Malignant Cells In Effusions Research Articles

Adaptation of a commercial fluorescent in situ hybridization test to the diagnosis of malignant cells in effusions

In effusion cytology, adjuvant techniques are often needed for the differentiation of reactive proliferating mesothelial cells and malignant cells. In the case of malignancy the further challenge is to distinguish metastatic tumors from the primary malignant mesothelioma. Fluorescence in situ hybridization (FISH) of cells in interphase is an accurate method to monitor the genetic status of cells, detecting aneuploid signals and gene deletions. Moreover, it has been proposed that a homozygous deletion of the p16 INK4A gene could more specifically identify malignant mesothelial cells among the exfoliated cells. The first objective of this study was to adapt the commercial FISH-test, UroVysion™ originally designed for the cytological diagnosis of bladder cancer, to the analysis of cells in effusions. The second objective was to test the clinical utility of the test. Sixty-eight pleural effusions were evaluated. The cytological diagnosis was malignant in 29 cases, inconclusive in 24 cases and benign in 15 cases. The independently verified final diagnoses were mesothelioma in 21 cases, metastatic cancer in 29 and benign in 18 cases. The algorithm for aneuploidy distinguished almost all tested malignant conditions from benign ones, also those with inconclusive cytology. The 9p21 locus, carrying the p16 INK4A gene, was homozygously deleted in two of the metastatic cancers, while this was seen in 12 of the 21 malignant mesotheliomas. Thus the commercial UroVysion™-test can be used to accurately distinguish malignant and reactive cells in effusions, particularly when cytology is inconclusive. The test may also indicate presence of MM.

Diagnostic Utility of CD44 Standard, CD44v6, and CD44v3-10 Expression in Adenocarcinomas Presenting in Serous Fluids

CD44 is an 85 to 90 kd integral transmembrane protein encoded by a single 20-exon gene located on the short arm of chromosome 11. In the standard form (CD44s), 10 of the 20 exons are transcribed. Multiple variant isoforms exist (CD44v1-10) which arise from alternate mRNA splicing of the remaining 10 exons. In contrast to the standard form of CD44, which is almost ubiquitously expressed, splice variants are highly restricted in their expression in normal or malignant tissues. The purpose of this study was to evaluate the extent to which metastatic adenocarcinomas in effusions express CD44s, CD44v6, and CD44v3-10 and to assess their diagnostic utility in distinguishing reactive mesothelial cells from adenocarcinomas. Archival paraffin-embedded cell blocks of serous fluids from 23 cases of benign effusions containing reactive mesothelial cells and 45 cases of malignant effusions with metastatic adenocarcinoma (18 ovarian, 11 pulmonary, 9 gastrointestinal, and 7 breast) were retrieved from the surgical pathology files. The cytopathology of all cases was reviewed to confirm the diagnosis. Immunohistochemistry was performed on all cases using antibodies for CD44s, CD44v6, and CD44v3-10 (Bender MedSystems, CA). Positive staining was defined as distinct linear membrane staining. Strong staining in at least 10% of the tumor cells was required to consider the case positive for the particular marker. In benign effusions mesothelial cells expressed CD44s in 22 cases (96%), CD44v6 in 1 cases (4%) and CD44v3-10 in 0 cases (0%). In contrast neoplastic cells in malignant effusions expressed CD44s in 11 cases (24%), CD44v6 in 21 cases (47%), and CD44v3-10 in 39 cases (87%). We concluded that CD44s and CD44v3-10 are useful markers that can be applied to cytologic specimens. CD44s immunostaining can be used as a reliable marker to identify reactive mesothelial cells, meanwhile CD44v3-10 immunostaining can detect majority of adenocarcinomas in malignant effusions.

