Lung carcinoma is the leading cause of cancer death in the U.S., with a predicted 180,000 deaths in 1997.1 Nonsmall cell lung carcinoma (NSCLC) comprises 70–80% of all lung carcinomas and has a 5-year survival rate of about 15%. Stage I disease (American Joint Committee on Cancer, International Staging System2) is associated with the best prognosis among all stages of NSCLC, but 30–40% of patients in Stage I experience recurrent disease despite complete surgical resection.2–4 Recent trials of Stage III NSCLC have suggested that chemotherapy and radiation added to surgery improves the survival of those patients.5–9 These preliminary studies also suggest that adjuvant chemotherapy and radiotherapy may be effective in reducing the recurrence risk for patients with Stage I disease who are undergoing a complete surgical resection.
Because chemotherapy and radiation add significant toxicity, many investigators are eager to identify prognostic factors for recurrent disease in the Stage I NSCLC population, so that these adjuvant therapies can be selectively applied. Many studies from numerous centers have been published, often with conflicting results, so that it is difficult to distinguish tumor features with true adverse prognostic significance.10, 11 Tumor features of Stage I NSCLC that have been identified in 2 or more large studies (>100 patients) with significant adverse prognostic influence include tumor size, histology types and subtypes, K-ras codon 12 mutation, abnormalities in p53, the presence of nerve cell adhesion molecule expression, DNA ploidy and proliferative activity of the tumors, the presence of neuroendocrine differentiation, HER-2/erb-B expression, blood vessel/lymphatic invasion, angiogenic index, blood group antigen expression, and mitotic index.12–17
Tumor cell metastasis occurs in a multistage process. Local invasion and destruction of extracellular matrix precedes entry of tumor cells into blood vessels, lymphatics, or other transport channels. The tumor cells must survive in the circulation, extravasate out of vascular channels into metastatic sites, and grow in the new location.18–20 Necessary functional attributes of tumor cells include production of angiogenic factors that lead to generation of fragile capillary structures, and elaboration of metalloproteinases and other compounds capable of degrading extracellular matrices.21–23 Moreover, tumor cell shape changes and locomotion are required in several stages of the metastatic process, and increased cell motility has been associated with metastatic potential in animal and human tumors.18, 24–26 However, few studies have investigated the correlation between the expression of intracellular proteins that are important for tumor cell motility and metastatic potential.
This article describes an analysis of the prognostic significance of the expression of three widely expressed proteins involved in the organization and regulation of the actin cytoskeleton in Stage I NSCLC: rac, ABP-280, and gelsolin. Rac is a 21 kDa GTPase and a member of the ras superfamily; it is thought to be a crucial intermediary in signalling events for ruffling and translocational motility.27–30 ABP-280 is a 280 kDa homodimeric protein that cross-links actin filaments into orthogonal networks.31 It is thought to be essential to the actin filament organization in the cell cortex that facilitates efficient locomotion of cultured melanoma cells, and its expression may correlate with invasive behavior of melanocytic tumors.32, 33 Gelsolin is an 80 kDa protein that regulates actin filament organization through complex effects on the dynamics of actin assembly into filaments, including the severing of actin filaments.34, 35 Increased expression of gelsolin in cultured fibroblasts increases motility,36, 37 although its expression is down-regulated in many human carcinomas, including those developing in the breast, colon, and bladder.38–42
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In preliminary studies, we confirmed the specificity of our monoclonal antibodies (data not shown) and examined the expression pattern of the three proteins—rac, ABP-280, and gelsolin—in normal lung (Fig. 1). Pulmonary macrophages and vascular smooth muscle cells showed strong staining for all three proteins. Normal alveolar epithelium showed weak or absent expression of all three proteins, whereas bronchiolar and bronchial epithelium showed high, moderate, and weak expression of rac, ABP-280, and gelsolin, respectively. However, moderate expression of gelsolin was seen in bronchial epithelium in areas of inflammation. In general, tumor cells showed moderate-to-strong expression of ABP-280 and rac (Fig. 2). Gelsolin staining was more variable, with the majority of cases showing absent or weak expression and a minority expressing gelsolin at moderate-to-high levels (Fig. 3). Seventeen percent of blocks showed variable staining for gelsolin, leading to an assignment of an average gelsolin expression grade and a focal gelsolin expression grade (see “Methods” for details).
