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AbstractMalignant histiocytosis (MH) is an extremely rare neoplasm of the macrophage–dendritic cell lineage. We report the clinical characteristics, molecular aberrations, treatments, and outcomes of patients with MH seen at two referral centers from January 2000 to May 2023. We identified 43 patients with MH, of which 26 had histiocytic sarcoma (MH‐H), 9 interdigitating dendritic cell sarcoma (MH‐IDC), and 8 Langerhans cell sarcoma (MH‐LC). The median age at diagnosis was 61 years (range, 3–83). Thirty‐three patients (77%) had multifocal disease, while 10 had unifocal involvement. Tumor specimens from 22 patients (51%) underwent targeted next generation sequencing, and 19 of 22 (86%) had at least one pathogenic mutation, including mutations in MAPK pathway genes (73%). The median overall survival (OS) among the entire cohort was 16 months (95% CI: 8–50). The outcomes of those with multifocal disease were significantly shorter than their unifocal counterpart: median OS of 10 months versus 50 months (p = .07). Patients with risk organ involvement (bone marrow, spleen, or liver) had significantly inferior outcomes. Chemotherapy and surgery were the most common first‐line treatments for multifocal and unifocal disease, respectively. While the outcome for patients with multifocal disease was poor, there was a subset of patients who had durable responses to treatment. Our study highlights that MH has heterogeneous clinical presentation, frequent oncogenic mutations, and prognosis, which is strongly tied to disease extent and type of organ involvement.

Background:Waldenström Macroglobulinemia (WM) is a rare, indolent B‐cell lymphoplasmacytic malignancy. Patient outcomes are highly heterogeneous, with a proportion of patients surviving less than 5 years (short survivors) and another proportion that survive longer than 10 years. The laboratory parameters in the currently used International Prognostic Scoring System for WM (IPSSWM) include hemoglobin, serum IgM, platelet count, and serum β2‐microglobulin.Aims:We aim to identify unique patient characteristics and prognostic features associated with short survival in WM.Methods:The medical records of patients with active WM that were diagnosed at Mayo Clinic Rochester, Arizona, and Florida between January 1st, 1996 and December 31st, 2013 were reviewed. The patients with smoldering WM were excluded. Cohorts of patients who either survived ≤ 5 years (Short Survival Group) or those with an overall survival (OS) ≥ 10 years (Long Survival Group) from the diagnosis of active disease were compared. Survivors with a follow‐up of ≤ 5 years were not included in the analyses. Two‐sided Wilcoxon rank sum test and Chi square/Fisher's exact test were used to compare the continuous and categorical variables, respectively. The variables that were statistically significant on univariate analysis (P < 0.05) were included in a multivariate logisitic regression analysis.Results:Of 893 consecutively seen WM patients (median follow‐up of 9 years [95% CI 8.4–9.7]), we identified 387 patients who could be categorized into either the Short Survival Group (n = 171 patients, 44%) or the Long Survival Group (n = 216, 56%). The Short Survival Group had a median OS of 2.4 years (2.1–2.8) and median age at diagnosis of 73 years (42–93), while the Long Survival Group (n = 216, 56%) had a median OS of 18.9 years (16‐NR) and median age at diagnosis of 62 years (31–85). The baseline characteristics and significant findings between the two groups are listed in the Table. Parameters significant on univariate analysis included age >65 years, albumin < 3.5 g/dL, β2‐microglobulin >3 μg/ml, and LDH > upper limit of normal (ULN). On multivariate analysis, albumin <3.5 g/dL and LDH > ULN were independently associated with short survival. Among the Long Survivors, 60 (28%) patients survived 15 years or more.Summary/Conclusion:Lactate dehydrogenase above the ULN and serum albumin <3.5 g/dL are independent biomarkers of short survival in WM. The MYD88 L265P status and the size of serum IgM are not prognostic in WM. Our data suggest the need for simplification of the current staging system with the use of widely available markers: serum albumin and lactate dehydrogenase.image

Background:Ibrutinib is approved in Europe and the US for the treatment of Waldenstrom macroglobulinemia (WM) with a median progression‐free survival (PFS) measured on several years. As PFS is commonly used for drug approval by regulatory entities, there is an unmet need of reliable surrogate markers for PFS that could help accelerating such process. This is of importance with novel BTK inhibitors currently in clinical trials.Aims:We hypothesized that the depth of response at 6 months can be predictive of PFS in patients with WM treated with the BTK inhibitor ibrutinib.Methods:We evaluated our hypothesis in two separate cohorts of WM patients treated with ibrutinib. The learning cohort was composed by participants of two prospective clinical trials at Dana‐Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center and Stanford Cancer Center. The validation cohort was composed by consecutive WM patients treated