Leukemia and Lymphornu, Vol. 8, pp. 87-96 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Hanvood Academic Publishers GmbH Printed in the United Kingdom
A Multiparametric Study of Malignant Lymphoma of Mucosa Associated Lymphoid Tissue (Malt) Leuk Lymphoma Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
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C. RIVAS, G. ECHEZARRETA, R. GARCiA, M. SANTOS, A. SANTON, M. ROBLEDO, J. BENITEZ, H. OLIVA, and G. DELSOL* Departments of Pathology, Immunology and Genetics, Fundacion Jimenez Diaz, Universidad Autonoma, Madrid, Spain, and *Laboratoire d'Anatomie Pathologique, C.H.U. Purpan. Toulouse, France. (Received 30 December 1991; in final form 12 February 1992)
A morphological, immunophenotypic and ultrastructural study, cell cycle estimation, DNA and cytogenetic analysis were performed in ten cases of B-MALT lymphomas. Five had low grade lymphoma and five had high grade. Low and high grade cases showed the same cells but in different percentages: These included centrocyte-like cells with occasional monocytoid cytoplasmic changes, and centroblast-like cells. However, in high grade cases more dysplastic and large cells were present. All cellular types showed an important development of rough endoplasmic reticulum. In all cases a large panel of monoclonal antibodies was employed t o study the B-cell immunophenotype. Ki-67 positivity ranged from 5% to 30% in low-grade cases and from 50% to 70% in high-grade cases. Gene rearrangement analysis showed rearrangement with Jh probe and half of the cases were also rearranged with the Kde probe (Kappa constant chain gene). A rearrangement banding pattern with TCR genes was not present in any of the cases. Cytogenetic study showed complex alterations in high grade cases and a normal karyotype in low grade lymphomas. Only one case had rearrangement for the bcl-2 probe. KEY WORDS:
B-MALT lymphomas immunophenotype ultrastructure gene rearrangement study flow cytometric analysis cytogenetic analysis.
follicles and consists of cells resembling centrocytes, the so called centrocyte-like (CCL) cells which may also resemble small lymphocytes or have a monocytoid appearance. Plasma cell differentiation is present in approximately a third of the cases and scattered larger transformed blasts are evident. The CCL cells characteristically invade glandular epithelium forming lymphoepithelial lesions (LEL)'. The distinctive features of high grade MALT lymphoma are not so well defined since LEL are absent and the tumour cells resemble centroblasts and immunoblasts as found in nodal high grade lymphomas. In a significant number of cases, however, the presence of areas of low grade lymphoma facilitates the identification of these tumours as of MALT type'.
INTRODUCTION Primary malignant lymphomas of mucosa associated lymphoid tissue (MALT) now constitute a distinct entity'+ which is not included in established classifications of nodal lymphomas, such as the Kiel clas~ification~-~. The architectural and cytological features of these malignancies have been well characterised in low grade lesions arising in the stomach. In these cases, the neoplastic infiltrate is centered around reactive
Address for correspondence: C. Rivas, Anatomia Patologica. Fundacion Jimenez Dim. Avda Reyes Catolicos 2-4.28040 Madrid, Spain. 87
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C. RIVAS et al.
In this report we have attempted to further in 2% osmium tetroxide and embedded in Vestopal characterize MALT lymphomas with respect to their W resin. Sections were stained with uranyl acetate and immunophenotype, ultrastructure, genetic character- lead citrate and observed using an Hitachi HU12A electron microscope. istics and proliferation rate. Flow cytometric analysis was carried out on cell suspensions from either fresh or paraffin embedded tissue prepared by pepsin digestion treatment MATERIALS AND METHODS according to the method of Hedley", using Surgical resection specimens from 10 cases of B-cell propidium iodine DNA stains. The analysis was M A L T lymphomas were studied. Five of these were undertaken using an EPICS-C flow cytometer low grade tumours (2 intestinal, 2 gastric and 1 (Coulter Hialeah Florida) and the cell cycle was thyroid) and 5 high grade (3 gastric and 2 intestinal). calculated using the PARA- 1 programme. Histograms Routine haematoxylin eosin stained sections were were considered diploid if only one symmetrical GO/G1 peak was present with a coefficient variation examined in all cases. Immunohistochemistry was carried using the (CV) less than 5% for fresh material and 10% for APAAP technique on frozen tissue and the Avidin paraffin embedded material. If the peak was Biotin immunoperoxidase technique on paraffin asymmetrical or two GO/Gl peaks were present, DNA embedded tissue with prior trypsinization of sections histograms were considered aneuploid. S + G2M where indicated. The panel of antibodies used is given phase was only measured in diploid samples. An in Table 19. S-phase in excess of 20% was considered as indicative Tissue for electron microscopy was fixed in 2.5% of a high proliferation rate. glutaraldehyde in cacodylate buffer (pH 7.4), postfixed For molecular studies, high molecular weight D N A
Table 1 Monoclonal Antibodies used. MoAbs
Source
Specificity
Frozen tissue CD3, Leu4 CD4, Leu3a CD5, Leu1 CD8,OKT8 CDlO, CALLA
Becton-Dickinson Becton-Dickinson Becton-Dickinson Ortho-diagnosis Becton-Dickinson
T-cells T-helper/inducer cells T-cell restrictive antigen (+ in some B low-grade lymphomas) T suppressor/cytotoxic Human common acute Lymphoblastic leukemia antigen (B-follicular centre lymphomas) B-cells (pan B) (B progenitor) B-cells (mature and DRC)
CD19, Leu12 CD20, B7, Leu14 CD30, Kil, BerH2*
Becton-Dickinson Ortho-diagnosis Becton-Dickinson DAKO
Ki67 MU (IgW Kappa Lambda LeuM5 Leu8 DRC
DAKO BRL diagnosis Becton-Dickinson Becton-Dickinson Becton-Dickinson Becton-Dickinson DAKO
Lymphoid activation marker (Hodgkin disease, some anaplastic high grade lymphomas) Nuclear proliferative marker B-cells B-cell~ B-cells Phagocytes Restrictive T-cell antigen mantle B-follicular Follicular dendritic cells
DAKO Biotest DAKO Biotest
T-cells Immature B cell, immature T-cell (T-B cell high grade lymphomas) B-cell marker B-cell marker
Immunotech-Delsol Immunotech-Delsol Immunotech-Delsol Becton-Dickinson
B-cell marker B-cell marker B-cell marker Human cytokeratin
Parafin embedded tissue UCHLl MTI L26 MB2 Experimental panel: (9) DBB42 DND53 DBA44 CAM 5.2
DRC = dendritic reticulum all.
