Histiocytoses are clonal hematopoietic disorders frequently driven by mutations mapping to the BRAF and MEK1 and MEK2 kinases. Currently, however, the developmental origins of histiocytoses in patients are not well understood, and clinically meaningful therapeutic targets outside of BRAF and MEK are undefined. In this study, we uncovered activating mutations in CSF1R and rearrangements in RET and ALK that conferred dramatic responses to selective inhibition of RET (selpercatinib) and crizotinib, respectively, in patients with histiocytosis.

Somatic mutations that increase the fitness of hematopoietic stem cells (HSCs) drive their expansion in clonal hematopoiesis (CH) and predispose individuals to blood cancers. Population variation in the growth rate and potential of mutant clones suggests that genetic factors may confer resilience against CH. Here, we identified a noncoding regulatory variant, rs17834140-T, that protects against CH and myeloid malignancies by selectively down-regulating the RNA-binding protein MSI2 in HSCs. By modeling variant effects and mapping MSI2 binding targets, we uncovered an RNA network that maintains human HSCs and influences CH risk. Variant rs17834140-T was associated with slower CH expansion, and stem cell MSI2 levels modified ASXL1-mutant HSC clonal dominance. These findings leverage natural resilience to illuminate posttranscriptional regulation in human HSCs, suggesting that inhibition of MSI2 or its targets could be rational strategies for blood cancer prevention.

ABSTRACT:Malignant histiocytic neoplasms (MHNs) are rare tumors derived from the mononuclear phagocyte system (MPS), encompassing histiocytic sarcoma, Langerhans cell sarcoma, interdigitating dendritic cell sarcoma, and other high-grade MPS lineage tumors. Despite advances in understanding histiocytic neoplasms, MHNs remain diagnostically challenging and lack standardized treatment algorithms. Current classification systems differ in lineage framing and fail to address mixed or ambiguous phenotypes, contributing to diagnostic uncertainty and inconsistent care. To address these gaps, the Histiocyte Society convened an international working group of pathologists and oncologists, including World Health Organization and International Consensus Classification contributors, to harmonize nomenclature, define minimum diagnostic criteria, and develop pragmatic treatment recommendations. Using a modified Delphi process and case-based review, the group formulated >40 consensus statements spanning classification, pathology, molecular testing, clinical evaluation, and therapeutic strategies. Key recommendations include the adoption of a unified MHN designation, use of a minimum immunophenotypic panel, integration of broad molecular profiling, and the documentation of previous hematopoietic malignancy. Treatment algorithms emphasize surgical resection for unifocal disease and targeted therapy or immune checkpoint inhibition for multifocal disease when actionable mutations or programmed death ligand 1 expression are present. These consensus recommendations aim to reduce diagnostic ambiguity, standardize reporting, and improve outcomes for both pediatric and adult patients. Future priorities include international registries to refine risk stratification and biomarker-driven trials exploring targeted therapy, immunotherapy, and combination approaches.

Somatic mutations that increase hematopoietic stem cell (HSC) fitness drive their expansion in clonal hematopoiesis (CH) and predispose to blood cancers. Although CH frequently occurs with aging, it rarely progresses to overt malignancy. Population variation in the growth rate and potential of mutant clones suggests the presence of genetic factors protecting against CH, but these remain largely undefined. Here, we identify a non-coding regulatory variant, rs17834140-T, that significantly protects against CH and myeloid malignancies by downregulating HSC-selective expression and function of the RNA-binding protein MSI2. By modeling variant effects and mapping MSI2 binding targets, we uncover an RNA network that maintains human HSCs and influences CH risk. Importantly, rs17834140-T is associated with slower CH expansion rates in humans, and stem cell MSI2 levels modify ASXL1-mutant HSC clonal dominance in experimental models. These findings leverage natural resilience to highlight a key role for post-transcriptional regulation in human HSCs, and offer genetic evidence supporting inhibition of MSI2 or its downstream targets as rational strategies for blood cancer prevention.

