Ten to 20 per cent of all human malignant primary bone tumors are chondrosarcomas, which are malignant cartilaginous lesions. Chondrosarcomas vary greatly with regard to biological behavior and to their propensity for local recurrence and metastasis. Histological grading traditionally has been used to assess the degree of malignancy of human chondrosarcoma and to predict the prognosis; however, histological grading is somewhat subjective, making it difficult to compare the findings of clinical studies. An objective, biochemical method to determine the degree of malignancy of a human chondrosarcoma (the propensity for recurrence and metastasis) would be of substantial value in the management of these patients3,7,9,12.
The matrix metalloproteinases, a class of enzymes, play an important role in the capacity of tumor cells to transverse tissue boundaries and metastasize, and they are directly involved in the processes of local invasion and metastasis5. Matrix metalloproteinase-1 (interstitial collagenase) degrades type-I, II, III, and X collagen and proteoglycans10,11. Matrix metalloproteinase-2 (seventy-two-kilodalton gelatinase A) degrades type-IV and denatured collagens, and its level is elevated in some malignant tumors11.
Under physiological conditions, remodeling and degradation of tissue are tightly regulated by a balance between the matrix metalloproteinases and the tissue inhibitors of metalloproteinases. Tissue inhibitor of metalloproteinase-1 inhibits matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-2 inhibits matrix metalloproteinase-216. The balance between the activity of the matrix metalloproteinases and that of their tissue inhibitors is directly related to the overall proteolytic activity, invasiveness, and metastatic potential of a particular tumor15,16. Investigators have shown that the expression of a matrix metalloproteinase and its tissue inhibitor is associated with the level of differentiation and the phenotype in human chondrosarcoma cell lines2. The ratio of matrix metalloproteinase to its tissue inhibitor has been related to the prognosis associated with several human malignant tumors, including cervical carcinoma13. Furthermore, it has been postulated that the levels of a matrix metalloproteinase and its tissue inhibitor are phenotypic markers of prognostic value for melanoma and colorectal carcinomas10.
Because matrix metalloproteinases are involved in the ability of a tumor to spread and to metastasize, the measurement of their expression in a tumor may be useful for predicting whether the tumor has the ability to penetrate tissue boundaries. Using reverse transcription-polymerase chain reaction, we demonstrated what we believe to be the first evidence of a relationship between the ratio of mRNA expression of matrix metalloproteinase-1 to that of tissue inhibitor of metalloproteinase-1 and the frequency of local recurrence, metastasis, and survival in human chondrosarcoma.
*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were the American Cancer Society, the Orthopaedic Research and Education Foundation, and Glaxo Wellcome.
†Division of Orthopaedic Surgery (K. R. B., A. P. T., J. M. H., and S. P. S.), Division of Diagnostic Pathology (L. J. L.), and Center for Clinical Effectiveness (L. A. H.), Duke University Medical Center, Durham, North Carolina 27710. E-mail address for Dr. Scully: scull002@mc.duke.edu.
Tumor Specimens
Sixteen paraffin-embedded archival pathological specimens that had been obtained during resections and amputations from 1975 through 1995 were selected from an established chondrosarcoma tumor bank. In selecting the specimens, one of us (L. J. L.) reevaluated the grade and the histological diagnosis of the tumors. The selection criteria included available follow-up records on the patient, details of the operative and adjuvant therapy, and appropriate processing of the resected specimen. In addition, an attempt was made to balance the number of low-grade (grade-1) tumors with the number of high-grade (grade-2 and 3)17 tumors. The clinical outcome and the grade of the tumor were not known to the investigators during the performance of the experiments.
All patients had wide excision of the primary tumor with negative operative margins. Each tumor was examined histologically and was assigned a grade17 at the time of resection. The patients were followed clinically for at least five years or until recurrence. No patient received adjuvant radiation therapy or chemotherapy before recurrence. The grade, histological characteristics, and stage of the tumor17; the status of the patient at the time of the study; and other possible prognostic factors were recorded (Table I). Eight of the sixteen tumors were grade 1, three were grade 2, and five were grade 3. Six patients had a good outcome, as defined by no recurrence or evidence of disease; nine patients died of disease; and one was alive with recurrent chondrosarcoma.
mRNA Isolation
Six sections, twenty micrometers thick, were cut from each paraffin-embedded specimen and placed into individual tubes with one milliliter of lysis buffer (Dynal, Lake Success, New York). Poly A+ mRNA was extracted from the lysate with use of 250 microliters of oligo(dT)25 Dynabeads (Dynal), as specified by the manufacturer. The beads were washed twice in a solution of ten-millimolar Tris-hydrochloric acid (pH 8.0), 150-micromolar lithium chloride, 0.1 per cent lithium dodecyl sulfate, and one-millimolar EDTA, followed by one wash in ten-millimolar Tris-hydrochloric acid (pH 8.0), 150-micromolar lithium chloride, and one-millimolar EDTA (all chemicals from Sigma Chemical, St. Louis, Missouri). Messenger RNA was eluted from the beads in water at 67 degrees Celsius, and reverse transcription was carried out immediately.
