American Journal of Pathology, Vol. 141, No. 4, October 1992 Copyright © American Association of Pathologzt
The Expression of the Hu (Paraneoplastic Encephalomyelitis/Sensory Neuronopathy) Antigen in Human Normal and Tumor Tissues Josep Dalmau,*t Henry M. Furneaux, Carlos Cordon-Cardo,t and Jerome B. Posner*t From the Departmnts of Neurology* and Pathologyt and the Cotzias Laboratory of Neuro-Oncology,* Memorial Sloan Kettenng Cancer Center, New York, New York
Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronalprotein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also e-xpress the antigen (Am J Pathol 1992, 141:881-886)
Paraneoplastic encephalomyelitis (PEM) and paraneoplastic sensory neuronopathy (PSN) are two rare, often overlapping neurological syndromes associated with cancer, usually small cell carcinoma of the lung (SCLC).1 2 Symptoms include dementia, cerebellar degeneration, brain stem encephalopathy, myelopathy, and sensory neuropathy.1 Pathologically, the disease is characterized by neuronal death, gliosis, and inflammatory infiltrates of the neuraxis.13 Patients with PEM/PSN and SCLC harbor in their serum and cerebrospinal fluid high titers of a highly specific antibody called anti-Hu.4 5 The anti-Hu antibody reacts with a set of protein antigens (molecular weight 35 to 40 kd) expressed in neurons and SCLC cells.356 The intrathecal synthesis of the antibody,7 and the detection of deposits of anti-Hu IgG3 and Hu-specific infiltrating lymphocytes in the nervous system and tumor,69 have suggested an immunologic origin of the disorder. According to this model, the expression of the Hu antigen by the tumor triggers an immune re-
sponse that is misdirected against similar proteins expressed in brain.9 Using a limited number of lung cancer cell lines, a previous study demonstrated the presence of the Hu antigen in all SCLCs whether or not the tumor came from a patient who made the antibody.'0 However, the spectrum of expression of the Hu antigen in tumor tissues from lung or other neoplasms is not known. To address this issue and to determine the value of the antiHu antibody as a diagnostic reagent to identify SCLC cells, we now undertook to study by immunohistochemistry and Western blot analysis in a series of normal human tissues and 1 15 tumors of different histologic types.
Materials and Methods
Normal and Tumor Tissues Normal human tissues were obtained either from biopsy or autopsy performed within 10 hours of the patient's death. One hundred six tumors of various histologic types were obtained from the tumor procurement service of
Memorial Sloan Kettering Cancer Center; the investigators had no knowledge of the patients' clinical states. In nine additional patients, the tumor was studied because the patient developed anti-Hu-associated PEM/PSN; two of these tumors were provided by the tumor procurement service of Memorial Sloan Kettering Cancer Center and seven were sent to us by physicians of other centers. Tissues were snap frozen in isopentane chilled by liquid nitrogen, embedded in Optimal Cutting Temperature Compound (Miles Inc, Elkhart, IN), and kept frozen at - 700C. Presented in part at the 43rd meeting of the American Academy of Neurology, Boston, Massachusetts, April 21-27, 1991. Supported by grants FISS 91/5722 Spain (JD), ACS PD-359 (HMF), and NIH NS 26064 (JBP). Accepted for publication April 16, 1992. Address reprint requests to Dr. Jerome B. Posner, Department of Neurology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.
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From seven SCLC and one chondromyxosarcoma, only tissue sections from paraffin blocks were available.
Results
Normal Tissues
Immunohistochemical Analysis of the Expression of the Hu Antigen Anti-Hu IgG was prepared from high-titer sera of patients with anti-Hu-associated PEM/PSN. The anti-Hu IgG was conjugated to biotin as previously described.11 By using the anti-Hu antibody conjugated with biotin, we avoided the use of a labeled secondary (anti-human IgG) antibody that would immunoreact with the nonspecific IgG of the tissue and obscure the results of the immunohistochemical study. Seven-micron-thick frozen tissue sections were fixed for 10 minutes in acetone and sequentially incubated with 0.3% hydrogen peroxide (to destroy tissue peroxidase activity) and 10% normal human serum (to prevent nonspecific binding of human IgG). The sections then were incubated with biotinylated anti-Hu IgG (2.5 ,ug/ml) for 12 hours at 4°C. Bound human antiHu IgG was visualized by incubation with avidin-biotin peroxidase and developed with 0.05% diaminobenzidine tetrahydrochloride. Incubation of the section with biotinylated normal human IgG served as negative control. Tissues that immunoreacted with the biotinylated antiHu IgG were considered to express the Hu antigen if the immunoreactivity was abolished by preincubation with another anti-Hu serum. Tissue sections from paraffin blocks were deparaffinated and rehydrated,6 and processed in the same way as described for the frozen sections.
