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Nucleolar Organizer Regions (NORs) Staining and Proliferating Cell Nuclear Antigen (PCNA) Immunostaining in Mucosa-Associated Lymphoid Tissue (MALT) Gastric Lymphomas
Path. Res. Pract. 189, 1004-1009 (1993)
Nucleolar Organizer Regions (NORs) Staining and Proliferating Cell Nuclear Antigen (PCNA) Immunostaining in Mucosa-Associated Lymphoid Tissue (MALT) Gastric Lymphomas ~~ P. Mikoul, P. Kanavaros2 , D. Aninosl, M. Tzardi 2 , A. Karamerisl,
B. Gorgoulisl, N. Papadopoulos3 , A. Lavergne4 and A. Galian 4 Departments of Pathology: 1401 General Army Hospital of Athens, Greece, 2University Hospital of Heraklion, Greece, 3University Hospital of Alexandroupolis, Greece, 4Lariboisiere Hospital, Paris, France
SUMMARY Nucleolar organizer region-associated proteins (AgNOR) and proliferating cell nuclear antigen (peNA) have been studied by means of a silver staining technique and immunohistochemistry, in paraffin-embedded, gastrectomy specimens of 12 low-grade and 13 high-grade gastric MALT lymphomas respectively. A significant difference was found between the AgNOR count and peNA index of low-grade lymphomas (mean AgNOR count 2.5 and mean peNA index 8.33 %) and high-grade lymphomas (mean AgNOR count 8.67 and mean peNA index 49.7 %). It is suggested that both methods are useful adjuncts to histopathology for the distinction between low and high grade gastric MALT lymphomas. We also found heterogeneity in AgNOR counts and peNA index among individual cases of either low or high grade MALT gastric lymphomas. This suggests that the AgNOR count and peNA index is helpful in the individual approach of the proliferation rate of each tumour, a parameter of potential importance for predicting the biological behaviour of the tumour and the prognosis of the disease.
Introduction The Nucleolar Organizer Regions (NORs) are loops of DNA (rDNA) in which ribosomal RNA (rRNA) is encodedl, 17, and during interphase they are located in the nucleoli of cells ll . NORs are also the sites of location of a peculiar group of acidic non-histonic proteins, which stain intensely with formic acid -silver nitrate 23 , 26, 28, 35,. The silver-stained NORs are defined as Ag-NORs and the argyrophilic proteins as Ag-NOR proteins. The method has been utilized by cytogeneticists for the evaluation of
* This work was supported by a grant from the Greek Anticancer Society. 0344·0338/93/0189-1004$3.50/0
certain genetic disorders, notably trisomies lo ,22. The usefulness of the method has been increased by means of a modification allowing the AgNOR reaction to run at room temperature 30 • The AgNOR method seems to be useful for diagnosis of malignancy, for evaluating the proliferation rate of a tumour37 , and even as a new prognostic parameter 12 • Proliferating cell nuclear antigen (peNA) is a 36 kd acidic non-histonic nuclear protein, the expression of which is correlated with the S-phase of the cell cycle 4 . This protein was originally detected during experiments with use of human autoantibodies in lupus patients 27 . It has been demonstrated to be an auxiliary protein to DNA polymerase delta 3 and its presence appears to be necessary © 1993 by Gustav Fischer Verlag, Stuttgart
NORs Staining and PCNA Immunostaining in MALT Gastric Lymphomas· 1005 for the initiation of cell proliferation24 • There is increasing evidence that PCNA assessment is an easy and useful tool to evaluate cell proliferation. Previous studies in lymphomas have shown good correlation between PCNA expression 36 , AgNOR count?, other proliferation indices 19 ,32 and histological grade. Accordingly we have investigated the significance of AgNOR enumeration and PCNA expression for grading and determining the proliferation rate in MALT lymphomas of the stomach.
