Congenital dyserythropoietic anemia type I: First report from the Congenital Dyserythropoietic Anemia Registry of North America (CDAR).

Congenital dyserythropoietic anemias (CDAs) are characterized by ineffective erythropoiesis and distinctive erythroblast abnormalities; the diagnosis is often missed or delayed due to significant phenotypic heterogeneity. We established the CDA Registry of North America (CDAR) to study the natural history of CDA and create a biorepository to investigate the pathobiology of this heterogeneous disease. Seven of 47 patients enrolled so far in CDAR have CDA-I due to biallelic CDAN1 mutations. They all presented with perinatal anemia and required transfusions during infancy. Anemia spontaneously improved during infancy in three patients; two became transfusion-independent rapidly after starting interferon-α2; and two remain transfusion-dependent at last follow-up at ages 5 and 30 y.o. One of the transfusion-dependent patients underwent splenectomy at 11 y.o due to misdiagnosis and returned to medical attention at 27 y.o with severe hemolytic anemia and pulmonary hypertension. All patients developed iron overload even without transfusions; four were treated with chelation. Genetic testing allowed for more rapid and accurate diagnosis; the median age of confirmed diagnosis in our cohort was 3 y.o compared to 17.3 y.o historically. In conclusion, CDAR provides an organized research network for multidisciplinary clinical and research collaboration to conduct natural history and biologic studies in CDA.

VPS4A Mutations in Humans Cause Syndromic Congenital Dyserythropoietic Anemia due to Cytokinesis and Trafficking Defects.

The Congenital Dyserythropoietic Anemia (CDA) Registry was established with the goal to facilitate investigations of natural history, biology, and molecular pathogenetic mechanisms of CDA. Three unrelated individuals enrolled in the registry had a syndrome characterized by CDA and severe neurodevelopmental delay. They were found to have missense mutations in VPS4A, a gene coding for an ATPase that regulates the ESCRT-III machinery in a variety of cellular processes including cell division, endosomal vesicle trafficking, and viral budding. Bone marrow studies showed binucleated erythroblasts and erythroblasts with cytoplasmic bridges indicating abnormal cytokinesis and abscission. Circulating red blood cells were found to retain transferrin receptor (CD71) in their membrane, demonstrating that VPS4A is critical for normal reticulocyte maturation. Using proband-derived induced pluripotent stem cells (iPSCs), we have successfully modeled the hematologic aspects of this syndrome in vitro, recapitulating their dyserythropoietic phenotype. Our findings demonstrate that VPS4A mutations cause cytokinesis and trafficking defects leading to a human disease with detrimental effects to erythropoiesis and neurodevelopment.

High-risk LCH in infants is serially transplantable in a xenograft model but responds durably to targeted therapy.

Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm characterized by a clonal proliferation of Langerhans-like cells. Genomic profiling has identified recurrent somatic activating mutations in the mitogen-activated protein kinase pathway, which are targetable by small-molecule inhibitors. However, key questions such as the curative potential of targeted therapy and the cell of origin remain unanswered. In this study, we describe clinical outcomes of a series of pediatric patients with multisystem BRAF V600E-mutant LCH, as well as the results of accompanying murine xenograft experiments. Four infants with LCH (range, 7-11 months at diagnosis) and secondary hemophagocytic lymphohistiocytosis were referred to our institution and subsequently treated with the BRAF V600E-specific inhibitor dabrafenib. All patients achieved complete clinical responses by 8 weeks of therapy, with remissions lasting a median of 36 months (range, 27-42 months). One infant successfully discontinued therapy long-term upon achieving a molecular response by real-time quantitative polymerase chain reaction (RT-qPCR). We further characterized the disease-propagating cell population in a subset of these patients by transplanting whole bone marrow into immunodeficient mice. Xenografted animals exhibited decreased survival with hematologic abnormalities, splenomegaly, and histiocytic infiltrates in the bone marrow resembling human disease. This process could also be secondarily transplanted, resulting in a comparable disease latency with similar histologic findings. These data further support the presence of a disease-initiating cell in the bone marrow compartment. We demonstrate that despite aggressive disease behavior in a xenograft model, these patients can achieve sustained clinical remissions with targeted monotherapy, with a select subset achieving molecular responses by RT-qPCR.

