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Keywords:

  • circulating 92-kilodalton (kDa) matrix metalloproteinase (MMP);
  • MMP-9;
  • tumor marker;
  • laryngeal carcinoma;
  • oropharyngeal carcinoma

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

Cancer lethality is usually the result of local invasion and metastasis of neoplastic cells from the primary tumor. Because of their ability to degrade extracellular matrix components (EMC), matrix metalloproteinases (MMPs) have been implicated in the breakdown of basement membranes and underlying stroma, thereby facilitating tumor growth and invasion.

METHODS

The authors quantitated, by gelatin zymography and densitometric analysis, MMP activity in the euglobulin plasma fraction of 50 healthy controls and 91 head and neck squamous cell carcinoma (HNSCC) patients (51 from the larynx and 40 from the oropharynx).

RESULTS

The median value for 92-kilodalton (kD) MMP (MMP-9) activity was increased significantly in laryngeal (Md 2.1 arbitrary units (AU)/mL plasma; range, 0.2–6.4) and oropharyngeal patients (Md 2.08 AU/mL; range, 0.0–5.0) with respect to the controls (Md 0.48 AU/mL; range, 0.0–1.8). Both groups of cancer patients showed a similar behavior. Multivariate analysis indicated that circulating 92-kD MMP activity was not predicted by the clinical-pathologic parameters such as tumor stage, histologic grade, and metastatic lymph nodes. There was no association between high levels of MMP-9 activity and either cigarette smoking or alcohol consumption, major risk factors for developing HNSCC.

CONCLUSIONS

The authors found a significant increase of MMP-9 plasma activity both in laryngeal and oropharyngeal squamous cell carcinoma patients as compared with healthy controls. Further studies are necessary to establish its usefulness in the clinical management of these patients. Cancer 2002;94:1483–91. © 2002 American Cancer Society.

DOI 10.1002/cncr.10356

Many steps during invasion and metastasis require specific interactions between tumor cells and the extracellular matrix (ECM).1 The invasive phenotype depends on the ability of tumor cells to attach to the ECM, degrade matrix components, and finally migrate through this partially degraded matrix. The repetitive cycling of these three processes enables tumor cells to invade host tissues.2 It is well known that tumor cells produce higher amounts of proteolytic enzymes than normal counterparts. Positive correlation between invasiveness properties and protease levels has been documented for all classes of proteinases both in animal tumor models and human tumors.3–5

The metalloproteinases (MMPs) comprise a large and ever-growing family of Zn2+ and Ca2+-dependent endopeptidases. Currently, it includes more than 20 members.6, 7 A group of four specific inhibitors have been identified for these enzymes and are referred to as the tissue inhibitors of MMPs (TIMPs).8 According to their substrate specificities and cellular location, the MMPs family comprises the interstitial collagenases, gelatinases, stromelysins, membrane type MMPs (MT-MMPs) and matrilysin-1 and -2.6, 7, 9 Several MMPs, including MMP-1, the gelatinases MMP-2 and MMP-9, and the stromelysins MMP-3 and MMP-10, have been implicated in cancer cell invasion and metastasis.10 Although a major function of MMPs in metastasis is to facilitate the breakdown of the ECM, including the basement membrane, they play substantial roles in the maintenance of a microenvironment that facilitates growth and angiogenesis of tumors at primary and metastatic sites. Metalloproteinases have been reported to play direct roles in other cellular activities such as differentiation, proliferation, and apoptosis.6

The collagenases Type IV, MMP-2 (gelatinase A, 72 kilodalton [kD]) and MMP-9 (gelatinase B, 92 kD), are also normal components of human plasma. Some studies have evaluated the diagnostic and/or prognostic utility of measuring these enzymes in plasma, serum, or other body fluids from cancer patients.11–15 Almost all these studies quantified the antigen concentration, and only few works studied the activity of MMPs. In a previous work we demonstrated, by quantitative zymography, that circulating MMP-9 activity is significantly elevated in the euglobulin plasma fraction of breast carcinoma patients, as compared with a group of benign mammary pathologies and healthy controls.16

Head and neck carcinoma is an important public health problem worldwide. It includes a large variety of malignant tumors arising from different tissues and localizations, therefore having distinct histopathology, biologic behavior, and prognosis. Head and neck squamous cell carcinoma (HNSCC) is the most frequent histologic type, both in tumors originating in the larynx or in the oropharynx. Although early stage disease is often curable with surgery or radiotherapy, most cases are diagnosed at an advanced stage, and this means a poor prognosis. Despite the evolution and refinement of multimodal treatments for head and neck carcinoma (surgery, radiation therapy, and chemotherapy), the overall survival rate in UICC TNM Stages III and IV is less than 40%.17, 18 Furthermore, patients cured of their initial early stage HNSCC are at high risk for development of second primary tumors, which pose the main threat to survival.19

