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Expression of Hypoxia-Inducible Cell-Surface Transmembrane Carbonic Anhydrases in Human Cancer

2001; Elsevier BV; Volume: 158; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)64038-2

1525-2191

Sergey V. Ivanov, Shu‐Yuan Liao, Alla Ivanova, Alla Danilkovitch‐Miagkova, Nadya I. Tarasova, Gregor Weirich, Marsha J. Merrill, Martin Proescholdt, Edward H. Oldfield, Joshua Lee, J Závada, Abdül Waheed, William S. Sly, Michael I. Lerman, Eric J. Stanbridge,

Nitric Oxide and Endothelin Effects

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors. An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors. The development of solid human tumors may be approximated by a two-stage model. In stage one, the malignant cells grow into small tumors, which then stop growing because of an inadequate supply of oxygen (hypoxia). 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3: 122-129Abstract Full Text Full Text PDF Scopus (116) Google Scholar, 15Kaelin Jr, WG Maher ER The VHL tumour-suppressor gene paradigm.Trends Genet. 1998; 10: 423-426Abstract Full Text Full Text PDF Scopus (200) Google Scholar pVHL is an integral part of a novel multiprotein ubiquitin ligase complex, termed VBC (VHL/Elongin B/Elongin C), that recruits important cellular proteins for rapid degradation by the ubiquitin-proteasome proteolysis system.16Tyers M Willems AR One ring to rule a superfamily of E3 ubiquitin ligases.Science. 1999; 284: 408-414Crossref Scopus (141) Google Scholar, 17Tyers M Rottapel R VHL: a very hip ligase.Proc Natl Acad Sci USA. 1999; 96: 12230-12232Crossref PubMed Scopus (45) Google Scholar The most plausible candidate targets for this proteolytic degradation include HIF1-α and HIF2-α.8Maxwell PH Wiesener MS Chang GW Clifford SC Vaux EC Cockman ME Wykoff CC Pugh CW Maher ER Ratcliffe PJ The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.Nature. 1999; 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In VHL-defective tumors, curiously enough, the two fundamental stages of tumor development occur either simultaneously or in reverse, first triggering the hypoxia-cellular response, followed by proliferation of transformed cells, consistent with the angiogenic phenotype of tumors seen in the VHL syndrome. Previously, we discovered that in VHL patients the CA9 and CA12 genes are overexpressed in tumors because of the absence of pVHL, and argued that these CA enzymes might be involved in sensing and maintaining the acidic tumor microenvironment.10Ivanov SV Kuzmin I Wei MH Pack S Geil L Johnson BE Stanbridge EJ Lerman MI Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes.Proc Natl Acad Sci USA. 1998; 95: 12596-12601Crossref PubMed Scopus (334) Google Scholar We have now extended these observations by analyzing the expression of CA IX and CA XII in a large sample of cancer cell lines and fresh or archival tumor specimens. Here we show that either one or both genes are overexpressed in many tumor types, suggesting that this is a common feature of cancer cells, consistent with the fundamental role of VHL in tumor development. We believe that overexpression of these enzymes contributes to the acidic tumor microenvironment and helps the cancer cells to grow and metastasize. We used 45 mRNA samples isolated from cancer cell lines provided by the Developmental Therapeutics Program, Frederick Cancer Research and Development Center, National Cancer Institute. Additional mRNA samples from central nervous system (CNS) tumor cell lines U105MG, U251MG, U373MG, and G4; leukemia cell lines U937 and HL60; the renal cell carcinoma cell line UM-RC-29; and the monocyte cell line THP-1 were kindly provided by Dr. Teizo Yoshimura (NCI-Frederick, Frederick, MD). Non-small cell carcinoma (H1373, H1264, H1693, H1944, H838, H1299, H157, H1466, H460, H727, and H28), small-cell carcinoma cell lines (H1184, H2081, H2227, H1086, H841, H69, H1820, H660, H1769, H446, H1238, H748, and H2552), cancer cell lines SCC-35, MCF-7-adr, SQ-20B, 510-HPV-18, and Scid (5020) were kindly