Detection of adenocarcinoma in peritoneal washings by staining with monoclonal antibody B72.3

Peritoneal washings are routinely performed in the staging evaluation of carcinomas of the ovary and in "second-look" explorations; major problems in the evaluation of these specimens continue to be the distinction between atypical mesothelium and adenocarcinoma and the identification in an otherwise inflammatory specimen of rare cells of adenocarcinoma, which may be undetected by the most trained individual. Monoclonal antibody (MAb) B72.3, reactive with an oncofetal, tumor-associated glycoprotein (termed TAG-72; MW greater than 1000 kd) expressed in a variety of epithelial malignancies but not generally expressed in benign or malignant mesothelium, was reacted with sections from the paraffin-embedded cell blocks of 185 peritoneal washings from 180 patients with extant cancer or a prior history of malignancy. One hundred four of the washings were initially interpreted as atypical mesothelium, with no evidence of malignancy; when reacted with MAb B72.3, 6 of these specimens demonstrated groups of metastatic adenocarcinoma cells not appreciated by the usual cytologic criteria. Of the 81 washings interpreted as showing cells of adenocarcinoma, 73 demonstrated expression of TAG-72 from both gynecologic and nongynecologic malignancies. In the remaining 49 cases without an associated malignant process, MAb B72.3 did not stain atypical mesothelium, but did react, however, with benign endometrial cells and müllerian inclusions in two cases. MAb B72.3 may be used as a diagnostic adjunct to the routine cytologic evaluation of malignancy in peritoneal washings; reactivity with MAb B72.3 may indicate the need for further evaluation to define the presence of a malignancy or an advanced cancer.     

Cytohistologic correlation of peritoneal washing cytology in gynecologic disease

The peritoneal washings and cul de sac aspirates from 204 patients undergoing 217 procedures for the evaluation of gynecologic disease were examined retrospectively and correlated with the histologic diagnoses. Of the 73 washings from patients with histologically benign genital disease, cytology diagnosed 64 (87.7%) as negative, 6 (8.2%) as inconclusive and 3 as malignant. One malignant washing was a true positive from a nongenital primary. False positives thus occurred in 2.7% of the benign cases on blind review. Of 144 cytologic examinations of washings from patients with histologically confirmed malignant disease of the female genital tract, 38 (26.4%) were considered positive after cytohistologic correlation. Four malignant cases (2.1%) were undercalled on blind review while 3 (2.0%) were considered overcalls. Eleven of 47 cases (23.4%) with biopsy-proven peritoneal disease had negative cytology after histologic correlation. Recurring problems in interpreting peritoneal washings included: (1) the differential diagnosis of the spectrum encompassed by reactive mesothelium, endosalpingiosis, borderline serous tumors and well-differentiated serous cystadenocarcinoma; (2) morphologic similarities between some tumor cells and mesothelial cells associated with treatment effects; and (3) a paucity of malignant cells in some washings, resulting in false-negative interpretations. Ineffective cytopreparation, particularly of bloody specimens, hampered interpretation of some specimens. Correlation with previous histology and cytology enhanced the accuracy of peritoneal washing cytology in this study.     

Peritoneal washings in ovarian tumors. Potential sources of error in cytologic diagnosis

Peritoneal washings were performed on 48 patients with suspected or known ovarian carcinoma. The procedure was part of the initial surgical staging in 27 patients with presumed stage I and II ovarian cancer and was performed during second-look operations in 21 other cases with proven ovarian malignancy. This paper presents the microscopic features of the washings, with particular emphasis on the cytologic differentiation between benign and malignant findings outside of the ovary. Thirty-four cases showed benign or reactive mesothelial cells and no evidence of peritoneal disease. The washings of six patient showed malignant cells, which were confirmed histologically. Notable atypia that mimicked ovarian carcinoma was found in eight patients who had benign or borderline lesions. These findings included papillary and glandlike epithelial structures, with varying degrees of cellular atypia and psammoma bodies. The histologic counterparts of these atypicalities were Müllerian inclusions, mesothelial proliferations and borderline serous tumors. The differential diagnosis between these entities is essential because false-positive cytologic diagnoses may alter postoperative treatment in some patients.     

