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Multiparametric flow cytometry assays are long recognized as an essential diagnostic test for leukemias and lymphomas. Lacking Food and Drug Administration–approved standardized tests, these assays remain laboratory developed tests. The recently published guidelines, CLSI H62, are the most detailed and up-to-date instructions for designing and validating clinical flow cytometry assays. This review provides a historical background for the current situation, summarizes key points from the CLSI guidelines, and lists practical points for assay development gained from personal experience.

Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression‐free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter‐laboratory variation in MM MRD reporting. Seventeen participants from 13 countries performed blinded analysis of the same eight de‐identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re‐analyzed for MRD and other bone marrow cells (Stage 2). Inter‐laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3). In Stage 1, participants agreed on MRD+/MRD− status 89% and 68% of the time respectively. Inter‐observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD− status improved to 74%. Better consistency enumerating all parameters among operators resulted in near‐unanimous agreement on sample adequacy. Uniform flow cytometry data analysis substantially reduced inter‐laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

Myeloid neoplasms occasionally occur in patients with sickle cell disease, and the underlying connection between the two diseases is unclear. Herein, we retrospectively analyzed four cases of sickle cell disease patients who developed myeloid neoplasm. Age at time of diagnosis ranged from 27 to 59 years with a median of 35.5 years. Two patients were treated with hydroxyurea and the other two with supportive care alone, with one out of the four patients receiving additional treatment with hematopoietic stem cell transplant. Three patients presented with leukocytosis, and the remaining patient presented with pancytopenia. Two patients were diagnosed with myelodysplastic syndrome/myeloproliferative neoplasm, one with myelodysplastic syndrome, and the other with acute myeloid leukemia. All four cases demonstrated certain degrees of myelodysplasia and complex cytogenetic abnormalities with − 7/7q- and/or − 5/5q- or with 11q23 (KMT2A) rearrangement. This cytogenetic profile resembles that seen in therapy-related myeloid neoplasm, suggesting that myeloid neoplasm in the setting of sickle cell disease may represent a subcategory of the disease distinct from de novo myeloid neoplasm in general. Extensive literature review further demonstrates this similarity in cytogenetic profile, as well as in other associated pathologic features. Potential etiology includes therapy for sickle cell disease, disease-related immunomodulation, or disease-related chronic inflammation. We hypothesize that constant hematopoietic hyperplasia, stimulated by a hemolysis-induced cytokine storm, may increase the chance of somatic mutations/cytogenetic aberrations, resulting in transformation of myeloid precursors. This group of myeloid neoplasms seems to herald a dismal clinical outcome, with median survival <1 year, although the exact pathogenesis and biology of the disease remain to be investigated by large cohorts in future studies.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2and PRDM1(Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

The IL-1 receptor-associated kinase 1 (IRAK-1) and IRAK-M are key signaling molecules in cellular responses to endotoxin initiated through the Toll-like receptors (TLRs). The aim of this study was to evaluate the effect of age on the modulation of TLRs and IRAK-1 and IRAK-M in peripheral blood mononuclear cells (PBMCs) exposed in vitroto endotoxin under conditions that could induce endotoxin tolerance. Peripheral blood mononuclear cells obtained from young (4- to 6-month-old) and aged (24- to 26-month-old) Brown Norway rats were treated with high-dose LPS, with or without priming with low-dose LPS. In comparison with younger rats, the intensity of TLR-4 expression was persistently high in monocytes from aged rats after stimulation with LPS and was not decreased by priming with low-dose LPS (P< 0.05). Messenger RNA (mRNA) for TLR-4 in PBMCs from aged rats did not show any decrease after priming with low-dose LPS as seen in PBMCs from young rats at 24 h (P= 0.01) after restimulation. In PBMCs from young rats, but not aged rats, preconditioning with low-dose LPS and subsequent stimulation with high-dose LPS resulted in markedly decreased IRAK-1 protein (P= 0.02) and decreased mRNA for IRAK-1 (P< 0.05). In contrast, PBMCs from aged rats treated in this manner continued to express measurable levels of IRAK-1 protein. Preconditioning with low-dose LPS caused an increase in both IRAK-M protein and mRNA (P= 0.05) after stimulation with high-dose LPS only in cells from young rats. These phenotypic characteristics of PBMCs from aged rats can interfere with their ability to develop tolerance to endotoxin.

