A RUNX1-FPDMM rhesus macaque model reproduces the human phenotype and predicts challenges to curative gene therapies.

Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

Natural history study of patients with familial platelet disorder with associated myeloid malignancy.

ABSTRACT: Deleterious germ line RUNX1 variants cause the autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematologic malignancies (HMs). We launched a FPDMM natural history study and, from January 2019 to December 2021, enrolled 214 participants, including 111 patients with 39 different RUNX1 variants from 45 unrelated families. Seventy of 77 patients had thrombocytopenia, 18 of 18 had abnormal platelet aggregometry, 16 of 35 had decreased platelet dense granules, and 28 of 55 had abnormal bleeding scores. Nonmalignant bone marrows showed increased numbers of megakaryocytes in 12 of 55 patients, dysmegakaryopoiesis in 42 of 55, and reduced cellularity for age in 30 of 55 adult and 17 of 21 pediatric cases. Of 111 patients, 19 were diagnosed with HMs, including myelodysplastic syndrome, acute myeloid leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and smoldering myeloma. Of those 19, 18 were relapsed or refractory to upfront therapy and referred for stem cell transplantation. In addition, 28 of 45 families had at least 1 member with HM. Moreover, 42 of 45 patients had allergic symptoms, and 24 of 30 had gastrointestinal (GI) symptoms. Our results highlight the importance of a multidisciplinary approach, early malignancy detection, and wider awareness of inherited disorders. This actively accruing, longitudinal study will genotype and phenotype more patients with FPDMM, which may lead to a better understanding of the disease pathogenesis and clinical course, which may then inform preventive and therapeutic interventions. This trial was registered at www.clinicaltrials.gov as #NCT03854318.

Efficacy of MEK inhibition in patients with histiocytic neoplasms.

Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway1,2. For the 50% of patients with histiocytosis who have BRAFV600 mutations3-5, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease6,7. However, no standard therapy exists for the remaining 50% of patients who lack BRAFV600 mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAFV600 mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73-100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1 (also known as MAP2K1) and MEK2 (also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling-and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.

Refining Diagnostic Subtypes of Peripheral T-cell Lymphoma Using a Multiparameter Approach.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous category, and many cases are unclassifiable and designated as PTCL-not otherwise specified (PTCL-NOS). Gene expression profiling (GEP) has delineated two prognostic subtypes within PTCL-NOS, PTCL-TBX21, and PTCL-GATA3, characterized by distinctive transcriptomes and a different prognosis. To further evaluate the pathologic features of these subgroups, 101 PTCL cases that did not meet specific criteria for well-defined T-cell lymphoma entities underwent detailed pathologic, immunophenotypic (including TFH biomarkers) and GEP analyses, separating them into PTCL-NOS (n=63) and PTCL-TFH (a.k.a. nodal PTCL-TFH, NOS, and TFH lymphoma, NOS) (n=38). PTCL-NOS cases were further categorized into PTCL-GATA3 (n=22; 34%) and PTCL-TBX21 (n=41; 66%), and a significant association (p < 0.02) with overall survival (OS) was reaffirmed. Histopathological assessment showed PTCL-GATA3 cases were characterized by monotonous medium-sized or large transformed cells with a minimal tumor microenvironment (TME) compared to PTCL-TBX21 cases, which consisted of pleomorphic cells in a polymorphous TME (p < 0.05). GEP analysis validated these TME distinctions. Immunophenotypic analysis showed that PTCL-GATA3 cases were predominantly CD4+CD8- and associated with significantly higher LEF1, MYC, and CD30 expression (p < 0.05). PTCL-TBX21 displayed a more diverse biomarker profile with two subgroups: one expressing cytotoxic antigens and enriched in CD8+CD4- or CD8-CD4- phenotype, and another lacking cytotoxic markers but showing a CD4+CD8- phenotype with increased ICOS expression, but devoid of other TFH markers. The PTCL-TFH cases correlated with an angioimmunoblastic T-cell lymphoma (AITL) gene signature, had more EBER-positive cells than the PTCL-GATA3 and PTCL-TBX21 cases, and a subset had some morphologic features of AITL (p < 0.01). This study highlights the unique morphologic and phenotypic variations within the newly-identified PTCL subtypes and should enable more precise diagnosis and tailored therapeutic strategies in the future.

