The prognostic value of PTEN, p53, and beta-catenin in endometrial carcinoma: a prospective immunocytochemical study.

Mutations of the PTEN, p53, and beta-catenin genes are the most frequent molecular defects in endometrial carcinomas. The aim of this study was to investigate their prognostic significance in this form of cancer. Imprint smears were obtained from 80 fresh endometrial tumor specimens and studied immunocytochemically for the expression of PTEN, p53, and beta-catenin proteins. The staining pattern was correlated with several well-established prognostic parameters, including 5-year survival. Positive staining of p53 was significantly correlated with increased stage (P < 0.0001), lymph node metastases (P = 0.001), and a nonendometrioid histology (P = 0.001). On the contrary, positive beta-catenin expression was significantly associated with decreased stage (P = 0.002), decreased grade (P = 0.007), and a negative lymph node status (P = 0.023). PTEN positivity was correlated with decreased stage (P = 0.002) and negative lymph nodes (P = 0.008). All the three markers affected survival significantly in univariate analysis but only beta-catenin had an independent prognostic impact. An independent prognostic significance was also shown for PTEN in the stage I subgroup of patients. The results of our study indicate that loss of beta-catenin expression is a strong and independent predictor of an unfavorable outcome in patients with endometrial carcinoma. Loss of PTEN may also be associated with a worse prognosis in patients with early-stage disease.

Prognostic value of DNA analysis of prostate adenocarcinoma: correlation to clinicopathologic predictors.

The ability to accurately predict tumor behavior and patient survival is a problem in managing patients with prostate cancer. DNA ploidy provides important information for the evaluation of the prognosis of prostate cancer. The aim of this study was to investigate the DNA ploidy in imprints from prostate adenocarcinomas in a group of 70 patients in relation to Gleason score, tumor differentiation, stage and PSA serum levels. The DNA content was studied in Feulgen-stained imprint smears through the image analysis technique using a SAMBA 2005 Image analyzer. According to our measurements, a strong correlation was observed between DNA ploidy status and tumor differentiation (p<0.001). A statistically significant difference was found between DNA aneuploidy and increased pretreatment PSA serum levels (>4 ng/ml) (p<0.001), as well as between ploidy pattern and stage of the disease (p<0.001). Our results conclude that DNA ploidy status appears to be an additional marker in the field of prognosis of prostatic adenocarcinoma and could provide useful information on the potential behavior of prostate cancer.

Expression of p120, Ki-67 and PCNA as proliferation biomarkers in imprint smears of prostate carcinoma and their prognostic value.

The cell proliferation markers p120, Ki-67 and proliferating cell nuclear antigen (PCNA) recognize nuclear antigens. The expression of these proteins by immunostaining methods was reported to be of value in determining the prognosis of patients with malignant diseases. In this study, we evaluated the prognostic significance of the expression of nuclear antigens p120, PCNA and Ki-67 in prostate cancer and compared the results with other prognostic factors. Imprint smear samples obtained from 70 patients immediately after radical prostatectomy for prostatic carcinoma were immunostained with monoclonal antibodies against p120, Ki-67 and PCNA. The immunostaining results were correlated with Gleason score, tumour differentiation, stage and prostatic specific antigen (PSA) levels. Our findings demonstrate that p120, Ki-67 and PCNA expression in prostatic carcinoma smears, correlated significantly with the degree of Gleason score (P < 0.001). When combining p120, Ki-67 and PCNA positivity with tumour differentiation there was a significant association among these parameters (P < 0.001). Overexpression of p120, Ki-67 and PCNA, was also associated with increased PSA serum levels (>4 ng/ml) (P < 0.001). The distribution of p120, Ki-67 and PCNA expression in prostate carcinomas was not statistically significant for Ki-67 (P = 0.69) and p120 (P = 0.22) but was significant for PCNA (P < 0.001) as far as the histological stage (T2a, T2b, T2c, T3a). P120, Ki-67 and PCNA expression had significant prognostic value for disease-free survival. Our results conclude that nuclear antigens p120, Ki-67 and PCNA appear to be additional markers in the field of prognosis of prostatic carcinoma.

Diagnostic approach of effusion cytology using computerized image analysis.