MN/CA9: a potential gene marker for detection of malignant cells in effusions

Many cancers cause malignant effusions. The presence of malignant cells in effusions has implications in diagnosis, tumour staging and prognosis. The detection of malignant cells currently presents a challenge for cytopathologists. New adjunctive methods are needed. Although the effusions provide excellent materials for molecular assay, the available molecular markers are extremely limited, which hinders its clinical application. MN/CA9 has proved to be a valuable marker in many cancers such as lung, breast, colon, kidney, etc. The present study was to evaluate MN/CA9 as a new molecular marker for the detection of cancer cells in pleural effusions. Seventy-one pleural effusions including 59 malignant effusions from patients with cancer, and 12 patients with benign diseases as a control, were subjected to RT-PCR for detection of MN/CA9 gene expression. MN/CA9 gene expression was detected in 53/59 (89.8%) pleural effusions from cancer patients (15/16 for breast cancers, 10/11 for lung cancers, 4/4 for ovary cancers, 2/3 for colon–rectal cancers, 5/6 for cancers of unknown site, 7/8 for mesothelioma and 10/11 for other cancers). Furthermore, MN/CA9 was positive in 13/18 (72.2%) of cytologically negative effusions of cancer patients. MN/CA9 was detected in only 1/12 (8.3%) effusions from the control patients (p<0.01). The sensitivity and specificity of MN/CA9 gene expression were, respectively, 89.8% and 91.7%. Our preliminary results suggest that MN/CA9 could be a potential marker for the detection of malignant cells in effusions. A large-scale study is needed to confirm these results.

Proteinase-Activated Receptor 1, 3 Expression in Primary Human Carcinoma Cells.

The coagulation system is regularly activated in patients with cancer, especially with lung cancer and gastrointestinal tumors. Up to 30% of these patients suffer from thromboembolic complications. Obviously, activation of thrombin occurs in the natural course of cancer. During tumor progression, many patients develop malignant effusions; treatment is complicated by increased resistance of tumor cells to chemotherapy. Thrombin-mediated activation of cellular receptor(s), such as proteinase-activated receptor-1(PAR-1), plays an important role in tumor growth, local progression, and distant metastasis. Our hypothesis is, that tumor cells that express PAR-1 have a survival benefit if they are activated by thrombin within the malignant effusion. We harvested primary cancer cells from pleural and peritoneal effusions of 110 patients with different carcinomas. In 22 cases we were able to establish long time cultures (2 pancreatic, 4 colon, 3 SCLC, 5 NSCLC, 3 mesothelioma, 1 gastric, 1 rectum, 1 breast, 1 melanoma and 1 NHL). We detected PAR-1, 3 expression using reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence activated cell sorting (FACS) scan. We found that in 76% the primary cancer cells were PAR-1 positive using RT-PCR assays, and in 70% the expression of PAR-1 receptor was positive using FACS scan. In contrast, we found that PCR for PAR-3 was positive in only 37.5% and the expression of the receptor in 6%. As a sign of thrombin activation, we regularly found increased levels of prothrombin fragment F1+2 in the malignant effusions compared to serum of patients (12.0 +/− 4.1 nmol/L vs 1.9 +/− 0.4 nmol/L).In summary, we found that most of the primary cancer cells in malignant effusions exhibit a positive PAR-1 expression, but PAR-3 expression is low. Also, thrombin is activated in most malignant effusions as shown by prothrombin fragment 1+2 elevation. Further experiments will show if these cell-biological mechanisms aggravate cellular resistance in primary tumor cells found in malignant effusion in end-stage cancer. Therapeutic consequences, such as therapeutic thrombin inhibition in addition to chemotherapy are discussed.

