Distribution of the 72-kd Type IV Collagenase in Nonneoplastic and Neoplastic Thyroid Tissue ELIAS CAMPO, MD, MARIA J. MERINO, MD, LANCE LIOTTA, MD, PHD, RONALD NEUMANN, AND WILLIAM STETLER-STEVENSON, MD, PHD The 72-kd type IV collagenase
(matrix metalloproteinase-2
21) is a neutrial metalloproteinase
[MMP-
that initiates the degradation
type IV collagen in basement membranes.
Its production
of
by tumor
cells has been correlated with the invasive and metastatic potential of neoplasms. Two recently developed against synth’etic peptides internal domain
(Ab48)
affinity-purified
from the amino terminus
of the molecule
of this enzyme in a variety
of thyroid tissues. All primary carcinomas
(20 papillary,
positive, with the more aggressive more reactive than the low-grade
tumors (tall cell variant of paptending to be
tumors (classic and microinvasive
papillary
carcinomas
Negative
or minimal positivity was found
and minimally
invasive
follicular
tumors).
in six cases of normal
one goiter, and two cases of Graves’
reactive follicular
seven fol-
as well as nine of 11 metastases were
illary carcinomas and invasive follicular carcinomas)
thyroid,
and an
were used to investigate
immunohisto~chemically the distribution licular, and t’hree medullary)
antibodies (Hl)
disease.
Immuno-
cells were seen focally in areas of inflammation,
fibrosis, and distortion of normal follicles, and in Hashimoto’s roiditis (four cases). Five of nine adenomas
thy-
showed positive cells.
but this could1be related to previous trauma to the area. We conclude that there is increased production (MMP-2)
of the 72-kd type IV collagenase
in thyroid cancer: however, this enzyme also is elevated
in benign conditions
that are undergoing
HUM PATHOI. 23:1395-1401.
Copyright
remodeling
and repair.
(G:‘,1992 by W.B. Saunders
Company
‘l‘lie rri;tligriariltransformatio~i of cellsis associated with changes ill their phenotype. However. the importance and Ggnific.ance of these phenotypic changes are not well understood. The invasion of adjacent tissues I)\ neoplastic (ells and their metastatic. clissemination &quire Itic:*prc )duction of extracellular matrix-degrading enzymes tliat allow the cells to progress through natural barriers, such as basement membranes (BMs) atid intersl.itiat collagen matrices.‘~” Invasive malignant tmllors have ;111abnormal distribution of the BM. with focal disruption ;r1~1 even complete absence in poorly in differentiated areas.‘.” ’ The loss of BM components
MD,
these cases may be a consequence of either a decrease in synthesis or, alternatively, an increase in BM degradation by specific enzymes produced by tumor cells. Several experimental models of molecular carcinogenesis have shown that the transformation to a malignant phenotype is associated with an increase in the production of extracellular matrix-degrading enzymes, particularly the 72-kd collagenase IV (matrix metalloproteinase-2 [MMP-2]).‘-‘” This enzyme is a neutral metalloproteinase that cleaves type IV collagen in a pepsinresistant domain. I I-” The catalytic degradation of type . . IV collagen is a crucial event in the progression of tumors since this molecule constitutes the structural scaffolding of the BM, cross-linking its difierent components.“.“’ It has been shown that the metastatic potential of different cell lines correlates with their ability to carry out enzymatic degradation of BM collagen. ” It also has been demonstrated that tumor progression is associated with an increase in the steady state of 72-kd type IV collagenase (MMP-2) mRNA.H,‘X Immunohistochemical and ultrastructural studies of thyroid lesions have demonstrated BM anomalies in Ileoplastic as well as in nonneoplastic diseases. ‘W The possible role of enzymatic activity such as the type IV collagenase (MMP-2) in these modifications of BM is not known. Recently, affinity-purified rabbit polyclonal antibodies were produced against different synthetic peptides from specific domains within the type IV collagenase (MMP-L’).“’ These antibodies recognize the 72kd type IV collagenase (MMP-2) in frozen as well as formalin-fixed, paraffin-embedded tissues and may be used in retrospective studies.‘” In this study we have evaluated the immunohistochemical distribution of type and IV collagenase (MMP-2) m . multiple nonneoplastic neoplastic thyroid lesions as well as in tumor metastasis; our results constitute the basis of this report.
-__ MATERIALS Tissues
1395
AND METHODS
HUMAN PATHOLOGY
Volume 23, No. 12 (December 1992)
mas. Eleven distant metastases to lymph nodes (five), bone (two), cervical soft tissues (two), contralateral thyroid lobule (one), and lung (one) also were studied. The 1 fi benign lesions included one nodular goiter, four cases of Hashimoto’s thyroiditis, two cases of Graves’ disease, and nine follicular adenomas. Sections from thyroid also were obtained from “normal” areas distant from the tumors in six patients. All tissues were rouCnely fixed in 10% huff’ered formalin and embedded in paraffin.
