Immunohistochemical labelling for prostate-specific antigen in breast carcinomas.

Immunohistochemical detection of prostate-specific antigen (PSA) is an aid in determining the prostatic origin of metastatic cells. However, small amounts of PSA have also been found in non-prostatic tissues and tumors, for example in some breast carcinomas, by highly sensitive immunofluorometric methods, but also by immunohistochemistry. Our aim was to evaluate the prevalence and prognostic value of histologically confirmed PSA immunoreactivity in breast carcinoma. Sections of formalin-fixed, paraffin-embedded samples from 171 breast carcinomas were immunostained for PSA. The staining results were compared with the mitotic activity, tumor size, histological grade, steroid receptors and follow-up data. For analysis the material was divided into subgroups according to the patients' age (pre- and postmenopausal). PSA was found by immunohistochemistry in 54 (32%) breast carcinomas. In survival analysis of the whole patient material PSA positivity did not show prognostic value. Among premenopausal patients concomitant estrogen receptor and PSA-negativity proved to be associated with high risk of breast cancer death (RR 6.2), also after adjustment for tumor size, histological grade, and axillary lymph node status. Among postmenopausal patients PSA positivity was associated with progesterone receptor positivity and high differentiation but not with age, nodal status, or mitotic activity. PSA can be detected by immunohistochemistry in a considerable number of breast carcinomas. PSA immunoreactivity alone does not seem to have any value as general prognosticator of breast carcinoma patients. However, concomitant absence of PSA and estrogen receptors was an indicator of unfavourable prognosis among premenopausal patients.

Phospholipase A2 in serum and colonic mucosa in ulcerative colitis.

Group II phospholipase A2 is involved in the pathogenesis of various inflammatory diseases and in the host defence against bacteria. The enzyme is expressed in the epithelial cells of colonic mucosa in ulcerative colitis. In this study, we measured the concentration of group II phospholipase A2 in serum and colonic mucosa of patients with ulcerative colitis of different severity and of control patients without any inflammatory disease. The activity of ulcerative colitis was assessed by endoscopy. The concentration of group II phospholipase A2 was measured with an immunoassay. The concentrations of group II phospholipase A2 in serum and colonic mucosa were significantly higher in patients with active and inactive ulcerative colitis than in controls. However, the group II phospholipase A2 levels did not separate patients with different disease activity. The concentration of group II phospholipase A2 in colonic mucosa corresponded with the mucosal inflammatory activity (higher in active colonic areas) intra-individually, but not between different patients with ulcerative colitis. Serum group II phospholipase A2 values were above the normal reference range more often than the values of 11 standard laboratory blood tests widely used for the follow-up of inflammatory activity in ulcerative colitis. These results indicate that the concentration of group II phospholipase A2 is increased in serum and colonic mucosa of patients with ulcerative colitis. The clinical value of the measurement of group II phospholipase A2 in the follow-up of ulcerative colitis remains to be clarified.

Group II phospholipase A2 in human male reproductive organs and genital tumors.

BACKGROUND: Group II phospholipase A2 (PLA2) is a lipolytic enzyme suggested to play a role in inflammation and antibacterial defence. In seminal fluid, the concentration of PLA2 is exceedingly high under normal circumstances (about 1,000 times the concentration in blood plasma of healthy humans). To elucidate the origin of the enzyme present in seminal plasma, we investigated the expression of group II PLA2 in male reproductive organs both at protein and mRNA levels. In addition, the presence of the enzyme was studied in common male genital tumors. METHODS: The methods used were immunocytochemistry, in situ hybridization, and Northern blotting. RESULTS: Northern blotting gave positive results for group II PLA2 mRNA in normal prostate, whereas other normal genital tissues gave negative results. Immunohistochemistry and in situ hybridization of group II PLA2 gave identical results. The enzyme was produced exclusively by the secretory epithelial cells of the prostatic gland. Surprisingly, expression was restricted to the posterior lobe and paraurethral glands of the prostate. Cells of prostatic adenocarcinoma expressed group II PLA2, whereas cells of other male genital tumors contained neither the enzyme protein nor the mRNA of group II PLA2. In some cases prostatic cancer cell seemed to express group II PLA2 at a higher rate than normal prostatic gland cells. CONCLUSIONS: The high content of group II PLA2 in seminal plasma is due to the local production and secretion of the enzyme by the epithelial cells of the prostatic glands. Group II PLA2 is expressed focally, suggesting that specialized prostatic glands secrete this enzyme. All prostatic adenocarcinomas tested expressed group II PLA2 in variable amounts.