provided by Dr. Bruce Johnson (NCI, Bethesda, MD). The human glioblastoma cell line, U87, was purchased from ATCC (Manassas, VA). RNA samples from other cancer cell lines were available on the Clontech MTN blot no. 7757-1, and samples from normal tissues on MTE Array no. 7775–1 blot (Clontech, Palo Alto, CA). Total RNA and mRNA isolation from cell lines and tumor tissues was done using commercially available kits (Invitrogen, Carlsbad, CA; Life Technologies, Inc., Grand Island, New York). Electrophoresis in formaldehyde gels and Northern blot analysis were performed according to published procedures.10Ivanov SV Kuzmin I Wei MH Pack S Geil L Johnson BE Stanbridge EJ Lerman MI Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes.Proc Natl Acad Sci USA. 1998; 95: 12596-12601Crossref PubMed Scopus (334) Google Scholar, 19Heiss JD Papavassiliou E Merrill MJ Nieman L Knightly JJ Walbridge S Edwards NA Oldfield EH Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor.J Clin Invest. 1996; 98: 1400-1408Crossref PubMed Scopus (225) Google Scholar Quantification of Northern hybridization signals was done as described previously10Ivanov SV Kuzmin I Wei MH Pack S Geil L Johnson BE Stanbridge EJ Lerman MI Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes.Proc Natl Acad Sci USA. 1998; 95: 12596-12601Crossref PubMed Scopus (334) Google Scholar or using a VE-1000 Video Camera System (Dage-MTI, Inc., Michigan City, IN) and NIH Image Version 1.6.1 software. The normal adult tissues from all organ sites and the corresponding benign and/or malignant neoplastic tissues were obtained from routine pathology specimens at St. Joseph Hospital (Orange, CA). These organs include the brain (cerebrum, cerebellum, ventricle, pons, pituitary gland), eyes, nose, throat, upper and lower respiratory system, heart, upper and lower gastrointestinal system (esophagus, stomach, small and large intestine), pancreas, liver, biliary system including gallbladder, female and male urogenital system (kidney, ureter, bladder, testicle, cervix, uterus, fallopian tube, ovary), adrenal gland, thyroid, parathyroid, salivary gland, spleen, bone, muscle, cartilage, skin, and the body cavity. All tissue samples were processed within 6 hours of surgical resection and fixed in 10% neutral-buffered formalin or snap-frozen. The formalin-fixed tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopic examination. The mouse monoclonal antibody (MN75) used to detect the MN/CA IX protein and the rabbit polyclonal antibody to CA XII protein have been described previously.20Zavada J Zavadova Z Pastorekova S Ciampor F Pastorek J Zelnik V Expression of MaTu-MN protein in human tumor cultures and in clinical specimens.Int J Cancer. 1993; 54: 268-274Crossref PubMed Scopus (180) Google Scholar, 21Karhumaa P Parkkila S Tureci O Waheed A Grubb JH Shah G Parkkila A Kaunisto K Tapanainen J Sly WS Rajaniemi H Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium.Mol Hum Reprod. 2000; 6: 68-74Crossref PubMed Scopus (79) Google Scholar Immunohistochemical staining of tissue sections with anti-CA IX and anti-CA XII antibodies was done using a peroxidase technique with microwave pretreatment, as described previously.22Liao SY Brewer C Zavada J Pastorek J Pastorekova S Manetta A Berman ML DiSaia PJ Stanbridge EJ Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial neoplasia and cervical carcinoma.Am J Pathol. 1994; 145: 598-609PubMed Google Scholar Known positive and negative tissue specimens were included in each run. For CA IX immunostaining, the primary antibody was used at a 1:10,000 dilution and the CA XII at a 1:500 dilution. The immunohistochemical results were semiquantitative based on the percentage of the positive cells seen in a total field of a single section. The pattern of staining was scored as diffuse when ≥40% of the cells stained and focal when <40% of the cells stained. A negative score was given to tissue sections that had no evidence of specific immunostaining. Renal clear-cell carcinoma cell line 786-0 and its derivative, expressing the