Figure 1. Expression of rac, ABP-280, and gelsolin is shown in normal lung. Arrows indicate the structures of interest: (a) alveolae; (b) terminal and respiratory bronchioles; and (c) tertiary, (d) secondary, and (e) primary bronchi. All three proteins are highly expressed in macrophages (M) and smooth muscle cells (SM), but weakly or not at all in alveolae. Rac is expressed highly, ABP-280 moderately, and gelsolin weakly in bronchiolar and bronchial epithelia. Heterogeneous expression of all three is seen in mucous glands (G).
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Figure 2. Expression of rac (A–C) and ABP-280 (D–F) is shown in lung cancer sections. M: macrophage. A and D show moderate expression, whereas B, C, E, and F show high expression.
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Figure 3. Expression of gelsolin is shown in lung cancer sections. M: macrophage. A: absent expression; B: moderate expression; C: low or average expression, high focal expression; D: low expression; E: high expression; F: normal bronchial mucosa.
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To explore the prognostic significance of expression of these proteins in NSCLC, we performed a pilot study in which tumors from ≥50 Stage I patients were examined. At least 25 of these patients had experienced recurrent disease, whereas the other 25 had remained cancer free; but they were otherwise chosen randomly. As seen in Table 1, the distribution of rac and ABP-280 expression among those who were and were not long term cancer free survivors was similar. In contrast, focal gelsolin expression was generally lower (P = 0.016) in the subset without recurrent disease compared with the group with cancer recurrence. Therefore, we extended the analysis of gelsolin expression to all tumor blocks available from a cohort of 244 patients with Stage I NSCLC that had been previously studied at our institution.
Table 1. Distribution of Expression of Rac, ABP-280, and Gelsolin in the Pilot Study of 50 or More Tumors
| Cancer free||0||6||7||12||25|
| Cancer relapse||0||1||11||18||30|
| P = 0.162|
| Cancer free||0||3||5||21||29|
| Cancer relapse||0||1||8||16||25|
| P = 0.663|
| Cancer free||8||13||3||1||25|
| Cancer relapse||5||6||9||5||25|
| P = 0.016|
Blocks from 229 patients from this previously studied cohort were available for gelsolin immunostaining, of which 74 had experienced cancer recurrence. In this 229-patient cohort, the median age was 66 years, there were 129 (56%) males and 100 (44%) females, and there were 57 (25%) squamous cell carcinomas, 132 (58%) adenocarcinomas, 28 (12%) large cell carcinomas, 6 (3%) mixed histologies, and 6 (3%) not specified. Considering focal gelsolin expression (Table 2, top), 28% of tumors expressed no gelsolin, 38% had low levels of gelsolin expression, and 34% expressed gelsolin at moderate or high levels. Considering average gelsolin expression (Table 2, bottom), the distribution of levels of expression were similar, but with the expected shift to lower levels of expression. In this larger population, significantly decreased cancer free survival was again associated with increasing focal gelsolin expression (P < 0.0001, Fig. 4A) and with increasing average gelsolin expression (P < 0.0001, Fig. 4B). Focal and average gelsolin expression were significantly correlated with lymphatic invasion (Table 3, P = 0.012 and 0.0065, respectively). Average gelsolin expression was significantly correlated with H-ras p21 expression and bcl-2 expression (Table 3, P = 0.027 and 0.056, respectively). There was no correlation between focal or average gelsolin expression and histology or any other molecular feature examined.
Table 2. Gelsolin Expression in the Entire 229 Patient Cohort
| ||Expression level|
| Cancer free||50 (32%)||64 (41%)||26 (17%)||15 (10%)||155|
| Cancer recurrence||15 (20%)||22 (30%)||20 (27%)||17 (23%)||74|
| Total||65 (28%)||86 (38%)||46 (20%)||32 (14%)||229|
| Cancer free||55 (36%)||70 (45%)||27 (17%)||3 (2%)||155|
| Cancer recurrence||15 (20%)||32 (43%)||17 (23%)||10 (14%)||74|
| Total||70 (31%)||102 (44%)||44 (19%)||13 (6%)||229|
Figure 4. Kaplan–Meier survival estimates for 229 patients are stratified by focal gelsolin expression (A) and average gelsolin expression (B). Tick marks reflect censored survival times for individual patients without cancer recurrence at last follow-up.
