The current TNM staging system relies solely on anatomic factors and is limited in its ability to discriminate a subset of patients with stage I disease with poor clinical outcome. In fact, for stage I lung ADC, tumor size is the only standard prognosticator available. In our study, we have demonstrated the prognostic power of immunologic parameters for stage I lung ADC, identifying three immune markers that are predictive of recurrence. This immunologic observation has both prognostic and therapeutic implications.
Tumor-infiltrating immune cells have shown prognostic value for several solid malignancies, including colorectal,1,2,7
and breast cancers4
(Appendix Table A2, online only). Galon et al1
have advocated, through their work in colorectal cancer, the use of three important parameters of tumor-infiltrating lymphocytes (TILs)—type, density, and location—to predict clinical outcome. In our study of a uniform cohort of patients with stage I lung ADC, we investigated eight markers of TILs and found the FoxP3/CD3 ratio in tumor-associated stroma to be significantly associated with recurrence. FoxP3 is a marker of regulatory T cells, a subset of lymphocytes known to suppress the host immune response. In patients with lung cancer, regulatory T cells are thought to play protumor roles,24
and their association with worse prognosis has been shown for all histologic types, including ADC.17,25
Interestingly, FoxP3 in the stroma only—and not in the tumor nest—was associated with recurrence, emphasizing the importance of assessing the location of TILs within the tumor microenvironment. In fact, the significance of immune cells in the tumor stroma has been shown in non–small-cell lung cancer. In patients with stages I to IIA disease, Dieu-Nosjean et al26
demonstrated the presence of tertiary de novo lymphoid structure in the tumor microenvironment, a structure they termed the tumor-induced bronchus-associated lymphoid tissue (Ti-BALT). The presence of mature dendritic cells in Ti-BALT correlated with prolonged overall and disease-free survival.
Furthermore, we demonstrate that in addition to type, density, and location, a fourth characteristic of TILs—the relative proportion of pro- and antitumor immune cells—is an important parameter. Our close examination of patients with high densities of stromal FoxP3 revealed that among these patients, a concurrent high density of stromal CD3 predicted better outcome. This suggests that even in the presence of high stromal FoxP3, a high density of CD3 may overcome the protumor effects of FoxP3-positive regulatory T cells. In addition to revealing prognostic value, this finding has significant implications for devising potential immunomodulatory therapy for patients with lung ADC; an intervention that decreases FoxP3 and increases CD3 would likely be beneficial. Of interest, cyclophosphamide has been shown to modulate the tumor immune microenvironment by depleting regulatory T cells.27
Also, in a murine melanoma model, activation of a T-cell costimulatory receptor, 4-1BB, has been shown to result in decreased tumor infiltration of regulatory T cells.28
Because FoxP3-positive regulatory T cells are thought to be a subset of CD4+ T cells, we also investigated stromal FoxP3 density and its relation to CD4 density. Although stromal densities of CD4 and FoxP3 showed significant correlation, combining CD4 and FoxP3 densities did not result in significant prognostic findings (Data Supplement).
In our analysis of chemokine expression on tumor cells, we found two to be of prognostic significance, one with antitumor associations (IL-12Rβ2) and one with protumor associations (IL-7R). IL-12Rβ2 is one of the two subunits that form the receptor for IL-12. Tumors expressing IL-12Rβ2 were associated with low-grade morphology, EGFR
mutation, and less recurrence. Given that IL-12Rβ2 expression is observed on normal lung epithelium,23
it is plausible that lung ADC progression is accompanied by loss of IL-12Rβ2 expression. In fact, in preclinical models, mice deficient in IL-12Rβ2 have been shown to spontaneously develop lung ADC.29
After observing that IL-12Rβ2 expression is associated with less-aggressive tumors, we investigated how tumor expression of IL-12Rβ2 is associated with the protumor stromal environment. In a group of patients identified as high risk by the stromal FoxP3 risk index, we observed that patients with high-level expression of IL-12Rβ2 (n = 91) experienced less-frequent recurrence than those with low-level expression (n = 25; 5-year RFP, 88% for high-level v
73% for low-level expression; P
= .086). This suggests that even in the presence of unfavorable stromal immune-cell infiltrates, high-level expression of IL-12Rβ2 on tumor cells may play a protective role. Thus, therapies targeted to maintain IL-12Rβ2 expression on tumor cells are of interest. In patients with stages I to IV lung ADC, methylation of the IL-12Rβ2
gene was shown to be associated with less mRNA expression in vivo and shorter survival.30
The recent publication of a phase I/II study of DNA methyltransferase inhibitor and a histone deacetylase inhibitor in patients with recurrent metastatic lung cancer is promising.31
Furthermore, in preclinical models, T cells genetically modified to secrete IL-12 have shown intrinsic resistance to regulatory T cells.32
IL-12–secreting T cells are of special interest, because this resistance could potentially overcome the protumor associations of regulatory T cells. In addition, the association of IL-12 and IL-12Rβ2 expression on tumor cells warrants further investigation.