The objective of this study is to investigate whether image cytometry is a sensitive and specific method for the differential diagnosis of equivocal cells in routine cytology of effusion smears. One hundred four effusion smears were studied from routine cytologic material. Cytologically 56 (53.8%) of the smears were classified as malignant, 26 (24%) as suspicious and 22 (21.1%) as benign. Two morphometric variables (nuclear major axis length and nuclear area) of the nuclei were measured by an image analysis system. Higher values for the area were found for malignant rather than benign and suspicious cells (p < 0.0005 and p < 0.005 respectively). The same result was extracted for the nuclear major axis length values (p < 0.0005 and p < 0.0005 respectively). Values of nuclear major axis length and nuclear area didn't differ significantly between benign and suspicious cells (p = 0.071 and p = 0.066 respectively). The results show that the range of the values for suspicious cells is closer to the range of the benign cells. Cytomorphometry of the effusion smear cells may provide important information for the differentiation of atypical mesothelial cells from malignant adenocarcinoma cells.

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas.

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant therapy.

Correlations between nuclear/cytoplasmic area ratio and classification of cervical smears.

The aim of this study was to assess the nuclear/cytoplasmic (N/C) ratio using computerized image analysis of cervical smears with intraepithelial neoplasia (CIN) grade I to III associated or not with cellular changes of human papillomavirus (HPV) in an attempt to determine if this method is more sensitive for the estimation of the grade of CIN. One hundred and ten cervical smears from women with a mean age 35.03 years were studied. The cytological diagnosis was as follows: CIN I + HPV (11), CIN II + HPV (11), CIN II + HPV (8), CIN I (7), CIN II (6), CIN III (8), Ca (22), HPV (32), CIN I-II + HPV (2) and CIN II-III + HPV (3). All cases were histologically examined: 93 cases were in agreement and 17 were under- or overestimated cytologically. The morphometric study of cervical smears was carried out by image analysis. Data were analysed by one way analysis of variance followed by Bonferroni test of multiple comparisons. Statistically significant differences were detected between the three grades of CIN or CIN HPV or only HPV (p<0.0001). The results demonstrated that the N/C ratio measured by image analysis on precancerous lesions of cervical smears could be considered as an additional tool for the classification of cervical smears, especially in determining the discrepancies between cytological and histological diagnoses.

Non-cryopreserved peripheral blood progenitor cells collected by a single very large-volume leukapheresis: a simplified and effective procedure for support of high-dose chemotherapy.

High-dose chemotherapy with autologous peripheral blood progenitor cell (PBPC) support has become a widely used treatment strategy. In order to simplify the procedure, a single very large-volume leukapheresis programme combined with short-term refrigerated storage of the PBPC was developed. Seventy-two patients suffering from various relatively chemosensitive malignancies received high-dose chemotherapy, consisting of agents with short in vivo half-lives and 24 to 48 hours later, the refrigerated PBPC were reinfused. A single very large-volume apheresis was sufficient to obtain at least 2 x 10(6)/kg CD34+ cells in 58 patients (81%), and 63% had at least 2.5 x 10(6) CD34+ cells/kg. Only two patients (3%) were transplanted with less than 1 x 10(6) CD34+ cells/kg. In three patients (4%) leukapheresis was repeated because of insufficient number of PBPC. The median CD34+ cell count was 3 x 10(6)/kg. A median of 38.5 L blood (range, 21 to 59) was processed, which accounted for a median of 9 x patient's total blood volume. Very large-volume leukapharesis was well tolerated with symptomatic hypocalcemia being the most common (18%) side-effect. The median time to neutrophils >1.5 x 10(9)/L, and to self-supporting platelet count >25 x 10(9)/L, was 10 and 12 days after reinfusion of PBPC graft, respectively. There were no treatment-related deaths. Our results indicate that this simplified approach of PBPC transplantation can be associated with prompt hematologic recovery in most patients and that it can be useful in settings where facilities are limited or for certain diseases where conditioning regimens with short half-life are appropriate. J. Clin. Apheresis, 15:236-241, 2000.

p53 protein expression and proliferative activity in ductal breast carcinomas.