Western Blot Analysis of the Expression of the Hu Antigen Serial 30-micron-thick frozen tissue sections were collected and washed in phosphate-buffered saline (PBS). Tissues then were homogenized in PBS with 0.1% Nonidet P-40 and centrifuged at 1 5,000g for 10 minutes, and the supernatant was retained. The protein content was measured according to the method of Bradford.12 The indicated amounts of protein were subjected to electrophoresis on 10% sodium dodecyl-polyacrylamide gel and transferred to nitrocellulose as described by Towbin et al.13 The nitrocellulose filters were blocked with 5% blotto and incubated with the indicated amount of anti-Hu sera for 6 hours at room temperature. The nitrocellulose filters then were washed with TRIS buffer (0.1 mol/l [molar] TRIS, pH 8.0; 0.2 mol/l sodium chloride; 0.2% Triton X-1 00; and 0.1% bovine serum albumin), incubated with 1251-labeled protein A (0.1 ,uCi/ml) for 1 hour at room temperature, washed with TRIS buffer, and exposed to X-ray film for 6 hours.
The expression of the Hu antigen in normal human tissues is shown in Table 1. Immunoreactivity was found highly restricted to the central and peripheral nervous tissue. All neurons tested reacted with the anti-Hu antibody. Immunoreactivity predominated in the nuclei, sparing the nucleoli, and with weaker staining of the cytoplasm (Figure 1A). The medullary chromaffin cells of the adrenal gland and ganglion cells of bronchus immunoreacted with the anti-Hu antibody with a pattern of immunostaining identical to neurons. All other normal tissues examined did not express the Hu antigen. Western blot analyses of homogenates of several normal human tissues are shown in Figure 2. The Hu antigen was found expressed only in brain tissue. Homogenate of bronchus was negative, indicating the low number of cells that were immunoreactive by immunohistochemistry. The 55-kd band observed in most of the tissues corresponds to the heavy chain of endogenous IgG. This band was present in the absence of added antibody (data not shown), and indicates direct binding of 1251labeled protein A.
Tumors The expression of the Hu antigen
was
studied in 115
tumors by immunohistochemistry (45 tumors), Western
blot (19 tumors),
or
both (51 tumors) (Table 2). Nine
Table 1. Expression of the HUAntigen
Tissue (#) Brain (5) Adrenal gland (medullary) (3) Bronchus (3) Lung (3) Ovary (3) Testis (3) Kidney (5) Prostate (3) Bladder (3) Spleen (4) Jejunum (4) Colon (4) Thyroid gland (3) Parathyroid gland (3) Liver (5) Pancreas (3) Muscle (5) Skin (3) (#)
=
by Normal Tissues
Immunohistochemistry + (chromaffin +
cells)
Western blot ND
(ganglion cells)
ND
+ +
(myenteric plexus) (myenteric plexus)
ND ND
number of samples examined for each type of tissue.
Expression of the Hu Antigen
883
AJP October 1992, Vol. 141, No. 4
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Figure 1. Immunohistochemical analysis of the expression of the HU antigen. A: Section of hippocampus (fascia dentata)from a neurologically normal individual incubated with biotinylated anti-Hu antibody. Immunoreactivity predominates in the nuclei of the neurons, with weaker staining of the cytoplasm, and sparing the nucleoli. Glial cells do not immunoreact with the anti-Hu antibody. B: A consecutive section of normal hippocampus incubated with biotinylated normal IgG sbows no immunoreactivity and serves as negative control. C: Metastatic lymph node from a patient with SCLC an anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. Immunoreactivity is observed in all the neoplastic cells; normal tissue contained in the lymph node[identified with Tand B cell markers (not shown)] does not immunoreact with the anti-Hu antibody. D: Chondromyxosarcomafrom apatient with anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. Immunoreactivity predominates in the nuclei of the tumor cells. E: Neuroblastoma from a patient without anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. Immunoreactivity predominates in the nuclei of the neoplastic cells. F: Adenocarcinoma of ovary from a patient without anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. No immunoreactivity is observed All sections have been counterstained with hematoxylin.
