Immunological Techniques to Differentiate Lymphoma from other Malignancies*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 22, No. 5 Copyright 1992, Institute for Clinical Science, Inc. Immunological Techniques to Differentiate Lymphoma from other Malignancies* EDWARD E. MORSE, M.D.,t HAROLD T. YAMASE, M.D.,t BERNARD R. GREENBERG, M.D.J JONATHAN R. SPORN, M.D., SHARON A. CORNISH, MT(ASCP),t and THOMAS R. KIRALY, M.A.t Departments of Laboratory Medicine, t Pathology,t and Medicine, The University of Connecticut Health Center/John Dempsey Hospital, Farmington, CT ABSTRACT Seven patients, who had lymph nodes or masses exam ined by both immunoperoxidase staining and flow cytometry, are presented to illustrate the value of each technique including a critical analysis of the current application of these techniques in the pathology laboratory. All seven patients had diagnoses established by immunoperoxidase staining using antibodies directed against: Leukocyte Common Antigen (LCA), Epithelial M embrane Antigen (EMA), Neuron Specific Enolase (NSE), Leu Ml5 B4 or chrom agrafin and synaptosin. Flow cytometry, w hich could be more rapidly performed, w hen sufficient cells could be separated from the node or mass, was diagnostic in two of the seven cases. Flow cytometry failed to show abnorm alities in Hodgkin s disease or solid tumors, but it was useful in rapid diagnosis of lymphoma, provided that the sample contained mostly involved tissue. N odes in w hich there was a m inor infiltration w ith lymphom a cells could only be detected by im m unoperoxidase technique. Introduction A num ber of new techniques are being applied to the diagnosis of lymphoma, particularly w hen the initial morphology shows large undifferentiated cells.1 2,3,4,5 * Send correspondence to: Edward E. Morse, M.D., Department of Laboratory Medicine, Room C2067A, MC #2225, The University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT Among the more rapid techniques are flow cytom etry (FC) and streptavidin im m u n o p ero x id ase sta in in g (SA IP). Critical analysis indicates some lim itation to these m ethods including sampling size, distribution variability of m alignancy within tissue biopsied, the need for fresh tissue and nonspecificity of antibodies used.6,7,9 E lectron m icroscopy (EM) may be helpful in isolated cases, and deoxyribonucleic acid (DNA) break point analysis appears to be the most spe /92/ $00.90 Institute for Clinical Science, Inc.

2 318 MORSE, YAMASE, GREENBERG, SPORN, CORNISH, AND KIRALY eific tech n iq u e w h en probes are availa b le 4,8 an d s u ffic ie n t c e lls can be obtained. In th e p re se n t p aper, seven cases are an aly zed th a t had b o th FC and SAIP stain attem pted and, w hen necessary, EM was added. N one of the c a se s r e q u ir e d D N A a n a ly s is for final diagnosis. T hese cases illustrate the problem s associated w ith processing tissues for single c e ll an aly sis and p ro v id e som e insight into the precautions that are necessary for appropriate interpretation of the techniques used. Methods Patients w ere selected because they had large, undifferentiated cells on frozen section of lymph node or other tissue biopsy and/or there was a suspicion of lym phom a from clinical presentation. Flow cytom etry (FC) was performed on cells se p ara te d from tissu es su s p en d ed in phosphate buffered saline (PBS) and centrifuged through Ficoll*. The separated buffy layer at the top of the ficoll band was w ashed twice in PBS, tagged w ith fluorescein-labelled antibodie s to s u rfa c e m a rk e rs in c lu d in g