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Expression and Prognostic Significance of Metalloproteinases and Their Tissue Inhibitors in Patients With Small-Cell Lung Cancer

From the Division of Medical Oncology and Hematology, Departments of Medical Biophysics, Laboratory Medicine and Pathobiology, and Biostatistics, Princess Margaret Hospital/Ontario Cancer Institute, The Toronto Hospital, and the University of Toronto, Toronto, Ontario, Canada.

Address reprint requests to F.A. Shepherd, MD, Division of Hematology Oncology, Princess Margaret Hospital, 5-104, 610 University Ave, Toronto, Ontario, Canada M5G 2M9; email fshepherd{at}torhosp .toronto.on.ca.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in tumor development and progression. MMP expression has been correlated with advanced clinical stage and poor survival in some tumors, but data for small-cell lung cancer (SCLC) are lacking. The aim of this study was to assess the expression of MMPs and TIMPs in SCLC and to evaluate their importance relative to standard prognostic factors.

PATIENTS AND METHODS: Expression of MMP-1, -2, -3, -9, -11, -13, and -14 and TIMP-1, -2, -3, and -4 was evaluated by immunohistochemistry (IHC). In situ hybridization was used to confirm expression of specific mRNAs. Clinical data collected included sex, tumor stage, performance status, weight loss, hematology (hemoglobin, WBC, platelets) and biochemistry (sodium, albumin, alkaline phosphatase, lactate dehydrogenase), treatment, and survival.

RESULTS: Samples from 46 patients were evaluated: 30 males, 16 females; 29 limited, 17 extensive stage; 35 Eastern Cooperative Oncology Group performance status 0-1. Positive IHC staining was evident for MMP-1 and -9 in 60% to 70% of tumor cells, and for MMP-11, -13, and -14 and TIMP-2 and -3 in 70% to 100% of tumor cells. Stromal staining of TIMP-1 to -3 was present in less than 30% of specimens. On multivariate analysis, only stage and decreased tumoral expression of TIMP-1 were significant for response (P = .043). Significant factors for survival were tumor stage (P = .0021); weight loss (P = .013); and high tumor cell expression of MMP-3 (P = .077), MMP-11 (P = .031), and MMP-14 (P = .019). MMP and TIMP expression did not differ significantly between stages.

CONCLUSION: MMPs and TIMPs are widely expressed in SCLC. Increased tumoral expression of MMP-3, -11, and -14 were independent negative prognostic factors for survival. The results support the evaluation of synthetic MMP inhibitors in patients with SCLC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE NEOPLASTIC CELL, by definition, has the ability to disseminate from its site of origin and to form metastatic colonies at distant sites. This activity depends on its capacity to penetrate the extracellular matrix (ECM) and the basement membrane and to induce angiogenesis. Tumor cells directly secrete or induce host stromal cells to elaborate proteinases that catalyze the degradation of the ECM, thereby facilitating the metastatic process.¹

Matrix metalloproteinases (MMPs) comprise the principal proteinase group involved in ECM degradation.² To date, 16 members of the family, which share common structural and functional characteristics, have been identified, and they have been classified into three broad groups based on their substrate specificities.³ MMPs are specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs).⁴ Four members of the TIMP family have been isolated, with variation in the specificity and affinity they exhibit for each MMP.^3,4

There is strong experimental evidence that implicates MMPs in the neoplastic process.^3,5,6 Tumor cell lines both in vitro and in experimental animal models have been shown to produce MMP-2 and -9.^7,8 MMPs have been detected in the serum, plasma, and urine of cancer patients and in human cancer tissues.⁵ Increased expression, including protein and mRNA of MMP-2, -9 and -11, have been detected in numerous solid organ types.^9-11 Their level of expression has been correlated with tumor agressiveness, as implied by increased histologic grade,^9,12 advanced clinical stage,^13-15 poor patient survival in gastric¹⁶ and lung adenocarcinoma,¹⁴ and increased relapse rate in colorectal cancer.¹⁷ Thus, in general, the expression of the various families of MMPs and the relative expression of any individual MMP increases with advancing tumor stage, and the level of overexpression correlates with clinical aggressiveness.³