with ibrutinib off clinical trial at DFCI and Mayo Clinic. Categorical responses at 6 months were assessed based on criteria from the 6thInternational Workshop for WM. PFS6 was defined as the time from the 6‐month response assessment until progression of disease or death from any cause. PFS6 curves were generated using the Kaplan‐Meier method and compared using the log‐rank test. Cox proportional hazard regression models were fitted for PFS6. Pvalues <0.05 were considered statistically significant.Results:The learning and validation cohorts were composed by 92 and 190 WM patients treated with ibrutinib, repsectively. There were no statistical differences between cohorts with regards to age, sex, serum IgM level, hemoglobin level, bone marrow involvement, prior treatment status, and MYD88 and CXCR4 mutational status (p>0.05 for all variables). The rates of partial response (PR) or better at 6 months in the learning and validation cohorts were 63% (54/86) and 72% (114/159), respectively (p = 0.15). The median PFS6 was not reached in the learning cohort and was 53 months (95% CI 44‐not reached) in the validation cohort. In the learning cohort, the median PFS6 for patients who attained a PR or better at 6 months was not reached, while for the patients who did not attained PR at 6 months was 48 months (95% CI 33‐not reached). The 3‐year PFS rate for patients who attained a PR or better at 6 months was 83% (95% CI 68–91%), while for patients who did not attained PR at 6 months was 58% (95% CI 33–76%) (Figure, left, log‐rank p = 0.02). Attaining PR or better at 6 months was associated with a hazard ratio (HR) or progressing or dying of 0.37 (95% CI 0.16–0.87; p = 0.02).In the validation cohort, the median PFS6 for patients who attained a PR or better at 6 months was 53 months (95% CI 44‐not reached), while for the patients who did not attained PR at 6 months was 32 months (95% CI 22‐not reached). The 3‐year PFS rate for patients who attained a PR or better at 6 months was 79% (95% CI 67–87%), while for patients who did not attained PR at 6 months was 50% (95% CI 26–70%) (Figure, right, log‐rank p = 0.04). Attaining PR or better at 6 months was associated with a hazard ratio (HR) or progressing or dying of 0.46 (95% CI 0.22–0.98; p = 0.04). Age, sex, serum IgM level, hemoglobin level, bone marrow involvement and prior treatment status were not prognostic of PFS6 in both cohorts.Summary/Conclusion:Attaining a PR or better at 6 months to ibrutinib is associated with longer median PFS and higher rates of 3‐year PFS in WM patients. Depth of response at 6 months could serve as a surrogate for PFS in clinical trials evaluating BTK inhibitors in WM patients.image

Introduction The discovery of MAPK/ERK pathway mutations has revolutionized the care for patients with histiocytic neoplasms. However, mutational analysis on biopsy specimens is often limited by low tumor cellularity leading to testing failure or disease involvement of difficult-to-reach sites (e.g., central nervous system). Recently, molecular analysis of cell-free DNA (cfDNA) has shown promise as a viable tool for discovering driver mutations in multiple cancers. A recent study of mainly BRAF-mutated non-Langerhans cell histiocytosis (LCH) showed 50% concordance between tumor and plasma cfDNA analysis for BRAF-V600E. However, it is unknown whether cfDNA has a role in the evaluation of cases that harbor mutations beyond BRAF-V600E. We conducted this study to address this unanswered question using a large cohort of individuals with diverse histiocytic neoplasms from a tertiary-care institution. Methods We included 40 cases with histiocytic neoplasms with stored plasma and germline DNA in this study. Tissue molecular analysis was conducted using a targeted next generation sequencing (NGS) using commercial assays or whole exome sequencing (WES) using an in-house assay. A custom panel of 226 genes, including exonic and intronic regions, was used for the cfDNA assay in this study. To avoid cfDNA false positives, germline mutations identified from normal DNA sequencing were filtered using a BostonGene ®-developed error reduction algorithm.The total length of the analyzed genomic regions was 1.56 MB. For samples with an input greater than 30 ng, a 0.2% variant allele frequency (VAF) limit of detection (LOD) was used; a 0.4% VAF LOD was used for samples less than 30 ng. Results The final analysis cohort included 38 patients with histiocytic neoplasms after exclusion of 2 cases (failure of germline DNA library preparation and mismatch of germline and somatic DNA, respectively). Disease types included Erdheim-Chester disease (ECD; n=13, 34%), Langerhans cell histiocytosis (LCH; n=13, 34%), and Rosai-Dorfman disease (RDD; n=12, 32%). 