Patient demographic data for the female patients in the Extragonadal GCT cohort (both Make-an-IMPACT and internal MSK patients, n=16)

The histiocytic and dendritic cell neoplasms encompass a clinically heterogeneous group of disorders leading to tissue damage secondary to the accumulation and infiltration of pathologic cells thought to be derived from the dendritic or monocytic lineages with accompanying inflammation. The pathophysiology of these disorders is poorly understood. Studies over the past 15 y have identified a high-frequency of BRAFV600E, MAP2K1, and other kinase alterations in the histiocytic neoplasms. This review highlights the onslaught of molecular advancements and discusses the impact these insights have had on our understanding of the molecular pathophysiology and therapeutic targets of these rare, enigmatic diseases.

Histiocytic neoplasms are clonal disorders of the monocyte/macrophage lineage defined by mutations activating mitogen-activated protein kinase (MAPK) signaling. Recently, the MEK1/2 inhibitor cobimetinib was FDA-approved for patients with adult histiocytoses. Here, aided by a prospective registry of patients with histiocytoses (NCT03329274), we identify that MEK1/2 mutations which constitutively activate MEK independently of RAF are associated with worse progression-free survival with MEK1/2 inhibition as compared to patients with other MEK1/2 mutational classes. The most common RAF-independent MEK1 mutation (MEK1E102_I103del) drove a lethal histiocytic-like neoplasm in mice, which was sensitive to the ERK1/2 inhibitor ulixertinib. We subsequently treated five MEK1E102_I103del-mutant patients with ulixertinib on prospective protocols, four of whom were refractory to MEK inhibition. Four of five patients experienced objective responses to ulixertinib. These data reveal the impact of oncogenic MEK mutations in vivo, identify patients with likelihood of resistance to MEK inhibition, and nominate ERK inhibition to overcome resistance to MEK inhibition in histiocytoses.

Genomically matched targeted therapies guided by MSK-IMPACT testing (n=22).

ABSTRACT:Non-Langerhans cell histiocytoses are a diverse group of histiocytic diseases. Different entities are defined based on clinical, histopathologic, and/or molecular characteristics. This study aimed to define NTRK-rearranged histiocytosis. Through international collaboration, we investigated 50 cases of histiocytosis with pan-tropomyosin receptor kinase (pan-TRK) expression and/or in-frame NTRK rearrangement. We also analyzed 45 control xanthogranulomas using pan-TRK immunohistochemistry and targeted RNA sequencing. Slides were centrally reviewed; clinical and molecular data were collected. The 50 cases comprised 30 children and 20 adults with a median age of 11.5 years (range, 0-73 years) and a male predominance (64%). Most patients (88%) had disease limited to the skin, including a single skin nodule in 41 patients and multiple skin lesions in 3 others. Four newborns presented with skin lesions, hepatomegaly, and thrombocytopenia that required transfusions. The 2 remaining patients had life-threatening lesions of the brain or bronchus. All cases displayed xanthogranuloma histology, often including foamy histiocytes and Touton giant cells. Histiocytes stained positive for pan-TRK in 50 of 50 cases, whereas all 45 control xanthogranulomas without in-frame NTRK fusions stained negative. NTRK1 fusion partners included IRF2BP2 (23/46), TPM3 (12/46), SQSTM1 (3/46), PRDX1 (3/46), NPM1 (2/46), LMNA (2/46), and ARHGEF2 (1/46). Clinical outcomes were favorable, including spontaneous disease regression in 3 of 4 newborns with systemic disease, and rapid clinical response in both patients with a brain or bronchial tumor treated with the TRK inhibitor larotrectinib. This study advances the molecular characterization of histiocytoses and may guide the diagnosis and personalized treatment of patients.

Clonality assessment of known oncogenic mutations in the female GCT cohort.

