Immunohistochemical diagnosis of prostatic cancer with metastasis

An immunohistochemical method for detecting prostatic acid phosphatase is described for the diagnosis of metastatic prostatic carcinoma. The specific antiserum against prostatic acid phosphatase was prepared from rabbit by injection of acid phosphatase purified from seminal fluid. This method gives a selective staining of the cytoplasm of the glandular epithelial cells of prostatic tissue specimens on paraffin section. Most of the non-prostatic tissues were negative except for occasional weak staining in granulocytes, islet cells of pancreas, parietal cells of stomach, tubular epithelial cells of kidney, and liver cells. Also examined were 50 consecutive cases of metastatic tumor involving the bone marrow and 5 cases of metastatic prostatic carcinoma involving the lymph node or lung. All 20 cases with prostatic primary lesion showed positive staining. All other cases were negative, except 5 of the 14 cases of metastatic breast carcinoma in women showing weakly positive results. The method is fairly specific for identification of metastatic prostatic carcinoma. Occasional positive staining in breast tumor needs further study to establish whether the staining is due to the same isoenzyme or to certain cross immunoreactivity.     

Histochemical and immunohistochemical characterization of surgically resected and heterotransplanted Wilms' tumor

Nine surgically resected Wilms' tumors (WIT) and nude mouse heterotransplants from one WIT were studied by histochemistry and immunohistochemistry. Histochemistry showed acid phosphatase in all cells, while alkaline phosphatase and gamma-glutamyl transpeptidase were present in only some tubules. Using immunohistochemistry, antibodies to the intermediate filaments cytokeratin and vimentin distinguished tubular epithelium and mesenchyme, respectively. WIT tubules were also identified using antibody against a structural component (epithelial membrane antigen) and a secretory product (uromucoid) associated with distal convoluted tubules of normal kidney. Basement membrane surrounding the tubules of WIT was demonstrated using antibody to type IV collagen plus laminin. Different blastema subpopulations were negative or stained positively with antibodies to cytokeratin and vimentin. Production of basement membrane by blastema was also shown. Fetal antigen expression in WIT was examined using the monoclonal PI 153/3 and J5 antibodies. The blastema and tubules of WIT were strongly stained by PI 153/3, which did not label normal adult kidney, and weakly stained by J5, which strongly labeled glomeruli and proximal convoluted tubules of normal kidney. These studies show that WIT blastema is heterogeneous in intermediate filament subtypes, while WIT tubules more closely resemble distal than proximal convoluted tubules of adult kidneys but also retain expression of fetal antigens.     

Expression of the epitope recognized by the monoclonal antibody 44-3A6 during human fetal development.

In this report we describe the expression of the adenocarcinoma associated antigen recognized by the monoclonal antibody 44-3A6, in various tissues during normal human fetal development. Conventional, formalin-fixed and paraffin-embedded sections of normal organs were examined from fetuses ranging from 9 to 42 weeks of gestation. Immunohistochemical localization of antigen-antibody complexes was accomplished using the avidin-biotin complex (ABC) method using horseradish peroxidase. The monoclonal antibody (MAb) 44-3A6 detects a cell surface 40 kD protein which is frequently expressed by adenocarcinomas and by select normal glandular tissues. Detectable expression of this protein was seen at different time periods during fetal development depending on the tissue. This expression was confined to a relatively small range of cell types and tissues; immunostaining was noted in select epithelial cells of the aerodigestive tract, exocrine pancreas, neural tissues, renal tubules, and transitional urothelium, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances, once expression occurred within a tissue, it continued through gestation. These data show that this tumor associated gene product is differentially expressed in a broad range of normal developing human fetal tissues.     

