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Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and platelets. The latter is important since this protein may work as negative regulator of granule release. Thus, we sought to begin to understand the structure of this protein in platelets. Flow cytometric analysis of this protein using region-specific (N-terminus, central region and C-terminus) monoclonal antibodies was performed. Antibody to the central region gave the strongest shift among all three antibodies, with the C-terminus having intermediate shift and N-terminus minimal shift. Western blotting using the same antibodies showed similar binding of all antibodies to α-synuclein. These results suggest a similar arrangement of this protein in platelets as seen in neurons. Future studies ought to look at the role that each protein region plays in platelets.

Objectives To examine flow cytometric (FCM) findings in clonal cytopenia of undetermined significance (CCUS) in relation to variant allele fraction (VAF) and mutation risk. Methods Nine FCM parameters, including 5 FCM metrics (Meyerson-Alayed scoring scheme [MASS] parameters) we previously used to identify myelodysplastic syndromes (MDS), were compared among 96 CCUS samples, 100 low-grade MDS samples and 100 samples from patients without somatic alterations (controls). Results FCM findings did not differ between CCUS samples with less than 20% VAF and controls. CCUS samples with more than 20% VAF (CCUS >20% VAF) demonstrated more than 1 abnormal FCM parameter at a frequency between MDS and controls. Abnormalities in CCUS with high-risk alterations (CCUS(hi)) were similar to MDS, with no statistical difference in the percentage of cases with more than 1 FCM abnormality or a positive MASS score. The positive predictive value (PPV) for clinically significant myeloid processes; MDS, CCUS(hi), and CCUS >20% VAF compared with other CCUS samples and controls was 94.8%, with 96.5% specificity and 61% sensitivity using a modified MASS score. A subset of MDS (43%) was distinguished from CCUS(hi) and CCUS >20% VAF using 3 parameters, with a 93.5% PPV and 83.3% specificity. Conclusions FCM abnormalities can distinguish high-risk CCUS based on VAF or alteration type from low-risk CCUS and MDS in many cases. The findings are of potential utility in the evaluation of patients with cytopenias.

Objectives We investigated the usefulness of a custom-designed 31-gene next-generation sequencing (NGS) panel implemented on a routine basis for the evaluation of low-grade lymphoproliferative disorders (LPDs). Methods In total, 147 blood, bone marrow, and tissue specimens were sequenced, including 81% B-cell, 15% T-cell, and 3% natural killer (NK)–cell neoplasms. Results Of the cases, 92 (63%) of 147 displayed at least one pathogenic variant while 41 (28%) of 147 had two or more. Low mutation rates were noted in monoclonal B-cell lymphocytoses and samples with small T- and NK-cell clones of uncertain significance. Pathogenic molecular variants were described in specific disorders and classified according to their diagnostic, prognostic, and potential therapeutic value. Diagnostically, in addition to confirming the diagnosis of 15 of 15 lymphoplasmacytic lymphomas, 10 of 12 T large granular lymphocytic leukemias, and 2 of 2 hairy cell leukemias (HCLs), the panel helped resolve the diagnosis of 10 (62.5%) of 16 challenging cases lacking a specified diagnosis based on standard morphology, phenotype, and genetic analysis. Conclusions Overall, implementation of this targeted lymphoid NGS panel as part of regular hematopathology practice was found to be a beneficial adjunct in the evaluation of low-grade LPDs.

Objectives Previously we demonstrated that a decreased percentage of CD177-positive granulocytes detected by flow cytometry (FCM) was associated with myelodysplastic syndrome (MDS). Here we expand on those findings to more rigorously evaluate the utility of CD177 for the detection of MDS. Methods Two hundred patient samples (100 MDS and 100 controls) were evaluated for granulocyte expression of CD177 and 11 other flow cytometric parameters known to be associated with MDS. Results We show that CD177, as a single analyte, is highly correlated with MDS with a receiver operating characteristic area under curve value of 0.8. CD177 expression below 30% demonstrated a sensitivity of 51% and a specificity of 94% for detecting MDS with a positive predictive value of 89.5%. In multivariate analysis of 12 MDS-associated FCM metrics, CD177 and the Ogata parameters were significant indicators of MDS, and CD177 increased sensitivity of the Ogata score by 16% (63%-79%) for predicting MDS. Finally, diagnostic criteria incorporating these parameters with a 1% blast cutoff level and CD177 resulted in a sensitivity of 90% and specificity of 91% for detecting MDS. Conclusions The findings indicate CD177 is a useful FCM marker for MDS.