C D 2,5,10,19,20, lam b d a an d kappa chains, and m yeloid and monocytic marke rst. T he stained cells w ere w ashed twice in PBS and analyzed on a Coulter Epics C t.7 Tissue sections from sim ilar tissues were also fixed in paraffin to slides, dehydrated, and stained w ith streptavidin immunoperoxidase-linked antibodies for tumor antigens AE-1, S100, CALLA, T and B antigens and light chainst.10 * Pharmacia Biotechnology Inc., 800 Centennial Ave., Piscataway, NJ t Coulter Electronics, 440 W. 20th Street, Hialeah, FL Í Dako Corp., 6392 Via Real, Carpintería, CA Electron microscopy (EM) was carried out on specim ens from two of the seven patients using gluteraldehyde-fixed tissues and a Philips (Holland) 300 electron microscope. Tissue was sectioned into one mm-sized cubes and fixed in cacodylate buffered four p ercen t glutaraldehyde, postfixed in cacodylate buffered two p ercen t osm ium tetroxide, d eh y drated and em bedded in poly bed 812. Sections one (Jim thick w ere cut and stained w ith to lu id in e b lu e for lig h t m icroscope evaluation, and areas w ere selected for thin sections. Final diagnosis was determ ined on thin sections fixed in formalin and stained with hematoxylin and eosin. In each case, techniques used w ere critically analyzed and a determ ination was made w hether or not they were: (1) diagnostic, (2) helpful in determ ining final diagnosis, (3) discrepant, or (4) tech nically inadequate. Case H istories Case 1. JH, a 10-year-old Caucasian male, presented with a rapidly enlarging scalp mass. Biopsy revealed a large cell infiltrate invading surrounding tissue. Staining by SAIP showed the large cells were CALLA and TdT positive. Flow cytometry showed 75 percent CALLA and 66 percent CD34, and the diagnosis of lymphoblastic lymphoma was made. Case 2. JD, a six-year-old Caucasian female, presented with rapidly enlarging neck nodes. Biopsy revealed a large cell infiltrate replacing much of the node. Staining by SAIP showed the large cells marked with pan B cell markers. Flow cytometry showed 65 percent B cells with a kappa/lambda ratio of 96/16, which was diagnostic of a monoclonal B cell population. Case 3. FC, a 77-year-old Caucasian female, presented with a tumor in the mouth along the tonsillar pillar which, on frozen section, initially appeared to be a squamous cell carcinoma. However, staining by SAIP showed the infiltrating cells were positive for Pan B and LCA, while they were negative for T cell markers, Ki-1, Leu Mi, keratin, and EA1. Flow cytometry showed only 30 percent B cells and kappa/lambda of 18/4. These findings, though not diagnostic, were helpful in confirming the diagnostic findings of SAIP. Electron microscopy further substantiated the lack of interepithelial junctions (desmosomes) that would have been found had this lesion been a squamous cell carcinoma. Case 4. RV, a 47-year-old Caucasian male, presented with a rapidly enlarging mediastinal mass.

3 IMMUNOLOGICAL TECHNIQUES TO DIFFERENTIATE LYMPHOMA FROM OTHER MALIGNANCIES 319 Biopsy showed the cells to be negative for LCA, while positive for epithelial antigen. Flow cytometry showed only 56 percent CD45/LCA and a normal mixture of T and B cells. Diagnosis was an aplastic carcinoma, probably lung. Case 5. HR, a four-year-old Hispanic male, presented with painless cervical adenopathy and had large undifferentiated cells staining positive for Leu Mi but negative for LCA by SAIP. Flow cytometry showed 91 percent CD45/LCA positive cells with a mix of T/B-75/14 percent and kappa/lambda 10/5. These apparently discrepant findings were due to the relative