Small-cell lung cancer (SCLC) is an aggressive malignancy. Systemic chemotherapy results in a significant improvement in median survival, but long-term cure is seldom achieved.¹⁸ Based largely on in vitro studies that document the importance of MMPs in tumor invasion and angiogenesis in other tumor types, clinical trials with synthetic MMP inhibitors have commenced in SCLC.¹⁹ However, there has been little evaluation of the expression of MMPs and TIMPs in SCLC,^11,20-23 nor have attempts been made to determine their prognostic significance in this malignancy. Thus, the basis to support the clinical use of these inhibitors in SCLC has not been established. For these reasons, we undertook this study to assess the cellular expression of MMPs and TIMPs and to correlate their expression with tumor stage, patient response, and survival in SCLC.

	PATIENTS AND METHODS

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Patients
Patients with SCLC were identified retrospectively from the database of The Toronto Hospital (Toronto, Ontario, Canada). Patients with adequate histologic material (obtained in the majority of cases by mediastinoscopy) and complete clinical data were subsequently entered onto the study. The following baseline data were obtained from patient charts: Eastern Cooperative Oncology Group (ECOG) performance status, history of weight loss, tumor stage, sites of metastases, baseline hemoglobin level, WBC and platelet counts, and biochemistry (serum sodium, albumin, alkaline phosphatase, lactate dehydrogenase). The type of systemic therapy, clinical response, and survival time were also recorded. The last date for follow-up was August 21, 1997.

Immunohistochemistry
Paraffin-embedded tissue specimens used for all immunohistochemistry (IHC) and in situ hybridization studies were retrieved from the Department of Pathobiology at The Toronto Hospital. Rabbit polyclonal antibodies to MMP-1, -2, -3, -9, -11, -13, and -14 and TIMP-1, -2, -3, and -4 were obtained from Triple Point Biologics (Forest Grove, OR). A second MMP-2 monoclonal antibody was also obtained from Oncogene Research Products (Calbiochem; Cambridge, MA). IHC was performed by the routine indirect peroxidase-antiperoxidase method.²⁴ Preliminary assessment was made after microwave antigen release.

The final dilution of each antibody was determined after preliminary serial dilution studies. These were chosen to yield an intermediate level of positive staining of bronchial epithelium in control slides of normal lung sections. Tumors that showed relatively diffuse or positive staining in tumor cells were scored as positive, whereas those that showed no or focal (< 10% of tumor cells) staining were scored as negative. The IHC scoring was performed independent of the knowledge of clinical and survival data. Stromal staining of TIMPs was assessed in fibroblasts and collagen stroma.

In Situ Hybridization
Preparation of the human TIMP-1 and -2 and MMP-1, -2, -9, and -11 cDNAs have been described previously.²¹ A 200-base pair fragment of the human TIMP-3 cDNA was generated by polymerase chain reaction.

Digoxygenin (DIG)-labeled riboprobes were prepared as described by the manufacturer (Boehringer Mannheim, Laval, Quebec, Canada) and the in situ hybridizations were performed as described previously.²⁵ Tissue specimens were dewaxed, incubated in 20 µg/mL proteinase K at room temperature for 5 minutes, washed, and acetylated by incubation in a mixture of 0.1 mol/L triethanolamine and 0.56% (vol/vol) acetic anhydride. Tissues were then prehybridized and hybridized with strand-specific DIG-labeled riboprobes for 16 hours. Sections were then washed and treated with anti-DIG antibody (Boehringer Mannheim). The sections were subsequently treated with color development solution according to vendors' instructions. Sections were dehydrated through an ethanol series, cleared by washing with xylene, and coverslipped.