Differential expression of the 67 kilodalton laminin receptor in epithelioid malignant mesothelioma and carcinomas that spread to serosal cavities

Expression of the 67-kd laminin receptor (67-kd LR) has been reported in a wide range of carcinomas, in many of which it correlated with poor differentiation, metastasis, disease progression, and poor survival. Malignant mesothelioma (MM) is a locally aggressive and highly lethal tumor of serosal cavities that is rarely associated with clinically detectable metastasis to distant organs. The aim of this study was to analyze the expression of the 67-kd LR in epithelioid MM and carcinomas in effusions. Effusions from patients diagnosed with ovarian (=24) and breast (=38) adenocarcinomas and MM (=24) (total = 86) were analyzed for 67-kd LR protein expression, using immunocytochemistry. The 67-kd LR mRNA expression was additionally analyzed in 21 MM effusions using reverse transcription-polymerase chain reaction (RT-PCR). Protein expression of the 67-kd LR was frequently detected in carcinomas (19/24 ovarian tumors, 79%; 15/38 breast tumors, 39%), but was rare in MM (2/24 cases, 8%), despite the presence of mRNA transcripts for the receptor in all 21 specimens studied using RT-PCR. Nine benign effusions that were additionally studied for protein expression were uniformly negative, as were all reactive mesothelial cells in malignant effusions. Our results suggest that the 67-kd LR may aid in the differential diagnosis between metastatic carcinoma, mainly of ovarian origin, and MM. They additionally suggest that the failure of MM to express the 67-kd LR protein, as opposed to the frequent expression in carcinomas with proven metastatic capacity, may be one of the factors contributing to the reduced ability of the former tumor to metastasize to distant organs.

Diagnostic value of reverse-transcriptase polymerase chain reaction for MAGE families expression in malignant thoracic pleural effusions

9533 Background: The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. Previous studies show that the MAGE genes families are only expressed in the malignant cells and not expressed in normal tissues except testis and placenta. The aim of this study was to improve tumor cell detection in effusions by reverse-transcriptase polymerase chain reaction (RT-PCR) for MAGE families expression. Methods: A total of 22 thoracic effusions from patients with lung cancer and 18 thoracic effusions from patients without an evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, RT-PCR for MAGE families expression and pleural biopsy under video assisted thoracoscopy. To improve the sensitivity of RT-PCR, we combined the results of RT-PCR for MAGE-1, 2, 3 and 4 expressions. Anyone of them expressed in the effusion was recorded as MAGE positive. Results: Tumor cell detection by cytology, RT-PCR for MAGE e...

FISH cytogenetics and prognosis in breast and non-small cell lung cancers.

Interphase cytogenetics by fluorescence in situ hybridization (FISH) has been demonstrated to be a valuable diagnostic tool in effusions from patients with solid tumors. As the next step, we investigated whether certain patterns of numeric aberrations in malignant effusion cells supply prognostic information. From a large series of effusions from patients with solid tumors, 55 effusions from breast cancer and 39 effusions from non-small cell lung cancer (NSCLC) were classified as malignant by cytology or FISH. Tumor cells were classified as FISH aneuploid for chromosome 11 and/or 17 or as not aneuploid. Predominant cytogenetic anomalies and patterns of intratumor cytogenetic heterogeneity were brought in relation to overall survival rate. There was no difference with respect to overall survival rate when effusions with or without aneuploidy for chromosomes 11 and 17 were compared. Likewise, in effusions with aneuploidy, there was no difference in overall survival rate among patients with different modal chromosome copy numbers (e.g., trisomy vs. tetrasomy 11) or among patients with a low or high grade of intratumor complexity (defined by the intratumor heterogeneity of FISH aneuploidy). In breast cancer, aneuploidy with gain of chromosome 11 was associated with a significantly superior survival rate, suggesting that amplification of chromosome 11 DNA is associated with a less aggressive phenotype. Simple chromosomal changes as determined by FISH, such as gain of chromosome 11 copy numbers in breast cancer, may be prognostic. Prospective studies in primary tumors that classify distinct prognostic groups by FISH cytogenetics are warranted.

Malignant effusions: from diagnosis to biology.

