DeepHeme: A generalizable, bone marrow classifier with hematopathologist-level performance.

Morphology-based classification of cells in the bone marrow aspirate (BMA) is a key step in the diagnosis and management of hematologic malignancies. However, it is time-intensive and must be performed by expert hematopathologists and laboratory professionals. We curated a large, high-quality dataset of 41,595 hematopathologist consensus-annotated single-cell images extracted from BMA whole slide images (WSIs) containing 23 morphologic classes from the clinical archives of the University of California, San Francisco. We trained a convolutional neural network, DeepHeme, to classify images in this dataset, achieving a mean area under the curve (AUC) of 0.99. DeepHeme was then externally validated on WSIs from Memorial Sloan Kettering Cancer Center, with a similar AUC of 0.98, demonstrating robust generalization. When compared to individual hematopathologists from three different top academic medical centers, the algorithm outperformed all three. Finally, DeepHeme reliably identified cell states such as mitosis, paving the way for image-based quantification of mitotic index in a cell-specific manner, which may have important clinical applications.

Highly sensitive single tube B-lymphoblastic leukemia/lymphoma minimal/measurable residual disease test robust to surface antigen directed therapy.

BACKGROUND: Measurement of minimal/measurable residual disease (MRD) in B-lymphoblastic leukemia/lymphoma (B-ALL) has become a routine clinical evaluation tool and remains the strongest predictor of treatment outcome. In recent years, new targeted anti-CD19 and anti-CD22 antibody-based and cellular therapies have revolutionized the treatment of the high-risk B-ALL. The new treatments raise challenges for diagnostic flow cytometry, which relies on the presence of specific surface antigens to identify the population of interest. So far, reported flow cytometry-based assays are developed to either achieve a deeper MRD level or to accommodate the loss of surface antigens post-target therapies, but not both. METHODS: We developed a single tube flow cytometry assay (14-color-16-parameters). The method was validated using 94 clinical samples as well as spike-in and replicate experiments. RESULTS: The assay was well suited for monitoring response to targeted therapies and reached a sensitivity below 10-5 with acceptable precision (coefficient of variation < 20%), accuracy, and interobserver variability (κ = 1). CONCLUSIONS: The assay allows for sensitive disease detection of B-ALL MRD independent of CD19 and CD22 expression and allows uniform analysis of samples regardless of anti-CD19 and CD22 therapy.

Clonal Characterization and Somatic Hypermutation Assessment by Next-Generation Sequencing in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Detailed Description of the Technical Performance, Clinical Utility, and Platform Comparison.

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches.

Abnormal B-lymphoblasts in myelodysplastic syndromes and myeloproliferative neoplasms other than chronic myeloid leukemia.

BACKGROUND: Lineage infidelity is characteristic of mixed phenotype acute leukemia and is also seen in blast phase of chronic myeloid leukemia (CML), myeloid/lymphoid neoplasia with eosinophilia and gene rearrangements, and subtypes of acute myeloid leukemia. Driver genetic events often occur in multipotent progenitor cells in myeloid neoplasms, suggesting that multilineage output may be more common than appreciated. This phenomenon is not well studied in myelodysplastic syndrome (MDS) and non-CML myeloproliferative neoplasms (MPN). METHODS: We systematically evaluated phenotypic lineage infidelity by reviewing bone marrow pathology and flow cytometry (FC) studies of 1262 consecutive patients with a diagnosis of MDS and/or non-CML MPN. We assessed B- and T-cells in these patients by FC. When abnormal B-lymphoblast (ABLB) populations were detected, we additionally evaluated immature B-cells using a high sensitivity FC assay for B-lymphoblastic leukemia/lymphoma (B-ALL). RESULTS: We identified 9 patients (7 MDS, 7/713, 1%; 2 non-CML MPN, 2/312, 0.6%; 0 in MDS/MPN) with low-level ABLB populations (0.012%-3.6% of WBCs in marrow) with abnormal immunophenotypes. Genetic studies on flow sorted cell populations confirmed that some ABLB populations were clonally related to myeloid blasts (4/6, 67%). On follow-up, ABLB populations in 8/9 patients remained stable or disappeared. Only 1 case progressed to B-ALL. CONCLUSIONS: These findings demonstrate that phenotypically detectable abnormal immature B lineage output occurs in MDS and non-CML MPN, albeit rarely. While presence of ABLB does not necessarily reflect blast crisis, the underlying disease biology of our findings may ultimately be relevant to patient management and warrants further investigation.

