Michael J. Cascio

Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism

Mutations in the calreticulin gene (CALR) were recently identified in approximately 70–80% of patients with JAK2‐V617F‐negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C‐terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium‐binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL‐3‐independent growth. Interestingly, expression of type I and type II mutant CALR in a nonhematopoietic cell line does not directly activate JAK/STAT signaling compared to wildtype CALR and JAK2‐V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll‐like receptor agonist. These effects are not dependent on the novel C‐terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2‐V617F‐negative essential thrombocytosis and primary myelofibrosis. Am. J. Hematol. 91:211–219, 2016.

De novo acute myeloid leukemia with 20-29% blasts is less aggressive than acute myeloid leukemia with ≥30% blasts in older adults: a Bone Marrow Pathology Group study.

It is controversial whether acute myeloid leukemia (AML) patients with 20–29% bone marrow (BM) blasts, formerly referred to as refractory anemia with excess blasts in transformation (RAEBT), should be considered AML or myelodysplastic syndrome (MDS) for the purposes of treatment and prognostication. We retrospectively studied 571 de novo AML in patients aged >50 years, including 142 RAEBT and 429 with ≥30% blasts (AML30), as well as 151 patients with 10–19% BM blasts (RAEB2). RAEBT patients were older and had lower white blood count, but higher hemoglobin, platelet count, and karyotype risk scores compared to AML30, while these features were similar to RAEB2. FLT3 and NPM1 mutations and monocytic morphology occurred more commonly in AML30 than in RAEBT. RAEBT patients were treated less often with induction therapy than AML30, whereas allogeneic stem cell transplant frequency was similar. The median and 4‐year OS of RAEBT patients were longer than those of AML30 patients (20.5 vs 12.0 months and 28.6% vs 20.4%, respectively, P = 0.003); this difference in OS was manifested in patients in the intermediate UKMRC karyotype risk group, whereas OS of RAEBT patients and AML30 patients in the adverse karyotype risk group were not significantly different. Multivariable analysis showed that RAEBT (P < 0.0001), hemoglobin (P = 0.005), UKMRC karyotype risk group (P = 0.002), normal BM karyotype (P = 0.004), treatment with induction therapy (P < 0.0001), and stem cell transplant (P < 0.0001) were associated with longer OS. Our findings favor considering de novo RAEBT as a favorable prognostic subgroup of AML. Am. J. Hematol. 89:E193–E199, 2014.

Cytogenetic Variation of B-Lymphoblastic Leukemia With Intrachromosomal Amplification of Chromosome 21 (iAMP21)A Multi-Institutional Series Review

OBJECTIVES B-lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21) is a relatively uncommon manifestation of acute leukemia and limited predominantly to the pediatric population. Case-specific information regarding flow cytometric, morphologic, and laboratory findings of this subtype of leukemia is currently lacking. METHODS We searched the databases of three large institutions for lymphoblastic leukemia with iAMP21 from 2005 through 2012 and analyzed the clinicopathologic features. RESULTS We identified 17 cases with five or more RUNX1 signals on interphase nuclei, 14 of which were consistent with the Childrens Oncology Group (COG) definition for iAMP21—namely, the presence of three or more RUNX1 signals on one marker chromosome. These cases showed a statistically significant lower peripheral WBC count and older age at diagnosis compared with all pediatric cases of B-ALL. We also identified three cases with increased RUNX1 signals scattered on multiple marker chromosomes that did not meet the COG definition of iAMP21 but showed similar 21q instability and older age at presentation. CONCLUSIONS Our findings not only demonstrate that B-ALL with iAMP21 is truly a distinct clinicopathologic entity but also suggest that a subset of cases of B-ALL with iAMP21 can show variable cytogenetic features.

