So I'm finally finished sorting through the abstracts from the 14th World Conference on Lung Cancer that I recently attended and this begins a series of posts of what I found of interest from the oral abstract presentations .
There were two presentations that focused on the immune cell response in the tumor microenvironment that were of particular interest to me in that I have a current project looking at CD3+ lymphocyte responses in resected NSCLC through my ongoing work with the Thoracic Oncology Research Group at Rush University Medical Center.
MO 22.01 from session VIII examined tissue microarrays constructed from 479 patients with stage 1 adenocarcinoma and a median follow-up of 4.3 years. They looked at a wide variety of different immune markers by immunohistochemistry and assessed localization (tumor vs. stromal) of immune cell infiltration, the grade of that immune cell infiltrate (low vs. high), and expression (sum of intensity of staining plus semi-quantitative assessment of distribution).
Results: The 5-yr relapse free survival (RFS) was 82% for the entire cohort. There was no independent prognostic significance for any subpopulation of immune cells. However, the ratio between stromal FOXP3+ (Treg cells) and CD3+ (total T cells) and tumor expression of IL7R and IL12Rbeta2 were found to be significant factors in predicting recurrence by univariate analysis. Tumor expression of IL12Rbeta2 and stromal FOXP3:CD3 were prognostic even in tumors less than 2 cm. By multivariate analysis, gender, stage (1A/1B), lymphatic invasion, tumor IL12Rbeta2 and stromal FOXP3:CD3 were independent predictors associated with recurrence.
This study highlights the importance of localization of immune cell response but also underscores the challenge for future studies to use more refined quantitative assessments of immune cell subpopulations and relative expression of IHC staining intesity (such as utilizing digital imaging and image analysis).
MO 22.04, also from session VIII, presented interesting complementary data to the above presentation. This study examined 196 resected stage 1-3A NSCLC using IHC for infiltrating CD8+ and FOXP3+ cells. They counted cells expressing these markers in tumor and stromal regions in five "randomly selected" high-power-fields and reported their data as the number of cells per mm2 for each region. They used median cell count as the cutoff to define patient subgroups.
Results: Increased tumor islet CD8+ and stromal FOXP3+ infiltration were associated with better prognosis, whilst increased stromal CD8+ and tumor islet FOXP3+ infiltration were associated with worse prognosis. Further, high CD8+ tumor islet/stromal infiltration was associated with better prognosis, whilst high FOXP3+ tumor islet/stromal infiltration was associated with worse prognosis.
Again, this study reinforces the crucial importance of identifying the microlocalization of immune cell subpopulations. But I note several critical issues with methodology in this study that are worth considering for future studies. First, the investigators did not clarify their methodology as to how high-power-fields were "randomly selected." Further, their use of the term "tumor islet" suggests a non-random selection of such foci (tumor budding?) for assessment. As a pathologist, I can safely say that this is quite unlikely "random"--which would undoubtedly lead to bias towards areas of the tumor that show higher concentrations of positively staining cells. In fact, our Rush group recently began re-evaluating EMT markers assessed by IHC in a group of NSCLC patients who underwent surgical resection to specifically assess and grade distribution and extent of staining at the tumor-stromal interface rather than the global expression of such staining. The data are being analyzed currently. Moreover, I have been assessing the lymphocytic response just by H&E and note an association between foci of tumor budding and intensity of lymphocytic response at the tumor invasive front. Second, manual counting of "events" just doesn't make it for this type of study and (again!) I must reiterate the need to use image analysis for this type of study.