Romiplostim for Treatment of Children and Young Adults With Severe Aplastic Anemia and Myelodysplastic Syndrome.

Thrombopoietin receptor agonists (TPO-RAs) induce trilineage hematopoiesis under conditions with acquired hematopoietic failure. We evaluated safety, tolerability, and preliminary efficacy of a TPO-RA, romiplostim (Nplate), with or without standard-of-care immunosuppressive therapy (±IST) for children (ages < 21 y) with newly diagnosed and relapsed/refractory severe aplastic anemia (SAA) and myelodysplastic syndrome (MDS). Data were collected from an observational study and a single arm interventional pilot study. The safety outcome was treatment-related adverse events (AEs). Efficacy was evaluated by complete hematopoietic response (CHR) at week 24. Romiplostim was commenced at 5 µg/kg/week, with dose escalation of 2.5 µg/kg/week (maximum, 20 µg/kg/dose) based on platelet response. Romiplostim was continued until CHR was observed. Ten subjects (SAA, 9 [IST, 4; without IST, 5]; MDS, 1) completed the study (median age: 9.2 y). Median romiplostim dose was 10 µg/kg/week (range: 5 to 17.5 µg/kg/week). The cumulative incidence of CHR was 70.4% (95% CI, 20.2%-92.6%). Among 21 AEs (Grade 1 to 3), 3 were attributed to romiplostim. At a median posttherapy follow-up of 10.9 months (range: 0.7 to 77.5), no clonal evolution, bone marrow fibrosis or mortality was reported. This proof-of-concept study provides data about short-term safety, tolerability, and preliminary efficacy of romiplostim (±IST) for treatment of pediatric SAA/MDS.

Utilizing digital pathology and immunohistochemistry of p53 as an adjunct to molecular testing in myeloid disorders.

TP53 mutation status guides early therapeutic decisions in the treatment of clonal myeloid disorders and serves as a simple means of monitoring response to treatment. We aim here to develop a standardized protocol for evaluating TP53 mutation status in myeloid disorders using immunohistochemistry assisted by digital image analysis and further compare this approach to manual interpretation alone. To accomplish this, we obtained 118 bone marrow biopsies from patients with hematologic malignancy and molecular testing for mutations associated with acute myeloid leukemia was performed. Clot or core biopsy slides were stained for p53 and digitally scanned. Overall mutation burden was assessed digitally using two different metrics to determine positivity, compared to the results of manual review, and correlated with molecular results. Using this approach, we found that digital analysis of immunohistochemistry stained slides performed worse than manual categorization alone in predicting TP53 mutation status in our cohort (PPV 91%, NPV 100% vs. PPV 100%, NPV 98%). While digital analysis reduced inter- and intraobserver variability when assessing mutation burden, there was poor correlation between the quantity and intensity of p53 staining and molecular analysis (R2 = 0.204). Therefore, digital image analysis of p53 immunohistochemistry accurately predicts TP53 mutation status as confirmed by molecular testing but does not offer a significant advantage over manual categorization alone. However, this approach offers a highly standardized methodology for monitoring disease status or response to treatment once a diagnosis has been made.

A Case of Secondary Hemophagocytic Lymphohistiocytosis Presenting with Severe Dyserythropoeisis in a Patient with Autoimmune Hemolysis.

The diagnosis of hemophagocytic lymphohistiocytosis (HLH) requires that several clinical criteria are met, and often relies on the identification of rare hemophagocytic cells in the bone marrow. Given the challenge in making the diagnosis, additional signs of immune dysregulation in the bone marrow would have practical clinical use in cases where overt hemophagocytosis is not seen. We present here a case of secondary HLH in a patient with autoimmune hemolysis ultimately diagnosed as Evans syndrome that initially presented with profound dyserythropoeisis in both the peripheral blood and bone marrow. We also explore an association between dyserythropoeisis and HLH in a series of cases previously seen at our institution.

Congenital Neutropenia with Specific Granulocyte Deficiency Caused by Novel Double Heterozygous SMARCD2 Mutations.

SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) is critical for myelopoiesis. Recently, bi-allelic SMARCD2 mutations have been reported in five children, causing autosomal recessive congenital neutropenia with specific granulocytes deficiency (CN-SGD); a syndrome resulting in G-CSF resistant neutropenia, recurrent infections, and dysplastic myelopoiesis. We report a new case with CN-SGD caused by two novel heterozygous pathogenic variants in the SMARCD2 gene (c.1081del (p.Gln361Argfs*15)), and (c.217C>T (p.Arg73*)). Treatment with the weekly dosing of thrombopoietin receptor agonist, Romiplostim, along with daily G-CSF transformed her clinical course, implying potential synergism. This report advances the understanding of CN-SGD caused by SMARCD2 mutations.

Gene Expression Signatures for the Accurate Diagnosis of Peripheral T-Cell Lymphoma Entities in the Routine Clinical Practice.

PURPOSE: Peripheral T-cell lymphoma (PTCL) includes heterogeneous clinicopathologic entities with numerous diagnostic and treatment challenges. We previously defined robust transcriptomic signatures that distinguish common PTCL entities and identified two novel biologic and prognostic PTCL-not otherwise specified subtypes (PTCL-TBX21 and PTCL-GATA3). We aimed to consolidate a gene expression-based subclassification using formalin-fixed, paraffin-embedded (FFPE) tissues to improve the accuracy and precision in PTCL diagnosis. MATERIALS AND METHODS: We assembled a well-characterized PTCL training cohort (n = 105) with gene expression profiling data to derive a diagnostic signature using fresh-frozen tissue on the HG-U133plus2.0 platform (Affymetrix, Inc, Santa Clara, CA) subsequently validated using matched FFPE tissues in a digital gene expression profiling platform (nCounter, NanoString Technologies, Inc, Seattle, WA). Statistical filtering approaches were applied to refine the transcriptomic signatures and then validated in another PTCL cohort (n = 140) with rigorous pathology review and ancillary assays. RESULTS: In the training cohort, the refined transcriptomic classifier in FFPE tissues showed high sensitivity (> 80%), specificity (> 95%), and accuracy (> 94%) for PTCL subclassification compared with the fresh-frozen-derived diagnostic model and showed high reproducibility between three independent laboratories. In the validation cohort, the transcriptional classifier matched the pathology diagnosis rendered by three expert hematopathologists in 85% (n = 119) of the cases, showed borderline association with the molecular signatures in 6% (n = 8), and disagreed in 8% (n = 11). The classifier improved the pathology diagnosis in two cases, validated by clinical findings. Of the 11 cases with disagreements, four had a molecular classification that may provide an improvement over pathology diagnosis on the basis of overall transcriptomic and morphological features. The molecular subclassification provided a comprehensive molecular characterization of PTCL subtypes, including viral etiologic factors and translocation partners. CONCLUSION: We developed a novel transcriptomic approach for PTCL subclassification that facilitates translation into clinical practice with higher precision and uniformity than conventional pathology diagnosis.

Utilization of anti-factor Xa levels to guide reversal of oral factor Xa inhibitors in the emergency department.

PURPOSE: Oral factor Xa inhibitors (FXaIs) are increasingly utilized for outpatient anticoagulation therapy; however, laboratory monitoring is not routinely used to assess the safety and efficacy of these agents. We aimed to evaluate the role of chromogenic anti-factor Xa (anti-Xa) assays in the emergency department (ED) in the setting of patients with an acute bleed or requiring emergent procedures. METHODS: A retrospective review of anti-Xa levels obtained in the ED between June 1, 2019, and April 30, 2020, was completed. Data were collected to describe the clinical setting of anti-Xa level collection, oral FXaIs used before admission, administration of reversal agents, and patient disposition to further characterize the role of anti-Xa levels in the management of rivaroxaban and apixaban reversal. RESULTS: Thirty anti-Xa levels were included in the final analysis. The median time from sample collection to anti-Xa assay result was 45.9 minutes (interquartile range, 35.3-54.7 minutes). Eleven patients (37%) received anticoagulation reversal after their anti-Xa levels were determined. Anticoagulation reversal agents included either activated prothrombin complex concentrates (aPCCs) or prothrombin complex concentrates (PCCs). Anti-Xa levels were collected in 2 patients who had received PCCs before arrival at our ED. Of the patients with anti-Xa levels below 30 ng/mL, none received aPCCs or PCCs after their anti-Xa levels were determined. Anti-Xa assays were used to rule out the presence of FXaIs in 3 patients. CONCLUSION: This study illustrates the novel role of anti-Xa levels in managing patients with an emergent need for reversal in the ED. The assay may be used to rule out the presence of oral FXaIs and avoid unnecessary administrations of anticoagulation reversal agents.