Double autophagy stimulation using chemotherapy and mTOR inhibition combined with hydroxychloroquine for autophagy modulation in patients with relapsed or refractory multiple myeloma

Multiple myeloma is an incurable plasma cell neoplasm for which novel agents have improved outcomes but therapeutic resistance is inevitable. Infusional cytotoxic chemotherapy is an effective cytoreductive strategy for aggressive relapse but is not curative. The autophagy pathway is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation that mediates therapeutic resistance (Online Supplementary Figure S1) as it is a reversible adaptive response that allows cancer cells to survive therapy-induced apoptosis. Autophagy inhibition with hydroxychloroquine, which blocks the clearance of autophagic vesicles, can augment the cytotoxicity of proteasome inhibitors, DNA damaging chemotherapy including cyclophosphamide, and inhibitors of the mechanistic target of rapamycin (mTOR). A series of clinical trials combining the autophagy inhibitor hydroxychloroquine with autophagy-inducing chemotherapy has been generally safe, but levels of autophagy induction have been relatively modest. We sought to improve the efficacy of this approach in myeloma by using two agents that induce autophagy through different mechanisms. Since the phosphatidylinositide 3-kinase (PI3K)AKTmTOR signaling pathway both serves as a potential resistance mechanism to cytotoxic chemotherapy in myeloma and when inhibited, induces autophagy, we hypothesized that adding rapamycin to standard alkylating agent cyclophosphamide would ‘doubly’ induce autophagy, providing an improved platform for the addition of an autophagy inhibitor. We conducted a safety pilot and phase I study with the primary objective to determine the maximum tolerated dose (MTD) of hydroxychloroquine and safety of the 4-drug combination. Adults with relapsed or refractory multiple myeloma were eligible for the study if they had received prior lenalidomide, bortezomib (Table 1). Full eligibility criteria are provided in the Online Supplementary Appendix. This was a 2-stage study: the 1 stage was an open label single center pilot study (July 2011-June 2012; Abramson Cancer Center, clinicaltrials.gov identifier: 01396200) to establish the safety of adding rapamycin and hydroxychloroquine individually to backbone chemotherapy (n=6). The 2 stage was a single center (January 2013September 2014; Knight Cancer Institute, clinicaltrials.gov identifier: 01689987) dose escalation phase I trial with 4 hydroxychloroquine dose levels: 400, 600, 800 and 1200 mg (n=18). The treatment schema displays the chemotherapy sequence which was designed to induce maximal mTOR inhibition (Figure 1A) (rapamycin half life = 60 hours) to chemo-sensitize cells to cyclophosphamide and dexamethasone and allow for measurement of autophagy and mTOR signaling after each component of therapy was added. Dose-limiting toxicity (DLT) was

Myeloid Cell Nuclear Differentiation Antigen (MNDA) Expression Distinguishes Extramedullary Presentations of Myeloid Leukemia From Blastic Plasmacytoid Dendritic Cell Neoplasm

Myeloid neoplasms constitute one of the most common malignancies in adults. In most cases these proliferations initially manifest in the blood and marrow; however, extramedullary involvement may precede blood or marrow involvement in a subset of cases, making a definitive diagnosis challenging by morphologic and immunohistochemical assessment alone. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive entity that frequently presents in extramedullary sites and can show morphologic and immunophenotypic overlap with myeloid neoplasms. Given that BPDCN and myeloid neoplasms may both initially present in extramedullary sites and that novel targeted therapies may be developed that exploit the unique molecular signature of BPDCN, new immunophenotypic markers that can reliably separate myeloid neoplasms from BPDCN are desirable. We evaluated the utility of myeloid cell nuclear differentiation antigen (MNDA) expression in a series of extramedullary myeloid leukemias (EMLs) and BPDCN. Forty biopsies containing EML and 19 biopsies containing BPDCN were studied by MNDA immunohistochemistry. The majority of myeloid neoplasms showed nuclear expression of MNDA (65%). In contrast, all cases of BPDCN lacked MNDA expression. These findings show that MNDA is expressed in the majority of EMLs and support the inclusion of MNDA immunohistochemistry in the diagnostic evaluation of blastic hematopoietic infiltrates, particularly when the differential diagnosis is between myeloid leukemia and BPDCN.