The immunocytochemical expression of p53 protein and Ki-67 labelling index in tumour cells of 100 ductal breast carcinomas of different histological grade and stage was evaluated in cytological material. In order to investigate p53 expression and Ki-67 expression an avidin-extravidin immunocytochemical technique was applied to imprints. Monoclonal antibody (MoAb) DO-p53 and proliferating cell monoclonal antibody were used as primary antibodies. A statistically significant difference was observed between p53 protein expression and grade of malignancy and clinical stage (P = 0.001, P < 0.001, respectively). A statistically significant difference was also observed between Ki-67 LI and histological grade and stage of the tumours (P < 0.001, P < 0.001 correspondingly). A correlation was observed between p53 protein expression and Ki-67 LI (P < 0.001). The immunocytochemical study of p53 protein and Ki-67 expression in cytological material represents a simple method which can be applied in routine cytological laboratories for the investigation of potential malignancy of ductal breast cancer.

Moc-31, fibronectin and CEA in the differential diagnosis of malignant effusions: an immunocytochemical study.

In discriminating benign and malignant origins of cytologically suspicious effusion smears a panel of antibodies against carcinoembryonic antigen (CEA), Fibronectin (F) and MOC-31 was used with immuno-cytochemical techniques. One hundred and thirty seven effusions were studied of which 107 had a malignant and 30 a benign aetiology as determined by clinical and histological examination. Cytologically 24 were diagnosed as benign, 97 as malignant and 14 as suspicious. Staining for F was positive in all effusions of benign and 3 of malignant origin. MOC-31 was positive in 95 (88.8%) of effusions of malignant origin but none of benign origin. Positive CEA was observed in 43% of effusions of malignant origin and in 10 of benign origin. The combination of MOC-31 positivity measured the sensitivity and specificity of the cytological examination in cases where the cytological examination result was suspicious as did F positivity improve the sensitivity for a benign origin of the effusion. Positivity or negativity for CEA is less valuable than the other parameters.

p53 protein expression and proliferative activity in imprints of superficial transitional cell carcinoma of the bladder.

OBJECTIVE: To investigate p53 protein expression and proliferative activity in imprints of tumor biopsies from superficial transitional cell carcinoma of the bladder in relation to the histologic grade of malignancy and recurrence status. STUDY DESIGN: The study group consisted of 70 cases of superficial transitional cell carcinoma of the bladder. In order to investigate p53 protein expression and Ki-67 expression, an immunocytochemical avidin-extravidin complex technique was performed using monoclonal antibodies p53 D0-7 and proliferating cells correspondingly. RESULTS: Thirty-seven percent of superficial transitional cell carcinoma cases showed positive expression of p53 protein. No correlation was found between p53 protein expression and grade of malignancy (P = .45). p53 Protein expression was statistically correlated with a high Ki-67 labeling index (LI) (P < .001) and recurrence status (P < .001). Forty-seven percent of cases showed a Ki-67 LI > 25%. No correlation was found between a high Ki-67 LI and grade of malignancy (P = .703). A significant difference in high Ki-67 LI between recurrent and nonrecurrent tumors of the same grade (P < .001) and between recurrent and nonrecurrent tumors was found independently of grade (P < .001). CONCLUSION: These results on cytologic material could provide useful information on the biologic behavior of superficial transitional cell carcinoma of the bladder at the time of diagnosis.

Cushing syndrome as the presenting feature of metastatic Leydig cell tumor of the testis.

We report a patient with a history of orchiectomy for Leydig cell tumor of the testis who developed Cushing syndrome. This syndrome was due to ectopic production of cortisol and was the primary feature of tumor recurrence.

DNA ploidy and p53 protein expression in superficial transitional cell carcinoma of the bladder.

Superficial transitional cell carcinoma of the bladder (STCCB) is a heterogeneous group of neoplasias with an unpredictable clinical course. In recent years many techniques have been used in order to predict the behaviour of these tumours at individual patient level. The aim of this study was to investigate in imprints from tumour biopsies the DNA ploidy and p53 protein expression in a group of 80 STCCB (pTa-pT1) patients in relation to histological grade and recurrence status. The DNA content was studied in Feulgen-stained imprints by the image analysis technique using a SAMBA 2005 analyser. In order to investigate p53 protein expression an avidin-extravidin immunocytochemical technique was used. According to our measurements a strong correlation was observed between recurrence status and DNA ploidy status (P < 0.001). No statistical difference was found in DNA ploidy status and grade of malignancy (P = 0.68). A statistically significant difference was found in p53 protein expression between recurrent and nonrecurrent tumours (P < 0.001). No statistically significant difference was found among tumours of grade I, grade II and grade III (P = 0.42). These results could provide useful information on the potential behaviour of STCCB.