SOLO tumors, obtained in frozen blocks, were studied by immunohistochemistry and Western blot analysis; the seven SCLC tumors available in paraffin blocks were studied by immunohistochemistry. All SCLC (16/16) expressed the Hu antigen. Only 1 of the 25 non-SCLC
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21, Figure 2. Western blot analysis of the expression of the HU antigen by normal tissues. Immunoblots of tissue homogenate (80 pg each) reacted with serum (diluted 1:4000) from a patient with anti-Hu associated PEM/PSN. C7X = brain cortex, OV = ovary, TES = testis, PRT = prostate, BLD = bladder, SPL = spleen, COL = colon, THY = thyroid gland, PTH = parathyroid gland, LG = lung, BRC = bronchus, PAN = pancreas.
ing sarcomas, one melanoma, one chondromyxosarcoma, one small cell carcinoma of stomach, and one undifferentiated carcinoma of the prostate. Immunoreactivity predominated in the nuclei of the neoplastic cells, with weaker staining of the cytoplasm (Figure 1 C, D, E). No immunoreactivity was observed when the tumors were incubated with biotinylated normal IgG. The anti-Hu antibody served to differentiate normal from neoplastic tissue in those tumors where normal and neoplastic cells were highly similar (Figure 1 C). For all the tumors, except one melanoma, results were identical using either immunohistochemistry or Western blot. The immunohistochemical study of the melanoma was considered negative, but the Western blot showed a weak band at 37 kd (data not shown). Furthermore, for all the Hu-positive tumors the pattern of Western blot analysis was similar, showing a positive discrete band at 37 kd (Figure 3). Medical records of the Hu-positive tumors obtained after the tumor study showed no history of paraneoplastic symptoms. Nine tumors were studied because the patients developed paraneoplastic symptoms. They included seven SCLC, one chondromyxosarcoma, and one undifferentiated tumor of the prostate. Clinical and immunopathologic studies of five of these patients have been reported elsewhere.314
Discussion We have studied the expression of the Hu antigen in 1 15 human tumors of different histologic types. In lung tumors, the Hu antigen was found selectively expressed in SCLC. The practical implication of this selective expression is that the anti-Hu antibody becomes a useful reagent to identify SCLC. Under normal circumstances, the Hu antigen is restricted in its expression to neurons of the central and peripheral nervous system. Because of its location and homology to the drosophila proteins embryonic lethal, abnormal visual system (ELAV) and sex lethal, the Hu is thought to be an RNA-binding protein that plays a role in neuronal development.9 In a very small number of patients with cancer, a high titer of antibody recognizing the Hu protein is found in the serum and cerebrospinal fluid and is associated with a neurologic disorder called paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/PSN).5615 A high titer (average 4600 U/ml) of antiHu antibody generally implies the presence of a SCLC, but rarely may be associated with other tumors, including neuroblastoma, sarcoma, or prostate carcinoma.14 A low titer of anti-Hu antibody (average, 80 U/ml) is found in
Expression of the Hu Antigen 885 AJP October 1992, Vol. 141, No. 4
15% of patients with SCLC but without neurologic dysfunction.5 Previous studies have shown the Hu antigen to be expressed in the SCLC of patients with paraneoplastic neurological disorders associated with the presence of the anti-Hu antibody,3'6 and in SCLC cell lines whether or not the tumor came from a patient who made the antibody.10 Using tumor tissues, we now demonstrate that in addition to all SCLC, a small percentage of tumors other than lung also express the Hu antigen. This is not surprising because many of the tumors that express the Hu antigen have already been shown to express other neuronal antigens and are thought to be neuroectodermal in origin.1619 Although the serum of these patients was not assayed for antibodies against the Hu antigen, we would not expect that they make antibodies because at least 85% of patients with SCLC (all of which express the Hu antigen) do not make a detectable antibody response.5 Thus, the crucial question is why a small number of patients with Hu-positive tumors make either a modest or a profound immunologic response to an antigen that is expressed in their tumor. One possible explanation is that most of these tumors evade immune surveillance by turning off the expression of major histocompatibility (MHC) class antigens.?-22 In preliminary experiments we have found that all the Hu tumors from paraneoplastic patients still have in their cell surface MHC class antigens (data not shown). This model, however, does not explain why a small number of patients mount an immune response against a putative normal neuronal protein. One theory is that neuron-specific proteins are not presented to the imTable 2. Expression of the HU antigen by Tumors HU positive/ total studied Tumor histology
Non-SCLC (A) Small cell cancer other than lung (#) Neuroblastoma Melanoma Merkel cell tumor Sarcoma (*) Adenoca. prostate Thymoma Renal carcinoma Breast adenoca. Seminoma Adenoca. ovary Bladder carcinoma Adenoca. colon Adenoca. pancreas
16/16 1/25 1/3 4/8 1/5 2/2 3/7 1/10 0/2 0/4 0/10 0/2 0/7 0/5 0/4 0/5
(A) = includes eight squamous, eight adenocarcinomas, two large cell, three bronchiolar, one mixed lung carcinoma, one anaplastic carcinoma of the lung (Hu +), and two carcinoid tumors; (#) includes one small cell cancer (SCC) of stomach (Hu +), one SCC of bladder, and one SCC of unknown origin. (*) = includes two osteogenic sarcomas, one osteosarcoma, one rabdomyosarcoma, one chondromyxosarcoma, and two Ewing tumors.