Education Case: Hereditary Thrombophilia With Double Heterozygous Factor V Leiden and Factor II c.*97G>A Mutations.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.1.

Up-front Treatment With Romiplostim in Children With Acquired Bone Marrow Failure: A Single Institutional Pediatric Case Series.

BACKGROUND: Thrombopoietin receptor agonists are emerging as a therapeutic option for patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). We report our experience of treating children with AA/MDS with romiplostim, thrombopoietin receptor agonist. OBSERVATIONS: Three children (AA, 2; MDS, 1) received romiplostim treatment at a median dose of 10 µg/kg/week (starting dose: 5 µg/kg/wk; 2.5 µg/kg/wk increment). Trilineage hematopoietic recovery occurred at a median of 13 weeks (range: 13 to 16 wk) without adverse events. Hematopoiesis continued to improve after therapy discontinuation (median follow-up: 2.8 y; range: 0.5 to 3.0). CONCLUSION: Our experience supports the short-term safety and efficacy of romiplostim in children with AA/MDS.

Discrepant Hemophilia A: An Underdiagnosed Disease Entity.

OBJECTIVES: The term discrepant hemophilia A (DHA) denotes the discrepancy between factor VIII activity (FVIII:C) measured by different assay methodologies in patients with nonsevere hemophilia A (HA). The objective was to review the characteristics and the current understanding of mechanisms contributing to assay discrepancy in DHA. METHODS: Characteristics of the DHA patients treated were examined by retrospective chart review. In addition, a literature review was performed to determine the current understanding of DHA. RESULTS: Three cases of DHA were diagnosed based on bleeding phenotype: 2 cases represented missed diagnoses of HA, and 1 represented misclassification of hemophilia severity. The revised diagnosis and classification of hemophilia directly affected clinical management. Review of the literature identified 18 articles with an estimated pooled prevalence of 36% (95% CI, 23%-56%; I2 = 85%; P < .01) among nonsevere HA. Furthermore, literature indicated that DHA is a feature of how different FVIII gene mutations affect FVIII:C activity within different assay methodologies. CONCLUSIONS: Our experience and literature review suggested that DHA is not only a laboratory phenomenon-it can affect clinical management in a subset of patients. A high index of suspicion for DHA is necessary while evaluating bleeding patients and/or classifying nonsevere HA.

t(15; 17) associated with primary myelofibrosis: a case report of an unusual clinical presentation and diagnostic dilemma.

BACKGROUND: primary myelofibrosis (PMF) is a myeloproliferative neoplasm which is associated with clonal molecular and cytogenetic abnormalities (CA) and varied clinical manifestations. While various CA have been previously described, t(15; 17) has not been reported in association with this condition. CASE PRESENTATION: A 69-year-old male presented with constitutional symptoms, cytopenias and bone marrow biopsy revealed immature blasts with fibrosis. Cytogenetic analysis showed a t(15;17) which initially suggested a diagnosis of acute promyelocytic leukemia (APL). However, flourescence in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR) studies were negative for transcripts promyelocytic leukemia (PML) gene and retinoic acid receptor alpha (RARA) or PML-RARA fusion. Along with these results, a second review of bone marrow histology, flowcytometry and the detection of a calreticulin gene (CALR) mutation helped with the correct diagnosis of PMF. Patient was then treated with ruxolitinib, a JAK (Janus kinase) 1 and 2 inhibitor, and eventually proceeded to receive a matched unrelated reduced intensity conditioning (RIC) allogeneic stem cell transplantation (ASCT) and has been doing well at the 6-month follow up. CONCLUSIONS: Our case highlights two points, that the t(15;17) is diagnostic of Acute Promyelocytic Leukemia (APL) in most cases, there are exceptions and it can be associated with other malignancies without causing any APL like features, as noted in this case. Also, that t(15; 17) by itself is never sufficient to diagnose APL without confirmation by other methods and relying solely on cytogenetics without timely confirmatory tests can lead to risks of delay in diagnosis and appropriate management.

