Robert P. Hasserjian

The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia

The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues was last updated in 2008. Since then, there have been numerous advances in the identification of unique biomarkers associated with some myeloid neoplasms and acute leukemias, largely derived from gene expression analysis and next-generation sequencing that can significantly improve the diagnostic criteria as well as the prognostic relevance of entities currently included in the WHO classification and that also suggest new entities that should be added. Therefore, there is a clear need for a revision to the current classification. The revisions to the categories of myeloid neoplasms and acute leukemia will be published in a monograph in 2016 and reflect a consensus of opinion of hematopathologists, hematologists, oncologists, and geneticists. The 2016 edition represents a revision of the prior classification rather than an entirely new classification and attempts to incorporate new clinical, prognostic, morphologic, immunophenotypic, and genetic data that have emerged since the last edition. The major changes in the classification and their rationale are presented here.

Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia

Mesenchymal cells contribute to the ‘stroma’ of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman–Bodian–Diamond syndrome—a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.

Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes

Recent genetic analyses of large populations have revealed that somatic mutations in hematopoietic cells leading to clonal expansion are commonly acquired during human aging. Clonally restricted hematopoiesis is associated with an increased risk of subsequent diagnosis of myeloid or lymphoid neoplasia and increased all-cause mortality. Although myelodysplastic syndromes (MDS) are defined by cytopenias, dysplastic morphology of blood and marrow cells, and clonal hematopoiesis, most individuals who acquire clonal hematopoiesis during aging will never develop MDS. Therefore, acquisition of somatic mutations that drive clonal expansion in the absence of cytopenias and dysplastic hematopoiesis can be considered clonal hematopoiesis of indeterminate potential (CHIP), analogous to monoclonal gammopathy of undetermined significance and monoclonal B-cell lymphocytosis, which are precursor states for hematologic neoplasms but are usually benign and do not progress. Because mutations are frequently observed in healthy older persons, detection of an MDS-associated somatic mutation in a cytopenic patient without other evidence of MDS may cause diagnostic uncertainty. Here we discuss the nature and prevalence of CHIP, distinction of this state from MDS, and current areas of uncertainty regarding diagnostic criteria for myeloid malignancies.

B-cell lymphomas with concurrent IGH-BCL2 and MYC rearrangements are aggressive neoplasms with clinical and pathologic features distinct from Burkitt lymphoma and diffuse large B-cell lymphoma.

B-cell lymphomas with concurrent IGH-BCL2 and MYC rearrangements, also known as “double-hit” lymphomas (DHL), are rare neoplasms characterized by highly aggressive clinical behavior, complex karyotypes, and a spectrum of pathologic features overlapping with Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and B-lymphoblastic lymphoma/leukemia (B-LBL). The clinical and pathologic spectrum of this rare entity, including comparison to other high-grade B-cell neoplasms, has not been well defined. We conducted a retrospective analysis of clinical and pathologic features of 20 cases of DHL seen at our institution during a 5-year period. In addition, we carried out case-control comparisons of DHL with BL and International Prognostic Index (IPI)-matched DLBCL. The 11 men and 9 women had a median age of 63.5 years (range 32 to 91). Six patients had a history of grade 1 to 2 follicular lymphoma; review of the prior biopsy specimens in 2 of 5 cases revealed blastoid morphology. Eighteen patients had Ann Arbor stage 3 or 4 disease and all had elevated serum lactate dehydrogenase (LDH) levels at presentation. Extranodal disease was present in 17/20 (85%), bone marrow involvement in 10/17 (59%) and central nervous system (CNS) disease in 5/11 (45%). Nineteen patients were treated with combination chemotherapy, of whom 18 received rituximab and 14 received CNS-directed therapy. Fourteen patients (70%) died within 8 months of diagnosis. Median overall survival in the DHL group (4.5 mo) was inferior to both BL (P=0.002) and IPI-matched DLBCL (P=0.04) control patients. Twelve DHL cases (60%) were classified as B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL, 7 cases (35%) as DLBCL, not otherwise specified, and 1 case as B-LBL. Distinguishing features from BL included expression of Bcl2 (P<0.0001), Mum1/IRF4 (P=0.006), Ki-67 <95% (P<0.0001), and absence of EBV-EBER (P=0.006). DHL commonly contained the t(8;22) rather than the t(8;14) seen in most BL controls (P=0.001), and exhibited a higher number of chromosomal aberrations (P=0.0009). DHL is a high-grade B-cell neoplasm with a poor prognosis, resistance to multiagent chemotherapy, and clinical and pathologic features distinct from other high-grade B-cell neoplasms. Familiarity with the morphologic and immunophenotypic spectrum of DHL is important in directing testing to detect concurrent IGH-BCL2 and MYC rearrangements when a karyotype is unavailable. The aggressive clinical behavior and combination of genetic abnormalities seen in these cases may warrant categorization as a separate entity in future classifications and call for novel therapeutic approaches.