Serous effusions are a frequently encountered clinical manifestation of metastatic disease, with breast, ovarian, and lung carcinomas and malignant mesothelioma (MM) leading the list. Recently, extensive research has resulted in expansion of the antibody panel that is available for effusion diagnosis, thereby reducing the risk for error. Despite this progress, relatively little has been done in way of understanding the biology of cancer cells in effusions, especially those of nonovarian origin. The diagnosis of a malignant effusion signifies disease progression and is associated with a worse prognosis regardless of the tumor site of origin. However, survival is much more variable with ovarian cancer compared with other tumors. Furthermore, cancer cells of different origins differ considerably in their biology and have unique phenotypic and genotypic characteristics. This review summarizes the current knowledge in this field and presents a model for the study of tumor metastasis and disease progression, through large comparative studies of malignant cells in effusions, primary tumors, and solid metastases. The case also is made for potential applications of this rapidly evolving body of knowledge in the diagnosis, classification, and prediction of biological behavior of processes resulting in cryptic effusions at the clinical level.

Malignant cell detection by fluorescence in situ hybridization (FISH) in effusions from patients with carcinoma

Cytological diagnosis of malignant cells in effusions is hampered by difficulties in the differentiation from reactive mesothelial cells. Because interphase cytogenetics by fluorescence in situ hybridization (FISH) might complement cytological evaluation, we determined the power of tumor cell detection using FISH and cytology in 201 effusions from patients with advanced cancer. Furthermore, 9 primary breast tumors were FISH-karyotyped, and chromosomal aberrations were compared with those of corresponding metastasic effusion cells. By using centromeric probes representing chromosomes 7, 8, 11, 12, 17, and 18, a rate of malignancy-associated aneusomy combined for the 6 chromosomes was detected in an overall of 44.8% of effusion specimens (range, 31.8% to 39.3% for the individual chromosome), comparable to cytology (43.3%). The combination of just 2 FISH probes (namely, representing chromosome pairs 8/11 and 8/17) was almost equally efficient in the identification of aneusomy. Approximately one fourth of the cytologically negative effusions were FISH positive and vice versa. From the initially FISH-negative effusions, 18.9% could be subsequently classified positive with dual-color FISH by visualization of intranuclear chromosomal complexity in rare aneuploid cells. Thus, “overall FISH analysis,” including dual-color evaluation, identified tumor cells in significantly more effusions (55.2%, P = .001) than conventional cytology, implying greater sensitivity. Finally, our finding that numerical aberration patterns in primary breast tumors and corresponding metastasic effusions are comparable indicates that FISH examination of primary tumors will indicate the centromeric probe(s) best suited for an efficient search for metastasis m the individual case.

Detection of cancer cells in effusions from patients diagnosed with gynaecological malignancies. Evaluation of five epithelial markers.

The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination.

Immunocytochemistry and DNA-Image Cytometry in Diagnostic Effusion Cytology I. Prevalence of Markers in Tumour Cell Positive and Negative Smears

To determine the prevalence of immunocytochemical positivities for a panel of antibodies in benign and malignant cells in effusions with known follow‐up in order to use these as diagnostic markers. Besides their ability to identify malignant epithelial cells their contribution to the differential diagnosis between carcinomatoses and mesotheliomas was investigated. 101 tumour cell positive and 53 negative effusions were stained with 12 different antibodies. Results were scored semiquantitatively per cell type. Furthermore, DNA‐image cytometry was performed. While prevalence of Ber‐EP4 positivity was 95.4% in metastatic carcinoma cells, it was 0% in those from mesotheliomas. No cell type reacted with this marker in benign effusions (0%). Ber‐EP4 correctly differentiated between metastatic carcinoma and mesothelioma in 98.0%. Prevalence of DNA‐aneuploidy was 95.4% in metastatic carcinomas, 57.1% in mesotheliomas and 0% in reactive effusions. Combining immunocytochemistry (Ber‐EP4 positivity) and DNA‐image cytometry (aneuploidy) results in a 100% detection of metastatic carcinomatoses and 57.1% of mesotheliomas. Both markers furthermore allowed a correct differentiation of these entities in 98%.