Primary lymphomas affecting the female reproductive system are uncommon but often pose a diagnostic challenge if their existence is not suspected. This article reviews the pathological and clinical features of lymphomas occurring in various sites in the female genital tract including the vulva, vagina, cervix, endometrium, fallopian tubes, and ovaries. Using the recent World Health Organization classification, the various types of lymphomas are identified as separate diseases and not as morphological variations of the same disease. The immunophenotypic and cytogenetics features of the major lymphomas are summarized. The incidence, presenting symptoms, gross and microscopic features, major differential diagnostic considerations, response to therapy, and expected outcome are discussed. Using published data on patient outcome, the International Federation of Obstetricians and Gynecologists and Ann Arbor staging systems are compared for their predictive value, and the difficulty in assigning primary and secondary status in extranodal lymphomas is emphasized. The observed differences in the behavior of some lymphomas in gynecological sites compared with their usual nodal location are presented. Finally, the possible etiology of these conditions is discussed in light of the emerging paradigm of mucosa-associated lymphoid tissue lymphomas.

We report a phase 1 study of pharmacokinetics, dosimetry, toxicity, and response of 131I anti-tenascin chimeric 81C6 for the treatment of lymphoma. Nine patients received a dosimetric dose of 370 MBq (10 mCi). Three patients received an administered activity of 1480 MBq (40 mCi), and 2 developed hematologic toxicity that required stem cell infusion. Six patients received an administered activity of 1110 MBq (30 mCi), and 2 developed toxicity that required stem cell infusion. The clearance of whole-body activity was monoexponential with a mean effective half-life of 110 hours (range, 90-136 hours) and a mean effective whole-body residence time of 159 hours (range, 130-196 hours). There was rapid uptake within the viscera; however, tumor uptake was slower. Activity in normal viscera decreased proportional to the whole body; however, tumor sites presented a slow clearance (T1/2, 86-191 hours). The mean absorbed dose to whole-body was 67 cGy (range, 51-89 hours), whereas the dose to tumor sites was 963 cGy (range, 363-1517 cGy). Despite lack of a “blocking” antibody, 1 of 9 patients attained a complete remission and 1 a partial remission. These data demonstrate this radiopharmaceutical to be an encouraging agent for the treatment of lymphoma particularly if methods to protect the normal viscera are developed.

In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival.The role of endothelial cell alteration in chronic rejection was examined in our model.

Injury has been hypothesized to cause inflammation through systemic release of lipopolysaccharide and pro-inflammatory cytokines, but this has proved difficult to demonstrate in humans. We looked for evidence of an inflammatory pattern of cytokine gene expression by peripheral blood mononuclear cells (PBM) in six polytraumatized patients (ISS = 25 ± 8) upon ER admission, and in six matched healthy controls. PBM tumor necrosis factor (TNF)-α, interleukin (IL)-1 β, IL-4, IL-6, IL-10, and interferon (IFN)-γ message was assessed by semi-quantitative reverse-transcription polymerase chain reaction. No increase in expression of any of the pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, or IL-6) was found after trauma, and IFN-γ tended to decrease. Of the immunosuppressive cytokines, IL-10 expression increased 5-fold (p < .05) but no change in IL-4 was discerned. This pattern is fundamentally different from the cytokine expression patterns expected with sepsis or exposure to lipopolysaccharide. These findings are inconsistent with the occurrence of systemic endotoxemia and subsequent global immunocyte activation early after trauma.

BACKGROUND: Trauma is believed to activate immunocytes but paradoxically also increases the risk of intraperitoneal infection. OBJECTIVE: To investigate these events by evaluating changes in the cytokine control networks of human peritoneal macrophages (PMø) early after trauma. DESIGN: Case-control study comparing cytokine messenger RNA (mRNA) expression by PMø from patients with extra-abdominal trauma with that of both peripheral blood mononuclear cells (PBM) and PMø obtained from healthy individuals. SETTING: Level I trauma center and basic science laboratory in a university hospital center. PATIENTS: Six patients with polytrauma (Injury Severity Score, ≥15) with clinically negative diagnostic peritoneal lavages performed on routine indications at admission to the emergency department and six healthy age- and sex-matched individuals undergoing inguinal herniorrhaphy under local anesthesia. INTERVENTIONS: Peritoneal macrophages were isolated from diagnostic peritoneal lavages in trauma patients. Identical lavages were performed through the hernia sac in the control group. MEASUREMENTS: Cellular RNA was assayed for tumor necrosis factor α (TNF-α), interleukin-1β, IL-6, and IL-10 message by semiquantitative reverse-transcription polymerase chain reaction. RESULTS: Normal PMø expressed high levels of TNF-α mRNA relative to PBM, but expression of the other proinflammatory cytokines was equivalent to that of PBM. Peritoneal macrophage expression of TNF-α mRNA was markedly (64-fold) decreased after trauma (P&lt;.001), when PBM expression of IL-10 mRNA was increased (P=.03). CONCLUSIONS: Human PMø constitutively show high levels of TNF-α message expression, and this is down-regulated by polytrauma. This might constitute a functionally "primed" state. If so, TNF-α down-regulation might contribute to functional PMø suppression after systemic injury.(Arch Surg. 1995;130:1186-1192)