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Table 3. Correlation between Gelsolin Expression and Other Tumor Characteristics
| ||Absent||Low||Moderate||High||P valuea|
|Focal gelsolin expression|
| Lymphatic invasion||0.012|
| Absent||47 (72%)||62 (72%)||23 (50%)||16 (50%)|
| Present||18 (28%)||24 (28%)||23 (50%)||16 (50%)|
|Average gelsolin expression|
| Lymphatic invasion||0.0065|
| Absent||49 (70%)||73 (72%)||21 (48%)||5 (38%)|
| Present||21 (30%)||29 (28%)||23 (52%)||8 (62%)|
| H-ras p21 expression||0.027|
| Absent||45 (65%)||76 (76%)||27 (61%)||5 (38%)|
| Present||24 (35%)||24 (24%)||17 (39%)||8 (62%)|
| bcl-2 expression||0.056|
| Absent||49 (71%)||58 (57%)||21 (48%)||6 (46%)|
|20 (29%)||43 (43%)||23 (52%)||7 (54%)|
Gelsolin was then included with 9 other factors, previously identified as conferring prognostic significance in the total population of 244 patients, in a Cox proportional hazards regression model to define independent risk factors for cancer free survival in the current cohort of 229 patients. The other factors included in this model were age >60 years, male gender, wedge resection versus lobectomy/pneumonectomy, adenocarcinoma subtype solid tumor with mucin, tumor dimension >4 cm, lymphatic invasion, p53 expression, K-ras codon 12 mutation, and lack of H-ras p21 expression. High focal gelsolin expression, seen in 32 patients (14%), compared with low or absent focal gelsolin expression, was found to carry the highest relative risk of cancer recurrence (4.04) among all measures tested (Table 4, top), and was the strongest independent risk factor for cancer recurrence. Moderate focal gelsolin expression, seen in 46 patients (20%), was also an independent risk factor compared with low or absent gelsolin expression, but at a lower risk ratio (2.26). Similar results were obtained considering average gelsolin expression levels (Table 4, bottom), for which the relative risk of cancer recurrence was 6.28 for high or average gelsolin expression compared with low or absent expression, and 1.88 for moderate compared with low or absent expression. Similar results were seen considering overall survival (Table 4, right lanes), and restricting the analysis to the subset of patients who had undergone a lobectomy or pneumonectomy.
Table 4. Multivariate Analysis of Independent Factors Associated with an Increased Risk of Cancer Recurrence in 229 Patients, Including Focal Gelsolin Expression (top half) and Including Average Gelsolin Expression (bottom half) (in Order of Relative Risk)
|Cox proportional hazards model||Cancer free survival analysis||Overall survival analysis|
|Variable||P value||Risk ratio||Lowera||High||Risk ratio|
|Focal gelsolin high vs. absent/low||0.0001||4.04||2.14||7.63||3.49|
| Wedge vs. lobectomy/pneumonectomy||0.0001||2.93||1.68||5.11||2.44|
| Solid tumor adenocarcinoma||0.0022||2.68||1.43||5.02||2.17|
| Low H-ras p21 expression||0.0042||2.32||1.30||4.12||1.98|
| Focal gelsolin moderate vs. absent/low||0.0074||2.26||1.24||4.09||1.85|
| Maximum tumor dimension ≥4 cm||0.035||1.89||1.05||3.43||1.74|
| Lymphatic invasion||0.013||1.86||1.14||3.05||1.79|
| K-ras codon 12 mutation||0.020||1.86||1.10||3.13||2.04|
| Age ≥60 years||0.071||1.77||0.95||3.30||2.11|
| p53 expression||0.029||1.74||1.06||2.85||1.41|
| Male gender||0.067||1.62||0.97||2.72||2.16|
|Average gelsolin high vs. absent/low||0.0001||6.28||2.76||14.32||5.15|
| Low H-ras p21 expression||0.0020||2.65||1.43||4.93||2.27|
| Wedge vs. lobectomy/pneumonectomy||0.0015||2.48||1.42||4.34||2.16|
| Solid tumor adenocarcinoma||0.0078||2.39||1.26||4.53||1.95|
| Lymphatic invasion||0.0094||1.92||1.17||3.15||1.81|
| Average gelsolin moderate vs. absent/low||0.037||1.88||1.04||3.41||2.05|
| Maximum tumor dimension ≥4 cm||0.051||1.81||1.00||3.28||1.69|
| Male gender||0.029||1.79||1.06||3.02||2.37|
| Age ≥60 years||0.077||1.75||0.95||3.24||1.93|
| K-ras codon 12 mutation||0.045||1.70||1.01||2.85||1.88|
| p53 expression||0.038||1.68||1.03||2.75||1.38|
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Tumor cell motility has long been recognized as an important malignant characteristic that facilitates metastatic progression of tumors both in vivo and in animal model systems.18, 20, 24–26, 46 Cell motility is a complex process involving multiple proteins that interact with and modulate the cortical actin network to induce its coordinated disassembly and reformation under tight spatiotemporal control.47, 48
We examined the expression of three proteins in early stage NSCLC that modulate the organization of the actin cytoskeleton, participating in the motility of multiple cell types. Rac and ABP-280 were expressed at appreciable levels in all tumor blocks examined, perhaps indicative of their important role in cell organization in all lung tumors. There was no correlation between low, moderate, or high level expression of these proteins and clinical outcome in a small pilot study. Gelsolin expression, on the other hand, was found to vary widely among the lung tumors examined and did not correlate with either rac or ABP-280 expression (data not shown). About one-third of lung tumor blocks showed no gelsolin expression, and somewhat more showed low gelsolin expression. The remainder showed significant gelsolin expression that was clearly increased compared with that seen in normal lung mucosa or normal stroma cells, and a fraction of those (14%) showed expression at relatively high levels.