Allogenic hematopoietic stem cell transplant (HCT) is a curative therapy for acute myeloid leukemia (AML). Relapse after HCT is the most common cause of treatment failure and is associated with poor prognosis. Early identification of which patients are at elevated risk of relapse may justify use of aggressive post-HCT treatment options, potentially preventing relapse and treatment failure. In this study, our goal was to predict relapse after HCT in AML patients using quantitative features extracted from digitized Wright-Giemsa stained posttransplant aspirate smears. We collected 39 aspirate specimens from a cohort of 39 AML patients after HCT, of which 25 experienced relapse, while 14 did not. Our approach comprised the following main steps. First, a deep learning model was developed to segment myeloblasts, a cell type in bone marrow that accumulates and characterizes AML. A total of 161 texture and shape descriptors were then extracted from these segmented myeloblasts. The top eight predictive features were identified using a Wilcoxon rank sum test over 100 iterations of 3-fold cross validation. A model was subsequently built employing these features and yielded an average area under the receiver operating characteristic curve of 0.80±0.05 in cross validation. The top eight features include four Haralick texture features and four fractal dimension features. The texture features appear to characterize chromatin patterns in myeloblasts while the fractal features quantify morphological irregularity and complexity of myeloblasts, in alignment with findings previously reported for AML patients post-treatment.

A decreased percentage of CD177pos neutrophils is frequently present in MDS and AML and is a useful flow cytometry (FCM) marker for the identification of MDS. The underlying mechanism leading to the low percentage of CD177pos neutrophils in MDS has not been explained. The aim of this study was to identify whether specific somatic mutations in myeloid neoplasms are associated with the low percentage of CD177pos neutrophils. 507 myeloid neoplasms with one or more pathogenic molecular abnormality identified by NGS and in which CD177 expression was assessed were evaluated. Correlation with CD177 expression was determined for 39 variables (including genes mutated, diagnostic groups and gender) using a 40 % cutoff level for low CD177 expression. In multivariate analysis mutations involving NPM1 (OD 0.26), RUNX1 (OD 0.39), TET2 (OD 0.58), and U2AF1 S34F (OD 0.25) were associated with low percentage of CD177pos neutrophils when all cases were evaluated. JAK2 (OD 2.5) alteration was associated with increased percentage of CD177pos neutrophils. Differences were noted between diagnostic subgroups with no single mutation associated with decreased CD177pos neutrophils in MDS and CCUS. The findings demonstrate an association between the percentage of CD177pos neutrophils and somatically acquired mutations involving several genes.

We describe a case of Omenn syndrome displaying exudative erythroderma and other characteristic features, including alopecia, absent B and naïve T cells, hyper immunoglobulin E levels, and eosinophilia. A pathogenic recombination-activating RAG1 homozygous genetic mutation confirmed the diagnosis. She required frequent antibiotics at both treatment and prophylactic doses, which alone did not control her erythroderma, but her high risk of infection precluded the use of systemic agents such as cyclosporine, which would further suppress her already severely compromised immune system. Thrice-weekly topical dilute hypochlorite compresses, combined with skin acidification with a low pH emollient, were initiated to control inflammation and for cutaneous bacterial prophylaxis. She demonstrated a marked improvement in her erythroderma within days after treatment initiation. Further improvement continued with the addition of systemic corticosteroids, with resolution of erythroderma after her first dose. This case reveals for the first time that dilute topical hypochlorite and skin pH restoration holds promise to control severe dermatitis associated with immunodeficiency and inflammatory syndromes with minimal side effects.

Background Myelodysplastic syndrome (MDS) represents a heterogenous spectrum of pre-leukemic conditions with a wide range of outcomes. Higher risk MDS as classified by the revised international prognostic scoring system (IPSS-R) score is associated with poor overall survival and up to 30% of patients progressing to acute myeloid leukemia. Hypomethylating agents (HMA) such as azacitadine can improve cytopenias and delay progression to leukemia in about 30% of patients, but these agents may take months to promote response and initially exacerbate cytopenias. Thus treatment related biomarkers that help predict eventual hematologic response are of interest. CD177 is expressed in neutrophils and plays a role in cellular adhesion. In healthy cells, it exhibits bimodal expression by flow cytometry that is stable over time within an individual. The percentage of CD177 positive neutrophils is often decreased in hematopoietic malignancies and myelodysplastic syndromes. Our group has demonstrated that CD177 has diagnostic utility in the identification of myelodysplastic syndromes. As transcription of CD177 is regulated by CpG methylation of its promotor, we hypothesized that treatment with HMAs may improve CD177 expression in clinical responders and potentially guide continuation of HMA therapy. Methods To interrogate the above, we performed a retrospective review of patients with a diagnosed with MDS or MDS/MPN overlap syndromes who received disease modifying therapy with HMA at our institution from 2015 to 2018. Inclusion criteria required documentation of serial bone marrow biopsies with aspirate flow cytometry analysis. CD177 positivity was determined by increase in mean florescence intensity compared to isotype controls. Data was analyzed with using cox multivariate and univariate analysis correlating to treatment response. Results Of the 237 patients, 27 patients met the above criteria. Their average age was 62 (21 to 77) at time of diagnosis with 20 men and 7 women. They exhibited a range of R-IPSS risk stratification with four very high risk, eight high risk, six intermediate risk, and four low risk. Five cases were MDS/MPN overlap. Patients received on average 10 months of HMA treatment with a wide range from 1 month to 42 months of treatment. Median baseline CD177 positivity was 16, 31, 28.5, and 72 percent respectively amongst R-IPSS groups. Of the 27 patients analyzed with repeat bone marrow biopsies, eight patients exhibited 20% or greater increase in CD177(+) neutrophils, ten exhibited a decrease in CD177(+) neutrophils of 20% or greater, and nine exhibited less than a 20% change in CD177(+) neutrophils. with similar distribution of R-IPSS risk stratification amongst groups. (CD177-decreased: 1 very high, 3 high, 1 intermediate, 2 low risk, CD177-stable 1 very high, 2 high, 2 intermediate, and 1 low, Improved-CD177 1 very high, 4 high, 2 intermediate and 1 low). Cox proportional hazard analysis suggests that patients exhibiting a decrease or stable CD177 were less likely to exhibit a treatment response with results trending to significance (OR= 0.13 p=0.099). Conclusion Our initial data suggests that change in CD177 may help predict HMA treatment response. More uniform prospective analysis is indicated to compared CD177 changes over initial treatment. Furthermore, CD177 in peripheral blood and bone marrow samples correlate excellently (R 2=0.95). Prospective studies are underway to correlate CD177 change and initial treatment response utilizing flow analysis of pre-treatment CBCs. Disclosures No relevant conflicts of interest to declare.