Image cytometry of breast carcinomas that are DNA diploid by flow cytometry: time to revise the concept of DNA diploidy?

OBJECTIVE: To investigate whether breast carcinomas found to be DNA diploid by flow cytometry (FCM) are still diploid if reassessed by image cytometry (ICM). STUDY DESIGN: In a series of 286 breast cancers analyzed by FCM there were 100 (35%) cancers that were classified as DNA diploid. Fourteen of the 100 diploid cases were selected for further analysis with ICM because the patient had died of breast cancer within 11-84 months after the diagnosis (a group with unfavorable outcomes), and 19 cases were selected at random from the cases who had no recurrence of cancer during follow-up of six or more years (a favorable group). RESULTS: Eleven (33%) of the 33 cases turned out to be DNA nondiploid, with a DNA index > or = 1.2 when analyzed by ICM. Nine of the 11 DNA aneuploid samples by ICM were found among the 14 patients with unfavorable prognoses and only 2 among the 19 patients with favorable outcomes (P = .002). The five-year survival rate of the women with DNA diploid cancer by both methods was 86%, whereas that of patients with DNA aneuploid cancer by ICM was 36% (P = .002). CONCLUSION: The results show that some breast carcinomas classified as DNA diploid based on FCM are not DNA diploid by ICM and that such carcinomas are associated with poorer outcomes than the ones that are DNA diploid also by ICM. The prognostic significance of DNA ploidy in breast cancer may need to be reexamined in studies where both FCM and ICM are used.

Immunohistochemical labelling for prostate specific antigen in non-prostatic tissues.

Immunohistochemical detection of prostate specific antigen (PSA) in metastases of adenocarcinomas is widely used as an aid to identify the prostatic origin of metastatic cells. However, on the one hand, PSA may not be expressed in some poorly differentiated prostatic carcinomas, while on the other, PSA immunoreactivity has been found in small amounts in non-prostatic tissues. The aim of the current study was to evaluate the prevalence of PSA immunoreactivity in normal non-prostatic tissues and in breast carcinoma. PSA was localized by immunohistochemistry with four commercial antibodies in 34 different normal human tissues, and in 15 ductal and seven apocrine breast carcinomas. Concentrations of PSA in tissue homogenates of prostate and nine non-prostatic tissues from autopsied subjects were measured by a two-site immunoradiometric assay. Weak PSA immunoreactivity was found by immunohistochemistry in kidney, parotid gland and pancreatic tissues. Variable PSA immunoreactivity was seen in three cases of ductal (20%) and two cases of apocrine breast carcinoma (28%). No consistent PSA immunoreactivity was found in homogenates of non-prostatic tissues by the immunoradiometric assay. We conclude that PSA is a quite specific marker of prostatic tissue. However, there are some non-prostatic neoplastic and normal tissues that express PSA. Therefore, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of immunolabelling for PSA alone.

Bleaching of melanin before image cytometry of the DNA content of pigmented skin tumors.

OBJECTIVE: To study the effect of bleaching of melanin with KMnO4 on the results of DNA image cytometry in pigmented skin tumors. STUDY DESIGN: Image cytometry of nuclear DNA content was performed on sections from 14 melanocytic skin tumors stained with Feulgen stain both with and without prior bleaching with KMnO4. RESULTS: The nuclear staining intensity of Feulgen stain was lower in the bleached sections, but this did not significantly affect the evaluation of ploidy. Heavy pigmentation caused some false peaks in the histograms (4 of 28 measurements made on unbleached slides). CONCLUSION: Bleaching of sections with KMnO4 can be useful when heavy melanin pigmentation would make DNA measurements impossible or difficult in image analysis cytometry. Bleaching is not advisable when only lightly pigmented tumors are analyzed if nuclei obscured by any pigment granules are to be avoided. In large series containing both bleached and nonbleached specimens, statistical analysis of these groups should be separated.

Detection of malignancy in endometrium by brush sampling in 1042 symptomatic patients.

Endometrial brush samples were taken from 1042 symptomatic hospital patients using Uterobrush(R), and the results were compared either to the histology of the endometrium obtained with dilatation and curettage (n = 313) or the patients' follow-up (n = 729). Only one cancer (100%) among patients 51 years of age (n = 365), 11 (91.7%) were detected by Papanicolaou classes III-V. One cancer was missed, whereas no false positive results were found. The diagnostic accuracy in this group of patients varied from 92.3% to 97.8%, depending on the group (true positive vs true negative) to which the Papanicolaou class III was placed. We conclude that endometrial cytology obtained by brushing is useful for symptomatic patients of all age groups and gives an indication for further examination. If cytology is normal and bleeding continues in postmenopausal patients, curettage is indicated.

