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.

An interactive e-learning module on peripheral blood smear analysis is an effective option for teaching pathology trainees.

OBJECTIVES: This study compares the effectiveness of an interactive e-learning module with a traditional text-based method for teaching peripheral blood smear analysis. METHODS: Pathology trainees at Accreditation Council for Graduate Medical Education residency programs were asked to participate. Participants completed a multiple-choice test on peripheral blood smear findings. Trainees were randomized into completing an e-learning module or a PDF reading exercise with the same educational content. Respondents rated their experience and completed a postintervention test composed of the same questions. RESULTS: In total, 28 participants completed the study; 21 improved their score in the posttest (mean, 21.6 correct answers) compared with the pretest (19.8; P < .001). This improvement was seen in both the PDF (n = 19) and interactive (n = 9) groups, with no difference in performance between the 2 groups. Trainees with less clinical hematopathology experience showed a trend of having the largest performance improvement. Most participants completed the exercise within 1 hour, rated the exercise as easy to navigate, were engaged, and reported learning new information about peripheral blood smear analysis. All participants indicated that they would likely complete a similar exercise in the future. CONCLUSIONS: This study suggests that e-learning is an effective tool for hematopathology education and equivalent to traditional narrative-based methods. This module could easily be incorporated into a curriculum.

Advances in myelodysplastic/myeloproliferative neoplasms.

The myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPN) category includes a heterogeneous group of diseases characterized by the co-occurrence of clinical and pathologic features of both myelodysplastic and myeloproliferative neoplasms. The recently published International Consensus Classification of myeloid neoplasms revised the entities included in the MDS/MPN category as well as criteria for their diagnosis. In addition to the presence of one or more increased peripheral blood cell counts as evidence of myeloproliferative features, concomitant cytopenia as evidence of ineffective hematopoiesis is now an explicit requirement to diagnose the diseases included in this category. The increasing availability of modern gene sequencing has allowed better understanding of the biologic characteristics of these myeloid neoplasms. The presence of specific mutations in the appropriate clinicopathologic context is now included in the diagnostic criteria for some of MDS/MPN entities. In this review, we highlight what has changed in the diagnostic criteria of MDS/MPN from the WHO 2016 classification while providing practical guidance in diagnosing these diseases.

The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma.

Genomic Features of Interstitial Deletions of Chromosome 9q in Acute Myeloid Leukemia.

Interstitial deletion in the long arm of chromosome 9 [del(9q)] is a fairly common cytogenetic finding associated with acute myeloid leukemia (AML), seen in approximately 2-5% of AML patients. However, the genomic features of the deletion remain largely unknown. Using chromosome analysis, single nucleotide polymorphism microarray, and next-generation sequencing, we characterized del(9q)s and other genomic alterations in 9 AML patients. We found several distinct features of the del(9q)s. The proximal breakpoints of the deletions are clustered within a 2.5-Mb region (chr9: 68,513,625-70,984,372; GRCh37) enriched with segmental duplications, which may represent a "hotspot" for genomic rearrangements. However, the distal breakpoints of the deletions vary significantly. In addition, the overall deleted region could be divided into a 14.4-Mb proximal constitutional region (chr9: 70,950,015-85,397,699; 9q21.11q21.32) and a 24.0-Mb distal oncogenic region (chr9: 85,397,700-109,427,261; 9q21.32q31.1). We further identified a 6.8-Mb common overlapped deletion region (CODR) in the distal region (chr9: 90,590,650-97,366,400). This CODR carries multiple genes that are reportedly involved in cancer pathogenesis. The prognostic value of the del(9q) in AML apparently depends on additional genomic alterations in the patients.

How I Diagnose Primary Myelofibrosis.