24 cases were multisystem (63%), and the remaining were single-system. Commonly involved organs included bone (n=25, 66%), kidney (n=12, 32%), skin (n=8, 21%), central nervous system (n=7, 18%), and pituitary (n=7, 18%). Most cases (n=31, 82%) underwent plasma collection pre-treatment, 5 cases underwent collection after histiocytosis-directed therapy, and 2 while on treatment. Tissue NGS/WES data were available for 28 (74%) cases; 5 cases (13%) had insufficient tissue cellularity, and 5 (13%) did not undergo any tissue sequencing. Among those who underwent successful tissue sequencing assays, the most common common driver alterations was BRAF V600E (n=5, 26%). Other alterations included those in MAP2K1 (n=4, 21%), BRAF-fusions (n=3, 10%; partners UBDT2, RNF11, and LMTK2, respectively), and single cases of KRAS, NRAS and BRAF indel, respectively. cfDNA analysis showed at least one variant in 23 cases (60%). Correlation between tissue and cfDNA for known driver mutations was poor and concordant findings were only seen in one case with ECD harboring BRAF V600E in tissue as well as plasma (VAF 0.5%). Among the 5 cases where tissue mutational analysis had failed, cfDNA showed mutations in MUTYH, PTCH2, NOTCH3 SDHD, and TMEM127, respectively (Figure 1). Among the 5 cases where tissue molecular assessment could not be undertaken due to various reasons, cfDNA showed alterations in 4 cases, encompassing NOTCH1, TET2, BRCA1, MSH6, PIK3CD, and FUS genes. Conclusions In a large cohort of adults with diverse histiocytic neoplasms, we found poor correlation between tumor and plasma cfDNA molecular findings. The reason for the lack of correlation may be related to low tumor shedding especially among non- BRAF V600E cases. cfDNA analysis may have a role to identify potential drivers of pathogenesis among cases that are unable to successfully undergo tissue molecular analysis. Further studies are underway to characterize other novel alterations that were discovered in the cfDNA analysis.

Purpose: The discovery of MAPK/ERK pathway mutations has led to the approval of MEK-inhibitor (MEKi) cobimetinib in histiocytic neoplasms. However, the pivotal clinical trial that led to the approval of cobimetinib included only 4 patients with Langerhans cell histiocytosis (LCH). Therefore, there is a need to define outcomes with MEKi therapy in patients with LCH. We undertook a multi-center collaboration through the Adult LCH Working Group for the Histiocyte Society to address this gap in knowledge. Methods: Adults with LCH who received MEKi therapy were included from five institutions. Objective response rate (ORR) was evaluated using PET/CT criteria, and CT/MRI if PET scan was not available; response categories were categorized into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Progression-free (PFS) and overall survival (OS) were calculated from the time of targeted therapy initiation. Risk organ disease was defined as liver, spleen, or bone marrow involvement. Results: 25 adults with LCH were included in this study; median age at MEKi initiation 43y (range, 18-85). Most common systems involved at diagnosis were bone (52%), pituitary (44%), and lung (36%). Risk organ involvement was observed in 7 (28%, 6 liver, 1 liver and spleen) and 2 (8%) patients had intra-axial CNS involvement. Median lines of prior therapy received were 2 (range: 1-5), and 4 (16%) patients received MEKi in first-line. MAPK/ERK pathway status was assessed via target capture next generation sequencing (NGS) in 22 (96%) cases. Amongst those who underwent NGS, alterations identified included: BRAF V600E (2/22, 9%), BRAF deletions (11/22 50%), MAP2K1 or MEK mutations (4/22, 18%), and MAPK1 or ERK2 (n=1, 5%). Overall, 5 patients also received BRAF inhibitors (4 patients as first therapy prior to MEKi and 1 concomitantly due to BRAF V600E positive melanoma). Specific MEKi agents utilized included cobimetinib (n=13) and trametinib (n=12). Among the response evaluable patients (n=23), ORR to MEKi treatment was 78% (9 CR, 9 PR, 4 SD, 1 PD). Among the 7 patients with liver involvement, responses were seen in 2 (29%) cases. ORR did not significantly differ according to mutation status among those with NGS data (78% MAPK mutated vs. 100% unmutated; p=1.0). In this subcohort, all of the 4 cases that did not respond to MEKi had liver involvement; 3 had BRAF deletions (2 SD and 1 PD), and one had MAPK1 mutation (SD). Median follow-up duration after MEKi initiation in the entire cohort was 18 months (95% CI 6 - 30). The median PFS and OS were not reached; 2-year PFS and OS were 78.1% and 79.9%, respectively (Figure 1A). The median PFS was much shorter in those with liver involvement (7 months [95% CI 0 - 14]) compared with those without liver disease (not reached [95% CI 19-not reached] P=0.047; Figure 1B). PFS did not differ significantly between MAPK pathway mutated and unmutated patients (p=0.34) Adverse events (AEs) were reported in 36% (n=9) and most commonly included rash (n=5) and fatigue (n=3). Four patients experienced grade 3-4 AEs including rash, fatigue, and thrombocytopenia. Common causes of MEKi discontinuation included AEs (n=3), progression (n=2), and drug holiday (n=2). At last follow-up, 5 patients died and 18 remained on MEKi. Death was attributable to LCH (n=3), concomitant malignancy (n=1), or unknown causes (n=1). Conclusions: In our multi-institutional study of adults with LCH, we found MEKi therapy to be highly efficacious. However, liver involvement correlated with a lack of response and worse PFS, while mutational status did not. Future studies are needed to evaluate optimum treatment for high-risk subgroups of LCH like liver disease.