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B-MALT LYMPHOMAS
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was extracted from fresh and frozen samples by appeared as a firm white infiltrating mass without standard methods. Complete digestion with the preservation of the normal parenchyma. All the high restriction enzymes: EcoR1, HindIII, BamH1, BglII, grade MALT cases showed bulky infiltrating tumours PstI of 5 pg of total DNA was followed by Southern with numerous adjacent enlarged lymph nodes. blot analysis. Blots were probed with 32-P labelled The histological study of low grade MALT DNA probes for genes encoding the T-cell receptor lymphomas confirmed previous descriptions of this Beta chain (p-TCR) and the heavy chain joining entity. Centrocyte-like cells illustrated the cytological region (Jh), kappa light chain (Kde), kappa constant spectrum previously described and included cells with region (Ck), lambda constant region (Cl) of the Ig gene a monocytoid appearance. Transformed blast forms and also probes encoding the c-myc and bcl-2 were scattered throughout the infiltrate and plasma oncogene (Table 2). Each filter was washed with cells were present in all cases. Lymphoepithelial 2 x SSC, 0.1% SDS at room temperature and lesions were prominent in all of these cases (Figure 1). High grade MALT cases showed extensive diffuse 0.1 x SSC, 0.1% SDS at 52°C. Autoradiography was infiltration with large nucleated tumour cells. These done at -80°C with intensifying screens. For karyotypic analysis portions of tumour were cells resembled centroblasts but had more abundant minced by mechanical methods followed by enzymatic cytoplasm and immunoblasts were also present. digestion with collagenase, 200 pl/ml for 1-2 hours at Lymphoepithelial lesions formed by cells seen in low 37°C. Cells were resuspended in RPMI colcemid and grade MALT were also present in 2 cases (cases 9 and a second culture of 72 hours with PKW mitogen was 10) (Figure 2). Immunohistochemistry confirmed the B cell nature performed. GTG banding techniques were done in all cases and each one showed immunoglobulin according to standard procedures. light chain restriction. The detailed immunophenotype is given in Table 3 as are the Ki67 scores which varied between 5 and 30% for the low grade cases RESULTS and between 50 and 70% for the high grade case. Ultrastructural findings (Table 4 and 5 ) showed the Results are summarized in Table 3. Macroscopically following cytological characteristics. In low grade low grade MALT gastrointestinal lymphomas were tumours centrocyte-like cells were medium sized characterized by flattened areas containing single or (10-12 pm in diameter) with indented, irregular oval multiple superficial ulcers. The thyroid tumour nuclei containing dense chromatin concentrated near Table 2 Probes used in the molecular study. the nuclear membrane. Single small nucleoli were noted. A moderate amount of cytoplasm was present Probe Size Enzyme Fragment size in which contained few organelles and variable developgerm line (Kb) ment of rough endoplasmic reticulum. Larger cells 12; 4 0.7 EcoRl with similar characteristics but with more diffuse T-cell receptor Hind111 7.3; 5.8; 3.3 beta chain chromatin were also present. Monocytoid B cells were 24 BamH 1 B-TCR larger than the centrocyte-like cells (12-15 pm in Heavy chain 2.5 HindIII 9.5 diameter) and contained nuclei which were rounder joining region BgIII 4 without significant membrane indentations. Nuclear JH 6 EcoR 1 17 BamH 1 16 chromatin was similar to that of other centrocyte-like 14 Kappa light 2.5 BamH 1 cells but the cytoplasm was more abundant and chain clearer. A well developed endoplasmic reticulum was Kde evident in these cells. 6.6 Kappa constant 2.5 Hind111 PstI 3.5 Lymphoepithelial lesions were characterized by region CK centrocyte-like and monocytoid cells which appeared Lambda constant 3.5 Pstl 1.75; 1 to enter the epithelium by way of pseudopod 4.3; 1.2; 1 BamH 1 region formation. The epithelium showed disruption of the CL basement membrane with disappearance of desmo9 EcoRl 20 c-myc somes. In addition, degenerate nuclear and cytobcl-2 3 PstI 16 BamH 1 18 plasmic changes were present, the most obvious of hind111 4 which was an increase of mitochondria giving rise to 18 EcoR 1 an oncocytic appearance (Figure 3).
F
M
F
F
M
M
F
4
5
6
7
8
9
10
With PKW mitogen; ** Frozen,material; 2 Paraffin; G = Germ line; R = Rearranged;
High grade Intestinal
-
27
65
-
-
68
50
-
1
25
5
30 14
20 36
24
73
44
7
1
4
4
A
D
D
A
A
D
A
D
D
D
G2W PLOID
8 14
18
80
76
5
11
S
87
83
Go/Gi
Cell cycle by pow cytometry gene
= Data non available.