OBJECTIVES: Primary myelofibrosis (PMF) is a BCR/ABL1-negative myeloproliferative neoplasm (MPN) with a shorter overall survival and a higher leukemic transformation than other BCR/ABL1-negative MPNs. Diagnosis of PMF can be challenging given its clinical, morphologic, molecular overlap with other myeloid neoplasms also associated with myelofibrosis, and reactive conditions. METHODS: We summarize and discuss the clinical, morphologic, and molecular features useful for diagnosing PMF as well as salient features helpful in distinguishing PMF from myelodysplastic syndrome with associated fibrosis and autoimmune myelofibrosis using a case-based approach. RESULTS: PMF in both its prefibrotic and fibrotic stages, the latter characterized by reticulin/collagen marrow fibrosis, is characterized by a proliferation of predominantly abnormal megakaryocytes and granulocytes in the bone marrow. Driver mutations in   JAK2, CALR, or MPLare seen in approximately 90% of PMF cases. In triple-negative cases, the presence of cytogenetic abnormalities and other somatic mutations identified by next-generation sequencing can help establish a diagnosis of PMF in the appropriate clinical and morphologic context. CONCLUSIONS: Given the significant difference in prognosis and treatment, integration of clinical, morphological, and molecular/genetic findings is essential in distinguishing PMF from other etiologies that can demonstrate myelofibrosis.

Distinct pathologic feature of myeloid neoplasm with t(v;11p15); NUP98 rearrangement.

Chromosome rearrangements involving NUP98 at 11p15 are rare but recurring abnormalities in acute myeloid leukemia (AML). Here we described 12 cases of myeloid neoplasms with t(v; 11p15); NUP98 rearrangement and characterized their pathologic features. Our patient cohort included 10 adults and 2 children with a median age of 51 years. They were predominantly AML (n = 10) including de novo AML, therapy-related AML, chronic myeloid leukemia with myeloid blast crisis, and mixed phenotype acute leukemia, as well as therapy-related myelodysplastic syndrome (MDS) and MDS/myeloproliferative neoplasm with increased blasts. The blasts shared some common features including pink/red cytoplasmic granules, presence of a perinuclear hof, Auer rods, and occasional bilobed nuclei, mimicking acute promyelocytic leukemia (APML). Flow cytometric studies showed blasts positive for MPO, CD117, CD13 and CD33, with a subset of cases negative for CD34 and/or HLA-DR and a subset of cases expressing monocytic markers. The translocations of 11p15 included t(7; 11) (p15; p15), t(2; 11) (q31; p15), t(9; 11) (p22; p15), t(5; 11) (q32; p15), and t(11; 12) (p15; q13). Three cases showed cryptic NUP98 rearrangement. These patients showed incomplete response to therapy with median overall survival of 17.5 months, a complete remission rate of 25% following chemotherapy induction and primary refractory disease of 58%. It is clinically important to recognize this group of diseases because the blasts can be misclassified as promyelocytes, and NUP98 rearrangement may be cryptic requiring fluorescence in situ hybridization (FISH) study. This case series highlights that NUP98-rearranged myeloid neoplasms are clinically, morphologically, and cytogenetically distinct and could be considered as a separate entity in the WHO classification defined by cytogenetic abnormality.

Detection of cryptogenic malignancies from metagenomic whole genome sequencing of body fluids.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) of body fluids is an emerging approach to identify occult pathogens in undiagnosed patients. We hypothesized that metagenomic testing can be simultaneously used to detect malignant neoplasms in addition to infectious pathogens. METHODS: From two independent studies (n = 205), we used human data generated from a metagenomic sequencing pipeline to simultaneously screen for malignancies by copy number variation (CNV) detection. In the first case-control study, we analyzed body fluid samples (n = 124) from patients with a clinical diagnosis of either malignancy (positive cases, n = 65) or infection (negative controls, n = 59). In a second verification cohort, we analyzed a series of consecutive cases (n = 81) sent to cytology for malignancy workup that included malignant positives (n = 32), negatives (n = 18), or cases with an unclear gold standard (n = 31). RESULTS: The overall CNV test sensitivity across all studies was 87% (55 of 63) in patients with malignancies confirmed by conventional cytology and/or flow cytometry testing and 68% (23 of 34) in patients who were ultimately diagnosed with cancer but negative by conventional testing. Specificity was 100% (95% CI 95-100%) with no false positives detected in 77 negative controls. In one example, a patient hospitalized with an unknown pulmonary illness had non-diagnostic lung biopsies, while CNVs implicating a malignancy were detectable from bronchoalveolar fluid. CONCLUSIONS: Metagenomic sequencing of body fluids can be used to identify undetected malignant neoplasms through copy number variation detection. This study illustrates the potential clinical utility of a single metagenomic test to uncover the cause of undiagnosed acute illnesses due to cancer or infection using the same specimen.