Material and Methods Specimens Twenty-five gastrectomy specimens of MALT lyphomas were examined, from the same number of patients. Only gastrectomy specimens were included because they permitted detailed histological analysis and grading. The histological analysis was performed on multiple sections and from different tumour areas. The lymphomas were classified histologically as high grade (13 cases) and low grade (12 cases) according to the criteria proposed by Chan et aP. In addition, the lymphomas were classified according to the updated Kiel classification 1988. In the group of low grade lymphomas there were 7 cases of centrocytic lymphomas of MALT type (cases no 1,2,3, 7, 8, 10, 12) and 5 cases of lymphoplasmacytic lymphomas of MALT type (cases no 4,5,6,9, 11). In the group of high grade lymphomas there were 4 cases of centroblastic centrocytoid lymphomas with component of centrocytic lymphoma of MALT type (cases no 1, 7, 11, 12), 8 cases of centro blastic polymorphic lymphomas with component of centrocytic lymphoma of MALT type (cases no 2, 3, 5, 6, 8, 9, 10, 13) and 1 case of immunoblastic lymphoma with plasma blastic differentiation and with component of lymphoplasmacytic lymphoma of MALT type (case no 4). These specimens had previously been phenotyped by standard immunohistochemical methods. All of them were of B-cell origin as assessed by using B (L26ICD20, 4KB5ICD45R, x and A light chains) and T (UCHLl/CD45RO and MTl/CD43) cell markers. Blocks were taken from these cases from routine files of the Departments of Pathology of Lariboisiere Hospital, 401 General Army Hospital, University Hospital of Heraklion and of Alexandroupoli. The tissue had been fixed in Bouin's fixative (14 cases) or in formalin 10 % (11 cases) and processed to paraffin wax.
Staining procedure Sections were cut at 5 !!m thickness from paraffin blocks. These were dewaxed in xylene, then hydrated through ethanols to distilled deionized water. a) AgNOR Staining: The AgNOR staining solution was prepared by dissolving 2 g of gelatin in 100 ml of 1 % aqueous formic acid (solution A), and 50 g of silver nitrate in 100 ml of distilled water (solution B). These solutions were mixed 1(A) : 2(B) volumes just prior to use. This was poured over the tissue sections and the slides were kept in a humidity chamber, protected from daylight for 30 minutes at room temperature. The specimens were then washed with distilled deionized water, dehydrated through alcohols to xylene, and mounted in DPX medium. No counterstain was performed. b) PC 10-1mmunohistochemistry: The slides were immersed for 10 min in 3 % H202 to block endogenous peroxidase and then taken to PBS (pH: 7.6). Immunostaining was performed using the PAP method (Dako). A dilution of 1 : 10 with one hour incubation
was found to be optimal. Very light haematoxylin counterstain was performed before mounting.
AgNOR counting procedure The slides were examined using a 100 oil-immersion lens so that AgNOR dots were counted at a magnification of 1000. One hundred lymphoid cells were randomly selected from each specimen and the AgNOR dots were counted individually, carefully focused, even when occurring in large aggregates. Special caution was taken in the study of low grade lymphomas so that AgNOR number in lymphoid cells from non-neoplastic reactive follicles were not counted. The reason was that, while in all 12 low grade gastric MALT lymphomas reactive follicles with varying degrees of colonization by small neoplastic cells were found, only one of the 12 cases displayed many follicular structures composed almost exclusively of small neoplastic cells. Thus, a comparative study between reactive and neoplastic follicles could not be performed due to the very limited number of cases with clearcut, unequivocal neoplastic follicles.
Evaluation of PCNA immunostaining The slides were examined using a 10 objective lens and ten fields were randomly selected. In the study of low grade lymphomas the lymphoid cells from non-neoplastic reactive follicles were not counted. Finally 1000 tumour cells from these fields were counted using a 40 objective lens. The PCNA index represented the percentage of cells with positive nuclear staining (regardless of the staining intensity) in the total number of tumour cells counted.
Results In all specimens clearly defined silver-stained dots were observed in all nuclei. These were separate or organized in small aggregates, and sometimes were placed in a different position than the haematoxylin-stained nucleoli or chromatin. Reactivity of PC10 was distinct although there was a gradation in the intensity of staining.
Low-grade lymphomas The neoplastic population, which was composed of small cells, possessed a mean number of 2.5 AgNORs per nucleus (Table 1), (Fig. 1). The mean PCNA index was 8.33 % (Table 1), (Fig. 2).
High-grade lymphomas The neoplastic population was mainly composed of large cells. In some cases, the presence of a distinct subpopulation of small neoplastic cells was obvious. This constituted a variable percentage (always small) of the neoplastic cell population. All neoplastic cells were taken under consideration in the counting procedure. The mean number was 8.67 AgNORs per nucleus (Table 2), (Fig. 3). The mean PCNA index was 49.6 % (Table 2), (Fig. 4). It was noticed that within each histological group of either high or low grade gastric MALT lymphomas there was a variation of AgNOR counts and peNA index among individual cases (Tables 1 and 2). Statistical analysis of the
1006 . P. Mikou et al.
Fig. 1. AgNOR staining in low-grade lymphoma (*400).