A Mr 58,000 glycoprotein specifically expressed in developing hamster pancreas and reexpressed in hamster and human pancreatic carcinomas

A glycoprotein with a molecular weight of 58,000 specifically expressed in exocrine hamster fetal pancreas was characterized using a monoclonal antibody (Mab B4). By immunoperoxidase, Mab B4 stained pancreatic tissue from the 10th day of gestation (6 days before delivery) until the 10th day after birth. The maximal expression of the Mab B4-specific protein called fetal pancreatic (FP) protein was reached between delivery and the 5th day of postnatal life. Endocrine pancreas was negative at any developmental stage. All adult pancreata examined were negative. Moreover, Mab B4 was tested against a wide variety of fetal and adult tissues; only immature pancreata were stained. Chemically induced pancreatic carcinomas were strongly stained by this Mab. On the contrary, other tumors (liver and kidney) appearing simultaneously with pancreatic carcinomas were negative. Using a nitrocellulose immunofixation assay, FP protein was found in all sera from hamsters bearing pancreatic tumors (23 cases tested). This protein was not detected in normal sera. Mab B4 cross-reacted with a protein in human fetal pancreas extracts, that behaves similarly to the hamster FP protein: it is present exclusively in exocrine fetal pancreas and is reexpressed in pancreatic adenocarcinomas. The high tissue specificity of this protein, its oncofetal character, and release into the blood circulation make the FP protein a potential tumor marker of pancreatic cancer.     

Acid phosphatase localization in prostatic carcinoma. A comparison of monoclonal antibody to heteroantisera

A series of 39 prostatic carcinomas was characterized in terms of grade, stage, histologic pattern, and serum acid phosphatase values. These cases were studied immunohistochemically with two different heteroantisera, a goat and rabbit antiserum, and with a monoclonal antibody to prostatic acid phosphatase (PAP). Eighty-three percent of carcinomas had some degree of PAP positivity when stained by the goat anti-PAP. Seventy percent were positive with the rabbit antiserum, and 59% showed positivity with the monoclonal antibody. Microacinar patterns were consistently the most positive for PAP, followed by cribriform patterns. The least positivity was observed in the undifferentiated, single-file and sheet-like patterns. Likewise, there was more PAP positivity in the lower Gleason and Mostofi grades. When the serum PAP positivity (done by counterimmunoelectrophoresis [CIEP]) was compared with tissue positivity (using the same goat antiserum), 37% were positive in both serum and tissue; 48% were negative in serum, but positive in tissue; and in only 9% the tissue sample was negative when the serum was positive. Based on these data, conclusions are drawn about the significance of the serum acid phosphatase elevations and the role of monoclonal antibodies and heteroantisera in clinical-diagnostic and research work.     

Location and distribution of difucoganglioside (VI3NeuAcV3III3Fuc2nLc6) in normal and tumor tissues defined by its monoclonal antibody FH6

The distribution of a novel difucoganglioside (6B ganglioside, NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer) in various normal adult and fetal tissues, as well as in cancer tissues, has been studied by immunoperoxidase staining with a specific monoclonal antibody, FH6, directed to this antigen. A large variety of embryonic and fetal tissues (stomach, colon, small intestine, pancreas, esophagus, lung, and heart) showed a diffuse, weakly positive staining, particularly in the epithelial layer, up to the 70th to 80th day of gestation. However, no staining was observed in various normal adult tissues, including gastrointestinal and glandular epithelial tissues which were stained positively by antibody N-19-9 (directed to sialyl-Lea) or CSLEXI (directed to sialyl-Lex). FH6-positive loci were limited to the proximal convoluted tubuli in kidney and granulocytes. In contrast, 44 of 76 cases of cancer tissue tested, including gastric, colonic, lung, breast, and renal cancers, showed clearly positive staining. The intensity of staining in gastric and colonic cancer tissues by FH6 antibody was weaker and less frequent, although the incidence of positive staining for lung (50%) and breast cancer (86%) was significantly higher than that of the antigen stained by monoclonal antibody FH4 (Y. Fukushi, S. Hakomori, and T. Shepard, J. Exp. Med., 159: 506-520, 1984), which is directed to the asialo core of the FH6 antigen. The antigen levels in the serum of patients with various cancers, inflammatory diseases, and normal subjects were determined by radioimmunoassay. The antigen level was found to be significantly higher in the serum of some patients with cancer, particularly lung, liver, and pancreatic cancers, as compared with the serum levels in other types of cancer, noncancerous diseases, and normal subjects.     

An immunohistological study of testicular germ cell tumours using two different monoclonal antibodies against placental alkaline phosphatase.