IgM, K,CD19, Ki67= 80% CD30 L26, MB2, DBB42, DND53*
M
3
Tumoral Ulcero-infiltrative
F
2
Immunophenotyge
IgM, L, CD19, Ki67 = 1&20% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 5% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 20% L26, MB2, DBB42, DND53* IgM, L, Cd19, Ki67 = 5% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 30% L26, MB2, DBB42, DND53* IgM, L, CD19, Ki67 = 5&70% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 50% L26, MB2, DBB42, DND53* IgM, L, CD19, Ki67 =60% L26, MB2, DBB42, DND53* IgM, L, CD19, Ki67 = 60% CALLA L26, MB2, DBB42, DND53*
Infiltrative
M
1
Site
Low grade Intestinal Infiltrative Low grade Gastric Ulcerated Low grade Gastric Infiltrative Low grade Thyroid Tumoral-infiltrative Low grade Intestinal Tumoral High grade Ulcero-infiltrative Gastric Ulcerated High grade Gastric Tumoral High grade Ulcero-infiltrative Gastric Tumoral-infiltrative High grade Intestinal
Macroscopy
Cnse Sex
Table 3 Immunophenotypic, Bow cytometric, gene rearrangement and cytogenic results.
G
G
G
G
G
G
R
G
R
R R
R
R
G
-
R
G R
G
-
R
G
G
G
G
G
G
G
G
R
G
G
-
-
-
G
G
R
R
G
G
G
G
R
G
G
G
-
G
Kde CK C
G
TCR JH
Rearrangement analysis
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** -
-
-
-
-
4&47,XY, -6, -7, -9, -10, 4647, XY, -6, -1, -9, - 10, -13, -13, -14, -16, +del lq(q32), +der 3q(q13), +der llq(q22). +der 12q (q43), +4 markers 4849, XX, -12, +13, -14, +15, -16, +17, +20, -21, -22 der 3q(q26), der 6q(qll) der 9p(p12), del 19q(q13) + micro, + 4 markers
no result
- * *
46,XY*
-
no result
no result
+ -
46,xx
no result -
-
-
-
-
-
-
-
-
-
c-myc bcl-2
Cytogenetic analysis
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B-MALT LYMPHOMAS
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Figure 1 Case 2. MALT B-low-grade. Characteristic cytology (Centrocyte-like = ccl. Monocytoid = mon. Centroblast = cb) in the tumour (Giemsa) and LEL (CAM 5.2- cytokeratin).
In high grade tumours large lymphoid cells (1420 pm in diameter) resembling centroblasts were characteristic. These cells contained round nuclei with finely dispersed chromatin and multiple nucleoli, close to the nuclear membrane. The cytoplasm was abundant with obvious ribosomes and a well
developed rough endoplasmic reticulum. Large anaplastic blast forms (20-25 pm in diameter) were present in smaller numbers (Figure 4). The flow cytometric, molecular genetic and cytogenetic results are summarised in Table 3 and illustrated in Figures 6 and 7.
Figure 2 Case 10. High grade. (a) LEL lesions in low grade area (b) proliferative marker. Ki-67 (c) intrasinusoidal growth (lymph node) with activation marker.
C. RIVAS et al.
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92
Figure 3 Case 2. MALT B-low-grude. Light and electron-microscopic correlation in a gland with LEL. Characteristic cells inside the epithelium (Centrocyte-like = ccl. Monocytoid = mon. Plasma cell = pc. Epithelial cell = ep) and in the surrounding tumor (Centroblast = cb. Lumen = L). x 7500.
Table 4 Percentage of different types of tumor cells, as seen by ultrastructure.
DISCUSSION
Low grade
The light microscopic and immunophenotypic features of the MALT cases in this study confirm those described in previous studies’ ‘-15. However, the ultrastructural characteristics of these lymphomas have not been previously investigated. Centrocyte-like cells differ from follicular centre centrocytes by showing a better developed rough endoplasmic
30% 20%
10% 204%
High grade
Centrocyte-like Monocytoid” Centroblast-like Large blasts Plasma cells Small lymphocytes
Few Few
“
25% 50% 5 YO
Table 5 Some electron microscopy findings.
Size
Nuclei
Nucleoli
Cytoplasm
R.E.R.
Cleaved dense chromatin Round dense cromatin
Single small
Small-medium
+/+ +
Single small
Medium large
+I+ +
Round dispersed chromatin Irregular dispersed chromatin
Single-multiple
Medium
+/+ +
Single-multiple
Medium
+I+ +
(run) Low grade Malt-CC (CC-like) Monocytoid High grode Malt-CB (CB-like) Large blasts
10-12 12-15
14-20 20-25
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B-MALT LYMPHOMAS
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Figure 4 Case 2. MALT B-low-grade. High magnificationof ultrastructural findings. (a) ccl and monocytoid cells among epithelial cells and under a gland. x 1O.OOO (b) MALT 1x1penetrating the basal membrane (BM). x2O.OOO.
Figure 5 Case 9. MALT B-high-grade. Blasts which resemble centroblast and large centrocytic-cellswith development of RER x 15.000. x 25.000.
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C. RIVAS et al.
wwbmb-w
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Figure 6 Flow cytometry. Two representative cases of cell cycle in MALT-L. (a) case 1. diploid with normal cycle (b)case 10. aneuploid.
reticulum. In addition the monocytoid variants of centrocyte-like cells are characterized by a larger amount of cytoplasm. The ultrastructural appearance of these cells is similar to those seen in marginal zone B-cells described in the spleen, supporting the suggestion that MALT lymphomas may be derived from these marginal zone cells. The larger transformed
cells present in low grade MALT lymphomas are ultrastructurally identical to those found in high grade MALT lymphomas. These cells, often called true centroblasts, differ from centroblasts by the presence of a larger amount of cytoplasm and a better developed rough endoplasmic reticulum. The ultrastructural appearances of lymphoepithelial lesions
Figure 7 Case 10. Representative karyotype from a stem line: 4849,XX, -3, +der 3q(q26), -6, +der 6q(qll), -9, +der 9p(p12), -12, +13, -14, +15, -16, +17, -19, +der (19), de119q(q13), +20, -21, -22, +micro, +4-5 markers with unknown origin. Arrows indicate chromosomal abnormalities.