Prospective comparative cytologic study of direct peritoneal smears and lavage fluids in patients with epithelial ovarian cancer and benign gynecologic disease

Direct peritoneal samples obtained by scraping or brushing (with a Cytobrush) were compared to peritoneal lavages (washings) for the cytologic evaluation of patients with gynecologic disease. The direct samples were obtained during laparotomy or laparoscopy, following saline lavage if that was performed, and were immediately smeared on glass slides and fixed in 95% alcohol. Only 9 of the direct peritoneal samples taken from 64 patients with benign gynecologic disease were unsatisfactory for cytologic interpretation while 19 of the 33 lavage specimens simultaneously collected from these patients were considered unsuitable for analysis (P less than .001). Two direct smears from cases with benign histology were reported as suspicious. Nineteen patients with epithelial ovarian cancer also had cytologic specimens collected by direct sampling and by washing. The direct smears were positive for malignancy in 12 cases, suspicious in 4 cases and negative in 3 cases while the lavage samples were positive in 9 cases, suspicious in 4 cases, negative in 4 cases and unsatisfactory in 2 cases. These results indicate that direct peritoneal sampling is a simple and reliable alternative to peritoneal lavage and produces a significantly lower incidence of unsatisfactory specimens.     

Peritoneal washing cytology. Uses and diagnostic criteria in gynecologic neoplasms

Review of a 20-month experience with 241 peritoneal washes performed on 191 patients showed that the use of these specimens has expanded greatly. Of the 19 patients with neoplastic cells in their peritoneal washing cytology specimens, 12 had primary ovarian neoplasms, 4 had primary uterine cervical neoplasms, 2 had primary endometrial neoplasms, and 1 had mammary carcinoma metastatic to the ovary. Gynecologic oncologists at this institution are now routinely obtaining peritoneal washing cytology specimens whenever there is intraabdominal surgery on patients known to have or suspected of having a pelvic neoplasm. The following criteria were found to be essential to the accurate evaluation of these specimens: (1) cells considered to be malignant should be present both singly and in groups and should be malignant by the usual cytologic criteria, (2) the patients must have or be known to have had a neoplasm whose cells are similar to those in the washing specimen, and (3) the cells considered to be neoplastic must be different from and not confused with reactive mesothelial cells. The last criterion is important because the peritoneal lavage traumatically removes mesothelium, which can appear atypical. These criteria make the cytologic interpretation of most peritoneal washing specimens straightforward; interesting diagnostic problems occur, however, including the evaluation of neoplasms of borderline malignancy, those "spilled" during surgery and second neoplasms found by peritoneal washing cytology.     

Qualitative and quantitative analysis of peritoneal fluids from women with gynecologic diseases. Comparison of cytology and flow cytometry for the detection of malignancy in lavage and ascitic fluids

A prospective study was undertaken to compare flow cytometric (FCM) analysis to conventional cytologic evaluation for the detection of malignant cells in peritoneal fluids (peritoneal lavages and ascitic fluids) from women with gynecologic diseases. The 94 peritoneal fluids analyzed came from 63 cancer patients (with epithelial ovarian carcinomas) and 31 control patients (with benign gynecologic diseases). The FCM DNA histograms were generated using propidium iodide as a DNA fluorochrome. Samples for cytologic analysis were stained with the standard May-Grünwald-Giemsa or Papanicolaou stains. Of the 94 samples, 90 were evaluable cytologically while 70 were suitable for FCM analysis. The sensitivities were 55% for FCM DNA analysis and 80% for cytologic analysis. FCM DNA analysis had a 30% false-positive rate; cytologic analysis produced no false-positive results. These results indicate that there is no advantage in employing FCM analysis instead of conventional cytologic evaluation for the detection of malignant cells in peritoneal fluids from gynecologic cases.     

