Performance evaluation of a novel artificial intelligence (AI)-assisted digital microscopy system for the routine analysis of bone marrow aspirates.

Bone marrow aspiration (BMA) smear analysis is essential for diagnosis, treatment and monitoring of a variety of benign and neoplastic hematological conditions. Currently this analysis is performed by manual microscopy. We conducted a multi-center study to validate a computational microscopy approach with an artificial intelligence (AI)-driven decision support system. A total of 795 BMA specimens (615 Romanowsky-stained and 180 Prussian blue-stained) from patients with neoplastic and other clinical conditions were analyzed, comparing the performance of the Scopio Labs X100 Full Field BMA system (test method) with manual microscopy (reference method). The system provided an average of 1385±536 (range 0-3131) cells per specimen for analysis. An average of 39.98±19.64 fields of view (range 0-140) per specimen were selected by the system for analysis, of them 87±21% (range 0-100%) were accepted by the qualified operators. These regions were included in an average of 17.62±7.24 regions of interest (range 1-50) per specimen. The efficiency, sensitivity, and specificity for primary and secondary marrow aspirate characteristics (maturation, morphology, and count assessment), as well as overall inter-user agreement, were evaluated. The test method showed high correlation with the reference method for comprehensive BMA evaluation, both on Romanowsky (90.85% efficiency, 81.61% sensitivity; specificity 92.88%) and Prussian blue (90.0% efficiency, 81.94% sensitivity; 93.38% specificity) stained samples. The overall agreement between the test and reference method for BMA assessment was 91.1%. For repeatability and reproducibility, all standard deviations and coefficients of variation values were below the pre-defined acceptance criteria both for discrete measurements (CV below 20%) and for differential measurements (SD below 5%). The high degree of correlation between the digital decision support system and manual microscopy demonstrates the potential of this system to provide a high-quality, accurate digital BMA analysis, -expediting expert review and diagnosis of BMA specimens, with practical applications including remote BMA evaluation, and possibly new opportunities for the research of normal and neoplastic hematopoiesis.

Mapping the cellular biogeography of human bone marrow niches using single-cell transcriptomics and proteomic imaging.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging.

The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

Hematological entities with plasmacytic differentiation: a case report.

INTRODUCTION: Plasmacytoma, a localized tumor of monoclonal plasma cells without any clinical, radiological or physical evidence of plasma cell neoplasm (PCN), is a rare entity that accounts for 1% of PCN. Immunoglobulin M (IgM) extramedullary plasmacytoma of mediastinal region has never been reported and is a diagnostic challenge considering other differential diagnoses. CASE PRESENTATION: We present the case of a 51-year-old African-American female with progressively increasing cough, dyspnea, and dysphagia for 6 months with a computed tomography (CT) scan revealing a subcarinal mass. The histopathological analysis of the mass reveals a diagnosis of lymphoma with plasma cell differentiation, with a differential of lymphoplasmacytic lymphoma and plasma cell neoplasm. The lymphoma panel via next-generation sequencing (NGS) and a myeloma-targeted fluorescent in situ hybridization (FISH) panel confirmed the diagnosis of IgM extramedullary plasmacytoma, an entity of rare occurrence. Treatment with radiation led to complete regression of the plasmacytoma with normal blood work-up. CONCLUSIONS: This report describes the challenges of diagnosing IgM extramedullary plasmactyoma. Our case report highlights the importance of cytogenetics and NGS in establishing a correct diagnosis that indeed has prognostic and therapeutic implications.

Host-Assisted Delivery of a Model Drug to Genomic DNA: Key Information from Ultrafast Spectroscopy and in silico Study.

Drug delivery to a target without adverse effects is one of the major criteria for clinical use. Herein, we have made an attempt to explore the delivery efficacy of SDS surfactant in a monomer and micellar stage during the delivery of the model drug, Toluidine Blue (TB) from the micellar cavity to DNA. Molecular recognition of pre-micellar SDS encapsulated TB with DNA occurs at a rate constant of k1 ∼652 s-1 . However, no significant release of encapsulated TB at micellar concentration was observed within the experimental time frame. This originated from the higher binding affinity of TB towards the nano-cavity of SDS at micellar concentration which does not allow the delivery of TB from the nano-cavity of SDS micelles to DNA. Thus, molecular recognition controls the extent of DNA recognition by TB which in turn modulates the rate of delivery of TB from SDS in a concentration-dependent manner.

Pembrolizumab for B-cell lymphomas relapsing after or refractory to CD19-directed CAR T-cell therapy.