The carcinogenesis process in head and neck carcinoma results from a dysregulation of cellular proliferation, differentiation, and cell death resulting in part from field-wide exposure of the upper aerodigestive tract to environmental agents such as tobacco smoking and alcohol consumption. Moreover, a synergic interaction between these factors was reported.20 However, the occurrence of this pathology in young adults and in nonusers of tobacco and alcohol, suggests a possible genetic predisposition as well.20

Several molecules, including CD44v6, bFGF, VEGF, MMP-2, and tumor specific genetic alterations in circulating DNA, have been studied in biologic fluids such as serum, plasma, or saliva in HNSCC patients, but their utility in clinical management requires further studies.21–26

Here, we measured 92-kD MMP activity in the euglobulin plasma fraction of HNSCC patients (n = 91) and compared their levels with a group of healthy controls (n = 50). The enzymatic activity was analyzed according to the clinical-pathologic features of these patients. Possible effects of smoking and alcohol consumption on plasma 92-kD MMP activity also were analyzed.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients and Control Subjects

The current study was performed with patients recruited from three hospitals of Buenos Aires (Roffo, Curie, and Pirovano), in the frame-shift of the Latin American multicentric collaborative study on risk factors for oropharyngeal and laryngeal carcinoma, coordinated by IARC (Lyon, World Health Organization).

Plasma MMP activity was measured in 91 patients with head and neck carcinoma (40 oropharyngeal and 51 laryngeal carcinomas [33 males and 7 females and 42 males and 9 females, respectively; median age, 64 {range, 44–78} and 57 {range, 31–74}, respectively]) and in a healthy population of 50 controls (34 males and 16 females; median age, 41 [range, 22–78]).

All patients included in this study had a single primary squamous cell carcinoma, and none had undergone any treatment before blood collection. Thirty-three of 91 patients presented regional lymph node involvement. Individuals who had smoked at least 1 cigarette daily during at least 1 year were classified as smokers. Individuals who had drank alcohol at least 1 time per month during at least 1 year were considered alcohol drinkers. Finally, those who had stopped smoking and/or drinking at least 1 year before the diagnosis were classified as ex-smokers and ex-drinkers.

Information about patients was recruited by specially trained interviewers, by the physicians direct assessment, and by review of their corresponding medical charts.

This study was approved by the ethical committees of the three mentioned hospitals. All patients signed an informed consent form.

Preparation of Euglobulins

Plasma was obtained from heparinized blood, aliquoted at 100 μL and frozen at −80 °C until processing.

Euglobulins were prepared by mixing 0.1 mL plasma with 0.9 mL cold deionized water and acidified to pH 5.5 with 40 μL 1% (v/v) acetic acid. This mixture was incubated for 60 minutes at 0 °C and centrifuged 10 minutes at 5000 rpm.27 Euglobulins then were dissolved in 1 mL phosphate-buffered saline solution, pH 7.4, so that the final dilution was 1:10.

Zymography and Densitometric Analysis

Freshly prepared euglobulins were analyzed on 9% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents.28 Gels were copolymerized with heat-denatured Type I collagen (gelatine; GIBCO BRL, Gaithersburg, MD) as enzyme substrate.29 Ten microliters of the final solution of euglobulin preparation was loaded onto each of the 15 lanes of the gelatin-polyacrylamide gels. The electrophoresis was performed with a Mini-Protean II (Bio-Rad Laboratories, Richmond, CA) at 100 V constant voltage during approximately 120 minutes.

After electrophoresis, gels were washed twice in 2.5% Triton X-100 in aqueous solution for 30 minutes each time to remove SDS and incubated in 0.15 M NaCl, 10 mM CaCl2, and 50 mM Tris-HCl buffer, pH 7.4, at 37 °C for 20 hours. Previous studies, using times of incubation between 6 and 72 hours demonstrated linearity in the 92-kD MMP activity up to 48 hours. When different aliquots of euglobulin preparation were analyzed by gelatin zymography, a linear correlation was observed between plasma volume (up to 20 μL) and MMP-9 activity.

