Ann Thorac Surg 1995;59:1069-1073
© 1995 The Society of Thoracic Surgeons
Immunostains for Blood Group Antigens Lack Prognostic Significance in T1 Lung Carcinoma
Carolyn M. Dresler, MD,
Jon H. Ritter, MD,
Mark R. Wick, MD,
Charles L. Roper, MD,
G. Alexander Patterson, MD,
Joel D. Cooper, MD
Divisions of Cardiothoracic Surgery and Anatomic Pathology, Washington University School of Medicine, St. Louis, Missouri
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Abstract
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Recent reports have suggested that the retention of blood group antigen expression on tumor cells may be an important prognostic factor for survival. From 1986 to 1991, 136 patients underwent operative resection for their T1 N0 non–small cell lung carcinoma. One hundred twenty tissue blocks were available for antigen testing, and the histologic types were as follows: adenocarcinoma (73 patients), squamous cell (39 patients), large cell/undifferentiated (7 patients), and mucoepidermoid (1 patient). Follow-up is complete for all patients (mean, 41 months). This distribution of patients among the blood groups was as follows: A, 56 (47%); O, 53 (44%); B, 9 (7.5%), and AB, 2 (1.7%). Immunostaining was performed for A, B, and H blood group antigens. The 5-year actuarial survival in the blood group A patients (53%) did not differ significantly from that in the blood group O patients (59%). Similarly, when tumors were examined for their respective antigens, no significant differences were found in the 5-year survival of either blood group A or O patients between the tumors that retain and those that lose blood group antigen expression. Retention or loss of blood groups A or O antigen expression does not predict survival in patients with early-stage lung carcinomas.
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Introduction
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Survival outcome in patients with non–small cell carcinoma of the lung is largely determined by the extent of disease. Complete surgical excision may be curative in selected patients. Accurate staging and the determination of resectability are critical in identifying those patients eligible for complete resection and potential cure. Nonetheless, treatment fails in some patients with limited disease who undergo seemingly curative surgical excision. Prognostic markers that would identify such patients prospectively may be of value so that more intensive adjuvant therapy can be given in those patients at higher risk for recurrent disease.
Blood group isoantigens of the ABH system are represented by a variety of glycoproteins and glycolipids, whose antigenic specificity is determined by variation in their constituent carbohydrate chains [1]. These determinants are expressed on red blood cells, endothelial cells, and many normal epithelial cells [1–4]. ABH antigens retain their reactivity in paraffin-embedded tissue, and thus the development of antibodies specific for these antigens has allowed for their subsequent detection by immunohistochemical testing. Previous investigations have shown that the pattern of expression of these isoantigens in normal tissues varies from the pattern in neoplastic tissues. The prognostic significance of a loss of or altered ABH antigen expression has been suggested for carcinomas in a variety of sites, including the urinary bladder and gastrointestinal tract [5–12].
In a recent report Lee and colleagues [13] demonstrated the prognostic significance of ABH expression in patients with non–small cell lung carcinoma. Patients whose tumors maintained the expression of blood group A isoantigens fared significantly better than did those with isoantigens of other blood types, or those with tumors that exhibited aberrant antigen expression. The 164 patients in Lee's report were in various stages of disease and were distributed among the four blood groups. It is difficult to extrapolate these overall results to patients with early-stage tumors for whom adjuvant therapy may be of more value. The current study was undertaken to explore the prognostic significance of the expression of ABH antigens in a group of patients undergoing resection of T1 N0 non–small cell lung cancers.
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Material and Methods
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All cases of primary non–small cell lung carcinoma dealt with during 1986 to 1991 were retrieved from the database of the Washington University School of Medicine, Division of Cardiothoracic Surgery. Only cases of T1 N0 M0 cancer were included, as determined by review of the operative and pathology reports. One hundred thirty-six such patients were identified who had undergone excision of their tumor by wedge resection, segmentectomy, lobectomy, or pneumonectomy. Tissue was available for 120 patients, and the pathology reports and the hematoxylin-eosin–stained sections for these patients were reviewed. The carcinoma was classified as adenocarcinoma (including bronchoalveolar), squamous cell carcinoma, or large cell/undifferentiated carcinoma. Patients with carcinoids or high-grade neuroendocrine neoplasms were excluded. Sections were also specifically evaluated to verify the lack of pleural invasion (as in T2 lesions). All lymph node sections were also reviewed to verify the lack of nodal metastases. Patient blood types were verified through blood bank records.