Prognostic significance of p53, bcl-2 and EGFR in carcinoma of the endometrium.

OBJECTIVE: To evaluate the part played by several parameters in the prognosis of patients with endometrial carcinoma. STUDY DESIGN: Eighty imprint smears from fresh endometrial tumor specimens were studied immunocytochemically for the expression of p53, bcl-2 and epidermal growth factor receptor. Also, the presence of estrogen receptor (ER) and progesterone receptor (PR) in the tumor tissue was measured. The data obtained were related to survival, and associations were sought between the parameters studied. RESULTS: Strong associations were found between advanced stage, high grade, lymph node metastases at diagnosis, nonendometrioid histology and p53 expression with poor survival. Bcl-2 expression was associated with good five-year survival. ER expression was associated marginally with good five-year survival, but PR expression was not. A strong association was found between p53 and advanced disease, stage and lymph node metastases at diagnosis. An association between EGFR positivity and survival was not found. CONCLUSION: p53 Expression of uterine tumors is an independent and strong indicator of poor prognosis. Even patients with stage I and II disease at surgery who have p53-positive tumors must be considered at high risk.

Changes of chromosomes 1, 3, 6, and 11 in metastatic effusions arising from breast and ovarian cancer.

This cytogenetic study deals with cell material obtained from 15 pleural fluids from 11 patients with breast cancer and 27 ascitic fluids from 16 patients with ovarian cancer; in addition, 8 pleural, 5 ascitic, and 1 pericardial fluid from patients with tuberculosis, liver cirrhosis, and heart insufficiency, were studied. Using mainly direct methods, as well as short-term cell cultures, the chromosome spreads were GTG-banded. Cancerous biopsies showed a plethora of numerical and structural chromosome anomalies and exhibited broad aneuploidy. Chromosomes participating more often in numerical and structural aberrations were 1, 3, 6, 7, 8, 9, 11, 12, and 17. This study provides further cytogenetic evidence for the involvement of these chromosomes in breast and ovarian malignancy.

Sequential administration of doxorubicin and paclitaxel followed by cyclophosphamide, methotrexate and 5-fluorouracil combination (CMF) in women with metastatic breast cancer.

Although the combination of paclitaxel with doxorubicin has yielded high response rates in metastatic breast cancer, severe cardiotoxic events have been reported in several patients. The rationale for our study was to evaluate the activity of paclitaxel/doxorubicin combination in patients with this disease but to avoid excessive cardiotoxicity. Therefore, we administered 4 cycles of doxorubicin/paclitaxel followed by 6 cycles of standard cyclophosphamide, methotrexate and 5-fluorouracil (CMF) regimen. Study medication consisted of doxorubicin 60 mg/m2 as a 15-min intravenous infusion followed by paclitaxel 175 mg/m2 as a 3-hour infusion. CMF regimen consisted of cyclophosphamide 600 mg/m2 as 1-hour intravenous infusion followed by methotrexate 40 mg/m2 and 5-fluorouracil 600 mg/m2 bolus injection. The main toxicity of doxorubicin/paclitaxel treatment phase was neutropenia (WHO grade 3/4, 58%), but we observed only one cardiac adverse event. Toxicities of the CMF treatment phase were not significant. Of 24 patients evaluable for response, 2 (8%) had complete responses and 11 (46%) achieved partial response. Ten additional patients (42%) had stable disease. The median time to progression was 12 months and the median overall survival was 18.5 months. The sequential administration of doxorubicin and paclitaxel followed by CMF appeared active and well tolerated in patients with metastatic breast cancer.

p53 and bcl-2 protein expression in non-small-cell lung carcinoma.

The diagnostic significance of p53 and bcl-2 proteins in epithelial non-small-cell lung cancers was examined, and the relationship between these proteins expression and other disease parameters, including stage of the disease and tumor differentiation, were studied. We analyzed p53 and bcl-2 proteins expression in 60 imprint smears of freshly resected lung tumors (37 squamous and 23 adenocarcinomas) using the immunocytochemical technique. There were seven patients with stage I disease, 24 with stage II, 23 with stage IIIa, and six with stage IIIb disease, according to the International Staging System classification. Sixteen of the tumors were bcl-2 positive and 25 were p53 positive. Twenty tumors were negative for both bcl-2 and p53 (33.3%). Statistical analysis showed no association between the incidence of p53 or bcl-2 positivity. Adenocarcinoma or squamous carcinoma analysis showed significant associations between p53 positivity and poor differentiation and advanced disease stage as well as bcl-2 and early disease stage and well-differentiated tumors. There was also an association between the stage of the disease and the degree of differentiation of the tumors. In conclusion, bcl-2 positivity must be considered a good prognostic sign. On the other hand, p53 positivity seems to indicate, even in tumors at a relatively early stage, that a serious aggressive tumor which will not be easily eradicated is present.