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-
Cl 25 26 27 28 '29 30' 31 32 33 34
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Cl C2 03 35 36 37 38 39 40 41 42 Figure 3. Western blot analysis of the expression of the HUantigen by tumors. Immunoblots of tissue homogenate (80 1'g each tumor) reacted with serum (diluted 1.4000) from a patient with anti-Hu associated PEM/PSN C = controls, Cl = crude neuronal preparation (rows 1, 3, 4,5), homogenate of brain cortex frow 2), C2 = homogenate of SCLC, C3 = adenocarcinoma of the lung. Lanes 1 to 5 = non-SCLC; 6-9 adenocarcinoma of the prostate; 10-13 renal cell carcinoma; 14-17 melanoma; 18-19 adenocarcinoma of the breast; 20-21 adenocarcinoma of the ovary; 22-24 neuroblastomas; 25-27 adenocarcinoma of the colon; 28-31 adenocarcinoma of the pancreas; 32-34 carcinoma of the bladder; 3538 sarcomas (37 Ewing); 39 thymoma; 40 Merkel cell tumor, 41 SCLC, 42 small cell metastasis (primary tumor unknown).
mune system during the establishment of immune toler-
ance,23 and that MHC proteins are not expressed in neurons.24 The de novo expression of the Hu protein in tumors (that express MHC) then may result in a profound immune reaction. According to this theory, however, one would expect a diverse immunoresponse against the other many neuronal-specific proteins whose expression has been demonstrated in a variety of tumors, especially SCLC.1 19 We believe that in addition to the coexpression of MHC class and Hu antigen by the tumor, other factors are involved in the pathogenesis of the immune disorder. One model would be that the sequence of the Hu protein is altered and perceived as foreign. This model can now be studied by analyzing the structure of the Hu mRNA in the tumors of patients with PEM/PSN.9 A practical implication of this model would be the development of monoclonal antibodies specifically directed against the tumor form of Hu antigen.
Acknowledgments The authors thank Dr. Myma R. Rosenfeld for helpful discussion and reading of the manuscript; Dr. Pilar Garin, who provided us
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with several tumors; and B. Nevins for patience and diligence in preparing the manuscript.
References 1. Henson RA, Urich H: Encephalomyelitis with carcinoma: Cancer and the nervous system. Oxford, England, Blackwell Scientific, 1982, pp 314-345 2. Furneaux HM, Posner JB: Paraneoplastic neurological syndromes. Proc Assoc Res Nerv Men Dis 1990, 68:187-219 3. Dalmau J, Furneaux HM, Rosenblum MK, Graus F, Posner JB: Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy. Neurology 1991, 41:1757-1764 4. Graus F, Cordon-Cardo C, Posner JB: Neuronal antinuclear antibody in sensory neuronopathy from lung cancer. Neurology 1985, 35:538-543 5. Dalmau J, Fumeaux HM, Gralla RJ, Kris MG, Posner JB: Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer: A quantitative Western blot analysis. Ann Neurol 1990, 27:544-552 6. Graus F, Elkon KB, Cordon-Cardo C, Posner JB: Sensory neuronopathy and small cell lung cancer: Antineuronal antibody that also reacts with the tumor. Am J Med 1986, 80: 45-52 7. Furneaux HM, Reich L, Posner JB: Central nervous system synthesis of autoantibodies in paraneoplastic syndromes. Neurology 1990, 40:1085-1091 8. Dalmau J, Furneaux HM, Posner JB: Identification of Huantigen specific lymphocytes in the tumor and brain of patients with paraneoplastic subacute sensory neuronopathy (abstr). Proc Am Assoc Cancer Res 1991, 32:272 9. Szabo A, Dalmau J, Manley G, Rosenfeld M, Wong E, Henson J, Posner JB, Fumeaux HM: HuD, a paraneoplastic encephalomyelitis antigen, contains RNA binding domains and is homologous to Elav and Sex-Lethal. Cell 1991, 67: 325-333 10. Budde-Steffen C, Anderson NE, Rosenblum MK, Posner JB: Expression of an antigen in small cell lung carcinoma lines detected by antibodies from patients with paraneoplastic dorsal root ganglionopathy. Cancer Res 1988, 48:430-434 11. Furneaux HM, Rosenblum MK, Dalmau J, Wong E, Woodruff P, Graus F, Posner JB: Selective expression of Purkinje cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration. N Engl J Med 1990, 322:18441851 12. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254
13. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979, 76:4350-4354 14. Dalmau J, Graus F, Rosenblum MK, Posner JB: Anti-Hu associated paraneoplastic encephalomyelitis/sensory neuronopathy: A clinical study of 71 patients. Medicine 1992, 71:59-72 15. Anderson NE, Rosenblum MK, Graus F, Wiley RG, Posner JB: Autoantibodies in paraneoplastic syndromes associated with small-cell lung cancer. Neurology 1988, 38:13911398 16. Cunningham JM, Lennon VA, Lambert EH, Scheithauer B: Acetylcholine receptors in small cell carcinomas. J Neurochem 1985, 45:159-167 17. Lloyd RV, Warner TF: Immunohistochemistry of neuronspecific enolase, Advances in Immunohistochemistry. Edited by RA DeLellis. New York, Masson Publishing USA, 1984, pp 127-140. 18. Lipinski M, Hirsch MR, Deagostini-Bazin M, Yamada 0, Tursz T, Goridis C: Characterization of neural cell adhesion molecules (NCAM) expressed by Ewing and neuroblastoma cell lines. Int J Cancer 1987, 40:81-86 19. Djakiew D, Delsite R, Pflug B, Wrathrall J, Lynch JH, Onoda M: Regulation of growth by a nerve growth factor-like protein which modulates paracrine interactions between a neoplastic epithelial cell line and stromal cells of the human prostate. Cancer Res 1991, 51:3304-3310 20. Doyle A, Martin WJ, Funa K, Gazdar A, Carney D, Martin SE, Linnoila I, Cuttitta F, Mulshine J, Bunn P, Minna J: Markedly decreased expression of class histocompatibility antigens, protein, and mRNA in human small cell lung cancer. J Exp Med 1985, 161:1135-1151 21. Tanaka K, Isselbacher KJ, Khoury G, Jay G: Reversal of oncogenesis by the expression of a major histocompatibility complex class gene. Science 1985, 228:26-30 22. Ruiter DJ, Mattijssen V, Broecker E-B, Ferrone S: MHC antigens in human melanomas: The role of MHC clones and 11 antigen expression in immune surveillance against tumors: I. Human tumors. Seminars in Cancer Biology 1991, 2:35-46 23. Medawar PB: Immunity to homologous grafted skin IlIl the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol 1948, 29:58-69 24. Graus F, Ribalta T, Campo E, et al: Immunohistochemical analysis of the immune reaction in the nervous system in paraneoplastic encephalomyelitis. Neurology 1990, 40: 21 9-222
The Expression of the Hu (Paraneoplastic Encephalomyelitis/Sensory Neuronopathy) Antigen in Human Normal and Tumor Tissues Josep Dalmau,*t Henry M. Furneaux, Carlos Cordon-Cardo,t and Jerome B. Posner*t From the Departmnts of Neurology* and Pathologyt and the Cotzias Laboratory of Neuro-Oncology,* Memorial Sloan Kettenng Cancer Center, New York, New York
Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronalprotein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also e-xpress the antigen (Am J Pathol 1992, 141:881-886)
Paraneoplastic encephalomyelitis (PEM) and paraneoplastic sensory neuronopathy (PSN) are two rare, often overlapping neurological syndromes associated with cancer, usually small cell carcinoma of the lung (SCLC).1 2 Symptoms include dementia, cerebellar degeneration, brain stem encephalopathy, myelopathy, and sensory neuropathy.1 Pathologically, the disease is characterized by neuronal death, gliosis, and inflammatory infiltrates of the neuraxis.13 Patients with PEM/PSN and SCLC harbor in their serum and cerebrospinal fluid high titers of a highly specific antibody called anti-Hu.4 5 The anti-Hu antibody reacts with a set of protein antigens (molecular weight 35 to 40 kd) expressed in neurons and SCLC cells.356 The intrathecal synthesis of the antibody,7 and the detection of deposits of anti-Hu IgG3 and Hu-specific infiltrating lymphocytes in the nervous system and tumor,69 have suggested an immunologic origin of the disorder. According to this model, the expression of the Hu antigen by the tumor triggers an immune re-
sponse that is misdirected against similar proteins expressed in brain.9 Using a limited number of lung cancer cell lines, a previous study demonstrated the presence of the Hu antigen in all SCLCs whether or not the tumor came from a patient who made the antibody.'0 However, the spectrum of expression of the Hu antigen in tumor tissues from lung or other neoplasms is not known. To address this issue and to determine the value of the antiHu antibody as a diagnostic reagent to identify SCLC cells, we now undertook to study by immunohistochemistry and Western blot analysis in a series of normal human tissues and 1 15 tumors of different histologic types.