Unstained sections were cut at 5-µm intervals and mounted on glass slides using tissue adhesive (Sta-On; Surgipath, Richmond, IL). Slides were stained with anti-A, anti-B, and anti-H antibodies using the avidin-biotin peroxidase complex procedure (Table 1
) [14]. Briefly, sections were deparaffinized in xylene and absolute ethyl alcohol, and endogenous peroxidase activity was quenched by a 30-minute incubation in 0.6% methanolic hydrogen peroxide. The sections were rehydrated in graded alcohols, water, and phosphate-buffered saline (pH, 7.4), then incubated with the specific antibodies overnight at 4°C. Antibody bridge assembly by the avidin-biotin peroxidase complex technique was carried out the following morning by two sequential 1-hour incubations. Chromogenic development was accomplished by immersion of the sections in 3,3`-diaminobenzidine solution (0.25 mg/mL with 0.003% hydrogen peroxide) for a maximum of 10 minutes. The sections were then dipped in 0.125% osmium tetroxide to enhance the chromogenic precipitate, followed by light counterstaining with Harris' hematoxylin.
Positive control sections consisted of stock tissues known to express the antigens of interest. Nonimmune murine ascitic fluid was substituted for the test antibody as a negative control. Internal positive control tissues consisted of bronchial epithelium, bronchial glands, and vascular endothelium. Results were considered positive when more than 5% of the tumor cells demonstrated intense staining. When only 1% to 5% of the cells were stained, this was considered an equivocal result, and for statistical analysis these cases were grouped with the negative cases, those which demonstrated no staining with appropriate internal controls. Examples of positive and negative staining are illustrated in Figures 1 and 2. Results were also correlated with those of known serologic blood types.
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Fig 1. . (A) Adenocarcinoma in patient with blood group A. There is positive staining of endothelium and erythrocytes by anti-A, with loss of tumor cell antigen expression. (B) Second patient of blood group A. Note the positive staining of endothelium and erythrocytes, with strong tumor cell expression. (Avidin-biotin-peroxidase method; original magnification, x20.)
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Fig 2. . Squamous cell carcinoma in patient with blood group B. (A) Positive staining of stromal vessels by anti-B, with no tumor cell reactivity. (B) Anti-H is expressed strongly in tumor cells, without vascular reactivity. (C) No labeling by anti-A. (Avidin-biotin-peroxidase method; original magnification, x20.)
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Patient follow-up was performed through the Washington University Tumor Registry. Any patients who were recorded as lost to follow-up were subsequently traced through either their physicians' offices or remaining family members. As of October 1993 follow-up was complete for 100% of the patients.
Statistical analysis was performed using Kaplan-Meier statistics for 5-year survivals. Comparisons were made using the Mantel log-rank test. Significance was set at a p value of 0.05 or less.
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Results
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One hundred twenty patients were evaluable, with a 100% follow-up for an average of 40.75 months (range, 0.3 to 92 months) postoperatively. Three patients were excluded from further statistical analysis because of operative death occurring within 1 month of surgical resection. Of the remaining 117 patients, adenocarcinoma (including histologic characteristics compatible with bronchoalveolar carcinoma) had been diagnosed in 72 (62%), squamous cell carcinoma in 37 (32%), large cell or undifferentiated carcinoma in 7 (6%), and mucoepidermoid carcinoma in 1 (0.9%) (Table 2). The blood group characteristics are presented in Table 3.
All pathology reports were reviewed to confirm the stage I status based on T determinations and the N status for resected lymph nodes. Not all patients had undergone mediastinoscopy before resection nor had intraoperative sampling of mediastinal lymph nodes been done in all. This was especially true for those who underwent a wedge resection.
The average age at the time of diagnosis was 69.2 years (range, 42 to 90 years), and there were 71 men and 49 women. Ninety-nine patients underwent a lobectomy (or pneumonectomy) and 21 underwent a wedge or segmental resection for treatment of their carcinoma.
The overall 5-year survival was 57%. There was no significant difference in survival between the patients with adenocarcinoma (61%) and those with squamous cell cancer (51%). Additionally, no survival difference was seen in terms of retention or loss of appropriate antigen expression when all blood groups were examined (Fig 3).
