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, 4 (4), 717-727

High-risk LCH in Infants Is Serially Transplantable in a Xenograft Model but Responds Durably to Targeted Therapy

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High-risk LCH in Infants Is Serially Transplantable in a Xenograft Model but Responds Durably to Targeted Therapy

Lynn H Lee et al. Blood Adv.

Abstract

Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm characterized by a clonal proliferation of Langerhans-like cells. Genomic profiling has identified recurrent somatic activating mutations in the mitogen-activated protein kinase pathway, which are targetable by small-molecule inhibitors. However, key questions such as the curative potential of targeted therapy and the cell of origin remain unanswered. In this study, we describe clinical outcomes of a series of pediatric patients with multisystem BRAF V600E-mutant LCH, as well as the results of accompanying murine xenograft experiments. Four infants with LCH (range, 7-11 months at diagnosis) and secondary hemophagocytic lymphohistiocytosis were referred to our institution and subsequently treated with the BRAF V600E-specific inhibitor dabrafenib. All patients achieved complete clinical responses by 8 weeks of therapy, with remissions lasting a median of 36 months (range, 27-42 months). One infant successfully discontinued therapy long-term upon achieving a molecular response by real-time quantitative polymerase chain reaction (RT-qPCR). We further characterized the disease-propagating cell population in a subset of these patients by transplanting whole bone marrow into immunodeficient mice. Xenografted animals exhibited decreased survival with hematologic abnormalities, splenomegaly, and histiocytic infiltrates in the bone marrow resembling human disease. This process could also be secondarily transplanted, resulting in a comparable disease latency with similar histologic findings. These data further support the presence of a disease-initiating cell in the bone marrow compartment. We demonstrate that despite aggressive disease behavior in a xenograft model, these patients can achieve sustained clinical remissions with targeted monotherapy, with a select subset achieving molecular responses by RT-qPCR.

Conflict of interest statement

Conflict-of-interest disclosure: A.R.K. received consultancy fees from Novimmune SA and Sobi for primary HLH therapy. The remaining authors declare no competing financial interests.

Figures

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Figure 1.
Figure 1.
Imaging and histologic features of patient responses to dabrafenib. (A) Representative fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography of patients 3 and 4 demonstrating responses to dabrafenib. Pretherapy imaging in patient 3 (leftmost panel) demonstrates significant thymic, cervical, periportal FDG avidity as well as diffuse bone marrow uptake, with near-complete resolution at 7 weeks after start of dabrafenib. Similarly, patient 4 exhibited diffuse, extensive FDG avidity of the marrow space, lymph nodes, liver, and spleen. Repeat imaging obtained after 6 weeks of dabrafenib (right) showed dramatic improvement of hypermetabolic areas. Bone marrow examination of patient 2 at diagnosis revealed an increased number of histiocytes (B; original magnification ×400, hematoxylin and eosin stain), which was confirmed by CD163 immunohistochemistry (C; original magnification ×400). The histiocytes are BRAF positive (red reaction product) as assessed by BRAF V600E–specific immunohistochemistry (D; original magnification ×400). The histiocytic hyperplasia persisted following dabrafenib therapy (E; original magnification ×400, hematoxylin and eosin stain), as confirmed by CD163 immunohistochemistry (F; original magnification ×400). BRAF immunohistochemistry (brown reaction product) labels some histiocytes but also smaller cells with myeloid morphology (G; original magnification ×400). In patient 1, despite dabrafenib therapy, hemophagocytic histiocytes remained evident in the bone marrow aspirate (H; original magnification ×1000). Bone marrow histiocytic hyperplasia was confirmed by CD163 immunohistochemistry (I, original magnification ×400; J, original magnification ×1000), which highlights scattered hemophagocytic forms (arrow). Numerous BRAF-positive cells were present, as assessed using BRAF V600E–specific immunohistochemistry (K, original magnification ×400; L, original magnification ×1000, brown reaction product), including histiocytes (L, black arrows) as well as cells with myeloid morphology (L, cross-hatched arrows).
Figure 2.
Figure 2.
Digital droplet PCR evaluation of patient samples at remission and reactivation. (A) Representative plots of ddPCR performed on RT-qPCR–negative samples for patients 4 (left) and 3 (right), with wild-type (WT) droplets on the vertical axis and mutant BRAF V600E on the horizontal axis. Both still exhibit a small population of BRAF V600E–mutant alleles, as evidenced by small cluster of FAM-positive droplets. (B-C) ddPCR performed on bone marrow 14 months after discontinuation of dabrafenib in patient 4 (B) and peripheral blood at time of relapse in patient 3 (C). a.u., arbitrary units.
Figure 3.
Figure 3.
Patient-derived bone marrow successfully engrafts primarily and secondarily in immunodeficient mice. (A) Representative flow cytometry scatterplots of human (h; x-axis) vs mouse (m; y-axis) engraftment of xenograft recipients following transplant of bone marrow from either RO-positive patients with hematopoietic involvement (BM+, right) or from an LCH patient without bone marrow involvement (BM−, left). (B) Survival curves of xenografted experimental animals. Survival between control and NSGS mice was statistically significant (*P < .01) by log-rank test. (C) Comparison between spleen sizes in control and experimental groups, with representative spleen images. (D) Hematologic abnormalities observed in BM+ and BM− transplanted animals. Error bars represent mean ± standard deviation. Statistical significance determined by Student t test. (E) Bone marrow histology from representative animals (original magnification ×400). (F) Representative flow scatterplots of bone marrow and spleens from a secondary transplant recipient animal. (G) Bone marrow from secondary transplant NSGS mouse shows a similar infiltration of histiocytes with abundant eosinophilic cytoplasm (left, original magnification ×400) and cytospins of splenocytes demonstrate frequent hemophagocytosis (white arrowheads) with numerous large, activated and vacuolated macrophages (right, original magnification ×1000). H&E, hematoxylin and eosin stain; WBC, white blood cell.

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