Lymphoma of the ocular adnexa: A study of 353 cases.

We studied the cases of 353 patients with lymphoma involving the ocular adnexa diagnosed at the Massachusetts General Hospital between 1974 and 2005. The patients included 153 males and 200 females, aged 7 to 95 years, with a mean age of 64 years. In 277 cases, there was no known history of lymphoma. Seventy-six patients had a history of lymphoma, with the ocular adnexa being involved at relapse or with progression of the previously diagnosed lymphoma. The patients had marginal zone lymphoma (182 cases), follicular lymphoma (80 cases), mantle cell lymphoma (18 cases), small lymphocytic lymphoma/chronic lymphocytic leukemia (13 cases), lymphoplasmacytic lymphoma (4 cases), splenic marginal zone lymphoma (2 cases), low-grade B cell, not subclassified (19 cases), precursor B lymphoblastic lymphoma (3 cases), diffuse large B-cell lymphoma (26 cases), and 1 case each of high-grade B-cell lymphoma, not subclassified, peripheral T-cell lymphoma, unspecified type, extranodal NK/T-cell lymphoma, nasal type, and Hodgkin lymphoma, nodular sclerosis type. Almost all marginal zone lymphoma patients (168 of 182, 92%) had primary ocular adnexal lymphoma. Fourteen marginal zone lymphoma patients (8%) had a prior history of lymphoma, usually arising in another extranodal site. Twenty-five of 80 (31%) follicular lymphoma patients had a prior history of lymphoma, usually arising in lymph nodes. Patients with mantle cell lymphoma, chronic lymphocytic leukemia, lymphoplasmacytic lymphoma, and splenic marginal zone lymphoma almost always had a prior history of lymphoma or were known to have widespread disease at the time of diagnosis of ocular adnexal lymphoma. A subset of the diffuse large B-cell lymphomas were associated with large destructive masses involving adjacent structures such as paranasal sinuses, raising the possibility that they may have arisen from one of the adjacent structures and involved the ocular adnexa by direct extension. The relatively high proportion of low-grade lymphoma, not subclassified, highlights the difficulty that may arise in distinguishing different types of low-grade lymphoma, particularly when biopsies are small and artifactually distorted. Ocular adnexal lymphoma is primarily a disease of older adults, with a slight female preponderance. Most lymphomas are low-grade B-cell lymphomas, with marginal zone lymphoma being by far the most common type. Marginal zone lymphoma typically involves the ocular adnexa primarily, whereas other types of low-grade B-cell lymphoma often involve the ocular adnexa secondarily. High-grade B-cell lymphomas only occasionally involve the ocular adnexa, and T-cell lymphoma, NK-cell lymphoma, and Hodgkin lymphoma are only rarely encountered in this site.

Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor.

Signaling through Notch receptors has been implicated in the control of cellular differentiation in animals ranging from nematodes to humans. Starting from a human expressed sequence tag-containing sequence resembling that of Serrate, the gene for a ligand of Drosophila melanogaster Notch, we assembled a full-length cDNA, now called human Jagged2, from overlapping cDNA clones. The full-length cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6% identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands that have subsequently been described. When in situ hybridization was performed, expression of the murine Jagged2 homolog was found to be highest in fetal thymus, epidermis, foregut, dorsal root ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb Jagged2 transcript was most abundant in heart, lung, thymus, skeletal muscle, brain, and testis. Immunohistochemistry revealed coexpression of Jagged2 and Notch1 within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts expressing human Jagged2 with murine C2C12 myoblasts inhibited myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment. Exposure of C2C12 cells to Jagged2 led to increased amounts of Notch mRNA as well as mRNAs for a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of Notchl in C2C12 cells also induced increased levels of the same set of mRNAs, suggesting positive feedback control of these genes initiated by binding of Jagged2 to Notch1. This feedback control may function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical cell fates.