Antibodies The synthetic dures were made (Biosearrh/Millipore
RESULTS The number of positive cells and the pattern of staining were similar with both antibodies, but occasionally Ab48 showed more granular cytoplasmic staining than AbH 1, A difference between both antibodies was observed in the smooth muscle cells of blood vessels, which were stained stronger with AbH 1 than with Ab48. Normal and Nonneoplastic
peptides used in the immunization proceon a Biosearch 9600 peptide synthesizer
Thyroid Lesions
to bovine sermn albumin using glutaraldehyde. Peptide Hl corresponds to the amino terminus of the latent 79-kd type IV collagenase (MMP-2) enzyme and has the sequence APSPIIKFPGDVAPKTDK. Pepride 48 corresponds to the activation locus and amino terminus of the (Z-kd activated form of the type IV collagenase (MMI’-2) and has the sequence TMRKPRCGNPDVANYNFFPRKPK, Rabbits for each antigen were immunized using appropriate bovine serum albumin-peptide conjugate in complete Freund’s adjuvant. Serum was used to prepare affinity-purified antipeptide antibodies as previously described.“’ Antibody H 1 (AbH 1) is directed against peptide H 1 and antibody 48 (Ab48) is directed against peptide 48. These antibodies have been characterized previously.‘5~‘H The); have been shown to react specifically with the 79-kd type 1V collagenase (MMP-9) by enzyme-linked imInurioahsorbent assav and western blot analysis.‘7.“”
As illustrated in Table 1, normal follicular cells were usually negative with both antibodies. Focal weak staining was observed in cells adjacent to foci of inflammation, such as palpation thyroiditis, lymphocytic thyroiditis, and atrophic areas. Focal immunoreactivity also was found in trapped follicular cells when sclerosis and fibrosis were present. These areas may represent an ongoing process of healing and repair, caused in many of the cases by previous damage to the site during fine-needle aspiration of nodules or by spontaneous hemorrhage, which is frequently seen in goiters. All Hashimoto’s thyroiditis cases showed variable immunoreactivity from weak to moderate in the clusters of oxyphilic cells. The normal follicular cells in these cases, however, were type IV collagenase (MMP-2) negative (Fig 1).
lmmunohistochemistry
Adenomas
Im~nunohistocherlli~~Il staining was performed using the avidin-hiotin-peroxidase complex technique (Vectastain ABC Kit, Vector Laboratories, Burhngame, CA).“’ The slides were dewaxed in xylene and then rehydrared. The endogenous peroxide was blocked with 0.3%~ H,O? in absolute methanol incubation fol- 30 minutes. The secCons werr next incubated with normal goat serun1 f’ol 20 minutes, followed by an overnight incubation with the primary antibody at 4°C. The primary antibodies were used at 2 to 5 pg/nil. diluted in 0.05 mol/L Tris-buffered saline, pH 7.6. The biotinylated goat anti-rabbit IgG and the avidill-biotin-peroxidase complex were diluted in the same buffer and incubated for 30 and 45 minutes, respectively. at room temperature. The slides were washed three times with Tris-buffered saline after each incubation. I’eroxidase activity was developed hy a solution of 5 mg of S-3’ di~uninobenLihir~e retrahydrochlori~~e (Sigma Chemicals, St 120uis. MO) dissolved in 1U ml, of 0.05 mol/L ‘l‘ris buffer, pH 7.6, and Cl.0395 ot H,,O,. The diaminobe~l~idi~~~ solution was filtered, and the sect&s were incubated under microscopic control. Harris’ hematoxylin was used to counterstain the slides. (Control slides were produced by replacing the primaq antibody with the IgC fraction of normal rabbit seruni. In addition. primary antibody absorbed with the synthetic peptide was usrd as the negative c.onIrol in selected cases. The immunoreactivit! was evaluated according to the intensity and extent of the staiIiing. The intensity of the iInmiilloreac.Tion was subjectively evaluated in three grades (I+, 2+. anti :I+). In cases showing variable intensity of the reaction an average score was adopted. The extent of the reac.Gon was considered as I + if the number- of reactive cells was less than 25Yo. 2+ when the number of reactive cells was 25% to 75Yc1,and Kf if more than 75% of the cells stained, The final score was obtained in each case by multiplying the intensity by the extent score. The iIriInunoI-eac,tivity of each case was then classified according to the combined score as weak (scores 1 and ‘L), moderate (scores 3 mti 4), or strong (scores 5 and higher).
Positive follicular cells were seen in five of nine adenomas (Table 1). In three cases the stained follicles were focally present in central areas of fibrosis. In these cases the reactivity adopted a diffuse c,yt,oplasmic pattern, The other two cases showed dlff use positivity throughout the adenoma. These lesions had all been aspirated previously.