Peritoneal washing cytology in cervical carcinoma. Analysis of 109 patients

The peritoneal washing cytologies of 109 patients (112 procedures) undergoing laparotomy for cervical carcinoma were evaluated retrospectively and compared with the clinical and pathologic findings. Nine patients (8.3%) had malignant peritoneal washings (including three of four with washings initially termed "inconclusive"). Four (4.9%) of the 82 patients with squamous carcinoma and 3 (16.7%) of 18 with adenocarcinoma had positive washings. Five (5.6%) of 90 washings obtained at initial explorations were positive, as compared with 4 (18.2%) of 22 washings obtained as follow-up operations in recurrent cases. The 111 peritoneal washing cytologies with a corresponding histologic evaluation of the peritoneal cavity showed a good correlation; peritoneal washing cytology had an efficiency of 91.0%, a sensitivity of 52.9% and a specificity of 100%. Two cases in which the cytologies were considered positive only after review had negative peritoneal histologies; both patients died of progressive disease within 11 months. Peritoneal washing cytology was positive in 5 (5.9%) of 84 cases with FIGO stage 1 cancers, 2 (18.2%) of 11 cases with stage 2 cancers, 1 (33.3%) of 3 cases with stage 3 cancers, and 1 (10%) of 10 cases with recurrent tumors. Eight (88.9%) of nine patients with malignant peritoneal washings died of disease from 3 to 15 months following surgery; one showed no evidence of disease at 9 months. These results suggest that: (1) cervical carcinomas are infrequently associated with a positive peritoneal washing; (2) peritoneal washing cytology is more likely to be positive in cases of adenocarcinoma than in cases of squamous carcinoma; (3) peritoneal washings obtained at the time of surgery for recurrence are more likely to contain malignant cells than are washings obtained during initial exploration; (4) nonkeratinizing malignant squamous cells may be confused with reactive mesothelial cells; and (5) peritoneal washing cytology is a relatively insensitive technique for detecting advanced cervical disease, but correlates with a poor prognosis when positive.     

Peritoneal fluid cytokines and the differential diagnosis of benign and malignant ovarian tumors and residual/recurrent disease examination

This study aimed to assess the potential value of peritoneal fluid cytokine examination for the differential diagnosis of ovarian tumors and for evaluating residual or recurrent disease after treatment. The cytokines that are commonly elevated in ovarian cancer, VEGF, IL-6, bFGF, IL-8 and M-CSF, and a reference ovarian tumor marker, CA 125, were measured in peritoneal fluids of 53 previously untreated patients with epithelial ovarian cancer, 18 ovarian cancer patients after surgical treatment and chemotherapy, and 17 patients with benign epithelial ovarian tumors. Non-parametric statistical analysis of data was performed. Ovarian cancer peritoneal fluids, as compared to peritoneal fluids of patients with benign ovarian tumors, contained significantly higher concentrations of IL-6, VEGF and CA 125, and significantly lower concentrations of bFGF and M-CSF, but only the levels of IL-6 and VEGF were significantly higher in peritoneal fluids of stage I and II ovarian cancer patients than of patients with benign ovarian conditions. IL-6 at the cutoff level of 400 pg/mL discriminated benign and malignant ovarian tumors with 92% sensitivity and 60% specificity, while VEGF at the cutoff of 400 pg/mL had 90% sensitivity and 80% specificity. At the cutoff level of 1200 pg/mL, IL-6 had 84% sensitivity and 87% specificity. A radical decrease in local cytokine and CA 125 levels in patients after treatment was independent of therapy outcome. IL-6 and VEGF measurements in peritoneal fluids might be useful for the differential diagnosis of malignant and benign ovarian conditions, but not for residual or recurrent disease examination.     

Cytology of bronchial benign granular-cell tumor

The cytologic features of a case with multiple bronchial benign granular-cell tumors are reported and compared with those of previously reported cases. Characteristic tumor cells were found in the bronchoscopic brushing smears and in cell block sections (but not smears) prepared from the washing fluid. These findings were confirmed by the bronchial biopsy and histologic study of the resected tumors. A cytologic diagnosis of bronchial granular-cell tumor should not be difficult because the cytologic appearance of the tumor cells is characteristic; however, the possibility of a concomitant tumor, such as adenocarcinoma or small-cell carcinoma, should be considered and excluded.     

Uterine Müllerian adenosarcoma with psammoma bodies: cytologic, histologic and ultrastructural studies of a case

Epithelial cells associated with psammoma bodies and undifferentiated sarcomatous tumor cells were found in peritoneal washings obtained during laparotomy for removal of metastatic lesions from a uterine stromal sarcoma. Histologic studies of the primary and recurrent tumors showed characteristics of Müllerian adenosarcoma. The cytologic, histologic and ultrastructural features are described.     

Cystosarcoma phyllodes. Diagnosis by fine needle aspiration cytology.

