Estimation of diagnostic value of TIMP2 expression in chosen childhood lymphomas and reactive lymphadenopathy

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1 PRACA ORYGINALNA Original Article Acta Haematologica Polonica 2008, 39, Nr 3, str MAŁGORZATA STOLARSKA 1, ELśBIETA SAŁACIŃSKA-ŁOŚ 2, KATARZYNA TARAN 3, JOANNA TRELIŃSKA 1, JÓZEF KOBOS 2 Estimation of diagnostic value of TIMP2 expression in chosen childhood lymphomas and reactive lymphadenopathy Ocena wartości diagnostycznej ekspresji TIMP-2 w wybranych chłoniakach i limfadenopatii odczynowej u dzieci 1 Oncohematology Unit, Department of Pediatrics, Medical University of Lodz, Poland 2 Department of Pathology of the Age of Development, Medical University of Lodz, Poland 3 Chair and Department of Pathology, Medical University of Lodz, Poland SUMMARY Pathology of the age of development is a very difficult and often surprising area of medicine and diagnosis differentiating between reactive conditions and malignancies in children appears an extremely important problem in everyday practice of both clinicians and pathologists. It is necessary to search for new tools which would allow to differentiate these two separate groups and to make prognosis for individual patients with malignant tumors. Tissue inhibitors of matrix metalloproteinases (TIMP-2) are currently evaluated and seem very promising markers of tumor growth and progression. The aim of our study was to examine the expression of TIMP-2 in different histologic types of children lymphomas and to estimate a possible diagnostic value of this marker by comparison with the expression of this marker in reactive lymphadenopathies. We observed TIMP-2 expression of examined tissue samples exclusively in lymphomas. We also noticed differences in TIMP-2 expression among histologic lymphoma types. We conclude that the role and significance of tissue inhibitors of metalloproteinases in children malignancies and reactive conditions need further research. The results of our study make us believe that TIMP-2 may be a useful marker in differential diagnosis between reactive lymphadenopathies and lymphomas. KEY WORDS: TIMP-2 Immunohistochemistry Lymphoma Children Differential diagnosis STRESZCZENIE Patologia wieku rozwojowego jest bardzo trudną i często zaskakującą dziedziną medycyny, a diagnostyka róŝnicowa zmian odczynowych i nowotworowych jest istotnym problemem w codziennej praktyce zarówno lekarzy klinicystów jak i patomorfologów. Dlatego teŝ poszukuje się nowych markerów diagnostycznych i rokowniczych, które pozwolą na określenie prognozy w jednostkowych przypadkach. Obecnie szczególna uwagę poświęca się badaniom czynników podścieliskowych, które ze względu na swoją rolę w procesach wzrostu i progresji nowotworów, uznawane są za obiecujące czynniki prognostyczne.