Fig. 2. PCNA (*400).
Table 1. AgNOR mean number and PCNA index in low grade lymphomas
Table 2. AgNOR mean number and PCNA index in high grade lymphomas
Case No.
LOW GRADE LYMPHOMAS AgNOR mean number PCNA index
Case No.
HIGH GRADE LYMPHOMAS AgNOR mean number PCNA index
1 2 3 4 5 6 7 8 9 10 11 12
2.2 2.76 3.22 2.44 1.85 2.15 2.1 3.11 2.5 2.14 2.26 3.3
12 % 7% 14 % 8% 4% 5% 7% 17 % 6% 9% 1% 10 %
1 2 3 4 5 6 7 8 9 10 11 12 13
8.69 8.71 10,48 10.15 7.54 8.31 8.06 9.63 8.22 8.56 7.64 7.96 8.3
mean value: 2.5 SD: 0.485 SE: 0.14
mean value: 8.33 % SD: 0.044 SE: 0.012
immunostaining
mean value: 8.67 SD: 0.916 SE: 0.07
in
low-grade
lymphoma
63% 50 % 61 % 73 % 75 % 65% 26 % 70 % 52 % 57 % 25 % 20 % 60 % mean value: 49.6 % SD: 0.186 SE: 0.052
NORs Staining and peNA Immunostaining in MALT Gastric Lymphomas· 1007
Fig. 3. AgNOR staining in high-grade lymphoma (*400).
Fig. 4. peNA (*400).
results, by means of t-test, showed that the difference between the two mean values was statistically very significant (p < 0.1 %) for both AgNOR count and reNA index (Table 3).
Table 3. Statistical analysis (Student's test)
Discussion The present study shows that both AgNOR counts and reNA immunostaining correlate well with histological grading of primary gastric MALT lymphomas. This is in keeping with previous findings that AgNOR counts can help in distinguishing high grade from low grade nonHodgkin nodal lymphomas7 and that reNA immunostaining correlates well with histological grade in nodal lymphomas and primary gastrointestinallymphomas25 ,36. The AgNOR count seems to represent a parameter of tumour proliferation rate 37 • It has been reported that AgNOR count has linear relationship with the percentage of S-phase cells, determined by means of DNA flowcytometry in high and low grade non-Hodgkin's lymphomas 9• This was also suggested by comparison of AgNOR values with Ki-67 immunoreactivity 20 and bromodeoxy-
immunostaining
In
high-grade
AgNOR count
peNA index
t-value: 21,276 DF: 23
t-value: 7.786 DF: 23
p < 0.1 %
p < 0.1 %
lymphoma
uri dine incorporation 29 . The augmentation of the AgNOR number per nucleus in hyperplastic or malignant cells has been reported to be also related to cell ploidy and transcriptional activity16, although this has been doubted lately14, and, the to the presence of a defect of nucleolar association 34 • Regarding reNA index, previous studies have shown that it correlates with the S-phase fraction of tumour cells determined by DNA flow cytometry 18,21,36. In addition, the proliferating fraction of tumour cells detected by reNA immunostaining correlates with that detected by Ki6725 , the latter marking cells in Gl and G2 phases in addition to those in S-phase6 , with thymidine labeling index 2 and with bromodeoxyuridine uptake in carcinoma celllines 6 •
1008 . P. Mikou et al.
In view of these data, taken together, the presence of high AgNOR count and high peNA index in high grade gastric MALT lymphomas could reflect a higher proliferation rate, higher transcriptional activity and more aneuploid cells than in low grade ones. In this study peNA index and AgNOR count do not correlate well with each other. It is likely that these methods quantify different aspects of proliferative activity but there is also evidence that peNA index and AgNOR count do not only measure the proliferation. Indeed, peNA expression does not correlate with the S-phase fraction determined by flow cytometry in some tumours 38 and AgNOR expression may be influenced by differentiation state, and proliferation rate 1S ,31. Nevertheless, it is particularly interesting that these two methods independently show a correlation with histological grading in gastric MALT lymphomas. In the present study, we found that within each histological group of either high or low grade gastric MALT lymphomas there was a variation of AgNOR counts and peNA index among individual cases. This variation indicates that within the generic groups of high and low grade gastric MALT lymphomas individual cases have a different proliferation rate and perhaps different biological behaviour, thereby suggesting that an individual approach based on the evaluation of the proliferation rate may be of importance for predicting the clinical outcome of these tumours. In this respect peNA immunostaining and/or AgNOR counts may be helpful in providing objective support for the histological grading of MALT lymphomas especially for cases of a borderline histology between low and high grade lymphomas. In conclusion, our preliminary data suggest that peNA index and AgNOR counts are useful adjuncts to histopathology for the histological grading of gastric MALT lymphomas. Moreover, they permit to detect variations of the proliferation rate among individual cases belonging to either low or high grade gastric MALT lymphomas. This could be useful in the individual approach of the proliferative activity and perhaps the clinical behaviour of each tumour. Further investigations are required to evaluate the prognostic significance of AgNOR count and peNA index in gastric MALT lymphomas, and to determine whether the variations of these parameters could reflect variations in the aggressiveness of tumours.
