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Ann Thorac Surg 1997;64:949-953
© 1997 The Society of Thoracic Surgeons

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Original Article: General Thoracic

Loss of v Integrin Expression and Recurrence in Node-Negative Lung Carcinoma

University of Pennsylvania Medical Center Thoracic Oncology Research Laboratory, Division of Cardiothoracic Surgery, Department of Surgery, Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Despite "curative" resection, metastases develop in many patients with node-negative (N0) non–small cell lung carcinoma. Alternative biologic markers for this tumor would be useful. Integrins are cell adhesion molecules that are thought to be important in tumor progression, and expression of these molecules previously has been shown to be altered in non–small cell lung carcinoma. We evaluated alterations in integrin expression and clinical outcome.

Methods. Immunohistochemical staining of tumor specimens was performed, and clinical data were reviewed retrospectively.

Results. Data were complete for 42 patients. Half of all patients (21/42) and 9 of 26 patients with negative nodes experienced tumor recurrence during follow-up. Neither histologic type nor tumor differentiation status correlated with recurrence. However, loss of the v integrin subunit was associated significantly with recurrence in the N0 group. Seventy-five percent of patients with negative nodes who exhibited recurrence lost v expression, compared with only 10% of patients with negative nodes who did not exhibit recurrence (p = 0.012). Alterations of other integrin subunits did not correlate significantly with prognostic follow-up variables.

Conclusions. Loss of v expression may serve as a marker for patients with node-negative non–small cell lung carcinoma who are at high risk for recurrence, potentially directing additional therapies.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
See also page 953.

The surgical staging and treatment of non–small cell lung carcinoma (NSCLC) unequivocally has improved the survival of patients with this common neoplasm, and the determination of the impact of hilar and mediastinal nodal disease on patient survival has engendered more rational treatment algorithms. However, although it is true that the presence or absence of NSCLC nodal spread carries with it significant prognostic data, it cannot predict with any degree of certainty whether patients will experience recurrence. In fact, despite "curative" resection, up to half of all patients at TNM stage I (including those with negative nodes) eventually experience recurrence and die of their disease, with most experiencing distant metastatic failure [1]. It is obvious that additional NSCLC prognostic biologic markers would be useful and could aid in decisions regarding the need for adjuvant and neoadjuvant therapies. For example, although nodal status is considered to be important in the staging of carcinoma of the breast, alternative markers such as hormonal receptor status, proliferative activity, and ploidy level have directed the use of chemotherapy in patients with node-negative disease [2]. The evaluation and implementation of reliable and reproducible biologic markers such as these are lacking for NSCLC, even though a number of possible candidates have been examined, including blood group, angiogenesis, oncogenes such as K-ras and p53, and ploidy levels [3–6].

As with other solid tumors, the growth and metastasis of carcinoma of the lung requires a complex sequence of events [7]. The primary focus of malignant cells must (1) escape local constraints on growth and invade the surrounding pulmonary parenchyma, (2) release from the primary tumor and move into the alveolar or bronchiolar capillaries or lymphatics, (3) survive within the circulation and interact with components of the clotting system and other serum proteins, (4) migrate into a secondary site, and (5) establish metastatic growth [8]. It is reasonable to assume that many, if not all, of these processes require alterations in the ability of lung carcinoma cells to adhere to themselves, normal surrounding cells, or the extracellular matrix.

A large body of experimental evidence suggests that alterations in the expression of one class of cellular adhesion molecules, the integrins, may be involved directly in malignant cellular growth and progression, or at least may serve as a marker of these processes. Integrins are cell surface glycoproteins that arrange themselves as noncovalently bonded alpha () and beta (ß) subunits. These molecules allow for cell–cell as well as cell–extracellular matrix (ie, collagen, fibronectin, vitronectin, fibrinogen, laminin) binding ("adhesion"). The possibility that observed changes in integrin expression may correlate with clinical variables in patients with epithelial malignancies is an attractive one. In this study, we evaluate the prognostic significance of integrin subunit expression in NSCLC.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Antibodies
Monoclonal antibodies (mAbs) recognizing 2, 3, and 5 were obtained from Telios Corporation (La Jolla, CA). Doctor Arnoud Sonnenberg provided the mAb directed against the 6 integrin [9], and Dr David Cheresh [10] provided the mAb LM142, which is directed against the vitronectin receptor subunit v. Doctors Joel Bennett and James Hoxie [11] provided the mAb SSA6, which is directed against platelet glycoprotein IIIa, the ß3 integrin subunit. The anti-ß4 mAb was provided by Dr Stephen Kennel [12]. Doctor Dean Sheppard [13] donated the anti-b6 mAb. Antibodies were diluted in phosphate-buffered saline/4% bovine serum albumin/0.02% azide. Antibodies were titered to obtain strong staining with minimum background. Purified antibodies were used at 5 to 20 mg/mL. Supernatants were used near or at dilutions of up to 1:5.