We recently reported that cross-linking the leukocyte common antigen (CD45) can rapidly induce aggregation of human peripheral blood mononuclear cells via lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) interactions. Herein, we have examined both T-cell-monocyte cellular interactions and the molecular signaling that are involved in this phenomenon. Experiments using highly purified T lymphocytes showed that CD45-induced aggregation requires the presence of both T cells and monocytes. Cross-linking CD45 only on T lymphocytes, but not on monocytes, initiated cellular clustering after reconstituting to the respective untreated cell type. By several criteria, CD45-induced clustering of T cells to autologous monocytes was shown to be Fc-receptor-in-dependent. When comparing intracellular signaling in leukocyte aggregation induced by CD45 cross-linking versus phorbol myristate-12-13-acetate (PMA) treatment, the former was found to be fivefold to 10-fold more sensitive to H-8, a reagent that effectively blocks cAMP- and cGMP-dependent protein kinases. On the other hand, reagents that increase intracellular cAMP levels (eg, dbcAMP, forskolin, and IBMX), protein kinase C (PKC) inhibitors (eg, staurosporine), and tyrosine kinase inhibitors (eg, herbimycin A and genistein) all readily inhibited PMA-induced, but not CD4S monoclonal antibody-induced, aggregation. We conclude that cross-linking the leukocyte common antigen on T cells induces LFA-1-/ICAM-1-dependent T-cell-monocyte aggregation through a unique signaling pathway independent of PKC, which involves instead cAMP-/cGMP-dependent protein kinases.

We recently demonstrated that different CD45 monoclonal antibodies (mAb) are able to induce cellular aggregation in human peripheral blood mononuclear cells (PBMC) through LFA-1/ICAM-1 interactions. Such interactions could be down-modulated by protein kinase (PK) A/G inhibitors, but were unaffected by inhibitors of PKC, suggesting the involvement of PKA or PKG in CD45 mAb-induced adhesion. In this study we show that after incubation of PBMC with several (but not all) mAb to CD45, CD45RO and CD45RA, intracellular cAMP, but not cGMP concentrations readily increase, reaching a maximum 30 min after start of activation. As evidenced by several lines of investigation cAMP accumulation was independent of Fc receptor-associated signaling as well as tyrosine phosphatase activity of CD45. In highly pure T lymphocytes, CD45 mAb were unable to induce cAMP synthesis, but readily did so after addition of autologous monocytes. After paraformaldehyde fixation of both quiescent or IFN-γ/TNF-α-preactivated monocytes, cAMP production was no longer detectable, suggesting monocytes as the cell of origin for the increased cAMP synthesis. Further, cAMP accumulation in monocytes occurred after reconstitution to T lymphocytes preincubated with CD45 mAb and extensively washed. Importantly, pretreatment of T lymphocyte/monocyte mixtures with LFA-1 mAb and/or ICAM-1 mAb down-regulated CD45 mAb-induced cAMP synthesis. Finally, we demonstrate that CD45 mAb are not only capable of inducing cAMP production, but also of directly stimulating PKA enzyme activity. Based on the data presented, we propose that CD45 signaling in T lymphocytes subsequently activates cAMP accumulation and PKA activation in monocytes via LFA-1/ICAM-1-dependent cellular interactions.

A novel cell aggregation-inducing characteristic of the leukocyte common antigen, CD45, is described and its underlying molecular mechanisms investigated. Formation of strong cell clusters was consistently observed in human PBMCs after crosslinking CD45 molecules with antibodies, directed to epitopes common for all CD45 isoforms (e.g., mAb ROS220 or NIH45-2) or the CD45RA (e.g., mAb Alb11) or the CD45RO isoform (e.g., mAb UCHLI). This phenomenon was not seen after PBMC treatment will CD45RA mAb HB11 or CD2 mAb 39C1.5. Identical to phorbol 12-myristate 13-acetate (PMA)-induced clustering, CD45-mediated aggregation was also suppressed by EDTA, by cytochalasin B, and by incubation at 4°C, all characteristics of adhesion mediated by integrins. The involvement of LFA-1 and ICAM-1 in CD45-mediated adhesion was supported by the observation that CD11a (LFA-1α) mAb R7.1, CD18 (LFA-1β) mAb R3.3, and CD54 (ICAM-1) mAb R6.1 or RR/1 all strongly inhibited CD45- and PMA-induced aggregation. Interestingly, highly pure T lymphocytes did not aggregate in response to CD45 mAb, but did after PMA treatment. These results indicate that triggering human PBMCs via CD45 can cause strong cell aggregation, largely through LFA-1/ICAM-1 interactions. Our findings support an important role of the CD45 antigen in signal transduction and intercellular interaction in human PBMCs. Copyright 1993, 1999 Academic Press

Introduction: A novel patient (pt) subset defined by RARA pathway activation was identified by super-enhancers (SEs) at the RARAand IRF8gene loci in AML and MDS. These SEs correlated with mRNA expression of each gene, as well as preclinical tumor cell differentiation and in vivoefficacy response to SY-1425 (tamibarotene), a potent and selective oral RARα agonist approved for treatment of relapsed/refractory (r/r) APL in Japan. This discovery formed the basis of a biomarker-directed phase 2 clinical study of SY-1425 as monotherapy and in combination with azacitidine for AML and MDS pts (NCT02807558).