Corp. Marlborough,
MA). The peptides
were conjugated
1396
Carcinomas
and Metastases
All carcinomas were positive for 72-kd type IV collagenase (MMP-2). The intensity of the imnlunostainir~g in the different histologic types is summarized in Table 2. P~pillnq (hciwmn. The reactivity in papillary carcinomas was weak in one case, moderate in 10, and strong in nine (Fig 2) (Table 2). Six of 10 tall cell carcinomas showed intense reactivity, while only three of the 10 non-tall cell carcinomas were similarly stained (Fig 3). One of these cases. however, had diffuse staining
TABLE 1. lmmunohistochemical Distribution of Type IV Collagenase in Normal and Benign Thyroid Conditions
TYPE IV COLLAGENASE
IN THYROID TISSUES
in the solid xeas and zones of squamous clifferentiation. ~l‘he rni~rc~~;~rc~inonla was weakly positive. Staining of fotticutar ;~reas was weaker than in the papillae. The staining pattern was usually difluse and intracytoplasmic, hut in s01iie c‘ascs an apical cy~oplasmic pattern also was noted. Positive inimuriorea~tivity was otxcrvrd in the intranrictex qtcq~tasniic inclusions. Eol/irrrior tkrcirwvw. Two minimally invxive fottic utar carcinomas showed weak to moderate staining of the fotticutar cells. The other two invasive poorly differentiatecl carcinomas showed strong, diffuse, cytoptasmic staining for type IV collagenase (MMl’-2) (Fig 4). Hiirthle tumors were moderately positive (Fig 5). Occasionall\;, inllnunoreacti~c tumor cells were seen within the \2sc~ritar luniina. :Llrrf~iinju C~arrir~t~a.s. At1 three med~~l1ary carciIIOIX~S wew ticjsitive, with a diffuse cytoplasmic pattern. :M~~tmI'u.sis. Line of 1 t metastases studied were ranging from moderate to positive. with intensities strong. The paltern of reactivity in all cases was diffuse inti~a~ytopl~~sIIii~. One negative case was a bone metastasis hm :I folticutar carcinoma that had been subjected to drc~ll~ific.atic,n chemistry before processing. The primar! tumor of’ this carcinoma was positive. The other negative mtxlstasis was an intrathyroidal metastasis of ;t fotlicutai- car-(.inonia. A distant metastasis of this case w,ts positive (Fig 6). The primary tumor of this patient was unavailable. In the lymph node metastases of two papillary carcinomas, nests of solid poorly differentiated ~11s were found alternating with well-differentiated foltic&u awi.4. In those cases, the reactivity was very strong in the solid areas, whereas the well-formed follicles wet-e Iqativ~ or only weakly stained. In addition to the lrnmunostaining of the folticulal cctls, wc chserved 72-kd type IV collagenase (MMY-2) rcsactivitv in hisCocvtcs and smooth muscle cells of the blood \;bssdc. E:n&thelial cells and fihrohtasts occa-
FIGURE 1. lmmunostaining for type IV collagenase in Hashimoto’s disease. Clusters of oxyphilic cells are positive whereas normal follicular cells are negative. (Immunoperoxidase; magnification - 150.)
.
i. ,.I
(Camp0 et al)
2. lmmunohistochemical Distribution of Type IV Collaaenase in Thvroid Carcinomas and Metastases
TABLE
sionatty were positive. The i~~ilii~i~iol~c’~t~.t i\.it!; of ttiest cells was stronger in areas of vascular neogenesis, such as in inllaniniator\; or granut;~tion tissue ;u’cW+. DISCUSSION Our findings support the hypottie5v5 that (1) an increaatl of type IV tx)ltageriase (%~I’-!?~ occurs in all histologic types ol. thyroid carcinomas and their metastases white the reactivity in normal thyroid tissue remain4 negative or negligihl~, and (2) a moderate inc.rease in enryme rspressioll can occur during r(.parativc processes. These findings are c.onsistent with other hiochemical and imnllunohisto~hellli~at studies that shou an increase in the productioIl of this e11nnw associated with the malignant transformation of ccljL;.‘.“.“‘.“” Tvpc
HUMAN PATHOLOGY
Volume 23, No. 12 (December
1992)
FIGURE 2. Papillary carcinoma, classic variant. The tumor cells show granular cytoplasmic immunoreactivity for type IV collagenase. (Immunoperoxidase; magnification x250.)
the enzyme. The fact that in our irnmunohistochemical study both antibodies disclosed similar reaction patterns suggests that the 72-kd type IV collagenase (MMP-2) is present in the latent form in these tumors or that the active enzyme and cleaved 80 amino acid peptide remain in close proximity after activation. How type IV rollagenase (MMP-2) contributes to the malignant phenotype of the cells is still not clear, but its mechanism may in-
IV collagenase (MMP-2). like other members of the family of metalloproteinases, is an enzyme secreted in a latent zymogen form that requires subsequent activation to degrade its substrate. This activation involves the loss of an 80 amino acid peptide from the amino terminus, ” This portion of the molecule is the fragment recognized by AbH 1. whereas Ab48 is directed against a peptide present in both the active and latent form of
FIGURE 3. Papillary carcinoma, tall cell variant, Type IV collagenase stained the cytoplasm of neoplastic cells strongly. (Immunoperoxidase; magnification X150.)