Then, the zymograms were stained with Coomassie Brilliant Blue R-250 and destained with 30% methanol and 10% acetic acid in distilled water. Bands of gelatine degradation could be seen as transparent areas against a blue background. Molecular weights were determined using standard prestained molecular weight markers (Bio-Rad). All reagents for electrophoresis were purchased from Sigma (St. Louis, MO). Gelatinolytic bands were measured with an image analyzer (Bio-Rad Densitometer, model GS-670) and referenced to a standard curve of bacterial collagenase (Sigma) at a range of concentrations from 0.025 to 0.25 AU/mL and corrected by the plasma dilution. All data were expressed as arbitrary units (AU) per milliliter of plasma.

As a control, a purified MMP-9 isolated from human blood (Boehringer-Mannheim Biochemicals, Indianapolis, IN) was also electrophoresed and analyzed by zymography in some assays.

Control of specific metal-dependent protease activity was conducted using an incubation buffer containing 25 mM EDTA. The proteinase inhibitors aprotinin and phenylmethyl sulphonyl fluoride (PMSF) were used to test the presence of serine protease activity.

The intraassay coefficient of variation of the samples (tested in duplicate) was approximately 5%, and the interassay coefficient of variation was approximately 10%.

An internal standard euglobulin fraction prepared from a pool of plasma from 20 healthy individuals was included on each gel for correction of intergel variation.

MMP Identification by Western Blot Analysis

Western blot analysis for MMP-9 and MMP-2 was conducted using a SDS-PAGE that was performed as described by Laemmli,30 using 10% separating and 4% stacking gels. It used a sample buffer containing 25 mM Tris, 10% v/v glycerol, 12.5 mg/mL SDS, 0.125 mg/mL bromophenol blue, and 1.25% 2B-mercaptoethanol (pH 6.8). After electrophoresis, gels were transferred to nitrocellulose membranes using an aquose electrical transfer (Bio-Rad Laboratories) for 1 hour. To detect MMP-9 and MMP-2 expression, we used anti-human MMP-9 and MMP-2 monoclonal antibodies from Calbiochem, Oncogene Research Product. An anti-mouse biotinylated IgG (GIBCO BRL) was used as second antibody and the detection system was streptavidin-phosphatase alkaline conjugate (Vector Laboratories, Burlingame, CA) and BCIP/NBT solution as substrate (Sigma).

Statistical Analysis

Differences in the level of MMP-9 among groups were compared using the Mann–Whitney U test, appropriate median test for even skewed data.

A receiver operator characteristic (ROC) curve31 was developed to determine the optimal reference value to differentiate patients with positive and negative circulating MMP-9 activity levels. Sensitivity, specificity, positive predictive value (PV+) and negative predictive value (PV−) were calculated using the optimal cutoff point.

Unconditional logistic regression was used to control for potential confounders (such as age, tumor size, presence or not of metastatic lymph nodes, and histologic grade) and to derive adjusted odd ratios (ORs) and 95% confidence intervals (CIs).

STATA software for windows (Statacop, 1997) was used for statistical analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

MMP Activity in Plasma Euglobulin Fraction

It was demonstrated previously that MMP activity was poorly detected by zymography in whole plasma samples either from healthy donors or from patients with benign diseases or malignant tumors. The acetic precipitation used to prepare the euglobulin plasma fraction was highly effective to enhance MMP detection allowing us to reveal several bands of circulating metal- dependent protease activity.16

As shown in Figure 1, all samples analyzed showed the gelatinolytic bands of 62 and 92 kD. In many samples, bands of 120 and 200 kD also were detected. These activities were completely suppressed by ethylenediamine tetraacetic acid (EDTA), but not by aprotinin or PMSF, confirming that the enzymes were MMPs. The commercial MMP-9, isolated from human blood, also showed three bands of 92,120 and 200 kD in the zymography (data not shown). Besides, Western blot analysis using specific anti-human antibodies determined that the band of 62 kD corresponded to MMP-2, whereas the 92-kD band was MMP-9.16

thumbnail image

Figure 1. Gelatin zymogram of plasma euglobulin fraction. The first three lanes correspond to the standard curve of bacterial collagenase. Laryngeal carcinoma patients (lanes A and B), oropharyngeal carcinoma patient (C), healthy control subjects (D and E), internal standard preparation (lane F). kDa: kilodalton.

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Plasma MMP Activity in Cancer Patients

Densitometric analysis of MMP-2 gelatinolytic band (Fig. 1) showed that its activity did not differ between controls and cancer patients.