Purpose: The understanding of cardiac-involved ECD (ECD-CV) and how this affects outcomes and pre-existing diseases remains limited. We aimed to determine the association, burden, and severity of pre-existing and de novo cardiovascular (CV) comorbidities with ECD-CV compared to patients (pts) without cardiac-involved ECD (ECD-noCV). Methods: Records of pts with ECD diagnosed from Jan-1990 to Dec-2021, consecutively seen at Mayo Clinic, AZ, FL, and MN were reviewed. Cardiac involvement was diagnosed by imaging. CV comorbidities were captured before and after ECD diagnosis. CTCAE v3.0 based grading system was used to assess CV comorbidities which graded the conditions as mild (grade 1), moderate (grade 2), severe (grade 3), and life-threatening or disabling (grade 4). Chi-square test was used to determine if having ECD-CV worsens pre-existing CV comorbidities in pts with ECD. As per the established methods, the cumulative CV disease burden was defined after considering 8 CV morbidities that are known to have direct structural and functional implications on the heart, including hypertension (HTN), coronary artery disease (CAD), myocardial infarction (MI), congestive heart failure (CHF), atrial fibrillation (afib), pericardial effusion, valvular disease, and conduction disorders. A composite CV morbidity grade was assigned based on the highest grade in one of the 8 CV comorbidities (Oeffinger, NEJM, 2006). Lastly, a multivariant analysis was conducted using the presence of BRAFV600E mutation, smoking, age ≥ 60 years (yr), conventional vs targeted therapy, and ECD-CV in pts with a composite grade of 4. Results: Among 106 ECD pts, 38% (n=40) had cardiac involvement. The median age at diagnosis of ECD was 57 yr (range: 49-67); 61% were males. The median follow-up was 5 yr (95% CI: 3.5-6.1), and 4.1 yr (95% CI: 3.2-6.5) for ECD-CV and ECD-noCV pts, respectively p=0.92. The median time of CV diagnoses for the entire cohort before and after ECD diagnosis was 4.8 yr (range: 0.06-42.5) and 3.4 yr (range: 0.05-18.7), respectively. BRAFV600E mutation was present in 65% (n=26) of ECD-CV vs 42% (n=28) of ECD-noCV, p=0.02. Eight pts with ECD-CV died; one from sudden cardiac death, while 9 ECD-noCV pts died, p=0.39. Smoking was present in 43% (n=18) of ECD-CV pts compared to 24% with ECD-noCV (n=16), p=0.05. In comparison to ECD-noCV pts, increased prevalence of pre-existing HTN [59% (n=23) vs 35% (n=23), p=0.016] and MI [10% (n=4) vs 2% (n=1), p=0.046] was seen in ECD-CV. There was no difference in hyperlipidemia, diabetes mellitus, CAD, CHF, valvular disease, afib, and conduction disorders among the two cohorts. After ECD diagnosis, de novo diagnosis of hyperlipidemia [67% (n=26) vs 38% (n=25); p=0.004] and CHF [35% (n=14) vs 7% (n=5); p=&lt;0.001] was seen more frequently in ECD-CV pts compared to ECD-noCV. Following the diagnosis of ECD, the severity of pre-existing conditions did not change in pts with or without ECD-CV. ECD-CV pts had a greater cumulative CV disease burden as the total number of CV morbidities was greater in ECD-CV pts both before and after ECD diagnosis (Table 1). Additionally, pts with ECD-CV had greater severity of CV morbidity (composite grade ≥4) than ECD-noCV before (35% v 3%; p=&lt;0.001) and after (42.5% vs 7.6%; p=&lt;0.001) ECD diagnosis. The composite grade of 4 before ECD diagnosis was most attributable to pericardial effusion (42% in pts with ECD-CV vs 0% in pts with ECD-noCV) and CAD (50% in pts with ECD-CV vs 100% in pts with ECD-noCV); after ECD diagnosis pericardial effusion (42% in pts with ECD-CV vs 0% in pts with ECD-noCV) and CAD (41% in pts with ECD-CV vs 100% in pts with ECD-noCV). After considering BRAFV600E mutation, age ≥60, smoking, ECD-CV, and first-line therapy (conventional vs targeted therapy), ECD-CV was the only factor independently associated with a composite grade of ≥4, before (OR 25.6, 95% CI: 4.8-137) and after (OR 9.7, 95% CI: 3.0-30.8) ECD diagnosis. Nonetheless, smoking, age &gt;60, and ECD-CV were independent factors associated when considered a composite grade of ≥3, before and after ECD diagnosis. Conclusion: ECD-CV did not prognosticate pts and did not worsen the pre-existing CV conditions compared to pts with ECD-noCV. However, ECD-CV pts had a higher cumulative CV disease burden and a higher grade of pre-existing and de novo CV morbidities compared to ECD-noCV. The presence of cardiac involvement in ECD was independently associated with a severe cumulative CV disease burden grade.