References 1 Alberts B, Bray D, Lewis], Raff M, Roberts K, Watson]D (1983) Molecular Biology of the Cell. New York Garland Publishing Inc. 2 Battersby S, Anderson T] (1990) Correlation of proliferative activity in breast tissue using PCNA/Cyclin. Hum Pathol21: 781 3 Bravo R, Frank R, Blundell PA, Macdonald-Bravo H (1987) CyclinlPCNA is the auxiliary protein of DNA polymerase-delta. Nature 326: 515-520 4 Celis ]E, Celis A (1985) Cell cycle-dependent variations in the distribution of the nuclear protein eyclin proliferating cell nuclear antigen in cultured cells: Subdivision of S-phase. Proc Nat! Acad Sci USA 82: 3262-3266
5 Chan ]K, Ng CS, Isaacson PG (1990) Relationship between high-grade lymphoma and low-grade B-cell mucosa-associated lymphoid tissue lymphoma (MALTOMA) of the stomach. Am] Pathol136: 1153-1164 6 Coltrera MD, Gown AM (1991) PCNA/Cyclin Expression and BrdU uptake define different subpopulations in different cell lines. ] Histochem Cytochem 39: 23-30 7 Crocker ], Nar P (1987) Nucleolar organizer regions in lymphomas.] Pathol151: 111-118 8 Crocker ], Skilbeck N (1987) Nucleolar organizer region associated proteins in cutaneous melanotic lesions: a quantitative study. ] Clin Pathol 40: 885-889 9 Crocker ], MaCartney ]C, Smith P] (1988) Correlation between DNA flow cytometric and nucleolar organizer region data in Non-Hodgkin's lymphomas. ] Pathol154: 151-156 10 De la Cruz FF, Gerald PS (1981) Trisomy 21. Baltimore University Paulo Press, 165-167 11 Derenzini M, Thiry M, Goessens G (1990) Ultrastructural cytochemistry of the mammalian cell nucleolus. ] Histochem Cytochem 38: 1237-1256 12 Derenzini M, Trere D (1991) Importance of interphase nucleolar organizer regions in tumour pathology. Virchows Arch [B] 61: 1-8 13 Derenzini M, Nardi F, Farabegoli F, Ottinetti A, Roncaroli F, Bussolati G (1989) Distribution of silver-stained interphase nucleolar organizer regions as a parameter to distinguish neoplastic from non-neoplastic reactive cells in human effusions. Acta Cytol33: 491-498 14 Derenzini M, Pession A, Farabegoli F, Trere D, Badiali M, Dehan P (1989) Relationship between interphase nucleolar organizer regions and growth rate in two neuroblastoma cell lines. Am] Pathol134: 925-932 15 Edwards S, Afford S, Crocker] (1991) The effect of inducing agents on the numbers of interphase fibrillar centers in the U937 pro monocytic cell line. Exp Cell Res 194: 118-121 16 Fakan S, Hernandez-Verdun D (1986) The nucleolus and the nucleolar organizer regions. Bioi Cell 56: 189-206 17 Gall ]G, Pardue ML (1969) Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc Nat! Acad Sci USA 64: 600-604 18 Garcia RL, Coltrera MD, Gown AM (1989) Analysis of proliferative grade using anti-PCNA/Cyclin in fixed, embedded tissues. Comparison with flow cytometric analysis. Am ] Pathol 134: 733-739 19 Hall PA, Richards MA, Gregory WM, d'Ardenne A], Lister TA, Stansfeld AG (1988) The prognostic value of Ki67 immunostaining in non-Hodgkin's lymphoma.] Patholl54: 223-235 20 Hall PA, Crocker ], Watts A, Stansfield AG (1988) A comparison of nucleolar organizer region staining and Ki-67 immunostaining in Non-Hidgkin's lymphoma. Histopathology 12: 373-381 21 Hall PA, Levison DA, Woods AL, et al (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. ] Pathol162: 285-294 22 Howell WM (1982) Selective staining of nucleolus organizer regions (NORs). In: Busch H, Rothblum L (Eds) The Cell Nucleus, 89-142. Academic Press, New York 23 Howell WM, Black DA (1980) Controlled silver-staining of nucleolar organizer regions with a protective colloidal developer: a I-step method. Experientia 36: 1014-1015 24 Jaskulski D, deRiel]K, Mercer WE, Calabretta B, Baserga R (1988) Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA Cyclin. Science 240: 1544-1546 25 Kamel OW, LeBrun DP, Davis RE, Berry G], Warnke RA (1991) Growth fraction estimation of malignant lymphomas in formalin-fixed paraffin-embedded tissue using anti-PCNA/Cyclin