On the other side, zymograms showed that 92-, 120-, and 200-kD gelatinolytic bands were larger in the euglobulin fraction of cancer patients as compared with healthy controls (Fig. 1). Because 120- and 200-kD bands were not constantly found in all samples, the following quantitative analysis was only performed with the MMP of 92 kD.

The control group showed a median of 0.48 AU/mL (range, 0.0–1.8) of 92-kD MMP activity in the plasma euglobulin fraction. A significant increase of this activity was observed both in the plasma of patients bearing laryngeal (2.1 [range, 0.2–6.4]) or oropharyngeal carcinoma (2.08 [range, 0.0–5.0]) (P < 0.01 vs. control group, Mann–Whitney U test). In Figure 2 we show the distribution of the 92-kD MMP activity in the individual euglobulin fraction of these subjects.

thumbnail image

Figure 2. Ninety-two–kilodalton MMP activity in individual euglobulin fraction samples of cancer patients. The line indicates the optimal cutoff value (1.14 AU/mL plasma). A higher number of individuals with elevated MMP-9 values was observed in the oropharyngeal and laryngeal carcinoma groups, as compared with healthy controls (chi-square test; P < 0.001). MMP: matrix metalloproteinase; AU: arbitrary units.

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The reference value of 1.14 AU/mL plasma was close to the inflection point on the ROC curve (Fig. 3), thereby maximizing sensitivity and specificity. This point corresponded to the 75th percentile value in normal control subjects. Then, values over this concentration of 92-kD MMP were defined as positive. On the basis of the optimal cutoff point, 78% (40 of 51) of patients with laryngeal carcinoma, 85% (34 of 40) of patients with carcinomas of the oropharynx, and 20% (10 of 50) of controls were positive (Fig. 2). A logistic regression analysis did not show association between MMP activity enhancement and known clinical-pathologic prognosis predictors (tumor stage, histologic grade, metastatic lymph nodes) in neither laryngeal nor in oropharyngeal carcinoma patients. Metalloproteinase positivity was not predicted by individual characteristics such as gender, age, and alcohol drinking or smoking status (Tables 1 and 2).

thumbnail image

Figure 3. The receiver operator characteristic (ROC) curve used to determine the value of the optimal reference point, to define 92-kilodalton MMP plasma positive levels.

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Table 1. Laryngeal Carcinoma Clinicopathologic Features of the Laryngeal Carcinoma Population in Relation to the Plasma 92-Kilodalton MMP Activity
FeaturePositive/negative (% of positive)Univariate analysisMultivariate analysisa
OR95% CIOR95% CI
  • MMP; matrix metalloproteinase; OR: odds ratio; CI: confidence interval.

  • a

    Including all the variables in the table.

  • b

    Reference value.

Age (yrs)
 < 50b10/4 (71)1
 51–5913/3 (81)1.70.3–9.6
 ≥ 6017/4 (81)1.70.3–8.31.50.6–3.7
Gender
 Maleb34/10 (77)1
 Female6/1 (86)1.80.2–16.41.30.2–16.4
Stage
 Ib4/1 (80)1
 II10/3 (77)0.80.1–10.6
 III15/3 (83)1.20.1–15.5
 IV11/4 (73)0.70.1–8.10.90.4–2.1
Histologic grade
 Well differentiatedb19/5 (79)1
 Moderatly differentiated16/6 (73)0.70.2–2.7
 Poorly differentiated5/0 (100)1.10.3–3.6
Lymph node metastases
 Negativeb26/8 (76)1
 Positive14/3 (82)1.40.3–6.31.80.3–10.4
Smoking status
 Current smokerb24/7 (77)1
 Ex-smoker12/4 (75)0.90.2–3.6
 Never smoker4/0 (100)0.90.4–2.0
Alcohol habits
 Current drinkerb17/3 (85)1
 Ex-drinker15/2 (88)0.50.1–2.3
 Never drinker2/1 (67)0.70.1–7.50.60.3–1.5
Table 2. Clinicopathologic Features of the Oropharyngeal Carcinoma Population in Relation to the Level of Plasma 92-Kilodalton MMP Activity
FeaturePositive/negative (% of positive)Univariate analysisMultivariate analysisa
OR95% CIOR95% CI
  • MMP: matrix metalloproteinase; OR: odds ratio; CI: confidence interval.

  • a

    Including all the variables in the table.

  • b

    Reference value.

  • c

    Eleven patients were ex-drinkers; all of them showed positive plasma MMP-9 activity levels.