Benign and Malignant Cells in Effusions: Diagnostic Value of Image DNA Cytometry in Comparison to Cytological Analysis

Identifying tumor cells in body cavity fluids reliably is a well-known diagnostic problem. Since cytometric quantitation of nuclear DNA content appears to be a promising new tool in the diagnosis and prognostic evaluation of many solid human tumors, we examined its validity in detecting malignant cells in cytologically positive effusions. For this purpose, image DNA cytometric measurements, including the evaluation of DNA-ploidy and the calculation of the DNA index (DI), were performed in 80 body cavity fluids. The results were correlated with cytology, clinical course and final histological diagnoses. We used aneuploidy, as shown by interactive image DNA cytometry, as a marker for the malignancy of cells that occur in body cavity fluids with a 100% specificity and 94.8% sensitivity. Cytological investigation showed a 92.3% specificity and 95.4% sensitivity. Combining both methods raised the specificity to 100% and the sensitivity to 98.5% and had a positive predictive value of 100% and a negative predictive value of 93.8%. The DNA-index (DI) was significantly higher in malignant effusions than in benign effusions: 1.5 +/- 0.74 (mean +/- SD) versus 1.11 +/- 0.26 (p < 0.05). Along with the difficult cytological evaluation of malignant cells in body cavity fluids, image DNA cytometry can be a helpful additional method for evaluating these cells. Combining the two techniques results in a highly specific and sensitive prediction of malignant cells. We, therefore, suggest using these methods for the reliable identification of tumor cells in effusions.

Interphase fluorescence in situ hybridization improves the detection of malignant cells in effusions from breast cancer patients.

In diagnostic evaluation of effusions, difficulties are encountered when atypical reactive mesothelial cells have to be differentiated from malignant cells. We tested the impact of fluorescence in situ hybridization (FISH) to identify metastatic cells in breast cancer effusions by detection of numerical chromosomal changes. Pleural and ascitic fluid samples (n=57) from 41 breast cancer patients were concomitantly evaluated by routine cytology and FISH, using centromere-specific probes representing chromosomes 7, 11, 12, 17 and 18. After setting stringent cut-off levels deduced from non-malignant control effusions (n=9), the rates of cells with true aneuploidy were determined in each effusion sample from breast cancer patients. The occurrence of aneuploid cells, as detected by FISH and indicative of malignancy, was correlated with the cytological findings. Routine cytology revealed malignancy in 60% of effusions. Using FISH, aneuploid cell populations could be observed in 94% of cytologically positive and in 48% of cytologically negative effusions, thus reverting diagnosis to malignancy. To confirm malignancy in cases with a low frequency of aneuploid cells, two-colour FISH was additionally performed and indeed showed heterogeneous chromosomal aneuploidy within single nuclei. We conclude that FISH is a valuable tool in the diagnosis of malignancy and may serve as an adjunct to routine cytological examination, as demonstrated here for breast cancer effusions.

Cytology of metastatic adenocarcinoma of the prostate in pleural effusions.

Malignant pleural effusions due to prostatic carcinoma are rare. We examined the cytologic and clinical presentations of 14 malignant pleural effusions caused by prostate cancer. These cases represented 2.3% of all positive pleural effusions at our institution. All patients (n = 10) had high grade, high stage tumors, including three with small cell anaplastic carcinoma. Three cases had clinically documented metastases to pleura, and in two cases, metastases were documented at autopsy. Most tumor cells had large nucleoli and were arranged in small, loosely cohesive groups. Fluids due to the small cell type of prostate carcinoma often contained a mixture of cells similar to those seen in small cell carcinoma of other sites such as the lung, as well as cells resembling the more typical type of prostate cancer. Prostatic specific antigen and prostatic acid phosphatase were positive in less than 50% of these malignant effusions. We conclude that prostatic carcinoma in pleural effusions occurs most commonly in high grade, high stage tumors and has a characteristic cytologic appearance. Negative staining for PSA and PAP does not rule out a prostatic source for malignant cells in effusions.