paucity of Reed-Stemberg cells (Leu M! positive) and the loss of architecture in processing of the samples for flow cytometry. Permanent sections clearly demonstrated the Reed- Sternberg variants and mixed cellularity Hodgkin s disease. Case 6. JD, An 80-year-old Caucasian male, presented with a rapidly developing mass of nodes in the neck. Staining by SAIP showed a large cell tumor infiltrate which stained with pan B antibody and was surrounded by T cells. Flow cytometry showed a mix of T and B cells and no clonal excess of kappa/lambda. Final diagnosis was diffuse large cell lymphoma. Case 7. MG, an 18-year-old Caucasian male, presented with pericardial effusion and mediastinal mass. Too few cells were obtained for FC, but enough tissue was available for staining by SAIP and showed reactions for neuron specific enolase, chromagrafin, and synaptosin, while that for LCA was negative. Electron microscopy confirmed neurosecretory granules and presence of neurofibrils. Diagnosis was paraganglioma. Results T he seven patients p resen ted here range in age from four years to 80 years, and all had biopsies from neck nodes, m ediastinal m asses, scalp, or tonsil. Frozen sections, if perform ed, generally showed a large cell infiltrate without further differentiation. Because therapy and prognosis w ere dependent upon a specific diagnosis, there was always pressure to determ ine the diagnosis as soon as possible, rather than w ait for perm a nent sections. Flow cytometry was capable of determ ining rapidly w hether or not th e lesio n was of B cell origin and w hether or not the cells were monoclonal, if there were enough cells. However, only in one patient (Case 2, table I) were the B cells sufficiently numerous and the kappa/lambda chain ratio sufficiently out of the range of normal cells to be diagnostic for lymphoma. In a second patient (Case 1, table I), antibody to CALLA dem onstrated that 75 percent cells were immature immunoblasts making up the majority of the cells. In these two cases, the diagnosis could have been made by flow cytometry alone. Studies with SAIP-linked antibodies on tissue slides confirm ed a m ajority of CALLA positive and TdT positive cells in case one and showed an infiltrate of large cells staining for pan B antigen surrounded by small lymphocytes staining for T cells in the tissues of the second case (table I). In Case 3, biopsy of a tonsillar node showed a subepithelial infiltrate of large cells thought at first to resem ble squamous cell carcinoma. Immunoperoxidase studies revealed the cells to be positive for LCA and pan B antigen, but negative for epithelial antigen (EA) and a num ber of other antibodies. Flow cytometry was helpful in confirming the presence of B cells, but the proportion was only 30 percent and kappa/lambda light chain ratio was only 4.5/1. These values w ere not clearly diagnostic. Studies by EM dem onstrated that these large cells contained no desmosomes expected in epithelial tumors and help ed establish the diagnosis of lymphoma. In Case 4, the biopsy from the m ediastinal mass dem onstrated large cells with g r e a t v a ria tio n in s iz e. Im m u n o peroxidase studies revealed the cells w ere negative for LCA but positive for EA indicating an epithelial (probably o rig in a tin g from lung) tum or. F low cytometry studies indicated 44 percent of the cells were negative for LCA, but the rest of the cells (56 percent) were a mix of T and B cells. W hile the finding that a large proportion of cells were not leukocytes is helpful in this case, the findings by FC were not considered diagnostic.

