Bone marrow lymphoid aggregates in myelodysplastic syndromes: incidence, immunomorphological characteristics and correlation with clinical features and survival

Leukemia Research 26 (2002) 525–530

Bone marrow lymphoid aggregates in myelodysplastic syndromes: incidence, immunomorphological characteristics and correlation with clinical features and survival Silvia M.M. Magalhães a,1 , F. Dário Rocha Filho b , José Vassallo c , Mariana P. Pinheiro d , Konradin Metze c , Irene Lorand-Metze d,∗ a

d

Department of Clinical Medicine, Hematology/Hemotherapy Center of Ceará (HEMOCE), Federal University of Ceará, Av. José Bastos 3390, BR 60436-160 Fortaleza, Ceará, Brazil b Department of Pathology and Forensic Medicine, Federal University of Ceará, Rua Monsenhor Furtado S/N–Rodolfo Teófilo, BR 60441-750 Fortaleza, Ceará, Brazil c Department of Pathology, Faculty of Medicine, State University of Campinas, P.O. Box 6111, BR 13083-970 Campinas, Brazil Department of Clinical Medicine, Hemocentro—State University of Campinas, P.O. Box. 6198, BR 13081-970 Campinas, SP, Brazil Received 13 June 2001; accepted 8 October 2001

Abstract Lymphoid aggregates (LA) are a common finding in bone marrow biopsies but little is known about their clinical implications and biological significance. We found LA in 51/206 patients with myelodysplastic syndromes (MDS). There was no correlation with age, disease progression or overall survival. The group with LA had lower hemoglobin values (P = 0.03), and was associated with an increase in reticulin fibres (P = 0.01). Although they were more frequent in RAEB, this did not reach statistical significance. Most LA had a benign morphology and showed CD20 expression in three distinct patterns: central, perinodular or diffuse. No evidence of an association with lymphoproliferative disease was observed. LA probably represent an ongoing immune stimulation and are probably related to an altered bone marrow microenvironment, with no impact on prognosis. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Myelodysplastic syndromes; Bone marrow; Lymphoid aggregates; Immunohistochemistry

1. Introduction Lymphoid aggregates (LA) are a common finding in bone marrow trephines and are usually considered to be a physiological feature, closely related to age and sex [1]. Benign LA are well distinguishable from surrounding hematopoietic tissue, non-paratrabecular and generally sharply circumscribed. They are predominantly composed of small well-differentiated lymphocytes admixed with varying numbers of plasma cells, histiocytes, mast cells, eosinophils and occasional larger lymphocytes. An increased frequency of benign bone marrow LA has been reported in association with chronic inflammatory Abbreviations: LA, lymphoid aggregates; MDS, myelodysplastic syndromes; H&E, hematoxylin and eosin; RA, refractory anemia; RARS, refractory anemia with ringed sideroblasts; RAEB, refractory anemia with excess of blasts; RAEB-t, refractory anemia with excess of blasts in transformation; CMML, chronic myelomonocytic leukemia ∗ Corresponding author. Fax: +55-19-3788-8600. E-mail address: [email protected] (I. Lorand-Metze). 1 Fax: +55-85-433-4420.

syndromes, infection, hemolysis, myeloproliferative disorders and autoimmune diseases [2]. In myelodysplastic syndromes (MDS) their reported incidence is about 10%, ranging from 2.5 to 25.0% in different series [1–6]. Little is known about LA characteristics, clinical implications and biological significance in this group of diseases. Several immunological abnormalities have been described in patients with MDS [7,8]. The impaired immune response is probably responsible for an increased susceptibility to infection, autoimmunity and an increased incidence of lymphoid malignancy [3,9]. A clear distinction between benign and malignant LA may frequently not be possible on morphological grounds only [10,11]. Immunohistochemical as well as molecular analysis may play a major role to establish a reliable distinction [11–13]. In the present study we analyzed the frequency and immunomorphological characteristics of bone marrow LA in patients with MDS. A possible association between LA and clinical or hematological features, as well as its implications for the course of the disease were also investigated. A retrospective follow-up study was performed to detect

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any possible clinical evidence of an association with a lymphoproliferative disease.

was calculated by the Kaplan–Meier method. A log-rank test was used to check the significance of the difference between survival distributions of both groups. Statistical tests were performed using the WinSTAT and SPSS softwares.