Materials and Methods
Normal and Tumor Tissues Normal human tissues were obtained either from biopsy or autopsy performed within 10 hours of the patient's death. One hundred six tumors of various histologic types were obtained from the tumor procurement service of
Memorial Sloan Kettering Cancer Center; the investigators had no knowledge of the patients' clinical states. In nine additional patients, the tumor was studied because the patient developed anti-Hu-associated PEM/PSN; two of these tumors were provided by the tumor procurement service of Memorial Sloan Kettering Cancer Center and seven were sent to us by physicians of other centers. Tissues were snap frozen in isopentane chilled by liquid nitrogen, embedded in Optimal Cutting Temperature Compound (Miles Inc, Elkhart, IN), and kept frozen at - 700C. Presented in part at the 43rd meeting of the American Academy of Neurology, Boston, Massachusetts, April 21-27, 1991. Supported by grants FISS 91/5722 Spain (JD), ACS PD-359 (HMF), and NIH NS 26064 (JBP). Accepted for publication April 16, 1992. Address reprint requests to Dr. Jerome B. Posner, Department of Neurology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.
881
882
Dalmau et al
AJP October 1992, Vol. 141, No. 4
From seven SCLC and one chondromyxosarcoma, only tissue sections from paraffin blocks were available.
Results
Normal Tissues
Immunohistochemical Analysis of the Expression of the Hu Antigen Anti-Hu IgG was prepared from high-titer sera of patients with anti-Hu-associated PEM/PSN. The anti-Hu IgG was conjugated to biotin as previously described.11 By using the anti-Hu antibody conjugated with biotin, we avoided the use of a labeled secondary (anti-human IgG) antibody that would immunoreact with the nonspecific IgG of the tissue and obscure the results of the immunohistochemical study. Seven-micron-thick frozen tissue sections were fixed for 10 minutes in acetone and sequentially incubated with 0.3% hydrogen peroxide (to destroy tissue peroxidase activity) and 10% normal human serum (to prevent nonspecific binding of human IgG). The sections then were incubated with biotinylated anti-Hu IgG (2.5 ,ug/ml) for 12 hours at 4°C. Bound human antiHu IgG was visualized by incubation with avidin-biotin peroxidase and developed with 0.05% diaminobenzidine tetrahydrochloride. Incubation of the section with biotinylated normal human IgG served as negative control. Tissues that immunoreacted with the biotinylated antiHu IgG were considered to express the Hu antigen if the immunoreactivity was abolished by preincubation with another anti-Hu serum. Tissue sections from paraffin blocks were deparaffinated and rehydrated,6 and processed in the same way as described for the frozen sections.
Western Blot Analysis of the Expression of the Hu Antigen Serial 30-micron-thick frozen tissue sections were collected and washed in phosphate-buffered saline (PBS). Tissues then were homogenized in PBS with 0.1% Nonidet P-40 and centrifuged at 1 5,000g for 10 minutes, and the supernatant was retained. The protein content was measured according to the method of Bradford.12 The indicated amounts of protein were subjected to electrophoresis on 10% sodium dodecyl-polyacrylamide gel and transferred to nitrocellulose as described by Towbin et al.13 The nitrocellulose filters were blocked with 5% blotto and incubated with the indicated amount of anti-Hu sera for 6 hours at room temperature. The nitrocellulose filters then were washed with TRIS buffer (0.1 mol/l [molar] TRIS, pH 8.0; 0.2 mol/l sodium chloride; 0.2% Triton X-1 00; and 0.1% bovine serum albumin), incubated with 1251-labeled protein A (0.1 ,uCi/ml) for 1 hour at room temperature, washed with TRIS buffer, and exposed to X-ray film for 6 hours.
The expression of the Hu antigen in normal human tissues is shown in Table 1. Immunoreactivity was found highly restricted to the central and peripheral nervous tissue. All neurons tested reacted with the anti-Hu antibody. Immunoreactivity predominated in the nuclei, sparing the nucleoli, and with weaker staining of the cytoplasm (Figure 1A). The medullary chromaffin cells of the adrenal gland and ganglion cells of bronchus immunoreacted with the anti-Hu antibody with a pattern of immunostaining identical to neurons. All other normal tissues examined did not express the Hu antigen. Western blot analyses of homogenates of several normal human tissues are shown in Figure 2. The Hu antigen was found expressed only in brain tissue. Homogenate of bronchus was negative, indicating the low number of cells that were immunoreactive by immunohistochemistry. The 55-kd band observed in most of the tissues corresponds to the heavy chain of endogenous IgG. This band was present in the absence of added antibody (data not shown), and indicates direct binding of 1251labeled protein A.
Tumors The expression of the Hu antigen
was
studied in 115
tumors by immunohistochemistry (45 tumors), Western
blot (19 tumors),
or
both (51 tumors) (Table 2). Nine
Table 1. Expression of the HUAntigen
Tissue (#) Brain (5) Adrenal gland (medullary) (3) Bronchus (3) Lung (3) Ovary (3) Testis (3) Kidney (5) Prostate (3) Bladder (3) Spleen (4) Jejunum (4) Colon (4) Thyroid gland (3) Parathyroid gland (3) Liver (5) Pancreas (3) Muscle (5) Skin (3) (#)
=
by Normal Tissues
Immunohistochemistry + (chromaffin +
cells)
Western blot ND
(ganglion cells)
ND
+ +
(myenteric plexus) (myenteric plexus)
ND ND
number of samples examined for each type of tissue.
