High-Risk Langerhans Cell Histiocytosis in Infants Exhibits Malignant Features in a Xenograft Model but Responds Durably to Targeted Therapy

Background: Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm primarily of childhood, characterized by a neoplastic proliferation of Langerhans-like cells. The clinical presentation of LCH is highly variable, and while some children are cured with combination chemotherapy, many will relapse and experience irreversible morbidity. Genomic profiling of LCH has identified recurrent somatic activating mutations in BRAF and MAP2K1, all of which culminate in activation of the mitogen-activated protein kinase pathway. However, key mechanistic and clinical questions such as the curative potential of targeted therapy and the cell of origin remain unanswered.

Methods: Four infants with risk-organ positive (RO+) multisystem BRAF V600E-mutant LCH and secondary hemophagocytosis were treated at our institution with the BRAF V600E-inhibitor dabrafenib after failing chemotherapy, or up front at the discretion of the treating physician. Response assessment was performed as indicated for good clinical care. Excess bone marrow obtained during routine clinical evaluation was collected under an IRB-approved protocol, and transplanted into busulfan-conditioned NOD/LtSz-SCID interleukin-2(IL2)RG^-/- (NSG) or NSG-SGM3 (NSGS) mice. Donor chimerism was assessed by flow cytometry for mouse and human CD45 and lineage reconstitution was determined with standard human markers. BRAF V600E detection was performed on extracted DNA from xenografts using a TaqMan Mutation Detection RTPCR assay.

Results: At presentation, all patients exhibited features of HLH (hemophagocytic lymphohistiocytosis) with fever, splenomegaly, pancytopenia, elevated markers of inflammation, hypofibrinogenemia, and hemophagocytosis on bone marrow examination. All patients displayed marked and rapid improvement following initiation of dabrafenib. With a median follow up of 21 months, all patients remain in complete clinical remission, and are thriving. None of the patients experienced significant adverse effects classically associated with dabrafenib. In two patients, despite clinical disease resolution, BRAF V600E was still detectable in bone marrow by RTPCR at 23 and 26 months respectively post initiation of dabrafenib. Remarkably, the other two patients who were treated upfront with dabrafenib achieved molecular remissions (defined as undetectable BRAF V600E allele in a previously positive patient) after 11 and 12 months respectively of therapy.

Among mice transplanted with patient bone marrow cells, the median survival was 9.6 weeks. A comparison arm consisting of mice transplanted with bone marrow from a low-risk LCH patient did not develop evidence of disease during the observation period. At necropsy, animals exhibited splenomegaly, thrombocytopenia and anemia. Flow cytometry of both bone marrow and spleens from mice demonstrated robust engraftment of human hematopoietic cells, and the BRAF V600E allele was detected from marrow and splenocytes in all mice. Frequent hemophagocytic forms were visible on bone marrow cytospins, and on histologic examination, bone marrow exhibited an abundance of CD1a-negative, CD163+ histiocytes, similar to findings observed in human disease.

Bone marrow and splenocytes from primary NSG xenograft recipients were transplanted into secondary NSGS mice, and two out of three animals examined similarly became ill with comparable disease latency, and achieved engraftment of human cells with detectable BRAF V600E in bone marrow and splenocytes.

Conclusions: For patients with LCH with hematologic involvement and secondary HLH, disease-initiating cells are present in the bone marrow and can engraft and recapitulate disease following primary and secondary transplant into immunodeficient mice. Targeted therapy with the BRAF V600E-specific inhibitor dabrafenib leads to durable, sustained remissions. Furthermore, we demonstrate that certain patients can achieve molecular remission with BRAF V600E-specific monotherapy alone. However, in at least a subset of patients with RO+ LCH, the persistence of mutated BRAF despite prolonged targeted therapy suggests that while quiescent, transformed disease-initiating cells are not eliminated by BRAF inhibition alone. Based on these results, we hypothesize that first-line therapy with BRAF inhibitors may be more likely to induce molecular remissions.