No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) has the highest incidence and prevalence of any leukemia in the United States. It has a characteristic immunophenotype, but variant forms (typically termed “atypical CLL”) are commonly encountered. It is not known if this immunophenotypic heterogeneity in CLL is clinically relevant. We hypothesized that the immunophenotype of CLL cells correlates with both biologic parameters and clinical outcomes.We performed a retrospective review of clinical, biologic, and flow cytometric data of patients undergoing an evaluation of an absolute lymphocytosis at the Duke University and Durham VA Medical Centers. Patients meeting immunophenotypic criteria for CLL based on cell surface expression of CD5, CD19, CD20, CD23, and surface immunoglobulin light chain (sIg) were included in this analysis. Atypical CLL was defined if any of the following were observed: (1) expression of CD5, CD20, CD23, or sIg that differed from a typical CLL immunophenotype (e.g. bright CD20 or low CD23; hereafter non-standard immunophenotype), (2) expression of T cell markers CD7 or CD8, or (3) expression of CD123. Through electronic chart review, clinical and biologic parameters were abstracted. These included age; gender; ethnicity; WBC, hemoglobin, platelet count, and Rai stage at diagnosis; time to first treatment; overall survival; CD38, ZAP70, IGHV mutation status, and interphase cytogenetics (FISH). A chi square test or Fisher’s exact test for small sample sizes was used to test association of categorical data. The central tendencies of continuous measurement were compared using the Wilcoxon rank sum test. Overall survival and time to treatment were calculated using the Kaplan-Meier product limit method. Survival curves were compared using the log-rank test. This study was approved by IRBs at Duke University and Durham VA Medical Centers.We reviewed 189 patients, including 119 (63%) with typical CLL immunophenotype and 70 (37%) with atypical. Among patients with atypical CLL, 93% (65 of 70) had a non-standard immunophenotype, 33% (23 of 70) had expression of T cell markers, and 33% (14 of 43) had expression of CD123. We observed expression of T cell markers in 12% (23 of 189) and CD123 in 12% (14 of 114) of the entire cohort, respectively. At the time of diagnosis, there was no association between typical or atypical CLL immunophenotype and age, gender, race/ethnicity, WBC, hemoglobin, platelet count, or Rai stage (p>0.05 for all comparisons). With regard to biologic parameters, there was no association of CLL immunophenotype with the proportion of patients with CD38 ≥30%; however, the central tendency of CD38 was higher in the atypical group when analyzed as a continuous variable (median value 38% vs. 10%, p=0.04). There was no difference in the proportion of ZAP70 positive cases between typical and atypical groups. Analysis of interphase cytogenetics (FISH) data showed that a higher percentage of typical than atypical immunophenotypes had del 13q as a sole abnormality and a lower percentage had trisomy 12 (p=0.02). Despite the increased frequency of del 13q as a sole abnormality, lower frequency of trisomy 12, and lower expression of CD38, there was no difference between groups in time to first treatment (median 5.3 years for typical vs. 2.1 years for atypical; p= 0.11) or in overall survival (median 13.6 yrs64 years for typical vs. 18.0 years for atypical; p=0.11) (Figure 1). However, when patients with non-standard CLL immunophenotype were compared to those with a standard immunophenotype, a significantly shorter time to first treatment was observed (median 6.0 years for standard immunophenotype vs. 2.2 years for non-standard, p=0.03).Different immunophenotypic subtypes are commonly encountered in the care of patients with CLL. These groups have different biological characteristics, including differences in expression of CD38 and cytogenetic abnormalities. Exploratory analysis also showed differences in clinical outcomes based on immunophenotypic parameters. Validation studies are currently underway.No relevant conflicts of interest to declare.