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suggest that they are formed as part of an active 7. Isaacson, P. G., Spencer, J. and Finn, T. (1986). Primary B-cell gastric malignant lymphoma. Human Path., 17, 72-82: process by centrocyte-like cells which induce dege8 Spencer, J., Finn, T., Pulfford, K. F., Mason, D. Y. and Isaacson, nerative changes in the e p i t h e l i ~ m ' ~ - ' ~ . P. G. (1985).The human gut contains a novel population of B While flow cytometry did not discriminate between lymphocytes which resemble marginal zone cells. Clin. Exp. Immunol., 62, 607-6 1 2. low and high grade MALT lymphomas, the presence Saati, T., Caspar, S., Brousset, P., Chittals, Caveriviere, P., of aneuploid cells in some cases of low grade MALT 9 Al Hounieu, H., Dastugue, N., Idoipe, J. B., Icart, J., Mazerolles, lymphoma confirmed the malignant nature of this C. and Delsol, G. (1989). Production of anti-B monoclonal antibodies (DBB42, DBASS, DNA7 and DND53) reactive on clinically indolent lesion'0*20'21.The proliferation rate paraffin embedded tissues with a new B-lymphoma cell line as detected by Ki67 staining clearly separates low and grafted into athymic nude mice. Blood, 74(7), 2476-85. high grade MALT lymphomas as previously de- 10. Hedley, D. W., Friedlander, M. L., Taylor, I. W., Rugg, C. A. and Musgrove, E. A. (1983). Method for analysis of cellular scribed for nodal lymphoma13. DNA content of paraffin-embedded pathological material using The results of our molecular genetic studies again flow cytometry. J Histochem and Cytochem, 31, 1333-1335. confirm previous The presence of a single 11. Addis, B., Hyjek, E. and Isaacson, P. G. (1988). Primary pulmonary lymphoma: a reappraisal of its histogenesis and its case showing bcl-2 gene rearrangement is consistent relationship to pseudolymphoma in lymphoid interstitial with sporadic reports of this rearrangement in MALT pneumonia. Hisropathology, 13, 1-17. lymphoma as in other report^^'-^'. No d istinctive 12. Myhre, Mh. and Isaacson, P. G. (1987). Primary B-cell gastric lymphoma: a reassessment of its histogenesis. J. Pathol. 152, karyotypic abnormalities were found in this study, this 1-1 1. is in contrast to results described by Wotherspoon et 13. Piris, M. A., Rivas, C., Morente, M., Martinez, M. C., Toledo, al, showing a variety of clonal cytogenetic abnormaliM. C., Orradre, J. L. and Oliva, H. Linfomas de celulas B. (1988). Estudio inmunohistologico con anticuerpos monoties in 9 of 14 MALT lymphoma2'. However no clonales en 197 casos. Med. Clin. (Barcelona), 90, 679-685. consistent abnormality was found by these authors 14. Piris, M. A., Rivas, C., Morente, M., Orradre, J. L., Rodriguez, who have suggested that rearrangements of chromoR., Cruz, M. A., Martinez, J. L., Rubio, C. and Oliva, H. (1990). Linfoma gastric0 de bajo grado originado en tejido linfoide some 1P and numerical abnormalities of chromoasociado a mucosas. Relacion de las celulas tumorales con la somes 3 and 7 may play a role in the genesis of MALT zona marginal y 10s linfocitos B monocitoides. Estudio inmunohistoquimico y ultraestructural. Med. Clinica (Barcelolymphomas.
Acknowledgements We thank Mrs Marilis Lacunar (+) for her technical help for electron-microscopy, Mrs Yolanda G. de Castro for the immunocytochemical work and the Photographic laboratory for their technical assistance. We also thank Prof. P. G. Isaacson for his kind help in reviewing the manuscript, prior to submission. This work was supported by three grants for research from DGICYT PB-87-0110, FISs 89/0797 and Becton-Dickinson SA.
REFERENCES 1. Isaacson, P., Wright, D. H., Judd, M. A. and Mepham, B. L. (1979).Primary gastrointestinal lymphomas. A classification of 66 cases. Cancer, 47, 1805-1819. 2. Isaacson, P. and Wright, D. H. (1983). Malignant lymphoma of mucosa-associated lymphoid tissue. A distinctive type of B-cell lymphoma. Cancer, 52, 1410-1416. 3 Moore, 1. and Wright, D. H. (1984).Primary gastric lymphoma. A tumour of mucosa-associated lymphoid tissue. A histological and immunohistochemical study of 36 cases. Histopathology, 8, 1025-1039. 4. Spencer, J., Finn, T. and Isaacson, P. G. (1985).Gut associated lymphoid tissue: a morphological and immunocytochemical study of the human appendix. Gut, 26,672-679. 5. Stansfeld A. G., Diebold, J., Kapanci, Y., Keleny, G., Lennert, K., Mioduszewska, O., Noel, H., Rilke, F., Sundstrom, C., Van unmik J. A. M. and Wright, D. H. (1988). Updated Kiel classification for lymphomas. Lancet, ii, 292-293. 6. Isaacson, P. G., Spencer, J. and Wright, D. H. (1988).Classifying primary gut lymphomas. Lancet, 11, 1148-1 149.