Background: Waldenström Macroglobulinemia (WM) is an indolent lymphoma with heterogeneous outcomes. The recently developed, Revised International Prognostic Scoring System for WM (rIPSS-WM) did not examine the prognostic impact of MYD88L265P, a somatic alteration present in most patients with WM. Moreover, rIPSS-WM could only be partially validated in an external cohort. Here, we report the performance of rIPSS-WM and propose a refined prognostic model with external validation. Methods: We reviewed medical records of treatment-naïve patients with active WM consecutively seen at Mayo Clinic, Rochester (MCR) between 01/01/1996 and 12/31/2017. We applied the rIPSS-WM model in patients with data available for all required parameters, including age, serum beta-2 microglobulin (β2M), serum lactate dehydrogenase (LDH) and serum albumin at diagnosis to assess the prognostic utility of this model. We then identified the dichotomized clinical and laboratory parameters prognostic for overall survival (OS) by univariate analyses (UVA) in our cohort. The significant parameters were analyzed to independently predict OS in a multivariable analysis (MVA) and to create a prognostic model, which was subsequently validated in an independent multi-institutional cohort. Results: We identified 889 patients with active WM at MCR, with a median follow-up of 8.2 [95% confidence interval (CI): 7.5-9] years. The estimated median OS for this cohort was 10.6 (95%CI: 9.7-11.4) years. Patients with available data in this cohort for the validation of rIPSS-WM (n=241) were risk stratified into very low (n=46), low (n=64), intermediate (n=58), high (n=46) and very-high risk groups (n=27) per rIPSS-WM, with the respective 5-year OS rates of 96%, 76%, 72%, 77% and 32%, demonstrating overlapping survival curves for the low, intermediate and the high-risk groups. Because rIPSS-WM could not be validated, we used the data from the MCR cohort and identified age at diagnosis of active WM, LDH &gt;upper limit of normal (ULN), β2M &gt;3µg/mL and albumin &lt;3.5g/dL as statistically significant prognostic markers for OS on UVA. Notably, the presence of MYD88 L265P did not impact OS [median 11.4 years (95%CI: 10.1-not reached (NR) for MYD88 L265P genotype (n=231) versus 10 years (95% CI: 8.1-NR) for MYD88 WT genotype (n=65); p=0.33]. On MVA, LDH &gt;ULN (HR 2.34), serum albumin &lt;3.5 g/dL (HR 1.5) and age groups 65-75 years (HR 1.4) and &gt;75 years (HR 2.6) were independently prognostic. Based on comparable HRs, we assigned a score of 1 point each to serum albumin &lt;3.5 g/dL and age group 66-75 years and similarly, 2 points each for age &gt;75 years and serum LDH &gt;ULN per their impact on OS. Patients with the composite scores of 0, 1 and 2 were categorized as low risk, low- intermediate risk and intermediate risk, respectively. Due to similar OS for the scores of 3 to 5, these were combined to form the high-risk group of our proposed model: Modified Staging System for WM (MSS-WM). The median OS was 14.6 years [95%CI: 9.1 years-NR] for low-risk (score 0, n=71, 21%), 11.2 years (95% CI: 9.1-15.2 years) for low-intermediate risk (score 1, n=110, 32%), 8.1 years (95% CI: 6.4-12.2) for intermediate risk (score 2, n=81, 24%) and 5.5 years (95 % CI: 3.9-11 years) for the high-risk group (score ≥3, n=79, 23%); p&lt;0.0001. In this derivation cohort, the 5-year OS rate was 93%, 82%, 69% and 55% and 10-year OS rate was 60%, 53%, 45% and 30% for the low, low-intermediate, intermediate, and high-risk cohort, respectively ( Figure 1A). We also validated MSS-WM using competing risk analysis (p=0.001) and in the cohort of rituximab treated patients (p&lt;0.0001). We then used data from an independent series of 335 treatment-naïve symptomatic patients from academic institutions (n=5) across the US and Europe for external validation of the MSS-WM model. The median follow-up for the validation cohort was 6.3 years (95% CI: 5.5-7.2 years). The 5-year OS was 93%, 90%, 75% and 57% and the estimated 10-year OS was 91%, 80%, 64% and 35% for the low risk (n= 86, 26%), low-intermediate (n=107, 32%), intermediate (n=86, 26%) and high-risk (n=59, 18%) cohorts, respectively (p&lt;0.0001, Figure 1B). Conclusions: Our proposed model, MSS-WM, is a simple, clinically useful, externally validated model that reliably risk stratifies previously untreated patients with active WM into four groups that have distinct outcomes based on the composite scores derived from the patients' age, serum albumin and serum LDH at diagnosis.