Treatment decisions for early re-induction of patients with de novo acute myeloid leukemia (AML) based on sub-optimal cytoreduction as seen on day 14 bone marrow (BM) biopsy is laden with controversy. The aim of our review was to correlate results of the day 14 BM biopsy to overall outcomes of induction therapy.Between November 1995 and July 2008, medical records for patients treated for de novo AML were retrospectively reviewed for the purpose of evaluating treatment decisions and outcomes based on day 14 BM biopsies. Of all patients treated during that period, 100 patients were categorized as de novo AML, and were treated with standard induction chemotherapy. Seventy-four (n=74) of these patients had paired BM biopsy reports. Response to therapy was based purely on morphology noted by the pathologist on the day 14 BM in this analysis, with indeterminate response (IR) defined as a hypocellular marrow (HM) with moderate increase in blasts above 5% in which the reviewing pathologist could not rule out the possibility of persistent leukemia. Residual disease (RD) was defined by the reviewing pathologist as grossly elevated percentages of abnormal populations of persistent blasts by morphology alone definitively consistent with residual disease (i.e. no flow cytometric or molecular adjuncts). Otherwise, the patients were classified as appropriate response with a HM and less than 5% blasts with no evidence of residual leukemia.Day 14 BM biopsies of the 74 patients (median age = 42 years, range 18 to 77) undergoing standard induction chemotherapy revealed that 45 patients (61%) had a HM with less than 5% blasts. Eleven patient's (15%) BM biopsies were classified as IR. Eighteen patients (24%) had morphologically definitive RD. In all, 29 patients (39%) had a sub-optimal response (SOR) to induction chemotherapy (IR+RD=SOR). The 45 patients with HM and low blast percentage were observed until count recovery as is the usual practice. However, 16 of 29 patients with SOR received re-induction chemotherapy (1 IR and 15 RD) of which 10 patients attained a morphologic CR (9 RD and 1 IR). The 13 remaining patients (3 RD and 10 IR) were observed without any re-induction therapy, and re-evaluated with a follow-up BM biopsy between days 21 and 42 of initial induction. Interestingly, 11 of these 13 patients had a morphologic CR on follow-up biopsy, including 2 of 3 patients initially categorized as RD. Analysis of the paired results from nadir to recovery for the 58 observed patients in this cohort revealed a positive predictive value (PPV) and negative predictive value (NPV) for the day 14 BM biopsy review of 15% and 93% respectively.Our data suggests that those patients with an IR on day 14 may not necessarily require re-induction chemotherapy, and may actually benefit from careful observation by avoiding the risks of reinduction and prolonged cytopenias. Thus, while the day14 BM is an important tool for the evaluation of response in AML patients undergoing induction therapy, it is not always a reliable test for residual leukemic burden, as illustrated by its low PPV. As such, future treatment decisions should be weighted by, but not based solely on this parameter. Our future plans are to evaluate this question prospectively in a larger cohort of de novo AML patients receiving induction therapy with centralized pathologic review, and also to look at the effect of cytogenetic and molecular mutation analysis at day 14 on treatment decision making and outcomes.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Chronic myelogenous leukemia (CML) can progress from an indolent chronic phase to an aggressive blast crisis phase but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi-Numb signaling axis. Specifically we find that chronic phase is marked by high and blast crisis phase by low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced phase disease in vivo. As a possible explanation for the decreased levels of Numb in blast crisis phase, we show that NUP98-HOXA9, an oncogene associated with blast crisis CML, can trigger expression of Musashi 2 (Msi2) an RNA binding protein which in turn represses Numb. Importantly, loss of Msi2 significantly impairs the development and propagation of blast crisis CML in vitroand in vivo. Finally, we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi-Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for therapy of aggressive leukemias.