1398
TYPE IV COLLAGENASE
IN THYROID TISSUES
(Camp0 et al)
FIGURE 4. I-ollicular carcinoma, widely for invasive. Diffuse immunoreactivity was noted. (lmtype IV (:ollagenase munoOer( zxbdase; magnification X 150.)
voke
the degl-adation of‘ BM to facilitate the invasion and dissemination of tumors.“’ Inhibition of this enzyme hv naturally occurring tissue inhibitors of metalloproteinases (TIMPs) has been shown to prevent tumor in\xion in vitro”’ and lung colony formation in viva.“’ The mechanisms of enzyme activation and its regulation in viva are not yet well understood. I” Collagenolytic activity usually correlates with metastatic potential
FIGURE 5. Hijrthle cell carcinoma. A mixture of positive and negative cells was observed in this tumor. (lmmunoperoxidase; magnification X250.)
in several muririe tumor models. “.“” In rtkr-transformed human bronchial epithelial cell lines, the amount of active enzyme correlated with the mRNA expression for the latent 72&d type IV collagenase (MMP-2) and with their invasive and metastatic behavior-s.‘,“” However, high levels of 72&d type IV collagenase also have been found in some normal and low metastasizing cell lines.“,“” In these cases the enzyme is found in the latent
HUMAN PATHOLOGY
Volume 23, No. 12 (December
1992)
FIGURE 6. (Left) Well-differentiated intrathyroidal follicular carcinoma negative for type IV collagenose. (Right) Distant metastasis of the same case to paraspinal fibrous tissue shows type IV collagenase immunoreactivity. (Immunoperoxidase: magnification x250.)
proenzyme form, suggesting that the malignant behavior of tumors may be correlated with the ratio of active to latent enzyme rather than the total amount of the enzyme.” Recently, an inhibitor of the 72-kd type IV collagenase (MMP-2) that binds to both the active and latent forms has been identified in several cell lines and human tun10rs.“i~“7 Given its homo10~gy to tissue inhibitor of metalloproteinase the inhibitor has been called TIMP2.“‘i This protein may play an important role in the regulation of the enzyme activity in vivo. Although there was an overlap in the intensity of the immunoreaction between the different carcinomas, the more aggressive variants, such as the tall cell papillary carcinoma’“.“H and the invasive follicular carcinomas, had stronger reactivity than the low-grade variants. Whether this increased expression of the enzyme in thyroid tumors is associated with an increase in its biologic activity is unclear. Papillary carcinomas and well-differentiated follicular carcinomas of the thyroid are tumors associated with excellent prognosis and long-term survival. The fact that these tumors had a lower immunoreactivity for type IV collagenase (MMP-2) seems to correlate with their indolent biologic behavior. Our findings suggest that in the early steps of the malignant transformation and in certain low-grade tumors the increased expression of this enzyme may not be associated with a similar increase in its biologic activity, although more studies will be needed to confirm this hypothesis. Immunohistochemica17,“‘.“!’ and ultrastructural”’ studies have shown that papillary and well-differentiated follicular carcinomas maintain an intact BM in the papillae and surrounding follicular structures. Similarly, “in situ” carcinomas of the breast that have a mostly preserved BM”.l” also can 1400
express high levels of the 72-kd type IV collagenase (MMP-2)” All but two of the metastases were positive for type 72-kd type IV collagenase. Negative results were obtained in a bone lesion that had been decalcified; therefore, we cannot exclude an artifactual result. In two follicular carcinomas the expression in the metastatic tissue was stronger than in the thyroid tumor, indicating a possible upregulation of this enzyme in metastases. In the lymph node metastases of two papillary carcinomas the poorly differentiated areas showed stronger immunoreactions than the well-differentiated follicular areas. The variable results of the staining in the spectrum of benign thyroid lesions tested may reflect the fact that many of these thyroid tissues had been previously traumatized either by palpation, aspiration, or spontaneous infarction and regeneration. It is reported that the BM of thyroid follicles in autoimmune thyroiditis may have a number of irreguand focal ruplarities, such as reduplications of type tures. “‘xcs’~ Our findings of focal expression IV collagenase (MMP-2) in this disease, as well as in the atrophic follicles associated with different grades of thyroid fibrosis, suggest that this enzyme may be involved in the remodeling processes of the gland. A cyclic phenomenon of enzymatic degradation and production of BM components has been reported to occur in remodeling processes associated with tissue development, involution, and repair. “‘-” In summary, our study demonstrates that malignant thyroid neoplasms and their metastases have increased production of the 72-kd type IV collagenase (MMP-P), with the more aggressive variants having stronger expression than low-grade tumors. The focal staining
TYPE IV COLLAGENASE
IN THYROID TISSUES
of folliculal- cells in inflammatory and atrophic thyroid tissues suggests a role for this enq ‘me in the remodeling processes of the gland as well. REFERENCE3 I I.iotta I..4, Kao
(matrix metalloproteinase-2
21) is a neutrial metalloproteinase
[MMP-
that initiates the degradation
type IV collagen in basement membranes.