na), 94, 601-606. 15. Lennert, K. and Feller, A. C. (1990). Non Hodgkin lymphome (Nach der aktualisierten Kiel-Klassification) Ed Springer Verlag. Heidelberg. pp. 141-150. Berlin. 16. Rivas, C. and Oliva, H. (1980). Linfomas no Hodgkin. Ed Salvat. pp. 50-106. Barcelona. 17. Kaiserling, E. (1978). Non Hodgkin Lymphome. Ed Gustav Fischer Verlag. Heidelberg. pp, 11Cb126. Berlin. 18. Rivas, C. Piris, M. A., Echezarreta, G., Sarasa, J. L., Rivas, F., Renedo, G. and Barat, A. (1992). Ultrastructural study of low grade B-MALT lymphomas. Arq Path (IAP). (In press). 19. Castrillo, J. M. and Rivas, C. (1990). El tejido linfoide asociado a mucosas y sus neoplasias. Rev. Clin. Esp., 187,213-215. 20. Friedlander, M. L., Hedley, D. W. and Taylor, I. W. (1984). Clinical and biological significance of aneuploidy in human tumors. J. Clin. Pathol., 37, 961-974. 21. Joensuu, H., Klemi, P. J. and Jalkanen, S. J. (1990). Biologic progression in non-Hodgkin's lymphoma. A flow cytometric study. Cancer, 65, 2564-2571. 22. Yanagi, Y., Chan, A., Chin, B., Minden, M. and Mak, T. (1985). Analysis of cDNA clones specific for human T cells and the beta and alfa chains of the T-cell receptor heterodimer from a human T-cell line. Proc. Natl. Acad. Sci. USA, 82,3430-3434. 23. Ravetch, J. V., Siebenlist, V., Korsmeyer, S. V., Waldmann, T. A. and Leder, P. (1981). The structure of the human immunoglobulin mu locus. Characterization of embrionic and rearranged V and D genes. Cell 27, 583-591. 24. Siminovitch, K. A,, Bakhshi, A., Goldman, P. and Korsmeyer, S. J. (1985).A uniform deleting element mediates the loss of K genes in human B cells. Nature, 316, 2W262. 25. Pan, L., Diss, T. C., Cunningham, D. and Isaacson, P. G. (1989). The bcl-2 gene in primary B cell lymphoma of mucosaassociated lymphoid tissue(MALT). Am. J. Pathol., 135,7-11. 26. Koduru, P. R. H., Filippa, D. A., Richardson, M. E., Jhanwar, S. C., Chaganti, S. R., Koziner, B., Clarkson, B. D., Lieberman, P. H. and Chaganti, R. S. K. (1987).Cytogenetic and histologic correlations in malignant lymphoma. Blood, 69, 97-102.
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27. Cunningham, D., Hickish, T., Rosin, R. D., Sauven, P., Baron, J. H., Farrell, P. J., Isaacson, P. G. (1989). Polymerase chain reaction for detection of dissemination in gastric lymphoma. The Lancet, 1, 695-697. 28. Van Krieken, J. H. J. M., Raffeld, M., Raghoebier, S., Jaffe, E. S., Van Ommen, G. J. B., Kluin, P. M. (1990). Molecular genetics of gastrointestinal non-Hodgkin’s lymphomas: un-
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0 1992 Hanvood Academic Publishers GmbH Printed in the United Kingdom
A Multiparametric Study of Malignant Lymphoma of Mucosa Associated Lymphoid Tissue (Malt) Leuk Lymphoma Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
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C. RIVAS, G. ECHEZARRETA, R. GARCiA, M. SANTOS, A. SANTON, M. ROBLEDO, J. BENITEZ, H. OLIVA, and G. DELSOL* Departments of Pathology, Immunology and Genetics, Fundacion Jimenez Diaz, Universidad Autonoma, Madrid, Spain, and *Laboratoire d'Anatomie Pathologique, C.H.U. Purpan. Toulouse, France. (Received 30 December 1991; in final form 12 February 1992)
A morphological, immunophenotypic and ultrastructural study, cell cycle estimation, DNA and cytogenetic analysis were performed in ten cases of B-MALT lymphomas. Five had low grade lymphoma and five had high grade. Low and high grade cases showed the same cells but in different percentages: These included centrocyte-like cells with occasional monocytoid cytoplasmic changes, and centroblast-like cells. However, in high grade cases more dysplastic and large cells were present. All cellular types showed an important development of rough endoplasmic reticulum. In all cases a large panel of monoclonal antibodies was employed t o study the B-cell immunophenotype. Ki-67 positivity ranged from 5% to 30% in low-grade cases and from 50% to 70% in high-grade cases. Gene rearrangement analysis showed rearrangement with Jh probe and half of the cases were also rearranged with the Kde probe (Kappa constant chain gene). A rearrangement banding pattern with TCR genes was not present in any of the cases. Cytogenetic study showed complex alterations in high grade cases and a normal karyotype in low grade lymphomas. Only one case had rearrangement for the bcl-2 probe. KEY WORDS:
B-MALT lymphomas immunophenotype ultrastructure gene rearrangement study flow cytometric analysis cytogenetic analysis.
follicles and consists of cells resembling centrocytes, the so called centrocyte-like (CCL) cells which may also resemble small lymphocytes or have a monocytoid appearance. Plasma cell differentiation is present in approximately a third of the cases and scattered larger transformed blasts are evident. The CCL cells characteristically invade glandular epithelium forming lymphoepithelial lesions (LEL)'. The distinctive features of high grade MALT lymphoma are not so well defined since LEL are absent and the tumour cells resemble centroblasts and immunoblasts as found in nodal high grade lymphomas. In a significant number of cases, however, the presence of areas of low grade lymphoma facilitates the identification of these tumours as of MALT type'.
