Rech, Karen L.

Background: Langerhans cell histiocytosis (LCH) is the most common histiocytic disorder that is now recognized as a neoplasm by the World Health Organization. It is generally classified based on the site and extent of disease involvement (single system or multisystem). Pulmonary LCH (pLCH), an uncommon interstitial lung disease associated with smoking, often presents as isolated pulmonary disease. It is unclear whether patients with clinically isolated pLCH have extra-pulmonary lesions at diagnosis or during the course of their disease. The role of [18F] fluorodeoxyglucose positron emission tomography-computed tomography (FDG-PET-CT) in the staging of pLCH remains unclear. Our study aims to better characterize the utility of radiographic staging studies at time of diagnosis and prevalence of extrapulmonary disease at follow up in pLCH. Methods: We conducted a retrospective study of patients presenting with clinical findings consistent with isolated pLCH seen at the Mayo Clinic from January 2000 to January 2020. All patients had a diagnosis of pLCH determined by chest imaging studies [computed tomography (CT) or high-resolution CT (HRCT)] or by histopathologic findings from surgical or transbronchial lung biopsy. Histopathologic findings for the diagnosis of pLCH required the presence of Langerhans cells (S100+/CD1a+/Langerin+). Patients were excluded if they had clinically apparent extra-pulmonary organ involvement at the time of diagnosis. BRAF V600E mutation was determined by immunohistochemistry (IHC) or cell-free DNA (cfDNA). Staging was defined as FDG-PET-CT or whole body CT imaging obtained within 3 months of diagnosis of pLCH. Extra-pulmonary LCH involvement was determined by imaging characteristics or by histopathologic findings. Descriptive statistics and overall survival (OS) were analyzed with JMP software, version 14 (SAS Institute Inc., Cary, NC). Results: A total of 112 patients with clinically isolated pLCH were identified. The median age at diagnosis was 45 years (range 21-73), and 48 (43%) were male. The majority (n=110, 99%) were former or current smokers with a median pack year of 25 (range 1-57). Three (3%) patients were noted to have occupational industrial exposure. Nine patients had a history of another cancer prior to their diagnosis and staging of pLCH [lung (n=4), breast (n=2), neuroendocrine (n=2), thyroid (n=1)]. Presenting symptoms included dyspnea on exertion 49 (44%), cough 18 (16%), chest pain 14 (13%), and 29 (28%) were incidentally discovered on imaging studies. On HRCT, the following characteristics were observed: 42 (38%) cystic, 32 (29%) cystic and nodular, 37 (33%) nodular, 1 (<1%) ground glass opacities. Seventy (63%) patients had lung biopsy confirmation, of which 11 (16%) patients underwent BRAF V600Emutation testing (7 by IHC, 4 by cfDNA). Six (55%) of these 11 patients were positive for BRAF V600E mutation. 34 (30%) patients underwent radiographic staging studies within 3 months of diagnosis of pLCH (25 FDG-PET-CT and 9 whole-body CT), and none of these imaging studies showed evidence of extra-pulmonary disease. Of the patients who had at least one year of follow-up (n=52), one (2%) developed extra-pulmonary disease. This patient did not undergo initial staging studies, developed headaches and was found to have a large calvarial lytic lesion within 2 months of pLCH diagnosis (BRAF V600E+). Among those who did not undergo staging at diagnosis (n=78), long term (>5 years) follow up data were available for 23 (29%), and none developed extra-pulmonary disease as determined by clinical notes or imaging studies. No patient had evidence of new extra-pulmonary involvement or second malignancy at the time of last known follow-up. After a median follow-up duration of 2.4 years (95% CI: 1.5-3.6, range: 0.1-17) ten (9%) patients died, of which 5 died of pLCH related complications. The median OS for entire cohort was 15 years (95% CI 9.1-not reached, Figure 1). Conclusion: Our study shows that adult patients with clinically isolated pLCH rarely present with extra-pulmonary manifestations at diagnosis or at follow up. These findings suggest a limited role of routine radiographic staging studies in pLCH unless clinically indicated. Studies on longer follow up of this cohort would provide further insights into the natural history of pLCH and are underway. Disclosures Bennani: Purdue Pharma: Other: Advisory Board; Verastem: Other: Advisory Board; Kite/Gilead: Research Funding; Affimed: Research Funding. Shah:Dren Bio: Consultancy.

