Non-sclerosing (T-cell) and sclerosing (B-cell) lymphocytic lobulitis in diagnostic breast biopsies: Clinical, imaging, and pathologic features.

Lymphocytic lobulitis (LL) is characterized by prominent lymphocytic infiltrates centered on lobules. Sclerosing lymphocytic lobulitis (SCLL) associated with diabetes mellitus (DM) or autoimmune disease (AI) was the first type to be described. Subsequently, non-sclerosing LL (NSCLL) was reported as an incidental finding in prophylactic mastectomies due to high risk germline mutations or a family history of breast cancer. The two types of LL were distinguished by stromal features and a predominant population of B-cells in the former and T-cells in the latter. In this study, 8 cases of NSCLL detected clinically or by screening were compared to 44 cases of SCLL. One case of NSCLL presented as a palpable mass, 2 as masses on screening, and 5 as MRI enhancement. In contrast, 80% of SCLL cases presented as palpable masses. Half the cases of NSCLL were associated with a BRCA1 or 2 mutation compared to 1 case of SCLL (2%). Three additional cases of NSCLL were associated with a strong family and/or personal history of breast cancer. Almost half (52%) of SCLL cases were associated with DM or AI, but only 25% of NSCLL. Immunoperoxidase studies confirmed a predominance of T-cells in NSCLL and B-cells in SCLL associated with DM or AI. It is important for pathologists to be aware of this new observation that NSCLL can be detected as a palpable mass or an imaging finding in diagnostic biopsies, as its presence can be indicative of a significant risk for breast cancer.

Cutaneous Manifestations of Myeloid Neoplasms Exhibit Broad and Divergent Morphologic and Immunophenotypic Features but Share Ancestral Clonal Mutations With Bone Marrow.

In this study, we performed a comprehensive molecular analysis of paired skin and peripheral blood/bone marrow (BM) samples from 17 patients with cutaneous myeloid or cutaneous histiocytic-dendritic neoplasms. The cutaneous manifestations included 10 patients with cutaneous acute myeloid leukemia (c-AML), 2 patients with full or partial Langerhans cell differentiation, 2 patients with blastic plasmacytoid dendritic cell neoplasms (BPDCN), 1 patient with both Langerhans cell differentiation and BPDCN, and 2 patients with full or partial indeterminate dendritic cell differentiation. Seven of the 10 c-AML patients (70%) exhibited concurrent or subsequent marrow involvement by acute myeloid leukemia, with all 7 cases (100%) demonstrating shared clonal mutations in both the skin and BM. However, clonal relatedness was documented in one additional case that never had any BM involvement. Nevertheless, NPM1 mutations were identified in 7 of the 10 (70%) of these c-AML cases while one had KMT2A rearrangement and one showed inv(16). All 3 patients (100%) with Langerhans cell neoplasms, 2 patients with BPDCN (100%), and one of the 2 patients (50%) with other cutaneous dendritic cell neoplasms also demonstrated shared mutations between the skin and concurrent or subsequent myeloid neoplasms. Both BM and c-AML shared identical founding drivers, with a predominance of NPM1, DNMT3A, and translocations associated with monocytic differentiation, with common cutaneous-only mutations involving genes in the signal transduction and epigenetic pathways. Cutaneous histiocytic-dendritic neoplasms shared founding drivers in ASXL1, TET2, and/or SRSF2. However, in the Langerhans cell histiocytosis or histiocytic sarcoma cases, there exist recurrent secondary RAS pathway hits, whereas cutaneous BPDCN cases exhibit copy number or structural variants. These results enrich and broaden our understanding of clonally related cutaneous manifestations of myeloid neoplasms and further illuminate the highly diverse spectrum of morphologic and immunophenotypic features they exhibit.

Optimization of Advanced Molecular Genetic Testing Utilization in Hematopathology: A Goldilocks Approach to Bone Marrow Testing.