Age (yrs)
 < 50b4/1 (80)1
 51–5910/2 (83)1.20.1–18.0
 ≥ 6020/3 (87)1.70.1–20.40.90.2–3.8
Gender
 Maleb27/4 (87)1
 Female7/2 (78)0.50.8–3.40.70.1–6.3
Stage
 Ib4/1 (80)1
 II2/1 (67)0.50.1–12.9
 III19/3 (86)1.60.1–19.4
 IV9/1 (90)2.20.1–45.71.90.5–6.3
Histologic grade
 Well differentiatedb19/2 (90)1
 Moderatly differentiated4/3 (57)0.10.0–1.1
 Poorly differentiated11/1 (92)1.10.1–14.31.00.3–3.1
Lymph node metastases
 Negativeb21/3 (87)1
 Positive13/3 (81)0.60.1–3.50.30.0–3.5
Smoking status
 Current smokerb17/3 (85)1
 Ex-smoker15/2 (88)1.30.2–9.0
 Never smoker2/1 (67)0.30.0–5.21.00.4–2.8
Alcohol habits
 Ever drinkersbc27/4 (87)1
 Never drinker7/2 (78)0.50.1–3.40.50.0–3.9

Because no difference in the levels of 92-kD MMP activity was observed, the two groups of head and neck patients were combined to summarize the following diagnostic test characteristics: sensitivity 83%, specificity 80%, PV+ 88%, and PV− 71%.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In murine and human models, tumor metastatic potential correlates with the degradation of basement membrane Type IV collagen and the levels of MMPs able to degrade the ECM.3 Overproduction of MMPs might result in increased levels of MMPs activity in body fluids, which, in turn, could have a potential utility as tumor markers.

Regarding HNSCC, some groups studied the expression of these enzymes and their natural inhibitors at the tissue protein and mRNA levels. Kurahara et al.32 reported that the expression of MMP-1, -2, -3, and -9 and of MT1-MMP increased with the enhancement of the metastatic potential of the tumor in 96 cases of oral squamous cell carcinoma by immunohistochemistry. They also found that the highest expression of TIMP-1 corresponded to the tumors with lymph node metastases. However, no significant relation was observed between the staining for TIMP-2 and the presence of lymph node involvement. Mueller et al.33 reported the overexpression of stromelysin 3 mRNA in the 95% of the analyzed HNSCC cases.

Other studies have analyzed circulating MMP, at the protein level, in other cancer patients. In this way, high levels of MMP-9 were detected in plasma of patients with both breast and colorectal carcinoma,11 and increased serum MMP-2 was observed in patients with lung12 and ovarian14 tumors. Besides, MMP-9/TIMP-1 complexes also were found increased in plasma of gastrointestinal and gynecologic carcinoma patients.13

Matrix metalloproteinase gelatinolytic activity in serum or plasma was studied in several animal tumor models,34, 35 whereas only a few works have studied the enzymatic activity of each circulating MMP in cancer patients. Hashimoto et al.36 reported elevated serum levels of Type IV collagenolytic activity in hepatocellular carcinoma patients, but the substrate degradation assay used did not allow to distinguish between MMP-2 and MMP-9. In this work, we analyzed the activity of individual circulating MMP after the precipitation of plasma proteins at pH 5.5 (regular euglobulin fraction) previous to an electrophoresis in SDS gels copolymerized with gelatin. In this way, four bands of gelatinolytic activity were detected as other authors described using a more complex methodology.37, 38 When whole plasma or serum were used, we could detect only slight MMP activity in comparison with euglobulins. Garbisa et al.12 reported a high variability when plasma MMP levels were measured. The method developed in our laboratory showed to be highly specific and reproducible.16

It was demonstrated that most plasma gelatinases circulate as latent enzymes.38 We do not know for certain why the acetic precipitation of plasma proteins is highly effective to reveal the activity of MMPs. As reported by several authors,39 both the acid treatment or the addition of SDS to the samples to run in a zymogram, convert latent MMPs to catalytically active forms, without proteolytic cleavage of the N-terminal inhibitory sequence. However, SDS-PAGE of whole plasma samples showed very low ability to activate MMPs. Perhaps, the acetic acid precipitation of the euglobulin may be increasing the activity through the concentration of the samples.16

We showed that the size and intensity of MMP bands of 92, 120, and 200 kD were markedly increased in the euglobulin fraction of cancer patients with respect to the control population. Because the striking differences between controls and cancer patients were mainly observed with the band of 92 kD, further analysis was performed on this MMP. The 92-kD band corresponded to MMP-9, according to its molecular weight and Western blot analysis.16