Expression of p53, bcl-2 and heat shock protein (hsp72) in malignant and benign ovarian tumours.

The occurrence of p53, bcl-2 and heat shock protein (HSP) expression in ovarian tumours was examined and the correlation was investigated between the expression of these proteins and other disease parameters, including FIGO stage, histological subtype, tumour differentiation and steroid hormone receptor status. We analysed p53, bcl-2 and HSP expression in 100 smears of patients with epithelial ovarian carcinomas, 16 smears of patients with borderline malignancy and 20 smears of patients with benign ovarian neoplasms by using immunocytochemical techniques. There were 29 patients with stage I disease, 24 with stage II disease, 40 with stage III disease and seven with stage IV disease according to the FIGO classification. The sensitivities and specificities of bcl-2, p53 and HSP for malignancy were 53% and 40%, 43% and 80%, and 37% and 90%, respectively. HSP was statistically significantly associated with malignant rather than benign tumours. Significant association was also observed between bcl-2 and p53, and p53 and HSP. The association of HSP with malignant tumours is confined to the premenopausal group of patients and in this group by itself there is also a significant association between p53 and malignancy. HSP and p53 were associated with undifferentiated carcinomas, bcl-2 and p53 expression is reduced as disease stage progresses in serous carcinomas and bcl-2 expression is increased as disease progresses in endometrioid carcinomas. There was no significant association between bcl-2 and ER/PR status. In conclusion, HSP has a high specificity for malignant ovarian tumours, bcl-2 and p53 have only moderate to low sensitivity and specificity. Changes in the frequency of bcl-2 and p53 overexpression between FIGO I and FIGO III stage disease of different ovarian carcinomas indicate a different role of these substances in cellular survival mechanisms in different carcinomas. bcl-2 probably is associated with cell proliferation but not with differentiation.

Expression of cathepsin D and pS2 in imprint smears of breast carcinoma.

The aim of this study was to add to existing information on the effects of certain tumour markers expressed by breast cancers on tumour malignancy as evidenced by size of primary and occurrence of lymph node invasion. One hundred freshly resected breast cancers were examined by immunocytochemical staining of imprint smears for Cathepsin D and pS2. Oestrogen receptor (ER) and progesterone receptor (PR) were tested for by dextrose-coated charcoal (DCC) assay and the results correlated with tumour size, histology and presence or absence of lymph node metastases at the time of surgery using chi(2) analysis. A significant positive correlation was demonstrated between Cathepsin D positivity and ER, PR and pS2 positivity. In tumours < 2 cm in diameter at surgery a positive correlation was observed between Cathepsin D positivity and the presence of lymph node metastases. The findings support the hypothesis that Cathepsin D may promote early metastasis, possibly by its proteolytic activity.

Cathepsin D immunoreactivity in ovarian cancer: correlation with prognostic factors.

In view of the somewhat inconclusive nature of the reports of the role of Cathepsin D (Cath D) in ovarian carcinoma and its relationship with various other parameters of malignancy the present study was performed to aid in the further clarification of this role. One hundred freshly resected primary ovarian carcinomas of various histological types were studied for ER, PR and Cath D status and the results examined with respect to menopausal status, histology, size and lymph node invasion. In our series Cath D positivity was more frequent in serous than in other types of ovarian cancer but this Cath D positivity was not related to the frequency of lymph node invasion regardless of the size of the tumor. Nor was any association observed between Cath D positivity and ER or PR status of the tumors or the menopausal state of the patients. The reported prognostic value of Cath D, ER and PR is discussed as well as the distinction between tumor invasion by lymphatic channels and direct interstitial infiltration. It was concluded that Cath D may not play a role in the former mode but, as might be expected from its proteolytic properties, in local spread by means of tissue destruction.