PURPOSE: This study investigated the effectiveness of algorithmic testing in hematopathology at the Brigham and Women's Hospital and Dana-Farber Cancer Institute (DFCI). The algorithm was predicated on test selection after an initial pathologic evaluation to maximize cost-effective testing, especially for expensive molecular and cytogenetic assays. MATERIALS AND METHODS: Standard ordering protocols (SOPs) for 17 disease categories were developed and encoded in a decision support application. Six months of retrospective data from application beta testing was obtained and compared with actual testing practices during that timeframe. In addition, 2 years of prospective data were also obtained from patients at one community satellite site. RESULTS: A total of 460 retrospective cases (before introduction of algorithmic testing) and 109 prospective cases (following introduction) were analyzed. In the retrospective data, 61.7% of tests (509 of 825) were concordant with the SOPs while 38.3% (316 of 825) were overordered and 30.8% (227 of 736) of SOP-recommended tests were omitted. In the prospective data, 98.8% of testing was concordant (244 of 247 total tests) with only 1.2% overordered tests (3 of 247) and 7.6% omitted tests (20 of 264 SOP-recommended tests; overall P < .001). The cost of overordered tests before implementing SOP indicates a potential annualized saving of $1,347,520 in US dollars (USD) in overordered testing at Brigham and Women's Hospital/DFCI. Only two of 316 overordered tests (0.6%) returned any additional information, both for extremely rare clinical circumstances. CONCLUSION: Implementation of SOPs dramatically improved test ordering practices, with a just right number of ancillary tests that minimizes cost and has no significant impact on acquiring key informative test results.

Initial Diagnosis of Classic Hodgkin Lymphoma With Skin Biopsy: A Rare Case and Review of Diagnostic Considerations.

ABSTRACT: Classic Hodgkin lymphoma (CHL) is a B-cell-derived lymphoma that classically displays a bimodal age distribution. CHL typically involves the mediastinum, lymph nodes, and other visceral organs. CHL is characterized histologically by the presence of a relatively paucicellular neoplastic cell population composed of large atypical cells (including Hodgkin and Reed-Sternberg forms) in a reactive mixed inflammatory background, often with prominent necrosis. CHL rarely occurs in the skin, and the associated mixed inflammatory infiltrate or necrotic appearance can create diagnostic uncertainty. Herein, we report the case of a 31-year-old man presenting with a painful dendritic rash of the anterior chest wall with axillary lymphadenopathy. After multiple nondiagnostic biopsies that revealed largely necrotic material, a chest wall skin biopsy was obtained. The skin biopsy was diagnostic of CHL, based on the presence of large atypical dermal cells, including Hodgkin and Reed-Sternberg forms, which expressed CD15, CD30 and Fascin, in a typical mixed inflammatory and necrotic background. Through the lens of this case, we discuss the characteristics and mechanisms of skin involvement of CHL, and the histopathologic and immunohistochemical pitfalls when considering the rare diagnosis of CHL in the skin.

Autopsy findings from patients diagnosed with COVID-19 demonstrate unique morphological patterns in bone marrow and lymph node.

AIMS: The identification of haemophagocytosis in bone marrow (BM) is recurrently identified in patients with severe COVID-19. These initial COVID-19 autopsy studies have afforded valuable insight into the pathophysiology of this disease; however, only a limited number of case series have focused on lymphoid or haematopoietic tissues. METHODS: BM and lymph node (LN) specimens were obtained from adult autopsies performed between 1 April 2020 and 1 June 2020, for which the decedent had tested positive for SARS-CoV-2. Tissue sections (H&E, CD3, CD20, CD21, CD138, CD163, MUM1, kappa/lambda light chains in situ hybridisation) were examined by two haematopathologists, who recorded morphological features in a blinded fashion. Haemophagocytic lymphohistiocytosis (HLH) was assessed based on HLH 2004 criteria. RESULTS: The BM demonstrated a haemophagocytic pattern in 9 out of 25 patients (36%). The HLH pattern was associated with longer hospitalisation, BM plasmacytosis, LN follicular hyperplasia and lower aspartate aminotransferase (AST), as well as ferritin at demise. LN examination showed increased plasmacytoid cells in 20 of 25 patients (80%). This pattern was associated with a low absolute monocyte count at diagnosis, lower white cell count and lower absolute neutrophil count at demise, and lower ferritin and AST at demise. CONCLUSIONS: Autopsy results demonstrate distinct morphological patterns in BM, with or without haemophagocytic macrophages, and in LN, with or without increased plasmacytoid cells. Since only a minority of patients met diagnostic criteria for HLH, the observed BM haemophagocytic macrophages may be more indicative of an overall inflammatory state.