PD-1 improves accurate detection of Sezary cells by flow cytometry in peripheral blood in mycosis fungoides/Sezary syndrome.

BACKGROUND: Accurate Sezary cell detection in peripheral blood of mycosis fungoides/Sezary syndrome (MF/SS) patients by flow cytometry can be difficult due to overlapping immunophenotypes with normal T cells using standard markers. We assessed the utility of programmed death-1 (PD-1/CD279), a transmembrane protein expressed in some hematopoietic cells, for identification and quantitation of circulating Sezary cells among established markers using flow cytometry. METHODS: 50 MF/SS and 20 control blood samples were immunophenotyped by flow cytometry. Principal component analysis (PCA) assessed contributions of antigens to separation of abnormal from normal T cell populations. PD-1 was assessed over time in blood and bone marrow of available MF/SS cases. RESULTS: Normal CD4+ T cells showed dim/intermediate to absent PD-1 expression. PD-1 in Sezary cells was informatively brighter (≥1/3 log) than internal normal CD4+ T cells in 39/50 (78%) cases. By PCA, PD-1 ranked 3rd behind CD7 and CD26 in population separation as a whole; it ranked in the top 3 markers in 32/50 (64%) cases and 1st in 4/50 (8%) cases when individual abnormal populations were compared to total normal CD4+ T cells. PD-1 clearly separated Sezary from normal CD4+ T cells in 15/26 (58%, 30% of total) cases with few and subtle alterations of pan-T cell antigens/CD26 and was critical in 6 (12% of total), without which identification and quantification were significantly affected or nearly impossible. PD-1 remained informative in blood/bone marrow over time in most patients. CONCLUSIONS: PD-1 significantly contributes to accurate flow cytometric Sezary cell assessment in a routine Sezary panel.

Role of Flow Cytometric Immunophenotyping for Classic Hodgkin Lymphoma in Small Biopsy and Cytology Specimens.

CONTEXT.­: The diagnosis of classic Hodgkin lymphoma (CHL) traditionally requires surgical tissue biopsy because of the paucity of diagnostic Hodgkin and Reed-Sternberg cells. Diagnosis can be challenging in small core needle and cytologic biopsies, which are increasingly used because of reduced costs and minimal invasiveness. Flow cytometric (FC) identification of Hodgkin and Reed-Sternberg cells is possible, but FC test efficacy is not well studied outside of validation settings, especially in small specimens. OBJECTIVE.­: To assess the testing efficacy of FC performed on small biopsy and cytology specimens for the diagnosis of CHL. DESIGN.­: We reviewed 131 patients with CHL and 459 patients without CHL during a 3-year period who underwent a small biopsy procedure, including core biopsy and/or cytology evaluation, with concurrent routine clinical FC testing for CHL, assessing performance of FC in small specimens. RESULTS.­: Evaluating testing efficacy, sensitivity was 95.4% and specificity was 98.2%, whereas positive and negative predictive values were 92.2% and 99.0%, respectively. Although there were more false-positive results than compared with published validation studies, expert review identified distinct diagnostic pitfalls; awareness of these may improve testing efficacy. CONCLUSIONS.­: Although FC diagnosis of CHL was historically considered unfeasible, our findings in a real-world clinical setting suggest that FC adds diagnostic value to small biopsy evaluation, reducing time to treatment, costs, and invasive excisional procedures.

A phase 2 biomarker-driven study of ruxolitinib demonstrates effectiveness of JAK/STAT targeting in T-cell lymphomas.

