Large tumors can be difficult to address not only due to their size, but also their internal complexity and heterogeneity. Building on their previous work, in which whole exome sequencing of 2-5 spatially different regions from resected localized lung adenocarcinomas from 11 patients revealed that mutational genomic intratumor heterogeneity (ITH) was linked to increased rates of postsurgical relapse in adenocarcinoma patients, Reuben et al. performed multi-region TCR profiling on the same samples.

The team first used the TCR sequencing data to examine the density of the T cell infiltrate in tumor samples and found that tumor infiltrating lymphocytes (TILs) accounted for anywhere between 11% and 85% of all cells per sample, with significant variation in density between patients, as well as between samples from different regions within the same tumor, suggesting ITH in TIL density between regions.

Next, the researchers turned to T cell clonality - a presumed metric of antigen-specific expansion or reactivity - and again observed significant ITH. Some regions showed fairly symmetrical distribution of T cell clones, while others showed that one or a few T cell clones were dominant. TCR ITH was observed within tumors from all patients, with clonality differences as large as 50% across regions. The authors also looked specifically at T cells relevant to tumors by comparing TILs to T lymphocytes from peripheral blood, and confirmed ITH for tumor-specific T cell clones. Comparing T cell clones across regions revealed that only 1-14% of clones were detectable in all regions of a single tumor.

When researchers honed in on the top 5 TCR clones in each patient, they saw that in most patients, the top clones were dominant in all regions, however, in a few patients the top overall clones were sometimes found at much lower frequencies in certain regions. The top 5 clones were always at least detectable in all regions, suggesting that the most immunogenic mutations were conserved and the most reactive T cells were preserved across regions.

To more broadly examine overlap in the overall repertoire between samples, researchers used the Morisita overlap index (MOI) and confirmed a total lack of overlap between patients. Within patients, they found that the complexity of overlap varied, with some regions in the same tumor having very similar overall repertoires, while others were more distinct.

Researchers also performed immune profiling by immunohistochemistry to determine the T cell subsets of the TILs, and results showed that CD4⁺ T cells were present at about a 2:1 ratio compared to CD8⁺ T cells. This higher frequency of CD4⁺ T cells could suggest that heterogeneity in neoantigens presented on MHC II molecules had a greater effect on the T cell response, but more definitive linkage of TCR sequence information and T cell subtype is required.

Comparing the newly obtained TCR data to the genomic data from the previous study revealed that a more complex genomic ITH is associated with a more complex TCR ITH, and that tumor heterogeneity in predicted neoantigens correlated to spatial differences in the T cell repertoire. The researchers hypothesized that the heterogeneity in mutations resulting in neoantigens in certain regions of a tumor leads to the increased clonal expansion of antigen-specific T cells in that region. They could not, however, rule out the possibility that the architecture of the tumor itself, including the vasculature and lymphatics, was contributing to the distribution of T cells within the tumor.

In light of the association previously found between genomic ITH and patient relapse, Reuben et al. also compared the T cell repertoire data to relapse rates, and, as expected, found that higher ITH in T cell clonality was observed in samples from patients who relapsed after surgery, and extended this correlation to more rapid disease progression and reduced disease-free survival. Interestingly, no significant associations were observed between postsurgical relapse and ITH of T cell density.

Overall, the positive correlation between genomic and TCR intratumoral heterogeneity and poor prognosis implicates high ITH as a biomarker for unfavorable outcomes. This research suggests that further investigation into ways to target antigens that are universally expressed within a tumor or to identify and target a broader set of mutations than detectable with a single biopsy may be needed for improved results.

by Lauren Hitchings