Using two monoclonal antibodies directed against placental alkaline phosphatase (H17E2 and D20L) the immunohistological staining of testicular germ cell tumours was compared with that of a wide range of normal and malignant tissues. All seminomas and malignant teratomas tested gave strong positive labelling with H17E2 but were either negative or only patchily positive with D20L. Neither antibody gave any positive reaction on the normal tissues tested. All other malignancies were negative with both antibodies apart from two cases of ovarian and one case of endometrical cancer (strongly stained by H17E2) and three cases of colonic carcinoma (weakly and patchily stained by both H17E2 and D20L). This indicates that germ cell neoplasms generally express a form of placental alkaline phosphatase recognised by antibody H17E2.     

Production of three monoclonal antibodies specifically reactive respectively with the ductal, acinar and islet cells of the human pancreas.

Three monoclonal antibodies, designated FP-1, FP-2 and FP-3, were obtained by immunizing a BALB/c mouse with dispersed human fetal pancreatic cells and using hybridoma technology. FP-1, FP-2 and FP-3 reacted specifically, respectively, with the ductal epithelial, acinar and islet cells, of the fetal pancreas as well as with adult human pancreatic tissue, suggesting a use in the discrimination of pancreatic cell types. Upon screening various tumor tissues, FP-1 was found to react with adenocarcinomas of the pancreas (16/23), stomach (7/8), colon (4/6) and gall bladder (2/2) as well as with the three malignant cell lines, PANC-1 (pancreas), HT-29 (colon) and LoVo (colon). In contrast to FP-1, FP-2 and FP-3 reacted with only 5% (2/39) and 0% (0/39), respectively, of these adenocarcinomas. Although FP-3 did not react with any of these adenocarcinomas, it did react with various APUDoma cells such as islet cell tumors of the pancreas (3/5), pheochromocytomas (2/2), medullary thyroid carcinomas (2/3) and gastrointestinal carcinoids (1/3). FP-3 thus appears to be the first monoclonal antibody with extremely high endocrine cell specificity.     

Immunohistological distribution of 5T4 antigen in normal and malignant tissues

A trophoblast cell surface antigen has been characterised by a monoclonal antibody (mAb) 5T4, raised following immunisation with solubilised wheat germ agglutinin binding glycoproteins from human syncytiotrophoblast plasma membrane (StMPM). The expression of the 72 kDa glycoprotein was assessed on cryostat sections of a range of neoplastic and non-neoplastic tissues, using an avidin-biotin immunoperoxidase technique. In products of conception, intense reactions were noted with villous syncytiotrophoblast membrane in normal early and term placenta, with weaker positivity of placental site trophoblast. Most normal or non-neoplastic tissues were negative, including liver, kidney, spleen, small intestine, ovary and testis. Faint or moderate positive reactions were present in some specialised epithelia. Of 115 neoplasms examined, 76 showed reactions with tumour cells including carcinomas of the bladder, breast, cervix, endometrium, lung, oesophagus, ovary, pancreas, stomach and testicular non-seminomatous germ cell tumours. Choriocarcinomas and placental site trophoblastic tumours were also positive. Most adenocarcinomas of colon and seminomas were negative as were all malignant melanomas and malignant lymphomas. A radioimmunoassay did not detect the antigen in either normal or pregnancy serum. The relatively low level of expression in normal tissues and reactivity with a wide range of carcinomas suggested that the antibody may be useful in diagnostic or targeting studies.     

Application of monoclonal antibody KH2 against lactosyl and neolactotetraosyl ceramide to immunohistochemical study of human cancers

Monoclonal antibody KH2 (IgM) reactive with lactosyl ceramide and neolactotetraosyl ceramide was applied for the first time to the staining of human cancerous and noncancerous tissues which were fixed with formalin and embedded in paraffin. Digestive-tract cancers originating from stomach, colon, pancreas, gallbladder and bile duct were clearly stained. The staining intensity was especially strong in the apical portion and the intraluminal content of adenocarcinomas. In noncancerous tissues, it was of interest that gastric intestinal metaplasia and certain fetal tissues were stained. A part of the tubules of the kidney, pancreatic ductal cells, lung alveolar epithelial cells and polymorphonuclear leukocytes were stained in normal adult tissues in our study. Enhancement of the intensity of immunostaining by neuraminidase treatment indicated that most of the antigenic determinants should be sialylated and the antigenicity exposed after neuraminidase treatment in several kinds of cancerous and noncancerous tissues. These data suggest that the monoclonal antibody KH2 may be useful in the immunohistologic diagnosis of formalin-fixed human cancerous tissues.     