A somatic mutation in erythro-myeloid progenitors causes neurodegenerative disease.

The pathophysiology of neurodegenerative diseases is poorly understood and there are few therapeutic options. Neurodegenerative diseases are characterized by progressive neuronal dysfunction and loss, and chronic glial activation. Whether microglial activation, which is generally viewed as a secondary process, is harmful or protective in neurodegeneration remains unclear. Late-onset neurodegenerative disease observed in patients with histiocytoses, which are clonal myeloid diseases associated with somatic mutations in the RAS-MEK-ERK pathway such as BRAF(V600E), suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet the expression of BRAF(V600E) in the haematopoietic stem cell lineage causes leukaemic and tumoural diseases but not neurodegenerative disease. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk-sac erythro-myeloid progenitors (EMPs) distinct from haematopoietic stem cells. We therefore hypothesized that a somatic BRAF(V600E) mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAF(V600E) in mouse EMPs results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder. This is associated with accumulation of ERK-activated amoeboid microglia in mice, and is also observed in human patients with histiocytoses. In the mouse model, neurobehavioural signs, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses and demonstrate that a somatic mutation in the EMP lineage in mice can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration and this offers opportunities for therapeutic intervention aimed at the prevention of neuronal death in neurodegenerative diseases.

High prevalence of myeloid neoplasms in adults with non-Langerhans cell histiocytosis.

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis that most commonly affects adults and is driven by a high frequency of mutations in BRAF, MAP2K1, and kinases promoting MAPK signaling. Because of the relative rarity of ECD, key clinical features of the disease may not be well defined. Across a multi-institutional cohort of 189 patients with ECD and ECD overlapping with Langerhans cell histiocytosis (so-called mixed histiocytosis [MH]), we identified an unexpected and heretofore undescribed frequent occurrence of myeloid neoplasms among patients with ECD and MH. Some 10.1% (19/189) of patients with ECD have an overlapping myeloid neoplasm, most commonly occurring as a myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), or mixed MDS/MPN overlap syndrome (including chronic myelomonocytic leukemia). Consistent with this, molecular analysis frequently detected hallmark driver mutations of myeloid neoplasms (such as JAK2V617F and CALR mutations) coexisting with those characteristic of histiocytosis (such as BRAFV600E and MAP2K1 mutations). Histiocytosis patients diagnosed with a concomitant myeloid malignancy were significantly older at diagnosis and more commonly presented with MH than those without a myeloid malignancy. In some cases, the presence of distinct kinase mutations in the histiocytosis and myeloid neoplasm resulted in discordant and adverse responses to kinase-directed targeted therapies. These data highlight the clinical importance of evaluating adults with histiocytosis for a concomitant myeloid neoplasm.

Functional evidence for derivation of systemic histiocytic neoplasms from hematopoietic stem/progenitor cells.

Langerhans cell histiocytosis (LCH) and the non-LCH neoplasm Erdheim-Chester disease (ECD) are heterogeneous neoplastic disorders marked by infiltration of pathologic macrophage-, dendritic cell-, or monocyte-derived cells in tissues driven by recurrent mutations activating MAPK signaling. Although recent data indicate that at least a proportion of LCH and ECD patients have detectable activating kinase mutations in circulating hematopoietic cells and bone marrow-based hematopoietic progenitors, functional evidence of the cell of origin of histiocytosis from actual patient materials has long been elusive. Here, we provide evidence for mutations in MAPK signaling intermediates in CD34+ cells from patients with ECD and LCH/ECD, including detection of shared origin of LCH and acute myelomonocytic leukemia driven by TET2-mutant CD34+ cell progenitors in one patient. We also demonstrate functional self-renewal capacity for CD34+ cells to drive the development of histiocytosis in xenotransplantation assays in vivo. These data indicate that the cell of origin of at least a proportion of patients with systemic histiocytoses resides in hematopoietic progenitor cells prior to committed monocyte/macrophage or dendritic cell differentiation and provide the first example of a patient-derived xenotransplantation model for a human histiocytic neoplasm.

The histopathology of Erdheim-Chester disease: a comprehensive review of a molecularly characterized cohort.