Tumor Specimens
Fresh tumor specimens were obtained from patients undergoing pulmonary resection and full intraoperative mediastinal nodal sampling for carcinoma of the lung by the staff of the Department of Surgery, Section of General Thoracic Surgery at the University of Pennsylvania Medical Center. The tumor specimens were embedded in OCT compound (Tissue Tek; Miles Diagnostics, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -70°C until sectioning. Thin (5- to 7-µm) sections were cut from the frozen blocks, fixed with cold acetone, and stained with hematoxylin and eosin for conventional histologic examination. Specimens were rejected for further analysis if there was disagreement between histologic diagnosis on comparison between the formal submitted tissue and the study specimen, a histologic diagnosis other than NSCLC, inadequate tumor tissue in the study specimen, or incomplete pathologic data. Tissue procurement was approved by the Institutional Human Studies Committee of the University of Pennsylvania School of Medicine.

Immunohistochemistry
After rehydration and blocking of nonspecific binding sites with appropriate 5% serum in phosphate-buffered saline solution/4% sodium azide, the sections were incubated at room temperature with the first antibody for 60 minutes. Each antibody was titered for optimum staining reactivity. After washing in phosphate-buffered saline solution, the bound primary antibody was detected with the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA) using 3-amino-9-ethylcarbazole as chromogen. No counterstain was applied to increase sensitivity. To determine the level of staining, two of the investigators (W.R.S. and S.M.A.) reviewed the slides independently. Frozen sections of normal human bronchus (areas remote from tumor) and human skin (neonatal foreskin) were used at each staining session as positive controls.

The staining intensity was graded on a three-point scale as no staining, weak staining (generalized weak staining or less than 50% of tumor cells staining strongly), or strong staining (uniform strong staining in 50% to 100% of tumor cells), taking the negative control and the most intensely stained section of the same series as the end points of the scale. All tests with negative staining results were repeated and, where available, normal human lung (taken from areas remote from tumor) and foreskin tissue (harvested at circumcision) were used as internal staining quality controls. Specimens were restained in cases where control tissue staining patterns appeared to be abnormal. Photographs of the specimens were taken by an Olympus (Lake Success, NY) Photomicroscope using Kodak (Rochester, NY) T-MAX 100 color slide film.

Clinical Follow-up
Patient follow-up data were obtained retrospectively from inpatient and clinical chart review, as well as direct communication with referring physicians and, when necessary, patients and patients' families. Information regarding tumor differentiation, histologic type, TNM stage, and specific nodal status were obtained by review of the formal pathology report. Hilar (N1) and mediastinal (N2) nodal groups were combined as N+. Recurrence was defined as radiographic or histologic documentation of new tumor lesions at any site (local or distant). Survival status, tumor-free survival, and cause and date of death were documented. Patients were disqualified from the study if any of these variables were incomplete, or could not be documented reliably.

Statistics
Integrin expression was scored as "downregulated" if the staining level was lower than that seen in normal bronchus. Integrin expression was scored as "upregulated" if the staining level was higher than that seen in normal bronchus. Continuous data in nominal categories were compared by ² analysis, with Fisher's exact test used where appropriate. A significance level of p = 0.05 was chosen. Statistical calculations were performed on a Macintosh IIsi personal computer (Apple Computer, Inc, Cupertino, CA), and Statview software (Abacus Concepts, Berkeley, CA) was used.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
General Patient and Specimen Characteristics
Of 51 original tumor specimens evaluated by immunohistochemistry [14], complete follow-up clinical data were available for 42 patients. Complete histopathologic characteristics were available in 40 of these specimens, with 29 adenocarcinomas and 11 squamous cell tumors identified. Differentiation was moderate to good in 14 specimens and poor in the remaining 26. Twenty-six specimens were staged as node-negative (N0) and 16 as node-positive (N1 or N2). Mean follow-up for all patients (including all those deceased as well as surviving up to this time) was 29.86 months.

Integrin Immunohistochemistry
The detailed integrin staining profile of all specimens has been reported previously. In brief, the normal bronchus was found to exhibit strong staining of 2, 3, 6, v, and ß4 (Fig 1). Negative staining was noted for 5, ß3, and ß6 integrin. A general downregulation of most collagen-laminin binding integrins, as well as the fibronectin-fibrinogen-vitronectin binding integrin v, was noted in tumors compared with normal bronchus. However, when these findings were correlated with nodal status (N0 or N+), no significant differences were observed in regard to the percentage that exhibited changes in integrin expression [14].