wtVHL transgene, were described earlier.10Ivanov SV Kuzmin I Wei MH Pack S Geil L Johnson BE Stanbridge EJ Lerman MI Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes.Proc Natl Acad Sci USA. 1998; 95: 12596-12601Crossref PubMed Scopus (334) Google Scholar They were grown to confluence in Dulbecco’s modified Eagle’s medium (cat. no. 11965-084; Life Technologies, Inc.) with 10% bovine fetal serum (Sigma, St. Louis, MO). Three independent experiments on pH and glucose measurements in the media were done using a Hanna Instruments microcomputer pH meter (model HI931000) and Infinity Glucose Reagent (Sigma Diagnostics, cat. no. 18-20). U87 cell culture and maintenance of hypoxia were performed as previously described.19Heiss JD Papavassiliou E Merrill MJ Nieman L Knightly JJ Walbridge S Edwards NA Oldfield EH Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor.J Clin Invest. 1996; 98: 1400-1408Crossref PubMed Scopus (225) Google Scholar Using MTE array blots containing 68 different mRNAs from a variety of normal adult tissues, we observed that the CA12 gene was highly expressed in kidney, colon and rectum, esophagus, brain, and the pancreas, whereas the mammary gland, bladder, uterus, trachea, and aorta showed low levels of expression. In the brain, only the corpus striatum [caudate nucleus (E2) and putamen (H2)] produced strong hybridization signals with the CA12 probe (Figure 1). Moderate expression of CA9 was observed in a limited number of tissues that included stomach and, to an even lesser extent, heart, liver, pancreas, and salivary gland (Figure 1). These data were consistent with hybridization results obtained with the MTN blots. In addition, low signals were also observed in brain and placenta (data not shown). We detected no significant differences in the distribution of CA12 expression in a variety of fetal tissues compared to normal adult tissues (Figure 1, column 11; and data not shown). In the case of CA9, however, fetal lung and muscle demonstrated high levels of expression whereas adult lung and muscle were negative (data not shown). Northern blot analyses of mRNAs obtained from a large number of cancer cell lines and freshly excised tumors are summarized in Table 1 and Table 2, and an example of a Northern blot is given in Figure 2. Among 87 RNA samples, representing tumors of the head and neck, lung, kidney, cervix, ovary, prostate, breast, colon, skin, and several leukemias, 50 (57%) expressed either one or both genes at variable intensity, whereas 37 were negative (Table 1 and Figure 2). Non-small cell lung cancer, colon and renal cell carcinoma cell lines, and tumors of the CNS showed the highest proportion of specimens expressing CA9 or CA12 (68, 78, 89, and 78%, respectively). Fourteen of the 18 tumors (78%) of the CNS and spinal cord showed expression of the genes (Table 2). Among these tumors 11 expressed both genes and three expressed either CA9 or CA12. It was previously shown and extensively documented in this study that expression of CA IX protein may serve as a diagnostic biomarker for clear cell renal cell carcinoma22Liao SY Brewer C Zavada J Pastorek J Pastorekova S Manetta A Berman ML DiSaia PJ Stanbridge EJ Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial neoplasia and cervical carcinoma.Am J Pathol. 1994; 145: 598-609PubMed Google Scholar, 23Liao SY Aurelio ON Jan K Zavada J Stanbridge EJ Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney.Cancer Res. 1997; 57: 2827-2831PubMed Google Scholar, 24McKiernan JM Buttyan R Bander NH Stifelman MD Katz AE Chen MW Olsson CA Sawczuk IS Expression of the tumor-associated gene MN: a potential biomarker for human renal cell carcinoma.Cancer Res. 1997; 57: 2362-2365PubMed Google Scholar and colorectal tumors,25Saarnio J Parkkila S Parkkila AK Haukipuro K Pastorekova S Pastorek J Kairaluoma MI Karttunen TJ Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation.Am J Pathol. 