Revealing the Tumor Ecosystem in Follicular Lymphoma By Mass Cytometry

In follicular lymphoma (FL), clinical progression/histologic transformation occurs as a result of emergence of biologically aggressive malignant clones that escape the immune response. Whether such clones are preexisting or arise as a consequence of spontaneous or therapy-induced DNA mutation/genomic instability remains an open question; however, it has become increasingly clear that it will be important to understand the multi-clonal structure of tumors in order to treat them more effectively. As well, tumor-infiltrating immune cells represent a complex and heterogeneous population and are hypothesized to either help or hinder tumor progression. Several studies have searched for prognostic value in various infiltrating immune cell subsets, but results have been inconsistent, likely in part because most studies focus only on a few, incompletely characterized subsets at a time. Furthermore, the potential for co-variance between malignant subclones and the complement of infiltrating immune cells has not yet been explored. We would propose that understanding the entire tumor "ecosystem" in FL is needed to make further strides in predicting biological behavior and ultimately in guiding selection of the most appropriate of available rational therapies.

In this study, we sought to use mass cytometry (CyTOF) to characterize both the clonal substructure of malignant populations and the infiltrating immune repertoire with unparalleled breadth, yet at single-cell resolution. We designed and optimized a 2-tube, 40-parameter CyTOF panel in which one tube is dedicated to characterizing population substructure among malignant B-cells and the other to profiling the composition of the infiltrating T-cell repertoire. We accessed viably frozen single cell suspensions from excess lymph node biopsy material remaining after diagnostic flow cytometry have been prospectively banked for the past 2 decades.

We stained 12 FL, 23 diffuse large B-cell lymphoma (DLBCL), and 5 reactive lymph node (rLN) samples using the B-cell marker tube, acquired CyTOF data, and performed analyses using tSNE and Complicity mapping algorithms, which led us to make the following observations: First, over half of FL samples contain at least two phenotypically distinct tumor subclones. In contrast, very few of the DLBCL samples that we have examined thus far exhibit definitive phenotypic subclones. Lower intra-tumoral heterogeneity in DLBCL may imply that these tumors represent outgrowth of a highly evolved, dominant clone as compared to a less evolved collection of "untested" clones in FL. Second, tSNE mapping of FL tumors revealed two distinct subtypes, one in which the individual tumors showed highly similar and partially overlapping phenotypes that localized in proximity to normal germinal center B cells ("GC" subtype), and the other which was composed of more phenotypically heterogeneous tumors that were localized more distantly from normal germinal center B cells ("non-GC" subtype).

We also stained 6 FL and 5 rLN samples using the T-cell marker tube, acquired CyTOF data, and performed analysis using tSNE and Scaffold mapping algorithms. We found the relative abundance of T-cells in FL samples was comparable to rLN (36.1% vs. 37.4% of total cells, p=0.90); however, most of the FL samples showed elevated CTL fractions (20.1% vs. 13.0% of total T-cells, p=0.02). Using Scaffold mapping, we were able to resolve a dramatic diversity of T and T/NK cell subsets, each of whose abundance varied substantially from one sample to the next. For example, the majority of CTL cells in rLN samples exhibited a relatively homogeneous, naïve-like phenotype, while CTL cells in FL typically contained a heterogeneous mixture of activated and exhausted subsets. The T-helper compartment exhibited a similar picture with relative population homogeneity within rLN samples, contrasting with heterogeneity within FL samples. Composite analysis correlating B- and T-cell features within the same FL specimen revealed that loss of HLA-DR and CD124 on malignant cells was correlated with CTL exhaustion, suggesting a potential immune escape mechanism.

Our findings illustrate that novel information is revealed by 40-dimensional CyTOF analysis and provide insight into important, but understudied aspects of intra-tumoral heterogeneity and variable host immune response in lymphoma.