Context.—Folate receptor α (FRA) is a glycosylphosphatidylinositol-anchored high-affinity folate receptor that localizes to the apical surface of epithelia when it is expressed in normal tissue. Unlike normal tissues, FRA may localize to the basolateral side in tumors. These features make FRA an attractive drug target, and several FRA-targeted drugs have been developed and are in phases of clinical testing. Folate receptor α protein expression shows intertumoral variability that may correlate with response to therapy and to clinicopathologic parameters. Using immunohistochemistry, a recent study of breast carcinomas found FRA protein expression was associated with triple-negative status and high histologic grade in breast cancer. Although a prior study of lung adenocarcinomas found the expression level of the gene encoding FRA, FOLR1, was significantly increased in low-histologic-grade tumors compared to high-histologic-grade tumors, the relationship between FRA protein expression and histologic grade has not been reported for lung adenocarcinomas.
Objective.—To investigate the relationship between FRA protein expression level and clinicopathologic parameters in lung adenocarcinomas, including histologic grade, by performing immunohistochemistry for FRA on a cohort of non–small cell lung carcinomas.
Design.—High-density tissue microarrays constructed from 188 non–small cell lung carcinomas and used in prior studies were immunostained with FRA-specific antibody clone 26B3. Folate receptor α membranous staining intensity was given a semiquantitative score from 0 to 3+ for triplicate cores of tumor and averaged for each tumor. An average semiquantitative score from 0 to 1.4 was considered low expression, and an average semiquantitative score greater than 1.4 was considered high expression.
Results.—The majority (60 of 78; 77%) of lung adenocarcinomas and a minority (4 of 41; 10%) of lung squamous cell carcinomas were positive for FRA. Folate receptor α expression in lung adenocarcinomas compared with squamous cell carcinomas was statistically different (P < .001, χ2 test). In lung adenocarcinomas, FRA expression level correlated with histologic grade (P = .005, χ2 test for trend), but no other clinicopathologic parameter. The majority (23 of 27; 85%) of grade 1 adenocarcinomas had high FRA protein expression, whereas approximately half of grade 2 (10 of 19; 53%) and grade 3 (12 of 25; 48%) adenocarcinomas had high FRA protein expression. Out of adenocarcinomas with lepidic growth pattern, 16 of 20 (80%) showed high FRA protein expression. Out of adenocarcinomas with solid growth pattern, 2 of 6 (33%) showed high FRA protein expression. In lung adenocarcinomas, FRA expression level did not correlate with thyroid transcription factor 1, napsin A, or survival.
Conclusions.—Folate receptor α protein was expressed in the majority of lung adenocarcinomas and a minority of lung squamous cell carcinomas. Folate receptor α protein expression correlated with histologic grade for lung adenocarcinomas, and the greatest difference was observed between grade 1 and grade 3. Our results indicate that poorly differentiated adenocarcinomas or focuses of poor differentiation in a heterogeneous tumor may lack FRA protein expression and be more likely to be resistant to FRA-targeting drugs.
Folate receptor α (FRA) is a glycosylphosphatidylinositol-anchored high-affinity folate receptor that is expressed in some normal tissues, including pneumocytes and renal tubules.1 Folate receptor α protein expression is also observed in tumors from a variety of sites, including breast,2 colon and rectum,3 endometrium,4,5 lung,1 and ovary.4 When expressed in normal tissues, FRA localizes to the apical/luminal surface. Unlike normal tissues, FRA may localize to the basolateral side, rendering it accessible to circulating biological factors. This feature has been exploited for in vivo molecular imaging of FRA-positive tumors.6 The abnormal localization of FRA in tumors makes it a rational drug target. A handful of FRA-targeted drugs have been developed and are in various phases of clinical testing for the treatment of FRA-positive tumors.7
Folate receptor α expression shows intertumoral variability that may correlate with clinicopathologic parameters such as histologic grade. A study of breast carcinomas showed FRA protein expression was associated with triple-negative status and high histologic grade.2 Conversely, a study of endometrial carcinomas showed FRA protein expression was associated with low histologic grade.4 No relationship between FRA protein expression and histologic grade was observed for ovarian and colorectal carcinomas.3,4 Studies of non–small cell lung carcinoma (NSCLC) cohorts have produced mixed results with respect to the relationship between FRA expression and histologic grade.1,8,9 Examining only lung adenocarcinomas, one study found FOLR1 (the gene encoding FRA) expression level was higher in well-differentiated compared with poorly differentiated tumors.10 However, this result has not been confirmed at the protein level by immunohistochemistry. In this study, we initially set out to confirm the FRA protein expression pattern in NSCLC using antibody clone 26B3 that had been previously reported.1 Because our cohort had good quantities of each histologic grade for lung adenocarcinomas, we were able to examine the relationship between FRA protein expression level and histologic grade.