Introduction Clonal cytopenia of undetermined significance (CCUS) represents a clonal group of cells with the presence of a hematologic malignancy-associated somatic mutation without meeting the morphologic criteria for MDS. CCUS patients have a variable risk of progression to MDS. The WHO requires at least 10% of a cell lineage have dysplastic morphology for the diagnosis of MDS, but CCUS patients have been described with no evidence of dysplasia (CCUS-ND) or with low level dysplasia (CCUS-D). We previously identified CCUS-D as biologically distinct with higher risk MDS mutations. We hypothesized that CCUS-D would be independently associated with the risk of progressive cytopenia over time and thus may have clinical relevance in the setting of CCUS. Methods Cases of CCUS were identified on review of bone marrow biopsies completed for the diagnosis of cytopenia from 8/2016 to 9/2018 with results of a custom 31-gene NGS hotspot panel to detect mutations in the following genes: ASXL1, BRAF, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, JAK3, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, and ZRSR2. The classification of CCUS-D or CCUS-ND required two or more board-certified hematopathologists to score the degree of morphologic dysplasia on bone marrow aspirates with at least one of the following morphologic criteria: >2%-50% erythroid cells with megaloblastoid changes and/or basophilic stippling, >5%- Statistical differences of baseline characteristics were evaluated by T-test for continuous variables, and Chi-square / Fisher's exact test were used for association between categorical variables. The repeated measures over time for various outcomes (WBC, ANC, hemoglobin, platelet) were visualized using scatter plot supervised with lowess smoother. The effects of dysplasia on various longitudinal outcomes were estimated using linear mixed-effect models. Results We identified 54 CCUS patients on review of 200 marrows completed for undiagnosed cytopenia. Twenty-six patients were identified with CCUS-D, and 28 patients with CCUS-ND. Baseline clinical characteristics including age, gender, race, smoking status, or presence of infection at time of initial diagnosis were similar. Hematology laboratory parameters including White Blood Cell Count (WBC), Absolute Neutrophil Count (ANC), Hemoglobin (Hgb), and Platelet Count (Plts) did not differ. (Table 1). Patients with CCUS-D had an average of 1.62 mutated genes detected per patient with the most common being TET2 (26.19%), SRSF2 (14.29%), and DNMT3A (14.29%). Patients with CCUS-ND had an average of 1.41 mutations per patient with the most common being TET2 (31.57%) and DNMT3A (31.57%). Patients were followed for a median of 32.2 months and a range from 1.0 to 48.8 months. On long term follow up, WBC, ANC, Hgb and plts trended lower in patients with CCUS-D compared to CCUS-ND, but the difference was marginally statistically significant (p = 0.09, 0.16, 0.13, 0.64 respectively). We also evaluated the rate of change in hematology parameters and found no significant difference in change rate of WBC, ANC, and hgb (p = 0.25, 0.13, and 0.28, respectively). There was a significant difference in the change rate of platelet counts between the two groups (p = 0.026). In particular, patients with CCUS-D had decreasing platelet counts at a rate of -0.04119 10E9/L/day and patients with CCUS-ND had increasing platelet counts at a rate of 0.01075 10E9/L/day. Two patients in the CCUS-D cohort progressed to MDS at an average of 938.5 days. One patient with CCUS-ND progressed to Large Granular Lymphocytic Leukemia at 805 days. Conclusions Low level (< 10%) dysplasia in cell lineages in CCUS is associated with a higher rate of decline in platelet counts but was not independently associated with increased risk of progressive neutropenia, leukopenia, or anemia. Longer follow up may be required to further understand the clinical significance of low level dysplasia in CCUS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Nodal marginal zone lymphoma (NMZL) is a rare non-Hodgkin lymphoma that arises from mature B-cells. We delineate outcomes, prognostic factors and treatment trends among a large cohort of patients with NMZL in the rituximab era. We identified 56 such patients treated at our institutions. The majority presented with advanced stage disease (78·6%). Over a median follow-up of 38·2 months, median progression-free survival (PFS) was 42·4 months and median overall survival (OS) was not reached. Kaplan-Meier estimates of OS at 120 months after diagnosis was 71·9%. High-risk follicular lymphoma international prognostic index (FLIPI) was associated with inferior PFS. Age >60 years and elevated serum lactate dehydrogenase (LDH) were associated with inferior OS. Transformation to diffuse large B-cell lymphoma occurred in 7 patients, 6 of who presented with advanced disease. OS was comparable to our previously reported extranodal MZL cohort. FLIPI score predicted for inferior PFS and OS when both cohorts were analysed together (n = 267). In summary, outcomes in NMZL are favourable with a large majority of patients surviving at 120 months. High risk FLIPI, age >60 years, and elevated serum LDH were associated with inferior outcomes.