Concentration and localization of carbohydrate antigen 19-9 in tissues of pancreatic cancer

Carbohydrate antigen (CA) 19-9 concentration in the tissue-extracts of cancerous and noncancerous pancreatic tissues were determined by radioimmunoassay. Pancreatic cancer tissues revealed significantly elevated CA 19-9 concentrations, when compared with chronic pancreatitis, normal adult pancreas, or fetal pancreas tissues. Metastatic liver tumors from pancreatic cancer also showed extremely high CA 19-9 concentrations, and there was no significant correlation between tissue CA 19-9 concentration and serum CA 19-9 level in patients with pancreatic cancer. In addition, positive localization of CA 19-9 was clearly observed in cancer cells of pancreatic cancerous tissues by immunohistochemical study, confirming the remarkable increase of CA 19-9 concentration in tissues of pancreatic cancer, although CA 19-9 was also partially found in non-malignant pancreatic tissues. The results indicated that CA 19-9 would be produced in great quantities by cancer cells in tissues of pancreatic cancer, and thus could be a valuable tumor associated antigen suggesting its clinical use as a tumor marker for cancer of the pancreas.     

Immunohistochemistry of carcino-embryonic antigen in the embryo, fetus and adult

This study concerns the immunohistologic distribution of carcino-embryonic antigen (CEA) in tissues and organs from 86 legal abortions, stillborn fetuses and perinatal deaths and from 5 adults without inflammatory disease or cancer. Monospecific antibodies to CEA of both polyclonal and monoclonal origin were applied to serial sections obtained from formalin-fixed, paraffin-embedded tissue blocks. Starting from the 9th week of gestational age, a positive staining reaction for CEA was found in the surface epithelium of the tongue, the tracheal mucosa and the following locations of the gastro-intestinal tract: the gastro-oesophageal junction, the pyloric antrum, the upper duodenum, throughout the colon and appendix. In the adult, CEA was also found at these sites. All other organs such as the central nervous system, lung, thyroid, thymus, liver, pancreas, gastric corpus, spleen, adrenals, kidney, ureter, bladder, gonads and breast were negative for CEA. Therefore, CEA appears to be a normal antigen in the gastro-intestinal tract at any age from fetal life onwards.     

Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies

With the aid of a highly specific murine monoclonal antibody, F12, an immunofluorescence method was elaborated that allowed sensitive and specific detection of the ganglioside antigen fucosyl-GM1 (IV2FucII3NeuAcGgOse4Cer) in different types of human lung cancer and normal tissues. Nineteen of 21 cases of small cell lung cancer were positive with the F12 immunofluorescence method as compared to 2 of 10 squamous epithelial cell lung cancers and 1 of 5 large cell lung cancer specimens. Specimens of lung adenocarcinoma (8 cases) and bronchial carcinoid (3 cases) were all negative, as were 2 examined cases of neuroblastoma. No fucosyl-GM1 could be detected in normal lung and bronchus. However, in thymus, spleen, and lamina propria of the small intestine sparsely distributed clusters of small round cells were stained as well as intramural ganglionic cells of the small intestine and islet cells of the pancreas. All other normal tissues tested were negative. Results obtained with immunofluorescence closely agreed with immunochemical determination of fucosyl-GM1 in lipid extracts of tissues. Our findings suggest that fucosyl-GM1 is strongly associated with small cell cancer of the lung and demonstrate that this tumor-associated antigen can be detected with high sensitivity and specificity with an immunofluorescence method based on the use of the F12 monoclonal antibody.     

Tamm-Horsfall protein in human renal tumours.