MATERIALS AND METHODS
Tissue Microarrays
Institutional review board approval of research protocols for this project was obtained through Houston Methodist Hospital Research Institute (Houston, Texas). Previously described tissue microarrays were used from a series of 188 NSCLCs from attempted curative surgical resections of lung cancers between 1974 and 1991 at Houston Methodist Hospital.11 Retrospective chart review was performed to obtain pathology reports, including original cell type diagnosis, and clinical data, including patient age, sex, smoking history, and survival status.
Tumors with an original diagnosis of bronchioloalveolar carcinoma were reclassified as adenocarcinoma with lepidic growth pattern per the 2015 World Health Organization criteria. Tumors with an original diagnosis of large cell carcinoma or NSCLC, not otherwise specified, were reclassified according to 2015 World Health Organization criteria as described in a prior study.12 Tumors that were reclassified as large cell carcinoma with null immunohistochemical phenotype were excluded from the study.
Histologic Grading
Retrospective chart review of pathology reports was performed to obtain degree of differentiation. Adenocarcinomas with lepidic growth pattern were considered grade 1. Well-, moderate-, and poorly differentiated adenocarcinomas were considered grades 1, 2, and 3, respectively. In the event of equivocal histologic grading, only the highest histologic grade was counted. Tumors that reclassified as adenocarcinoma with solid pattern were considered grade 3.
Immunohistochemistry
Recut sections 4 μm thick were obtained from tissue microarray blocks and immunostained with prediluted anti-FRA antibody clone 26B3 (Biocare Medical, Concord, California) as previously described.13 Folate receptor α membranous staining intensity was given a semiquantitative score from 0 to 3+ for triplicate cores of tumor, then averaged for each tumor. An average semiquantitative score from 0 to 1.4 was considered low expression and an average semiquantitative score greater than 1.4 was considered high expression. All immunohistochemistry results were evaluated against a negative control.
For reclassifying tumors with an original diagnosis of large cell carcinoma or NSCLC, not otherwise specified, and for determining thyroid transcription factor 1 (TTF-1) and p40 expression, recut sections 4 μm thick were obtained from tissue microarray blocks and stained with 2 dual-antibody cocktails for DG3 + CK5/napsin A and p40/TTF-1 (prediluted; napsin A and p40, rabbit polyclonal; DG3 + CK5 and TTF-1, mouse monoclonal; Biocare Medical) as previously described.14
Statistics
Statistical calculations were performed using GraphPad Prism version 6 software (GraphPad Software, Inc, La Jolla, California).
RESULTS
Out of 188 tumors in the tissue microarray, 119 tumors (63%), including 78 adenocarcinomas and 41 squamous cell carcinomas, had adequate tissue present for FRA immunohistochemistry. The majority (60 of 78; 77%) of adenocarcinomas were positive for FRA (Table 1). Of the adenocarcinomas, the most frequent FRA semiquantitative score was 3+ (29 of 78; 37%). The majority (37 of 41; 90%) of squamous cell carcinomas were negative for FRA (Table 1). Folate receptor α expression level in adenocarcinomas compared with squamous cell carcinomas was statistically different (P < .001, χ2 test).
Next, we investigated the clinicopathologic characteristics of lung adenocarcinomas compared with FRA expression level. Age, sex, smoking status and pack-years, histologic grade, TNM, and stage grouping were compared and showed no statistically significant differences, with the exception of histologic grade (Table 2). The majority (23 of 27; 85%) of grade 1 adenocarcinomas had high FRA expression, whereas approximately half of grade 2 (10 of 19; 53%) and grade 3 (12 of 25; 48%) adenocarcinomas had high FRA expression (Table 2). Because histologic grade is considered to be a progressive spectrum, a χ2 test for trend was used to compare histologic grade and FRA expression and the resulting P value was statistically significant (P = .005). Considering histologic subtypes on opposite ends of the grading spectrum, adenocarcinomas with lepidic growth pattern were mostly (16 of 20; 80%) positive for FRA, whereas only 2 of 6 adenocarcinomas with solid pattern (33%) were positive for FRA. Representative images of adenocarcinoma with lepidic and solid histologic patterns are shown in Figure 1, A and B.
Clinicopathologic Characteristics of Lung Adenocarcinomas Compared With Folate Receptor α (FRA) Expression Level
Representative images of adenocarcinoma with lepidic histologic pattern that demonstrate 3+ folate receptor α staining intensity (A) and adenocarcinoma with solid histologic pattern with negative folate receptor α staining (B) (original magnification ×100).
Figure 2. Disease-specific survival for lung adenocarcinomas following attempted curative surgical resection. P = .95, log-rank test; hazard ratio (log rank): 1.03, 95% confidence interval 0.47–2.26. Abbreviation: FRA, folate receptor α.