Expanding the Immunophenotypic Spectrum of Neoplastic and Reactive Plasmacytoid Dendritic Cells.

OBJECTIVES: Targeted therapies for blastic plasmacytoid dendritic cell neoplasm (BPDCN) have presented a diagnostic dilemma for differentiating residual BPDCN from reactive plasmacytoid dendritic cells (pDCs) because these conditions have a similar immunoprofile, necessitating discovery of additional diagnostic markers. METHODS: Fifty cases of BPDCN involving bone marrow (26/50) and skin (24/50) as well as other hematologic malignancies (67) and nonneoplastic samples (37) were included. Slides were stained using a double-staining protocol for the following immunohistochemical marker combinations: TCF4/CD123, TCF4/CD56, SOX4/CD123, and IRF8/CD123. RESULTS: The nuclear marker SOX4 is expressed in neoplastic pDCs; in our cohort, SOX4/CD123 showed 100% sensitivity and 98% specificity in distinguishing BPDCN from reactive pDCs and other neoplasms. TCF4/CD56 had a 96% sensitivity and 100% specificity for BPDCN. IRF8 is a nonspecific marker that is positive in BPDCN and pDCs as well as other myeloid malignancies. CONCLUSIONS: The novel immunohistochemical combination SOX4/CD123 distinguishes BPDCN, including CD56-negative BPDCN, from both reactive pDCs and other neoplasms. Because of their high diagnostic sensitivity and specificity, the double-staining marker combinations TCF4/CD123, TCF4/CD56, and SOX4/CD123 can be used to confirm lineage in BPDCN cases and detect minimal/measurable residual disease in tissue specimens.

Myeloid sarcoma with NPM1 mutation may be clinically and genetically distinct from AML with NPM1 mutation: a study from the Bone Marrow Pathology Group.

Myeloid sarcoma (MS) is currently considered equivalent to de novo acute myeloid leukemia (AML); however, the relationship between these entities is poorly understood. This retrospective multi-institutional cohort study compared 43 MS with NPM1 mutation to 106 AML with NPM1 mutation. Compared to AML, MS had more frequent cytogenetic abnormalities including complex karyotype (p = .009 and p = .007, respectively) and was enriched in mutations of genes involved in histone modification, including ASXL1 (p = .007 and p = .008, respectively). AML harbored a higher average number of gene mutations (p = .002) including more frequent PTPN11 mutations (p < .001) and mutations of DNA-methylating genes including DNMT3A and IDH1 (both p < .001). MS had significantly shorter overall survival (OS) than AML (median OS: 44.9 vs. 93.2 months, respectively, p = .037). MS with NPM1 mutation has a unique genetic landscape, and poorer OS, compared to AML with NPM1 mutation.

First study comparing genetic profiles of MS and AML with a common disease-defining lesion.NPM1^Mut MS may be genetically distinct from NPM1^Mut AML.NPM1^Mut MS may have inferior overall survival compared to NPM1^Mut AML.

Cardiac megakaryocytes in SARS-CoV-2-positive autopsies.