Signaling through JAK1 and/or JAK2 is common among tumor and nontumor cells within peripheral T-cell lymphoma (PTCL). No oral therapies are approved for PTCL, and better treatments for relapsed/refractory disease are urgently needed. We conducted a phase 2 study of the JAK1/2 inhibitor ruxolitinib for patients with relapsed/refractory PTCL (n = 45) or mycosis fungoides (MF) (n = 7). Patients enrolled onto 1 of 3 biomarker-defined cohorts: (1) activating JAK and/or STAT mutations, (2) ≥30% pSTAT3 expression among tumor cells by immunohistochemistry, or (3) neither or insufficient tissue to assess. Patients received ruxolitinib 20 mg PO twice daily until progression and were assessed for response after cycles 2 and 5 and every 3 cycles thereafter. The primary endpoint was clinical benefit rate (CBR), defined as the combination of complete response, partial response (PR), and stable disease lasting at least 6 months. Only 1 of 7 patients with MF had CBR (ongoing PR > 18 months). CBR among the PTCL cases (n = 45) in cohorts 1, 2, and 3 were 53%, 45%, and 13% (cohorts 1 & 2 vs 3, P = .02), respectively. Eight patients had CBR > 12 months (5 ongoing), including 4 of 5 patients with T-cell large granular lymphocytic leukemia. In an exploratory analysis using multiplex immunofluorescence, expression of phosphorylated S6, a marker of PI3 kinase or mitogen-activated protein kinase activation, in <25% of tumor cells was associated with response to ruxolitinib (P = .05). Our findings indicate that ruxolitinib is active across various PTCL subtypes and support a precision therapy approach to JAK/STAT inhibition in patients with PTCL. This trial was registered at www.clincialtrials.gov as #NCT02974647.

Plasmacytoid dendritic cell expansion defines a distinct subset of RUNX1-mutated acute myeloid leukemia.

Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon-producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.

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

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

Genetic Basis of Extramedullary Plasmablastic Transformation of Multiple Myeloma.

In patients with multiple myeloma, plasmablastic transformation in the bone marrow is rare and associated with poor outcomes. The significance of discordant extramedullary plasmablastic transformation in patients with small, mature clonal plasma cells in the bone marrow has not been well studied. Here, we report the clinicopathologic, cytogenetic, and molecular features of 10 such patients (male/female: 6/4, median age: 65 y, range: 48 to 76 y) with an established diagnosis of multiple myeloma in the bone marrow composed of small, mature plasma cells in parallel with a concurrent or subsequent extramedullary plasmablastic transformation. Eight patients with available survival data showed an overall aggressive clinical course with a median survival of 4.5 months after the diagnosis of extramedullary plasmablastic transformation, despite aggressive treatment and even in patients with low-level bone marrow involvement. Pathologically, the extramedullary plasmablastic myeloma were clonally related to the corresponding bone marrow plasma cells, showed high levels of CMYC and/or P53 expression with a high Ki-67 proliferation index by immunohistochemistry and harbored more complex genomic aberrations including frequent mutations in the RAS pathway and MYC rearrangements compared with their bone marrow counterparts. In summary, although genetic and immunohistochemical studies were not uniformly performed on all cases due to the retrospective nature of this study, our data suggest that discordant extramedullary plasmablastic transformation of multiple myeloma has an aggressive clinical course and is characterized by frequent mutations in the RAS pathway and more complex genomic abnormalities.

Genetic basis for iMCD-TAFRO.

TAFRO syndrome, a clinical subtype of idiopathic multicentric Castleman disease (iMCD), consists of a constellation of symptoms/signs including thrombocytopenia, anasarca, fever, reticulin fibrosis/renal dysfunction, and organomegaly. The etiology of iMCD-TAFRO and the basis for cytokine hypersecretion commonly seen in iMCD-TAFRO patients has not been elucidated. Here, we identified a somatic MEK2P128L mutation and a germline RUNX1G60C mutation in two patients with iMCD-TAFRO, respectively. The MEK2P128L mutation, which has been identified previously in solid tumor and histiocytosis patients, caused hyperactivated MAP kinase signaling, conferred IL-3 hypersensitivity and sensitized the cells to various MEK inhibitors. The RUNX1G60C mutation abolished the transcriptional activity of wild-type RUNX1 and functioned as a dominant negative form of RUNX1, resulting in enhanced self-renewal activity in hematopoietic stem/progenitor cells. Interestingly, ERK was heavily activated in both patients, highlighting a potential role for activation of MAPK signaling in iMCD-TAFRO pathogenesis and a rationale for exploring inhibition of the MAPK pathway as a therapy for iMCD-TAFRO. Moreover, these data suggest that iMCD-TAFRO might share pathogenetic features with clonal inflammatory disorders bearing MEK and RUNX1 mutations such as histiocytoses and myeloid neoplasms.