The activity of 92-kD MMP was significantly enhanced in the euglobulin fraction of HNSCC patients with respect to the healthy population. This finding seems to be confined to some tumor pathologies because, as we previously reported,16 patients bearing colon and brain tumors presented values of plasma 92-kD MMP activity that were not significantly elevated with respect to those observed in the control group. Using a cutoff point of 1.14 AU/mL of plasma, we found that approximately 78% of larynx carcinoma patients and 85% of patients with malignant tumors of the oropharynx showed high values, whereas only approximately 20% of healthy controls did. Regarding the high sensitivity of the test to detect patients with HNSCC, we must take into account that our estimations may have a little bias for being obtained from the same data used to pick up the value of the cutoff point. Up to now, our results suggest that plasma MMP-9 activity is a good marker for the presence of HNSCC tumors. Further studies are necessary to establish whether high MMP values could be associated with local recurrence or metastatic dissemination.

Several tumor specific genetic alterations in the plasma and serum of head and neck carcinoma patients have been reported. Among these ones, loss of heterozygosity and microsatellite instability have been described in tumor derived circulating DNA.23, 24 Promoter hypermethylation of key genes in critical pathways appears to be frequent in HNSCC. In this sense, Sanchez-Caspedes et al. and Lopes Bittencourt Rosas et al.25, 26 demonstrated a promoter hypermethylation pattern of p16, MGMT (O6-methylguanine-DNA-methyltransferase) and DAK-kinase (death-associated protein kinase) genes in HNSCC. In many cases, they found the same epigenetic changes in the paired serum25 and saliva26 samples.

Recently, Dietz et al.22 published the results of a pilot study in 26 Stage IV HNSCC patients, where they concluded that serum bFGF levels, but not VEGF nor MMP-2, had prognostic relevance, being independent of other prognostic factors like tumor site, age, total tumor volume, and response to therapy. Van Hal et al.21 reported that plasma levels of CD44v6 were not useful to distinguish between HNSCC and the group of controls.

We did not find any association between the main clinical-pathologic prognostic parameters, such as tumor size, tumor stage, histologic grade and metastatic regional lymph nodes, and the levels of 92-kD MMP activity in the plasma of HNSCC patients, indicating that MMP-9 could be an independent tumor marker. The results did not change when the effects of smoking or alcohol drinking were considered.

The most important factor in the prognostic evaluation of HNSCC is the TNM classification, the lymph node stage being the most relevant. However, it is well known that the same TNM classification for two patients does not always guarantee similar outcome; there is significant prognostic variation within each TNM classification. This has led to propose several categories within each stage with similar outcomes regardless of TNM classification.18 We found that MMP-9 activity behaves independently of this clinical parameter. Probably high amounts of MMP-9 identified some particular biologic characteristics of the cancer cells such as the invasive behavior40 or the ability to metastasize.41 Future research in this field will determine whether plasma MMP-9 activity could be an useful predictor of recurrence free or overall survival. This, in turn, could help to define suitable therapy.

It is important to develop new molecular targets to be used as diagnostic markers, to monitor the response to therapy and as prognostic indicators. As our follow-up study of 60 breast carcinoma patients during 53 months suggested, the levels of plasma 92-kD MMP, measured every 3 months, may be useful to determine the effectiveness of a primary treatment and to predict recurrence of the disease (S.M. Ranucolo, personal communication). We estimate it may be relevant to perform a similar study in a new group of HNSCC patients.

In conclusion, using a specific and reproducible test to quantify the activity of individual MMPs in plasma, we found a remarkable increase of 92-kD MMP activity in the plasma of HNSCC patients, that was independent of the known clinical-pathologic parameters. Further studies are necessary to establish whether this molecule could be a useful tool for the oncologists in managing HNSCC patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors acknowledge the collaboration of the following professionals: L. Adam, R. Adam, L. Califano, A. Gonzalez, R. Pradier, P. Saco, G. Urrutía, and A. Voogd from the Institute of Oncology “Angel H. Roffo,” University of Buenos Aires; C. Cenoz, J. M. Garau, C. Ries Centeno, and V. Thompson from Pirovano Hospital; N. Frascino, O. Gonzalez Aguilar, D. Osimkin, H. Pardo, A. Rossi, A. Rubino, and A. Vamelli from the Oncology Hospital “Marie Curie,” Municipaly of Buenos Aires, and they also acknowledge the interviewers G. Solarz and M. E. Cambas.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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