NORs Staining and PCNA Immunostaining in MALT Gastric Lymphomas . 1009 19A2. Correlation with Ki-67 labeling. Am J Pathol 138: 1471-1477 26 Lischwe MA, Smetana K, Olson MOJ, Busch H (1979) Proteiris C23 and B23 are the major nucleolar silver staining proteins. Life Sci 25: 701-708 27 Miyachi K, Fritzier MJ, Tan EM (1978) Autoantibody to a nuclear antigen in proliferating cells. J Immunol121: 2228-2234 28 Ochs RL, Busch H (1984) Further evidence that phosphoprotein C23 (1l0kD/pI5,1) is the nucleolar silver staining protein. Exp Cell Res 152: 260-265 29 Orita T, Kajiwara K, Nishizaki T, Ikeda N, Kamiryo T, Aoki H (1990) Nucleolar organizer regions in meningioma. Neurosurgery 26: 43-46 30 Ploton D, Bobichon H, Adnet JJ (1982) Ultrastructural localization of NOR in nucleoli of human breast cancer tissues using a one-step Ag-NOR staining method. Bioi Cell 43 : 229-232 31 Reeves BR, Casey G, Honeycombe JR, Smith S (1984) Correlation of differentiation state and silver staining of nucleolar organizers in the promyelocytic leukaemia cell line. 13: 159-166 32 Roos G, Dige U, Lenner P, Lindh J, Johansson H (1985) Prognostic significance of DNA analysis by flow cytometry in non-Hodgkin's lymphoma. Haematol Oncol 233-242
33 Smith J, Crocker J (1988) Evaluation of nucleolar organizer region associated proteins in breast malignancy. Histopathology 12: 113-121 34 Underwood JCE, Giri DD (1988) Nucleolar organizer regions as diagnostic discriminants of malignancy. J Patholl55: 95-96 35 Williams MA, Kleinschmidt JA, Krohne G, Franke WW (1982) Argyrophilic nuclear and nucleolar proteins of Xenopus laevis oocytes identified by gel electrophoresis. Exp Cell Res 137: 341-351 36 Woods AL, Hall PA, Shepherd NA, Hanby AM, Waseem NH, Lane DP, Levison DA (1991) The assessment of proliferating cell nuclear antigen (PCNA) immunostaining in primary gastrointestinal lymphomas and its relationship to histological grade, S+G2 + M phase fraction (flow cytometric analysis) and prognosis. Histopathology 19: 21-27 37 Wooseley J (1991) Measuring cell proliferation. Arch Pathol Lab Med 115: 555-557 38 Yu CCW, Hall PA, Fletcher CDM, et al (1991) Haemangiopericytomas: the prognostic value of immunohistochemical staining with a monoclonal antibody to proliferating cell nuclear antigen (PCNA). Histopathology 19: 29-33
Received October 13, 1992 . Accepted in revised form February 24, 1993
Key words: NORs - peNA - MALT lymphoma P. Mikou, MD, 76, Papanastasiou St, Agios Dimitrios, 17342, Athens, Greece
Nucleolar Organizer Regions (NORs) Staining and Proliferating Cell Nuclear Antigen (PCNA) Immunostaining in Mucosa-Associated Lymphoid Tissue (MALT) Gastric Lymphomas ~~ P. Mikoul, P. Kanavaros2 , D. Aninosl, M. Tzardi 2 , A. Karamerisl,
B. Gorgoulisl, N. Papadopoulos3 , A. Lavergne4 and A. Galian 4 Departments of Pathology: 1401 General Army Hospital of Athens, Greece, 2University Hospital of Heraklion, Greece, 3University Hospital of Alexandroupolis, Greece, 4Lariboisiere Hospital, Paris, France
SUMMARY Nucleolar organizer region-associated proteins (AgNOR) and proliferating cell nuclear antigen (peNA) have been studied by means of a silver staining technique and immunohistochemistry, in paraffin-embedded, gastrectomy specimens of 12 low-grade and 13 high-grade gastric MALT lymphomas respectively. A significant difference was found between the AgNOR count and peNA index of low-grade lymphomas (mean AgNOR count 2.5 and mean peNA index 8.33 %) and high-grade lymphomas (mean AgNOR count 8.67 and mean peNA index 49.7 %). It is suggested that both methods are useful adjuncts to histopathology for the distinction between low and high grade gastric MALT lymphomas. We also found heterogeneity in AgNOR counts and peNA index among individual cases of either low or high grade MALT gastric lymphomas. This suggests that the AgNOR count and peNA index is helpful in the individual approach of the proliferation rate of each tumour, a parameter of potential importance for predicting the biological behaviour of the tumour and the prognosis of the disease.