Predominance of CD4+ T Cells in T-Cell/Histiocyte-Rich Large B-Cell Lymphoma and Identification of a Subset of Patients With Peripheral B-Cell Lymphopenia

Objectives T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a morphologic variant of large B-cell lymphoma whose flow cytometry findings are not well characterized. Methods Nineteen cases with flow cytometric immunophenotyping were identified from the case records of four institutions between 2001 and 2016. Results In most cases, neoplastic B cells were not detected by flow cytometry. Overall, cases showed a predominance of CD4+ T cells, which in some cases was marked. Significant coexpression of CD57 was seen on CD4+ T cells where this marker was analyzed, which correlated with PD-1 expression. Two cases also showed a profound systemic B-cell lymphopenia, which was associated in one case with hypogammaglobulinemia. Conclusions Overall, our work challenges previous findings that cases of THRLBCL are rich in CD8+ T cells and highlights parallels between THRLBCL and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). Also, an association of THRLBCL with systemic B-cell lymphopenia has not been previously reported but may represent an underrecognized manifestation.

An analysis of blastic plasmacytoid dendritic cell neoplasm with translocations involving the MYC locus identifies t(6;8)(p21;q24) as a recurrent cytogenetic abnormality

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive neoplasm with leukaemic features and frequent skin involvement. Translocations involving the MYC locus have been recently identified as recurrent cytogenetic abnormalities in this entity. The aim of this study was to assess the clinicopathological, immunophenotypic and genetic features in MYC‐rearranged BPDCN cases.

TdT-positive Infiltrate in Inflamed Pediatric Kidney: A Potential Diagnostic Pitfall

We encountered a patient with infantile nephrotic syndrome associated with a dense interstitial inflammatory infiltrate and prominent extramedullary hematopoiesis. Immunohistochemical analysis revealed numerous terminal deoxynucleotidyl transferase (TdT)-positive cells, which may raise concern for lymphoblastic lymphoma. Thus, we further characterized a group of pediatric kidneys with inflammation. TdT-positive nuclei were quantitated, and dual immunostains for TdT/CD79a, TdT/CD3, and TdT/CD43 were performed in a subset of cases; flow cytometry was performed in 1 case. TdT-positive nuclei were present in inflamed pediatric kidneys in 40 of 42 patients. TdT counts (average of 3 maximal high-power fields) ranged from 1 to >200, with a mean of 47. The presence and number of TdT-positive nuclei showed a strong association with younger patient age. Extramedullary hematopoiesis was identified in 11/42 patients, all under the age of 1. The presence of extramedullary hematopoiesis did not correlate with TdT count (P=0.158). Dual immunostaining and flow cytometric analysis in 1 case showed weak expression of B-cell markers and favored normal precursor B cells. Although TdT is a common marker of lymphoblastic lymphoma, we have demonstrated that TdT-positive cells may be part of the inflammatory milieu in infant kidneys. Together with cytologic, architectural, and clinical features, these data can help to avoid misinterpretation of involvement by lymphoblastic lymphoma/leukemia.

Anemia: Evaluation and Diagnostic Tests

Anemia is among the most common medical problems and clinical and laboratory evaluation need to be approached logically. The complete blood count with red cell indices offers clues to diagnosis. Many anemias have characteristic red cell morphology. The reticulocyte count serves as a useful screen for hemolysis or blood loss. Testing for specific causes of the anemia is performed. Occasionally, examination of the bone marrow is required for diagnosis. Molecular testing is increasingly being use to aid the diagnostic process. This article reviews diagnostic tests for anemia and suggests a rational approach to determining the etiology of a patients anemia.

Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm is an aggressive malignant neoplasm originating from precursor plasmacytoid dendritic cells. Blastic plasmacytoid dendritic cell neoplasm shows a characteristic immunophenotype in which the tumor cells express CD4, CD56, TCL1, and bright CD123, and lack expression of lineage-specific myeloid and lymphoid antigens. Although cytogenetic and molecular aberrations are common, diagnostic recurrent abnormalities have not been identified. In this chapter, we summarize the clinical, pathologic, and genetic abnormalities identified to date. We explore the current concepts in treatment and the potential role of targeted therapies in the management of this rare disorder.