Thromboembolic phenomena are an important complication of infection by severe acute respiratory coronavirus 2 (SARS-CoV-2). Increasing focus on the management of the thrombotic complications of Coronavirus Disease 2019 (COVID-19) has led to further investigation into the role of platelets, and their precursor cell, the megakaryocyte, during the disease course. Previously published postmortem evaluations of patients who succumbed to COVID-19 have reported the presence of megakaryocytes in the cardiac microvasculature. Our series evaluated a cohort of autopsies performed on SARS-CoV-2-positive patients in 2020 (n = 36) and prepandemic autopsies performed in early 2020 (n = 12) and selected to represent comorbidities common in cases of severe COVID-19, in addition to infectious and noninfectious pulmonary disease and thromboembolic phenomena. Cases were assessed for the presence of cardiac megakaryocytes and correlated with the presence of pulmonary emboli and laboratory platelet parameters and inflammatory markers. Cardiac megakaryocytes were detected in 64% (23/36) of COVID-19 autopsies, and 40% (5/12) prepandemic autopsies, with averages of 1.77 and 0.84 megakaryocytes per cm2 , respectively. Within the COVID-19 cohort, autopsies with detected megakaryocytes had significantly higher platelet counts compared with cases throughout; other platelet parameters were not statistically significant between groups. Although studies have supported a role of platelets and megakaryocytes in the response to viral infections, including SARS-CoV-2, our findings suggest cardiac megakaryocytes may be representative of a nonspecific inflammatory response and are frequent in, but not exclusive to, COVID-19 autopsies.

Global assessment of IRF8 as a novel cancer biomarker.

Interferon regulatory factor 8 (IRF8) is a member of the IRF family that is specific to the hematopoietic cell and is involved in regulating the development of human monocytic and dendritic-lineage cells, as well as B-cells. Because its utility as a sensitive and specific monoblast marker in the context of acute monocytic leukemias has been recently demonstrated, we hypothesized that it may also be useful as a novel immunohistochemical marker in myeloid sarcomas and blastic plasmacytoid dendritic cell neoplasms (BPDCNs) with respect to their differential diagnoses. In this retrospective study, we analyzed the IHC expression pattern of IRF8 in 385 patient samples across 30 types of cancers, referenced to their mRNA expression data available through The Cancer Genome Atlas. In addition, we assessed IRF8 in 35 myeloid sarcomas and 15 BPDCNs. Twenty-four of 35 cases of myeloid sarcomas (68.5%) showed positivity for IRF8, with six cases (17.1%) demonstrating IRF8 expression in the absence of CD34 and MPO. All 15 of 15 BPDCNs (100%) showed strong uniform expression of IRF8 and were occasionally more definitive than CD123. IRF8 was negative in all desmoplastic small round cell tumors, Ewing sarcomas, synovial sarcomas, and undifferentiated pleomorphic sarcomas, as well as all epithelial malignancies tested except for 2 triple negative breast cancers that showed subset weak staining. In conclusion, IRF8 is a novel marker helpful in identifying extranodal hematopoietic tumors that can otherwise be difficult to diagnose given the broad differential diagnoses and frequent loss of more common lineage-defining markers.

Skeletal Muscle and Peripheral Nerve Histopathology in COVID-19.

OBJECTIVE: To explore the spectrum of skeletal muscle and nerve pathology of patients who died after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to assess for direct viral invasion of these tissues. METHODS: Psoas muscle and femoral nerve sampled from 35 consecutive autopsies of patients who died after SARS-CoV-2 infection and 10 SARS-CoV-2-negative controls were examined under light microscopy. Clinical and laboratory data were obtained by chart review. RESULTS: In SARS-CoV-2-positive patients, mean age at death was 67.8 years (range 43-96 years), and the duration of symptom onset to death ranged from 1 to 49 days. Four patients had neuromuscular symptoms. Peak creatine kinase was elevated in 74% (mean 959 U/L, range 29-8,413 U/L). Muscle showed type 2 atrophy in 32 patients, necrotizing myopathy in 9, and myositis in 7. Neuritis was seen in 9. Major histocompatibility complex-1 (MHC-1) expression was observed in all cases of necrotizing myopathy and myositis and in 8 additional patients. Abnormal expression of myxovirus resistance protein A (MxA) was present on capillaries in muscle in 9 patients and in nerve in 7 patients. SARS-CoV-2 immunohistochemistry was negative in muscle and nerve in all patients. In the 10 controls, muscle showed type 2 atrophy in all patients, necrotic muscle fibers in 1, MHC-1 expression in nonnecrotic/nonregenerating fibers in 3, MxA expression on capillaries in 2, and inflammatory cells in none, and nerves showed no inflammatory cells or MxA expression. CONCLUSIONS: Muscle and nerve tissue demonstrated inflammatory/immune-mediated damage likely related to release of cytokines. There was no evidence of direct SARS-CoV-2 invasion of these tissues. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that muscle and nerve biopsies document a variety of pathologic changes in patients dying of coronavirus disease 2019 (COVID-19).