Its production
of
by tumor
cells has been correlated with the invasive and metastatic potential of neoplasms. Two recently developed against synth’etic peptides internal domain
(Ab48)
affinity-purified
from the amino terminus
of the molecule
of this enzyme in a variety
of thyroid tissues. All primary carcinomas
(20 papillary,
positive, with the more aggressive more reactive than the low-grade
tumors (tall cell variant of paptending to be
tumors (classic and microinvasive
papillary
carcinomas
Negative
or minimal positivity was found
and minimally
invasive
follicular
tumors).
in six cases of normal
one goiter, and two cases of Graves’
reactive follicular
seven fol-
as well as nine of 11 metastases were
illary carcinomas and invasive follicular carcinomas)
thyroid,
and an
were used to investigate
immunohisto~chemically the distribution licular, and t’hree medullary)
antibodies (Hl)
disease.
Immuno-
cells were seen focally in areas of inflammation,
fibrosis, and distortion of normal follicles, and in Hashimoto’s roiditis (four cases). Five of nine adenomas
thy-
showed positive cells.
but this could1be related to previous trauma to the area. We conclude that there is increased production (MMP-2)
of the 72-kd type IV collagenase
in thyroid cancer: however, this enzyme also is elevated
in benign conditions
that are undergoing
HUM PATHOI. 23:1395-1401.
Copyright
remodeling
and repair.
(G:‘,1992 by W.B. Saunders
Company
‘l‘lie rri;tligriariltransformatio~i of cellsis associated with changes ill their phenotype. However. the importance and Ggnific.ance of these phenotypic changes are not well understood. The invasion of adjacent tissues I)\ neoplastic (ells and their metastatic. clissemination &quire Itic:*prc )duction of extracellular matrix-degrading enzymes tliat allow the cells to progress through natural barriers, such as basement membranes (BMs) atid intersl.itiat collagen matrices.‘~” Invasive malignant tmllors have ;111abnormal distribution of the BM. with focal disruption ;r1~1 even complete absence in poorly in differentiated areas.‘.” ’ The loss of BM components
MD,
these cases may be a consequence of either a decrease in synthesis or, alternatively, an increase in BM degradation by specific enzymes produced by tumor cells. Several experimental models of molecular carcinogenesis have shown that the transformation to a malignant phenotype is associated with an increase in the production of extracellular matrix-degrading enzymes, particularly the 72-kd collagenase IV (matrix metalloproteinase-2 [MMP-2]).‘-‘” This enzyme is a neutral metalloproteinase that cleaves type IV collagen in a pepsinresistant domain. I I-” The catalytic degradation of type . . IV collagen is a crucial event in the progression of tumors since this molecule constitutes the structural scaffolding of the BM, cross-linking its difierent components.“.“’ It has been shown that the metastatic potential of different cell lines correlates with their ability to carry out enzymatic degradation of BM collagen. ” It also has been demonstrated that tumor progression is associated with an increase in the steady state of 72-kd type IV collagenase (MMP-2) mRNA.H,‘X Immunohistochemical and ultrastructural studies of thyroid lesions have demonstrated BM anomalies in Ileoplastic as well as in nonneoplastic diseases. ‘W The possible role of enzymatic activity such as the type IV collagenase (MMP-2) in these modifications of BM is not known. Recently, affinity-purified rabbit polyclonal antibodies were produced against different synthetic peptides from specific domains within the type IV collagenase (MMP-L’).“’ These antibodies recognize the 72kd type IV collagenase (MMP-2) in frozen as well as formalin-fixed, paraffin-embedded tissues and may be used in retrospective studies.‘” In this study we have evaluated the immunohistochemical distribution of type and IV collagenase (MMP-2) m . multiple nonneoplastic neoplastic thyroid lesions as well as in tumor metastasis; our results constitute the basis of this report.
-__ MATERIALS Tissues
1395
AND METHODS
HUMAN PATHOLOGY
Volume 23, No. 12 (December 1992)
mas. Eleven distant metastases to lymph nodes (five), bone (two), cervical soft tissues (two), contralateral thyroid lobule (one), and lung (one) also were studied. The 1 fi benign lesions included one nodular goiter, four cases of Hashimoto’s thyroiditis, two cases of Graves’ disease, and nine follicular adenomas. Sections from thyroid also were obtained from “normal” areas distant from the tumors in six patients. All tissues were rouCnely fixed in 10% huff’ered formalin and embedded in paraffin.