2 478 M. STOLARSKA et al. Celem naszych badań była ocena ekspresji tkankowego inhibitora metaloproteinaz (TIMP-2) w dwóch diametralnie róŝniących się rokowaniem grupach zmian patologicznych węzłów chłonnych odczynowych rozrostach oraz chłoniakach. Stwierdziliśmy ekspresję TIMP-2 w połowie badanych przypadków chłoniaków, a nie stwierdziliśmy w Ŝadnym przypadku odczynowej limfadenopatii. Zaobserwowaliśmy takŝe róŝnice w ekspresji TIMP-2 pomiędzy poszczególnym podtypami histopatologicznymi chłoniaków. Wyniki przeprowadzonych przez nas badań wskazują, Ŝe rola tkankowych inhibitorów metaloproteinaz w odczynowych i nowotworowych rozrostach węzłów chłonnych wymaga dalszych badań oraz pozwalają przypuszczać, Ŝe ocena ekspresji tkankowych inhibitorów metaloproteinaz moŝe okazać się przydatnym narzędziem w diagnostyce róŝnicowej przyczyn limfadenopatii w wieku rozwojowym. SŁOWA KLUCZOWE: TIMP-2 Immunohistochemia Chłoniak Dzieci Diagnostyka róŝnicowa INTRODUCTION Despite advances in modern pediatric oncology, poor results of instituted treatment are still observed. Proper histologic diagnosis which has always been the base of therapy may become a new prognostic marker for many children malignancies. Unfortunately differential diagnosis of malignant neoplasms of childhood still poses a very difficult problem for clinicians and pathologists. It is necessary to search for new diagnostic markers which would allow us not only to establish the proper diagnosis of malignant tumors but first of all to differentiate reactive conditions from malignancies. One of the most common groups of children malignancies are lymphomas. Currently, thanks to immunohistochemistry it is much easier to classify lymphoma histologic subgroups and to make prognosis in individual cases. Unfortunately there is still no tool which allows to differentiate reactive conditions from lymphomas in microscopic examination of removed lymph nodes in doubtful cases. The extracellular matrix is remodeled constantly by matrix metaloproteinases and its activity depends on tissue inhibitors of matrix metalloproteinases action. Tumor growth and progression depends on interactions of tumor cells specially on the front of the lesion with extracellular matrix. Tissue inhibitors of matrix metalloproteinases (TIMPs) are multifunctional proteins with growth-regulatory activity. An imbalance between metalloproteinases and TIMPs plays a crucial role in tumor progression. Tumor invasion and metastases can be inhibited by up-regulation of TIMP expression in tumor cells. TIMPs inhibit the growth of primary tumor, too. Conversely, downregulation of TIMPs contribute to the invasive potential (1, 2, 3). TIMP-2 is one of the best known tissue inhibitor of proteinases and its part in tumor growth and progression is now under careful evaluation. It is proved that TIMP-2 is mitogenic for both normal and tumor cells and that it also has an antiapoptotic effect on lymphocytes. The latest research suggests a possible use of TIMP-2 in differential diagnosis of lymphadenopathies (2, 3, 4, 5, 6).

3 Estimation of diagnostic value of TIMP2 479 MATERIALS AND METHODS We selected for our study 34 formalin-fixed and paraffin-embedded tissue sections of childhood lymphomas and reactive lymph nodes from the files of the Department of Pathology of the Age of Development, Medical University of Lodz. Tumor tissue samples were reclassified according to current WHO Classifications of Tumours of Haematopoietic and Lymphoid Tissues. From these tissue samples paraffin blocks about the thickness of 3 4 micrometers were prepared and stained with hematoxylin and eosin (HE) and were used for immunohistochemical examination with the use of tissue inhibitor of proteinases TIMP-2 (Novocastra) and with immunoperoxidase reaction according to Hsu (EnVision+ System, Peroxidase DAB). The expression of the investigated marker was estimatal with computer image analysis system (Multi Scan Base v Computer Scanning System, Ltd.). In immunohistochemical study we assumed cytoplasmic type of reaction brown color of the cytoplasm of neoplastic cells and we rated TIMP-2 expression as: weak (less than 10% of positive tumor cells), of intermediate degree (from 10% to 60%) or strong (more than 60%). RESULTS In microscopic examinations of the tissue samples we diagnosed as follows: 13 Hodgkin lymphoma cases, 8 non-hodgkin lymphoma cases and 13 reactive lymphadenopathies. Details of histologic examination of malignancies are represented in Table 1. In reactive conditions the etiologic factor was proved in 5 cases (2 cases of cat scratch disease, 2 cases of toxoplasmosis, 1 case of mononucleosis) in other 8 cases the causative factor remained unknown despite clinical examination and laboratory tests. In immunohistochemical examination of 34 tissue samples, TIMP-2 expression was found in 17 cases exclusively in lymphomas. We did not notice TIMP-2 expression in any of the reactive conditions. TIMP-2 expression observed in lymphomas was rated as: strong 1 case, of intermediate degree 5 cases, and weak 11 cases. In Hodgkin lymphomas, TIMP-2 expression was found in 12 of 13 tumor tissue samples. Details of immunohistochemical findings are shown in Table 1 and Photos 1 3. Table 1. Results of histologic and immunohistochemical research in examined group of childhood lymphomas Tabela 1. Wyniki oceny histologicznej I immunohistochemicznej badanej grupy chłoniaków wieku dziecięcego Case N Histologic diagnosis according WHO TIMP-2 expression in tumor cells 1 HL nodular sclerosis II classical HL weak 2 HL nodular sclerosis I classical HL intermediate 3 HL nodular sclerosis II classical HL intermediate 4 HL nodular sclerosis I classical HL intermediate 5 HL nodular sclerosis II classical HL intermediate