The cytologic features of 10 benign, 2 borderline and 5 malignant phyllodes tumors were studied, and an attempt was made to correlate the cytologic findings with corresponding histologic categories. Seventy-five percent of the benign and borderline tumors were interpreted as benign cystosarcoma phyllodes on fine needle aspiration cytology. Eighty percent of the malignant phyllodes tumors were identified as malignant lesions cytologically. The cytologic features assessed were the epithelial:stromal ratio and morphology of the stromal component, including the degree of atypia, mitotic activity, capillary vessels traversing the stromal fragments, presence of foamy macrophages, histiocytic giant cells and bipolar naked nuclei. A diagnosis of phyllodes tumor was suggested cytologically by the presence of both epithelial and stromal elements; the stroma was present as cellular "phyllodes fragments" and isolated mesenchymal cells. The parameters suggesting malignancy were extreme paucity or absence of epithelial elements and stromal cells in diffuse sheets and clusters less cohesive than normal, with marked stromal atypia and mitotic activity.     

Peritoneal cytology in uterine papillary serous carcinoma.

OBJECTIVE: To highlight the significance of positive peritoneal cytology in uterine papillary serous carcinoma (UPSC). STUDY DESIGN: Seventeen consecutive UPSC cases with peritoneal cytology from 1993 to 1997 were reviewed and compared with the original cytologic diagnosis and extent of tumor involvement in tissues. RESULTS: Of the 17 post-menopausal women with UPSC, 11 had early-stage tumors (clinical stage I and II); three cases (27%) with positive peritoneal cytology were upgraded from at least International Federation of Gynecologists and Obstetricians stage IA to IIIA. No change in surgical stage was noted in four of six (67%) advanced cases with positive peritoneal cytology. The review diagnoses of peritoneal cytology did not differ from the original diagnoses. CONCLUSION: The features of UPSC in peritoneal cytology are those of a high grade malignancy and may be shared by tumors with similar histology from other sites. The malignant features are readily identified, but the site of origin may not be completely ensured. Positive peritoneal cytology upgrades the surgical stage of early-stage UPSC cases and helps with prognostication and treatment. One case with positive washings but without residual tumor probably represented early spread and/or multicentric origin of the tumor.     

Information, discrimination and divergence in cytology. III. Optimization of classification of Papanicolaou smears

The performance of a cytology laboratory can be objectively quantitated as the total discrimination, a defined quantity of information. The total discrimination is dependent on the number of categories used in gynecologic cytology and on the corresponding histologic states; over-classification results in a higher rate of misinformation and reduced total discrimination. Total divergence is another measure of the association between cytologic categories and histologic states; in contrast to the total discrimination, the total divergence does not require a one-to-one correspondence between the cytologic categories and the histologic states. Using data from the Gynecologic Cytology Laboratory of the University of Minnesota, the total discrimination was maximized when gynecologic cytology used three categories of diagnosis, consisting of (1) normal, atypical benign or reactive atypia, (2) cervical intraepithelial neoplasia (CIN) and (3) all malignancies. The use of four categories, (1) normal, atypical benign or reactive atypia, (2) mild or moderate dysplasia, (3) severe dysplasia or squamous carcinoma in situ and (4) all malignancies, was almost equally informative. Observations on the total divergences resulted in similar conclusions. These findings generally support the recommendation of the consensus workshop sponsored by the National Cancer Institute (the Bethesda System nomenclature) to group all degrees of CIN into two large categories.     

"Collagen balls" in peritoneal washings. Prevalence, morphology, origin and significance.

Peritoneal washings obtained at laparotomy from women undergoing surgery for neoplasms of the genital tract may contain "collagen balls," consisting of tissue fragments composed of collagen covered with mesothelial cells. Collagen balls were found in 19 (4.5%) of 418 peritoneal washings and were more prevalent in specimens labeled pelvic washings (17 of 294, or 5.8%) than in those labeled peritoneal washings (2 of 124, or 1.6%). In 15 of the 19 cases in which we found collagen balls, at least one ovary was available for microscopic examination. In 14 of the 15 cases minute nodular papillary stromal projections covered with mesothelium were found on the surface of the ovaries. We conclude that collagen balls, a nonspecific entity, most probably originate on the surface of the ovaries. Their significance lies in their being mistaken for mucin-distended cells exfoliated from a neoplasm or from detached fragments of a papillary ovarian neoplasm.     