SummaryHistiocytic neoplasms are diverse clonal haematopoietic disorders, and clinical disease is mediated by tumorous infiltration as well as uncontrolled systemic inflammation. Individual subtypes include Langerhans cell histiocytosis (LCH), Rosai–Dorfman–Destombes disease (RDD) and Erdheim–Chester disease (ECD), and these have been characterized with respect to clinical phenotypes, driver mutations and treatment paradigms. Less is known about patients with mixed histiocytic neoplasms (MXH), that is two or more coexisting disorders. This international collaboration examined patients with biopsy‐proven MXH with respect to component disease subtypes, oncogenic driver mutations and responses to conventional (chemotherapeutic or immunosuppressive) versus targeted (BRAF or MEK inhibitor) therapies. Twenty‐seven patients were studied with ECD/LCH (19/27), ECD/RDD (6/27), RDD/LCH (1/27) and ECD/RDD/LCH (1/27). Mutations previously undescribed in MXH were identified, including KRAS, MAP2K2, MAPK3, non‐V600‐BRAF, RAF1 and a BICD2‐BRAF fusion. A repeated‐measure generalized estimating equation demonstrated that targeted treatment was statistically significantly (1) more likely to result in a complete response (CR), partial response (PR) or stable disease (SD) (odds ratio [OR]: 17.34, 95% CI: 2.19–137.00, p = 0.007), and (2) less likely to result in progression (OR: 0.08, 95% CI: 0.03–0.23, p < 0.0001). Histiocytic neoplasms represent an entity with underappreciated clinical and molecular diversity, poor responsiveness to conventional therapy and exquisite sensitivity to targeted therapy.

Abstract Myeloproliferative neoplasms (MPNs) are characterized by aberrant activation of the JAK-STAT pathway. Ruxolitinib (RUX) is a JAK1/2 inhibitor used to treat myelofibrosis (MF) and hydroxyurea-resistant/intolerant polycythemia vera. While RUX has demonstrated important clinical benefits, a large proportion of patients have persistent disease manifestations despite therapy. CDK8 regulates phosphorylation of STAT proteins in a RUX-independent manner; thus, CDK8 inhibition may alter downstream STAT target gene expression. RVU120 is a highly selective and potent CDK8/19 inhibitor that inhibits STAT1/5 phosphorylation. We hypothesized that the combination of RUX and RVU120 would act in a cooperative manner to attenuate JAK-STAT signaling and reduce MPN phenotypes in vivo. Using JAK2V617F mutant leukemic cell lines, we identified dose-responsive changes in viability with RUX and RVU120 in all cell lines. Pharmacodynamic analysis validated that STAT5 protein phosphorylation was decreased by RUX/RVU120, although at different phosphorylation sites by RUX (pTYR694) and RVU120 (pSER726/731); exposure to RUX+RVU120 inhibited phosphorylation at both sites. Notably, analysis of combinatorial effects of RUX+RVU120 on viability demonstrated drug synergies (combinatorial index <1) in RUX-naïve and resistant SET-2 cells. Nascent RNA expression data exploring JAK-STAT and mediator-related pathways of pathogenesis will be presented at the meeting. For in vivo studies, we used two previously-described murine models of MF and PV. Briefly, retrovirally-transfected (MSCV-MPLW515L-IRES-GFP) or Jak2V617F-conditional knock-in bone marrow (BM) was intravenously injected into lethally-irradiated congenic recipients. Mice were randomized and treated for several weeks with vehicle (VEH), single-agent RUX/RVU120, or RUX+RVU120 arms. Spleen weights between VEH and RUX/RVU120 treated mice at time of terminal sacrifice were significantly reduced across both models (p⇐0.009). In MPLW515L mice, WBCs and PB GFP% were significantly reduced (p<0.002) and histopathology demonstrated near-elimination of BM reticulin fibrosis and improved trilineage hematopoiesis between RUX+RVU120 and RUX arms. Data on overall survival, cytokine profiles, and the impact of RVU120 on dynamic gene expression will be presented at the meeting. The combination of RVU120 and RUX demonstrated biochemical synergy and differential inhibition of STAT5 phosphorylation in vitro. Further, in vivo treatment with RVU120/RUX+RVU120 resulted in significant reductions of disease manifestation (splenomegaly, WBC, fibrosis scoring, hematopoiesis) when compared to VEH/RUX. These data nominate JAK1/2 and CDK8/19 inhibition as a potential novel therapeutic strategy in MPNs; further work on nascent RNA expression and cytokine profiles will potentially elucidate additional mechanisms of action. Citation Format: Zachary Zaroogian, Elżbieta Adamczyk, Adrianna Moszyńska, Marta Obacz, Urszula Pakulska, Benjamin Durham, Milena Mazan, Shoron Mowla, Katarzyna Wnęk, Amritha Varshini Hanasoge Somasundara, Beata Barczuk, Aniela Gołas, Ross Levine, Tomasz Rzymski, Raajit Rampal. Combination JAK1/2 and CDK8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7225.