MicroRNAs are 18-22 nucleotide-long RNA molecules that regulate expression of genes. We and others have previously demonstrated a role for microRNAs in the pathogenesis of B cell malignancies. Computational predictions suggest that the human genome encodes several thousand microRNAs. Thus far, about 700 microRNAs have been discovered in humans, including over 200 new microRNAs in the past year alone. The ongoing discovery of microRNAs makes it difficult to comprehensively study their role in a disease group. The advent of high throughput sequencing allows the simultaneous identification of millions of transcripts, thereby providing a sensitivity that is several orders of magnitude higher than conventional methods. We hypothesized that high throughput sequencing would be an effective tool to comprehensively identify microRNAs in normal and malignant B cells.While there is an overlap between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) in morphology, immunophenotype and cytogenetics, distinguishing between BL and DLBCL is critical because there are important differences in their clinical management. We investigated whether microRNA expression could be used to reliably distinguish BL from DLBCL.We carefully chose 31 human samples to represent the spectrum of normal and malignant B cells including FACS-sorted naive, germinal center, memory, plasma cells, EBV transformed and activated B cells. Samples derived from B cell malignancies included B-lymphoblastic lymphoma, chronic lymphocytic leukemia (immunoglobulin gene mutated and unmutated), mantle cell lymphoma, marginal zone lymphomas, HIV-related lymphoma, BL, DLBCL (activated and germinal center type), primary mediastinal B cell lymphoma, Hodgkin lymphoma, and multiple myeloma.We applied massively parallel, high-throughput sequencing of the 18-22 nt RNAs from these cases and generated a total of 255,624,785 sequences (∼5 billion bases). Using a computational approach that we have previously validated with normal B cells, we identified the expression of 429 known microRNAs in normal and malignant B cells, a number that is over three times higher than previously recognized in any tissue type. We also identified the expression of 302 novel microRNAs in normal and malignant B cells. The vast majority of these microRNAs were highly conserved in multiple species.As a proof of principle, we generated a custom microarray that included all the known human, and viral microRNAs, as well as 302 novel microRNAs identified by sequencing, and applied it to the clinically important distinction of BL from DLBCL. Biopsy samples were collected from 104 patients (BL, N=25, DLBCL, N=79) treated at 9 institutions that comprise an international consortium. All cases were reviewed for pathology diagnosis and profiled for microRNA expression. We constructed a Bayesian predictor to distinguish BL from DLBCL based on the microRNA expression. The predictor performance was tested using leave-one-out cross-validation. We also applied gene expression profiling to the cases of DLBCL to identify the molecular subsets of DLBCL: activated B cell like and germinal center B cell like DLBCL. The microRNA profiles of these cases were equally efficacious in distinguishing the DLBCL subsets.The predictor constructed based on microRNA expression was over 90% accurate in distinguishing BL from DLBCL, using pathology diagnosis as the gold standard. Further, microRNA-based predictor was also over 90% accurate in the distinction of the molecular subsets of DLBCL, compared to the gold standard of gene expression-profiling.As additional validation, we performed in situ hybridization of selected microRNAs to directly visualize their expression using methods that are easily accessible in conventional pathology laboratories. We found excellent concordance between the expression results derived from microarrays and in situ hybridization suggesting a ready path to clinical translation.Our study represents the first comprehensive delineation of microRNA expression in B cell malignancies using high throughput sequencing. Our data suggest that microRNAs are a promising marker for the distinction of aggressive lymphomas.No relevant conflicts of interest to declare.

Background: Mature B cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells are common and constitute the majority of leukemias and lymphomas. MicroRNAs are known to play a role in oncogenesis, lineage-selection, and immune cell function, including early B cell differentiation. However, the full extent and function of microRNA expression during mature B cell differentiation and in B cell malignancies are not known. Methods: From normal young patients undergoing tonsillectomies, we sorted the mature B cell subsets (naive, germinal center, memory and plasma) using FACS, based on their expression of CD19, CD38, IgD and CD27. These sorted B cells were profiled for microRNA expression using a highly sensitive multiplexed real-time PCR assay, as well as for gene expression at the whole genome level using Affymetrix U133plus microarrays. miRNA targets can be predicted based on seed sequence matching of their 2–8 nt to the 3′UTR of gene transcripts. For each B cell stage, we experimentally validated microRNA regulation of predicted target genes of interest, LMO2, MYBL1 and PRDM1, by microRNA over-expression experiments and luciferase assays. Results: We found that microRNAs have a characteristic expression pattern that defines each mature B cell stage. Examination of both microRNA and mRNA expression showed that in each B cell population, the target genes predicted based on seed matching were expressed at lower levels, results that were highly significant (P<1E-10). We found that differential microRNA expression is important at every B cell stage transition, and differentially expressed microRNAs frequently target differentially expressed transcription factors. In the naive to germinal center B cell and germinal center B cell to memory cell transitions, we found that miR-223 had an inverse relationship with its predicted target genes LMO2 and MYBL1. To test this relationship predicted based on seed pairing, in Germinal Center-derived BJAB cells, we over-expressed miR-223 by introducing its precursor, and saw a subsequent knockdown of LMO2 and MYBL1 at both the mRNA and protein level. We confirmed seed sequence specificity by comparing miR-223 knockdown of luciferase reporter activity on the LMO2 3′UTR compared to its seed sequence mutant. We further found that miR-9 and miR-30 family members directly regulate PRDM1 (blimp1), a master regulator of the GC to PC transition. In U266 cells (PC-derived), introduction of miR-9 and miR-30 family precursor resulted in decreased PRDM1 protein expression, although transcript levels were not changed, consistent with previous evidence that miRNA can regulate at the post-transcriptional steps. We further profiled over 50 tumors derived from various B cell malignancies (small lymphocytic lymphoma, Burkitt lymphoma, and the molecular subsets of diffuse large B cell lymphoma) and found that these malignancies maintain the expression patterns of their respective lineage; microRNA expression profiles of normal B cells could correctly classify the lineage of these tumors in over 80% of the cases. In contrast to other malignancies, common lymphomas do not down-regulate microRNAs, but rather maintain the microRNA-expression patterns of their normal B-cell counterparts. Conclusion: Through concomitant microRNA and mRNA-profiling, we demonstrate a regulatory role for microRNAs at every stage in mature B-cell differentiation. Further, we have experimentally identified a direct role for the microRNA-regulation of key transcription factors in B-cell differentiation: LMO2, MYBL1 and PRDM1 (Blimp1). Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B-cells.

