Paul Hosking

ATP Synthase Is Responsible for Maintaining Mitochondrial Membrane Potential in Bloodstream Form Trypanosoma brucei

ABSTRACT The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F1-ATPase α and β subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the β subunit decreased the mRNA and protein levels for the β subunit, as well as the mRNA and protein levels of the α subunit. Similarly, RNAi of α subunit decreased the α subunit transcript and protein levels, as well as the β-subunit transcript and protein levels. In contrast, α and β RNAi knockdown resulted in a 60% increase in the F0 complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F0-32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for α or β subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of α and β RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form T. brucei.

Immunophenotypes of Chronic Myelomonocytic Leukemia (CMML) Subtypes by Flow Cytometry A Comparison of CMML-1 vs CMML-2, Myeloproliferative vs Dysplastic, De Novo vs Therapy-Related, and CMML-Specific Cytogenetic Risk Subtypes

OBJECTIVES We sought to immunophenotype blasts, monocytes, and granulocytes in chronic myelomonocytic leukemias (CMMLs) and compare CMML subtypes, to identify if significant antigen expression differences existed. METHODS Bone marrow blasts, monocytes, and granulocytes from CMML subgroups (n = 30; World Health Organization types 1/2, proliferative/dysplastic, therapy related/de novo, and low/intermediate/high cytogenetic risk) were immunophenotypically compared by flow cytometry with 10 nonneoplastic control marrows. RESULTS Aberrancies were present in blasts of 26 (87%) of 30 CMMLs (26 diagnostic; four follow-up) and six (60%) of 10 controls (P = .089), monocytes of 28 (93%) of 30 CMMLs and six (60%) of 10 controls (P = .026), and granulocytes of eight (28%) of 29 CMMLs and zero of 10 controls (P = .166). Underexpression of CD14 and CD15 on monocytes was more common in CMMLs compared with controls (P = .008 and P = .043). Statistical analysis showed no significant difference in antigen expression between the CMML subgroups on blasts or monocytes; granulocytes demonstrated more common HLA-DR expression in CMML-2 vs CMML-1. CONCLUSIONS These findings confirm heterogeneity within CMML subgroups and find no specific qualitative or quantitative findings characteristic of a subgroup.

Pure Erythroid Leukemia and Erythroblastic Sarcoma Evolving From Chronic Myeloid Neoplasms

OBJECTIVES Pure erythroid leukemia (PEL) is an extremely rare entity that may, even more rarely, evolve from a preexisting chronic myeloid neoplasm (CMN); there is minimal literature regarding this latter phenomenon. METHODS We describe 14 patients with PEL that represented progression from a preexisting myelodysplastic syndrome (MDS, n = 8) or myeloproliferative neoplasm (MPN, n = 6), three of which manifested as erythroblastic sarcoma (EBS), a rare entity. These patients had a highly complex karyotype with prominent clonal evolution and a very aggressive clinical course. RESULTS Patients with PEL from MDS showed a more rapid progression time to PEL and had lower platelet counts compared with PEL from MPN. No other significant differences were found between the two groups. CONCLUSIONS These data represent the largest cohort of patients with PEL and an antecedent CMN, as well as the largest series of EBS reported to date, and underscore the unique morphologic, cytogenetic, immunophenotypic, and clinical features of this uncommon entity.

CD30 Expression in Monomorphic Posttransplant Lymphoproliferative Disorder, Diffuse Large B-Cell Lymphoma Correlates With Greater Regulatory T-Cell Infiltration

Objectives CD30 is a protein thought to promote cell proliferation/survival and downregulate the immune response. Twenty percent to 40% of de novo diffuse large B-cell lymphomas (DLBCLs) express CD30, and some patients have been treated with the anti-CD30 agent brentuximab. In the solid organ transplant setting, allograft regulatory T cells (Tregs) have been shown to be modulated via CD30 signaling. Methods Posttransplant lymphoproliferative disorders (PTLDs) constitute a heterogeneous group of lymphomas, and since CD30 expression has been rarely formally assessed in PTLDs, we analyzed a cohort of PTLDs. Results We found that 26 (79%) of 33 PTLDs were CD30+. Of these, 17 (77%) of 22 DLBCL monomorphic PTLDs were CD30+ compared with 56 (38%) of 148 de novo DLBCLs (P = .009). The median FoxP3+ Treg count was higher in CD30+ than in CD30- PTLDs, 3.0 vs 0 (P = .012). Conclusions These findings suggest a pathophysiologic link between CD30 activity and Tregs and may indicate differential expression of CD30 in B-cell lymphomas arising in the setting of immune dysregulation.

Carcinocythemia: A rare entity becoming more common? A 3‐year, single institution series of seven cases and literature review

Carcinocythemia is a rare phenomenon defined as morphologically identifiable, circulating tumor cells in the peripheral blood. No modern case series of carcinocythemia exists in the literature.

Early Fluorescence in situ Hybridization Assessment during Acute Myeloid Leukemia Induction Chemotherapy

The standard of care for upfront remission induction therapy for “fit” adults with de novo acute myeloid leukemia (AML) is continuous-infusion cytarabine with concurrent anthracycline (i.e., “7+3” regimen) [1]. Fourteen days after the initiation of chemotherapy, bone marrow is re-evaluated to identify patients who may benefit from immediate receipt of an additional cycle of remission induction chemotherapy. Current NCCN guidelines specify that a second cycle of induction chemotherapy should be administered prior to count recovery if (1) more than 5% of the cells in the day-14 bone marrow sample are morphologically consistent with myeloblasts and (2) the clinical status of the patient permits safe administration of cytotoxic therapy [2].