We found that high level expression of gelsolin conferred a significantly worse prognosis on the patients from whom such tumors were removed, and that high level gelsolin expression conferred the highest risk of cancer relapse among all molecular, clinical, and pathologic features evaluated in this population (22 distinct features).43 Although the number of patients studied here is substantial, these findings should be confirmed by other independent studies in well-characterized Stage I NSCLC populations.
Gelsolin is a multifunctional actin-modulating protein, which is activated by micromolar Ca2+ or lowered pH (<6.5) and inactivated by polyphosphoinositides, and can nucleate (accelerate) actin filament assembly, sever actin filaments, and cap the barbed end of filaments.35, 49, 50 The importance of high gelsolin expression as an indicator of recurrence risk in Stage I NSCLC is consistent with in vitro studies in which gelsolin expression in fibroblasts has been positively correlated with translocational motility, ruffling activity, and pinocytosis.36, 37, 51 Gelsolin has also been identified as a substrate for caspace-3, an aspartate specific protease activated during apoptosis.52 The gelsolin cleavage product is constitutively activated for severing actin filaments and mediates, in part, the morphologic changes of apoptosis in some cell types. However, in Jurkat cells gelsolin also appears to function as an inhibitor of apoptosis by an undefined mechanism.53 Thus, the role of gelsolin in apoptosis may also contribute to its prognostic significance in NSCLC. However, we suspect that it is gelsolin's role in motility that causes NSCLC tumors with high gelsolin expression to be associated with a poorer prognosis. The finding that both focal and average gelsolin expression correlated with lymphatic invasion is consistent with enhanced motility in the gelsolin-expressing lung carcinoma cell.
Loss of gelsolin expression has been observed during bladder, breast, lung, and colon carcinogenesis, both in cell lines and through immunohistochemical analysis of human tumors.54–57 Although frequent, loss of gelsolin is not universal in cancers from these tissues. Gelsolin transfection into transformed NIH 3T3 cell lines and bladder carcinoma cell lines leads to a reversion of transformation in assays in vitro and in limited studies in vivo.54, 58 However, multiple other cytoskeletal and other proteins, when expressed in the NIH3T3 model system, also have tumor suppression properties.59, 60 Although the majority of the lung tumors examined here had no or low gelsolin expression, consistent with these previous studies in other epithelial cell malignancies54–56 and lung carcinoma,57 a significant fraction expressed gelsolin at high levels and had a generally worse prognosis. It is difficult to reconcile all of these observations into a simple model for the action of gelsolin during malignant epithelial cell transformation. We hypothesize that gelsolin expression is generally extinguished during transformation, but in some tumors later events lead to and permit its expression, and in those tumors it facilitates metastasis by increasing tumor cell motility.
Although tumor cell motility is recognized as an important contributor to the multistep metastatic process, relatively little investigation has been carried out regarding its importance as a prognostic factor. We propose that surrogate measures of tumor cell motility, such as high gelsolin expression, may have broad prognostic significance to human cancer. This concept is supported by recent studies indicating the importance of thymosin β15 expression in prostate carcinoma metastasis.61