B-cell lymphomas with concurrent MYC and BCL2 abnormalities other than translocations behave similarly to MYC/BCL2 double-hit lymphomas.

Large B-cell lymphomas with IGH@BCL2 and MYC rearrangement, known as double-hit lymphoma (DHL), are clinically aggressive neoplasms with a poor prognosis. Some large B-cell lymphomas have concurrent abnormalities of MYC and BCL2 other than coexistent translocations. Little is known about patients with these lymphomas designated here as atypical DHL. We studied 40 patients of atypical DHL including 21 men and 19 women, with a median age of 60 years. Nine (23%) patients had a history of B-cell non-Hodgkin lymphoma. There were 30 diffuse large B-cell lymphoma (DLBCL), 7 B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma, and 3 DLBCL with coexistent follicular lymphoma. CD10, BCL2, and MYC were expressed in 28/39 (72%), 33/35 (94%), and 14/20 (70%) cases, respectively. Patients were treated with standard (n=14) or more aggressive chemotherapy regimens (n=17). We compared the atypical DHL group with 76 patients with DHLand 35 patients with DLBCL lacking MYC and BCL2 abnormalities. The clinicopathologic features and therapies were similar between patients with atypical and typical DHL. The overall survival of patients with atypical double-hit lymphoma was similar to that of patients with double-hit lymphoma (P=0.47) and significantly worse than that of patients with DLBCL with normal MYC and BCL2 (P=0.02). There were some minor differences. Cases of atypical double-hit lymphoma more often have DLBCL morphology (P<0.01), less frequently expressed CD10 (P<0.01), and patients less often had an elevated serum lactate dehydrogenase level (P=0.01). In aggregate, these results support expanding the category of MYC/BCL2 DHL to include large B-cell lymphomas with coexistent MYC and BCL2 abnormalities other than concurrent translocations.

Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma.

We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)-DLBCL compared with activated B-cell (ABC)-DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the "gold standard" GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL.

Primary central nervous system Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly: a clinicopathologic study of five cases.

We report five cases of primary central nervous system (CNS) Epstein-Barr virus (EBV)-positive lymphoma of the elderly. This represented an incidence of 4 % of primary CNS diffuse large B-cell lymphoma (DLBCL) after EBV screening in 134 cases. All five patients were 65 years or older with no previous history of congenital or iatrogenic immune deficiencies. The histologic morphology of all the cases was DLBCL, with variable amounts of necrosis. The cell of origin (COO) as determined by the Hans algorithm disclosed germinal center type in 2 cases and non-germinal center type in 3 cases. MYC translocation was not detected, and MYC overexpression was detected in only one case. Three patients died shortly after diagnosis, and the remaining 2 patients were in complete remission for 2 and 10 years, respectively. We conclude that EBV+ DLBCL among the elderly is uncommon in primary CNS lymphoma in the Eastern United States. The patients usually present with a single mass lesion with headache and sensorimotor symptoms. The histologic morphology is DLBCL, but clonal T-cell gene rearrangement may be detected. The outcome varies from case to case and appears to be unrelated to the COO or MYC status.

MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell lymphoma.

Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9 angioimmunoblastic T-cell lymphomas, 11 peripheral T-cell lymphomas not otherwise specified (PTCLNOS), and normal T cells, and demonstrated that ALCLs express many of the miRNAs that are highly expressed in normal T cells with the prominent exception of miR-146a. Unsupervised hierarchical clustering demonstrated distinct clustering of ALCL, PTCL-NOS, and the AITL subtype of PTCL. Cases of ALK(+) ALCL and ALK(-) ALCL were interspersed in unsupervised analysis, suggesting a close relationship at the molecular level. We identified an miRNA signature of 7 miRNAs (5 upregulated: miR-512-3p, miR-886-5p, miR-886-3p, miR-708, miR-135b; 2 downregulated: miR-146a, miR-155) significantly associated with ALK(+) ALCL cases. In addition, we derived an 11-miRNA signature (4 upregulated: miR-210, miR-197, miR-191, miR-512-3p; 7 downregulated: miR-451, miR-146a, miR-22, miR-455-3p, miR-455-5p, miR-143, miR-494) that differentiates ALK(-) ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs associated with ALK expression. Of these, the miR-17∼92 cluster and its paralogues were also highly expressed in ALK(+) ALCL and may represent important downstream effectors of the ALK oncogenic pathway.