4 320 MORSE, YAMASE, GREENBERG, SPORN, CORNISH, AND KIRALY TABLE I Summary of Patients Studied Patient Biopsy/Diagnosis Frozen Section SAIP Electron Flow Cytometry Microscopy 1 JH Scalp mass Lymphoblastic Lymphoma 2 JD Lymph node Large cell Lymphoma 3 FD Tonsillar node Large cell Lymphoma 4 RV Mediastinal mass Large cell carcinoma of lung 5 HR Lymph node Hodgkins disease Large cell infiltrate await Permanent sections Large cell infiltrate await Pernament sections Large cell Infiltrate? Squamous cell Carcinoma large cell infiltrate await Permanent sections 6 JD Mass of neck nodes diffuse Large cell lymphoma 7 MG Mediastinal mass Paraganglioma CALLA & TdT (*) Pan B antigen (*) L26 on large cells Surrounding small lymph on sections T & B cells Pan B/LCA (*) Negative T, Leu-Ml. Ki-1 Keratin, epithelial antigen Negative LCA (*) Positive epithelial antigen 8c mucin Large cells (*) Leu M1 positive LCA negative Pan B antigen (*) in large tumor cells surrounding Lymph T markers Neuron specific (*) Enolase, chromagrafin and synaptophysin Positive Negative for LCA CALLA 75% (*) CD34/66% B1/B4 66/58C) K/A. 92/16 CALLA Neg B1/B4 29/30 K/X 18/4 (h) 56% CD45/LCA (h) Mix of T & B cells 91% CD45/LCA (d) Mix of T8t B 75/14% K/X 10/5 Mix of T & B cells (d) No clonal excess K/X No Intercellular junctions Technical difficulties Neuron Too few cells specific fibrils SAIP = Streptavidin immunoperoxidase staining (*) Diagnostic finding (h) Helpful (d) Discrepant = Not performed In Case 5, the node biopsy showed an in filtra te of large abnorm al cells w hich w ere p o sitiv e w ith im m unop e ro x id a s e -lin k e d a n ti-l e u M j b u t LCA negative. This finding is consistent w ith the perm anent sections showing R e e d -S te rn b e rg v a ria n ts in n o d u lar sclerosing H odgkin s disease. In this case, flow cytometry revealed 91 percent LCA positive cells and a mix of T (75 percent) and B (14 percent) cells. The B cells showed a kappa/lambda ratio of 10/5. The FC findings could rep resen t reactive hyperplasia or a normal lymph node and w ere discrepant or nondiagnostic. The biopsy in Case 6 stained w ith SAIP was diagnostic showing a B cell tumor surrounded by small lymphocytes m arking as T cells. Flow cytom etry was not diagnostic show ing a mix of

5 IMMUNOLOGICAL TECHNIQUES TO DIFFERENTIATE LYMPHOMA FROM OTHER MALIGNANCIES 321 B and T cells without excess kappa or lam bda chains and could be co n sid ered discrepant. Finally, in Case 7, immunoperoxidase studies showed tumor cells negative for LCA but positive for neuron specific enolase, chrom agrafin, and synaptosin consistent w ith a diagnosis of paraganglioma in a biopsy of a m ediastinal mass. Flow cytometry studies were difficult to carry out because too few cells could be separated from the tissue for technically adequate exam ination. Studies by EM dem o n strated n eu ro n specific fibrils and granules to help confirm the diagnosis (table 1). Discussion T hese cases illustrate that tissue staining w ith SA IP-linked antibodies selectively applied can be very sensitive in detecting lymphoma and differentiating it from epithelial tumors in lymph nodes and other biopsy tissues. Specificity was not tested in the present series, but it has been shown to be virtually 100 percent in another series.4 Michie et al,4 using three antibodies to EA1, S100 and LCA, studied 120 biopsies from patients with large u ndifferentiated cells. T hey found no overlap betw een the antibodies (100 percent specificity) but 17/120 failed to react with any of these antibodies (86 percent s e n s itiv ity ). A d d in g an a n tib o d y 5 directed against m esenchym al cells or neural tum ors m ight increase the sensitivity of this combination. In the present series, flow cytometry showed diagnostic levels of B cells, m onoclonal kappa/ lam bda ratio (6/1) or presence of CALLA (75 percent) in two of the seven patients. In two others, flow results were helpful but not diagnostic. In two patients the flow results w ere not diagnostic and could be considered m isleading since they suggested normal nodes or hyperplasia in cases of H odgkin s disease and large cell