Limitations of HLH-2004 criteria in distinguishing malignancy-associated hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by dysregulated immune activation.  Primary HLH involves hereditary deficits in cytotoxic lymphocytes while secondary HLH is triggered by extrinsic factors. The HLH-2004 criteria are widely used for clinical diagnosis, yet their specificity for HLH or their ability to differentiate primary from secondary disease is unclear, potentially leading to inappropriate treatment. We describe several cases where fulfillment of HLH-2004 criteria obscured the diagnoses of underlying malignancies and delayed curative management. These issues are remedied without waiting for genetic testing results through an alternative diagnostic approach using flow cytometry-based immunologic assays and a thorough investigation for malignancy.

Immature lymphoid cells are a consistent feature of chylothorax in neonates and infants: cytologic features and flow cytometry aid in its distinction from lymphoblastic leukemia/lymphoma.

Chylothorax is rare in young children and occurs secondary to an array of etiologies. In this report, we describe the cytologic and immunophenotypic features of chylothorax in three neonates and infants. In each case, lymphocytes were the predominant cell type and had atypical cytologic features that closely resembled those of malignant lymphoblasts, including larger size, irregular nuclear contours, immature chromatin, presence of nucleoli, and high nuclear-to-cytoplasmic ratios. Flow cytometry (FC) was performed in two cases and revealed a predominant T-cell population with normal expression of T-lineage antigens and no expression of CD34, CD10, or CD1a or coexpression of CD4 and CD8. In summary, chylothorax in neonates and infants are comprised of cytologically immature lymphoid cells. In such cases, FC is useful for distinction between chylothorax and a lymphoblastic malignancy.

Pan myeloid antigen-negative pediatric acute megakaryoblastic leukemia.

Acute megakaryoblastic leukemia (AMKL) is a relatively common type of acute myeloid leukemia in children. We describe two unusual cases of AMKL that by flow cytometry (FC) lacked expression of any commonly evaluated myeloid antigens. One case presented as a periorbital myeloid sarcoma and clinically was thought to be a solid tumor. In both cases, the leukemic blasts were variably positive for the megakaryocytic marker CD61. Cytogenetics confirmed the presence of the t(1;22) in one case. Cytogenetics and inclusion of megakaryocytic markers in FC panels when evaluating pediatric specimens is critical for appropriate diagnosis for myeloid antigen negative AMKL.

Diagnostic usefulness and prognostic impact of CD200 expression in lymphoid malignancies and plasma cell myeloma.

The membrane glycoprotein MRC OX-2 (CD200) is expressed in several lymphoid malignancies. However, the diagnostic usefulness and potential prognostic importance of CD200 expression have not been rigorously examined. We show that CD200 is uniformly expressed in chronic lymphocytic leukemia (CLL) and absent in mantle cell lymphoma (MCL). It is important to note that expression of CD200 is retained even in CLLs with immunophenotypic aberrancies, making CD200 a particularly useful marker for discrimination between these cases and MCL. CD200 is expressed in nearly all precursor B-lymphoblastic leukemias, with aberrant overexpression or underexpression compared with normal B-cell progenitors in 55% of cases. More than 70% of plasma cell myelomas (PCMs) expressed CD200, and loss of CD200 expression in PCM may be associated with more clinically aggressive disease. CD200 is expressed in several hematolymphoid neoplasms. Analysis of its expression has several diagnostic and potentially prognostic applications in the flow cytometric evaluation of lymphoid malignancies.

Plasma cell myeloma and related neoplasms.