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Fig 3. . Kaplan-Meier survival results for all blood groups. (neg = tumor negative for appropriate blood group antigen; pos = tumor positive for appropriate blood group antigen; SE = standard error.)
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The 5-year actuarial survival for blood group A patients was 53%. Tissue was available for antigen testing in 55 patients with blood group A surviving more than 1 month. The 5-year survival rate for the patients with blood group A and tumor positive for A antigen (n = 42) was 52%, versus 34% in those who had lost the A antigen (n = 13) (Fig 4), but this difference was not statistically significant (p = 0.737).
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Fig 4. . Kaplan-Meier survival results for patients with blood group A. (neg = negative for antigen; pos = tumor positive for antigen A; SE = standard error.)
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The 4-year actuarial survival for blood group O patients was 65%. Tissue was available for antigen testing in 51 patients with blood group O surviving more than 1 month. Tumor tissue from 28 patients showed retention of the O antigen and their 4-year survival was 63%. In the remaining 23 patients, the expression of O antigen was lost, and their 4-year survival was 69% (Fig 5), but this difference was not statistically significant (p = 0.437).
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Fig 5. . Kaplan-Meier survival results for patients with blood group O. (neg = negative for antigen; pos = positive for antigen; SE = standard error.)
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Comment
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Previous studies have investigated alterations in the expression of blood group antigens as a prognostic factor in patients with various carcinomas. The hypothesis of these investigations is that, as malignant cells become more poorly differentiated, they may lose the ability to synthesize normal ABH antigens. This has been most widely studied in the setting of transitional cell carcinomas of the urinary bladder, in which the loss of ABH expression indeed correlates with an adverse prognosis [7–9, 11, 12, 15]. Similar studies have been carried out to investigate this hypothesis in the setting of carcinomas of the stomach, colon, larynx, and lung [5, 6, 10, 13, 15–18].
The ABO blood group system consists of several genes [1]. The A and B alleles of the ABO locus code for specific glycosyltransferases, which then modify the oligosaccharide that is the product of the glycosyltransferase of a second gene, H. The O phenotype reflects the presence of unmodified H oligosaccharide, whereas the A and B phenotypes represent different terminal modifications of the H oligosaccharide. The molecules are present as constituents of a number of different glycoproteins and glycolipids. Cells bearing these characteristic markers include the endothelium, most epithelia, and red blood cells. In addition to the major A, B, and O divisions, there are numerous subgroups of A: A1, which is the most common, A2, and many less common A variants. These variants are typed as A in standard serologic tests, but may also react with anti-H sera [24].
Our study findings demonstrate that loss of the blood group antigen A on the tumor cell does not alter the clinical survival of patients with early-stage lung carcinoma. Lee and associates [13] have previously examined the loss of blood group A antigen in 164 patients with disease distributed throughout stages I to IIIb. They concluded that loss of the A antigen conferred a significantly worse prognosis, and that it was additive to age, nodal status, and the mitotic rate. This worse prognosis showed no correlation with age, sex, tumor stage, the presence of vascular invasion, or nuclear grade. When they examined only patients with stage I disease for loss of the blood group A antigen, they still found a significant difference in survival, although there were only 27 such patients. When they examined their patients with stage II or III disease, they found the ability of antigen A status to predict survival decreased.
The ability to predict tumor prognosis is most critical for the early-stage patients. Patients with stage I or II disease have the greatest probability of 5-year survival. Several studies are underway to examine the effect of adjuvant or neoadjuvant therapy. Therefore it would be of great assistance to be able to identify the patients who potentially face a worse prognosis and who would thus be candidates for more aggressive therapy.
In another recent study conducted by Matsumoto and colleagues [18], 89 patients with lung cancer distributed among all four stages (including small cell) were examined for their blood group antigen status. Similar immunohistochemistry staining as that done in our study was performed to identify retention or loss of the blood group antigens. Their results suggested a worse prognosis for patients whose tumor did not express their specific blood group antigen. They found a statistical correlation only for loss of the B group antigen in a study group consisting of 22 patients.