4 480 M. STOLARSKA et al. 6 HL nodular sclerosis II classical HL weak 7 HL nodular sclerosis II classical HL intermediate 8 HL nodular sclerosis I classical HL weak 9 HL nodular sclerosis I classical HL weak 10 HL nodular sclerosis I classical HL weak 11 HL - nodular sclerosis I classical HL weak 12 HL nodular sclerosis II classical HL no expression 13 HL lymphocyte depleted classical HL no expression 14 ALCL weak 15 ALCL strong 16 Burkitt lymphoma weak 17 Burkitt lymphoma weak 18 Burkitt lymphoma weak 19 T-cell lymphoma no expression 20 T-cell lymphoma no expression 21 T-cell lymphoma weak HL Hodgkin lymphoma, ALCL Anaplastic Large Cell Lymphoma Photo 1. Intermediate degree of TIMP-2 expression in nodular sclerosis classical Hodgkin Lymphoma type II, H&E. Oryg. magn. 200 Fot. 1. Ekspresja TIMP-2 o pośrednim stopniu nasilenia w komórkach chłoniaka Hodgkina (typ II włóknienia guzkowego), H&E, 200

5 Estimation of diagnostic value of TIMP2 481 Photo 2. Strong TIMP-2 expression in Anaplastic Large Cell Lymphoma, H&E. Oryg. magn. 200 Fot. 2. Silna ekspresja TIMP-2 w komórkach anaplastycznego chłoniaka wielkokomórkowego, H&E, 200 Photo 3. Weak TIMP-2 expression in Burkitt Lymphoma, H&E. Oryg. magn. 200 Fot. 3. Słaba ekspresja TIMP-2 w komórkach chłoniaka Burkitta, H&E, 200

6 482 M. STOLARSKA et al. DISCUSSION Enlargement of peripheral lymph nodes is a common problem in childhood. Most of the cases are symptomatic, due to infections, allergy and other nononcologic causes. Lymph nodes enlargement appears a natural reaction of child organism to contact with any kind of antigens. Unfortunately lymph nodes enlargement may be also the symptom of lymphoma. Lymphomas, currently classified together with leukemias, are the most common group of malignant neoplasms of childhood. In some cases histologic differentiation between reactive conditions (e.g. due to all kinds of infectious agents) and lymphomas is very difficult and poses a problem in pathologists routine work. Therefore, scientists search for new markers to make the final diagnosis easier and more precise, especially that prognosis in those two discussed groups of pathology is totally different. The results of research on the role of extracellular matrix in tumor growth and progression seem to be very promising and bring hope to find new diagnostic and prognostic markers for everyday differential diagnosis in doubtful cases including causes of lymphadenopathies. Extracellular matrix takes part in many physiological processes where tissue remodeling is involved, but also in tumor invasion and metastasizing. Metalloproteinases (MMPs) and their inhibitors (TIMPs) appear the most important agents of extracellular matrix which are involved in neoplastic disease. The role of metaloproteinases and their inhibitors in pathological processes has been proved in many malignancies, specially in lymphomas (7). Overproduction and unrestrained activity of MMPs are the reasons of malignant transformation of tumor cells, so TIMPs play a crucial role in tumor growth (1, 2). First of all TIMPs inhibit the MMPs activity, they also regulate cell growth and are involved in apoptosis, with both TIMP-1 and TIMP-2 showing an antiapoptotic effect. We focused on TIMP-2 because its expression and action in many malignancies has been already discovered but still remains unclear, specially in lymphomas (2, 4, 5, 6, 8, 9). It is interesting that in all the examined reactive conditions there was no expression of TIMP-2 and opposite to most of examined lymphomas at least weak expression of TIMP-2 was observed. The lack of TIMP-2 expression in reactive lesions needs further explanation, especially in relation to MMPs expression. However it is important to underline that even in our not so numerous group of cases the difference in the expression of TIMP-2 in reactive lymphadenopathies and lymphomas appeared distinct and pointed to its potential utility in the differential diagnosis. Analyzing the expression of TIMP-2 in different histological types of lymphomas we found it particularly characteristic of Hodgkin lymphomas which is in agreement with other authors results. (7, 11, 12). Among all the examined Hodgkin lymphomas TIMP-2 expression was stronger in nodular sclerosis type II than in type I but without statistically significant correlations. In non-hodgkin lymphomas the expression of the investigated marker was weak and often limited to tumor border cells only. In addition we noticed two different patterns of TIMP-2 expression in lymphoma tissue samples. In some of the investigated cases the expression was visible only in tumor border cells, which seems to be easy to explain. The peripheral part of the tumor