Local Disease Control in Ocular Adnexal Lymphoproliferative Disorders: Comparative Outcomes of MALT Versus Non-MALT Histologies

Micro‐Abstract The efficacy of involved field radiotherapy (IFRT) in the outcomes of patients with different subtypes of ocular adnexal lymphoproliferative disorder is lacking. We retrospectively analyzed and compared the outcomes of patients with mucosa‐associated lymphoid tissue (MALT) and non‐MALT ocular adnexal lymphoproliferative disorder after being treated with IFRT. Our results reveal that IFRT provided excellent disease control with superior failure‐free survival in the MALT cohort when compared with the non‐MALT cohort. Introduction: Ocular adnexal lymphoproliferative disorders (OALDs) are almost exclusively of B‐cell origin, with the majority being extra‐nodal marginal zone lymphomas of mucosa‐associated lymphoid tissue (MALT). The comparative efficacy of involved field radiation therapy (IFRT) in MALT vs. non‐MALT OALDs is not known. Materials and Methods: We present a single‐center, large cohort, retrospective study of the efficacy of IFRT in OALDs. Failure‐free survival (FFS), complete remission, and local, regional, and distant failure were determined for 112 patients with MALT OALDs (n = 71) and non‐MALT OALDs (n = 41) cohorts. Results: Fifty‐six patients with MALT OALD and 26 patients with non‐MALT OALD received IFRT only (without any planned concurrent or sequential systemic chemothereapy or chemo‐immunotherapies). Among the OALD cohorts treated with only IFRT, complete remission was achieved in 49 (87.5%) patients in the MALT cohort and 23 (88.4%) in the non‐MALT cohort (P = .99). Clinically, resolution of symptoms occurred in 83.3% and 93.3% of the patients in the MALT and non‐MALT cohorts, respectively. Local failure occurred in 4 (7.1%) patients in the MALT cohort, compared with 4 (15.3%) patients in the non‐MALT cohort (P = .24). Regional failure (or extra‐orbital failure) occurred in 5 (8.9%) patients in the MALT cohort and in 3 (11.5%) patients in the non‐MALT cohort (P = .71). Distant failure was reported in 1 (1.7%) and 2 patients (7.6%) in the MALT and non‐MALT cohorts, respectively (P = .18). The median follow‐up of survivors was 5.1 years (range, 0.1‐22.5 years) in the MALT cohort and 3.9 years (range, 0.1‐22.9 years) in the non‐MALT cohort. The 5‐year and 10‐year FFS was 95% (95% confidence interval [CI], 88%‐100%) and 83% (95% CI, 70%‐98%) for the ocular MALT and 67% (95% CI, 48%‐94%) and 56% (95% CI, 34%‐91%) for the non‐MALT cohorts, respectively (log rank for P = .025). On multivariate analyses, age (hazard ratio [HR], 1.06; 95% CI, 1.10‐1.12; P = .03), presence of non‐MALT histology (HR, 13.9; 95% CI, 2.05‐94.4; P = .007), and radiation dose < 30.6 Gy (HR, 5.27; 95% CI, 1.14‐24.3; P = .03) were associated with worse FFS. The 5‐year and 10‐year overall survival was 92% (95%, CI 83%‐100%) and 80% (95% CI, 66%‐96%) for the MALT and 78% (95% CI, 61%‐100%) and 62% (95% CI, 38%‐100%) for the non‐MALT cohorts, respectively (P = .80). Conclusion: Our results reveal that IFRT provided excellent disease control with superior FFS in the MALT cohort when compared with the non‐MALT group.

Evaluation of CD43 expression in non-hematopoietic malignancies

OBJECTIVES CD43 is normally expressed only on the surface of leukocytes, and is considered a sensitive and specific marker for hematologic malignancies. As such, it may have diagnostic utility in confirming hematolymphoid lineage in cases that are negative for CD45. Aberrant CD43 expression has been described in non-hematopoietic tumors, although literature data on this topic is variable and sometimes contradictory. To clarify and expand on existing literature findings, we evaluated CD43 expression by immunohistochemistry (IHC) in a large cohort (307) of non-hematopoietic neoplasms, including poorly differentiated malignancies. METHODS 17 tissue microarrays and sections from 19 individual cases were stained with CD43 (clone DF-T1) monoclonal antibody. The proportion of positive cells, stain localization (nuclear, cytoplasmic or membranous), and intensity (compared to internal leukocyte controls) were recorded in all cases. RESULTS There were 98/307 (32%) positive cases, that showed focal weak nuclear staining in 1-25% of cells, including 23/25 (92%) pancreatic ductal adenocarcinomas; 31/34 (91%) breast invasive ductal carcinomas; 13/15 (87%) papillary thyroid carcinomas; 3/4 (75%) follicular thyroid carcinomas; 6/15 (40%) renal cell carcinomas; 9/28 (32%) lung adenocarcinomas; 1/13 (8%) lung squamous cell carcinomas (SCCs); 2/8 (25%) prostate adenocarcinomas; 8/62 (13%) colon adenocarcinomas; and 2/21 (10%) neuroendocrine neoplasms. None of the positive cases demonstrated strong, membranous CD43 expression comparable to that seen in background mature lymphocytes or segmented neutrophils. Negative cases included 11 cervical SCCs, 12 cervical adenocarcinomas, 19 urothelial carcinomas, 10 lung small cell carcinomas, 11 sarcomas, and 19 poorly differentiated carcinomas from various tissue sites. CONCLUSIONS In our cohort, most non-hematopoietic neoplasms are negative for CD43 expression, with a subset showing focal, weak nuclear positivity. This data indicates that uniform and strong membranous staining appears to be specific to hematopoietic neoplasms.