distinguishing them from 'true' near-diploid peaks have not been established. Normal thyroid (n = 4) and kidney (n = 1) tissue were allowed to autolyze over a fixed period of time and DNA content was analyzed by flow cytometry (FCM). Autolysis was associated with the development of distinct separate G0/G1 peaks which had low DNA indices (1.09-1.18) and showed decreased forward light scatter (FSC) when compared to fresh tissue. Using DNA content and FSC measurements similar false DNA aneuploid peaks were identified in 29/94 surgical specimens. These cases included both benign and malignant lesions from thyroid (n = 63) with the remaining 31 neoplastic cases being from breast (16), lymphoma (8), sarcoma (4), lung (2) and uterine (1) tissue. In addition, false DNA multiploidy was identified. None of these cases showed histological evidence of necrosis. In a parallel comparison study using image cytometry (ICM) on the thyroid nodules, the presence of false DNA aneuploidy was supported. Investigators should routinely employ quality control criteria to identify possible cases of false DNA aneuploidy when measuring DNA content using FCM.

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.

The use of sequential fine-needle aspiration biopsy with flow cytometry to monitor radiation induced changes in breast carcinoma.

Thirteen patients with locally advanced or recurrent breast carcinoma were monitored during radiation therapy by multiple, sequential, fine-needle aspiration biopsies (FNAB) with flow cytometry. The material was analyzed for qualitative cytomorphological evidence of radiation effect and for DNA content and cell cycle alterations. DNA ploidy was not affected by the radiation therapy, although the aneuploid tumors showed an increased frequency of cell cycle alterations. The most common change seen was an increase in S-G2M (58%). Other changes included a decrease in the proliferative/growth fraction (17%) and no significant redistribution of cells (25%). There was a relationship between the initial proliferative activity of the tumors and the type of cell cycle change which occurred. Flow cytometric analysis was a better predictor of early clinical response than was cytomorphological assessment. Sequential FNAB with flow cytometry is an effective method of monitoring the response of breast cancer to radiation therapy.

The role of DNA flow and image cytometry in the evaluation of body cavity fluids.

In addition to conventional cytomorphologic study, 50 body cavity fluid specimens (benign and malignant) were analyzed by both flow cytometry (FCM) and image cytometry (ICM) in order to evaluate the potential application of these techniques in the diagnosis of malignancy. While 88% of the fluids were similarly classified by FCM and ICM as being either diploid (66%) or nondiploid (22%), with similar DNA index values, nondiploid peaks were identified by ICM alone in 12% of the fluids. Aneuploid populations were present in 92% of the cytologically positive fluids and in 15% of the cytologically negative fluids. All of the latter fluids came from patients who clinically had tumors involving the respective body cavities. These results show that (1) both FCM and ICM are useful adjuvant techniques in the evaluation of body cavity fluid specimens and (2) ICM is superior to FCM in the identification of small aneuploid subpopulations. The identification of aneuploid populations by either method is highly suggestive of malignancy.

Incidental glomus coccygeum. When a normal structure looks like a tumor.

The glomus coccygeum is located at the tip of the coccyx; it measures several millimeters in diameter. We describe two glomera coccygea incidentally discovered in pilonidal sinus excision specimens. Review of several reports of coccygeal glomus tumors indicates that most of them probably represent normal glomera coccygea misdiagnosed as tumors. Pathologists should be aware of this normal structure.

Fine needle aspiration of schwannomas. Value of electron microscopy and immunocytochemistry in the preoperative diagnosis.

In the absence of Verocay bodies, the cytologic appearance of schwannomas can be quite nonspecific, with the differential diagnosis including a number of spindle cell lesions. The diagnostic accuracy can be enhanced by the use of electron microscopy (EM) and immunocytochemistry; both techniques are easily applicable to aspirated material. Six cases of schwannoma, diagnosed as such preoperatively by the application of these techniques, are reported. Only two of the cases showed Verocay bodies on light microscopy. Electron microscopy, performed on four cases, revealed a complex arrangement of long cell processes, with scattered desmosomelike structures, a prominent focally reduplicated basal lamina and long-spacing collagen. All of the cases showed positive staining for S-100 protein.

Epithelial proliferation in thymic hyperplasia. An immunohistochemical study and correlation with the developing fetal thymus.

Thymic hyperplasia is a B-cell lymphoid proliferation in which an epithelial component has not, to our knowledge, been previously described. We present a case of thymic hyperplasia in which numerous lymphoid follicles with germinal centers were partially surrounded by small sheets of spindle and epithelioid cells. Electron microscopy confirmed the epithelial nature of these cells. Immunostaining was performed using antibodies to keratins, S100 protein, and two B-cell markers, LN1 and MB2. The proliferated epithelium stained only for high-molecular-weight keratin, whereas the lymphoid tissue stained positively for both B-cell markers. To determine the origin of the proliferated epithelium, the staining was compared with that of the developing fetal and normal adult thymus. We have shown that during fetal development, the keratin composition of thymic epithelium changes from staining predominantly with low- to high-molecular-weight keratin. The immunostaining characteristics of the epithelium in this case of thymic hyperplasia suggest an origin from adult-type epithelium. Furthermore, the association of S100-positive interdigitating reticulum cells with the proliferated epithelium suggests that it is of medullary origin. Our results indicate that epithelial proliferation can be an important component of thymic hyperplasia.