Introduction The Nucleolar Organizer Regions (NORs) are loops of DNA (rDNA) in which ribosomal RNA (rRNA) is encodedl, 17, and during interphase they are located in the nucleoli of cells ll . NORs are also the sites of location of a peculiar group of acidic non-histonic proteins, which stain intensely with formic acid -silver nitrate 23 , 26, 28, 35,. The silver-stained NORs are defined as Ag-NORs and the argyrophilic proteins as Ag-NOR proteins. The method has been utilized by cytogeneticists for the evaluation of
* This work was supported by a grant from the Greek Anticancer Society. 0344·0338/93/0189-1004$3.50/0
certain genetic disorders, notably trisomies lo ,22. The usefulness of the method has been increased by means of a modification allowing the AgNOR reaction to run at room temperature 30 • The AgNOR method seems to be useful for diagnosis of malignancy, for evaluating the proliferation rate of a tumour37 , and even as a new prognostic parameter 12 • Proliferating cell nuclear antigen (peNA) is a 36 kd acidic non-histonic nuclear protein, the expression of which is correlated with the S-phase of the cell cycle 4 . This protein was originally detected during experiments with use of human autoantibodies in lupus patients 27 . It has been demonstrated to be an auxiliary protein to DNA polymerase delta 3 and its presence appears to be necessary © 1993 by Gustav Fischer Verlag, Stuttgart
NORs Staining and PCNA Immunostaining in MALT Gastric Lymphomas· 1005 for the initiation of cell proliferation24 • There is increasing evidence that PCNA assessment is an easy and useful tool to evaluate cell proliferation. Previous studies in lymphomas have shown good correlation between PCNA expression 36 , AgNOR count?, other proliferation indices 19 ,32 and histological grade. Accordingly we have investigated the significance of AgNOR enumeration and PCNA expression for grading and determining the proliferation rate in MALT lymphomas of the stomach.
Material and Methods Specimens Twenty-five gastrectomy specimens of MALT lyphomas were examined, from the same number of patients. Only gastrectomy specimens were included because they permitted detailed histological analysis and grading. The histological analysis was performed on multiple sections and from different tumour areas. The lymphomas were classified histologically as high grade (13 cases) and low grade (12 cases) according to the criteria proposed by Chan et aP. In addition, the lymphomas were classified according to the updated Kiel classification 1988. In the group of low grade lymphomas there were 7 cases of centrocytic lymphomas of MALT type (cases no 1,2,3, 7, 8, 10, 12) and 5 cases of lymphoplasmacytic lymphomas of MALT type (cases no 4,5,6,9, 11). In the group of high grade lymphomas there were 4 cases of centroblastic centrocytoid lymphomas with component of centrocytic lymphoma of MALT type (cases no 1, 7, 11, 12), 8 cases of centro blastic polymorphic lymphomas with component of centrocytic lymphoma of MALT type (cases no 2, 3, 5, 6, 8, 9, 10, 13) and 1 case of immunoblastic lymphoma with plasma blastic differentiation and with component of lymphoplasmacytic lymphoma of MALT type (case no 4). These specimens had previously been phenotyped by standard immunohistochemical methods. All of them were of B-cell origin as assessed by using B (L26ICD20, 4KB5ICD45R, x and A light chains) and T (UCHLl/CD45RO and MTl/CD43) cell markers. Blocks were taken from these cases from routine files of the Departments of Pathology of Lariboisiere Hospital, 401 General Army Hospital, University Hospital of Heraklion and of Alexandroupoli. The tissue had been fixed in Bouin's fixative (14 cases) or in formalin 10 % (11 cases) and processed to paraffin wax.