2. Materials and methods Bone marrow biopsies performed at the time of diagnosis in patients with MDS were retrospectively drawn from the files of the Federal University of Fortaleza, Ceará (UFC— northeast region) and the State University of Campinas (UNICAMP—southeast region) over the period between 1995 and 1999. The diagnosis of MDS was based on FAB criteria [14]. Peripheral cytopenias were always associated with cell atypias in at least two hemopoietic cell lines in BM. Deficiency anemias, renal and hepatic failure, thyroid dysfunction, hepatitis, cytomegalovirus and HIV infections were excluded, as well as inflammatory and autoimmune diseases. From each case that entered the study, we collected age, sex, blood counts at diagnosis, survival time and progression in FAB type. In all cases a transiliac bone marrow biopsy using a Jamshidi needle was carried out, as a part of the initial diagnostic evaluation. Bone marrow specimens were fixed in Bouin’s solution and decalcified with nitric acid (UFC cases) or fixed and decalcified in Zenker’s solution, acetic acid (UNICAMP cases). Tissues from all cases were embedded in paraffin. The presence of LA in sections stained with hematoxylin and eosin (H&E) was investigated by two independent observers. Each examination included the following histological features: (a) number of LA in each section: single or multiple; (b) histotopography: central, paratrabecular, or intertrabecular (if centrally located, LA occasionally touched the trabeculae); (c) defined or ill defined borders; (d) cytological features: polymorphous (collection of small lymphocytes interspersed with plasma, mast and reticulum cells and eosinophils) or monomorphous (one cell type only). The specimens stained with Gömöri’s silver impregnation were examined with regard to increase and thickening of the reticulin network in the hematopoietic tissue. Immunohistochemical staining was carried out by avidin–biotin complex method [15] with 3,3-diaminobenzidine as chromogen and hematoxylin as a counterstaining. Microwave heating was used as an optimized antigen retrieval procedure [16]. Antibodies to CD20, CD45RO, CD3 and Bcl-2 (Dakopatts) were employed in each specimen. Sections to be stained with CD3 were previously treated with 0.1% trypsin. The omission of primary antibodies was used as negative controls and appropriate positive controls (tonsils) were processed in parallel with each group of slides. Initially, we compared the patients from both institutions in order to detect geographical differences in MDS in both regions. This was done by the t-test. The clinical and laboratory features of the patients with and without LA were also compared by the t-test. To examine the influence of FAB type on the frequency of LA we used the χ 2 -test. Correlations were examined by the Spearman test. Survival analysis

3. Results Among the 206 cases of MDS that were reviewed, 98 were from UNICAMP and 108 from UFC. Both groups were similar for all parameters studied except for hemoglobin (mean 9.33 g/dl, UNICAMP and mean 8.07 g/dl, UFC) and neutrophil count (1.89 × 109 /l, UNICAMP and mean 2.63 × 109 /l, UFC).Therefore, both groups were analyzed together. At least one lymphoid aggregate was found in diagnostic BM sections from 51 (24.7%) cases. Clinical and hematological features of the cases, according to presence of LA in BM are detailed on Table 1. According to the FAB classification 129 patients had refractory anaemia (RA), 29 had refractory anaemia with ringed sideroblasts (RARS), 27 refractory anaemia with excess of blasts (RAEB), 4 RAEB-t, 11 chronic myelomonocytic leukaemia (CMML) and one case MDS not classified. Five cases were diagnosed as hyperfibrotic MDS. The frequency of LA per FAB type was: 31/129 RA (24%), 5/29 RARS (17%), 9/27 RAEB (33%), 2/4 RAEB-t, 1/11 CMML (9%) and 3/5 in MDS, hyperfibrotic type. Although the frequency was higher in RAEB, the ␹2 -test did not reach significance (P = 0.09). The presence of LA in BM biopsy was associated with an increase in reticulin fibres (P = 0.01). Patients presenting LA did not differ according to age and sex distribution. Concerning peripheral blood counts, patients presenting LA had significantly lower hemoglobin values (P = 0.03). However, there was no influence of LA on survival (P = 0.79) (Fig. 1). This was also the case when survival was analyzed within each FAB type. Progression or Table 1 Features of the cases according to the presence of lymphoid aggregates in bone marrow biopsies