Classic hairy cell leukemia (HCL) is an indolent lymphoid malignancy characterized by the accumulation of neoplastic mature B-like cells in the peripheral blood, bone marrow, and spleen. Leukemic cell infiltration of the bone marrow typically causes bone marrow failure and fibrosis as well as splenomegaly. Virtually all cases of HCL feature the somatic BRAFV600E mutation, which originates in hematopoietic stem cells (HSCs). Despite advances in treatment and bulk gene expression studies, HCL remains incurable, and its pathogenesis remains incompletely understood. Hence, we sought to comprehensively analyze the gene expression and chromatin accessibility of HCL cells at single-cell resolution and in concert with BRAFV600E mutational status within individual cells. We applied single cell RNA sequencing to bone marrow mononuclear cells from patients who were newly diagnosed with HCL and previously untreated (n = 24,181 cells across 3 patients) and compared to age-matched bone marrow samples from healthy controls (n = 41,853 across 4 patients). HCL cells (n = 11,756 cells) formed a distinct, relatively homogeneous cluster, as confirmed through expression of HCL-defining immunophenotypic markers, scoring of a 17-gene expression signature specific to HCL, light chain monoclonality, and copy number aberrations. In 2 out of 3 HCL patients, we noted two distinct subgroups of hairy cells that were IgM+IgD+IgGlow or IgM-IgD-IgGhigh, suggesting differences in class switching. Compared to normal memory B cells, HCL cells showed an increased inflammation signature (IL2/STAT5 signaling, IFNg response) and were predicted to interact more extensively with T and NK cells through inference of receptor-ligand interactions with CellPhoneDB. This analysis recapitulated known interactions involved in HCL growth or bone marrow fibrosis including FLT3L-FLT3, FGF2-FGFR1, TGFB1-TGFB receptors, and identified pathways involved in general immune dysregulation and inflammation including CD86-CTLA4 and ICOSLG-ICOS, respectively. We also noted that CD8+ T effector cells had increased gene expression signatures of dysfunction and exhaustion, which may contribute to HCL pathogenesis. In addition to analyzing HCL cells, we examined HSCs from HCL samples, which, compared to HSCs from control samples, showed a significantly increased signature of BRAF-mutant kinase activity and were more primed towards the lymphoid (p < 0.001) lineage than to the myelo-monocytic (p < 0.001) or myelo-erythroid (p < 0.001) lineages. We next applied single-cell ATAC sequencing to bone marrow mononuclear cells from patients who were newly diagnosed with HCL and previously untreated (n = 41,247 cells across 4 patients). Hairy cells formed a single relatively homogeneous cluster. Differential transcription factor motif activity analysis between HCL (n = 430) and normal (n = 946) memory B cells revealed increased activity of transcription factors that promote memory B and suppress plasma cell development such as BACH2 (q < 0.001) and BHLHA15 (MIST1) (q < 0.001) and decreased activity of transcription factors that drive plasma cell development such as IRF4 (q < 0.001). To link genotype to phenotype at single-cell resolution in these primary HCL samples, we also performed BRAFV600E mutation identification in single cells with Genotyping of Transcriptomes (GoT) and Genotyping of Targeted loci with Chromatin Accessibility (GoTChA). With GoT, we observed expected enrichment of BRAFV600E in hairy cells and depletion in T, natural killer, and myeloid cells. This pattern was also observed through GoTChA, which enabled 8% genotyping. Altogether, we have analyzed HCL at single-cell resolution and shown that hairy cells are relatively homogeneous transcriptionally and epigenetically, resembling memory B cells. In the inflammatory and fibrotic microenvironment, HCL cells have extensive interactions with other immune cells. HCL HSCs showed an increased BRAF-mutant pathway activity signature and an increased lymphoid and decreased myeloid lineage potential, contributing to HCL pathogenesis. This work is ongoing, and future studies are warranted to examine the effect of cladribine or BRAF inhibitor treatment on cell type-specific lineage bias as well as immune dysfunction and their association with clinical outcomes.