Entrustable Professional Activities in Hematopathology Pathology Fellowship Training: Consensus Design and Proposal.

Hematopathology fellowship education has grown in complexity as patient-centered treatment plans have come to depend on integration of clinical, morphologic, immunophenotypic, molecular, and cytogenetic variables. This complexity is in competition with the need for timely hematopathology care with stewardship of patient, laboratory, and societal resources. Accreditation Council for Graduate Medical Education Milestones provide a guidance document for hematopathology training, but fellows and their educators are in need of a simple framework that allows assessment and feedback of growth toward independent hematopathology practice. Entrustable professional activities provide one such framework, and herein, we provide proposed Hematopathology Fellowship Entrustable Professional Activities based on review of pertinent guidelines and literature, with multiple rounds of expert and stakeholder input utilizing a modified mini-Delphi approach. Ten core entrustable professional activities deemed essential for graduating hematopathology fellows were developed together with skills and knowledge statements, example scenarios, and corresponding Accreditation Council for Graduate Medical Education Milestones. Application of these entrustable professional activities in program design, fellow evaluation, and decisions regarding level of supervision is discussed with consideration of benefits and barriers to implementation. These entrustable professional activities may be used by hematopathology fellowship directors and faculty to provide fellows with timely constructive feedback, determine entrustment decisions, provide the Clinical Competency Committee with granular data to support Milestone evaluations, and provide insight into areas of potential improvement in fellowship training. Fellows will benefit from a clear roadmap to independent hematopathology practice with concrete and timely feedback.

New developments in non-Hodgkin lymphoid malignancies.

The revised fourth edition of the World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues (2017) reflects significant advances in understanding the biology, genetic basis and behaviour of haematopoietic neoplasms. This review focuses on some of the major changes in B-cell and T-cell non-Hodgkin lymphomas in the 2017 WHO and includes more recent updates. The 2017 WHO saw a shift towards conservatism in the classification of precursor lesions of small B-cell lymphomas such as monoclonal B-cell lymphocytosis, in situ follicular and in situ mantle cell neoplasms. With more widespread use of next generation sequencing (NGS), special entities within follicular lymphoma and mantle cell lymphoma were recognised with recurrent genetic aberrations and unique clinicopathological features. The diagnostic workup of lymphoplasmacytic lymphoma and hairy cell leukaemia has been refined with the discovery of MYD88 L265P and BRAF V600E mutations, respectively, in these entities. Recommendations in the immunohistochemical evaluation of diffuse large B-cell lymphoma include determining cell of origin and expression of MYC and BCL2, so called 'double-expressor' phenotype. EBV-positive large B-cell lymphoma of the elderly has been renamed to recognise its occurrence amongst a wider age group. EBV-positive mucocutaneous ulcer is a newly recognised entity with indolent clinical behaviour that occurs in the setting of immunosuppression. Two lymphomas with recurrent genetic aberrations are newly included provisional entities: Burkitt-like lymphoma with 11q aberration and large B-cell lymphoma with IRF4 rearrangement. Aggressive B-cell lymphomas with MYC, BCL2 and/or BCL6 rearrangements, so called 'double-hit/triple-hit' lymphomas are now a distinct entity. Much progress has been made in understanding intestinal T-cell lymphomas. Enteropathy-associated T-cell lymphoma, type II, is now known to not be associated with coeliac disease and is hence renamed monomorphic epitheliotropic T-cell lymphoma. An indolent clonal T-cell lymphoproliferative disorder of the GI tract is a newly included provisional entity. Angioimmunoblastic T-cell lymphoma and nodal T-cell lymphomas with T-follicular helper phenotype are included in a single broad category, emphasising their shared genetic and phenotypic features. Anaplastic large cell lymphoma, ALK- is upgraded to a definitive entity with subsets carrying recurrent rearrangements in DUSP22 or TP63. Breast implant-associated anaplastic large cell lymphoma is a new provisional entity with indolent behaviour. Finally, cutaneous T-cell proliferations include a new provisional entity, primary cutaneous acral CD8-positive T-cell lymphoma, and reclassification of primary small/medium CD4-positive T-cell lymphoma as lymphoproliferative disorder.