Antibodies The synthetic dures were made (Biosearrh/Millipore
RESULTS The number of positive cells and the pattern of staining were similar with both antibodies, but occasionally Ab48 showed more granular cytoplasmic staining than AbH 1, A difference between both antibodies was observed in the smooth muscle cells of blood vessels, which were stained stronger with AbH 1 than with Ab48. Normal and Nonneoplastic
peptides used in the immunization proceon a Biosearch 9600 peptide synthesizer
Thyroid Lesions
to bovine sermn albumin using glutaraldehyde. Peptide Hl corresponds to the amino terminus of the latent 79-kd type IV collagenase (MMP-2) enzyme and has the sequence APSPIIKFPGDVAPKTDK. Pepride 48 corresponds to the activation locus and amino terminus of the (Z-kd activated form of the type IV collagenase (MMI’-2) and has the sequence TMRKPRCGNPDVANYNFFPRKPK, Rabbits for each antigen were immunized using appropriate bovine serum albumin-peptide conjugate in complete Freund’s adjuvant. Serum was used to prepare affinity-purified antipeptide antibodies as previously described.“’ Antibody H 1 (AbH 1) is directed against peptide H 1 and antibody 48 (Ab48) is directed against peptide 48. These antibodies have been characterized previously.‘5~‘H The); have been shown to react specifically with the 79-kd type 1V collagenase (MMP-9) by enzyme-linked imInurioahsorbent assav and western blot analysis.‘7.“”
As illustrated in Table 1, normal follicular cells were usually negative with both antibodies. Focal weak staining was observed in cells adjacent to foci of inflammation, such as palpation thyroiditis, lymphocytic thyroiditis, and atrophic areas. Focal immunoreactivity also was found in trapped follicular cells when sclerosis and fibrosis were present. These areas may represent an ongoing process of healing and repair, caused in many of the cases by previous damage to the site during fine-needle aspiration of nodules or by spontaneous hemorrhage, which is frequently seen in goiters. All Hashimoto’s thyroiditis cases showed variable immunoreactivity from weak to moderate in the clusters of oxyphilic cells. The normal follicular cells in these cases, however, were type IV collagenase (MMP-2) negative (Fig 1).
lmmunohistochemistry
Adenomas
Im~nunohistocherlli~~Il staining was performed using the avidin-hiotin-peroxidase complex technique (Vectastain ABC Kit, Vector Laboratories, Burhngame, CA).“’ The slides were dewaxed in xylene and then rehydrared. The endogenous peroxide was blocked with 0.3%~ H,O? in absolute methanol incubation fol- 30 minutes. The secCons werr next incubated with normal goat serun1 f’ol 20 minutes, followed by an overnight incubation with the primary antibody at 4°C. The primary antibodies were used at 2 to 5 pg/nil. diluted in 0.05 mol/L Tris-buffered saline, pH 7.6. The biotinylated goat anti-rabbit IgG and the avidill-biotin-peroxidase complex were diluted in the same buffer and incubated for 30 and 45 minutes, respectively. at room temperature. The slides were washed three times with Tris-buffered saline after each incubation. I’eroxidase activity was developed hy a solution of 5 mg of S-3’ di~uninobenLihir~e retrahydrochlori~~e (Sigma Chemicals, St 120uis. MO) dissolved in 1U ml, of 0.05 mol/L ‘l‘ris buffer, pH 7.6, and Cl.0395 ot H,,O,. The diaminobe~l~idi~~~ solution was filtered, and the sect&s were incubated under microscopic control. Harris’ hematoxylin was used to counterstain the slides. (Control slides were produced by replacing the primaq antibody with the IgC fraction of normal rabbit seruni. In addition. primary antibody absorbed with the synthetic peptide was usrd as the negative c.onIrol in selected cases. The immunoreactivit! was evaluated according to the intensity and extent of the staiIiing. The intensity of the iInmiilloreac.Tion was subjectively evaluated in three grades (I+, 2+. anti :I+). In cases showing variable intensity of the reaction an average score was adopted. The extent of the reac.Gon was considered as I + if the number- of reactive cells was less than 25Yo. 2+ when the number of reactive cells was 25% to 75Yc1,and Kf if more than 75% of the cells stained, The final score was obtained in each case by multiplying the intensity by the extent score. The iIriInunoI-eac,tivity of each case was then classified according to the combined score as weak (scores 1 and ‘L), moderate (scores 3 mti 4), or strong (scores 5 and higher).
Positive follicular cells were seen in five of nine adenomas (Table 1). In three cases the stained follicles were focally present in central areas of fibrosis. In these cases the reactivity adopted a diffuse c,yt,oplasmic pattern, The other two cases showed dlff use positivity throughout the adenoma. These lesions had all been aspirated previously.