INTRODUCTION Primary malignant lymphomas of mucosa associated lymphoid tissue (MALT) now constitute a distinct entity'+ which is not included in established classifications of nodal lymphomas, such as the Kiel clas~ification~-~. The architectural and cytological features of these malignancies have been well characterised in low grade lesions arising in the stomach. In these cases, the neoplastic infiltrate is centered around reactive
Address for correspondence: C. Rivas, Anatomia Patologica. Fundacion Jimenez Dim. Avda Reyes Catolicos 2-4.28040 Madrid, Spain. 87
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In this report we have attempted to further in 2% osmium tetroxide and embedded in Vestopal characterize MALT lymphomas with respect to their W resin. Sections were stained with uranyl acetate and immunophenotype, ultrastructure, genetic character- lead citrate and observed using an Hitachi HU12A electron microscope. istics and proliferation rate. Flow cytometric analysis was carried out on cell suspensions from either fresh or paraffin embedded tissue prepared by pepsin digestion treatment MATERIALS AND METHODS according to the method of Hedley", using Surgical resection specimens from 10 cases of B-cell propidium iodine DNA stains. The analysis was M A L T lymphomas were studied. Five of these were undertaken using an EPICS-C flow cytometer low grade tumours (2 intestinal, 2 gastric and 1 (Coulter Hialeah Florida) and the cell cycle was thyroid) and 5 high grade (3 gastric and 2 intestinal). calculated using the PARA- 1 programme. Histograms Routine haematoxylin eosin stained sections were were considered diploid if only one symmetrical GO/G1 peak was present with a coefficient variation examined in all cases. Immunohistochemistry was carried using the (CV) less than 5% for fresh material and 10% for APAAP technique on frozen tissue and the Avidin paraffin embedded material. If the peak was Biotin immunoperoxidase technique on paraffin asymmetrical or two GO/Gl peaks were present, DNA embedded tissue with prior trypsinization of sections histograms were considered aneuploid. S + G2M where indicated. The panel of antibodies used is given phase was only measured in diploid samples. An in Table 19. S-phase in excess of 20% was considered as indicative Tissue for electron microscopy was fixed in 2.5% of a high proliferation rate. glutaraldehyde in cacodylate buffer (pH 7.4), postfixed For molecular studies, high molecular weight D N A
Table 1 Monoclonal Antibodies used. MoAbs
Source
Specificity
Frozen tissue CD3, Leu4 CD4, Leu3a CD5, Leu1 CD8,OKT8 CDlO, CALLA
Becton-Dickinson Becton-Dickinson Becton-Dickinson Ortho-diagnosis Becton-Dickinson
T-cells T-helper/inducer cells T-cell restrictive antigen (+ in some B low-grade lymphomas) T suppressor/cytotoxic Human common acute Lymphoblastic leukemia antigen (B-follicular centre lymphomas) B-cells (pan B) (B progenitor) B-cells (mature and DRC)
CD19, Leu12 CD20, B7, Leu14 CD30, Kil, BerH2*
Becton-Dickinson Ortho-diagnosis Becton-Dickinson DAKO
Ki67 MU (IgW Kappa Lambda LeuM5 Leu8 DRC
DAKO BRL diagnosis Becton-Dickinson Becton-Dickinson Becton-Dickinson Becton-Dickinson DAKO
Lymphoid activation marker (Hodgkin disease, some anaplastic high grade lymphomas) Nuclear proliferative marker B-cells B-cell~ B-cells Phagocytes Restrictive T-cell antigen mantle B-follicular Follicular dendritic cells
DAKO Biotest DAKO Biotest
T-cells Immature B cell, immature T-cell (T-B cell high grade lymphomas) B-cell marker B-cell marker
Immunotech-Delsol Immunotech-Delsol Immunotech-Delsol Becton-Dickinson
B-cell marker B-cell marker B-cell marker Human cytokeratin
Parafin embedded tissue UCHLl MTI L26 MB2 Experimental panel: (9) DBB42 DND53 DBA44 CAM 5.2
DRC = dendritic reticulum all.
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was extracted from fresh and frozen samples by appeared as a firm white infiltrating mass without standard methods. Complete digestion with the preservation of the normal parenchyma. All the high restriction enzymes: EcoR1, HindIII, BamH1, BglII, grade MALT cases showed bulky infiltrating tumours PstI of 5 pg of total DNA was followed by Southern with numerous adjacent enlarged lymph nodes. blot analysis. Blots were probed with 32-P labelled The histological study of low grade MALT DNA probes for genes encoding the T-cell receptor lymphomas confirmed previous descriptions of this Beta chain (p-TCR) and the heavy chain joining entity. Centrocyte-like cells illustrated the cytological region (Jh), kappa light chain (Kde), kappa constant spectrum previously described and included cells with region (Ck), lambda constant region (Cl) of the Ig gene a monocytoid appearance. Transformed blast forms and also probes encoding the c-myc and bcl-2 were scattered throughout the infiltrate and plasma oncogene (Table 2). Each filter was washed with cells were present in all cases. Lymphoepithelial 2 x SSC, 0.1% SDS at room temperature and lesions were prominent in all of these cases (Figure 1). High grade MALT cases showed extensive diffuse 0.1 x SSC, 0.1% SDS at 52°C. Autoradiography was infiltration with large nucleated tumour cells. These done at -80°C with intensifying screens. For karyotypic analysis portions of tumour were cells resembled centroblasts but had more abundant minced by mechanical methods followed by enzymatic cytoplasm and immunoblasts were also present. digestion with collagenase, 200 pl/ml for 1-2 hours at Lymphoepithelial lesions formed by cells seen in low 37°C. Cells were resuspended in RPMI colcemid and grade MALT were also present in 2 cases (cases 9 and a second culture of 72 hours with PKW mitogen was 10) (Figure 2). Immunohistochemistry confirmed the B cell nature performed. GTG banding techniques were done in all cases and each one showed immunoglobulin according to standard procedures. light chain restriction. The detailed immunophenotype is given in Table 3 as are the Ki67 scores which varied between 5 and 30% for the low grade cases RESULTS and between 50 and 70% for the high grade case. Ultrastructural findings (Table 4 and 5 ) showed the Results are summarized in Table 3. Macroscopically following cytological characteristics. In low grade low grade MALT gastrointestinal lymphomas were tumours centrocyte-like cells were medium sized characterized by flattened areas containing single or (10-12 pm in diameter) with indented, irregular oval multiple superficial ulcers. The thyroid tumour nuclei containing dense chromatin concentrated near Table 2 Probes used in the molecular study. the nuclear membrane. Single small nucleoli were noted. A moderate amount of cytoplasm was present Probe Size Enzyme Fragment size in which contained few organelles and variable developgerm line (Kb) ment of rough endoplasmic reticulum. Larger cells 12; 4 0.7 EcoRl with similar characteristics but with more diffuse T-cell receptor Hind111 7.3; 5.8; 3.3 beta chain chromatin were also present. Monocytoid B cells were 24 BamH 1 B-TCR larger than the centrocyte-like cells (12-15 pm in Heavy chain 2.5 HindIII 9.5 diameter) and contained nuclei which were rounder joining region BgIII 4 without significant membrane indentations. Nuclear JH 6 EcoR 1 17 BamH 1 16 chromatin was similar to that of other centrocyte-like 14 Kappa light 2.5 BamH 1 cells but the cytoplasm was more abundant and chain clearer. A well developed endoplasmic reticulum was Kde evident in these cells. 6.6 Kappa constant 2.5 Hind111 PstI 3.5 Lymphoepithelial lesions were characterized by region CK centrocyte-like and monocytoid cells which appeared Lambda constant 3.5 Pstl 1.75; 1 to enter the epithelium by way of pseudopod 4.3; 1.2; 1 BamH 1 region formation. The epithelium showed disruption of the CL basement membrane with disappearance of desmo9 EcoRl 20 c-myc somes. In addition, degenerate nuclear and cytobcl-2 3 PstI 16 BamH 1 18 plasmic changes were present, the most obvious of hind111 4 which was an increase of mitochondria giving rise to 18 EcoR 1 an oncocytic appearance (Figure 3).