Tamm-Horsfall Protein (THP) is found to be localised in the ascending loop of Henle and the distal tubules of normal and foetal kidneys. We have used an anti-THP monoclonal antibody (D2) to stain foetal, normal and kidney tumours. The dinitrophenyl hapten staining technique was the most specific and sensitive staining procedure. The staining of Tamm-Horsfall Protein in foetal and normal kidney was in agreement with most other investigators. The majority of renal cell carcinoma (22/24) and rhabdoid renal tumours (3/4) stained positively for THP. None of the Wilms' tumours, mesoblastic nephromas or bone metastasising renal tumours of childhood gave positive staining. The findings would suggest that renal cell carcinomas and rhabdoid kidney tumours arise from ascending loop of Henle or distal tubules. The lack of positive staining in the majority of childhood renal tumours raises the possibility of a different histogenesis for these neoplasms.     

Murine monoclonal antibodies to colon-specific antigen p

A previous report described the production of monoclonal antibodies Mu-1, -2, -3, and -4 against colon-specific antigen p. We now report the tissue distribution of the reactive epitopes. Each of the monoclonal antibodies was reactive with epitopes shared by the entire length of the intestine, with Mu-2 and Mu-3 determinants being present in the stomach as well. Mu-1 and Mu-4 appeared to have a more restricted distribution than did Mu-2 and Mu-3, which were present in several nongastrointestinal tissues. Unfortunately, these MAbs were not ideal for in vivo use against colorectal carcinoma. Mu-1 was not present in neoplastic colon tissue, Mu-2 and Mu-4 were reactive with granulocytes, and Mu-3 was reactive with kidney tubules and connective tissue. An additional fusion was performed from which Mu-9 was selected for further study. Among normal adult tissues this monoclonal antibody gave reactivity restricted to the intestines. The stomach and all nonintestinal tissues were negative by immunoperoxidase. Among colon tumors 60% were positive for the Mu-9 epitope. The data suggest Mu-9 may be a good candidate for in vivo targeting of diagnostic and therapeutic agents to colon cancer.     

A new cancer-associated antigen defined by a monoclonal antibody against a synthetic carbohydrate chain

Carbohydrate antigens can be designed by referring to previously defined carbohydrate structures. We have generated a novel monoclonal antibody (MAb) (Flα-75) against an artificially designed antigen (Flα), using organic-synthetic chemistry methods and hybridoma technology. Flα (GalβI → 4GlcNAcβI → 6GalNAαI → Ser/Thr) belongs to core type 6 of O-linked glycans, which has not been previously reported in human cancers. To produce antibodies against Flα, a glycolipid was synthesized which carries the carbohydrate portion of Flα on a ceramide foundation (GalβI → 4GlcNAcβI → 6GalNAcαI → Cer). The MAbs we obtained (Flα-75, Flα-87) specifically recognized Flα and had only a very weak or no cross-reactivity with other glycolipids similar to Flα. We investigated the expression of Fin in human tissues, including 110 gastric cancers, 73 colon cancers and 42 pancreatic cancers. Flα was found in human cancerous tissues but not in normal adult tissues. The rate of positive staining with Flα-75 was 80.0% for gastric cancer, 52.4% for pancreatic cancer and 38.4% for colon cancer. Flα-75 also reacted with the tissues neighboring gastric and pancreatic tumors but not intensely. Among fetal tissues, Flα-75 reacted with the pyloric glands of the stomach, the centro-acinar cells of the pancreas, the convoluted tubules of the kidney and the terminal bronchioles of the lung.     

Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues

Reduced drug accumulation due to overexpression of individual members of the ATP binding cassette (ABC) superfamily of membrane transporters has been investigated as a cause of multidrug resistance and treatment failure in oncology. This study was designed to develop an immunohistochemical assay to determine the expression and localization of the 72kDa ABC half-transporter ABCG2 in normal tissues. Formalin-fixed, paraffin embedded archival tissue from 31 distinct normal tissues with an average of eight separate tissue samples of each were immunostained with rabbit-anti-ABCG2 antibody 405 using a modified avidin-biotin procedure. As a negative control, each sample was also stained with antibody pre-adsorbed with peptide to assess background staining. As a means of verification, selected tissues were also stained with the commercially available monoclonal antibody 5D3. ABCG2 positivity was consistently found in alveolar pneumocytes, sebaceous glands, transitional epithelium of bladder, interstitial cells of testes, prostate epithelium, endocervical cells of uterus, squamous epithelium of cervix, small and large intestinal mucosa/epithelial cells, islet and acinar cells of pancreas, zona reticularis layer of adrenal gland, kidney cortical tubules and hepatocytes. Placental syncytiotrophoblasts showed both cytoplasmic and surface staining. Our results support a hypothesis concluding that ABCG2 plays a role in the protection of organs from cytotoxins. However, many of the cell types expressing ABCG2 have a significant secretory function. These data suggest a dual function for ABCG2 in some tissues: the excretion of toxins and xenobiotics including anti-cancer agents and a potential, as-yet undefined role in the secretion of endogenous substrates.     