Expression of the Hu Antigen
883
AJP October 1992, Vol. 141, No. 4
:,
t."
.'IL &
1A
".:
Figure 1. Immunohistochemical analysis of the expression of the HU antigen. A: Section of hippocampus (fascia dentata)from a neurologically normal individual incubated with biotinylated anti-Hu antibody. Immunoreactivity predominates in the nuclei of the neurons, with weaker staining of the cytoplasm, and sparing the nucleoli. Glial cells do not immunoreact with the anti-Hu antibody. B: A consecutive section of normal hippocampus incubated with biotinylated normal IgG sbows no immunoreactivity and serves as negative control. C: Metastatic lymph node from a patient with SCLC an anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. Immunoreactivity is observed in all the neoplastic cells; normal tissue contained in the lymph node[identified with Tand B cell markers (not shown)] does not immunoreact with the anti-Hu antibody. D: Chondromyxosarcomafrom apatient with anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. Immunoreactivity predominates in the nuclei of the tumor cells. E: Neuroblastoma from a patient without anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. Immunoreactivity predominates in the nuclei of the neoplastic cells. F: Adenocarcinoma of ovary from a patient without anti-Hu associated PEM/PSN, incubated with biotinylated anti-Hu antibody. No immunoreactivity is observed All sections have been counterstained with hematoxylin.
SOLO tumors, obtained in frozen blocks, were studied by immunohistochemistry and Western blot analysis; the seven SCLC tumors available in paraffin blocks were studied by immunohistochemistry. All SCLC (16/16) expressed the Hu antigen. Only 1 of the 25 non-SCLC
(
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H
0 IZe QJ~4> l~4C0TO( '[i#'
46..
21, Figure 2. Western blot analysis of the expression of the HU antigen by normal tissues. Immunoblots of tissue homogenate (80 pg each) reacted with serum (diluted 1:4000) from a patient with anti-Hu associated PEM/PSN. C7X = brain cortex, OV = ovary, TES = testis, PRT = prostate, BLD = bladder, SPL = spleen, COL = colon, THY = thyroid gland, PTH = parathyroid gland, LG = lung, BRC = bronchus, PAN = pancreas.
ing sarcomas, one melanoma, one chondromyxosarcoma, one small cell carcinoma of stomach, and one undifferentiated carcinoma of the prostate. Immunoreactivity predominated in the nuclei of the neoplastic cells, with weaker staining of the cytoplasm (Figure 1 C, D, E). No immunoreactivity was observed when the tumors were incubated with biotinylated normal IgG. The anti-Hu antibody served to differentiate normal from neoplastic tissue in those tumors where normal and neoplastic cells were highly similar (Figure 1 C). For all the tumors, except one melanoma, results were identical using either immunohistochemistry or Western blot. The immunohistochemical study of the melanoma was considered negative, but the Western blot showed a weak band at 37 kd (data not shown). Furthermore, for all the Hu-positive tumors the pattern of Western blot analysis was similar, showing a positive discrete band at 37 kd (Figure 3). Medical records of the Hu-positive tumors obtained after the tumor study showed no history of paraneoplastic symptoms. Nine tumors were studied because the patients developed paraneoplastic symptoms. They included seven SCLC, one chondromyxosarcoma, and one undifferentiated tumor of the prostate. Clinical and immunopathologic studies of five of these patients have been reported elsewhere.314
Discussion We have studied the expression of the Hu antigen in 1 15 human tumors of different histologic types. In lung tumors, the Hu antigen was found selectively expressed in SCLC. The practical implication of this selective expression is that the anti-Hu antibody becomes a useful reagent to identify SCLC. Under normal circumstances, the Hu antigen is restricted in its expression to neurons of the central and peripheral nervous system. Because of its location and homology to the drosophila proteins embryonic lethal, abnormal visual system (ELAV) and sex lethal, the Hu is thought to be an RNA-binding protein that plays a role in neuronal development.9 In a very small number of patients with cancer, a high titer of antibody recognizing the Hu protein is found in the serum and cerebrospinal fluid and is associated with a neurologic disorder called paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/PSN).5615 A high titer (average 4600 U/ml) of antiHu antibody generally implies the presence of a SCLC, but rarely may be associated with other tumors, including neuroblastoma, sarcoma, or prostate carcinoma.14 A low titer of anti-Hu antibody (average, 80 U/ml) is found in