Erdheim-Chester disease is a rare, non-Langerhans cell histiocytosis histologically characterized by multi-systemic proliferation of mature histiocytes in a background of inflammatory stroma. The disease can involve virtually any organ system; most commonly the bones, skin, retroperitoneum, heart, orbit, lung, and brain are affected. Although a histiocytic proliferation is the histological hallmark of the disease, a wide range of morphological appearances have been described as part of case studies or small series. A comprehensive review of histopathological features in clinically and molecularly defined Erdheim-Chester disease has yet to be characterized. To address this issue and help guide clinical practice, we comprehensively analyzed the pathological spectrum of Erdheim-Chester disease in a clinically and molecularly defined cohort. We reviewed 73 biopsies from 42 patients showing involvement by histiocytosis from a variety of organ systems, including bone (16), retroperitoneum (11), skin (19), orbit (6), brain (5), lung (6), cardiac structures (2), epidural soft tissue (3), oral cavity (2), subcutaneous soft tissue (2), and testis (2). In eight patients, one or more bone marrow biopsies were performed due to clinical indication and an accompanying myeloid neoplasm was detected in six of them. Thirty-eight cases were investigated for genetic abnormalities. Somatic mutations involving BRAF (25/38), MAP2K1 (6/38), ARAF (2/38), MAP2K2 (1/38), KRAS (1/38), and NRAS (1/38) genes were detected. One of the cases with a MAP2K1 mutation also harbored a PIK3CA mutation. We have observed marked heterogeneity in histology and immunophenotype, identified site-specific features, overlap with other histiocytic and myeloid disorders and potential diagnostic pitfalls. We hope that broadening the spectrum of recognized pathologic manifestations of Erdheim-Chester disease will help practicing clinicians and pathologists to diagnose Erdheim-Chester disease early in the disease course and manage these patients effectively.

Acute leukaemia with a pure erythroid phenotype: under-recognized morphological and cytogenetic signatures associated universally with primary refractory disease and a dismal clinical outcome.

AIMS: Pure erythroid leukaemia (PEL) is an extremely rare and aggressive subtype of acute myeloid leukaemia defined by the World Health Organization (WHO) as a neoplastic proliferation of immature cells committed exclusively to the erythroid lineage, comprising >80% of bone marrow cells and not meeting the criteria of other well-defined myeloid neoplasms. The aim of this study was to describe the clinicopathological features of acute leukaemias with a pure erythroid phenotype (ALPEP) irrespective of their WHO classification and to determine if ALPEP represents a distinct clinicopathological entity. METHODS AND RESULTS: We identified seven cases of ALPEP, in which immature cells fulfilled WHO morphological and immunophenotypical criteria for PEL. All patients except one were male, with a median age of 60 years. Three cases represented de novo PEL, three were therapy-related myeloid neoplasms and one was a blast phase of a myeloproliferative neoplasm. Extensive tumour necrosis was present in five cases (71%). Five cases with available modal karyotypes all demonstrated a complex karyotype involving the TP53 gene locus, with three cases (60%) also showing a monosomy 5 or deletion 5q and additional material on chromosome 19q13. All patients died of their disease, with a mean overall survival of 189 and 64.7 days in cases without and with necrosis on the initial biopsy, respectively. CONCLUSIONS: We describe previously unreported but relatively common findings of extensive tumour necrosis and recurring cytogenetic abnormalities in ALPEP. Our findings suggest strongly that ALPEP represents a distinct clinicopathological entity regardless of its WHO classification.

Novel insights into the early histopathogenesis of immunodeficiency-associated Burkitt lymphoma: a case report of Burkitt microlymphoma arising within HIV lymphadenitis.

AIMS: To report a case detailing the early histopathogenesis of Burkitt lymphoma (BL), Epstein-Barr virus (EBV) positive, in the clinical setting of HIV infection. METHODS AND RESULTS: We describe a 57 year-old woman with newly diagnosed HIV infection who presented with unilateral localized axillary lymphadenopathy. Histopathological, immunohistochemical, and flow cytometric analyses were performed and showed EBV-positive nests of Burkitt cells, including rare nests also positive for cytomegalovirus, exclusively located within hyperplastic monocytoid B-cell areas of HIV lymphadenitis. Due to positron emission tomography/computed tomography findings of persistent localized lymphadenopathy with markedly increased fludeoxyglucose avidity a core needle biopsy was performed. Histopathological, immunohistochemical, fluorescence in situ hybridization, and flow cytometric analyses confirmed BL. CONCLUSIONS: Early histopathogenesis of HIV-associated BL occurred as nests of EBV-positive Burkitt cells within prominent monocytoid B-cell areas in our case. The results equip the pathologist to identify a very subtle Burkitt microlymphoma which would allow for prompt and appropriate chemotherapeutic intervention and may promote research into the possible relationship between monocytoid B cells and BL.