miR-15a/16-1 deletion in activated B cells promotes plasma cell and mature B-cell neoplasms.

Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.

Peripheral host T cells survive hematopoietic stem cell transplantation and promote graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67+) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Multiparametric in situ imaging of NPM1-mutated acute myeloid leukemia reveals prognostically-relevant features of the marrow microenvironment.

Ancillary testing during the initial workup of acute myeloid leukemia (AML) is largely performed using aspirated materials. We utilized multiplex immunofluorescence (MIF) imaging with digital image analysis to perform an in situ analysis of the microenvironment in NPM1-mutated AML using diagnostic bone marrow biopsy tissues (N = 17) and correlated these findings with diagnostic next-generation sequencing (NGS, N = 17), flow cytometry (FC, N = 14), and first remission (CR1) NPM1-specific molecular MRD (n = 16) data. The total CD3-positive T-cell percentages correlated positively between FC and MIF (r = 0.53, p = 0.05), but were significantly lower by MIF (1.62% vs. 3.4%, p = 0.009). The percentage of mutant NPM1-positive (NPM1c+) cells ranged from 9.7 to 90.8% (median 45.4%) and did not correlate with the NPM1 mutant allele fraction by NGS (p > 0.05). The percentage of CD34+/NPM1c+ cells ranged from 0 to 1.8% (median 0.07%). The percentage of NPM1c+ cells correlated inversely (34% vs. 62%, p = 0.03), while the percentages of CD3-/NPM1c- cells (64% vs. 35%, p = 0.03), and specifically CD3-/CD4-/NPM1c- cells (26% vs. 13%, p = 0.04), correlated positively with subsequent MRD. Discordances between MIF and FC/NGS data suggest that aspirate materials are likely an imperfect reflection of the core biopsy tissue. Furthermore, increased numbers of NPM1 wild-type cells within the microenvironment at diagnosis correlate with the subsequent presence of MRD.

Detection of the KITD816V mutation in myelodysplastic and/or myeloproliferative neoplasms and acute myeloid leukemia with myelodysplasia-related changes predicts concurrent systemic mastocytosis.

Greater than 90% of cases of systemic mastocytosis (SM) harbor pathogenic KIT mutations, particularly KITD816V. Prognostically-significant pathogenic KIT mutations also occur in 30-40% of core binding factor-associated acute myeloid leukemia (CBF-AML), but are uncommonly associated with concurrent SM. By comparison, the occurrence of SM in other myeloid neoplasms bearing pathogenic KIT mutations, particularly those with a chronic course, is poorly understood. Review of clinical next-generation sequencing (NGS) performed at our institutions in patients with known or suspected hematologic malignancies over an 8-year period revealed 64 patients with both a pathogenic KIT mutation detected at one or more timepoints and available bone marrow biopsy materials. Patients with KITD816V-mutated myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), or overlap MDS/MPN (n = 22) accounted for approximately one-third of our cohort (34%). Comprehensive morphologic and immunophenotypic characterization revealed that nearly all cases (n = 20, 91%) exhibited concurrent SM. In contrast, of the 18 patients (28%) with AML and KITD816V, only eight (44%) showed evidence of SM at any point in their disease course (p = 0.0021); of these eight, the AML component was characterized as AML with myelodysplasia-related changes (AML-MRC) in all but one instance (n = 7, 87%). Twelve patients (19%) had pathogenic KIT mutations other than p.D816V, all in the setting of AML (CFB-AML, n = 7; AML, not otherwise specified, n = 2; AML-MRC, n = 1; acute promyelocytic leukemia, n = 1); only two of these patients (17%), both with CBF-AML, exhibited concurrent SM. The remaining 12 patients (19%) had SM without evidence of an associated hematological neoplasm (AHN). For nearly one-third of the 30 SM-AHN patients in our cohort (n = 9, 30%), the SM component of their disease was not initially clinicopathologically recognized. We propose that identification of the KITD816V mutation in patients diagnosed with MDS, MPN, MDS/MPN, or AML-MRC should trigger reflex testing for SM.