Divergent clonal evolution of a common precursor to mantle cell lymphoma and classic Hodgkin lymphoma.

Clonal heterogeneity and evolution of mantle cell lymphoma (MCL) remain unclear despite the progress in our understanding of its biology. Here, we report a 71-yr-old male patient with an aggressive MCL and depict the clonal evolution from initial diagnosis of typical MCL to relapsed blastoid MCL. During the course of the disease, the patient was diagnosed with classic Hodgkin lymphoma (CHL) and received a CHL therapeutic regimen. Molecular analysis by next-generation sequencing of both MCL and CHL demonstrated clonally related CHL with characteristic immunophenotype and PDL1/2 gains. Moreover, our data illustrate the clonal heterogeneity and acquisition of additional genetic aberrations including a rare fusion of SEC22B-NOTCH2 in the process of clonal evolution. Evidence obtained from our comprehensive immunophenotypic and genetic studies indicates that MCL and CHL can originate from a common precursor by divergent clonal evolution, which may pose a therapeutic challenge.

Transformation of monoclonal B lymphocytosis to Epstein-Barr virus-positive large B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and classic Hodgkin lymphoma.

Transformation of chronic lymphocytic leukemia (CLL) to an aggressive lymphoma, so-called Richter syndrome (RS), usually includes diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma (CHL). The transformation can be clonally related to the underlying CLL, and is often Epstein-Barr virus (EBV)-associated. Here we report an 86-year-old female with a newly identified CLL-like monoclonal B-lymphocytosis (MBL) who developed diffuse lymphadenopathy. Biopsy of the left axillary lymph node showed EBV-positive large B-cell lymphoma with morphologic and immunophenotypic features intermediate between DLBCL and CHL, so-called gray zone lymphoma. Comprehensive immunophenotypic, cytogenetics and molecular studies demonstrate a clonal relatedness that suggests a transformation from MBL to EBV+ gray zone lymphoma.

PHF6 and DNMT3A mutations are enriched in distinct subgroups of mixed phenotype acute leukemia with T-lineage differentiation.

The genetic aberrations that drive mixed phenotype acute leukemia (MPAL) remain largely unknown, with the exception of a small subset of MPALs harboring BCR -ABL1 and MLL translocations. We performed clinicopathologic and genetic evaluation of 52 presumptive MPAL cases at Memorial Sloan Kettering Cancer Center. Only 29 out of 52 (56%) cases were confirmed to be bona fide MPAL according to the 2016 World Heath Organization classification. We identified PHF6 and DNMT3A mutations as the most common recurrent mutations in MPAL, each occurring in 6 out of 26 (23%) cases. These mutations are mutually exclusive of each other and BCR-ABL1/MLL translocations. PHF6- and DNMT3A-mutated MPAL showed marked predilection for T-lineage differentiation (5/6 PHF6 mutated, 6/6 DNMT3A mutated). PHF6-mutated MPAL occurred in a younger patient cohort compared with DNMT3A-mutated cases (median age, 27 years vs 61 years, P < .01). All 3 MPAL cases with both T- and B-lineage differentiation harbored PHF6 mutations. MPAL with T-lineage differentiation was associated with nodal or extramedullary involvement (9/15 [60%] vs 0, P = .001) and a higher relapse incidence (78% vs 22%, P = .017) compared with those without T-lineage differentiation. Sequencing studies on flow-cytometry-sorted populations demonstrated that PHF6 mutations are present in all blast compartments regardless of lineage differentiation with high variant allele frequency, implicating PHF6 as an early mutation in MPAL pathogenesis. In conclusion, PHF6 and DNMT3A mutations are the most common somatic alterations identified in MPAL and appear to define 2 distinct subgroups of MPAL with T-lineage differentiation with inferior outcomes.