Introduction: Waldenström macroglobulinemia (WM) is a rare B-cell lymphoma that may be preceded by an asymptomatic IgM-monoclonal gammopathy of undetermined significance (MGUS). The mechanisms underlying the progression from IgM-MGUS to WM remain to be fully elucidated; however, the stepwise accumulation of genomic abnormalities has been suggested as a potential driver. Currently, the role of epigenetic regulation of DNA remains underexplored in this disease spectrum. Aberrant DNA methylation is a hallmark of oncogenesis that plays an important role in hematologic malignancies. The aim of this study was to assess differential DNA methylation in WM compared to IgM-MGUS and identify hyper- and hypomethylated pathways that may alter key cellular functions. Methods: This study included 15 patients with WM and 6 with IgM-MGUS. Bone marrow samples were prospectively collected and CD19+ and/or CD138+ B cells were positively selected. Subsequently, genome-wide DNA methylation analysis was performed. CpG methylation ratios were segmented into 200-bp regions and differentially methylated regions (DMRs) comparing WM to IgM-MGUS were identified. DMRs with a Q-value &lt;0.05 and absolute methylation difference of ≥20% were considered significant and utilized for analysis. DMRs were annotated according to genomic regions of interest (promotors, 3'UTR, 5'UTR, introns, exons, CpG islands, CpG shores). Next, genes with significant promotor hyper- or hypomethylation were assessed using the Ingenuity Pathway Analysis. Pathways with a corrected p-value&lt;0.05 were considered statistically significant and a Z-value was calculated based on the degree of differential methylation. Results: Genome-wide DNA methylation analysis revealed a significant number of DMRs (n= 15691, hyper: 8248; hypo: 7443) in WM compared to IgM-MGUS. Assessing genomic regions, promotors (hyper: 1379; hypo: 2403), introns (hyper: 1548; hypo: 2292), CpG shores (hyper: 587; hypo: 849) and 3'UTR (hyper: 66; hypo: 193) regions were observed to have a significantly greater number of hypomethylated DMRs (all comparisons, p&lt;0.001) in WM compared to IgM-MGUS (Figure A). While exons (hyper: 1686; hypo: 1378), CpG islands (hyper: 2128; hypo: 82), and 5'UTR (hyper: 854; hypo: 246) regions had a significantly greater number of hypermethylated DMRs (all comparisons, p&lt;0.001) (Figure A). Next, promotor methylation-based pathway analysis revealed differential methylation of multiple cellular pathways important in WM pathogenesis (Figure B). These included pathways involved in intracellular signaling and the regulation of cellular proliferation, such as the STAT3 and CXCR4 pathways which were hypomethylated, and the PTEN signaling pathway which was hypermethylated. Of interest, we have previously demonstrated the transcription factor, STAT3, to have an increased expression in WM and to be implicated in IgM secretion (Elsawa, 2011). Next, multiple cytokine signaling pathways were observed to be hypomethylated in WM compared to IgM-MGUS including members of the interleukin (IL) family (IL-1, IL-8, IL-15, IL-23), VEGF, and TGF-β. Of relevance, previous studies have demonstrated the elevation of IL-8, IL-15, and increased expression of angiogenic cytokines, including VEGF, in patients with WM (Lewicki, 2010; Anagnostopoulos, 2007). Furthermore, our group has also shown miRNA-based epigenetic regulation of IL-8 and IL-15 in WM compared to IgM-MGUS (Chohan, 2023). Lastly, several pathways critical in the regulation of the cell structure and the extracellular matrix (ECM) were found to be hypomethylated including the FGF, actin cytoskeleton, PAK, and FAK signaling pathways. Of note, the FGF/FGFR axis has been demonstrated to be dysregulated in WM, and its targeting has been shown to halt WM cell growth (Sacco, 2021). Conclusions: This study demonstrates differential DNA methylation of key genomic regions in WM compared to IgM-MGUS. Promotor methylation-based analysis reveals hypomethylation of several important cellular pathways involved in cytokine signaling, cell structure/ECM, and intracellular signaling. These results underscore the influence of epigenetics in the IgM-gammopathy disease spectrum and suggest the potential role of aberrant DNA methylation underlying the malignant progression from IgM-MGUS to WM.

