Timothy P. Singleton

Unrelated Cord Blood Transplantation in Adult and Pediatric Acute Lymphoblastic Leukemia: Effect of Minimal Residual Disease on Relapse and Survival

Data on pretransplantation minimal residual disease (MRD) and outcomes of umbilical cord blood transplantation (UCBT) are limited. Out of the 143 patients with acute lymphoblastic leukemia (ALL) who underwent UCBT at the University of Minnesota between 2004 and 2010, we evaluated 86 patients with available MRD assessment data by 4- and 8-color flow cytometry analysis immediately before transplantation. Ten patients (11.6%) were MRD-positive, and 76 were MRD-negative (88.4%). Most of the patients (82%) received myeloablative conditioning. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. In multivariate analysis, age, disease status (complete remission [CR] 1 versus CR2/CR3), disease group (precursor B cell ALL versus Philadelphia chromosome-positive ALL versus T cell ALL), and time to transplantation had no impact on relapse. Patients with MRD before UCBT had a greater incidence of relapse at 2 years (relapse rate, 30%; 95% confidence interval [CI], 4%-56%) and lower 3-year disease-free survival (30%; 95% CI, 7%-58%) compared with those without MRD (relapse rate, 16%; 95% CI, 8%-25%; P = .05; disease-free survival, 55%; 95% CI, 43%-66%; P = .02). Our data suggest that in patients with ALL, achieving an MRD-negative state before UCBT improves outcomes.

Spectrum of Clonal Large Granular Lymphocytes (LGLs) of αβ T Cells: T-Cell Clones of Undetermined Significance, T-Cell LGL Leukemias, and T-Cell Immunoclones.

OBJECTIVES Clones of T-cell large granular lymphocytes (LGLTs) were detected by flow cytometry. Disease associations are described. METHODS Flow cytometry on blood or marrow detected clonal LGLTs by analyzing variable regions of the T-cell receptor β chain. RESULTS LGLT clones were detected in 20% (54/264) of tested patients. The clone sizes were less than 2.0 × 10(9)/L in the blood in 73% and less than 10% of marrow space in 94%. Blood counts showed cytopenias. Clinical associations included B-cell clones, myeloid neoplasms, nonneoplastic disorders of blood or marrow, transplants, systemic immune disorders, carcinomas, or hypothyroidism. Twelve patients had LGLT leukemia. Most (76%) had small LGLT clones with limited impact on the clinical management. CONCLUSIONS Most of the LGLT clones detected by flow cytometry were small and did not change the clinical management. We propose the following terminology: T-cell clones of undetermined significance, LGLT leukemias, and T-cell immunoclones.

Pleural primary effusion lymphoma in an elderly patient

An 87-year-old man, recent immigrant from Korea, was admitted to the hospital due to decreased oral intake of food and fluids and abdominal discomfort. The patient had a history of severe dementia, diabetes, hyperlipidemia, hypertension, and chronic renal insufficiency. No history of malignancy, blood transfusions, drug abuse, high-risk sexual behavior, HIV, hepatitis B, or hepatitis C infection was obtained; however, no testing for HIV or other viruses was performed during this admission. Initial admission laboratory examinations were unremarkable except for hypernatremia, which was corrected by fluid administration, and an elevated lactate dehydrogenase (1,566 u/l, normal range 325–750). A computed tomography (CT) scan was largely unremarkable except for small bilateral renal cysts and a moderate-sized right-sided pleural effusion with associated compressive atelectasis as well as a pericardial effusion. No masses or lymphadenopathy were identified. A thoracentesis was performed and yielded 1 l of clear, dark-orange fluid. Fluid analysis showed a pH of 7.2, total protein levels of 5.2 g/dl, glucose levels of 43 mg/dl, triglycerides of 30 mg/dl, amylase levels < 30 U/l, and a lactate dehydrogenase level of 11771 u/l. Aerobic and anaerobic cultures as well as acid-fast bacteria (AFB) and fungal cultures were negative. Pleural fluid cell counts were 4,140 nucleated cells/microliter, of which 4% were neutrophils, 3% lymphocytes, and 93% other (malignant) cells. Wright-stained cytospin preparations, Papanicolaoustained Surepath preparations, and hematoxylin and eosin stained cellblock preparations were made from the pleural fluid. All preparations showed a dyshesive population of large pleomorphic cells with plasmablastic features in a background of red blood cells, small lymphocytes, and neutrophils. Many apoptotic bodies and occasional mitotic figures were present. In Wright-stained cytospin preparations (Fig. C-1) most cells measured 14–16 l, with a range of 11–30 l. Their nuclei were single, round, with smooth to slightly irregular membranes, measured 12–14 l (range 9–18 l) and harbored one to three round or irregularly shaped macronucleoli ranging from 3 to 5 l in size. The cytoplasm was variable, from a narrow rim around the nucleus to more abundant, pale to dark blue in the Wright-stained preparations, with a peripheral accentuation. Neoplastic cells with more abundant cytoplasm had eccentrically placed nuclei and a pale-staining perinuclear area reminiscent of the hof of plasma cells (Figs. 2C-A and C). Occasional cytoplasmic vacuoles were seen. Papanicolaou-stained (Figs. 2C-B and D) and H&E-stained preparations (Fig. 3C-A) accentuated the plasmacytoid appearance of the cells and the presence of macronucleoli and highlighted the thick chromatinic rim and coarse granularity of the nuclear chromatin. Flow cytometry was performed on the pleural fluid and showed that the large cells were negative for CD2, CD4, CD5, CD7, CD10, CD14, CD19, CD20, CD56, as well as for j and k immunoglobulin light chains. They showed dim expression of CD45, CD3, CD38, and CD138; however, the sample was considered not interpretable due to the poor viability of the neoplastic cells. Immunoperoxidase stains performed on the cellblock preparation showed that the neoplastic cells were negative for cytokeratin AE1/AE3 and S100, as well as for all B-cell markers (CD20, CD79a, PAX5) and most T-cell markers (CD2, CD4, CD5, CD7, CD8, CD45RO) applied. They were also negative for j and k light chains, CD10, bcl2, bcl6, and ALK1, but were strongly positive for CD3 Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota *Correspondence to: Stefan E. Pambuccian, M.D., Professor, Department of Laboratory Medicine and Pathology, Director of Cytopathology, University of Minnesota Medical Center, Fairview, C422 Mayo MMC 76, 420 Delaware Street SE, Minneapolis, MN 55455, USA. E-mail: pambu001@umn.edu Received 31 March 2011; Revision 2 June 2011; Accepted 22 June 2011 DOI 10.1002/dc.21793 Published online 19 September 2011 in Wiley Online Library (wileyonlinelibrary.com).