Session 1 of the 2009 Workshop of the Society for Hematopathology/European Association of Haematopathology, Cleveland, OH, focused on plasma cell neoplasms. This report summarizes the salient diagnostic, clinical, and genetic features of plasma cell myeloma (PCM) and related neoplasms. Based on the cases submitted to the workshop, we highlight common diagnostic issues and unusual manifestations of plasma cell neoplasms, such as t(11;14)+ PCM, plasma cell leukemia, and nonsecretory plasmacytoma, as well as plasmablastic transformation of PCM. Additional issues repeatedly raised at the workshop included the differential diagnosis of extramedullary dissemination of PCM vs primary extramedullary plasmacytoma and plasmablastic lymphoma; systemic plasma cell neoplasms in immunocompromised people; and Epstein-Barr virus-associated plasma cell neoplasms. Difficult cases with borderline features presented by submitters emphasized the necessity of integrating clinical, immunophenotypic, and genetic features for appropriate classification of these disorders.

Plasmablastic lymphoma and related disorders.

B-cell lymphomas with plasmablastic features are a heterogeneous group of lymphomas. While they may share overlapping morphologic or immunophenotypic features, distinct clinicopathologic or molecular genetic features exist for some that have allowed their recognition as distinct entities. Session 2 of the 2009 Society for Hematopathology/European Association for Haematopathology Workshop dealt with the theme of plasmablastic lymphomas (PBLs) and related disorders. Topics included human herpesvirus 8-associated Castleman disease and PBLs, PBLs occurring in the setting of HIV infection, anaplastic large cell lymphoma kinase+ diffuse large B-cell lymphoma, and other lymphomas with plasmablastic or plasmacytic features. In this report, we review PBLs and related disorders in the context of submitted cases and illustrate key diagnostic points, highlight controversial areas, and provide recommendations on features that should be assessed and terminology that might be used when dealing with these lymphomas.

Extragenitourinary malakoplakia in a patient with myeloma clinically mimicking extramedullary myelomatous disease.

Malakoplakia is a rare granulomatous disorder of unknown etiology and usually affects patients with underlying immunosuppression. This disorder usually involves the genitourinary tract but has been reported in a wide array of anatomical sites. We present a case of extragenitourinary malakoplakia, developing in a patient with a history of plasma cell myeloma, which clinically mimicked recurrent extramedullary myelomatous involvement. Radiologically, this lesion was a 10-cm soft-tissue mass located in the left flank and iliacus muscle. Excisional biopsy revealed a histiocytic infiltrate with histologic features diagnostic of malakoplakia. This case demonstrates the clinical and pathologic diagnostic challenges of malakoplakia arising outside the genitourinary tract. Given that it can closely mimic malignancy in such settings, malakoplakia should be considered in the differential diagnosis of soft-tissue masses developing in patients with hematologic malignancy and iatrogenic immunosuppression. This case highlights the importance for awareness on the part of clinicians, radiologists, and pathologists that malakoplakia can present as a soft-tissue mass.

Update on the Diagnosis and Classification of the Plasma Cell Neoplasms.

The plasma cell neoplasms are malignancies of the most terminally differentiated cells in B-cell ontogeny and are usually associated with the production of a monoclonal immunoglobulin molecule or M protein. These malignancies include tumors whose clinical manifestations are directly attributable to the end-organ damage induced by the dysregulated proliferation of neoplastic plasma cells. In contrast, disorders, such as primary amyloidosis, have a paradoxically low burden of neoplastic plasma cells, rendered highly pathogenic by the end-organ damage induced by deposition of the secreted paraprotein. In this article, discussion focuses on plasma cell myeloma. The molecular pathogenesis of plasma cell myeloma is reviewed and the diagnosis of the plasma cell neoplasms discussed.

Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression.

The Runx1/core binding factor-beta (CBFbeta) transcriptional complex is required for the establishment of hematopoiesis during development. Despite its critical role during development, a detailed analysis of Runx1 expression within specific lineages and developmental stages of the adult hematopoietic system is lacking. To address this, we have developed a Runx1-green fluorescent protein (GFP) knock-in mouse. We show that Runx1 is expressed in several hematopoietic lineages, including myeloid, B-lymphoid, and T-lymphoid cells. By contrast, Runx1 is weakly expressed in early erythroid cells, and its expression is rapidly extinguished during later stages of erythropoiesis. Runx1 expression is induced during early B-cell development and is expressed at a uniform level during all subsequent stages of B-cell development. Within the thymus, Runx1 is expressed at the highest level in CD4-CD8- double-negative thymocytes. In peripheral T cells, Runx1 is differentially expressed, with CD4+ T cells expressing 2- to 3-fold higher levels of Runx1 than CD8+ cells. Taken together, these findings indicate that although widely expressed in the hematopoietic system, the expression of Runx1 is regulated in a cell type- and maturation stage-specific manner. In addition, the Runx1-IRES-GFP knock-in mouse strain should prove valuable for investigation of Runx1 function in adult hematopoiesis.