View larger version (144K): [in this window] [in a new window]   Fig 1. . Photomicrograph of normal bronchial epithelium with underlying non–small cell lung carcinoma (adenocarcinoma). Note strong (red) immunostaining with v monoclonal antibody at both the progenitor bronchial epithelium and the tumor cells. (NBE = normal bronchial epithelium; S = submucosal tissue; TC = tumor cells.) (x100 before 40% reduction.)

A significant correlation between loss of integrin v immunostaining and the presence of recurrent disease in patients with node-negative disease during the follow-up period was observed. Specifically, 75% (6/9) of patients with negative nodes who experienced NSCLC recurrence exhibited a loss of v immunostaining, compared with only 10% (1/10) of those who did not experience recurrence (p = 0.012 by Fisher's exact test) (Fig 2).

View larger version (12K): [in this window] [in a new window]   Fig 2. . Percentage of non–small cell lung carcinoma specimens from patients with negative nodes that exhibited downregulation or complete loss of v expression versus the recurrence of non–small cell lung carcinoma during the clinical follow-up period.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

Despite our best efforts at surgical and combined therapy, NSCLC continues to be a disturbingly common and relatively untreatable disease. This neoplasm is now responsible for approximately one third of all male and one fifth of all female cancer deaths in this country [15]. Perhaps more alarming is the fact that although most patients with NSCLC present with advanced disease, even those with aggressively treated lower-stage lesions succumb to the tumor 30% to 50% of the time [1]. The current TNM nodal staging system first proposed and clinically validated by Mountain [16] has added much to our ability to predict patient outcome and tailor therapy. However, the fact that so many patients with only hilar metastases (N1) or no nodal metastases at all (N0) eventually do so poorly suggests that attempts to identify additional biologic markers of prognosis are warranted.

In an earlier study, we found no correlation of integrin expression with mediastinal or hilar nodal metastasis [14], even though several other studies have demonstrated such a relation in other epithelial tumor types [17]. Other groups have examined integrin expression in NSCLC, albeit in smaller numbers. Damjanovich and colleagues [18] noted strong v subunit staining of normal bronchial epithelium, and they also noted a trend toward downregulation of this integrin in the 11 NSCLC tumor specimens they analyzed. A few other investigators have evaluated integrin expression in NSCLC; however, either the number of tumors studied has been small or immunostaining for the v integrin subunit has not been performed [19, 20]. Most importantly, those studies did not examine the correlation of integrin expression with either short-term (nodal status, differentiation) or long-term (tumor recurrence, survival) clinical variables.

Why would diminished expression of the v integrin subunit portend a poorer prognosis in patients with NSCLC? In contrast to our findings, several groups have observed a correlation between increased, rather than decreased, expression of v and tumorigenesis. Danen and associates [21] demonstrated a gain in vß3 integrin heterodimer expression related to in situ tumor progression in an animal model of melanoma. These effects may be due to increased tumor angiogenesis, because others have shown that v expression is linked to the expression of vascular endothelial growth factors, and also that angiogenesis and neointimal hyperplasia may be blocked by monoclonal antibodies and blocking polypeptides directed against vß3 [22]. Perhaps integrin heterodimers other than vß3 are operative in this study's findings. In addition to the observation of increased expression of vß3 and its positive relation to melanoma tumorigenesis, expression of the vß5 integrin heterodimer was found to be downregulated in the study by Danen and associates [21]. In another report examining the relation between integrin expression and nodal status in breast carcinoma, loss of expression of both v and ß5 were associated with positive nodal spread [17]. We did not evaluate expression of the ß5 subunit in this study; however, it is possible that the heterodimer that is downregulated in patients with node-negative NSCLC is vß5. In an earlier study by Freidrichs and co-workers [23], which correlated 6 expression with poorer survival in patients with breast cancer, and in this study, integrin expression has been shown to correlate with important clinical outcome variables. Although previous studies evaluating adjuvant therapy for stage I NSCLC have not shown a significant treatment benefit [23], one theoretically could argue that because of the lack of reliable prognostic markers for these patients, those who were most likely to benefit were not identified.

It is important to note that immunohistochemical techniques in studies such as this have both advantages and disadvantages. Changes in levels of integrin protein expression at the cell surface as detected by mAbs could result from any number of steps in protein synthesis, including alterations in gene transcription, translation, posttranslational protein processing, protein packaging, and intracellular transport. It is obvious that further study is required both to validate these findings in larger numbers of patients and to determine the basic molecular or cellular mechanisms involved in altered integrin expression.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This project was funded in part by a development grant from the University of Pennsylvania Cancer Center. Doctor Smythe was the recipient of an American Cancer Society Clinical Fellowship Award. We thank Ms Mildred Daise for her excellent technical assistance.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Presented at the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997.

Address reprint requests to Dr Smythe, University of Pennsylvania Medical Center, 3400 Spruce St, Philadelphia, PA 19104.

	References

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