Clearly the issue is unresolved. In the present study consisting of a larger cohort of patients, we were unable to find a significant correlation between loss of blood group antigens, specifically A and H (the numbers of patients with blood group B were too small to evaluate), and 5-year survival in patients with T1 N0 cancer. This retrospective study is flawed because the staging may not be precise, as not all patients underwent preoperative mediastinoscopy or thorough intraoperative lymph node dissections. However, most patients (83%) underwent a lobectomy together with lymph node removal, with results demonstrating their probable N0 status. It is in this group of patients with early-stage disease who undergo complete resection that the ability to predict the likelihood of or improve the 5-year survival is so critical. If we can identify reliable predictors of worse survival, the patients so identified should be recommended for adjuvant therapy in study protocols. At present, the loss of blood group antigens is not a reliable predictor of poorer survival.
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Footnotes
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Presented at the Poster Session of the Thirty-first Annual Meeting of The Society of Thoracic Surgeons, Palm Springs, CA, Jan 30–Feb 1, 1995.
Address reprint requests to Dr Dresler, Department of Surgery, Fox Chase Cancer Center, 7701 Burholme Ave, Philadelphia, PA 19111.
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References
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- Watkins WM. Biochemistry and genetics of the ABO, Lewis, and P blood group systems. In: Harris H, Hirschhorn K, eds. Advances in human genetics, Vol 10. New York: Plenum, 1980.
- Race RR, Sanger R. Blood groups in man, 6th ed. Oxford: Blackwell, 1975.
- Boorman KE, Dodd BE, Lincoln PJ. Blood group serology, 6th ed. Edinburgh: Churchill Livingstone, 1988.
- Issitt PD. Applied blood group serology, 3rd ed. Miami: Montgomery Scientific Publications, 1985.
- Slocombe G, Berry C, Swettenham K. The variability of blood group antigens in gastric carcinoma as demonstrated by the immunoperoxidase technique. Virchows Arch A Pathol Anat Histol 1980;387:289–300.[Medline]
- Lin F, Liu PI, McGregor DH. Isoantigens A, B, and H in morphologically normal mucosa and in carcinoma of the larynx. Am J Clin Pathol 1977;68:372–6.[Medline]
- Bergman S, Javadpour N. The cell surface antigen A, B, or O(H) as an indicator of malignant potential in stage A bladder carcinoma: preliminary report. J Urol 1978;119: 49–51.[Medline]
- Limas C, Lange PH, Fraley EE, Vessella RL. A,B,H antigens in transitional cell tumors of the urinary bladder. Cancer 1979;44:2099–107.[Medline]
- Newman AJ, Carlton CE, Johnson S. Cell surface A, B, or O(H) blood group antigens as an indicator of malignant potential in stage A bladder carcinoma. J Urol 1980;124:27–9.[Medline]
- Wiley E, Mendelsohn G, Eggelston J. Distribution of carcino-embryonic antigens and blood group substances in adenocarcinoma of the colon. Lab Invest 1981;44:507–13.[Medline]
- Cuadrado E, Rodriguez-Trinidad A, Blasco E, Torrado J, Garcia JAL, Arozena F. Blood group isoantigens ABO(H) in transitional carcinoma of the bladder: a clinicopathologic study. J Urol 1986;114:409–15.
- Yamada T, Fukui I, Yokokawa M, Oshima H. Changing expression of ABH blood group and cryptic T-antigens of non-invasive and superficially invasive papillary transitional cell carcinoma of the bladder from initial occurrence to malignant progression. Cancer 1988;61:721–6.[Medline]
- Lee JS, Ro JY, Aysegul AS, et al. Expression of blood group antigen A-a favorable prognostic factor in non-small cell lung cancer. N Engl J Med 1991;324:1084–90.[Abstract]
- Hsu SM, Raine L, Fanger HA. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase procedures. J Histochem Cytochem 1981;29:577–80.[Abstract]
- Limas C. Quantitative interrelations of Lewis antigens in normal mucosa and transitional cell bladder carcinoma. J Clin Pathol 1991;44:983–9.[Abstract/Free Full Text]
- Langkilde NC, Wolf H, Orntoft TF. Lewis antigen expression in benign and malignant tissues from RBC Le(a-b-) cancer patients. Br J Haematol 1991;79:493–9.[Medline]
- Miyake M, Taki T, Hitomi S, Hakomori S. Correlation of expression of H/Le/Le antigens with survival in patients with carcinoma of the lung. N Engl J Med 1992;327:14–8.[Abstract]
- Matsumoto H, Muramatsu H, Shimotakahara T, et al. Correlation of expression of ABH blood group carbohydrate antigens with metastatic potential in human lung carcinomas. Cancer 1993;72:75–81.[Medline]
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Abstract
Introduction