F
M
F
F
M
M
F
4
5
6
7
8
9
10
With PKW mitogen; ** Frozen,material; 2 Paraffin; G = Germ line; R = Rearranged;
High grade Intestinal
-
27
65
-
-
68
50
-
1
25
5
30 14
20 36
24
73
44
7
1
4
4
A
D
D
A
A
D
A
D
D
D
G2W PLOID
8 14
18
80
76
5
11
S
87
83
Go/Gi
Cell cycle by pow cytometry gene
= Data non available.
IgM, K,CD19, Ki67= 80% CD30 L26, MB2, DBB42, DND53*
M
3
Tumoral Ulcero-infiltrative
F
2
Immunophenotyge
IgM, L, CD19, Ki67 = 1&20% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 5% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 20% L26, MB2, DBB42, DND53* IgM, L, Cd19, Ki67 = 5% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 30% L26, MB2, DBB42, DND53* IgM, L, CD19, Ki67 = 5&70% L26, MB2, DBB42, DND53* IgM, K, CD19, Ki67 = 50% L26, MB2, DBB42, DND53* IgM, L, CD19, Ki67 =60% L26, MB2, DBB42, DND53* IgM, L, CD19, Ki67 = 60% CALLA L26, MB2, DBB42, DND53*
Infiltrative
M
1
Site
Low grade Intestinal Infiltrative Low grade Gastric Ulcerated Low grade Gastric Infiltrative Low grade Thyroid Tumoral-infiltrative Low grade Intestinal Tumoral High grade Ulcero-infiltrative Gastric Ulcerated High grade Gastric Tumoral High grade Ulcero-infiltrative Gastric Tumoral-infiltrative High grade Intestinal
Macroscopy
Cnse Sex
Table 3 Immunophenotypic, Bow cytometric, gene rearrangement and cytogenic results.
G
G
G
G
G
G
R
G
R
R R
R
R
G
-
R
G R
G
-
R
G
G
G
G
G
G
G
G
R
G
G
-
-
-
G
G
R
R
G
G
G
G
R
G
G
G
-
G
Kde CK C
G
TCR JH
Rearrangement analysis
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** -
-
-
-
-
4&47,XY, -6, -7, -9, -10, 4647, XY, -6, -1, -9, - 10, -13, -13, -14, -16, +del lq(q32), +der 3q(q13), +der llq(q22). +der 12q (q43), +4 markers 4849, XX, -12, +13, -14, +15, -16, +17, +20, -21, -22 der 3q(q26), der 6q(qll) der 9p(p12), del 19q(q13) + micro, + 4 markers
no result
- * *
46,XY*
-
no result
no result
+ -
46,xx
no result -
-
-
-
-
-
-
-
-
-
c-myc bcl-2
Cytogenetic analysis
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Figure 1 Case 2. MALT B-low-grade. Characteristic cytology (Centrocyte-like = ccl. Monocytoid = mon. Centroblast = cb) in the tumour (Giemsa) and LEL (CAM 5.2- cytokeratin).
In high grade tumours large lymphoid cells (1420 pm in diameter) resembling centroblasts were characteristic. These cells contained round nuclei with finely dispersed chromatin and multiple nucleoli, close to the nuclear membrane. The cytoplasm was abundant with obvious ribosomes and a well
developed rough endoplasmic reticulum. Large anaplastic blast forms (20-25 pm in diameter) were present in smaller numbers (Figure 4). The flow cytometric, molecular genetic and cytogenetic results are summarised in Table 3 and illustrated in Figures 6 and 7.
Figure 2 Case 10. High grade. (a) LEL lesions in low grade area (b) proliferative marker. Ki-67 (c) intrasinusoidal growth (lymph node) with activation marker.
C. RIVAS et al.
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Figure 3 Case 2. MALT B-low-grude. Light and electron-microscopic correlation in a gland with LEL. Characteristic cells inside the epithelium (Centrocyte-like = ccl. Monocytoid = mon. Plasma cell = pc. Epithelial cell = ep) and in the surrounding tumor (Centroblast = cb. Lumen = L). x 7500.
Table 4 Percentage of different types of tumor cells, as seen by ultrastructure.
DISCUSSION
Low grade
The light microscopic and immunophenotypic features of the MALT cases in this study confirm those described in previous studies’ ‘-15. However, the ultrastructural characteristics of these lymphomas have not been previously investigated. Centrocyte-like cells differ from follicular centre centrocytes by showing a better developed rough endoplasmic
30% 20%
10% 204%
High grade
Centrocyte-like Monocytoid” Centroblast-like Large blasts Plasma cells Small lymphocytes
Few Few
“
25% 50% 5 YO
Table 5 Some electron microscopy findings.
Size
Nuclei
Nucleoli
Cytoplasm
R.E.R.