Representative images of adenocarcinoma with lepidic histologic pattern that demonstrate 3+ folate receptor α staining intensity (A) and adenocarcinoma with solid histologic pattern with negative folate receptor α staining (B) (original magnification ×100).
Figure 2. Disease-specific survival for lung adenocarcinomas following attempted curative surgical resection. P = .95, log-rank test; hazard ratio (log rank): 1.03, 95% confidence interval 0.47–2.26. Abbreviation: FRA, folate receptor α.
To examine the possibility that FRA expression correlates with TTF-1 or napsin A, contingency tables were constructed. The percentages of TTF-1– and napsin A–positive tumors were comparable for both low and high FRA expression groups (Table 3). The Fisher exact test returned no statistically significant difference between low and high FRA expression groups (P = .74 and P > .99, respectively).
Thyroid Transcription Factor 1 (TTF-1) and Napsin A Expression in Lung Adenocarcinomas Compared With Folate Receptor α (FRA) Expression Level
To determine if FRA expression level is a prognostic factor, Kaplan-Meier survival analysis was performed (Figure 2). Comparing high and low FRA expression level groups of the entire cohort of adenocarcinomas resulted in no statistically significant difference (P = .95, log-rank test; univariate hazard ratio [log rank], 1.03; 95% confidence interval, 0.47–2.26).
COMMENT
In this study, the majority (60 of 78; 77%) of lung adenocarcinomas and a minority (4 of 41; 10%) of lung squamous cell carcinomas were positive for FRA (Table 1). This result is in remarkable agreement with 2 prior studies using the same antibody clone, 26B3, and a positive/negative cutoff.1,13 Using the same tissue microarray used in this study and a different antibody clone (mAb343), a higher percentage of lung squamous cell carcinomas (36 of 71; 51%) stained positive for FRA.11 The reason for discrepancy of FRA immunostaining in the same cohort is not certain, but it is likely due to differences in epitope specificity between antibody clones.
The major finding of this study was that FRA protein expression correlates with histologic grade in lung adenocarcinomas (P = .005, χ2 test for trend). Consistent with the statistical result, the majority (16 of 20; 80%) of adenocarcinomas with lepidic growth pattern were positive for FRA, whereas a minority (2 of 6; 33%) of adenocarcinomas with solid pattern were positive for FRA. Comparing histologic grades, most of the difference in FRA expression was observed between grade 1 and grade 3 adenocarcinomas. Prior studies that examined the relationship between FRA expression and histologic grade in NSCLC had mixed results.1,8,9 Compared with these 3 studies, we included only adenocarcinomas and had sufficient quantities of each histologic grade for a valid χ2 test. In support of our result, Iwakiri et al,10 examining only lung adenocarcinomas, found increased FOLR1 expression in well-differentiated compared with poorly differentiated tumors.
In our study, FRA expression was not a prognostic indicator for lung adenocarcinomas (Figure 2). Studying a lung adenocarcinoma cohort, O'Shannessy et al1 found high FRA expression detected by the 26B3 clone was associated with improved overall survival. The reason for the discrepancy with our result is not certain because both studies consisted of patients that did not receive adjuvant therapy, and detailed follow-up, including cause of death, was obtained in both studies. However, the 2 studies are based on patient cohorts from different time periods, which may introduce other factors that impact patient care and therefore survival. Two studies of NSCLC cohorts treated with pemetrexed have shown FRA expression detected by the 26B3 clone is associated with improved overall survival.8,9 One study of an NSCLC cohort treated with pemetrexed found FRA expression was not associated with survival; however, this study used an unspecified noncommercial antibody clone.15 The prognostic impact of FRA protein expression deserves continued exploration.
Currently, there is not an established grading system for lung adenocarcinomas, and this is a limitation for our study.16,17 In our study, the grade 1 adenocarcinomas included those with lepidic growth and with well-differentiated glandular architecture. The higher-grade adenocarcinomas included those with solid growth pattern and with poorly differentiated glandular architecture. In this way, histologic grade refers to a spectrum of morphologic differentiation. This means the morphology of the adenocarcinoma correlated with FRA protein expression level in our study, and furthermore adenocarcinoma morphologies might be a predictor of FRA protein expression level. It is reasonable to speculate that poorly differentiated adenocarcinomas or focuses of poor differentiation in a heterogeneous tumor may lack FRA protein expression, and therefore may be resistant to FRA-targeted drugs.
In conclusion, we have examined the FRA immunostaining pattern of an NSCLC cohort and found most lung adenocarcinomas were positive for FRA, whereas most lung squamous cell carcinomas were negative for FRA. Folate receptor α protein expression level correlated with histologic grade in the adenocarcinomas, and the greatest difference was observed between grade 1 and grade 3 adenocarcinomas.