B-cell neoplasms and Hodgkin lymphoma in the spleen.

B-cell lymphoma of spleen may be primary (most commonly splenic diffuse large B-cell lymphoma) or secondary (typically low-grade non-Hodgkin lymphoma). Depending on the specific lymphoma subtype, there may be a predominantly white pulp pattern of involvement, a predominantly red pulp pattern or a focal nodular pattern. Splenectomy is the ideal specimen for a multiparametric integrative diagnosis of splenic lymphoma, as it allows for a combined study of morphology, immunohistology, flow cytometry, cytogenetics, and molecular genetic techniques. This review article describes the clinicopathologic characteristics of all the relevant B-cell neoplasms that may be encountered in a splenic biopsy or a splenectomy specimen.

Myeloid, mast cell, histiocytic and dendritic cell neoplasms and proliferations involving the spleen.

Splenic involvement and consequent splenomegaly are usually seen as part of systemic involvement by myeloid neoplasms as well as mast cell and histiocytic neoplasms. Primary splenic involvement by these neoplasms is rare. Splenectomy is usually not performed for establishing a diagnosis of these entities. However, in rare instances, the pathologist may need to evaluate the spleen secondary to splenic rupture or palliative splenectomy to alleviate symptoms related to splenomegaly. This review article describes the clinicopathologic features of a broad group of myeloid, mastocytic, and histiocytic proliferative and neoplastic disorders.

Genomic analysis in myeloid sarcoma and comparison with paired acute myeloid leukemia.

Myeloid sarcoma (MS) is a rare manifestation of acute myeloid leukemia (AML) characterized by extramedullary proliferation of myeloid blasts. Owing to the rarity of MS, the clonal evolution of cell populations giving rise to MS is not well understood. To study the genomic signature of MS, we used a capture-based next-generation sequencing panel targeting 479 cancer genes to interrogate the genetic variants present in MS samples and compared their genetic profiles with their paired AML samples from a cohort of seven individuals. We identified a spectrum of single-nucleotide variants (SNVs) and a spectrum of copy number alterations in MS. Our study found that variant profiles observed in MS were generally similar to AML from the same individual, supporting the notion that these tumors are derived from a common precursor, rather than de novo tumors in a susceptible host. In addition, MS cases with a higher number of SNVs show worse clinical outcomes than MS with a lower number of SNVs. Identification of these abnormalities could potentially contribute to improved prognostic classification and identify new therapeutic targets for MS.

Molecular and Cytogenetic Education in Hematopathology Fellowship.

OBJECTIVES: Given the increased complexity of molecular and cytogenetic testing (MOL-CG), the Society for Hematopathology Education Committee (SH-EC) was interested in determining what the current expectations are for MOL-CG education in hematopathology (HP) fellowship training. METHODS: The SH-EC sent a questionnaire to HP fellowship program directors (HP-PDs) covering MOL-CG training curricula, test menus, faculty background, teaching, and sign-out roles. These findings were explored via a panel-based discussion at the 2018 SH-EC meeting for HP-PDs. RESULTS: HP fellows are expected to understand basic principles, nomenclature, and indications for and limitations of testing. Interpretation of common assays is within that scope, but not necessarily proficiency in technical troubleshooting of testing or analysis of complex raw data. CONCLUSIONS: The consensus was that HP fellows should understand the components of MOL-CG testing necessary to incorporate those results into an accurate, clinically relevant, and integrated HP report.

Venetoclax in Combination with Decitabine for Relapsed T-Cell Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Cell Transplant.