Statistical Analysis
The Cox proportional hazards model was used in two steps to assess the prognostic value of the clinical, laboratory, and IHC values. First, the clinical factors and laboratory values were considered. Stepwise selection techniques were used to determine which of these were significant at P < .05. In the second step, the clinical and laboratory values considered significant in the first step were kept in the model, and the IHC values for each MMP and TIMP were then added one by one. The clinical factors considered were tumor stage (extensive v limited), ECOG performance status (0-1 v 2-3), age (continuous), and weight loss (yes v no). The hematologic and biochemical parameters were considered as continuous variables. The IHC variables were MMP-1, -3, -9, -11, -13, and -14 and TIMP-1, -2, -3, and -4 (tumoral and stromal staining).

Survival was measured from the date of diagnosis to the date of death or last follow-up before study closure. The Kaplan-Meier product limit method was used to estimate the overall survival for the group and to illustrate the effect of each of the variables on survival. The log-rank test was used to find the difference between the curves. When the variable was continuous, the population was categorized in two groups based on the known clinical cutoff point (abnormal v normal); otherwise, the median was chosen as the cutoff point.

The logistic regression model was used to analyze the prognostic factors for response. The same technique used for survival end points was used to choose the significant clinical factors and test IHC variables for significance.

	RESULTS

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Patient Population
Complete clinical, laboratory, and IHC data were obtained for 46 patients with SCLC who presented to the Division of Medical Oncology at The Toronto Hospital from March 1982 to August 1996. Patient characteristics are listed in Table 1. Because the pathologic material was predominantly from mediastinoscopy procedures, the majority of patients (29 of 46) had limited disease, and 76% had an ECOG performance status of 0 to 1. Twenty-six percent of patients were hyponatremic, 37% had elevated lactate dehydrogenase levels, and 24% had an elevated leukocyte count (Table 1).

The systemic chemotherapy regimens administered and the results of treatment are listed in Table 2. All but nine patients received cisplatin-based chemotherapy. The overall response rate was 76%, with complete and partial response rates of 46% and 30%, respectively. At the closing date, only four of 46 patients were still alive. The median survival time for the group was 1.13 years, and the 2-year survival rate was 14% (Fig 1).

View larger version (9K): [in this window] [in a new window]   Fig 1. Overall survival curve of patients with SCLC (n = 46).

Distribution of MMP and TIMP Expression by IHC
IHC staining results for the MMPs and TIMPs are listed in Table 3. Tumoral expression of MMPs was common, and all except MMP-3 were expressed in more than 50% of specimens. MMP-13 and -14 expression was noted in more than 80% of cases. Tumoral expression of MMP-2 was not found, despite the use of two commercially available anti–MMP-2 antibodies. Tumoral expression of TIMP-2 and -3 was found in more than 70% of the specimens. Although we noted positive staining in reactive fibroblasts and infiltrating lymphocytes for some MMPs, stromal cell expression was not specifically evaluated and quantitated. Stromal cell expression of TIMP-3 and -4 was found in less than 40% of cases and of TIMP-1 and -2 in less than 20%.

Statistical analysis failed to identify significant coexpression between the individual MMPs or the MMPs and TIMPs. There was no statistically significant difference in the expression of MMPs and TIMPs based on limited or extensive stage.

In Situ Hybridization
Initially, the use of a probe for abundant small nuclear RNA confirmed the feasibility of performing in situ hybridization on the formalin-fixed tissue and optimized proteinase K digestion conditions. In situ hybridization primarily was used to validate the presence of individual MMP and TIMP mRNAs and to identify cell types that expressed these genes in SCLC specimens. Therefore, normal lung tissue and a limited number of specimens with a moderate to high level of IHC signal were subjected to DIG-labeled MMP-9 and -14 and TIMP-1 and -2 riboprobes. Signals were detected with antisense riboprobes, and the lack of signal on the adjacent sections with the sense riboprobe verified the specificity of the signal.