Background: Burkitt lymphoma (BL) is a highly aggressive lymphoma that can be cured in up to 80% of patients when treated with intensive multi-agent chemotherapy. The distinction between BL and diffuse large B-cell lymphoma (DLBCL) is critical because there are important differences in their clinical management. However, the distinction can be difficult because of an overlap between DLBCL and BL in morphology, immunophenotype and cytogenetics. Previous work has shown that gene expression profiling can distinguish these entities with a high degree of certainty. Our previous work has demonstrated that microRNAs play a direct role in regulating key transcription factors in normal and malignant B cells. We investigated whether microRNA expression could be used to reliably distinguish BL from DLBCL. Methods: Biopsy samples were collected from 104 patients with a diagnosis of either sporadic BL (N=25) or DLBCL (N=79). All cases were reviewed for pathology diagnosis and profiled for microRNA expression using microarrays. Using the 30 most highly differentially expressed microRNAs with the highest t-statistic, we applied singular value decomposition to identify the 10 most predictive microRNAs. Using those 10 microRNAs, we constructed a Bayesian predictor to distinguish BL from DLBCL. The predictor performance was tested using leave-one-out cross-validation. We further applied gene expression profiling to 52 cases of DLBCL to identify the molecular subsets of DLBCL: activated B cell type and germinal center B cell type DLBCL. We constructed a Bayesian predictor to distinguish these molecular subsets based upon their gene expression. A separate predictor was created from the microRNA profiles of these DLBCL subsets and tested using leave-one-out cross-validation. In order to understand the effects of lineage-specific microRNAs in B cell lymphomas, we applied FACS-sorting to isolate mature B cell subsets including naïve B cells, germinal center B cells, plasma cells and memory cells. We compared the microRNAs involved in germinal center differentiation to those expressed highly in Burkitt lymphoma. Results: The predictor constructed based on microRNA expression was 90% accurate in distinguishing Burkitt lymphoma from DLBCL, using pathology diagnosis as the standard. The microRNA-based predictor was also over 90% accurate in the distinction of the molecular subsets of DLBCL, compared to the gold standard of gene expression-profiling. Further, we found that the Burkitt lymphoma cases consistently expressed microRNAs related to normal germinal center B cell differentiation, suggesting that they also maintain expression of B cell stage-specific microRNAs. Conclusion: This study demonstrates that the microRNA expression profiles can be used to accurately distinguish Burkitt lymphoma from DLBCL. The ability of the predictor to identify the molecular subsets of patients with DLBCL and those with BL suggests that it will be useful in the diagnosis and management of patients with Burkitt lymphoma. Further, the patterns of microRNA expression and their target genes suggests a role for microRNAs in the pathophysiology of these tumors.

To minimize toxicity and monitor immune recovery without interference of long term use of immunosuppressive agents, we have investigated T-cell depleted, nonmyeloablative allogeneic therapy using matched family member donors. Methods: Seventy five patients who were not candidates for ablative therapy due to age or comorbid diseases received fludarabine 30mg/sq m and cyclophosphamide 500mg/sq m IV qd x 4 with alemtuzumab 20mg IV qd x 5 followed by stem cell infusion. No other post transplant immunomodulation was provided. Results: Patient diagnoses included lymphoma/myeloma (20), leukemias/MDS (30), myelofibrosis/aplasia (7) and metastatic solid tumours (18). The median age was 50 (range 18–17) with a median follow up of 18 months. The median CD34+ cell dose collected was 13.4 x 106/kg. Engraftment occurred in 100% of patients with a median of 97% donor cells responsible for patient hematopoiesis by 3 months. Forty six also had a DLI (range 106–108 CD3+ cells/kg). Overall, Grade III–IV acute GVHD occurred in only 5/75 (7%) and 18 (29%) had grade II–IV. Four patients developed chronic GVHD. The transplant regimen was well tolerated with 4% 100 day treatment related mortality. In those with hematologic malignancies, only 7% started in remission, though 45 (60%) attained a CR. The most common cause of death in this group was progressive disease (28%), followed by infections (5%), and GVHD (5%). A subgroup of 21 of these older, more infirm patients who had high risk AML in 1–2nd CR or PR or ≥2 chronic stable phase CML, partially responding lymphoma, or severe myelofibrosis have been followed for at least 1 year. At 1 year, 13/21 (62%) remain alive and in continuing remission. Phenotypic analysis of lymphocyte subsets (measured by flow) revealed recovery at 6 months. Figure Figure T cell VBeta family recovery (spectratype analysis using PCR) revealed robust recovery by 6 months as well (first figure pre and second is 6 months post transplant), despite T depletion. Figure Figure TRECs analysis reveals little, if any, recovery in these patients for at least 12–18 months. Conclusions: Nonablative allogeneic transplantation using alemtuzumab for T cell depletion results in reliable engraftment with little acute GVHD or TRM. Immune recovery assays reveal that despite T cell purging, T cell subset and VBeta families have significant recovery by 6 months and TRECS results show the recovery is primarily from peripheral expansion of transplanted donor cells, not education of new T cells. These data possibly reflect the advantage of low dose donor lymphocyte boosts without planned long term use of immunosuppressive agents. The future challenge will to be to develop strategies to further improve immune recovery in this setting.