Extranodal marginal zone lymphoma (EMZL) is a B-cell lymphoma arising from mucosa-associated lymphoid tissue (MALT). The disease characteristics, clinical course and treatment vary considerably based on site of involvement. Because long-term outcome data for EMZL are limited, we sought to describe the clinical details of a large number of patients with EMZL evaluated at the Case Comprehensive Cancer Center over a 12-year period to identify prognostic markers including the impact of site of involvement. We identified 211 cases of EMZL involving the stomach (30%), ocular adnexa (19%), lungs (16%) and intestines (9%). Initial treatment included antibiotics (18%), radiation (21%), rituximab (20%), chemotherapy (3%), rituximab + chemotherapy (7%), surgery (17%) or observation (8%). After a median follow-up of 44·3 months (range 2·2-214·9), median progression-free survival (PFS) was 68·2 months (95% confidence interval [CI] 54·5-111·3) and median overall survival (OS) has not been reached. Age >60 years, elevated lactate dehydrogenase level (LDH), ≥4 lymph node groups involvement, and high follicular lymphoma international prognostic index (FLIPI) were associated with inferior PFS/OS. In summary, patients with EMZL have excellent prognosis with median OS in excess of 10 years. Age, elevated LDH, advanced disease, and high FLIPI score are associated with worse outcomes.

Background & Aim Background In allogeneic stem cell transplantation (HSCT), relapse of hematologic malignancies is a leading cause of post-transplant morbidity. In the early post-transplant period, when immune reconstitution is pending, residual leukemic clones can proliferate, resulting in disease relapse with bleak prognosis and limited treatment options. Donor lymphocyte infusions (DLI) are often used as treatment of post-transplant leukemia relapse but this practice is limited by graft versus host disease (GVHD) as a complication. Adoptive transfer of selected lymphocyte subsets that effect tumor cytotoxicity without inducing GVHD, such as natural killer (NK) cells and TCRγδ T cells, may provide early donor-derived surveillance for residual or early relapse disease after HSCT. With this background, a Phase I clinical trial (CASE1Z19) was designed to explore the prophylactic administration of donor lymphocytes depleted of TCR-αβ T cells and CD19+ B cells early in the post-transplant course. A maximum threshold of Methods, Results & Conclusion Methods Three qualification runs were conducted using the Miltenyi CliniMACS Plus system to deplete TCR-αβ T and CD19+ B cells from non-mobilized, peripheral blood mononuclear cells (PBMC) in leukapheresis products sourced by a commercial vendor. Each product was analyzed for lymphocyte subset composition and stability post-processing. Results The mean starting total white blood cell count was 6.4 × 109 cells from which TCR-αβ T cells and CD19+ B cells were maximally depleted by >99.8% with mean Log10 reductions of 3.10 and 3.06 respectively (Table1). The TCR-αβ T cell content in products generated from each run were 0.2-0.5 × 105/kg, based on an average 60kg adult. The yield of NK cells and TCRγδ T cells from each manufacturing ranged from 50-97% and 86-100% respectively. All products were stable post-processing, at room temperature for up to 6 hours, with a mean cell viability of 95.1%. Conclusion A donor lymphocyte product sufficiently purified of TCRαβ T and CD19+ B cells with subsequent enrichment of NK cells and TCR-γδ T cells can consistently be generated for prophylactic adoptive transfer in the post-transplant setting. Additional analysis characterizing NK and TCR-γδ T cell subsets for function and activation is underway.

Analysis of the bone marrow remains a basic tool in the evaluation of patients affected by hematopoietic disorders. This chapter reviews the normal morphologic features of the marrow as well as outlining practical aspects in the evaluation of the marrow aspirate smear and core biopsy. We further review the bone marrow aspirate, biopsy, and collection for therapeutic intent procedures. We present guidelines for submitting marrow samples for research. Ancillary diagnostic tests such as flow cytometry, immunohistochemistry, and cytogenetic and molecular studies important for defining the nature of marrow disorders briefly are mentioned. The chapter will give the reader a fundamental understanding of the bone marrow as an organ involved in hematopoiesis and disease processes.