Megakaryoblastic disorders in children.

Acute leukemia is the most common neoplasm in children, of which approximately one fourth of cases are acute myeloid leukemia. To a large extent, the clinicopathologic spectrum of acute myeloid leukemia seen in the pediatric age group is similar to that in adults. However, megakaryoblastic proliferations, including transient myeloproliferative disorder and acute megakaryoblastic leukemia, occur exclusively or predominantly in children. Although the clinical and pathologic features of these disorders have been well characterized, until recently their pathogenetic mechanisms have remained poorly understood. Recent advances have provided important insight into the pathogenesis of these disorders, and they provide a molecular basis for a clinically meaningful classification of primary megakaryoblastic disorders in the pediatric age group.

Terminal deoxynucleotidyl transferase-positive lymphoid cells in reactive lymph nodes from children with malignant tumors: incidence, distribution pattern, and immunophenotype in 26 patients.

The presence of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursors in benign lymph nodes from children has been characterized insufficiently. By using single- and double-labeling immunohistochemical analysis, we examined the frequency, distribution, morphologic features, and immunophenotype of TdT-positive cells in benign lymph nodes from 26 consecutive pediatric patients (4 boys, 22 girls; age, 10 weeks-17 years; median, 4.5 years), 23 of whom had a history of malignant neoplasm. We identified TdT-positive lymphoid cells in all 26 cases. These cells were found adjacent to medullary and cortical sinuses, with a frequency of 1 to 180 cells per high-powerfield (median, 20 cells), and were present singly and in small clusters. They were morphologically heterogeneous and showed a precursor B-cell immunophenotype including colocalization with CD34 by single-antibody immunohistochemical analysis and coexpression of variable levels of CD79a and CD10 and lack of CD3 expression by double immunostaining. These features should aid in the evaluation of pediatric lymph nodes for partial involvement by lymphoblastic lymphoma/leukemia.

Clinicopathologic analysis of follicular lymphoma occurring in children.

Follicular lymphoma is a rare lymphoid malignancy in pediatric patients and consequently remains poorly characterized, particularly with respect to its immunophenotype and molecular pathogenesis. A total of 23 pediatric patients with follicular lymphoma were identified, with a median age of 11 years and a male-to-female ratio of 2.3:1. Of the 19 patients for whom presenting clinical features were available, 15 patients had stage I, 1 had stage II, and 3 had stage III or IV disease. All tumors had a follicular architecture, and 74% of cases had grade 2 or 3 histologic features. All patients expressed CD20 and bcl-6, and 80% were positive for CD10. Bcl-2 expression was detected in only 5 of 16 cases. Consistent with this finding, bcl-2 gene rearrangements were detected in only 2 of 16 cases by polymerase chain reaction. These patients were treated primarily with cyclophosphamide, doxorubicin, vincristine, and prednisone-based chemotherapy; 4 patients also received involved-field irradiation. Of the 13 patients with available clinical follow-up, all but 2 achieved durable clinical remission. Importantly, all 4 patients with tumors diffusely positive for bcl-2 either presented with stage III/IV disease or had disease refractory to therapy, whereas patients with bcl-2-negative tumors uniformly had stage I disease, achieved complete remission, and experienced no relapses. These findings indicate that, in contrast to adult follicular lymphomas, dysregulated bcl-2 expression does not play a significant pathogenetic role in most pediatric follicular lymphomas. However, bcl-2 expression in pediatric follicular lymphoma identifies a subset of patients in whom disease is often disseminated at clinical presentation and is more refractory to combination chemotherapy.