ERGIC3, which is regulated by miR‐203a,is a potential biomarker for non‐small cell lung cancer

In a previous study, we found that ERGIC3 was a novel lung cancer‐related gene by screening libraries of differentially expressed genes. In this study, we developed a new murine monoclonal antibody (mAb) against ERGIC3. This avid antibody (6‐C4) is well suited for immunohistochemistry, immunoblotting and solid‐phase immunoassays. Furthermore, we systematically investigated expressions of ERGIC3 in a broad variety of normal human tissues and various types of tumors by immunohistochemistry. In normal human tissues, 6‐C4 reacted only in some epithelial cells, such as hepatocytes, gastrointestinal epithelium, ducts and acini of the pancreas, proximal and distal tubules of the kidney, and mammary epithelial cells; however, most normal human tissues were not stained. Moreover, almost all carcinomas that originated from the epithelial cells were positive for 6‐C4, whereas all sarcomas were negative. Notably, 6‐C4 strongly stained non‐small cell lung cancer (NSCLC) cells but did not react with normal lung tissues. Hence, ERGIC3 mAb could be used in histopathological diagnosis and cytopathological testing to detect early‐stage NSCLC. We also studied the mechanisms of ERGIC3 regulation in vitro and in vivo by means of bioinformatics analysis, luciferase reporter assay, miRNA expression profiling and miRNA transfection. Results showed that miR‐203a downregulation induced ERGIC3 overexpression in NSCLC cells.     

Common antigens found on fetal cells and viral transformed adult prostatic tissue.

In vivo and in vitro immunologic cross reactivity between SV40 virus transformed prostatic tissue and fetal antigens of LVG/LAK strain hamsters has been studied. The hamsters immunized with fetal antigens demonstrated significant resistance to tumor growth. Complement-dependent humoral cytotoxicity of target SV40 tumor cells in microtest plates was used to demonstrate the presence of significant antibody titer in fetal-immunized hamsters. When the immune sera were absorbed with SV40 transformed prostatic tissue, cytotoxicity indices were markedly lower than with the unabsorbed immune sera. The transformed prostatic tissue was found to have an elevated tartrate inhibited acid phosphatase indicative of the presence of epithelial components in the transformed tissue. These experiments demonstrate in vivo and in vitro cross reactviity between fetal antigens and tumor associated antigens of transformed prostatic tissue. This study suggests a similarity between fetal antigens and antigens of a specific organ tissue (prostate) transformed by a DNA oncogenic virus.     

Monoclonal antibody (anti-Leu 7) directed against natural killer cells reacts with normal, benign and malignant prostate tissues

The reactivity of monoclonal antibody (MAb) anti-leu 7 (HNK-1) on formalin-fixed sections of prostate tissues was determined by an immunoperoxidase assay. Anti-Leu 7 was found to react with both primary and metastatic tissues from 6/6 normal prostate, 15/15 benign prostatic hyperplasia, and 37/39 prostate carcinoma samples. Anti-Leu-7 reactivity was localized in the cytoplasm of the supranuclear region of prostatic epithelial cells and the fibromuscular stroma did not stain. Myelinated nerve fibers in the stroma were stained with anti-Leu 7 and this served as an internal control. Anti-Leu 7 did not bind to prostatic acid phosphatase (PAP) or prostate-specific antigen (PSA) in direct or competitive binding radioimmunoassays. Anti-Leu 7 was more effective (5/5) in the identification of metastatic tumors of prostate origin than an MAb to PSA (2/5). The differential pattern of reactivity of anti-Leu 7 compared to anti-PSA on serial sections of prostate tissues suggests that Leu 7 may be a useful diagnostic and prognostic marker of prostate cancer.