Number of cases Age (years) Sex (male/female) Hemoglobin (g/dl) Leucocytes (×109 /l) Neutrophils (×109 /l) Platelets (×109 /l) FAB types RA RARS RAEB RAEB-t CMML Hyperfibrotic MDS Increase in reticulin fibres Transformation to AML

Cases with LA

Cases without LA

P

51 56 (25–88) 18/33 7.3 3.1 1.98 107

155 60 (15–89) 75/80 8.8 3.6 1.55 94

NS NS 0.01 NS 0.03 NS

31 (24%) 5 (17%) 9 (33%) 2 1 (9%) 3 26/51 10/51

98 24 18 2 10 2 46/155 36/155

0.01 NS

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Fig. 1. Survival curves of patients with myelodysplastic syndromes with and without bone marrow lymphoid aggregates (P = 0.79 by the log-rank test).

transformation into acute leukemia was not more frequent in these patients. All patients were followed for at least 19 months (median 21 months) with no clinical evidence of a lymphoproliferative disorder. One patient with sideroblastic anemia and paratrabecular LA has now a 37 months follow-up and do not present any evidence of a lymphoproliferative disorder. The majority of the LA (39/51) were centrally located, four of which occasionally touched the bone trabeculae and only two cases showed a paratrabecular distribution. Third defined nodules were detected in seven cases (13%) but they were centrally located (Fig. 2). The cytological appearance was polymorphous in all cases. No germinative centres were found. In 26 cases immunostaining could not be evaluated, either because no LA was found in the new sections, or for substantial loss of tissue during the antigen retrieval process. Therefore, only 25 bone marrow specimens were studied for their immunohistochemical features. These data are summarized in Table 2. In all except two patients the aggregates showed positivity for CD20. Three distinct staining patterns could be identified according to the topography of positive cells: central (10 cases), perinodular (3 cases) and diffuse (12 cases) (Fig. 3A). Positivity with one of the selected T cell related antibodies (CD3 and/or CD45RO) was observed in 13 cases (Fig. 3B). Among them, only one was CD20 negative. In the other cases, those cells in the LA without expression of CD20 did not express CD3 or CD45RO. Immunohistochemical staining for Bcl-2 was evident in only two nodules, both composed by B and T cells.

Fig. 2. Bone marrow biopsy from a patient with MDS presenting a large centrally located lymphoid aggregate H&E, 100×.

4. Discussion Reactive LA in the bone marrow have been reported in a variety of disorders including hematopoietic clonal disorders such as myeloproliferative disorders and MDS. The reported incidence of LA in MDS varies from 2.5 to 25.0% [1–6]. They were a common finding in our series. However, no difference in age was found between cases presenting LA and those who did not present this feature. The pathopysiological meaning of LA in BM is not well understood,

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Table 2 FAB sub-groups and immunoreactivity patterns of bone marrow aggregates in patients with myelodysplastic syndromes Patients

FAB sub-group

Immunoreactivity patterns CD3

CD45RO

CD20 Central

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

RA RAEB-t RAEB RAEB-t RA RA CMML RA RA Hyperfibrotic RAEB RA UCa Hyperfibrotic RA RA Hyperfibrotic RA RA RA RA RA RA RA RARS a

+ − − − − − + + − + − − − − + − − − − − − − − − −

+ − − − + − − − − + + + − − − − − − − + − + + + −

Bcl-2 Perinodular

+ +

+ +

+ + +

+ + +

+

Diffuse + + + + + +

+ +

+

+ + +

− − − − − − + − − − + − − − − − − − − − − − − − −

UC: unclassifiable MDS.

