--DRUK 06 Stolarska str. 477-484

Acta Haematologica Polonica 2008, 39, Nr 3, str. 477–484
PRACA ORYGINALNA – Original Article
MAŁGORZATA STOLARSKA1, ELśBIETA SAŁACIŃSKA-ŁOŚ2,
KATARZYNA TARAN3, JOANNA TRELIŃSKA1, JÓZEF KOBOS2
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
Department of Pathology of the Age of Development, Medical University of Lodz, Poland
3
Chair and Department of Pathology, Medical University of Lodz, Poland
2
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.
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).
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. 8.08 – 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˚
1
2
3
4
5
Histologic diagnosis according WHO
HL – nodular sclerosis II classical HL
HL – nodular sclerosis I classical HL
HL – nodular sclerosis II classical HL
HL – nodular sclerosis I classical HL
HL – nodular sclerosis II classical HL
TIMP-2 expression in tumor cells
weak
intermediate
intermediate
intermediate
intermediate
480 M. STOLARSKA et al.
6
HL – nodular sclerosis II classical HL
7
HL – nodular sclerosis II classical HL
8
HL – nodular sclerosis I classical HL
9
HL – nodular sclerosis I classical HL
10
HL – nodular sclerosis I classical HL
11
HL - nodular sclerosis I classical HL
12
HL – nodular sclerosis II classical HL
13
HL – lymphocyte depleted classical HL
14
ALCL
15
ALCL
16
Burkitt lymphoma
17
Burkitt lymphoma
18
Burkitt lymphoma
19
T-cell lymphoma
20
T-cell lymphoma
21
T-cell lymphoma
HL – Hodgkin lymphoma, ALCL – Anaplastic Large Cell Lymphoma
weak
intermediate
weak
weak
weak
weak
no expression
no expression
weak
strong
weak
weak
weak
no expression
no expression
weak
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×
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×
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
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 502-11-323
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Received 16.01.2008 r. and accepted 8.07.2008 r.
Communicating Author:
dr n. med. Malgorzata Stolarska
Klinika Chorób Dzieci
ul. Sporna 36/50
91-738 Łódź
[email protected]