Aplastic Anemia and Monosomy 7–Associated Dysmegakaryocytopoiesis

Aplastic anemia (AA) is marrow failure due to an inadequate number of hematopoietic cells in the marrow. Prior reports have described a more aggressive clinical course in aplastic anemia with monosomy 7. We report 3 pediatric cases of AA with normal cytogenetics followed by acquisition of monosomy 7. Bone marrow biopsies were initially diagnostic of AA but later showed monosomy 7 and an increased number of megakaryocytes with small hypolobated nuclei. Immunohistochemical stains for CD61 highlighted the marked dysmegakaryocytopoiesis. The marrow blast percentage was increased in only 1 patient with 4.6% blasts. The 3 patients underwent bone marrow transplantation, and each has remained disease free for 7 to 18 months after transplantation. Pediatric patients with AA and normal cytogenetics may develop monosomy 7 with a myelodysplastic syndrome, unclassified. Patients with AA and monosomy 7 should be evaluated for dysmegakaryocytopoiesis.

Transformed large B-cell lymphoma in rituximab-allergic patient with chronic lymphocytic leukemia after allogeneic stem cell transplant: successful treatment with ofatumumab

Transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) to a more aggressive lymphoma, including large B-cell (LBCL) or Hodgkin disease, is a well-recognized complication termed Richter transformation (RT) [1]. Its occurrence following allogeneic hematopoietic stem cell transplant (alloHCT) is extremely rare, with only one patient having been reported to date [2]. This patient was treated successfully with withdrawal of immunosuppressive drugs and donor lymphocyte infusion (DLI). Here, we present a case of RT following alloHCT in a 43-year-old female with a history of severe anaphylactic reaction to rituximab, a drug commonly used in patients with relapse or post-transplant lymphoproliferative disorder (PTLD) [3–5]. The patient was successfully treated with withdrawal of immunosuppressive drugs and ofatumumab, a fully human anti-CD20 monoclonal antibody with high efficacy in CLL and indolent lymphomas [6,7]. To our knowledge, ofatumumab has not been reported in either a post-transplant or LBCL setting. In 1998, a 43-year-old female was diagnosed with CLL/SLL by lymph node (LN) and bone marrow (BM) biopsies. Cytogenetics revealed a 14q32 deletion in three out of 17 metaphases. She was followed for 1 year without therapeutic intervention. In October 1999, she developed symptomatic lymphadenopathy and was treated with fludarabine and prednisone for 6 months. Rituximab could not be given for more than two cycles due to severe anaphylactic reactions. Starting in 2003, she intermittently received multiple chemotherapies, including fludarabine, cyclophosphamide, vincristine, steroids and most recently bendamustine. Given the short duration of responses, emerging cytogenetic aberrations (trisomy 12) and recurrent infections (primarily in the lung and sinuses), the patient was referred for consideration of alloHCT in April 2011. Prior to transplant, her BM had a prominent diffuse, interstitial lymphoid infiltrate comprising 80–90% cellularity [Figure 1(A)]. The cells were small to intermediate in size with scant cytoplasm on hematoxylin and eosin (H&E) staining [Figure 1(B)], and had the usual characteristics of CLL/SLL, including scant cytoplasm and clumped chromatin, on Wright–Giemsa staining [Figure 1(C)]. Flow cytometry demonstrated a lambda-monotypic B-cell population that expressed CD5, dim to absent CD20 and uniform CD23, without CD79b, further supporting a diagnosis of CLL/SLL. Cytogenetics confirmed the persistence of del 14 and trisomy 12. Figure 1 Pre-transplant bone marrow sampling. (A, B) H&E stained sections of the trephine biopsy at (A) × 10 and (B) × 100 oil

Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma

MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis. Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations. Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting. Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity.

Abnormal immunophenotype of the T-cell-receptor beta Chain in follicular-helper T cells of angioimmunoblastic T-cell lymphoma.

Analysis for 24 variable regions of the T‐cell‐receptor beta chain by flow cytometry (Vbeta) is a new technique to detect clonal alpha‐beta T lymphocytes and is characteristically performed on peripheral blood. Angioimmunoblastic T‐cell lymphoma (AITL) has increased neoplastic follicular‐helper T cells (FHT), which often express CD10; but nonneoplastic, CD10‐positive T cells may be associated with reactive lymphadenopathy and with B‐cell lymphomas. This study documents the utility of Vbeta analysis of FHT in specimens of AITL from blood, from marrow, and from lymph nodes.

Cellular intrinsic mechanism affecting the outcome of AML treated with Ara-C in a syngeneic mouse model.

The mechanisms underlying acute myeloid leukemia (AML) treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.