Cell proliferation in dimethylbenz(A)anthracene(DMBA)-induced rat mammary carcinoma treated with antiestrogen toremifene.

Cell proliferation during antiestrogen toremifene treatment was studied using the DMBA-induced rat mammary carcinoma model. The volume corrected mitotic index (M/V INDEX) and the S-phase fraction (SPF) determined by flow cytometry (FCM) were used as proliferation markers. Two series of rats (A and B) treated with two dose levels of toremifene were used. The two series of tumors appeared to have different growth properties. In series A the tumors were rapidly growing with high proliferation rate. In this series, toremifene (3 mg/kg for 4 weeks) reduced significantly the mean MV/INDEX, but the slight reduction of the mean SPF was not significant. In series B the tumors grew slowly and had low levels of proliferation markers. One-third of the tumors were spontaneously stable in the untreated group. Higher dose of toremifene was used in this series (12 mg/kg for 4 weeks), and the number of regressing or stable tumors was 58% compared with 31% in series A. Taking into consideration the high number of spontaneously stable tumors in series B, it may be concluded that about one-third of the tumors regressed or remained stable due to toremifene treatment in both series. The reduction of the M/V INDEX was significant only when the regressing treated tumors were compared with the growing controls. The reduction of the SPF was not significant. We think that the M/V INDEX is a more appropriate method to measure cell proliferation than is the SPF in this tumor model, where the tumors are heterogenous and, e.g., spontaneous apoptosis is known to be frequent.

Long-term survival after pancreatic adenocarcinoma--often a misdiagnosis?

Prognosis of adenocarcinoma of the pancreas has remained poor, but a few patients are reported to live 5 years or longer after the diagnosis. Using the data of the Finnish Cancer Registry, we could identify only 78 patients (1.3%) who had survived for longer than 5 years after the diagnosis of pancreatic cancer among 5,837 patients diagnosed in Finland in 1975-1984. However, in 33 of the 78 cases a histological diagnosis of pancreatic cancer had never been made, and the majority of the remaining 45 patients turned out not to have pancreatic adenocarcinoma after a review. The results suggest that the majority of patients with long-term survival following the diagnosis of pancreatic cancer have never had pancreatic adenocarcinoma. Taking a biopsy from a suspected pancreatic neoplasm and careful histological evaluation may prohibit misdiagnosis of this highly lethal disease.

DNA content analysis of fine needle aspirates obtained from lymphatic tissue.

DNA ploidy and S-phase fraction (SPF) of 69 lymphomas and 50 samples from benign lymphatic tissue were determined by flow cytometry from cells obtained by fine needle aspiration biopsy. An aneuploid histogram with a DNA index (DI) > 1.05 was obtained from 15 (22%) lymphomas. However, six (12%) benign lymph nodes also showed a bimodal DNA histogram, with the DI ranging from 1.06 to 1.15. The benign nature of these lymph nodes was confirmed by histology, follow-up of > 5 years or both. The median size of the SPF was 3.2%, 3.6%, 4.2%, 12.1% and 15.5% in benign lymph nodes, Hodgkin's disease and in low, intermediate and high grade non-Hodgkin's lymphomas, respectively. Only 8 (18%) of the benign tumors had an SPF > 5% as compared with 39 (62%) of the lymphomas (P < .0001). An SPF of > 12% was never obtained from benign tissue. The size of the SPF as determined from a fine needle aspirate correlates with lymphoma histology, and a bimodal DNA histogram with a small DI obtained from lymphatic tissue does not necessarily indicate the presence of lymphatic malignancy.

Flow and image cytometric study of pancreatic neuroendocrine tumours: frequent DNA aneuploidy and an association with the clinical outcome.