The applicability of a keratin 7 monoclonal antibody in routinely Papanicolaou-stained cytologic specimens for the differential diagnosis of carcinomas.

The monoclonal antibody OV-TL 12/30, which detects keratin 7, was tested for its usefulness in cytologic diagnosis by reincubating previously Papanicolaou-stained slides. For this purpose malignant effusions of 73 patients with histologically confirmed cancers of the colon, ovary, mesothelium, breast, lung, esophagus, pancreas, urinary bladder, stomach, kidney, and prostate were used. All malignant cells from ovarian adenocarcinomas were positive, whereas malignant cells from colonic adenocarcinomas and malignant mesotheliomas were negative. Adenocarcinomas of gastric, renal, pancreatic, esophageal, and mammary origin demonstrated variable staining. Transitional cell carcinomas were positive, whereas squamous and small cell lung carcinomas were negative. Because OV-TL 12/30 does not react with normal and atypical mesothelial cells in these preparations, this reagent is a valuable tool in distinguishing benign mesothelial cells and adenocarcinoma cells. The authors' results demonstrate that this antibody is an excellent tool in the differential diagnosis of malignant cells in effusions and can be used in routinely stained cytologic specimens to determine primary tumor localization. In addition to its ability to distinguish between ovarian and colonic adenocarcinomas, its negativity in mesotheliomas may prove helpful in several diagnostic considerations.

DNA analysis of malignant effusions: Comparison with cytologic diagnosis and carcinoembryonic antigen content : Pinto MM. Department of Pathology, Bridgeport Hospital, 267 Grant Street, Bridgeport, CT 06616. Anal Quant Cytol Histol 1992;14:222-6

Twenty-three consecutive malignant effusions from 19 patients submitted for cytologic examination were analyzed for carcinoembryonic antigen (CEA) content and for DNA analysis by flow cytometry. The study was undertaken to determine if the addition of DNA analysis would improve the sensitivity of cytologic diagnosis and CEA assay. CEA examination was performed on Papanicolaou-stained smears and hematoxylin-and-eosin-stained cell blocks. Final diagnoses were correlated with histologic examination (four patients), clinical and radiologic studies, and follow-up. The malignant effusions in 19 patients were secondary to carcinoma of the breast (5), lung (5), ovary (1), endometrium (1), mucinous carcinoma of the colon (1), unknown primary (1), extraovarian papillary carcinoma (1), mesothelioma (2) and large cell lymphoma (2). The sensitivity of cytologic diagnosis was 100% and specificity 100%. DNA aneuploidy, defined as the presence of two separate peaks in the histogram, was present in 7 of 23 fluids (sensitivity, 30%). Four fluids had insufficient cells for analysis, and one histogram showed debris (following chemotherapy). DNA aneuploidy was detected in effusions secondary to carcinoma of the breast (4), lung (1) and lymphoma (2). Using 5 ng/mL as the cutoff, the sensitivity of CEA was 68%. DNA analysis of cells in malignant effusions is less sensitive than cytologic diagnosis, and CEA assay and is not recommended for routine use in the diagnosis of malignant effusions.

The characterization of peritoneal and pleural exudate cells from malignant effusions

The immune function of peripheral blood cells and cells from the pleural and abdominal effusions of patients with advanced cancer was compared to that of peripheral blood cells from controls. The parameters examined included lymphocyte subsets, natural killer (NK) cell activity, and anti-Daudi and lymphokine-activated killer (LAK) cell activity. The percentage of CD4+ pleural and peritoneal exudate cells (PEC) was significantly higher than the percentage of peripheral blood mononuclear cells (PBMC) in the patients. The percentage of CD8+CD11+ PEC and PBMC, being the suppressor T-cells, of the patients was increased compared with controls, while the percentage of CD8+CD11- PEC, being the cytotoxic T-cells, was identical to the PBMC of both patients and controls. The NK activity of PEC was significantly lower than that of PBMC in both patients and controls, and there was no correlation between the NK activity of PBMC and PEC. Although the anti-Daudi activity of PEC was markedly low, LAK cells with high activity could be induced by culture with interleukin-2 for 4 days. These results suggest that the immune function of cells in malignant effusions may be depressed due to a low population of cytotoxic T cells, low NK activity and increased suppressor T cells, while the local administration of interleukin-2 may induce LAK cells. Therefore, effective local immunotherapy for malignant effusions should not only augment effector cells, but also inhibit suppressor cells.