7 Estimation of diagnostic value of TIMP2 483 is usually the place of intensive proliferation of neoplastic cells, so the presence of MMPs and their inhibitors should be noticeable (10). The other pattern of expression found in other lymphoma tissue samples is much harder to explain. In those cases TIMP-2 expression was observed in the centre of tumor mass, especially strong in one of the anaplastic large cell lymphoma cases. Lymphomas, especially ALCL are found as a very aggressive type of malignancies, so high expression of TIMP-2 is surprising. A possible explanation of observed reaction is antiapoptotic activity of TIMP-2 and high expression of this marker as its result. However this hypothesis needs further investigations and comparative studies on pro- and antiapoptotic markers. We conclude that very complicated mechanisms of MMPs/TIMPs action in reactive conditions and lymphomas of the age of development limit the possibility of clear explanation and univocal conclusions of study results. However we believe that our study may be good inspiration to further research on MMPs and TIMPs in childhood malignancies. Differences in TIMP-2 expression in lymphoma histologic types should be confirmed and correlated with other factors, specially antiapoptotic and proapoptotic markers. Our study indicates also that evaluation of TIMPs activity seems to be a very promising tool in the differential diagnosis of causes of lymphadenopathies in doubtful cases. Supported by grant of Medical University of Lodz REFERENCES 1. Bosman FT, Stamenkovic I. Functional structure and composition of the extracellular matrix. J.Pathol 2003; 200: Jiang Y, Wang M, Celiker MY et al. Stimulation of Mammary Tumorigenesis by Systemic Tissue Inhibitor of Matrix Metalloproteinases 4 Gene Delivery. Cancer Res 2001; 15: Balcer AH, Dylan R, Murphy G. Metalloproteinase inhibitors: biological action and therapeutical opportunities. J Cell Science 2002; 115: Guedez L, Stetler-Stevenson WG, Wolff L et al. In vitro suppression of programmed cell death of B-cells by tissue inhibitor of metalloproteinases. J.Clin.Investig. 1998; 102: Hayakawa T, Yamashita K, Ohuchi E, Shinagawa A. A cell-growth promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2). J Cell Sci 1994; 107: Hagemann T, Gunawan B, Schulz M et al. mrna expression of matrix metalloproteinases and their inhibitors differs in subtypes of renal cell carcinomas. Eur J Cancer 2001; 37: Pennanen H, Kuittinen O, Turpeenniemi-Hujanen T. Plasma MMP-2-TIMP-2 complex levels measured during follow-up predict a risk of relapse in patients with malignant lymphoma. Eur.J Haematol 2008; 80: Kossakowska AE, Urbański SJ, Watson A et al. Patterns of expression of metalloproteinases and their inhibitors in human malignant lymphomas. Oncol Res 1993; 5: Suminoe A, Matsuzaki A, Hattori H et al. Expression of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) genes in blasts of infant acute lymphoblastic leukemia with organ involvement. Leukemia Res 2007; 31: Rust R, Blokzijl T, Harms G et al. TIMP-1 expression in anaplastic large cell lymphoma is usually restricted to macrophages and only seldom observed in tumour cells. J Pathol 2005; 8:

8 484 M. STOLARSKA et al. 11. Pennanen H, Kuittinen O, Soini Y, Turpeenniemi-Hujanen T. Clinicopathological correlation of TIMP-1 and TIMP-2 in Hodgkin s lymphoma. Eur J Haematol 2004; 72: Oelmann E, Herbst H, Zuhlsdorf M et al. Tissue inhibitor of metalloproteinases 1 in an autocrine and paracrine survival factor, with additional immune-regulatory functions, expressed by Hodgkin/Reed- Sternberg cells. Blood 2002; 99: Received r. and accepted r. Communicating Author: dr n. med. Malgorzata Stolarska Klinika Chorób Dzieci ul. Sporna 36/ Łódź m.stolarska@wp.pl