but Faulkner-Jones et al. showed that 22% of the patients presenting this feature were later diagnosed as having a lymphoproliferative disease (mean follow-up of 21.9 months) [9]. Morphological criteria have been used to distinguish benign from malignant LA in the bone marrow. Benign aggregates are well circumscribed with clearly defined margins, have usually a central location and may exhibit germinal centers [2,10,17–22]. They are usually composed of a mixture of T and B cells with polyclonal expression of immunoglobulins. Monoclonality may be detected by monotypic expression of immunoglobulins or clonal rearrangement in the immunoglobulin heavy chain or T cell receptor genes. Even these techniques give equivocal results in at least 20% [11]. Bcl-2 expression has been claimed to be an evidence for malignancy, but it has also been described in benign lymphoid nodules [23,24]. For all these reasons, in lymphoid infiltrates of uncertain origin, a sure diagnosis may only be made considering clinical, morphological, immunological and, if possible, molecular data. In the present study, LA showed morphologic features of benign nodules, although in some cases they were large, ill defined or touching trabeculae. In none of the patients however, a lymphoid neoplasm could be demonstrated by immunohistochemistry. Expression of Bcl-2 was evident in only two cases in which a mixed T and B cell composition

was detected. After a median follow-up of almost 2 years none of the patients showed evidence of lymphoma. In the present study we analyzed patients with MDS from two Brazilian institutions located at different geographical regions. Fortaleza is located at the equator, at the coast. Campinas is located at the Capricorn tropic and belongs to one of the most heavily industrialized regions in the country. As the etiology of MDS is related to environmental mutagens [25], or perhaps viruses [26–29], one would expect that MDS could have different characteristics in different regions. Surprisingly, no major differences were found for the parameters analyzed between patients of the two institutions. A high frequency of RA was found. However, all patients fullfilled the minimal criteria for diagnosis of MDS [30] and a systematic exclusion of non-clonal disorders had been made. A variety of immunological abnormalities have been found in MDS [7,8]. Impaired lymphoid function could be due to involvement of the lymphoid system in the malignant clone but this was proven only in few cases [21,29]. As the monocyte/macrophage system is derived from the abnormal clone in most cases, it could be assumed that it is functionally abnormal, presenting disregulation of the production of cytokines and leading to a persistent immune stimulation. This could give rise to lymphoma. On the other hand, Barrett et al. [8] have recently postulated that an immune mechanism, mostly due to myelossupressive T cells is

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uniform increase in bone marrow B cells has been observed in infiltrates of B cell lymphomas [18,19]. A significant increase in T cells in MDS was observed by Horny et al. [19]. In our study, the B cells exhibited three different patterns of distribution: central, perinodular and diffuse. T cells were more difficult to demonstrate, as already found in other studies [31]. Although, immunohistochemical techniques have been used with consistent satisfactory results on BM specimens [32], in half of the cases this analysis was not possible, mostly because the new sections failed to show LA. In few cases however, possibly due to technical problems, antigen retrieval process repeatedly led to detachment of sections from slides. In conclusion, LA in our MDS patients were heterogeneous in their immunomorphological characteristics and showed no clinical evidence of association with lymphoid malignancy. A longer follow-up and confirmation with other series are required to draw firm conclusions.

Acknowledgements S.M.M. Magalhães provided the concept, design, collected the data as well as interpreted it, drafted the paper, and provided study materials. F.D. Rocha Filho provided technical support and helped with data interpretation. J. Vassallo provided funding and contributed to the design as well as interpretation of the data. M.P. Pinheiro collected the data. K. Metze provided statistical epertise. I. Lorand-Metze contributed to the study design, interpretation of the data, provided the critical revision and gave final approval. References

Fig. 3. (A) Lymphoid aggregate with CD20 expression on lymphoid cells. (streptavidin–biotin peroxidase, 400×), (B) centrally located lymphoid aggregate mostly formed by CD3 positive cells (streptavidin–biotin peroxidase, 400×).

responsible, at least in part, for the bone marrow failure seen in MDS. In our study, LA were more frequently observed in high risk patients with MDS, presenting a more altered microenvironment and increase in reticulin fibres. This is in keeping with the hypothesis that they could be an expression of a more severe damage to the hemopoietic stroma due to the presence of a viral infection or another immune stimulation. Therefore, LA could be an expression of the deranged immune function in MDS. In the present study, LA were not associated with more severe cytopenias, nor had an influence on disease progression or overall survival of the patients. There have been a number of reports concerning immunophenotype of normal and reactive lymphocytes in human bone marrow [18,19]. Most reactive lymphoid aggregates contain a mixture of T and B cells whereas a significant

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