Staining procedure Sections were cut at 5 !!m thickness from paraffin blocks. These were dewaxed in xylene, then hydrated through ethanols to distilled deionized water. a) AgNOR Staining: The AgNOR staining solution was prepared by dissolving 2 g of gelatin in 100 ml of 1 % aqueous formic acid (solution A), and 50 g of silver nitrate in 100 ml of distilled water (solution B). These solutions were mixed 1(A) : 2(B) volumes just prior to use. This was poured over the tissue sections and the slides were kept in a humidity chamber, protected from daylight for 30 minutes at room temperature. The specimens were then washed with distilled deionized water, dehydrated through alcohols to xylene, and mounted in DPX medium. No counterstain was performed. b) PC 10-1mmunohistochemistry: The slides were immersed for 10 min in 3 % H202 to block endogenous peroxidase and then taken to PBS (pH: 7.6). Immunostaining was performed using the PAP method (Dako). A dilution of 1 : 10 with one hour incubation
was found to be optimal. Very light haematoxylin counterstain was performed before mounting.
AgNOR counting procedure The slides were examined using a 100 oil-immersion lens so that AgNOR dots were counted at a magnification of 1000. One hundred lymphoid cells were randomly selected from each specimen and the AgNOR dots were counted individually, carefully focused, even when occurring in large aggregates. Special caution was taken in the study of low grade lymphomas so that AgNOR number in lymphoid cells from non-neoplastic reactive follicles were not counted. The reason was that, while in all 12 low grade gastric MALT lymphomas reactive follicles with varying degrees of colonization by small neoplastic cells were found, only one of the 12 cases displayed many follicular structures composed almost exclusively of small neoplastic cells. Thus, a comparative study between reactive and neoplastic follicles could not be performed due to the very limited number of cases with clearcut, unequivocal neoplastic follicles.
Evaluation of PCNA immunostaining The slides were examined using a 10 objective lens and ten fields were randomly selected. In the study of low grade lymphomas the lymphoid cells from non-neoplastic reactive follicles were not counted. Finally 1000 tumour cells from these fields were counted using a 40 objective lens. The PCNA index represented the percentage of cells with positive nuclear staining (regardless of the staining intensity) in the total number of tumour cells counted.
Results In all specimens clearly defined silver-stained dots were observed in all nuclei. These were separate or organized in small aggregates, and sometimes were placed in a different position than the haematoxylin-stained nucleoli or chromatin. Reactivity of PC10 was distinct although there was a gradation in the intensity of staining.
Low-grade lymphomas The neoplastic population, which was composed of small cells, possessed a mean number of 2.5 AgNORs per nucleus (Table 1), (Fig. 1). The mean PCNA index was 8.33 % (Table 1), (Fig. 2).
High-grade lymphomas The neoplastic population was mainly composed of large cells. In some cases, the presence of a distinct subpopulation of small neoplastic cells was obvious. This constituted a variable percentage (always small) of the neoplastic cell population. All neoplastic cells were taken under consideration in the counting procedure. The mean number was 8.67 AgNORs per nucleus (Table 2), (Fig. 3). The mean PCNA index was 49.6 % (Table 2), (Fig. 4). It was noticed that within each histological group of either high or low grade gastric MALT lymphomas there was a variation of AgNOR counts and peNA index among individual cases (Tables 1 and 2). Statistical analysis of the
1006 . P. Mikou et al.
Fig. 1. AgNOR staining in low-grade lymphoma (*400).
Fig. 2. PCNA (*400).
Table 1. AgNOR mean number and PCNA index in low grade lymphomas
Table 2. AgNOR mean number and PCNA index in high grade lymphomas
Case No.
LOW GRADE LYMPHOMAS AgNOR mean number PCNA index
Case No.
HIGH GRADE LYMPHOMAS AgNOR mean number PCNA index
1 2 3 4 5 6 7 8 9 10 11 12
2.2 2.76 3.22 2.44 1.85 2.15 2.1 3.11 2.5 2.14 2.26 3.3
12 % 7% 14 % 8% 4% 5% 7% 17 % 6% 9% 1% 10 %
1 2 3 4 5 6 7 8 9 10 11 12 13
8.69 8.71 10,48 10.15 7.54 8.31 8.06 9.63 8.22 8.56 7.64 7.96 8.3
mean value: 2.5 SD: 0.485 SE: 0.14
mean value: 8.33 % SD: 0.044 SE: 0.012
immunostaining
mean value: 8.67 SD: 0.916 SE: 0.07
in
low-grade
lymphoma
63% 50 % 61 % 73 % 75 % 65% 26 % 70 % 52 % 57 % 25 % 20 % 60 % mean value: 49.6 % SD: 0.186 SE: 0.052
NORs Staining and peNA Immunostaining in MALT Gastric Lymphomas· 1007
Fig. 3. AgNOR staining in high-grade lymphoma (*400).