Rosai-Dorfman disease (RDD) is a rare histiocytosis with heterogenous clinical features. In this study, we characterized the histologic and phenotypic features in 33 RDD patients to better define the pathologic diagnosis. Cases included 24 patients with extracutaneous disease (“R” group), and 9 patients with lesions limited to the skin or subcutaneous tissue (“C” group). We identified OCT2 as a novel marker for the monocyte-macrophage phenotype of RDD, expressed in 97% of RDD cases. In contrast, OCT2 expression was seen in 0% of Erdheim-Chester disease cases and 6.7% of Langerhans cell histiocytosis cases. Other markers useful in the diagnosis of RDD included S100 (100%), CD163 (88%), and cyclin D1 (97%). In a subset of cases, RDD showed moderate to strong expression of factor 13a (30%), p16 (64%), and phosphorylated extracellular signal-regulated kinase (45%); RDD was uniformly negative for ZBTB46, CD1a, and langerin. Within the “R group” of RDD, increased expression of factor 13a or phosphorylated extracellular signal-regulated kinase showed a statistically significant association with multifocal disease ( P <0.05). Identification of the unique monocyte-macrophage phenotype of RDD with OCT2 expression furthers our understanding of this complex disease and allows for more uniform classification.

Anaplastic large cell lymphomas (ALCLs) are broadly classified into ALK-positive and ALK-negative. ALK-negative ALCL is composed of DUSP22-rearranged, TP63-rearranged, and triple-negative cases. While lymphoid enhancer–binding factor (LEF1) plays a crucial role in T-cell maturation, limited data exist on its expression in T-cell lymphomas, including ALCL. We characterized the expression of LEF1 in ALCL by immunohistochemistry. LEF1 nuclear expression in the neoplastic cells was graded as negative (0), weak (1+), intermediate (2+), or strong (3+), with the percentage of LEF1-positive neoplastic cells recorded. A total of 45 ALCL cases were evaluated, of which 16 were DUSP22-rearranged. About 93.8% (15/16) DUSP22-rearranged cases showed strong expression of LEF1 in >75% tumor cells, compared with 3.4% (1/29) non–DUSP22-rearranged ALCL (P<0.0001). The striking association of LEF1 protein overexpression with DUPS22 rearrangement in ALCL was further confirmed by a gene expression profiling study which revealed significantly higher LEF1 expression in DUSP22-rearranged ALCL compared with other ALCL subtypes (P=0.0001). Although LEF1 is a nuclear mediator of the Wnt/β-catenin pathway, CTNNB1 RNA and protein levels were not overexpressed in LEF1-positive cases, suggesting the LEF1 overexpression in ALCL may not be involved in the Wnt/β-catenin pathway. The strong and uniform LEF1 expression pattern has a high positive predictive value (93.8%) and high negative predictive value (96%) for DUSP22 rearrangement in ALK-negative ALCL. The combination of characteristic morphologic and molecular features of DUSP22-rearranged cases with the high LEF1 expression further emphasizes that DUSP22-rearranged ALCL represents a distinct clinicopathologic subset of ALCL.

Introduction: Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell lymphomas classified into ALK-positive and ALK-negative. The ALK-negative ALCLs are a genetically heterogeneous group that includes DUSP22-rearranged, TP63-rearranged and triple-negative cases. The DUSP22-rearranged ALCLs characteristically grow in a diffuse sheet-like pattern and are composed of intermediate-sized monomorphous hallmark cells, admixed with smaller cells with prominent nuclear indentations and occasional central nuclear pseudo inclusions (King RL et al, Am J Surg Pathol. 2016). Further, DUSP22-rearranged ALCL is a molecularly distinct subset of ALCL with a unique immunogenic phenotype that portends a favorable prognosis (Luchtel RA et al. Blood 2018). Lymphoid enhancer binding factor (LEF1) is a crucial nuclear mediator of the Wnt/ β-catenin pathway and a transcription factor involved in T-cell maturation. However, limited data exists on the association of LEF1 in T-cell lymphomas, including ALCL. The aims of this study were to explore LEF1 expression by immunohistochemistry in different subsets of ALCLs and to analyze the association of Wnt/ β-catenin pathway in this cohort. Methods: After approval by the institutional review board, we screened pathology reports from January 1, 1995 to December 31, 2019 and identified patients with a diagnosis of ALCL using our institutional database. Details on fluorescence in situ hybridization studies in ALK-negative cases were documented. Immunostaining for LEF1 and β catenin was performed on all cases where formalin fixed paraffin embedded tissue was available. The presence or absence of β catenin nuclear staining was recorded. LEF1 nuclear staining in the neoplastic ALCL cells was graded as negative (0), weak (1+), intermediate (2+) or strong (3+) and the percentage of LEF1-positive neoplastic cells was detailed (Figure 1). LEF1 scoring was performed by manual counting of the stained nuclei out of all tumor nuclei by two pathologists (A.R. and M.S.) and the grading was reviewed by another pathologist (P.J.K.). Results: We evaluated 45 ALCL cases, including 41 (91.1%) ALK-negative and 4 (8.9%) ALK-positive ALCLs. Among the ALK-negative ALCLs, there were16 DUSP22-rearranged, and 25 non-DUSP22-rearanged (7 TP63-rearranged and 18 triple-negative) cases. Fifteen (94%) of 16 DUSP22-rearranged ALCLs showed strong expression of LEF1 in &gt;75% tumor cells (Figure 2). In contrast, this strong and uniform LEF1 overexpression was only detected in 1 of 29 (3%) of the remaining ALCL cases (P &lt;.0001) (Table 1). We previously characterized a distinctive molecular signature in ALK-negative ALCLs with DUSP22-rearrangements using gene expression microarray analysis on frozen ALCL tissue samples (Luchtel RA et al. Blood 2018). On examining LEF1 gene expression in this previously published dataset, DUSP22 -rearranged cases had significantly higher LEF1 expression than any other genetic subtype (triple-negative, TP63-rearranged, or ALK-positive). Overall, mean LEF1 expression was 6.3-fold higher in ALCLs with DUSP22 rearrangements than in ALCLs without DUSP22-rearrangements (P=0.0001). LEF1 is a coactivator of the Wnt/β-catenin pathway; therefore, it is reasonable to hypothesize the overexpression of LEF1 may promote Wnt/β-catenin signaling. However, all LEF1-positive cases were negative for β-catenin by immunohistochemistry, suggesting that LEF1 overexpression is independent of the Wnt/β-catenin pathway signaling in these cases. Conclusions: There is a striking association between high LEF1 expression and DUSP22-rearrangements in ALCLs. The strong and uniform LEF1 expression pattern has a high positive predictive value (94%) and high negative predictive value (96%) for DUSP22- rearrangement in ALK-negative ALCLs (Table 2). The combination of characteristic morphologic and molecular features of DUSP22-rearranged cases with the unique LEF1 expression further emphasizes that DUSP22-rearranged ALCL represents a distinct clinicopathologic subset of ALCL. Disclosures No relevant conflicts of interest to declare.

Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (FFPE)-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p &lt; 0.001) [Fig 1A]. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p&lt;0.01, Fig 1C). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and CHK2 (T68) (data not shown). This finding was further confirmed by WB analyses (Fig1DI). In addition, decreased of total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY19 and OCI-LY17 cell lines (Fig 1DI). Immunoprecipitation experiments with anti-PD1 antibody showed that PD-1 was in the same complex with SHP-1 and SHP-2 in OCI-LY7 cells but only with SHP-2 in OCI-LY19 cells (Fig 1DII). In parallel, mRNA sequencing was performed on 11 nodal tissues from either RT (n=8) or progressive CLL (n=3) after they developed clinical progression. The expression of PD-1 (PDCD1) was positively correlated with the expression of SHP-1 (PTPN6, r=0.56) and Cyclin E1 (CCNE1, r=0.62), implicating a direct role of PD-1 in regulating cellular proliferation and the induction in phosphatase expression in RT and CLL. Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

Abstract Langerhans cell histiocytosis (LCH) is a rare histiocytic neoplasm. To date, there is a lack of U.S. Food and Drug Administration–approved treatments in adult LCH to establish optimal first-line therapy. We conducted a retrospective, single-center case series evaluating the use of BRAF inhibitors in adult patients with BRAFV600E- LCH proven by biopsy. Our case series is the first to report the use of BRAF inhibitors as first-line therapy in adults with LCH. We also report the efficacy with single-agent dabrafenib in adult LCH. All but one of our patients had favorable response to targeted therapy.

Abstract Objectives Crystal-storing histiocytosis (CSH) is rare in plasma cell dyscrasias, with only 3 cases reported in the setting of amyloid. No cases of crystal-negative histiocytosis coincident with multiple myeloma and amyloidosis have been reported previously. Methods A 58-year-old woman presented with pain due to destructive bone lesions and was found to have plasma cell myeloma (PCM) and marrow amyloid deposition associated with crystal-negative histiocytosis. Differential diagnoses included Langerhans cell histiocytosis, Erdheim-Chester disease, and Rosai Dorfman disease. BRAF mutations were negative, and there was no evidence of paraprotein crystals, arguing against typical CSH. Results The patient was treated with bortezomib, cyclophosphamide, and dexamethasone, and she subsequently underwent autologous stem cell transplant and ixazomib maintenance. She achieved complete remission with improvement of her symptoms and preserved remission after following up at 60 months. Conclusions We describe a case of crystal-negative histiocytosis associated with PCM. CSH is a rare disorder associated with paraprotein-producing conditions in which immunoglobulins aggregate as intracellular crystals in the lysosomes of organ-specific phagocytic macrophages. Light chain tropism in PCM can also lead to the development of amyloid deposition in organs and, in rare cases, is associated with light chain aggregation as intracellular crystals in macrophages.