Cleaved dense chromatin Round dense cromatin
Single small
Small-medium
+/+ +
Single small
Medium large
+I+ +
Round dispersed chromatin Irregular dispersed chromatin
Single-multiple
Medium
+/+ +
Single-multiple
Medium
+I+ +
(run) Low grade Malt-CC (CC-like) Monocytoid High grode Malt-CB (CB-like) Large blasts
10-12 12-15
14-20 20-25
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Figure 4 Case 2. MALT B-low-grade. High magnificationof ultrastructural findings. (a) ccl and monocytoid cells among epithelial cells and under a gland. x 1O.OOO (b) MALT 1x1penetrating the basal membrane (BM). x2O.OOO.
Figure 5 Case 9. MALT B-high-grade. Blasts which resemble centroblast and large centrocytic-cellswith development of RER x 15.000. x 25.000.
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C. RIVAS et al.
wwbmb-w
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Figure 6 Flow cytometry. Two representative cases of cell cycle in MALT-L. (a) case 1. diploid with normal cycle (b)case 10. aneuploid.
reticulum. In addition the monocytoid variants of centrocyte-like cells are characterized by a larger amount of cytoplasm. The ultrastructural appearance of these cells is similar to those seen in marginal zone B-cells described in the spleen, supporting the suggestion that MALT lymphomas may be derived from these marginal zone cells. The larger transformed
cells present in low grade MALT lymphomas are ultrastructurally identical to those found in high grade MALT lymphomas. These cells, often called true centroblasts, differ from centroblasts by the presence of a larger amount of cytoplasm and a better developed rough endoplasmic reticulum. The ultrastructural appearances of lymphoepithelial lesions
Figure 7 Case 10. Representative karyotype from a stem line: 4849,XX, -3, +der 3q(q26), -6, +der 6q(qll), -9, +der 9p(p12), -12, +13, -14, +15, -16, +17, -19, +der (19), de119q(q13), +20, -21, -22, +micro, +4-5 markers with unknown origin. Arrows indicate chromosomal abnormalities.
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suggest that they are formed as part of an active 7. Isaacson, P. G., Spencer, J. and Finn, T. (1986). Primary B-cell gastric malignant lymphoma. Human Path., 17, 72-82: process by centrocyte-like cells which induce dege8 Spencer, J., Finn, T., Pulfford, K. F., Mason, D. Y. and Isaacson, nerative changes in the e p i t h e l i ~ m ' ~ - ' ~ . P. G. (1985).The human gut contains a novel population of B While flow cytometry did not discriminate between lymphocytes which resemble marginal zone cells. Clin. Exp. Immunol., 62, 607-6 1 2. low and high grade MALT lymphomas, the presence Saati, T., Caspar, S., Brousset, P., Chittals, Caveriviere, P., of aneuploid cells in some cases of low grade MALT 9 Al Hounieu, H., Dastugue, N., Idoipe, J. B., Icart, J., Mazerolles, lymphoma confirmed the malignant nature of this C. and Delsol, G. (1989). Production of anti-B monoclonal antibodies (DBB42, DBASS, DNA7 and DND53) reactive on clinically indolent lesion'0*20'21.The proliferation rate paraffin embedded tissues with a new B-lymphoma cell line as detected by Ki67 staining clearly separates low and grafted into athymic nude mice. Blood, 74(7), 2476-85. high grade MALT lymphomas as previously de- 10. Hedley, D. W., Friedlander, M. L., Taylor, I. W., Rugg, C. A. and Musgrove, E. A. (1983). Method for analysis of cellular scribed for nodal lymphoma13. DNA content of paraffin-embedded pathological material using The results of our molecular genetic studies again flow cytometry. J Histochem and Cytochem, 31, 1333-1335. confirm previous The presence of a single 11. Addis, B., Hyjek, E. and Isaacson, P. G. (1988). Primary pulmonary lymphoma: a reappraisal of its histogenesis and its case showing bcl-2 gene rearrangement is consistent relationship to pseudolymphoma in lymphoid interstitial with sporadic reports of this rearrangement in MALT pneumonia. Hisropathology, 13, 1-17. lymphoma as in other report^^'-^'. No d istinctive 12. Myhre, Mh. and Isaacson, P. G. (1987). Primary B-cell gastric lymphoma: a reassessment of its histogenesis. J. Pathol. 152, karyotypic abnormalities were found in this study, this 1-1 1. is in contrast to results described by Wotherspoon et 13. Piris, M. A., Rivas, C., Morente, M., Martinez, M. C., Toledo, al, showing a variety of clonal cytogenetic abnormaliM. C., Orradre, J. L. and Oliva, H. Linfomas de celulas B. (1988). Estudio inmunohistologico con anticuerpos monoties in 9 of 14 MALT lymphoma2'. However no clonales en 197 casos. Med. Clin. (Barcelona), 90, 679-685. consistent abnormality was found by these authors 14. Piris, M. A., Rivas, C., Morente, M., Orradre, J. L., Rodriguez, who have suggested that rearrangements of chromoR., Cruz, M. A., Martinez, J. L., Rubio, C. and Oliva, H. (1990). Linfoma gastric0 de bajo grado originado en tejido linfoide some 1P and numerical abnormalities of chromoasociado a mucosas. Relacion de las celulas tumorales con la somes 3 and 7 may play a role in the genesis of MALT zona marginal y 10s linfocitos B monocitoides. Estudio inmunohistoquimico y ultraestructural. Med. Clinica (Barcelolymphomas.
Acknowledgements We thank Mrs Marilis Lacunar (+) for her technical help for electron-microscopy, Mrs Yolanda G. de Castro for the immunocytochemical work and the Photographic laboratory for their technical assistance. We also thank Prof. P. G. Isaacson for his kind help in reviewing the manuscript, prior to submission. This work was supported by three grants for research from DGICYT PB-87-0110, FISs 89/0797 and Becton-Dickinson SA.
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