Eighteen pancreatic neuroendocrine (NE) tumours were analysed for nuclear DNA content by image cytometry (ICM) and flow cytometry (FCM). The DNA indices (DIs) obtained by ICM were somewhat higher than those obtained by FCM, but a major disagreement was present only in 1 case. Thirteen patients had been followed up at least for 6 years after the diagnosis or until death. At 6 years of follow-up all 4 patients with a tumour with a DI greater than or equal to 1.8 by ICM had died from their NE tumour or had metastatic disease, whereas all 9 patients with a smaller DI had no evidence of the disease (P = 0.001). The DIs calculated from the FCM data also correlated well with the final outcome (P = 0.01). A high incidence of DNA aneuploidy was found by both methods in histologically and clinically benign NE tumours; 12 (67%) were DNA aneuploid by FCM and 16 (89%) by ICM. It is concluded that pancreatic NE tumours are frequently DNA aneuploid, and both cytometric DNA methods give prognostic information in these tumours. The presence of DNA aneuploidy should not be considered as a sign of malignant behaviour in pancreatic NE tumours, whereas a large DI is associated with poor prognosis.

DNA ploidy in pancreatic neuroendocrine tumors.

The nuclear DNA content of 17 pancreatic neuroendocrine tumors was measured from paraffin-embedded tissue with flow cytometry. The tumors were classified by immunostaining with antisera for synaptophysin, insulin, gastrin, glucagon, pancreatic polypeptide, somatostatin, and vasoactive intestinal polypeptide. Eight (47%) of the 17 tumors were aneuploid, and two (12%) were multiploid (had two aneuploid stemlines of cells). Seven of the eight insulinomas, one of the four gastrinomas, and two of the four nonspecified neuroendocrine tumors had an abnormal nuclear DNA content. The DNA indices of the aneuploid and multiploid cases ranged from 1.13 to 1.93, and three cases had a DNA index greater than 1.50. During the follow-up for up to 16 years (mean, 7 years), one patient with diploid nonspecified tumor died of the disease, another patient with a multiploid gastrinoma had metastatic disease develop, and a third patient with a multiploid nonspecified tumor was alive with the disease. The authors conclude that many neuroendocrine tumors of the pancreas have an abnormal nuclear DNA content as measured by DNA flow cytometry. DNA multiploid pancreatic neuroendocrine tumors may be associated with a less favorable clinical course, but this needs to be confirmed in additional studies.

Clinical significance of nuclear DNA content in pancreatic carcinoma.

The nuclear DNA content of 62 pancreatic adenocarcinomas was analysed by flow cytometry from paraffin-embedded material. Radical surgery could be performed in 12 of the 24 cases with diploid carcinoma, but only in 3 of the 38 cases with a non-diploid tumour (P = 0.0002); the radically resected carcinomas also had a lower fraction of cells in the S-phase (P = 0.009). Non-diploid nuclear DNA content (38 cases, 61 per cent) was associated with advanced stage (P = 0.002), poor histological differentiation (grade II or III, P = 0.004), and primary tumour site in the body or the tail as compared with the head (P = 0.01). The median survival time of the patients with diploid carcinoma was 13 +/- 3 (SE) months, and that of the patients with non-diploid carcinoma 3 +/- 1 months (P = 0.0001). The DNA index with the cutoff value 1.4 was a slightly more powerful prognostic factor than DNA ploidy, and it was the most important independent prognostic factor in Cox's multivariate analysis (P less than 0.001) followed by histological grade (P less than 0.03). We conclude that diploid pancreatic carcinomas are associated with a longer survival than the non-diploid ones, and that radically operable carcinomas form a special subgroup with frequent diploidy and less aggressive biological behaviour.

Evidence for false aneuploid peaks in flow cytometric analysis of paraffin-embedded tissue.

It has recently been shown that bimodal histograms with false aneuploid peaks may be obtained by DNA flow cytometry from histologically normal tissue allowed to autolyze. To investigate if such peaks can be generated from surgically excised archival tissue, 198 paraffin blocks from 179 patients containing histologically normal spleen (n = 65), liver (n = 26), thyroid (n = 32), pancreas (n = 19), salivary gland (n = 49), or lymph node tissue (n = 7), obtained from the archives of two university pathology departments, were analyzed for nuclear DNA content. The great majority (n = 160, 83.8%) of the 191 interpretable histograms had a single symmetrical G1 peak; and 8 histograms, all produced from liver tissue had a tetraploid pattern. A slight or a prominent repeatable deviation in the G1 peak outline was present in 14 (7.3%) cases. A peak resembling an aneuploid G1 peak with a DNA index (DI) ranging from 1.14 to 1.38 was repeatedly produced from 9 (4.7%) blocks containing histologically normal or inflamed splenic (n = 3), pancreatic (n = 3), liver (n = 1), thyroid (n = 1), or lymph node (n = 1) tissue. The three abnormal peaks produced from pancreatic tissue were rounded in shape and resembled closely the ones that can be obtained from autolytic pancreatic tissue, and the six remaining extra peaks were all fused with the "diploid" peak. In conclusion, false peaks, probably caused by degradation of the nuclear contents during formalin fixation or before it, may rarely be obtained from surgical paraffin-embedded samples.