Flow cytometry is a technique of cell analysis commonly used in laboratory hematology and immunology. The basic operational principles of a flow cytometer are reviewed, and its uses in diagnostic hematology and immunology are discussed.

Induction chemotherapy with cytarabine and an anthracycline (7+3) remains the standard of care for acute myeloid leukemia (AML).We retrospectively analyzed 183 newly diagnosed AML patients to compare the utility of rapid peripheral blast clearance (PBC), day of peripheral blast disappearance, residual blasts, and cellularity at day 14 bone marrow biopsy (D14BM) in predicting clinical response to 7+3 induction, overall survival (OS), and relapse-free survival (RFS).In multivariable logistic regression analysis, day 2 PBC85% [P = .0016] was the only predictor of remission status, with sensitivity and specificity of 75%. Peripheral blast disappearance within 5 days after induction and 10% cellularity in D14BM predicted superior OS and RFS in multivariate analysis. Median follow-up of patients was 28 months since diagnosis. Two-year OS and RFS for patients with ≤ 10% versus10% cellularity at D14BM was 60.6% [95% confidence interval (CI), 50.8%-72.2%] versus 32.5% [95% CI, 23.0%-45.8%], and 51.9% [95% CI, 41.9%-64.3%] versus 28.8% [95% CI, 19.1%-43.4%], respectively [P = .0003 for OS and .002 for RFS].Rapid PBC after 7+3 induction showed a significant improvement in specificity compared with D14BM, with similar sensitivity. Neither of these methods were reliably specific tools for the decision of early reinduction, despite their prognostic value. Our findings indicate that morphological cellularity in D14BM is an independent prognostic factor for OS and RFS, regardless of blast percentage, and that ≤ 10% cellularity defines D14BM hypoplasia.

Background Neoplasms derived from plasmacytoid dendritic cells (PDCs) are currently divided into two broad categories: mature PDC proliferations associated with myeloid neoplasms (MPDMN) and blastic plasmacytoid dendritic cell neoplasm (BPDCN); only BPDCN is recognized in the WHO 2016 classification of hematopoietic neoplasms. We present seven patients with high grade myeloid neoplasms (MNs), mostly acute leukemias, having a spectrum of PDC differentiation and not fitting with MPDMN or BPDCN. Methods We analyzed seven MN cases having increased myeloblasts and prominent CD56-negative PDC proliferations comprising 5-26% of bone marrow or blood cellularity as measured by flow cytometry. The cases included five acute myeloid leukemia (three FAB M4 subtype, two unclassified), one mixed phenotype acute leukemia, and one case of unclassified MN. Results Six cases demonstrated immunophenotypic evidence of PDC differentiation from leukemic blasts, based on variable expression of CD34, CD45, CD123, and CD304 by the leukemic cells. Four cases had circulating PDC populations in blood. None of the cases met clinical or pathologic criteria for BPDCN. Morphologic review was available for four acute leukemia cases and demonstrated either nodular or interstitial infiltrates of PDCs. All cases had an aggressive clinical course, and three cases had FLT3 ITD mutation. Conclusions These cases demonstrate that high grade MNs, in particular AML, can exhibit PDC differentiation, with or without monocytic differentiation, in a manner distinct from MPDMN or BPDCN. The existence of MNs with immature PDC proliferations suggests that there is a broader spectrum of PDC-associated neoplasms than currently recognized. © 2019 International Clinical Cytometry Society.

Fanconi anemia (FA) is an autosomal recessive, progressive bone marrow failure disorder characterized by congenital defects and marked cancer predisposition. Hematopoietic stem cell transplant is the therapy of choice for FA patients with progressive pancytopenia. These patients receive multiple transfusions for cytopenias. Oxymetholone has been used with variable success to improve cytopenias. Eltrombopag has been shown to induce bilineage or trilineage hematopoiesis in aplastic anemia and patients with myelodysplastic marrow. We report a case of FA where eltrombopag in conjunction with oxymetholone induced trilineage hematopoiesis and eliminated transfusion requirement before transplant, thereby enhancing favorable outcome after hematopoietic stem cell transplant.

Splenic marginal zone lymphoma (SMZL) is a rare non-Hodgkin lymphoma. We sought to identify prognostic factors and define outcomes in a cohort of 64 patients with SMZL who were treated at two large academic medical centers in North America in the rituximab era.Over a median follow-up of 37.8 (range 6-167.1) months, Kaplan-Meier estimate of median OS was 156.3 months and median PFS was 52.9 months. On univariate analysis, baseline hemoglobin12 g/dl was associated with inferior OS (p = 0.045). High-risk FLIPI score was associated with inferior PFS when compared with intermediate/low risk (p = 0.05) and marginally significant with regard to OS (p = 0.056). Splenectomy was not predictive of OS or PFS (p = 0.563 and 0.937, respectively). Transformation to diffuse large B-cell lymphoma occurred in four (6.3%) patients during the observation period. OS was comparable to contemporaneous cohorts of patients with extranodal and nodal marginal lymphomas and FLIPI score was highly predictive for inferior PFS and OS when all three cohorts were analyzed together.Outcomes of SMZL, in our series, were excellent, with a median OS of13 years. Low hemoglobin and high-risk FLIPI were associated with inferior outcomes.