CD19-directed chimeric antigen receptor-modified (CAR T) T cells achieve durable remissions in about 30% to 40% of relapsed/refractory large B-cell lymphomas. T-cell exhaustion and/or an immunosuppressive tumor microenvironment may contribute to CAR T-cell failure. Pembrolizumab, an anti-PD1 immune checkpoint inhibitor, may reverse T-cell exhaustion after CAR T-cell therapy. We treated 12 patients with B-cell lymphomas who were either refractory to (n = 9) or relapsed after (n = 3) CD19-directed CAR T-cell (4-1BB-costimulated) therapy with pembrolizumab 200 mg IV every 3 weeks. Median time from CAR T-cell infusion to first pembrolizumab dose was 3.3 months (range, 0.4-42.8 months). Pembrolizumab was well tolerated, and the only grade ≥3 adverse events related to pembrolizumab were neutropenia (n = 3; 25%). Best overall response rate after pembrolizumab was 25% (3 of 12 patients; 1 complete response; 2 partial responses). One (8%) patient had stable disease; thus, 4 of 12 (33%) patients had clinical benefit. After pembrolizumab, 4 patients with clinical benefit had an increase in percentage of CAR T cells by mass cytometry by time of flight (CyTOF); 3 of 4 of these patients also had increases in CAR19 transgene levels by quantitative polymerase chain reaction. Deep immune profiling using CyTOF revealed increased CAR T-cell activation and proliferation and less T-cell exhaustion in clinical responders. Together, PD1 blockade with pembrolizumab after CD19-directed CAR T-cell therapy appears safe and may achieve clinical responses in some patients with B-cell lymphomas refractory to or relapsed after CAR T-cell therapy. This trial was registered at www.clinicaltrials.gove as #NCT02650999.

Targeted massively parallel sequencing of mature lymphoid neoplasms: assessment of empirical application and diagnostic utility in routine clinical practice.

Massively parallel sequencing (MPS) has become a viable diagnostic tool to interrogate genetic profiles of numerous tumors but has yet to be routinely adopted in the setting of lymphoma. Here, we report the empirical application of a targeted 40-gene panel developed for use in mature lymphoid neoplasms (MLNs) and report our experience on over 500 cases submitted for MPS during the first year of its clinical use. MPS was applied to both fresh and fixed specimens. The most frequent diagnoses were diffuse large B-cell lymphoma (116), chronic lymphocytic leukemia/small lymphocytic lymphoma (60), marginal zone lymphoma (52), and follicular lymphoma (43), followed by a spectrum of mature T-cell neoplasms (40). Of 534 cases submitted, 471 generated reportable results in MLNs, with disease-associated variants (DAVs) detected in 241 cases (51.2%). The most frequent DAVs affected TP53 (30%), CREBBP (14%), MYD88 (14%), TNFRSF14 (10%), TNFAIP3 (10%), B2M (7%), and NOTCH2 (7%). The bulk of our findings confirm what is reported in the scientific literature. While a substantial majority of mutations did not directly impact diagnosis, MPS results were utilized to either change, refine, or facilitate the final diagnosis in ~10.8% of cases with DAVs and 5.5% of cases overall. In addition, we identified preanalytic variables that significantly affect assay performance highlighting items for specimen triage. We demonstrate the technical viability and utility of the judicious use of a targeted MPS panel that may help to establish general guidelines for specimen selection and diagnostic application in MLNs in routine clinical practice.

Pathology and genetics of anaplastic large cell lymphoma.

Anaplastic large cell lymphomas are a rare subtype of peripheral/mature T-cell lymphomas which are clinically, pathologically and genetically heterogeneous. Both ALK-positive (ALK+) and ALK-negative (ALK-) ALCL are composed of large lymphoid cells with abundant cytoplasm and pleomorphic features with horseshoe-shaped and reniform nuclei. ALK+ ALCL were considered as a definite entity in the 2008 World Health Organization classification of hematopoietic and lymphoid tissues. ALK-ALCL was included as a provisional entity in the WHO 2008 edition and in the most recent 2017 edition, it is now considered a distinct entity that includes cytogenetic subsets that appear to have prognostic implications (e.g. 6p25 rearrangements at IRF4/DUSP22 locus). ALK+ ALCLs are distinct in epidemiology and pathogenetic origin and should be distinguished from ALK-ALCL, cutaneous ALCL and breast implant associated ALCL which have distinct clinical course and pathogenetic features. Breast implant-associated ALCL is now recognized as a new provisional entity distinct from other ALK-ALCL; notably that it is a noninvasive disease associated with excellent outcome. In this article, we will provide an overview of the salient themes relevant to the pathology and genetic mechanisms in ALCL.

Lymphoid neoplasms of the oral cavity with plasmablastic morphology-a case series and review of the literature.

Plasmablastic lymphoma (PBL) is a rare aggressive variant of large B-cell lymphoma defined as a proliferation of large neoplastic plasmablasts/immunoblasts. PBL was first described as a distinct entity in a group of 16 patients with lymphoma of the oral cavity. Most patients are HIV-positive men. The disease has also been reported in other patient groups, often in association with primary or other acquired immunodeficiency. PBL shows a predilection for the oral cavity, although extraoral involvement also occurs. Because of its rarity, unique clinical features, and overlapping morphologic/immunophenotypic features, care must be taken to distinguish PBL from diffuse large B-cell lymphoma and plasma cell neoplasms with plasmablastic features. We report 3 cases of neoplasms with plasmablastic histomorphology involving the oral cavity. The relevant clinical, morphologic, and immunophenotypic features and treatment are presented, along with a review of the literature.

NF2 and ATRX gene copy number losses on a case of ovarian ependymoma.