Autolysis is a potential source of false aneuploid peaks in flow cytometric DNA histograms.

Serial flow cytometric nuclear DNA content analyses were performed from normal human spleen, thyroid, liver, and pancreas removed from ten patients at autopsy and stored for up to 8 d without any preservative to study the effect of autolysis on DNA histograms. Fine needle aspiration biopsy (FNAB) samples were taken in diluted ethanol and tissue biopsies from the same area in formalin for embedding into paraffin at the time of autopsy and serially thereafter. Histograms obtained from samples taken within 10 h after death had a symmetrical G1 peak with a small coefficient of variance (CV) except histograms produced from paraffin-embedded pancreatic tissue, but bimodal distributions similar to those seen in aneuploid tumors were obtained from many samples stored longer than for 20 h. The DNA indices of the bimodal histograms were usually less than 1.3. The false peaks were more prominent in FNAB samples than in paraffin-embedded samples. The time of appearance of the false aneuploid peaks varied individually, and they were usually first seen in samples taken from the pancreas, followed by the liver, the thyroid and the spleen. Because neoplasms may become necrotic in vivo and fixation of fresh surgical samples may be slow and incomplete, increased DNA staining caused by autolysis may be a source of false aneuploid peaks in DNA content analysis.

Co-existence of two aneuploid stemlines in benign adenomas. A report of three cases with stemline heterogeneity.

The co-existence of 2 or more aneuploid stemlines (DNA multiploidy) has been described in malignant human neoplasms and such cancers have often been found to be associated with a poor prognosis. Here 3 benign human adenomas with 2 co-existing aneuploid stemlines are described. Despite DNA stemline heterogeneity and large DNA indices up to 2.8 none of the adenomas recurred or gave rise to metastases after a simple excision during the follow-up of 8, 10 and 11 years. Two adenomas were hormonally active. Marked cellular atypia and frequent mitoses were seen in 1 of the adenomas but the other 2 tumours had little atypia. The present cases indicate that DNA stemline heterogeneity may occur in benign adenomas, and not even the presence of 2 aneuploid stemlines with greatly increased nuclear DNA content can be regarded as a conclusive sign of malignancy.

Comparison of fresh, ethanol-preserved, and paraffin-embedded samples in DNA flow cytometry.

Fresh, ethanol-preserved, and formalin-fixed and paraffin-embedded samples taken from the same part of 15 human tumors, and from one normal spleen and one pancreas were analyzed for nuclear DNA content by flow cytometry. The coefficient of variation (CV) values of the G1 peaks were smaller in the fresh than in the other samples (P less than 0.001). The DNA ploidy of the tumors was the same in all types of samples. The DNA indices (DIs) measured from either ethanol-preserved or formalin-fixed tissue correlated strongly with those obtained from fresh tissue (P less than 0.001), although they tended to be somewhat smaller in the fresh samples. The S-phase fractions measured from all types of samples were of the same order of magnitude in most cases (P less than 0.001). Uninterpretable histograms were most often obtained from fresh samples. Identical DI values and rather constant S-phase fractions were obtained from ethanol-preserved samples stored at 4 degrees C for up to 5 months. It is concluded that all three types of samples are suitable for the determination of DNA ploidy, DI, and S-phase fraction and that 50% ethanol is suitable for long-term preservation of flow cytometric samples.

A simple preservative for flow cytometric DNA analysis.

A solution containing citric acid buffered saline (CABS) and 99% ethanol (E) 1:1 was used for preserving cells for flow cytometric DNA analysis. DNA histograms obtained from fine needle biopsy aspirates and preserved in CABS+E had a similar mean coefficient of variation (CV) as was obtained from aspirates taken in CABS (3.3 vs. 3.4%) and a clearly smaller mean CV than was obtained from aspirates preserved in 50% ethanol (mean 4.8%, P less than .0001). Aspirates taken in CABS more often contained a small (less than 3,000) number of cells as compared with aspirates preserved either in CABS+E or ethanol (P less than .0001). Since preservation of cells in CABS+E allows long-term storage of samples and results in a decreased number of insufficient samples as compared with buffered saline and in an enhanced resolution as compared with 50% ethanol, CABS+E is recommended for preservation of cytological samples to be analyzed for DNA content with flow cytometry.