Indeterminate DC histiocytosis is distinct from LCH and often associated with other hematopoietic neoplasms.

Indeterminate dendritic cell histiocytosis (IDCH) is a rare and poorly understood entity characterized by accumulation of CD1a+/S100+ histiocytes (as Langerhans cell histiocytosis (LCH)), but with reduced-absent expression of Langerin/CD207. We assembled 43 cases of IDCH (defined by CD1a+/CD207<20% immunophenotypic profile) examining the clinical, pathologic, and molecular landscape. Median age at presentation was 70 years (IQR: 44-80 years) with cutaneous (31/43; 72%) and nodal (11/43; 26%) involvement predominating. Eighteen (42%) individuals had an associated non-histiocytic hematopoietic neoplasm ('secondary' IDCH) while 7 of 43 (16%) had a concurrent non-IDCH histiocytosis ('mixed' histiocytosis). Most cases exhibited morphology indistinguishable from LCH but with a CD1c+/CSF1R(CD115)-phenotype, mirroring the signature of normal indeterminate cells and conventional dendritic cell type 2. Mutational analysis revealed frequent KRAS (13/32; 41%), and BRAF p.V600E (11/36, 31%) mutations that were nearly mutually exclusive. RNAseq analysis uncovered ETV3::NCOA2 fusion in 6 other patients presenting as a sole genetic alteration without any other concurrent histiocytic or hematopoietic neoplasm. BRAF and MAP2K1 alterations were significantly associated with partial/retained (1-20%) Langerin expression (P = .005) and mixed histiocytosis (P = .002). Remarkably, myeloid alterations (DNMT3A, TET2 and SRSF2) co-occurred in IDCH tissues of several individuals. Paired sequencing of IDCH and concurrent non-IDCH hematopoietic neoplasm in four individuals revealed shared mutations. Age at diagnosis and any nodal involvement at diagnosis predicted inferior overall survival, but BRAF/RAS pathway alterations did not impact outcome. These data have implications for the diagnostic evaluation, classification, and therapeutic management of IDCH.

Localized Peritumoral AL Amyloidosis Associated With Mantle Cell Lymphoma With Plasmacytic Differentiation.

Immunoglobulin light chain (AL) amyloidosis is characterized by the deposition of amyloid fibers derived from pathologic immunoglobulin light chains. Although systemic plasma cell neoplasms are the most common cause of AL amyloidosis, a subset of cases is caused by B-cell lymphoproliferative disorders such as lymphoplasmacytic lymphoma or extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue. Recently, SOX11-negative IGH hypermutated mantle cell lymphoma (MCL) is recognized to show frequent plasmacytic differentiation and indolent clinical course. Here, we report 3 cases of peritumoral AL amyloidosis associated with SOX11-negative MCL. All 3 cases showed cyclin D1 expression by immunohistochemistry and CCND1 translocation as detected by fluorescence in situ hybridization analysis. Peritumoral AL amyloidosis was observed at the biopsy sites in the gastrointestinal tract, a supraclavicular lymph node, and a cervical lymph node, and all presented with marked plasmacytic differentiation of lymphoma cells. None of the cases showed evidence of bone marrow involvement by morphology and immunophenotyping. None of the patients had distant organ involvement with systemic amyloidosis. All 3 patients had an indolent clinical course and are alive with disease at the time of the last follow-up (range: 48 to 74 mo). Our findings show that MCL with plasmacytic differentiation can cause amyloid deposition and CCND1 abnormalities should be performed in all cases of extramedullary AL amyloidosis. Recognition of indolent MCL as a cause of peritumoral AL amyloidosis may have important clinical management implications.

Neurological manifestations of Erdheim-Chester Disease.

