High-throughput sequencing of archival cerebrospinal fluid specimens defines B-lymphoblastic leukemia clonal composition.

Detailed characterization of the B-lymphoblastic leukemia (B-ALL) cells which invade the central nervous system (CNS) has been limited by practical challenges. To test whether the clonal composition of the cerebrospinal fluid (CSF) reflects the primary B-ALL tissue, we applied immunoglobulin (Ig) high-throughput sequencing (HTS) of archival CSF cytospin preparations from six patients with morphologically defined CNS involvement. We discovered that most CSF clones are detectable at some timepoint in the primary tissue, but that shifting clonal abundance is prevalent across tissue sites between diagnosis and relapse. Ig HTS of CSF cytospins may improve understanding of sanctuary site dissemination in B-ALL.

Acute lymphoblastic leukemia clonal distribution between bone marrow and peripheral blood.

Acute lymphoblastic leukemia (ALL) is often composed of numerous subclones. Here we test whether the clonal composition of the blood is representative of the bone marrow at leukemia onset. Using ultra-deep IGH sequencing, we detected 28 clones across 16 patients; 5/28 were only in the marrow. In four patients, the most abundant clones differed between sites, including three in which the dominant medullary clones were minimally detectable in the blood. These findings demonstrate that the peripheral blood often underrepresents the genetic heterogeneity in a B-ALL and highlight the potential impact of tissue site selection on the detection of minor subclones.

DC-SIGN binding to mannosylated B-cell receptors in follicular lymphoma down-modulates receptor signaling capacity.

In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell-cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca2+ response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.

Patient-derived xenografts of low-grade B-cell lymphomas demonstrate roles of the tumor microenvironment.

To discern features of non-Hodgkin lymphomas (NHL) that are autonomous from those that are shaped by the tumor environment (TE), we used patient-derived xenografts (PDX) to probe the effects on neoplastic cells of manipulating the TE. Properties of neoplastic cells that are often considered to be autonomous include their relative independence from stromal support, their relative survival and/or proliferation advantages compared with nonneoplastic cells, and their state of differentiation. Prior approaches to creation of PDX models likely select for neoplasms, which are the most capable of engraftment, potentially masking the effects of the TE. To overcome this bias, we developed a robust protocol that rapidly produced xenografts with more than 85% of unselected, cryo-preserved, B-cell NHL specimens, including low-grade tumors such as follicular and marginal zone lymphoma. To discern features that are shaped by the TE, we extensively studied 4 low-grade lymphoma specimens. B-cell engraftment required components of the native TE; specifically, CD4+ cells. The relative survival of neoplastic compared with nonneoplastic B cells was not autonomous in 2 specimens; specifically, neoplastic B cells from 2 specimens showed a greater dependence on the TE than normal B cells for engraftment. Furthermore, the differentiation of neoplastic B cells was dependent on the TE; mature B-cell neoplasms converted to plasmacytoma-like lesions in the grafts. These results highlight the central and patient-specific roles of the TE in maintaining the relative survival of neoplastic cells compared with normal cells and in controlling the differentiation of neoplastic cells.