Purpose: Advances in targeted therapeutics for histiocytic neoplasms have led to a growing survivor population, especially for Erdheim-Chester disease (ECD), Langerhans cell histiocytosis (LCH), and Rosai-Dorfman disease (RDD). This growing population is at risk of adverse health-related quality of life (HRQoL) as a result of the disease or treatments received. Patient-reported outcomes in this population remain understudied, and we aim to define their HRQoL using a validated tool. Methods: We enrolled patients from 2 histiocytosis centers and an international conference from Aug 2022-April 2023 to complete the PROMIS © (Patient-Reported Outcomes [PROs] Measurement Information System) 29+2 profile v2.1, which captures 8 domains of physical/mental health and functioning. Domains are reported as T-scores, with 50 being the general population mean. We calculated summary T-scores for Physical Health (PH) and Mental Health (MH), and differences of 3 from general population were considered clinically meaningful. We then used these T-scores to calculate each patient's PROMIS-Preference (PROPr) score, which is a health-utility score that measures overall HRQoL. A mean PROPr score difference of 0.4 from the US population mean of 0.52 was considered meaningful. We ran independent-samples t-tests to examine differences in PH/MH summary scores and PROPr between sub-cohorts. Finally, we performed a multinomial logistic regression analysis to identify factors associated with worse scores, and calculated odds ratios (OR) for each factor. Results: We included a total of 77 participants. Specific diagnoses included ECD (n=33, 42.9%), LCH (n=21, 27.3%), RDD (n=19, 24.7%), and other/unknown (n=4, 5.2%). Median age at diagnosis was 50y (9-79) and at survey was 54y (18-82), with a median 4 year difference between diagnosis and survey completion. Racial demographics included white (n=64, 83.1%), Black (n=12, 15.6%), and Asian (n=1, 1.3%). 4 patients (5.2%) identified as having a Hispanic ethnicity. Disease extent included unifocal (n=13, 16.9%), single system multifocal (ss-M, n=8, 10.4%), single system lung LCH (ss-L, n=3, 3.9%), or multisystem (n=53, 68.8%) disease. The most commonly involved organs included bone (n=44, 57.1%), kidney (n=24, 31.2%), skin (n=24, 31.2%), CNS (n=24, 31.2%), and pituitary (n=19, 24.7%). PH and MH summary scores were lower in various diagnostic categories as compared with the US population, and PROPr scores were reduced in all diagnostic categories. Bone involvement was associated with worse PH (-6.16, 95%CI -10.81, -1.52, [p=0.005]), MH (-7.96, 95%CI -12.48, -3.44, [p=&lt;0.001]), and PROPr (-0.184, 95%CI -0.313, -0.056, [p=0.003]) scores. CNS involvement was associated with worse PH (-4.78, 95%CI -9.14, -0.42, [p=0.016]). We initially constructed a multinomial regression model using diagnosis, race, age at survey completion, and CNS and bone involvement. This model was not statistically significant, and in particular, diagnosis and age did not significantly contribute to the model. We then removed diagnosis and age and constructed a new model using the remaining variables, which was statistically significant for all 3 scores (p&lt;0.05). We found that factors associated with worse PH score were disease involvement of bone (OR=6.19, 95%CI 1.88, 20.43 [p=0.003]) and CNS (OR=10.36, 95%CI 2.01, 53.39 [p=0.005] Figure 2). Factors associated with worse MH scores included female sex (OR=4.12, 95%CI 1.24, 13.66 [p=0.021]) and CNS involvement (OR=10.74, 95%CI 2.47, 46.79 [p=0.002]). Having race/ethnic group other than white/Non-Hispanic or Black was associated with improved MH. However, as this group had only 4 members, the significance of this is uncertain. Finally, CNS involvement was found to be associated with worse odds of having a low PROPr score (OR=10.46, 95%CI 2.17, 50.37 [p=0.003]). Conclusions: Several PROs were worse in patients with histiocytic neoplasms as compared with the general population. We further found that CNS involvement was independently associated with worse HRQoL, regardless of the diagnosis type. Additionally, female sex was associated with worse MH scores, and bone involvement with worse PH scores. PROPr score was particularly low among patients with ECD. Future research is warranted to assess the impact of treatments on HRQoL, as well as the economic loss resulting from loss of productivity as highlighted by low PROPr scores.