Bone marrow fibrosis: pathophysiology and clinical significance of increased bone marrow stromal fibres

In bone marrow biopsies, stromal structural fibres are detected by reticulin and trichrome stains, routine stains performed on bone marrow biopsy specimens in diagnostic laboratories. Increased reticulin staining (reticulin fibrosis) is associated with many benign and malignant conditions while increased trichrome staining (collagen fibrosis) is particularly prominent in late stages of severe myeloproliferative diseases or following tumour metastasis to the bone marrow. Recent evidence has shown that the amount of bone marrow reticulin staining often exhibits no correlation to disease severity, while the presence of type 1 collagen, as detected by trichrome staining, is often associated with more severe disease and a poorer prognosis. It was originally thought that increases in bone marrow stromal fibres themselves contributed to the haematopoietic abnormalities seen in certain diseases, but recent studies suggest that these increases are a result of underlying cellular abnormalities rather than a cause. A growing body of evidence suggests that increased deposition of bone marrow stromal fibres is mediated by transforming growth factor‐β and other factors elaborated by megakaryocytes, but it is likely that other cells, cytokines and growth factors are also involved. This suggests new avenues for investigation into the pathogenesis of various disorders associated with increased bone marrow stromal fibres.

Oncogenic Forms of NOTCH1 Lacking Either the Primary Binding Site for RBP-Jκ or Nuclear Localization Sequences Retain the Ability to Associate with RBP-Jκ and Activate Transcription

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (ΔE) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-Jκ response elements within DNA. Both truncated forms also bound to the transcription factor RBP-Jκ in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-Jκ-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jκ-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jκ, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, ΔE localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membrane-bound ΔE. Transcriptional activation by this mutant form of membrane-bound ΔE was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jκ at promoter sites.

Evaluation of bone marrow reticulin formation in chronic immune thrombocytopenia patients treated with romiplostim

Romiplostim is a thrombopoietin receptor agonist that increases platelet counts in patients with chronic immune thrombocytopenia (ITP). Thrombopoietin receptor agonists are reported to increase the risk for reticulin fiber deposition within bone marrow. This report describes bone marrow findings from romiplostim-treated rats, a retrospective analysis of reticulin observed in romiplostim ITP clinical trials, and a prospective clinical study of the effects of romiplostim on bone marrow morphology. In rats, romiplostim produced a dose-dependent increase in bone marrow fibrosis that resolved after treatment withdrawal. Of 271 ITP patients in romiplostim clinical trials, 10 were reported to have reticulin deposition; reticulin grade was increased in 4 of 5 patients with both pretreatment and on-treatment bone marrow results. Reticulin grade often decreased soon after romiplostim discontinuation. In the prospective study, reticulin grade during romiplostim treatment remained within the normal range for all patients and was increased in only 1 of 6 patients with pretreatment and on-treatment bone marrow results. This report suggests that romiplostim produces reversible, dose-dependent bone marrow changes in rats and produces modest increases in bone marrow reticulin in some ITP patients that decrease when therapy is discontinued. These studies were registered at www.clinicaltrials.gov as #NCT00102323, #NCT00102336, #NCT00861224, and #NCT00116688.

Quantitation of Bacteria in Bone Marrow from Patients with Typhoid Fever: Relationship between Counts and Clinical Features

ABSTRACT Enteric fever is the only bacterial infection of humans for which bone marrow examination is routinely recommended. A prospective study of the concentrations of bacteria in the bone marrow and their relationship to clinical features was conducted with 120 Vietnamese patients with suspected enteric fever, of whom 89 had confirmed typhoid fever. Ninety-three percent of the Salmonella entericaserovar Typhi samples isolated were resistant to ampicillin, chloramphenicol, and co-trimoxazole. For 81 patients with uncomplicated typhoid and satisfactory bone marrow aspirates, the number of serovar Typhi CFU in bone marrow aspirates was a median value of 9 (interquartile range [IQR], 1 to 85; range, 0.1 to 1,580) compared to 0.3 (IQR, 0.1 to 10; range, 0.1 to 399) CFU/ml in simultaneously sampled blood. The ratio of individual blood counts to bone marrow counts was 10 (IQR, 2.3 to 97.5). The number of bacteria in blood but not bone marrow was correlated inversely with the duration of preceding fever. Thus, with increasing duration of illness the ratio of bone marrow-to-blood bacterial concentrations increased; the median ratio was 4.8 (IQR, 1 to 27.5) during the first week compared with 158 (IQR, 60 to 397) during the third week. After lysing the host cells, the median ratio of viable bone marrow to blood increased, reflecting the higher concentration of intracellular serovar Typhi in the bone marrow. Effective antibiotic pretreatment had a significantly greater effect in reducing blood counts compared to bone marrow counts (P < 0.001). Thus, bacteria in the bone marrow of typhoid patients are less affected by antibiotic treatment than bacteria in the blood. The numbers of bacteria in bone marrow correlated negatively with the white blood cell (R = −0.3, P = 0.006) and platelet counts (R = −0.32, P = 0.01) and positively with fever clearance time after treatment (R = 0.4,P < 0.001). The bacterial load in bone marrow therefore may reflect the clinical course of the infection, and high levels may suppress neutrophil proliferation.