Prognostic Factor Analysis
On univariate analysis, the significant clinical factors that adversely affected survival were extensive stage (P = .004), failure to achieve complete response (P = .0012), and pretreatment weight loss (P = .033). The laboratory factors predictive for poor survival were hypoalbuminemia (P = .0471) and hyponatremia (P = .003). Tumoral expression of MMP-11 (P = .025) and MMP-14 (P = .042) were also significant negative prognostic factors on univariate analysis (Table 4; Fig 2).

View larger version (21K): [in this window] [in a new window]   Fig 2. (A) Overall survival curve of patients with SCLC expressing MMP-11 (— —; n = 36) relative to nonexpression (· · · · ·; n = 10). (B) Overall survival curve of patients with SCLC expressing MMP-14 (— —; n = 37) relative to nonexpression (· · · · ·; n = 8).

Clinical, laboratory, and IHC factors were also assessed for their prognostic influence on survival by multivariate analysis (Table 4). The same adverse clinical factors held as for univariate analysis: extensive stage (P = .0021; risk ratio, 2.92; 95% confidence interval [CIR, 1.47 to 5.77), and weight loss (P = .0134; risk ratio, 2.32; 95% CI, 1.19 to 4.53). For the IHC variables, the tumoral expression of MMP-11 (P = .0315; risk ratio, 2.97) and MMP-14 (P = .0191; risk ratio, 3.93) persisted as significant independent negative prognostic factors when accounting for these clinical factors. The tumoral expression of MMP-3 reached borderline significance (P = .0767). The tumoral and stromal expression of TIMP-1, -2, -3, and -4 had no significant influence on survival.

For patients who achieved a complete or partial response (35 of 46), on multivariate analysis, hyponatremia (P = .0027) was a significant negative prognostic factor for survival. The tumoral expression of MMP-11 (P = .0545) and MMP-14 (P = .0702) was of borderline significance for poor survival, thus showing a tendency similar to that for the entire population.

Multivariate analysis was also used to determine prognostic factors significant for response to systemic therapy. Of the clinical factors associated with response, tumor stage was found to be significant, with limited stage associated with greater likelihood of achieving response (P = .0432). Decreased tumoral expression of TIMP-1 was a significant independent factor associated with the likelihood of achieving a complete or partial response (P = .0351; odds ratio, 0.19; 95% CI, 0.04 to 0.89).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
SCLC is an aggressive and highly invasive malignancy, and the majority of patients present with metastatic disease.¹⁸ Combination chemotherapy results in response rates of 70% to 80%, although prolonged survival remains elusive.^18,26 This study shows extensive expression of MMPs and TIMPs in human SCLC and is the first to document that tumoral expression of MMP-11 and -14 is an independent negative prognostic factor for survival, while accounting for accepted clinical and laboratory factors.

MMP and TIMP expression in SCLC has been sparsely reported. In the largest series from Japan, 15 SCLC specimens were assessed by IHC for MMP-2 and -7 and TIMP-2 as part of a wider assessment of lung neoplasms.²³ In SCLC specimens, high-level expression in tumor cells was noted for MMP-2 (42%), TIMP-2 (60%), and MMP-7 (13%). MMP-9, -11, and -14 and TIMP-2 mRNA have also been detected in SCLC tissues by other investigators,^21,22 although Rouyer et al,¹¹ did not detect the expression of the MMP-11 gene in any of nine human small-cell carcinomas examined.