Human MYD88L265P is insufficient by itself to drive neoplastic transformation in mature mouse B cells.

MYD88 L265P is the most common mutation in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) and one of the most frequent in poor-prognosis subtypes of diffuse large B-cell lymphoma (DLBCL). Although inhibition of the mutated MYD88 pathway has an adverse impact on LPL/WM and DLBCL cell survival, its role in lymphoma initiation remains to be clarified. We show that in mice, human MYD88L265P promotes development of a non-clonal, low-grade B-cell lymphoproliferative disorder with several clinicopathologic features that resemble human LPL/WM, including expansion of lymphoplasmacytoid cells, increased serum immunoglobulin M (IgM) concentration, rouleaux formation, increased number of mast cells in the bone marrow, and proinflammatory signaling that progresses sporadically to clonal, high-grade DLBCL. Murine findings regarding differences in the pattern of MYD88 staining and immune infiltrates in the bone marrows of MYD88 wild-type (MYD88WT) and MYD88L265P mice are recapitulated in the human setting, which provides insight into LPL/WM pathogenesis. Furthermore, histologic transformation to DLBCL is associated with acquisition of secondary genetic lesions frequently seen in de novo human DLBCL as well as LPL/WM-transformed cases. These findings indicate that, although the MYD88L265P mutation might be indispensable for the LPL/WM phenotype, it is insufficient by itself to drive malignant transformation in B cells and relies on other, potentially targetable cooperating genetic events for full development of lymphoma.

High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia.

Acute myeloid leukemia (AML) with mutated NPM1 is a newly recognized separate entity in the revised 2016 WHO classification, and is associated with a favorable prognosis. While previous studies have evaluated NPM1 in a binary fashion, we recently demonstrated a significant independent negative prognostic effect of high NPM1 mutant allele burden (VAF) at diagnosis in a cohort of de novo AML patients. Although the importance of minimal residual disease (MRD) monitoring in NPM1-mutated AML has been well characterized, the potential relationship between diagnostic allele burden and MRD is unknown. We retrospectively evaluated for MRD at first remission (CR1). We used either next-generation sequencing (NGS) [n = 71], and/or immunohistochemistry (IHC) for mutant NPM1 (NPM1c) [n = 60], in a subset of patients from our recently examined cohort. We identified a statistically significant positive correlation between the VAF at diagnosis, and at CR1 (Spearman r = 0.4, P = .006), and enrichment for MRD in high diagnostic VAF patients (P = .05), as previously defined. IHC-positivity also correlated significantly with a higher median diagnostic NPM1 VAF (0.42 vs 0.39, P = .02), and with the VAF at CR1 (Spearman r = 0.7, P = .003). In multivariable analyses, both high diagnostic VAF (P = .003) and MRD (P = .02) were independent predictors of shorter event-free survival (EFS). Our findings suggest a relationship between the NPM1 mutant allele burden at diagnosis, and the presence of MRD at first remission. Our findings support IHC as a potentially useful adjunctive tool for disease monitoring.