Long-term disease-free survival in adults with T-cell acute lymphoblastic leukemia (T-ALL) remains poor, particularly after relapse, with few available salvage options. Preclinical data suggest that inhibition of the antiapoptotic protein BCL-2 (B-cell lymphoma 2) either alone or in combination with other agents, may be a unique therapeutic approach for the treatment of T-ALL. We present a case of a young male with T-ALL, relapsed after allogeneic hematopoietic stem cell transplant, who achieved a second complete remission following salvage therapy with combined venetoclax and decitabine. Assessment of measurable residual disease by next generation sequencing showed no evidence of residual disease of a sensitivity of 1 × 10-6. While the combination of venetoclax and hypomethylating agents has shown promise in the treatment of relapsed/refractory AML, and to our knowledge, this is the first report of this combination demonstrating clinical activity in relapsed/refractory T-ALL.

KSHV viral cyclin interferes with T-cell development and induces lymphoma through Cdk6 and Notch activation in vivo.

Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.

Spleens of myelofibrosis patients contain malignant hematopoietic stem cells.

Cancer stem cell behavior is thought to be largely determined by intrinsic properties and by regulatory signals provided by the microenvironment. Myelofibrosis (MF) is characterized by hematopoiesis occurring not only in the marrow but also in extramedullary sites such as the spleen. In order to study the effects of these different microenvironments on primitive malignant hematopoietic cells, we phenotypically and functionally characterized splenic and peripheral blood (PB) MF CD34+ cells from patients with MF. MF spleens contained greater numbers of malignant primitive HPCs than PB. Transplantation of PB MF CD34+ cells into immunodeficient (NOD/SCID/IL2Rγ(null)) mice resulted in a limited degree of donor cell chimerism and a differentiation program skewed toward myeloid lineages. By contrast, transplanted splenic MF CD34+ cells achieved a higher level of chimerism and generated both myeloid and lymphoid cells that contained molecular or cytogenetic abnormalities indicating their malignant nature. Only splenic MF CD34+ cells were able to sustain hematopoiesis for prolonged periods (9 months) and were able to engraft secondary recipients. These data document the existence of MF stem cells (MF-SCs) that reside in the spleens of MF patients and demonstrate that these MF-SCs retain a differentiation program identical to that of normal hematopoietic stem cells.

Splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms: heterogeneous morphology and cytological composition.

We studied 24 spleens with extramedullary hematopoietic proliferation (EMHP), a key feature of advanced-stage Philadelphia chromosome-negative myeloproliferative neoplasms, obtained from 24 patients (14 primary myelofibrosis, 7 polycythemia vera and 3 unclassifiable). Hematoxylin and eosin, reticulin and trichrome stains, and immunohistochemical stains for myeloperoxidase, glycophorin-C, CD42b, CD34, CD117, CD8, nerve growth factor receptor and smooth muscle actin were evaluated. Clinical information was correlated with the morphological findings. Three distinct histological patterns of EMHP were recognized: diffuse (12), nodular (5), and mixed-nodular and diffuse (7). The preponderant lineage was granulocytic in diffuse, trilineage in nodular and erythroid in mixed EMHP. Erythropoiesis was largely intravascular, granulopoiesis was within the splenic cords and megakaryopoiesis was observed in both locations. The stromal changes paralleled the histological pattern with preservation of the splenic stromal and vascular architecture in the diffuse areas as opposed to areas of nodular EMHP. The morphological features of the splenic EMHP did not correlate with specific subtypes of myeloproliferative neoplasms. The mean duration of follow-up from initial diagnosis was 80 months. A total of 15 of the 24 patients died of disease: 8 of 12 (67%) with diffuse, 2 of 5 (40%) with nodular and 5 of 7 (71%) with mixed growth patterns. The mean duration from diagnosis to splenectomy was shorter in patients with diffuse (83 months) as compared with those with nodular EMHP (127 months). Our study demonstrates that splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms shows distinct histological patterns that do not correlate with disease subtypes, but appear to suggest a trend between the histological patterns and clinical behavior. These results suggest a different biology of the disease in the nodular and diffuse extramedullary hematopoietic proliferation groups.