Distinguishing malignant mesothelioma from pulmonary adenocarcinoma: an immuno-histochemical approach using a panel of monoclonal antibodies.

A panel of six monoclonal antibodies (MAbs) was employed to evaluate antigen expression in pulmonary adenocarcinomas and mesotheliomas. Monoclonal anti-human milk fat globulin (HMFG-2), anti-carcinoembryonic antigen (NP-2), anti-epithelial membrane antigen (EMA), anti-cytokeratin (PKK-1), anti-tumor-associated antigen 72 (B72.3), and anti-human myelomonocytic antigen (Leu M-1) antibodies were used to localize their respective antigens in formalin-fixed, paraffin-embedded tumors by using the avidin-biotin-complex immunoperoxidase technique. In all, 28 mesotheliomas obtained from Ohio State University Anatomic Pathology files and from a Southwest Oncology Group (SWOG) protocol were compared to 22 pulmonary adenocarcinomas by using this MAb panel. None of the mesotheliomas demonstrated positive staining with MAbs NP-2 (anti-CEA) or Leu M-1. However, 95% (21/22) of adenocarcinomas stained with one of these two antibodies. Although neither of these two MAbs stained all adenocarcinomas, each antibody demonstrated positive immunostaining in more than 90% of the adenocarcinomas studied. Therefore, MABs NP-2 and Leu M-1 are, individually, quite useful for distinguishing mesothelioma from adenocarcinoma. However, in our study, no single MAb could be used to distinguish these two tumor types in every case. MAb B72.3 stained 91% (20/21) adenocarcinomas but also stained 7% (2/28) of mesotheliomas. MAb HMFG-2 reacted positively with 95% of adenocarcinomas, but also stained 39% of the mesotheliomas, usually in a membranous pattern. MAbs EMA and PKK-1 were not found useful in distinguishing mesothelioma from adenocarcinoma. We conclude that MAbs Leu M-1 and NP-2 were both useful in distinguishing mesothelioma from pulmonary adenocarcinoma in that positive staining was demonstrated in adenocarcinomas and not mesotheliomas.

Radioimmunoguided surgery using monoclonal antibody.

The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery.

Intraoperative radioimmunolocalization of colorectal carcinoma with a hand-held gamma probe and MAb B72.3: comparison of in vivo gamma probe counts with in vitro MAb radiolocalization.

A new intraoperative approach to tumor localization using radiolabelled monoclonal antibody (MAb) B72.3 involves the use of a hand-held gamma-detecting probe (GDP) by the surgeon and, subsequently, the pathologist. We report here the use of 125I-labelled MAb B72.3 IgG and a GDP to localize primary and metastatic colorectal cancer in 31 patients. The patients were administered radiolabelled MAb i.v., and all underwent surgical exploration 5 to 35 days post-injection. In vivo localization of the MAb was evaluated using a GDP, with tumor and normal tissue counts being obtained. In each case, the subsequent tumor and normal tissue that were resected were analyzed in vitro for MAb localization; this was evaluated by calculating the radiolocalization index, i.e., the ratio of the injected dose per gram localized to tumor versus that of normal tissue. When the GDP was used intraoperatively, MAb B72.3 localized tumors in 68% (21/31) of the patients; the arbitrary criterion of tumor-to-normal tissue ratios higher than or equal to 2.0:1 in vivo being taken as positive. Resected tumor radiolocalization indices ranged from 0.5 to 543, and 71% (22/31) of the patients studied had tumors with radiolocalization indices higher than or equal to 3. Of 50 carcinoma biopsies, 34 that were probe-positive were antigen-positive when B72.3 was used in immunoperoxidase assays, while 4 carcinoma biopsies that were probe-negative were also antigen-negative. Twelve of 50 biopsies were probe-negative and antigen-positive, but many of these lesions only contained a few antigen-positive cells; none of the 50 was probe-positive and antigen-negative. Tumors of all histologic grades localized injected MAb and, in general, higher in vivo probe ratios and radiolocalization indices were obtained from patients who underwent surgery 20 to 35 days following injection of the MAb. MAb B72.3 localized tumor in all sites to which colon carcinoma commonly metastasizes, including mesenteric and peri-aortic lymph nodes, liver, lung, and peri-rectal soft tissue. There was a strong statistical correlation (p = 0.001) between detecting MAb B72.3 localization to tumors using the GDP intraoperatively and subsequent in vitro analysis of cpm/g for tumor versus normal tissues. These studies thus further validate the use of 125I-labelled MAb B72.3 IgG and of a hand-held gamma probe for the intraoperative detection of carcinomatous lesions.