Nonaspiration-needle smear preparations of pulmonary lesions. A comparison of cytology and histology

Material for cytologic smears was obtained from pulmonary lesions in 146 patients at the Ohio State University between 1979 and 1984 using Rotex or Lee screw needles. Corresponding histologic specimens were available for comparison in 77 of these cases. Diagnoses of malignant neoplasms made by cytologic evaluation (55 cases) were confirmed by the corresponding histologic specimens in 93% of those cases. Possible explanations for the cytologic false-positive diagnoses of malignancy are presented. Correlations between the cytologic and histologic diagnoses of the morphologic type of tumor were 100% for adenocarcinoma, 75% for squamous-cell carcinoma and 20% for large-cell undifferentiated carcinoma. The correlation was 100% for small-cell carcinoma when the histology specimen represented the tumor. Nonneoplastic benign lesions diagnosed cytologically had corresponding benign histologic diagnoses in 94% of the cases. These results compare favorably with those reported for other fine needle aspiration studies of pulmonary lesions. The advantages of using Rotex needles as compared to fine needle aspiration are discussed.     

Diagnostic value of hair shafts and squamous cells in peritoneal washing cytology

OBJECTIVE: Little attention has been given to hair shafts and squamous cells in peritoneal fluid. To investigate their diagnostic value in peritoneal washing specimens, we reviewed peritoneal washing cytology preparations from 83 cases of ovarian tumors. STUDY DESIGN: We reviewed peritoneal washing specimens and histologic sections of 86 cases of ovarian tumors and tumorous conditions, including 22 teratomas, 16 serous adenocarcinomas, 10 clear cell adenocarcinomas, 9 endometrioid adenocarcinomas, 5 cases of endometriosis, 4 mucinous adenomas, 3 serous cystadenocarcinomas and 17 other tumors. RESULTS: We observed both squamous cells and hair shafts surrounded by inflammatory cells in 5 of the 22 cases of ovarian teratoma. Rupture of an ovarian teratoma was clinically and histologically found in one of the five cases. Hair shafts were not observed in the other tumors or in nonneoplastic conditions. The diameter of hair shafts in peritoneal washing specimens ranged from 10 to 28.8 microns (average, 16.6), and such hair shafts were present within an ovarian teratoma examined histologically. The diameter of hair shafts from six normal adults who were examined as controls ranged from 61.5 to 118.6 microns (average, 89.4). CONCLUSION: Hair shafts and squamous cells surrounded by inflammatory cells in peritoneal washing specimens are a diagnostic clue to ovarian teratoma and can be observed even when rupture of the tumor is not detected clinically or microscopically.     

Expression of matrix metalloproteinases-2 and -9 by cells isolated from the peritoneal fluid of women with ovarian carcinoma

OBJECTIVE: To investigate the level of expression of matrix metalloproteinases (MMP)-2 and -9 by cells isolated from the peritoneal fluid of women with ovarian carcinoma. STUDY DESIGN: Tumor tissue specimens and cells isolated from peritoneal fluid from 20 patients with epithelial ovarian carcinoma were examined for MMP-2 and -9 expression using immunostaining. Six benign peritoneal effusions containing mesothelial cells were also included in the study. RESULTS: Expression of both MMP-2 and -9 was noted in cancer cells in peritoneal fluid of all cases studied. Peritoneal fluid cancer cells showed increased expression of both MMP-2 and -9 relative to mesothelial cell expression of these MMPs. Positive immunoreactivity of these MMPs in primary tumor tissues was confirmed by immunohistochemistry. CONCLUSION: Our findings suggest that both MMP-2 and -9 are frequently overexpressed in ovarian cancer cells disseminated in the peritoneal cavity and that determination of cellular MMP-2 and -9 expression could be useful in distinguishing cancer cells from mesothelial cells in peritoneal fluid cytologic specimens from women with ovarian epithelial carcinoma.     