In contrast to IL-12Rβ2 expression, IL-7R expression was associated with aggressive tumor features: high-grade morphology, lymphovascular invasion, larger tumor size, KRAS
mutation, and more-frequent recurrence. IL-7 and IL-7R are implicated in lung cancer lymphangiogenesis via c-Fos/C-Jun–dependent vascular endothelial growth factor D (VEGF-D) upregulation.33
In breast cancer, IL-7R has been shown to induce tumor growth and lymphangiogenesis through upregulation of VEGF-D.34,35
Its ligand, IL-7, is produced by stromal and epithelial cells and plays a central role in T-cell development, in addition to providing a potent lymphocyte-survival factor through the JAK-STAT pathway.36
The role of the IL-7/IL-7R axis in the tumor immune microenvironment in lung ADC warrants more investigation.
The prognostic significance of the immune markers was further strengthened by their ability to stratify within currently known prognosticators—stage (IA and IB; A to F), tumors ≤ 2 cm (G to I), and morphologic grade (Data Supplement). Patients with stage IB disease with a high stromal FoxP3 risk index or high-level expression of IL-7R experienced outcomes similar to those of patients with stage II disease, for whom adjuvant chemotherapy is recommended. The ability to prognosticate within tumors ≤ 2 cm is especially important, because this represents a population in which incidence is expected to increase with the adoption of more-widespread CT screening, but in which a standardized prognostic factor is lacking. When we assessed overall survival as an end point, IL-7R remained a significant prognosticator in both the training (P = .007) and validation cohorts (P = .02), whereas findings were not significant for FoxP3/CD3 risk index (P = .21) or IL-12Rβ2 (P = .51). The ability of IL-7R to prognosticate both recurrence-free and overall survival merits further investigation of its biologic role (Data Supplement).
Fig 3. Recurrence-free probability (RFP) by three prognostic immune markers for patients with (A-C) stages IA and (D-F) IB disease and for (G-I) tumors ≤ 2 cm. (A) Among patients with stage IA disease, stromal forkhead box P3 (FoxP3) risk index score (more ...)
One limitation of our study is the semiquantitative nature of immunohistochemical scoring. Although a digital analysis was attempted using the Aperio ScanScope XT (Aperio, Vista, CA), the results were confounded by anthracotic pigments picked up as positive stains, a unique problem encountered in lung specimens. Accurate analysis and discrimination of the tumor from the tumor-associated stroma were best achieved by direct visualization under the microscope.
Our findings shed light on the complex tumor immune microenvironment in stage I lung ADC. First, we demonstrated that in the tumor-associated stroma, immune infiltrates rich in FoxP3-positive regulatory T cells create a protumor environment, and this environment may be overcome when there is a concurrently high density of CD3+ lymphocytes. Second, IL-12Rβ2 is expressed on normal lung epithelium and on less-aggressive tumors but to a lesser extent on more-aggressive tumors. Furthermore, tumors expressing IL-12Rβ2 tend to do well despite an unfavorable immune environment. Third, IL-7R expression on tumors is associated with aggressive features.
In conclusion, we have identified prognostic immune factors in this first, to our knowledge, large-scale study of the tumor immune microenvironment in patients with stage I lung ADC, a population anticipated to increase with widespread CT screening. For a population in which tumor size is currently the main prognostic factor, our results provide important prognostic tools and demonstrate the feasibility of using a multidisciplinary approach to advance beyond the limitations of the current staging system. More importantly, these findings provide a crucial foundation for future investigations into immunomodulatory therapies for lung ADC.