A 34-year-old female with sickle cell anemia (hemoglobin SS disease) and severe iron overload presented to our institution with the subacute presentation of recurrent pain crisis, fever of unknown origin, pancytopenia, and weight loss. A CT scan demonstrated both lung and liver nodules concerning for granulomatous disease. Subsequent biopsies of the liver and bone marrow confirmed the presence of noncaseating granulomas and blood cultures isolatedMycobacterium aviumcomplex MAC. Disseminated MAC is considered an opportunistic infection typically diagnosed in the immunocompromised and rarely in immunocompetent patients. An appreciable number of mycobacterial infection cases have been reported in sickle cell disease patients without immune dysfunction. It has been reported that iron overload is known to increase the risk for mycobacterial infection in vitro and in vivo studies. While iron overload is primarily known to cause end organ dysfunction, the clinical relationship with sickle cell disease and disseminated MAC infection has not been reported. Clinical iron overload is a common condition diagnosed in the sub-Saharan African population. High dietary iron, genetic defects in iron trafficking, as well as hemoglobinopathy are believed to be the etiologies for iron overload in this region. Patients with iron overload in this region were 17-fold more likely to die fromMycobacterium tuberculosis. Both experimental and clinical evidence suggest a possible link to iron overload and mycobacterial infections; however larger observational studies are necessary to determine true causality.

We identified 11 patients with CD10(+) cutaneous T-cell lymphoma by flow cytometry. All cases were CD4(+) and CD8(-). Three patients had extensive lymphadenopathy, systemic symptoms and an aggressive clinical course consistent with angioimmunoblastic T-cell lymphoma or peripheral T-cell lymphoma. However, 8 of the 11 patients had a prolonged disease course with gross morphology, histology and tumor cell phenotype indistinguishable from mycosis fungoides or Sezary syndrome. Immunohistochemical studies confirmed CD10 expression in seven of the eight cases and revealed the lymphoma cells were Bcl-6(+), PD-1(+), and EBV(-). Two had significant expression of CXCL-13(+). The findings indicate that lymphoma cells from mycosis fungoides or Sezary syndrome may express follicular center helper T-cell markers CD10, Bcl-6, and PD-1 and occasionally CXCL-13. The expression of these markers in some cases of mycosis fungoides/Sezary syndrome suggests follicular center helper T-cell differentiation and may lead to confusion in distinguishing mycosis fungoides/Sezary syndrome from other follicular center helper T-cell marker positive T-cell lymphomas with cutaneous manifestations.

Human mesenchymal stromal cells (MSC) have been shown to support the growth and differentiation of hematopoietic stem cells (HSC). We hypothesized that intra-osseous (IO) co-transplantation of MSC and umbilical cord blood (UCB) may be effective in improving early HSC engraftment, as IO transplantation has been demonstrated to enhance UCB engraftment in NOD SCID-gamma (NSG) mice. Following non-lethal irradiation (300rads), 6 groups of NSG mice were studied: 1) intravenous (IV) UCB CD34+ cells, 2) IV UCB CD34+ cells and MSC, 3) IO UCB CD34+ cells, 4) IO UCB CD34+ cells and IO MSC, 5) IO UCB CD34+ cells and IV MSC, and 6) IV UCB CD34+ and IO MSC. Analysis of human-derived CD45+, CD3+, and CD19+ cells 6 weeks following transplant revealed the highest level of engraftment in the IO UCB plus IO MSC cohort. Bone marrow analysis of human CD13 and CD14 markers revealed no significant difference between cohorts. We observed that IO MSC and UCB co-transplantation led to superior engraftment of CD45+, CD3+ and CD19+ lineage cells in the bone marrow at 6 weeks as compared with the IV UCB cohort controls. Our data suggests that IO co-transplantation of MSC and UCB facilitates human HSC engraftment in NSG mice.

Neuropilin-1 (NRP-1)/CD304 is a marker for plasmacytoid dendritic cells. We determined the distribution of NRP-1/CD304 expression on normal hematopoietic cells and in 167 acute leukemias by flow cytometry. NRP-1/CD304 surface expression was frequent in precursor B-cell acute lymphoblastic leukemia (36/51 [71%]) and uncommon in acute myeloid leukemia (22.9%). In acute myeloid leukemia, expression was noted in all (4/4) acute myeloid leukemias with the M4eo subtype and in 50% of specimens (6/12) with complex cytogenetics. On hematopoietic cells, NRP-1/CD304 was expressed on normal erythroid progenitors, plasma cells, and B-cell progenitors, as well as plasmacytoid dendritic cells. Expression was not consistently detected on other hematopoietic cell types. Owing to this distribution of expression, the detection of NRP-1/CD304 alone on a hematopoietic cell cannot be used to determine plasmacytoid dendritic cell differentiation. Finally, we show that NRP-1/CD304 is overexpressed in 30% of precursor B-cell acute lymphoblastic leukemia samples compared with normal B-cell progenitors, allowing for its potential use as a marker for the detection of minimal residual disease.