Our study represents a more comprehensive evaluation of MMP and TIMP expression in this malignancy. Despite the use of two commercially available antibodies to MMP-2, we failed to detect MMP-2 expression in any of the 45 specimens assessed. However, the same antibodies detected positive staining for MMP-2 in 40% of primary non-SCLC cases studied in our laboratory (Liu et al, manuscript submitted for publication). MMP-2 may be more specific for non-SCLC than SCLC. Alternatively, the lack of detection in the SCLC sections may represent either the poor sensitivity of the antibodies used or heterogeneity of expression within the tumor specimens examined.²⁷

In contrast to reports of MMP expression in other tumor types,^13-15 we found no significant difference in the spectrum of MMP and TIMP expression between limited and extensive stage. This may be a function of the excess number of patients with limited disease in this cohort, or alternatively may reflect a similar biology between the two stages.

A major aim of this study was to assess the prognostic importance of MMP and TIMP expression on survival and response to therapy, while accounting for generally accepted significant clinical and laboratory factors. The latter have been well documented by several large analyses.^26,28-32 An evaluation of MMP and TIMP expression and their prognostic significance in this malignancy is timely and extremely relevant given the use of synthetic MMP inhibitors in clinical trials.^33-36 Phase III trials have now commenced in various malignancies, including at least two trials of adjuvant treatment after response to first-line therapy in SCLC.

In our univariate analysis, extensive stage and weight loss at presentation, as expected, were significant for poorer survival and indicate that our cohort is representative of SCLC in general. In the multivariate analysis, after accounting for the relevant clinical and laboratory factors, the tumoral expression of both MMP-11 and -14 were both independent negative prognostic factors for survival (P = .0315 and .0191; risk ratios, 2.97 and 3.93, respectively), along with extensive stage and weight loss. The tumoral expression of MMP-3 was of borderline significance. Although there is strong evidence that implicates MMP-11 and -14 in the invasive process, their prognostic significance has been less well examined. Expression of MMP-14 has been correlated with an increased propensity for lymph node metastases in non-SCLC.²² Northern blot and IHC analysis performed on 88 primary bronchopulmonary carcinomas for MMP-2, -11, and -14 and TIMP-1, -2, and -3 showed a significant increase in the expression of MMP-14 with increasing stage (P = .0038).³⁷ The synthetic inhibitors presently studied in clinical trials have varying levels of activity against the currently identified MMPs, and their degree of specificity for the enzymes is also variable. In general, however, most have shown strong activity against MMP-2 and -9, with lesser activity against other MMPs, including MMP-3, -11, and -14.

In the present study, MMPs and TIMPs were also assessed for their prognostic value in terms of response to therapy. Limited stage and a decreased tumoral expression of TIMP-1 were associated with a greater likelihood of response. The latter finding seems to be contradictory, but the exact role of TIMPs is not well understood.⁶ Evidence points to TIMPs retarding tumor development by inhibition of tissue invasion and angiogenesis. Malignant tumors would be expected to have an excess of MMPs relative to TIMPs, and a number of reports support this hypothesis. However, an equal number of reports have shown that malignant tumors have high TIMP levels.⁶ Increased TIMP-1 mRNA expression has been observed in advanced colorectal cancer³⁸ and correlated with poorer survival in non-SCLC.³⁹ In breast cancer cells, TIMP-2 has been shown to be complexed with MMP-14 in the activation of MMP-2.⁴⁰ There is in vitro evidence that TIMPs have a cell-signaling and growth stimulatory role independent of their metalloproteinase inhibition.⁴¹

In conclusion, this study has confirmed the extensive expression of MMPs and TIMPs in specimens of humanSCLC. In multivariate analysis, this study has documented that tumoral expression of MMP-11 and MMP-14 are independent negative prognostic factors for survival. Therefore, these findings are the first to provide support for the current clinical trials of adjuvant synthetic MMP inhibitors in patients with SCLC after response to first-line chemotherapy.

	ACKNOWLEDGMENTS

 
The MMP-13 and -14 cDNAs were provided by Dr S. Apte, Cleveland Clinic Foundation, Cleveland, OH, and the MMP-3 cDNA was provided by Dr L. Matrisian, Vanderbilt University, Nashville, TN.

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Submitted June 17, 1998; accepted February 2, 1999.

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