Limits of sensitivity for the radioimmunodetection of colon cancer by means of a hand held gamma probe.

This study was undertaken to define the limits for the radioimmunodetection of minimal deposits of colorectal cancer cells using a hand held gamma probe. 125I labeled monoclonal antibody 17-1A and its F(ab')2 fragments were reacted in vitro with cells of the human colorectal cancer line SW 1116. The limits of sensitivity of the probe were determined by injecting doubling dilutions of 125I-antibody coated SW 1116 cells ranging from 10(7) to 3.9 x 10(4) subserosally at 2 cm intervals into 60 cm segments of freshly obtained autopsy or surgical specimens of human colon. A linear relationship was observed between the number of cells injected and the number of counts obtained with either the probe or well counter. As few as 6.25 x 10(5) 125I-antibody coated cells (less than 1 mm3) were detected under experimentally defined conditions by an earlier version of the probe, and 3.9 x 10(4) coated cells (much less than 1 mm3) could be detected by the currently available model. Although the count rates were less than 5% of those obtained by well counter, nevertheless, these were 10-25 times greater than background and allowed the detection of tumor cell deposits that otherwise would not have been discernible by either palpation or external scintigraphy. These findings, in conjunction with ongoing clinical studies, suggest that the hand held gamma probe may increase the usefulness of monoclonal antibodies for the radioimmunodetection of cancer.

Tumor-associated antigen expression of primary and metastatic colon carcinomas detected by monoclonal antibody 17-1A.

Murine monoclonal antibody (MAb) 17-1A has been used in radioimmunodetection and immunotherapy trials of intestinal adenocarcinoma in humans. Tumor heterogeneity of antigen expression has been recognized as a potential limiting factor in such studies. The authors report a study designed to evaluate the degree of heterogeneity of 17-1A antigen expression among primary and metastatic human colon carcinomas. All 141 specimens, including 74 primary or metastatic colonic adenocarcinomas, were evaluated with the use of an avidin-biotin complex immunoperoxidase technic on briefly fixed frozen tissue sections. All of these showed at least focal staining with MAb 17-1A. However, well- or moderately differentiated tumors generally showed diffuse cytoplasmic immunostaining, whereas poorly differentiated tumors showed minimal immunostaining with no detectable antigen in most areas. In 16 cases that had both primary and metastatic adenocarcinomas or multiple metastatic tumors, 17-1A antigen expression was similar among the tumor sites except for one case. This case showed variation in tumor differentiation and corresponding variation in 17-1A antigen expression. Of 36 additional malignant tumors that were not of colonic epithelial origin, adenocarcinomas of the stomach, duodenum, endometrium, ovary, and breast showed 17-1A antigen expression.

Radioimmunoguided surgery using the monoclonal antibody B72.3 in colorectal tumors.

The authors have developed a hand-held gamma-detecting probe (GDP) for intraoperative use that improves the sensitivity of external radioimmunodetection. Radiolabeled monoclonal antibody (MAb) B72.3 was injected in six patients with primary colorectal cancer and 31 patients with recurrent colorectal cancer an average of 16 days preoperatively. The GDP localized the MAb B72.3 in 83 percent of sites. The technique, known as a radioimmunoguided surgery (RIGS) system did not alter the surgical procedure in patients with primary colorectal cancer but did alter the approach in 26 percent (8/31) of patients with recurrent colorectal cancer. Two patients avoided unnecessary liver resections and two underwent extraabdominal approaches to document their disease. The RIGS system may influence the short-term morbidity and mortality of surgery for colorectal cancer. Larger series and longer follow-up are needed to determine whether the RIGS system confers a survival advantage to the patient with colorectal cancer.

A unique familial lobular glomerulopathy.

Five members of a family (four brothers and their father) were found to have proteinuria and microhematuria. One of the affected brothers developed chronic renal failure. Although a renal biopsy was never performed, this brother has subsequently received a renal transplant from a clinically unaffected sister. Renal biopsy was performed on four of the siblings (three brothers and the sister). Renal tissue from all three brothers who underwent biopsy showed similar glomerular lesions, characterized by striking cloverleaf expansion of glomerular lobules by an acellular hyaline material and segmental mesangial cell proliferation. Electron microscopy showed massive mesangial and subendothelial deposits and occasional areas of glomerular basement membrane splitting with round inclusions. The renal biopsy specimen of the sister was normal on light and electron microscopy and negative for immunoglobulins and complement on immunofluorescent staining. Immunofluorescence microscopy on one of the male siblings was negative for immunoglobulins and complement. No other physical or serologic abnormalities could be established. To our knowledge, this is a unique form of familial glomerulopathy that has not been previously described.