Reverse Transcription
First-strand cDNA synthesis was performed in a volume of twenty-microliter reverse-transcription buffer containing Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Division of Roche Molecular Systems, Foster City, California), ribonuclease inhibitor, and deoxynucleotide triphosphate, and oligo(dT)16 was used as a primer in a reaction.
Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction was performed on the sixteen paraffin-embedded operative specimens to evaluate the expression of cDNA for matrix metalloproteinase-1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2. Oligonucleotide primers for matrix metalloproteinases 1 and 2 and their tissue inhibitors were designed with use of RNA sequences available in the GenBank database and a computer program (Primer Premier, version 3.1; Premier Biosoft International, Palo Alto, California). Primers were synthesized by Custom Primers (Palo Alto, California). Gel electrophoresis yielded single bands of the expected product sizes. Product identity was confirmed by restriction enzyme digestion.
Digoxigenin-labeled polymerase chain reaction products were subjected to electrophoresis on 3:1 agarose gels (NuSieve; FMC BioProducts, Rockland, Maine) and transferred to nylon membranes in a ten-times-standard concentration of saline-sodium citrate solution. The membranes were air-dried, and chemiluminescent detection was carried out with use of a Wash and Block Buffer Kit and CSPD substrate (Boehringer Mannheim, Indianapolis, Indiana). The membranes were exposed to film (Eastman Kodak, Rochester, New York) for five hours, and chemiluminescent signals then were detected.
Densitometry and Statistical Analysis
The band intensity on autoradiographs was quantitated by densitometry with use of Image software (Wayne Rasband, Research Services, National Institutes of Health, Bethesda, Maryland). The ratios of the matrix metalloproteinases to their tissue inhibitors were determined for each sample by comparing the band intensity for matrix metalloproteinase-1 with that for tissue inhibitor of metalloproteinase-1 and the band intensity for matrix metalloproteinase-2 with that for tissue inhibitor of metalloproteinase-2. The individual ratios were grouped according to the outcome (disease-free survival or recurrence), and the mean ratios were compared with use of the two-tailed Student t test. Kaplan-Meier curves were plotted to compare the survival of patients who had high ratios with the survival of those who had low ratios. The ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 was defined as high when it was more than 0.8, the value that was approximately one standard deviation from the means for both outcome groups. The ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 was defined as high when it was more than 1.41, which was the median value for the specimens. Prognostic significance was determined with log-rank analysis.
Multifactorial Analysis
The possible prognostic variables that were examined included the size, location, and grade of the tumor; the age and gender of the patient; and the ratios of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 and of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2. Univariate and bivariate statistical analyses were performed with use of the chi-square test, the Fisher exact test, the t test, and analysis of variance. The predictive variables suggested by the bivariate analysis then were used for the stepwise logistic-regression analysis. Decrement to chi-square testing was used to determine the optimum model. Independent significance was calculated with use of logistic-regression analysis, and the 95 per cent confidence intervals were determined. Statistical analyses were performed with use of Statistica software (StatSoft, Tulsa, Oklahoma).
The ratios of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 were calculated from the relative band intensities of cDNA (Fig. 1). The ratio for each tumor was plotted, as were the mean ratios (and standard deviation) for the two outcome groups (patients who had recurrence and those who were free of disease) (Fig. 2-A). The mean ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 was significantly higher (p < 0.003) in patients who had local recurrence or metastasis (0.939 ± 0.148) than in those who were free of disease (0.703 ± 0.070). With the numbers available, we could find no significant difference between the mean ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 in the patients who were free of disease and the mean ratio in those who had recurrence (Fig. 2-B).
Kaplan-Meier survival curves were constructed with use of the ratios of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 (Fig. 3-A). Patients who had a low ratio survived without evidence of disease for significantly longer (p = 0.0015) than patients who had a high ratio (more than 0.8). The survival curves showed a trend toward an association between a higher ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 (more than 1.41) and a worse prognosis (Fig. 3-B), but this was not found to be significant, with the numbers available.
Two of the patients who had a grade-1 neoplasm died of metastatic chondrosarcoma. The ratio of matrix metalloproteinase-1 to its tissue inhibitor was very high (1.058) in one tumor (Case 1) and was low (0.647) in the other (Case 4).