OBJECTIVE: To characterize the spectrum of neurologic involvement in Erdheim-Chester Disease (ECD), a treatable inflammatory neoplasm of histiocytes. METHODS: Sixty-two patients with ECD were prospectively enrolled in a natural history study that facilitated collection of clinical, imaging, laboratory, neurophysiologic, and pathologic data. RESULTS: Ninety-four percent of the patients had neurologic abnormalities on examination or imaging, and 22% had neurologic symptoms as the initial presentation of ECD. The most common neurologic findings were cognitive impairment, peripheral neuropathy, pyramidal tract signs, cranial nerve involvement, and cerebellar ataxia. Imaging revealed atrophy and demyelination along with focal lesions that were located throughout the nervous system, dura, and extra-axial structures. The BRAF V600E variant correlated with cerebral atrophy. Brain pathology revealed lipid-laden, phagocytic macrophages (histiocytes) accompanied by demyelination and axonal degeneration. INTERPRETATION: In patients with ECD, neurologic morbidity is common and contributes significantly to disability. Since neurologic symptoms can be the presenting feature of ECD and, given the mean delay in ECD diagnosis is 4.2 years, it is critical that neurologists consider of ECD and other histiocytosis in patients with inflammatory, infectious, or neoplastic-appearing white matter. Furthermore, given the broad spectrum of neurologic involvement, neurologists have an important role in a team of specialists treating ECD patients.

Neurologic and oncologic features of Erdheim-Chester disease: a 30-patient series.

BACKGROUND: Erdheim-Chester disease (ECD) is a rare histiocytic neoplasm characterized by recurrent alterations in the MAPK (mitogen-activating protein kinase) pathway. The existing literature about the neuro-oncological spectrum of ECD is limited. METHODS: We present retrospective clinical, radiographic, pathologic, molecular, and treatment data from 30 patients with ECD neurohistiocytic involvement treated at a tertiary center. RESULTS: Median age was 52 years (range, 7-77), and 20 (67%) patients were male. Presenting symptoms included ataxia in 19 patients (63%), dysarthria in 14 (47%), diabetes insipidus in 12 (40%), cognitive impairment in 10 (33%), and bulbar affect in 9 (30%). Neurosurgical biopsy specimens in 8 patients demonstrated varied morphologic findings often uncharacteristic of typical ECD lesions. Molecular analysis revealed mutations in BRAF (18 patients), MAP2K1 (5), RAS isoforms (2), and 2 fusions involving BRAF and ALK. Conventional therapies (corticosteroids, immunosuppressants, interferon-alpha [IFN-α], cytotoxic chemotherapy) led to partial radiographic response in 8/40 patients (20%) by MRI with no complete responses, partial metabolic response in 4/16 (25%), and complete metabolic response in 1/16 (6%) by 18F-fluorodeoxyglucose (FDG)-PET scan. In comparison, targeted (kinase inhibitor) therapies yielded partial radiographic response in 10/27 (37%) and complete radiographic response in 14/27 (52%) by MRI, and partial metabolic response in 6/25 (24%) and complete metabolic response in 17/25 (68%) by FDG-PET scan. CONCLUSIONS: These data highlight underrecognized symptomatology, heterogeneous neuropathology, and robust responses to targeted therapies across the mutational spectrum in ECD patients with neurological involvement, particularly when conventional therapies have failed.

Clonal hematopoiesis in angioimmunoblastic T-cell lymphoma with divergent evolution to myeloid neoplasms.

TET2 and DNMT3A mutations are frequently identified in T-cell lymphomas of T follicular helper cell origin (TCL-TFH), clonal hematopoiesis (CH), and myeloid neoplasms (MNs). The relationships among these 3 entities, however, are not well understood. We performed comprehensive genomic studies on paired bone marrow and tissue samples as well as on flow cytometry-sorted bone marrow and peripheral blood subpopulations from a cohort of 22 patients with TCL-TFH to identify shared CH-type mutations in various hematopoietic cell compartments. Identical mutations were detected in the neoplastic T-cell and myeloid compartments of 15 out of 22 patients (68%), including TET2 (14/15) and DNMT3A (10/15). Four patients developed MNs, all of which shared CH-type mutations with their TCL-TFH; additional unique genetic alterations were also detected in each patient's TCL-TFH and MN. These data demonstrate that CH is prevalent in patients with TCL-TFH and that divergent evolution of a CH clone may give rise to both TCL-TFH and MNs.