1998; 53: 279-285Abstract Full Text Full Text PDF Scopus (221) Google Scholar, 26Saarnio J Parkkila S Parkkila AK Waheed A Casey MC Zhou XY Pastorekova S Pastorek J Karttunen T Haukipuro K Kairaluoma MI Sly WS Immunohistochemistry of carbonic anhydrase isozyme IX (MN/CA IX) in human gut reveals polarized expression in the epithelial cells with the highest proliferative capacity.J Histochem Cytochem. 1998; 46: 497-504Crossref PubMed Scopus (144) Google Scholar whereas CA XII has been identified as a biomarker for non-small-cell lung cancer (10; US patent no. 5,589,579).Table 1Expression of CA9/CA12 mRNA in Human Cancer Cell LinesTumor typeCA12-positiveCA9-positiveCA9/CA12-negativeHead and Necknot doneSCC-35(2), SQ-20B(2)Lung non-small cell carcinomaNCI-H1264(1), NCI-J460(3), NCI-H1944(1), NCI-H157(1), NCI-H28(1), A549(10), EKVX(1), HOP62(1), HOP92(2)NCI-H1373(1), NCI-H1264(1), NCIH1466(1), NCI-H460(1), NCI-H727(2)NCI-H1693, NCI-H838, NCI-H1299, NCI-H226, NCI-H23, NCI-H522Lung small cell carcinomaNCI-H2081(1), NCI-H69(1), NCI-H1769(1)NCI-H1184, NCI-H2227, NCI-H1086, NCI-H660, NCI-H1820, NCI-H841, NCI-H446, NCI-H1238, NCI-H748, NCI-H2552Renal clear cell carcinomaUM-RC-6(3), UM-RC-29(2*In these cell lines truncated mRNA was observed.), 786-0 (2), ACHN(1), RXF-393(3), TK-10(1)786-0 (3), A498(1), RXF-393(2)CAKI-1 (VHL†VHL, this cell line expresses active pVHL.), UO-31CNS tumorsU105MG(1), U373MG(2), G4(2), SF-295(1), SF-539(2), SNB75(2)U105MG(2), U373MG(3), U251MG(2), G4(1), SF-539(1), SNB19(2), SNB75(2)SF-268LeukemiaMOLT-4(1), K-562(1*In these cell lines truncated mRNA was observed.), SR(3)K-562(1)HL60, U937, CCRF-CEM, Burkitt's lymphoma RajiColon tumorsHT29(2), KM12(1)COLO 205(2), HCC-2998(1), HCT-15(3), HT29(3), KM12(1), SW480(1)HCT-116Cervix tumorsHeLa S3(2)HeLa S3(2)Mammary tumorsT 47D(2)MDA-N, MDA-MB-435, MDA-MB-231, MCF-7, NCI/ADR-RES, HS 578TOvary tumorsOVCAR-5 (1), SK-OV-3 (2)SK-OV-3 (1)OVCAR-3, OVCAR-4, OVCAR-8Prostate tumorsPC-3(1)PC-3(1)DU-145MelanomaMALME-3M (1)LOX IMVI, M14, SK-MEL-2Expression intensity in arbitrary units (in parentheses).* In these cell lines truncated mRNA was observed.† VHL, this cell line expresses active pVHL. Open table in a new tab Table 2CA9/CA12 mRNA Expression in CNS and Spinal Cord Tumors, Based on Northern Blot AnalysisSample numberTumor diagnosisVHL statusCA12 expression*Intensity of expression is shown in arbitrary units and based on β-actin calibration.CA9 expression*Intensity of expression is shown in arbitrary units and based on β-actin calibration.1Meningiomawt—12Anaplastic astrocytomawt——3Meningiomawt124Meningiomawt125Glioblastomawt4106Glioblastomawt——7Glioblastomawt——8Astrocytomawt149Glioblastomawt2—10Cerebellar hemangioblastomawt——11Meningiomawt1—12Hemangioblastomamut2213Cerebellar hemangioblastoma (VHL-)mut6414Cerebellar hemangioblastoma (VHL-)mut6315Spinal hemangioblastoma (VHL-)mut2116Spinal hemangioblastoma (VHL-)mut3417Cerebellar hemangioblastoma (VHL-)mut5418Hemangioblastoma (VHL-)mut56* Intensity of expression is shown in arbitrary units and based on β-actin calibration. Open table in a new tab Expression intensity in arbitrary units (in parentheses). High levels of CA IX expression were consistently observed in the basal cells in and near the infundibulum and medulla of the hair follicle, mesothelial cells, and coelomic epithelium of the body cavities. In the visceral organs, high levels of CA IX expression in the epithelium were identified but limited to rete ovarii, rete testis, ductular efferens, bile ducts, pancreatic ducts, and gallbladder. In the gastrointestinal tract, diffuse CA IX immunoreactivity was observed in the gastric mucosa, ductal cells of Brunner’s glands, and crypt cells of the duodenum, jejunum, and, to a lesser degree, in the terminal ileum and appendix. In the peripheral and central nervous systems, CA IX expression was limited to the ventricular lining cells and the choroid plexus. Interestingly, mesodermal cells of the amniotic/chorionic plate of the placenta and cartilaginous tissues from joint spaces also showed variable degrees of CA IX protein expression. CA XII was variably expressed in mesothelial cells and the coelomic epithelium of the body