Corp. Marlborough,
MA). The peptides
were conjugated
1396
Carcinomas
and Metastases
All carcinomas were positive for 72-kd type IV collagenase (MMP-2). The intensity of the imnlunostainir~g in the different histologic types is summarized in Table 2. P~pillnq (hciwmn. The reactivity in papillary carcinomas was weak in one case, moderate in 10, and strong in nine (Fig 2) (Table 2). Six of 10 tall cell carcinomas showed intense reactivity, while only three of the 10 non-tall cell carcinomas were similarly stained (Fig 3). One of these cases. however, had diffuse staining
TABLE 1. lmmunohistochemical Distribution of Type IV Collagenase in Normal and Benign Thyroid Conditions
TYPE IV COLLAGENASE
IN THYROID TISSUES
in the solid xeas and zones of squamous clifferentiation. ~l‘he rni~rc~~;~rc~inonla was weakly positive. Staining of fotticutar ;~reas was weaker than in the papillae. The staining pattern was usually difluse and intracytoplasmic, hut in s01iie c‘ascs an apical cy~oplasmic pattern also was noted. Positive inimuriorea~tivity was otxcrvrd in the intranrictex qtcq~tasniic inclusions. Eol/irrrior tkrcirwvw. Two minimally invxive fottic utar carcinomas showed weak to moderate staining of the fotticutar cells. The other two invasive poorly differentiatecl carcinomas showed strong, diffuse, cytoptasmic staining for type IV collagenase (MMl’-2) (Fig 4). Hiirthle tumors were moderately positive (Fig 5). Occasionall\;, inllnunoreacti~c tumor cells were seen within the \2sc~ritar luniina. :Llrrf~iinju C~arrir~t~a.s. At1 three med~~l1ary carciIIOIX~S wew ticjsitive, with a diffuse cytoplasmic pattern. :M~~tmI'u.sis. Line of 1 t metastases studied were ranging from moderate to positive. with intensities strong. The paltern of reactivity in all cases was diffuse inti~a~ytopl~~sIIii~. One negative case was a bone metastasis hm :I folticutar carcinoma that had been subjected to drc~ll~ific.atic,n chemistry before processing. The primar! tumor of’ this carcinoma was positive. The other negative mtxlstasis was an intrathyroidal metastasis of ;t fotlicutai- car-(.inonia. A distant metastasis of this case w,ts positive (Fig 6). The primary tumor of this patient was unavailable. In the lymph node metastases of two papillary carcinomas, nests of solid poorly differentiated ~11s were found alternating with well-differentiated foltic&u awi.4. In those cases, the reactivity was very strong in the solid areas, whereas the well-formed follicles wet-e Iqativ~ or only weakly stained. In addition to the lrnmunostaining of the folticulal cctls, wc chserved 72-kd type IV collagenase (MMY-2) rcsactivitv in hisCocvtcs and smooth muscle cells of the blood \;bssdc. E:n&thelial cells and fihrohtasts occa-
FIGURE 1. lmmunostaining for type IV collagenase in Hashimoto’s disease. Clusters of oxyphilic cells are positive whereas normal follicular cells are negative. (Immunoperoxidase; magnification - 150.)
.
i. ,.I
(Camp0 et al)
2. lmmunohistochemical Distribution of Type IV Collaaenase in Thvroid Carcinomas and Metastases
TABLE
sionatty were positive. The i~~ilii~i~iol~c’~t~.t i\.it!; of ttiest cells was stronger in areas of vascular neogenesis, such as in inllaniniator\; or granut;~tion tissue ;u’cW+. DISCUSSION Our findings support the hypottie5v5 that (1) an increaatl of type IV tx)ltageriase (%~I’-!?~ occurs in all histologic types ol. thyroid carcinomas and their metastases white the reactivity in normal thyroid tissue remain4 negative or negligihl~, and (2) a moderate inc.rease in enryme rspressioll can occur during r(.parativc processes. These findings are c.onsistent with other hiochemical and imnllunohisto~hellli~at studies that shou an increase in the productioIl of this e11nnw associated with the malignant transformation of ccljL;.‘.“.“‘.“” Tvpc
HUMAN PATHOLOGY
Volume 23, No. 12 (December
1992)
FIGURE 2. Papillary carcinoma, classic variant. The tumor cells show granular cytoplasmic immunoreactivity for type IV collagenase. (Immunoperoxidase; magnification x250.)
the enzyme. The fact that in our irnmunohistochemical study both antibodies disclosed similar reaction patterns suggests that the 72-kd type IV collagenase (MMP-2) is present in the latent form in these tumors or that the active enzyme and cleaved 80 amino acid peptide remain in close proximity after activation. How type IV rollagenase (MMP-2) contributes to the malignant phenotype of the cells is still not clear, but its mechanism may in-
IV collagenase (MMP-2). like other members of the family of metalloproteinases, is an enzyme secreted in a latent zymogen form that requires subsequent activation to degrade its substrate. This activation involves the loss of an 80 amino acid peptide from the amino terminus, ” This portion of the molecule is the fragment recognized by AbH 1. whereas Ab48 is directed against a peptide present in both the active and latent form of
FIGURE 3. Papillary carcinoma, tall cell variant, Type IV collagenase stained the cytoplasm of neoplastic cells strongly. (Immunoperoxidase; magnification X150.)