Cytologic findings in peritoneal washings associated with benign gynecologic disease

In order to define the problems in the interpretation of peritoneal washing cytology specimens, 149 benign cases were examined retrospectively and the cytologies compared with the correlating histologies. There were problems in the cytologic interpretation on blind review in 18 cases (12.1%). Difficulties in interpretation of peritoneal washing cytology included conditions associated with (1) mesothelial reactions and adhesion formation, (2) rupture of cystic structures, (3) fallopian tube epithelium and (4) combinations of these factors. It is anticipated that familiarity with these problems will enhance accuracy in peritoneal washing cytology, particularly in those cases in which a malignancy is present but has not penetrated into the peritoneal cavity.     

Combined analysis of serum lactic dehydrogenase levels and isozyme patterns in ovarian neoplasms

A total of 65 cases of ovarian tumor admitted for surgical intervention were prospectively enrolled in this study, including 40 cases with benign ovarian tumors and 25 cases of ovarian cancer. The 25 malignancies were comprised of 22 cases of common epithelial origin and three cases with Krukenberg tumors. Serum total LDH level and its isozyme activity were measured preoperatively in each patient. The mean value (+/-SD of serum LDH in the malignant group was 876.3 (+/- 450.4) IU/1, which was significantly higher than 364.8 (+/- 87.9) IU/1 in the benign group. Twenty-two cases of ovarian malignancy (88%) and 6 cases of the benign group (15%) had elevated serum LDH levels above 450 IU/1. Analysis of the LDH isozyme pattern demonstrated that there was a significant shift to LDH-4 and LDH-5 fractions in ovarian malignancy when compared to the benign group (with the mean value of 9.5%, 14.6% vs. 6.5% and 4.6%, respectively). The elevated LDH level could yield 88% and 85% in sensitivity and specificity for detecting ovarian malignancy, while the abnormal isozyme pattern could yield 84% and 77.5%, respectively. From combined analysis of total LDH levels and isozyme patterns, the true positive detection rate and true negative detection rate could both reach 100%. In conclusion, the present findings imply that total serum LDH level and isozyme activity measurement may provide significant aids in evaluation and detection of human ovarian cancers among patients with ovarian tumors.     

Presence of simian virus 40 sequences in malignant mesotheliomas and mesothelial cell proliferations

Malignant mesotheliomas (MMs) are pleural‐, pericardial‐, or peritoneal‐based neoplasms usually associated with asbestos exposure. Mesothelial cells are biphasic and may give rise to epithelial and sarcomatous MMs. In addition, benign or atypical proliferations of mesothelial cells may occur in response to many stimuli. There have been recent reports of simian virus 40 (SV40) DNA large T antigen (Tag) sequences in pleural MMs. To further understand the relationship between SV40, MMs, and mesothelial proliferations, we studied 118 MMs from multiple sites in Germany and North America, including 93 epithelial pleural, 14 sarcomatous or mixed pleural MMs, and 11 peritoneal MMs. In 12 pleural MMs, adjacent noninvasive tumor foci were identified and studied separately. Information about asbestos exposure (detailed history and/or microscopic examination for asbestos bodies) was available from 43 German patients. In addition, 13 examples of reactive mesothelium and 20 lung cancers from the United States were tested. DNA was extracted from frozen tumor and adjacent nontumorous tissues or after microdissection of archival formalin‐fixed, paraffin‐embedded microslides. Two rounds of PCR were performed with primers SVFor 3 and SVRev, which amplify a 105 bp region specific for SV40 Tag. The specificity of the PCR product was confirmed in some cases by sequencing. Our major findings were: 1) Specific SV40 viral sequences were present in 57% of epithelial invasive MMs, of both pleural and peritoneal origin. No significant geographic differences were found, and frozen and paraffin‐embedded tissues were equally suitable for analysis. 2) There was no apparent relationship between the presence of SV40 sequences and asbestos exposure. 3) SV40 sequences were present in the surface (noninvasive) components of epithelial MMs. 4) SV40 sequences were not detected in MMs of sarcomatous or mixed histologies. 5) Viral sequences were present in two of 13 samples (15%) of reactive mesothelium. 6) Lung cancers lacked SV40 sequences, as did non‐malignant tissues adjacent to MMs. Our findings demonstrate the presence of SV40 sequences in epithelial MMs of pleural and peritoneal origin and their absence in tumors with a sarcomatous component. Viral sequences may be present in reactive and malignant mesothelial cells, but they are absent in adjacent tissues and lung cancers. J. Cell. Biochem. 76:181–188, 1999. © 1999 Wiley‐Liss, Inc.