Lack of expression of MTAP in uncommon T-cell lymphomas.

UNLABELLED: The majority of peripheral T-cell lymphomas were found to lack methylthioadenosine phosphorylase, an enzyme that is essential for the salvage of adenine from methylthioadenosine, a product of polyamine synthesis. Importantly, tumors that lack this enzyme have been shown to be more sensitive to inhibitors of de novo purine synthesis (6-thioguanine, methotrexate). BACKGROUND: T-cell lymphomas, in particular peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL), have only limited and noncurative treatment options. PATIENTS AND METHODS: We report here that a high percentage of PTCL, AITL, and ALCL lack the enzyme methylthioadenosine phosphorylase (MTAP), as do T-cell leukemia and T-cell lymphoblastic leukemia. MTAP-deficient cells cannot cleave endogenous methylthioadenosine to adenine and 5-methylthioribose-1-phosphate, a precursor of methionine, and as a result have enhanced sensitivity to inhibitors of de novo purine biosynthesis. A recently introduced antifolate, pralatrexate, which has been shown to inhibit de novo purine biosynthesis, has been approved for treatment of PTCL and may have an increasing role in therapy. An alternative strategy involving coadministration of methylthioadenosine and high-dose 6-thioguanine has been proposed and may prove to be selectively toxic to MTAP-deficient uncommon lymphomas. CONCLUSION: Thus the consequences of MTAP deficiency suggest that new therapeutic interventions for T-cell lymphoma may be feasible.

Genome-wide miRNA profiling of mantle cell lymphoma reveals a distinct subgroup with poor prognosis.

miRNA deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1-positive MCL (n = 30) and cyclin D1-negative MCL (n = 7) and compared them with small lymphocytic leukemia/lymphoma (n = 12), aggressive B-cell lymphomas (n = 138), normal B-cell subsets, and stromal cells. We identified a 19-miRNA classifier that included 6 up-regulated miRNAs and 13 down regulated miRNA that was able to distinguish MCL from other aggressive lymphomas. Some of the up-regulated miRNAs are highly expressed in naive B cells. This miRNA classifier showed consistent results in formalin-fixed paraffin-embedded tissues and was able to distinguish cyclin D1-negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from small lymphocytic leukemia/lymphoma, dominated by 23 up-regulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL patients demonstrated a cluster characterized by high expression of miRNAs from the polycistronic miR17-92 cluster and its paralogs, miR-106a-363 and miR-106b-25, and associated with high proliferation gene signature. The other clusters showed enrichment of stroma-associated miRNAs, and also had higher expression of stroma-associated genes. Our clinical outcome analysis in the present study suggested that miRNAs can serve as prognosticators.

Composite biclonal marginal zone lymphoma of lung and chronic lymphocytic leukemia: pathologic, phenotypic, cytogenetic, and molecular study.

The simultaneous diagnosis of marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is rare. This study reports a patient with composite synchronous biclonal occurrence of MALT lymphoma of the lung and CLL/SLL. The morphology of the lung and peripheral blood showed features of MALT lymphoma and CLL, respectively. The cytogenetic evaluation of the lung specimen revealed a t(1;14) (p22;q32), a frequent genetic abnormality in MALT lymphoma. Flow cytometry analysis of the lung tissue showed features of MALT lymphoma and CLL/SLL with different light chain restriction, whereas the blood showed phenotypic evidence of CLL/SLL. Fluorescence in situ hybridization study of the blood showed a deletion of 13q14 and 17p13. Immunoglobulin heavy chain (IgH) gene rearrangement study of the lung tissue and blood showed a monoclonal IgH gene rearrangement with distinct light chain restriction, suggesting that the immunophenotypically different cell populations originated from separate clones.