We have investigated the use of partially matched family member donors in combination with alemtuzumab and a non-myeloablatvie preparative regimen in an effort to expand the population of patients who may benefit from allogeneic immunotherapy. Methods: Sixty three patients received fludarabine 30mg/sq m, cyclophosphamide 500mg/sq m IV qd x 4 days and alemtuzumab 20mg IV qd x 5 days followed by infusion of partially matched family member donor stem cells. Mycophenolate alone or in combination with cyclosporine was given for 6–8 weeks following transplantation. Results: Patient diagnoses included lymphoma/myeloma n= 16, leukemias/MDS n=33, myelofibrosis/aplasia n=3 and metastatic solid tumours n=11. The median age was 48 (range 17–66) with a median follow up of 28 months. The donor matching was 5/6 in 8, 4/6 in 22, and 3/6 in 33 cases. The median CD34+ cell dose collected was 13.47 x 106/kg (SD= 5.10) with nearly 2 logs of T cells depletion. Engraftment occurred in 92% of patients with a median of 87% donor cells responsible for patient hematopoiesis by 6 weeks following therapy. Twenty patients also received a DLI (range 105–107 CD3+ cells/kg). Grade III–IV acute GVHD occurred in only 8/63 (13%) with 14 (22%) experiencing grade II–IV. Nine patients developed chronic GVHD or failure to thrive (15%). The transplant regimen was well tolerated with 10% TRM. In those with hematologic malignancies, only 6 (12%) started in remission, though 38 (73%) attained a CR. The most common cause of death in this group was progressive disease (33%), followed by infections (19%), and GVHD (12%). Overall 1 year survival for this high risk group of 63 patients was 26%. A subgroup had aplastic anemia/myelofibrosis or leukemia/lymphoma in second or greater CR, PR and had a median disease free and overall survival of 3 years. Figure Figure Phenotypic analysis of lymphocyte subsets (measured by flow), and T cell VBeta family recovery (spectratype analysis using PCR) revealed robust recovery by 6 months in those without GVHD (dark bars) compared to those with GVHD (gray bars), despite T depletion. Figure Figure Further, the measured T cell recovery is from peripheral expansion of residual transplanted T cells as newly educated T cells are not measurable (using TRECs analysis) for at least 1 year, if at all. Conclusions: The results demonstrate reasonable tolerance and reliable engraftment using T cell depleted, partially matched family member donors in a non-myeloablative setting with low treatment related mortality and severe GVHD. The future challenge will to be to develop strategies to improve immune recovery to enhance immune-mediated graft-versus-tumour effect and to minimize the risk of infections.

Posttransplant lymphoproliferative disorders (PTLDs) represent a spectrum ranging from Epstein-Barr virus (EBV)-driven polyclonal lymphoid proliferations to EBV+ or EBV– malignant lymphomas. Central nervous system (CNS) PTLDs have not been characterized fully. We reviewed the clinical, radiologic, and pathologic features of 12 primary CNS PTLDs to define them more precisely. Patients included 10 males and 2 females (median age, 43.4 years) who were recipients of kidney (n = 5), liver (n = 2), heart (n = 2), peripheral blood stem cells (n = 2), or bone marrow (n = 1). All received immunosuppressive therapy. CNS symptoms developed 3 to 131 months (mean, 31 months) after transplantation. By neuroimaging, most showed multiple (3 to 9) intra-axial, contrast-enhancing lesions. Histologic sections showed marked expansion of perivascular spaces by large, cytologically malignant lymphoid cells that were CD45+, CD20+, EBV+ and showed light chain restriction or immunoglobulin gene rearrangement. In distinction to PTLDs in other organ systems, CNS PTLDs were uniformly high-grade lymphomas that fulfilled the World Health Organization criteria for monomorphic PTLDs. Extremely short survival periods were noted for each CNS PTLD that followed peripheral blood stem cell transplantation. Survival of others with CNS PTLD varied; some lived more than 2 years.