Extra-adrenal paraganglioma and pulmonary chondroma: a case report and review of the literature.

This report describes the findings of an extra-adrenal paraganglioma and chondroma of the lung in an elderly man. Tissue analysis of the paraganglioma showed a high level of catecholamines, as well as the presence of somatostatin and small amounts of other peptide hormones. Immunoperoxidase staining demonstrated the distribution of peptide hormones in the tumor. This case illustrates two important points. First, it confirms the ability of paraganglionic tissue to secrete peptide hormones. Secondary, it adds yet another case to the unusual group of patients described by Carney (Carney JA: Cancer 43:374-382, 1979).

Diagnostic histochemical and immunohistochemical studies in malignant mesothelioma.

This study was undertaken to evaluate the relative utility of histochemical and immunohistochemical stains in diagnosing malignant mesothelioma of the thorax. We performed a battery of histochemical stains, including periodic acid Schiff (PAS) with and without diastase, mucicarmine, colloidal iron (Coll Fe) with and without hyaluronidase, and immunohistochemical stains for keratin and carcinoembryonic antigen (CEA) on 12 pleural mesothelioma specimens obtained from 11 patients, five primary pulmonary adenocarcinomas, and one metastatic adenocarcinoma each to pleura and pericardium. All diagnoses were established by autopsy or thorough clinical and surgical evaluation. The diagnosis of mesothelioma was established following rigid anatomic criteria. All tissue was formalin fixed and paraffin embedded. Commercially available reagents and antisera were used in all cases. Results showed a high rate of positivity for keratin and hyaluronidase-sensitive Coll Fe in the mesotheliomas while adenocarcinomas were uniformly positive for CEA and keratin and generally positive for PAS-D (diastase) and mucicarmine. Mesotheliomas were negative for CEA, mucin, and PAS-D. Positive keratin staining was also seen in the spindle cell components of mesotheliomas. Immunohistochemical stains often added significantly to our ability to establish the diagnosis of mesothelioma with confidence, since they were more frequently and more clearly positive than histochemical stains.

Radioimmunodetection of implanted tumors with gamma probe.

Tumor uptake of 125I- and 131I-radiolabeled anti-CEA antibodies was compared in female Swiss nude mice, each bearing a CEA-producing human colon adenocarcinoma xenografted in one flank. Counts from the tumor and contralateral flank were recorded with a manipulatable, cadmium-telluride crystal gamma detector at 24, 48, and 72 hours following injection. The animals were killed, and the tumors and other organs were removed, weighed, and then assessed in an automatic gamma counter. The cadmium-telluride counter was more efficient at counting 125I-labeled antibodies than 131I antibodies. The tumor to contralateral flank ratios improved with the use of a monoclonal anti-CEA and polyclonal anti-CEA in combination compared with the single antibodies. The investigation of the external counting characteristics of the portable gamma detector demonstrated the potential of the adjunctive use of intraoperative detection with external radioimmunoscintigraphy for detection and localization of gastrointestinal tumors.

Intraoperative probe-directed immunodetection using a monoclonal antibody.

To assess monoclonal antibody (MAb) 17-1A and its F(ab')2 fragment in intraoperative radioimmunodetection and to evaluate further the clinical usefulness of a hand-held gamma-detecting probe (GDP), we injected radiolabeled monoclonal antibody 17-1A three to six days preoperatively or its F(ab')2 fragment two to three days preoperatively into 18 patients with colorectal cancer. Intraoperative GDP counts with tumor-tissue ratios of 1.5:1 or greater were obtained from 15 (75%) of 20 tumor sites, with ratios averaging 2.3:1 for fragments and 3.4:1 for whole antibody. The GDP counts contributed to intraoperative decision making in three patients, either by localization of tumor not identified by inspection or palpation or by mapping margins of resection with histologic confirmation of a local/regional recurrence. These preliminary data demonstrate that probe-directed, intraoperative radioimmunodetection can assist the surgeon in detecting subclinical tumor deposits and thus better evaluate the extent of primary or recurrent colorectal cancers intraoperatively.

The response of a non-functional VIP- and somatostatin-containing tumour to tolbutamide in vitro.

A patient with a tumour containing clinically non-expressive somatostatin (SRIF) and vasoactive intestinal peptide (VIP) was studied in vivo with basal and tolbutamide-provoked SRIF and VIP measurements and failed to respond to tolbutamide infusion. An acute cell dispersion model was used to study this tumour after resection. Incubation of tumour cells in tolbutamide (2 mg/ml) resulted in increases in intracellular SRIF but not in the levels of SRIF released into the incubating medium. In contrast, incubation of tumour cells with tolbutamide decreased supernatant (extracellular) and total (intracellular) VIP by 50%, suggesting a local peptide-peptide modulation of VIP release by high intracellular levels of SRIF or, alternatively, suppression of VIP synthesis and/or release by tolbutamide. Failure of 'nonfunctional' tumours to produce symptoms or abnormal plasma peptide levels may be due to defects in peptide release or complex paracrine peptide-peptide interactions.