lymphoma. In one case, technical difficulties led to too few cells for adequate examination. If technical inadequacy is not considered, flow cytometry showed a sensitivity of 67 percent (4/6). In our laboratory, specificity of flow cytometry has been 100 p ercen t.6 In m ost reported studies3,5,6 7, as in ours, electron microscopy was used only for selected difficult cases. Electron microscopy was helpful in two cases showing neuron specific granules and neurofibrils in one case of paraganglioma and lack of desmosomes in a suspected squamous cell carcinoma that was, in fact, a B cell lymphoma. It seems clear that immunoperoxidase staining w ith specific antibodies has several advantages over other techniques. First, tissue architecture is m aintained in the biopsy specimen; second, individual cells can be visualized, so that even a small population of neoplastic cells can be observed in tissue sections; third, specific antibodies can determ ine w hether a m inor population is ectoderm al, m esodermal or endoderm al to define origin. In the case of B cell lymphoma, monoclonal light chains can be specifically exam ined in the abnorm al population and all of this can be done using frozen stored or fixed tissues. In contrast, flow cytometry, which can be performed more quickly in most laboratories, m ust be perform ed on cells separated from fresh tissues and washed to remove debris. The single cell suspension m ust contain a major proportion of neoplastic cells unless a tumor specific antibody is available to detect a m inor population, e.g., CALLA. With such loss of architectural relationships, a majority of reactive lymphocytes tends to obscure th e m in o r p o p u la tio n, e.g., R e e d - Sternberg cells of H odgkin s disease. It is recom m ended that pathologists in terested in defining diseases using th ese expensive tec h n iq u es recognize

6 322 MORSE, YAMASE, GREENBERG, SPORN, CORNISH, AND KIRALY the strengths and weakness of each and prepare for the advent of still new er techniques that will further supplant current procedures, e.g., probes w hich detect alteration in DNA.1,3 The present cases illustrate the value of architectural relationships m aintained in tissue sections, which are lost in the processing of tissues for flow cytometry. References 1. Ba c c h i, C. E., D o r f m a n, R. F., H o p p e, R. T., C h a n, J. K. C., and W a r n k e, R. A.: Metastatic carcinoma in lymph nodes simulating syncytial variant of nodular sclerosing Hodgkin s disease. Am. J. Clin. Pathol. 96: , B o r o w i t z, M. T., B o u s v a r o s, A., B r y n e s, R. K., C o u s e r, J. B., C r is s m a n, J. C., W h i t c o m b, C. C., Ke r n s, B. J., and B y r n e, G. E.: Monoclonal antibody phenotyping of B cell non Hodgkin lymphomas. Am. J. Pathol. 121: , K r i e k e n, J. H. J. M., M a d e i r o s, J., P a l s, S. T., Ra f f e e d, M., and Kl u i n, P. H. M.: Diffuse aggressive B-cell lymphomas of the gastrointestinal tract. Am. J. Clin. Pathol. 97: , M i c h i e, S. A., S p a g n o l o, D. V., D u n n, K. A., W a r n k e, R. A., and R o u s e, R. V.: A panel approach to the evaluation of the sensitivity and specificity of antibodies for the diagnosis of routinely processed histologically undifferentiated human neoplasms. Am. J. Clin. Pathol. 88: , M i l l e r, R. T.: Im m u n o h is to c h e m istry in th e c o m m u n ity p ra c tic e o f p a th o lo g y. L a b. M e d. 22: , M o r s e, E. E., Ya m a s e, H. T., G r e e n b e r g, B. R., S p o r n, J. R., C o r n i s h, S. A., Kir a l y, T. R., Z ie m b a, R. A., and F a l l o w, M. A.: The role of flow cytometry in the diagnosis of lymphoma. A critical analysis. (Submitted Am. J. Clin. Path.). 7. N a d ji, M. and G a n je i, P.: Immunochemistry in diagnostic cytology: A 12-year perspective. Am. J. Clin. Pathol. 94: , PARKER, J. W.: Flow cytometry in the diagnosis of hematologic diseases. Ann. Clin. Lab. Sci. 16: , P r e u d h o m m e, J. L.: Lymphocyte markers: Diagnostic help or costly vogue? Diagn. Immunol. 2: , Sw a n s o n, P. E. and W ic k, M. R.: Commercial avidin-biotin-peroxidase complex kits. Am. J. Clin. Pathol. 91 (Suppl l): , 1989.