cavity. In general, high levels of CA XII expression were found in certain tissues that were CA IX-negative. These tissues were the distal convoluted tubules and the intercalated cells of the collecting duct of the kidney, sweat glands of the skin, the epithelium of the breast, some of the proliferative endometrial glands, and seminal vesicles. Low levels of CA XII expression were also found focally in ductal cells and mucous cells of the salivary glands and submucosal glands of the upper respiratory system, epithelial cells of Schneider’s membrane of the nose, and acinar cells of the pancreas. Limited numbers of positive epithelial cells were also found in the prostate, vas deferens, and transitional mucosa of the renal pelvis. In the gastrointestinal tract, CA XII expression was observed, but was limited to the surface glands of the large intestine only. Very weak immunoreactivity of CA XII was also found in the gastric glands. In the peripheral and central nervous system, the CA XII immunoreactivity was restricted to the posterior lobe of the pituitary glands, remnant of Rathke’s pouch, the choroid plexus and limited numbers of ganglion cells in the cortex. In the placenta, limited numbers of syncytiotrophoblasts were immunoreactive. The only normal tissues co-expressing CA IX and CA XII were mesothelial cells, ductular efferens, and, to a lesser degree, the choroid plexus and gastric glands. A summary of the distribution of expression in normal tissues is given in Table 3, and selected normal tissues with high expression of CA IX and/or CA XII are illustrated in Figure 3.Table 3The Distribution of CA IX and CA XII Protein Expression in Normal Adult Human TissuesCA IXCA XIILining cells of body cavity Mesothelial cells (serous membrane)DiffuseFocal Coelomic epithelium (surface of the ovary)DiffuseFocal Reactive mesothelial cellsDiffuseDiffuse Underling stellate stromal cellsFocalFocalOral cavity/upper respiratory system Ductal and mucous cells of submucosal glandsNegativeFocal Ductal cells of salivary glandsFocalFocal Mucous cells of salivary glandsNegativeFocal Epithelial cells of Schneider's membraneNegativeFocal Reactive reserve cells of respiratory epitheliumRareRareLower respiratory system Reactive reserve cells of respiratory epitheliumRareRareGastrointestinal system Gastric pitsFocalNegative Gastric fundus/pyloric glandsDiffuseFocal Ductal cells of the Brunner's glands of the duodenumDiffuseNegative Crypt cells of duodenum, small intestine, appendixDiffuse/FocalNegative Crypt cells of large intestineRareNegative Surface glandular cells of large intestineNegativeFocalPancreas Ductal cellsFocalNegative Acinar cellsNegativeFocalGallbladder/biliary tractDiffuseNegativeGenito-urinary system Kidney Distal convoluted ductsNegativeDiffuse Intercalated cells of the collecting ductNegativeDiffuse Renal pelvis Transitional cellsNegativeFocal Prostate gland Ducts and glandsNegativeRare Seminal vesiclesNegativeFocal Testis Ductular efferensDiffuseDiffuse Rete testisDiffuseNegative Uterine cervix Basal cells of squamous mucosaNegativeFocal Reactive Reserve cells of the glandsRareRare Uterine endometrial glands Proliferative phaseNegativeFocal Secretory phaseNegativeNegative Ovary Surface coelomic epitheliumDiffuseFocal Rete ovariiDiffuseNegativeBreast Lobular and ductal unitsNegativeFocalSkin Basal cells of epidermisNegativeFocal Basal cells of hair follicleDiffuseNegative Sweat glandsNegativeDiffuseSkeletal system Cartilaginous tissues near joint spacesFocalNegative Skeletal muscleFocalNegativeCentral nervous system Neuron of the cerebellum and cerebrumNegativeRare Choroid plexusDiffuseDiffuse Lining cells of the ventricleFocalNegative Posterior lobe of pituitary glandNegativeDiffuse Remnant of Rathke's pouchNegativeDiffusePlacenta Mesodermal cells of amniotic/chorionic plateFocalNegative SyncytiotrophoblastsNegativeFocalAll normal adult human tissues were examined. Those not listed in the table were negative for both CA IX and CA XII protein expression.Diffuse, ≥40% of cells within a field stain pos