Methods to Elucidate Hematopoietic Clonal Organization in Bone Marrow

Every tissue in the human body becomes a mosaic of somatic mutations throughout life. Each cell in the body contains a record of the genetic alterations occurring from conception until that individual cell came into existence through cell division. Most of these changes are benign, but cells with alterations that endow a selective advantage can preferentially divide and expand. Already, this process has been described in a diverse set of phenotypically normal tissues including skin, esophagus and colon (Colom et al., 2021; Lee-Six et al., 2019; Martincorena et al., 2015). In these solid organs, clonal mutations are geographically constrained within contiguous regions of tissue. In the blood, these somatic mutations are termed clonal hematopoiesis (CH) (Steensma et al., 2015). Previous work has extensively characterized the burden of CH in humans, showing an increased prevalence with age and increased risk for hematologic malignancy and all-cause mortality (Genovese et al., 2014; Jaiswal et al., 2014; Young et al., 2016; Young et al., 2019). Despite our detailed understanding of CH in the blood, little is known about how these hematopoietic clones exist in the bone marrow (BM) and how they interact with the BM niche.

We hypothesize that the spatial restriction of clones observed in solid organs also exists in the BM. To characterize the spatial and clonal organization within the BM niche, we combined single cell proteogenomics with novel methods for spatially aware variant detection. We characterized the BM clonal organization of a patient with polycythemia vera (PV)-a myeloproliferative neoplasm (MPN). MPNs are blood disorders characterized by abnormal hematopoiesis and an increased risk of transformation to aggressive leukemia. Studying a patient with PV was ideal as this is an oligoclonal disease, and we had prior knowledge that the patient's disease was driven by a large JAK2 V617F mutated clone and a presumed DNMT3A R882Cmutated subclone.

Single BM cells were prepared for proteogenomics using the Tapestri platform (Mission Bio). From the BM aspirate, 1,486 individual live BM cells were assayed using the TotalSeq-D Heme Oncology Cocktail V1.0 (BioLegend) for immunophenotyping and the 45 gene myeloid panel (Mission Bio) for genomic characterization. Analysis identified the known JAK2 V617F and DNMT3A R882C mutations, and previously unknown TET2 L1081* and GNAS R844H mutations. The single cell assay disentangled the clonal complexity of this patient's disease-a large JAK2 V617F homozygous clone, a small TET2 L1081* heterozygous subclone, a small JAK2 V617F heterozygous clone, and independent GNAS R844H heterozygous and DNMT3A R882C heterozygous clones. Proteomic analysis revealed that the cells clustered into four groups including; B-lymphocytes marked by CD19, T-lymphocytes marked by CD3, the presumptive MPN cells highly expressing CD141 (thrombomodulin) and CD71 (transferrin receptor 1), and a mixed population of largely CD38 positive hematopoietic progenitors.

To further characterize the spatial organization of these hematopoietic clones in the BM, the patient's femur head was sliced into 2mm sections using a band-saw and then further partitioned with hand tools into approximately 400 BM fragments, of which 96 were selected for droplet digital PCR (ddPCR) targeting the mutations identified by single-cell and bulk sequencing. The ddPCR assay identified each of these mutations in all assayed BM fragments, albeit at a wide range of variant allele fractions (VAFs). Interestingly, this suggested a heterogeneous distribution of somatically mutated clones spatially in the BM. Clustering of these bone marrow fragments identified regions marked by high JAK2 V617F and TET2 L1081* VAF (cluster 2), high JAK2 V617F VAF alone (cluster 3), and lower VAF for any mutations (clusters 1 and 2; Figure 1).

These findings provide a comprehensive characterization of spatial organization and clonal hierarchy within the BM. Surprisingly, the clonal mutations were not well mixed in the BM with regions of high clonal diversity millimeters away from regions with a paucity of these mutated clones. Future work will expand this platform to describe BM clonal architecture in a larger set of bone marrow specimens and a wide variety clinical scenarios to help further explain the role of the BM niche in CH, MPNs and leukemia.