Expression of c-myc, c-Ha-ras1, and c-erbB-2 Proto-oncogenes in Normal and Malignant Human Breast Epithelial Cells

Short-term cultures of normal human mammary epithelial cells were used to determine the extent to which c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon short-term tissue culture. In every sample, proto-oncogene transcript levels increased upon short-term culture of the epithelial cells. These levels often exceeded by 10-fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the proto-oncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for proto-oncogene expression. The effusion metastasis sample expressed high levels of c-erbB-2 messenger RNA, in accord with its amplified gene copy number; otherwise, the levels of proto-oncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these proto-oncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated proto-oncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer.

Reactivity of tumor cells in malignant effusions with a panel of monoclonal and polyclonal antibodies.

A panel of 13 mouse monoclonal antibodies (mAb) and 1 rabbit polyclonal antibody was tested for reactivity with tumor cells in 26 effusions obtained from patients with carcinoma of ovary, breast, or mesothelioma, using an immunoperoxidase staining reaction. Specific staining of tumor cells but not reactive mesothelial cells was demonstrated with some of the mAb in the panel. In 4 of 26 effusions no evidence of malignancy was obtained after routine cytological staining, but this was reversed on the basis of immunoperoxidase staining of tumor cells with the mAb in the panel. Serial effusions were evaluated in 4 patients during the course of chemotherapy, allowing an assessment of effect of therapy on the antigenic characteristics of the tumor cells. In another 4 patients, the results of immunoperoxidase staining of effusions were compared with those obtained by applying the same antibody panel to solid tumor nodules. There was a tendency to develop changes in the pattern of reactivity during therapy, and the pattern of reactivity was more restricted in tumor nodules than in effusions. One of the mAb in our panel (2G3) was consistently shown to produce strong staining of a high proportion of tumor cells in effusions and tumor nodules from patients with ovarian or breast cancer and may be of value in immunocytochemical diagnosis and therapy of epithelial malignancies.

Diagnostic and Therapeutic Applications of Monoclonal Antibodies to Small Cell Carcinoma of the Lung

For diagnosis and treatment of small cell lung cancer (SCLC), we have developed two different types of monoclonal antibodies, one of which is useful for specific diagnosis of SCLC. The other has been used for purging tumor cell infiltrates from bone marrow in high-dose chemotherapy followed by autologous bone marrow rescue. The monoclonal antibody, TFS-4, is highly specific for SCLC. It reacts with SCLC but is unreactive with squamous cell carcinoma or adenocarcinoma of the lung. It can distinguish SCLC from non-SCLC in histologic diagnosis at transbronchial biopsy, and in cytological identification of tumor cells in malignant effusions and in infiltrated bone marrow. Radiolabeled TFS-4 specifically accumulated into SCLC tumors and depicted a clear-demarcated tumor image with a gamma scintillation camera. The TFS-4 labeled with 131I inhibited the growth of SCLC tumors transplanted into nude mice. TFS-2 antibody demonstrated "pancarcinoma" reactivity, showing binding to SCLC, non-SCLC, and carcinomas derived from other organs such as the colon, pancreas, and stomach. This antibody failed to react with a variety of normal tissues including lung, bone marrow, spleen, stomach, colon, and pancreas. TFS-2 showed complement-mediated cytolysis of target cells. TFS-2 did not inhibit the growth of hemopoietic progenitors committed to granulocyte-macrophage, or erythroid lineage. In mixed cell populations of tumor cells and bone marrow cells, the antibody effectively killed the tumor cells.