Introduction: Very advanced age (≥80 years) DLBCL patients have worse prognosis. These unfavorable outcomes are largely considered to be a result of the combined effects of increased comorbidities, frailty, diminished tolerance and access to effective chemoimmunotherapy. It is not clear yet whether DLBCL patients of very advanced age have disease that is intrinsically more aggressive. Methods: We accessed the Stem Cell Transplant and Hematologic Malignancies database of University Hospitals Seidman Cancer Center for DLBCL patients diagnosed between 2000 and 2016. Information collected included demographic characteristics, baseline laboratory and disease information, as well as treatment details. Progression free survival and overall survival were calculated from time of diagnosis using Kaplan Meier methodology, comparisons were done with log rank. Cumulative incidence of relapse with death as competing risk was calculated for patients who underwent treatment, comparisons between age groups were done with the Gray test. Results: A total of 542 DLBCL patients were identified, 85 (16%) were older than 80 years. Table 1 shows the baseline demographic and disease characteristics. Older patients had a higher incidence of comorbidities, specifically cardiovascular comorbidities, prior renal insufficiency, and hyperlipidemia. Expectedly, very elderly patients had higher R-IPI, with a trend towards worse performance status that did not reach statistical difference. The proportion of patients diagnosed with non - germinal center DLBCL was not different than younger DLBCL patients, and there was no statistical difference in the incidence of double expressor or double hit lymphomas, possibly secondary to the small number of patients tested. A smaller proportion of patients ≥ 80 years received antineoplastic therapy (89% vs. 98%, p = 0.001), and use of less intense therapy was more common (table 2). Sixty one percent of patients, however, were still treated with R-CHOP (38.8%) or R-miniCHOP (22.2%). The overall response rate (ORR) for any therapy was 68.2%, lower than the ORR for younger patients (85.9%, p = 0.007). When only patients treated with RCHOP/R-miniCHOP were included, the difference in ORR was smaller, though still statistically significant (77.5% vs. 89.8%, p = 0.021). Median number of cycles was similar (5 vs. 6 cycles, respectively). Patients of all ages treated with single agent rituximab presented an ORR of 43%. After a median of 40 months follow up, estimated 4-year overall survival was 42% (95% CI 31-54%) for patients ≥ 80 years, 67% for patients aged between 60 and 79 years (95% CI 61-74%) and 78% for patients < 60 years (95% CI 73-84%) (Figure 1). Four - year survival for patients ≥80 years treated with R-CHOP or R-miniCHOP chemoimmunotherapy was 50% (95% CI 36-65%) vs. 75% for younger patients (95% CI 70-79%) (p Conclusions: Very elderly (≥80 years of age) DLBCL patients have significantly worse overall survival, progression free survival, and treatment response rates than younger patients. However, when chemoimmunotherapy is feasible, disease relapse rates are comparable to those of younger patients, and increased mortality does not appear to be a result of increased disease relapse. Additional research is needed to establish more widely applicable, better tolerated effective treatment regimens for this patient population. Disclosures Lazarus: Pluristem Ltd.: Consultancy. Malek:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Tomlinson:Foundation Medicine: Consultancy. Caimi:Genentech: Other: Advisory Board PArticipation, Research Funding; Kite Pharma: Other: Advisory Board Participation; Celgene: Speakers Bureau; Kite Pharma: Other: Advisory Board Participation.

Purpose Africa is burdened by the AIDS epidemic and attendant increase in HIV/AIDS-related malignancies. Pragmatic approaches to therapeutic intervention could be of great value. Dose-modified oral chemotherapy for AIDS-related non-Hodgkin's lymphoma is one such approach. Patients and Methods The oral regimen consisted of lomustine 50 mg/m2 on day 1 (cycle 1 only), etoposide 100 mg/m2 on days 1 to 3, and cyclophosphamide/procarbazine 50 mg/m2 each on days 22 to 26 at 6-week intervals (one cycle) for two total cycles in HIV-infected patients with biopsy-proven non-Hodgkin's lymphoma. Results Forty-nine patients (21 in Uganda and 28 in Kenya) were treated. The majority of patients were female (59%) and had a poor performance status (63%); 69% of patients had advanced-stage disease; and 18 patients (37%) had access to antiretroviral therapy. In total, 79.5 cycles of therapy were administered. The regimen was well tolerated, had modest effects (decline) on CD4+ lymphocyte counts (P = .077), and had negligible effects on HIV-1 viral replication. Four febrile neutropenia episodes and three treatment-related deaths (6% mortality rate) occurred. The overall objective response rate was 78% (95% CI, 62% to 88%); median follow-up time was 8.2 months (range, 0.1 to 71 months); median event-free and overall survival times were 7.9 months (95% CI, 3.3 to 13.0 months) and 12.3 months (95% CI, 4.9 to 32.4 months), respectively; and 33% of patients survived 5 years. Conclusion Dose-modified oral chemotherapy is efficacious, has comparable outcome to that in the United States in the pre–highly active antiretroviral therapy setting, has an acceptable safety profile, and is pragmatic in sub-Saharan Africa. The international collaboration has been highly successful, and subsequent projects should focus on strategies to optimize combination antiretroviral therapy and chemotherapy and follow-up tissue correlative studies.

