A Patient-Derived Xenograft Model of Marginal Zone and Follicular Lymphomas Demonstrates a Key Role of CD4+ T Cells

As we previously described, intra-peritoneal injection of single cell suspensions (including non-neoplastic cells) from >90% of primary B cell non-Hodgkin's lymphoma specimens results in lymphoma cell growth in the omentum of the NSG mouse strain (NOD-scid IL2rg null) (ASH Meeting on Lymphoma Biology 2014; abstract #144). To date, we have shown reproducible engraftment of 17/20 specimens (including 9 follicular, 5 marginal zone, 4 diffuse large B cell, 2 mantle cell). Molecular markers specific for each patient's tumor (including clone specific features of the IGK, IGH, BCL2 and BCL1 loci) provided verification that all 17 of the engrafted tumors contained the same clone identified in the original specimen. Each tumor was implanted and engrafted in at least 3 mice (range 3-41) and each had a characteristic and often distinctive time course of engraftment, propensity to disseminate, and histologic features, indicating that the behaviors of the xenografts reflected intrinsic properties of the original tumors.

B cell engraftment required CD4+ T cells. B cells were purified by negative selection from three specimens with particularly robust engraftment, a FL (J1) and two MZL (M1 and W1). Depletion of non-B cells entirely ablated engraftment for all 3 tumors; mock depletion had no effect. Selective depletion of CD4+ but not CD8+ T cells ablated engraftment of both tumors assessed (J1 and M1). Finally, purified CD4+ cells (but not CD8+) fully reconstituted engraftment of purified B lymphoma cells from tumor M1.

In addition, the xenograft model highlighted two unexpected features of these tumors:

1) The neoplastic B cells of some tumors were relatively resistant to engraftment compared to non-neoplastic B cells present in the same specimen. For 5 tumors (2 MZL and 3 FL), the number of human cells and the ratio of neoplastic to non-neoplastic B cells in the xenografts at two weeks were determined by performing bar-coded, ultra-deep sequencing on the entire mesentery including the omentum. For the 2 MZL, the fraction of B cells that was clonal was > 90% at 2 weeks ; in contrast, for 3 FL specimens, the fraction of B cells that were neoplastic dropped from >60% at time of implantation to less than 5%. These results indicate that neoplastic B cells do not necessarily have an engraftment advantage over non-neoplastic B cells. Where non-neoplastic cells can respond to proliferation/survival cues, neoplastic cells in the same FL specimens apparently resist the same signals.

2) We observed that B cells, including neoplastic B cells, have a striking propensity to differentiate into plasma cells when engrafted in the mice. In all specimens at the time of injection, neoplastic B cells comprised >60% of the cells and plasma cells were negligible. In contrast, at 2-4 weeks the number of CD20+ B cells fell to <20% and plasma cells comprised >20% of the cells in all xenografts. 4/17 xenografts (including 2 FL and 2 MZL tumors) assayed showed monoclonality of the plasma cells at two weeks (based on kappa:lambda light chain ratio). These results indicate that the neoplastic B cells of some tumors have a developmental plasticity in the xenograft that is not typically apparent in patients.

In summary, a xenograft model reveals several features of low grade B cell lymphomas that are not apparent from static observations: 1) CD4+ T cells are essential to engraftment, suggesting that CD4+ T cells may be critical to lymphomagenesis; 2) neoplastic B cells of some tumors do not respond to the proliferation/survival cues to which non-neoplastic B cells are sensitive; 3) neoplastic B cells of some tumors differentiate into post-mitotic plasma cells, potentially eliminating these cells from the proliferative pool.