Spatial Organization of Hematopoietic Clones in the Bone Marrow of a Patient with Polycythemia Vera

All humans become genetic mosaics over time as our stem cells acquire somatic mutations throughout life. In the hematopoietic compartment, this phenomenon is termed clonal hematopoiesis (Steensma et al. 2015). Clonal hematopoiesis (CH) increases in prevalence with age and is associated with an increased risk for hematologic malignancy (Genovese et al. 2014; Jaiswal et al. 2014; Xie et al. 2014). CH harboring JAK2 mutations are frequently observed in healthy individuals; and yet JAK2 mutations are also the most frequent drivers of myeloproliferative neoplasms (MPNs) such as polycythemia vera (PV). MPNs are clonal diseases marked by dysregulated hematopoiesis and can progress to a secondary myelodysplastic syndrome or acute leukemia. There is increasing evidence that the clonal mutations driving MPNs arise early in life-potentially in utero-often years before the development of symptoms (Williams et al. 2022). While the intrinsic factors driving clonal expansion are increasingly known (Watson et al. 2020), the extrinsic factors driving the expansion of clones harboring these MPN driver mutations are not well characterized. Prior work has shown that clonal mutations are geographically constrained in solid organs such as skin, colon and esophagus (Lee-Six et al. 2018; Martincorena et al. 2015, 2018). Strikingly, in the esophagus non-malignant NOTCH1 mutant clones can even outcompete nascent malignant clones (Colom et al. 2021). In the hematopoietic compartment, the spatial organization of clones harboring these mutations and their interaction with the niche and each other is largely unknown. We hypothesized that clonal mutations are geographically constrained in the bone marrow niche as is seen in solid organs. To answer these questions, we developed methods for spatially-aware clonal mutation detection.

As proof of principle, we characterized the hematopoietic clonal organization within a femur head explant from a 50 year-old man with PV undergoing hip arthroplasty. Briefly, the femur head was sectioned into 2mm slices, which were then further partitioned into approximately 300 bone marrow fragments (2mm³). Approximately 20% of the fragments were snap frozen and pulverized for DNA extraction. The remainder were formalin fixed and decalcified for correlative imaging. Targeted error-corrected sequencing was performed using a myeloid-specific panel (Duncavage et al. 2018) on 24 bone marrow fragments, and bulk peripheral blood and bone marrow aspirate. In the peripheral blood, JAK2 V617F (0.24 VAF) and TET2 L1081* (0.02 VAF) mutations were identified. In the bone marrow aspirate, the same JAK2 (0.62 VAF) and TET2 (0.05 VAF) mutations were identified in addition to a DNMT3A R882C mutation (0.02 VAF). Interestingly, peripheral blood sequencing two years prior identified the JAK2 and DNMT3A mutations, but not the TET2 mutation, suggesting recent clonal expansion of TET2 mutant clone.

From the 24 sequenced bone marrow fragments, the geographical organization of clonal mutations in the bone marrow was determined (Figure 1). The JAK2 V617F clone was identified in all bone marrow fragments sequenced (0.12-0.69 VAF). The TET2 L1081* clone was identified in 11/24 bone marrow fragments with some regional constraint (0.03-0.05 VAF). The DNMT3A R882C mutation was detected in only a single bone marrow fragment (0.02 VAF) and co-localized with the JAK2 V617F and TET2 L1081* mutations. A previously undetected clonal SETBP1 E616V mutation was identified in two adjacent bone marrow fragments from sequential slices of the femur head (0.05 VAF and 0.10 VAF). Together this provides the first characterization of spatial organization of clonal mutations in the bone marrow niche for an MPN patient.

Currently, we are utilizing digital droplet PCR targeting these detected mutations in ~60 additional bone marrow fragments to accurately quantify clone size across the tissue. We plan to employ multiplex fluorescent imaging of bone marrow fragments adjacent to detected clusters of mutations to identify the surrounding bone marrow niche elements in those regions. These findings suggest that bone marrow biopsy from a small section of bone marrow may not accurately describe disease state and clonal mutation burden in myeloid neoplasms. Future applications of these tools will help describe the extrinsic factors and local microenvironment that drive clonal evolution over time in the bone marrow.

Spencer:Wugen Inc.: Consultancy. Oh:Celgne/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Challen:Incyte: Consultancy, Other: Sponsored Research agreements.