Background Complete blood count (CBC) is the most common hematology test performed. Reference limits, aka normal values, are known to vary from one hospital to another. However, little is known about the values of the upper (UL) and lower (LL) limits of common elements reported in the CBC and how they differ among facilities. The objectives of our study were to report the UL and LL of CBC elements from various hospitals in the US, analyze the variances, and describe their clinical significance. Methods This study was approved by the Mayo Clinic institutional review board. We obtained the reference limits of CBCs from various hospitals using the Epic electronic medical records Care Everywhere health information exchange. CBCs from 2020-2023 were included. We included the following CBC elements: hemoglobin (Hgb; male and female), mean cell volume (MCV), white blood cell count (WBC), and platelet count (PLT). We performed descriptive statistics and calculated the coefficients of variation (COVs). In addition, we analyzed the data for clinical context using: A) World Health Organization (WHO) major criterion for polycythemia vera of Hgb &gt;16.5 g/dL and &gt;16.0 g/dL for males and females, respectively; B) WHO major criterion for essential thrombocytosis of PLT &gt;450 x109/L; and C) common textbook reference limits for MCV (80-100 fL) and WBC (5-10 x109/L). Results We collected CBC reference limits from 143 hospitals across 43 states and 99 cities. Most (54.5%) hospitals were community-based, while the rest (45.5%) were academic. The statistics are shown in the Table. In general, there was minimal variance in the UL and LL of CBC elements, with COVs mostly under 8.0%. The one exception was WBC LL, in which the COV was 10.5%. There was no significant difference in the CBC reference limits between academic and community hospitals. The median Hgb UL and LL for males were 17.5 (range, 16.3-18.1) and 13.5 (range, 12.0-14.3), respectively. Similarly, the median Hgb UL and LL for females were 15.7 g/dL (range, 14.6-16.3) and 12.0 g/dL (range, 11.0-12.5), respectively. Majority (93.5%) of the hospitals' male Hgb UL exceeded 16.5 g/dL. In contrast, only few (3.6%) of the hospitals' female Hgb UL exceeded 16.0 g/dL (Figure A). The median MCV UL and LL were 99.0 fL (range, 92.2-103.0) and 80.0 fL (range, 77.0-88.0), respectively. A substantial minority (19.6%) of the hospitals' MCV UL exceeded 100 fL (Figure B). The median WBC UL and LL were 10.8 x109/L (range, 8.9-13.0) and 4.0 x109/L (range, 3.1-5.4), respectively. Most (80.4%) hospitals' WBC UL exceeded 10 x109/L (Figure C). The median PLT UL and LL were 400 x109/L (range, 317-500) and 150 x109/L (range, 125-172), respectively. Over a quarter (27.5%) of the hospitals' PLT UL were at or exceeded 450 x109/L (Figure D). Conclusions: There was minimal variance in CBC reference limits across hospitals in the US with WBC LL having the highest COV. Most MCV UL and WBC UL exceeded common textbook reference limits. Almost all male Hgb UL and over a quarter of PLT UL exceed the WHO major criteria of polycythemia vera and essential thrombocytosis, respectively. Since our findings suggest that there could be a risk of underdiagnosing these myeloproliferative neoplasms due to the commonly used reference limits, clinical context must be used when interpreting normal CBC results, especially when there is a suspicion of polycythemia vera or essential thrombocytosis.