1398
TYPE IV COLLAGENASE
IN THYROID TISSUES
(Camp0 et al)
FIGURE 4. I-ollicular carcinoma, widely for invasive. Diffuse immunoreactivity was noted. (lmtype IV (:ollagenase munoOer( zxbdase; magnification X 150.)
voke
the degl-adation of‘ BM to facilitate the invasion and dissemination of tumors.“’ Inhibition of this enzyme hv naturally occurring tissue inhibitors of metalloproteinases (TIMPs) has been shown to prevent tumor in\xion in vitro”’ and lung colony formation in viva.“’ The mechanisms of enzyme activation and its regulation in viva are not yet well understood. I” Collagenolytic activity usually correlates with metastatic potential
FIGURE 5. Hijrthle cell carcinoma. A mixture of positive and negative cells was observed in this tumor. (lmmunoperoxidase; magnification X250.)
in several muririe tumor models. “.“” In rtkr-transformed human bronchial epithelial cell lines, the amount of active enzyme correlated with the mRNA expression for the latent 72&d type IV collagenase (MMP-2) and with their invasive and metastatic behavior-s.‘,“” However, high levels of 72&d type IV collagenase also have been found in some normal and low metastasizing cell lines.“,“” In these cases the enzyme is found in the latent
HUMAN PATHOLOGY
Volume 23, No. 12 (December
1992)
FIGURE 6. (Left) Well-differentiated intrathyroidal follicular carcinoma negative for type IV collagenose. (Right) Distant metastasis of the same case to paraspinal fibrous tissue shows type IV collagenase immunoreactivity. (Immunoperoxidase: magnification x250.)
proenzyme form, suggesting that the malignant behavior of tumors may be correlated with the ratio of active to latent enzyme rather than the total amount of the enzyme.” Recently, an inhibitor of the 72-kd type IV collagenase (MMP-2) that binds to both the active and latent forms has been identified in several cell lines and human tun10rs.“i~“7 Given its homo10~gy to tissue inhibitor of metalloproteinase the inhibitor has been called TIMP2.“‘i This protein may play an important role in the regulation of the enzyme activity in vivo. Although there was an overlap in the intensity of the immunoreaction between the different carcinomas, the more aggressive variants, such as the tall cell papillary carcinoma’“.“H and the invasive follicular carcinomas, had stronger reactivity than the low-grade variants. Whether this increased expression of the enzyme in thyroid tumors is associated with an increase in its biologic activity is unclear. Papillary carcinomas and well-differentiated follicular carcinomas of the thyroid are tumors associated with excellent prognosis and long-term survival. The fact that these tumors had a lower immunoreactivity for type IV collagenase (MMP-2) seems to correlate with their indolent biologic behavior. Our findings suggest that in the early steps of the malignant transformation and in certain low-grade tumors the increased expression of this enzyme may not be associated with a similar increase in its biologic activity, although more studies will be needed to confirm this hypothesis. Immunohistochemica17,“‘.“!’ and ultrastructural”’ studies have shown that papillary and well-differentiated follicular carcinomas maintain an intact BM in the papillae and surrounding follicular structures. Similarly, “in situ” carcinomas of the breast that have a mostly preserved BM”.l” also can 1400
express high levels of the 72-kd type IV collagenase (MMP-2)” All but two of the metastases were positive for type 72-kd type IV collagenase. Negative results were obtained in a bone lesion that had been decalcified; therefore, we cannot exclude an artifactual result. In two follicular carcinomas the expression in the metastatic tissue was stronger than in the thyroid tumor, indicating a possible upregulation of this enzyme in metastases. In the lymph node metastases of two papillary carcinomas the poorly differentiated areas showed stronger immunoreactions than the well-differentiated follicular areas. The variable results of the staining in the spectrum of benign thyroid lesions tested may reflect the fact that many of these thyroid tissues had been previously traumatized either by palpation, aspiration, or spontaneous infarction and regeneration. It is reported that the BM of thyroid follicles in autoimmune thyroiditis may have a number of irreguand focal ruplarities, such as reduplications of type tures. “‘xcs’~ Our findings of focal expression IV collagenase (MMP-2) in this disease, as well as in the atrophic follicles associated with different grades of thyroid fibrosis, suggest that this enzyme may be involved in the remodeling processes of the gland. A cyclic phenomenon of enzymatic degradation and production of BM components has been reported to occur in remodeling processes associated with tissue development, involution, and repair. “‘-” In summary, our study demonstrates that malignant thyroid neoplasms and their metastases have increased production of the 72-kd type IV collagenase (MMP-P), with the more aggressive variants having stronger expression than low-grade tumors. The focal staining
TYPE IV COLLAGENASE
IN THYROID TISSUES
of folliculal- cells in inflammatory and atrophic thyroid tissues suggests a role for this enq ‘me in the remodeling processes of the gland as well. REFERENCE3 I I.iotta I..4, Kao