Comprehensive genomic sequencing is becoming a critical component in the assessment of hematologic malignancies, with broad implications for patients' management. In this context, unequivocally discriminating somatic from germline events is challenging but greatly facilitated by matched analysis of tumor:normal pairs of samples. In contrast to solid tumors, in hematologic malignancies conventional sources of normal control material (peripheral blood, buccal swabs, saliva) could be highly involved by the neoplastic process, rendering them unsuitable. In this work we describe our real-world experience using cell-free DNA (cfDNA) isolated from nail clippings as an alternate source of normal control material, through the dedicated review of 2,610 tumor:nail pairs comprehensively sequenced by MSK-IMPACT-heme. Overall, we found that nail cfDNA is a robust germline control for paired genomic studies. In a subset of patients, nail DNA may be contaminated by tumor DNA, reflecting unique attributes of the hematologic disease and transplant history. Contamination is generally low level, but significantly more common among patients with myeloid neoplasms (20.5%; 304/1,482) than among those with lymphoid diseases (5.4%; 61/1,128) and particularly enriched in myeloproliferative neoplasms with marked myelofibrosis. When identified in patients with lymphoid and plasma-cell neoplasms, mutations commonly reflected a myeloid profile and correlated with a concurrent/evolving clonal myeloid neoplasm. Donor DNA was identified in 22% (11/50) of nails collected after allogeneic stem-cell transplantation. In this cohort, an association with a recent history of graft- versus-host disease was identified. These findings should be considered as a potential limitation to the use of nails as a source of normal control DNA but could also provide important diagnostic information regarding the disease process.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in posttreatment settings can be crucial for relapse risk stratification in patients with B-cell and plasma cell neoplasms. Prior studies have focused on validation of various technical aspects of the MRD assays, but more studies are warranted to establish the performance characteristics and enable standardization and broad utilization in routine clinical practice. Here, the authors describe an NGS-based IGH MRD quantification assay, incorporating a spike-in calibrator for monitoring B-cell and plasma cell neoplasms based on their unique IGH rearrangement status. Comparison of MRD status (positive or undetectable) by NGS and flow cytometry (FC) assays showed high concordance (91%, 471/519 cases) and overall good linear correlation in MRD quantitation, particularly for chronic lymphocytic leukemia and Blymphoblastic leukemia/lymphoma (R = 0.85). Quantitative correlation was lower for plasma cell neoplasms, where underestimation by FC is a known limitation. No significant effects on sequencing efficiency by the spike-in calibrator were observed, with excellent inter- and intra-assay reproducibility within the authors' laboratory, and in comparison to an external laboratory, using the same assay and protocols. Assays performed both at internal and external laboratories showed highly concordant MRD detection (100%) and quantitation (R = 0.97). Overall, this NGS-based MRD assay showed highly reproducible results with quantitation that correlated well with FC MRD assessment, particularly for Bcell neoplasms. (J Mol Diagn 2024, 26: 168-178; https://doi.org/10.1016/j.jmoldx.2023.11.009)

Langerhans cell histiocytosis (LCH), juvenile xanthogranuloma (JXG), and Rosai-Dorfman-Destombes disease (RDD) can each manifest as a focal lesion, as multiple lesions, or as a widespread systemic disorder with organ involvement. Erdheim-Chester disease (ECD) is a rare systemic disease process in children, with more frequent adult presentations. New distinct emerging entities covered include ALK-positive histiocytosis and post-leukemia/lymphoma histiocytic lesions. These histiocytic lesions are now best classified as myeloid-derived inflammatory neoplastic disorders composed of clonal dendritic- or macrophage-/monocyte-derived cells that infiltrate tissues and are driven by recurrent kinase-activating alterations, most often in the mitogen-activated protein kinase (MAPK), PI3K-AKT, and receptor tyrosine kinase (RTK) signaling pathways, which have all had a long history of being associated with human neoplasia, with ERK overexpression noted in many of these neoplasms (Fig. 28.1; Table 28.1) (1–3). Furthermore, these oncological signaling cascades are critical to the intranuclear regulation of transcription factors that serve as key factors influencing cellular proliferation, survival, and differentiation (4).

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide ameans to enhance response to checkpoint blockade that is readily translatable to the clinic.

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.