Univariate analysis revealed that the grade of the tumor (p = 0.00048), male gender (p = 0.0013), and a high ratio of matrix metalloproteinase-1 to its tissue inhibitor (p = 0.000086) were possible predictors of outcome. The size and location of the tumor, the age of the patient, and the ratio of matrix metalloproteinase-2 to tissue inhibitor of metalloproteinase-2 were not found to be predictors of outcome (p > 0.1). All of the patients had negative margins after wide or radical excision, so the effect of that variable on recurrence could not be assessed. Stepwise logistic-regression analysis revealed that a high ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 was a moderately significant independent predictor of poor outcome (a = 0.07) and that this ratio was a better model for predicting poor outcome than the grade of the tumor or the grade of the tumor and the ratio (Table II). As expected, the histological grade of the chondrosarcoma also was significant.
The present study demonstrated that a low ratio of mRNA expression of matrix metalloproteinase-1 to that of tissue inhibitor of metalloproteinase-1 was significantly related to disease-free survival of patients who had chondrosarcoma. This relationship may be useful for identifying patients at risk for metastasis and to assess survival of patients who have chondrosarcoma.
Metastasis is a dynamic cascade that begins early in the growth of a primary neoplasm, and it is determined by the ability of tumor cells to invade tissues and escape the compartment of origin by penetrating basement membranes, degrading extracellular matrix, and gaining access to the vascular system8. Central to each event in the cascade is the ability of the cell to actively transgress tissue boundaries. Matrix metalloproteinases and their inhibitors are involved in the degradation and remodeling of tissue in many physiological and pathological conditions, including metastasis4,6,10,18. The expression or overexpression of matrix metalloproteinases has been associated with the invasiveness of many neoplasms such as squamous-cell carcinomas, colorectal tumors, and malignant pulmonary tumors1,10,13,14. Elevated levels of matrix metalloproteinase-2 have been found in primary cancers of the breast, prostate, and colon16. Each of the enzymes in the matrix metalloproteinase family has unique high-specificity substrates on which they act. Matrix metalloproteinase-1 degrades fibrillar collagen (types I, II, and III), type-X collagen, and proteoglycans. Matrix metalloproteinase-2 is active against type-IV, V, VII, X, and XI collagens as well as gelatins and elastin11.
Many authors have postulated that the ratio of matrix metalloproteinases to their endogenous inhibitors is associated with invasion and metastasis1,5,6,13,14,16. The ratios of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 in the tumors in the present study were significantly related to the clinical outcome. In tumors with a high ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1, the increased proteolytic activity against type-I, II, and III collagen and the reduced expression of tissue inhibitor of metalloproteinase-1 could be expected to promote matrix degradation and remodeling, a feature central to the metastasis of chondrosarcoma15. These data suggest that the expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in chondrosarcoma is directly related to the progression of the disease, and the ratios may provide a basis for future grading and staging systems.
In conclusion, the ratio of matrix metalloproteinase-1 to its tissue inhibitor appears to be significantly related to the degree of malignancy, local recurrence, metastasis, and survival in human chondrosarcoma. A high ratio indicates that a tumor is more likely to progress to matrix tissue degradation, local invasion, and distant metastasis. It appears that the activity of matrix metalloproteinase-1 may be more critical than that of matrix metalloproteinase-2 in the progression of these tumors. The histological grade of the tumor also was found to be a significant prognostic indicator of outcome, but current grading systems remain subjective3,7,9. Therefore, the use of the ratio of matrix metalloproteinase-1 to its tissue inhibitor as an objective measure of prognosis may be beneficial12.
The methods used in the present study are rapid and accurate and may be applicable to tissue samples obtained at the time of biopsy. The clinical importance of such techniques should be confirmed with randomized, prospective studies of a larger number of patients. Many investigators have suggested that the modulation of tissue inhibitor of metalloproteinase-1 and, thus, the activity of matrix metalloproteinase-1, could play a role in the treatment of cancer4-6,15. Because chondrosarcoma is considered to be resistant to traditional radiation therapy and chemotherapy, patients who have this type of tumor may benefit from such novel therapeutic approaches. Also, the role that matrix metalloproteinases play in the mechanisms underlying metastasis may be a useful focus for the development of effective adjuvant therapy. Furthermore, measurement of matrix metalloproteinase and tissue inhibitors of metalloproteinase in order to monitor tumor responses to new therapies may be crucial to the development of such novel strategies.
NOTE: The authors thank Jason Ku for generating the Kaplan-Meier plots.