Immunohistochemical evaluation of human placental implantation: an initial study.

Immunohistochemical staining for human chorionic gonadotropin and factor VIII-related antigen with the avidin-biotin complex immunoperoxidase technique was used as a marker for syncytiotrophoblast and endothelial cells, respectively, in the human placental bed. Material from placental implantation sites at varying stages of gestation (8 weeks to term) was studied. Trophoblastic invasion of the uterine stroma and blood vessels were evaluated. Syncytiotrophoblasts lining placental villi and anchoring villi were positive for human chorionic gonadotropin at all stages of gestation studied. Endothelial cells lining maternal uterine blood vessels were positive for factor VIII-related antigen. At early stages of intrauterine placentation (8 and 11 weeks) trophoblastic invasion of uterine blood vessels and trophoblastic incorporation in the walls of dilated vessels were present. An unexpected finding, however, was the large number of giant cells in the superficial placental bed which had morphology suggestive of syncytiotrophoblast but which were negative for human chorionic gonadotropin. In addition, many enlarged, rather pleomorphic cells lining superficial blood vessels were found to be positive for factor VIII-related antigen, which identified them as endothelial cells and not migrating trophoblastic elements. This study demonstrates that human chorionic gonadotropin and factor VIII-related antigen immunoperoxidase staining is a helpful adjunct in evaluating human placentation and suggests extension of the technique with use of other antibodies to evaluate components of the placental bed.

Prostatic carcinoma metastatic to bone: sensitivity and specificity of prostate-specific antigen and prostatic acid phosphatase in decalcified material.

Decalcified bone marrow biopsies containing metastatic tumor from 36 patients were stained for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) using the avidin biotin complex (ABC) immunoperoxidase technique. Of these patients, 22 had known prostate primaries, ten had known nonprostatic, and four female patients had unknown primaries. Prostate-specific antigen was identified in 86% (19/22) of the metastatic prostatic carcinomas. Prostatic acid phosphatase was present in only 36% (8/22). None of the patients with nonprostatic primaries or unknown primaries showed positive staining for either antigen (0/14). This study indicates that immunoperoxidase staining for PSA is very sensitive and specific in the diagnosis of metastatic prostate carcinoma, while PAP was less sensitive using decalcified bone marrow specimens. We believe that immunostaining with PSA should be of great value in diagnosis of prostatic carcinoma metastatic to the bone.

Monoclonal antibodies directed against preneoplastic and neoplastic murine mammary lesions.

We have produced a panel of monoclonal antibodies directed against a dimethylbenzanthracene-induced murine mammary tumor. Five rat-mouse hybridomas produced antibodies that bound to some murine mammary tumors, but not to normal renal adherent cells, lymphocytes, 3T3 fibroblasts, red blood cells, or mammary gland. One of these antibodies, designated AMT8, was selected for further evaluation based on its relatively strong reactivity, as determined by immunofluorescence. Indirect immunofluorescent studies on frozen histological tissue sections and quantitative immunofluorescent binding studies on cultured normal and tumor cells revealed that AMT8 was bound to certain murine mammary tumors and their preneoplastic hyperplastic nodules, but not to normal murine organs including normal mammary glands. Two tumors and their hyperplastic alveolar nodule counterparts that contained the antigen recognized by AMT8 did not express functional estrogen and progesterone receptors, indicating that antigen expression was not dependent on functional receptors. The antigen recognized did not cap, was found to modulate slowly, and was reexpressed in the presence of excess AMT8. From these findings, we conclude that AMT8 may prove to be a valuable tool for the study of early mammary tumorigenesis.

Granular-cell tumor of the breast. A cytologic, immunohistochemical and ultrastructural study of two cases.

Two primary granular-cell tumors of the breast are reported. Cytologically and histologically, these tumors had the classic features of small, eccentrically located nuclei and numerous periodic acid-Schiff-positive cytoplasmic granules. The characteristic cytologic features were best appreciated in touch imprints, not in frozen sections. Immunohistochemically, the tumors demonstrated diffuse cytoplasmic staining for S-100 protein but negative staining for myoglobin. The significance of these immunohistochemical staining characteristics, particularly in evaluating the possible histogenesis of these tumors, is discussed. The ultrastructural features of these two tumors are also presented and compared to findings reported by other investigators.