Fig. 4. peNA (*400).
results, by means of t-test, showed that the difference between the two mean values was statistically very significant (p < 0.1 %) for both AgNOR count and reNA index (Table 3).
Table 3. Statistical analysis (Student's test)
Discussion The present study shows that both AgNOR counts and reNA immunostaining correlate well with histological grading of primary gastric MALT lymphomas. This is in keeping with previous findings that AgNOR counts can help in distinguishing high grade from low grade nonHodgkin nodal lymphomas7 and that reNA immunostaining correlates well with histological grade in nodal lymphomas and primary gastrointestinallymphomas25 ,36. The AgNOR count seems to represent a parameter of tumour proliferation rate 37 • It has been reported that AgNOR count has linear relationship with the percentage of S-phase cells, determined by means of DNA flowcytometry in high and low grade non-Hodgkin's lymphomas 9• This was also suggested by comparison of AgNOR values with Ki-67 immunoreactivity 20 and bromodeoxy-
immunostaining
In
high-grade
AgNOR count
peNA index
t-value: 21,276 DF: 23
t-value: 7.786 DF: 23
p < 0.1 %
p < 0.1 %
lymphoma
uri dine incorporation 29 . The augmentation of the AgNOR number per nucleus in hyperplastic or malignant cells has been reported to be also related to cell ploidy and transcriptional activity16, although this has been doubted lately14, and, the to the presence of a defect of nucleolar association 34 • Regarding reNA index, previous studies have shown that it correlates with the S-phase fraction of tumour cells determined by DNA flow cytometry 18,21,36. In addition, the proliferating fraction of tumour cells detected by reNA immunostaining correlates with that detected by Ki6725 , the latter marking cells in Gl and G2 phases in addition to those in S-phase6 , with thymidine labeling index 2 and with bromodeoxyuridine uptake in carcinoma celllines 6 •
1008 . P. Mikou et al.
In view of these data, taken together, the presence of high AgNOR count and high peNA index in high grade gastric MALT lymphomas could reflect a higher proliferation rate, higher transcriptional activity and more aneuploid cells than in low grade ones. In this study peNA index and AgNOR count do not correlate well with each other. It is likely that these methods quantify different aspects of proliferative activity but there is also evidence that peNA index and AgNOR count do not only measure the proliferation. Indeed, peNA expression does not correlate with the S-phase fraction determined by flow cytometry in some tumours 38 and AgNOR expression may be influenced by differentiation state, and proliferation rate 1S ,31. Nevertheless, it is particularly interesting that these two methods independently show a correlation with histological grading in gastric MALT lymphomas. In the present study, we found that within each histological group of either high or low grade gastric MALT lymphomas there was a variation of AgNOR counts and peNA index among individual cases. This variation indicates that within the generic groups of high and low grade gastric MALT lymphomas individual cases have a different proliferation rate and perhaps different biological behaviour, thereby suggesting that an individual approach based on the evaluation of the proliferation rate may be of importance for predicting the clinical outcome of these tumours. In this respect peNA immunostaining and/or AgNOR counts may be helpful in providing objective support for the histological grading of MALT lymphomas especially for cases of a borderline histology between low and high grade lymphomas. In conclusion, our preliminary data suggest that peNA index and AgNOR counts are useful adjuncts to histopathology for the histological grading of gastric MALT lymphomas. Moreover, they permit to detect variations of the proliferation rate among individual cases belonging to either low or high grade gastric MALT lymphomas. This could be useful in the individual approach of the proliferative activity and perhaps the clinical behaviour of each tumour. Further investigations are required to evaluate the prognostic significance of AgNOR count and peNA index in gastric MALT lymphomas, and to determine whether the variations of these parameters could reflect variations in the aggressiveness of tumours.
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Received October 13, 1992 . Accepted in revised form February 24, 1993
Key words: NORs - peNA - MALT lymphoma P. Mikou, MD, 76, Papanastasiou St, Agios Dimitrios, 17342, Athens, Greece