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15% of patients with SCLC but without neurologic dysfunction.5 Previous studies have shown the Hu antigen to be expressed in the SCLC of patients with paraneoplastic neurological disorders associated with the presence of the anti-Hu antibody,3'6 and in SCLC cell lines whether or not the tumor came from a patient who made the antibody.10 Using tumor tissues, we now demonstrate that in addition to all SCLC, a small percentage of tumors other than lung also express the Hu antigen. This is not surprising because many of the tumors that express the Hu antigen have already been shown to express other neuronal antigens and are thought to be neuroectodermal in origin.1619 Although the serum of these patients was not assayed for antibodies against the Hu antigen, we would not expect that they make antibodies because at least 85% of patients with SCLC (all of which express the Hu antigen) do not make a detectable antibody response.5 Thus, the crucial question is why a small number of patients with Hu-positive tumors make either a modest or a profound immunologic response to an antigen that is expressed in their tumor. One possible explanation is that most of these tumors evade immune surveillance by turning off the expression of major histocompatibility (MHC) class antigens.?-22 In preliminary experiments we have found that all the Hu tumors from paraneoplastic patients still have in their cell surface MHC class antigens (data not shown). This model, however, does not explain why a small number of patients mount an immune response against a putative normal neuronal protein. One theory is that neuron-specific proteins are not presented to the imTable 2. Expression of the HU antigen by Tumors HU positive/ total studied Tumor histology
Non-SCLC (A) Small cell cancer other than lung (#) Neuroblastoma Melanoma Merkel cell tumor Sarcoma (*) Adenoca. prostate Thymoma Renal carcinoma Breast adenoca. Seminoma Adenoca. ovary Bladder carcinoma Adenoca. colon Adenoca. pancreas
16/16 1/25 1/3 4/8 1/5 2/2 3/7 1/10 0/2 0/4 0/10 0/2 0/7 0/5 0/4 0/5
(A) = includes eight squamous, eight adenocarcinomas, two large cell, three bronchiolar, one mixed lung carcinoma, one anaplastic carcinoma of the lung (Hu +), and two carcinoid tumors; (#) includes one small cell cancer (SCC) of stomach (Hu +), one SCC of bladder, and one SCC of unknown origin. (*) = includes two osteogenic sarcomas, one osteosarcoma, one rabdomyosarcoma, one chondromyxosarcoma, and two Ewing tumors.
46-
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:
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30- 0 46
2
3 4
5 6
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8
9
12 13 14161 10 1020 1|I11
Cl C2 C3 10 11 12 13 14 1S 16 17
3730-
Cl C2 C3 182 2 29 3021 22 23 24 4637
30
-
-
Cl 25 26 27 28 '29 30' 31 32 33 34
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Cl C2 03 35 36 37 38 39 40 41 42 Figure 3. Western blot analysis of the expression of the HUantigen by tumors. Immunoblots of tissue homogenate (80 1'g each tumor) reacted with serum (diluted 1.4000) from a patient with anti-Hu associated PEM/PSN C = controls, Cl = crude neuronal preparation (rows 1, 3, 4,5), homogenate of brain cortex frow 2), C2 = homogenate of SCLC, C3 = adenocarcinoma of the lung. Lanes 1 to 5 = non-SCLC; 6-9 adenocarcinoma of the prostate; 10-13 renal cell carcinoma; 14-17 melanoma; 18-19 adenocarcinoma of the breast; 20-21 adenocarcinoma of the ovary; 22-24 neuroblastomas; 25-27 adenocarcinoma of the colon; 28-31 adenocarcinoma of the pancreas; 32-34 carcinoma of the bladder; 3538 sarcomas (37 Ewing); 39 thymoma; 40 Merkel cell tumor, 41 SCLC, 42 small cell metastasis (primary tumor unknown).
mune system during the establishment of immune toler-
ance,23 and that MHC proteins are not expressed in neurons.24 The de novo expression of the Hu protein in tumors (that express MHC) then may result in a profound immune reaction. According to this theory, however, one would expect a diverse immunoresponse against the other many neuronal-specific proteins whose expression has been demonstrated in a variety of tumors, especially SCLC.1 19 We believe that in addition to the coexpression of MHC class and Hu antigen by the tumor, other factors are involved in the pathogenesis of the immune disorder. One model would be that the sequence of the Hu protein is altered and perceived as foreign. This model can now be studied by analyzing the structure of the Hu mRNA in the tumors of patients with PEM/PSN.9 A practical implication of this model would be the development of monoclonal antibodies specifically directed against the tumor form of Hu antigen.
Acknowledgments The authors thank Dr. Myma R. Rosenfeld for helpful discussion and reading of the manuscript; Dr. Pilar Garin, who provided us
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with several tumors; and B. Nevins for patience and diligence in preparing the manuscript.
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