Ovarian ependymomas are rare glial neoplasms that typically occur in women on their third to fourth decades of life. They are histologically similar to ependymomas of the central nervous system but may have a broader immunophenotype. We describe a 27-year-old woman who presented to the emergency department with a 3-week history of cough and shortness of breath. Further workup disclosed a left pelvic mass and extensive intra-abdominal metastases. Pathology revealed sheets of monomorphic cells within a fibrillary stroma, papillary projections, true ependymal rosettes, and pseudorosettes consistent with an ependymoma of ovarian origin. Next-generation sequencing showed ATRX and NF2 copy number losses. Fluorescence in situ hybridization for EWSR1 demonstrated monosomy of 22q in greater than 90% of cells. These molecular alterations have not been previously reported in ovarian or extra-central nervous system ependymomas.

T-Cell Large Granular Lymphocytic Leukemia and Coexisting B-Cell Lymphomas: A Study From the Bone Marrow Pathology Group.

OBJECTIVE: T-cell large granular lymphocytic (T-LGL) leukemia is associated with B-cell lymphomas (BCLs), especially small BCLs. We aimed to explore and expand upon its association with BCLs. METHODS: We retrospectively studied clinicopathologic features of T-LGL leukemia patients with coexisting BCL from January 2001 to December 2016. RESULTS: Among 432 patients with T-LGL leukemia, 22 (5.1%) had an associated B-cell non-Hodgkin lymphoma. Thirteen (59%) patients had large and nine (41%) had small BCL. T-LGL leukemia occurred synchronously with BCL in five, preceded BCL in three, and followed BCL in 14 patients. Anemia was the most common cytopenia (68%). Only one patient had a history of rheumatoid arthritis. CONCLUSION: To our knowledge, this is the first multicenter study looking at the spectrum and incidence of BCLs in patients with T-LGL leukemia and highlights its association with large BCLs (3% of T-LGL leukemias).

Oncocytic Adrenocortical Neoplasm Diagnosed after Robot-Assisted Adrenalectomy.

Oncocytic tumors, composed of eosinophilic, mitochondria-rich cells, can occur in several locations throughout the body. These tumors can occur in the adrenal cortex and are rarely malignant. We report a case of a patient presenting with an incidental adrenal mass which was later diagnosed as a oncocytic adrenocortical neoplasm (OAN). The patient is a 53-year-old man found to have a 7.2 cm right adrenal mass, incidentally found by computed tomography (CT). After metabolic workup was negative, a right robotic adrenalectomy (RA) was performed. Pathologic analysis revealed clusters of large cells with abundant eosinophilic and granular cytoplasm, consistent with OAN. This pathology is rare, with only about 150 cases described in the literature. It occurs in females 2.5 times more frequently and more commonly on the left side. Diagnosis is usually made by imaging criteria, typically with CT or magnetic resonance imaging (MRI). Treatment is generally surgical, since OAN can be malignant in some cases. Differentiation between benign and malignant OAN is done based on the Lin-Weiss-Bisceglia criteria and can be difficult. If malignancy is diagnosed, recurrence is common and close surveillance should be performed.

A case of primary cutaneous mucormycosis caused by minor trauma.

We present the case of a 66-year-old neutropenic man with mantle-cell lymphoma who presented for evaluation of a rapidly expanding necrotic eschar after a minor cutaneous injury. Histopathology revealed infection with Rhizopus indicating primary cutaneous mucormycosis. Our case reviews the presentation and management of this condition as well highlights the potential for minor cutaneous injuries in the hospital to lead to this dangerous infection.

Testicular seminomatous mixed germ cell tumor with choriocarcinoma and teratoma with secondary somatic malignancy: a case report.

INTRODUCTION: Testicular tumors are a heterogeneous group of neoplasms exhibiting diverse histopathology and can be classified as seminomatous and non-seminomatous germ cell tumor types. Mixed germ cell tumors contain more than one germ cell component and various combinations have been reported. Here, we present a rare case of a mixed germ cell tumor composed of seminoma, choriocarcinoma and teratoma with a secondary somatic malignancy. CASE PRESENTATION: A 31-year-old Caucasian man presented with splenic rupture to our hospital. A right-sided testicular swelling had been present for 6 months and his alpha-fetoprotein, beta-human chorionic gonadotropin, and lactose dehydrogenase were increased. An ultrasound of his scrotum revealed an enlarged right testis with heterogeneous echogenicity. Multiple hypervascular lesions were noted in his liver and spleen. He underwent transcatheter embolization therapy of his splenic artery followed by splenectomy and right-sided orchiectomy. A computed tomography scan also showed metastasis to both lungs. During his last follow up after four cycles of cisplatin-based chemotherapy, the level of tumor markers had decreased, decreases in the size of his liver and pulmonary lesions were noted but new sclerotic lesions were evident in his thoracolumbar region raising concern for bony metastasis. CONCLUSIONS: Prognosis of testicular tumor depends mainly on the clinical stage, but emergence of a sarcomatous component presents a challenge in the treatment of germ cell tumors and the histological subtype of this component can be used as a guide to specific chemotherapy in these patients.