Pseudolymphomatous folliculitis is a rare entity. We present a 62-year-old man with a recurrent solitary nodule on his nose requiring multiple excisions. Microscopic examination of the excisions showed a dense lymphocytic infiltrate containing numerous histiocytes and S100+, CD1a+ dendritic cells that surrounded and infiltrated hypertrophic hair follicles. Diffuse sheets of CD3+ T cells and nodular clusters of CD20+ B cells were also seen. There was normal reactive pattern of follicular centers. Light chain restriction was not detected. T-cell receptor and immunoglobulin heavy chain gene rearrangements by polymerase chain reaction revealed negative findings. A diagnosis of pseudolymphomatous folliculitis was made based on the hypertrophic hair follicles, periadnexal S100+ and CD1a+ dendritic cells, and negative clonal gene rearrangement study findings. This case of recurrent pseudolymphomatous folliculitis is instructive because of the resemblance to cutaneous lymphomas and cutaneous lymphoid hyperplasias, and the need for correct diagnosis to avoid overtreatment of this indolent condition.

Allogeneic transplantation of multiple umbilical cord blood units in adults: role of pretransplant-mixed lymphocyte reaction to predict host-vs-graft rejection

The dose of graft-nucleated cells and CD34(+) haematopoietic progenitor cells are predictors of allogeneic engraftment and survival in umbilical cord blood (UCB) recipients. In this single institution prospective phase II trial, flow cytometric analyses of CD34(+) progenitor and lymphocyte populations in unmodified single unit human leucocyte antigen (HLA)-disparate UCB grafts infused into 31 consecutive adults (median age 41 years, range 20-64) receiving myeloablative conditioning were compared with clinical outcomes. Median infused UCB graft-nucleated cells and CD34(+) dose was 2.2 x 10(7)/kg and 1.2 x 10(5)/kg respectively. Day to absolute neutrophil count >/=0.5 x 10(9)/l with full donor chimerism averaged 27 d (range 12-41). Univariate analyses demonstrated that UCB graft-infused cell doses of CD34(+) (P = 0.015), CD3(+) (P = 0.024) and CD34(+)HLADR(+)CD38(+) progenitors (P = 0.043) correlated with neutrophil engraftment. This same analysis did not demonstrate a correlation between CD34(+) (P = 0.11), CD3(+) (P = 0.28) or CD34(+)HLADR(+)CD38(+) (P = 0.108) cell dose and event-free survival (EFS). High-resolution matching for HLA-class II (DRB1) resulted in improved EFS (P = 0.02) and decreased risk for acute graft-versus-host disease (GVHD) (P = 0.004). Early mortality (prior to post-transplant day +28) occurred in three patients, while 26 patients achieved myeloid engraftment. These results suggest that UCB graft matching at DRB1 is an important risk factor for acute GVHD and survival, while higher UCB graft cell doses of CD34(+), committed CD34(+) progenitors and CD3(+) T cells favourably influence UCB allogeneic engraftment.

Context.—Despite the widespread practice of pathologist review of blood and body fluid smears, little is known about its impact on improving patient care. Objective.—To assess the clinical usefulness of pathologist review of blood and body fluid smears. Design.—Survey study. Pathology residents contacted the ordering physician after pathologist reviews were reported to assess their clinical impact. Results.—Ninety-six pathologist reviews met criteria for study inclusion, and 64 ordering physicians were successfully contacted during the 2-month study period. Of the 64 cases, 19 reviews (30%) had been seen by the physician within 24 to 48 hours after the report was issued and 33 (51%) had not been seen; in 4 (6%) instances, physicians did not remember whether they had seen the review. Eight reviews (13%) were considered urgent enough to warrant immediate communication by the pathologist. Of the 27 reviews that were seen or directly communicated, 23 (85%) contributed to clinical diagnosis and/or patient management. Conclusions.—This study demonstrates the contribution of pathologist reviews of blood and body fluids to clinical diagnosis and patient management. The results also highlight the problem of a lack of physician awareness of clinical pathology results.

We examined pre-mobilization blood CD34+ cell count to predict ability to mobilize adequate peripheral blood progenitor cells (PBPC) in 106 cancer patients and 36 allogeneic donors. Mean pre-mobilization therapy blood CD34+ cell count was 3.1 cells x 10(6)/l (s.d. = 3.9, r = 0.3-37) and mean CD34+ cells collected were 5.3 x 10(6) cells/kg/leukapheresis procedure (s.d. = 7.0, r = 0.03-53). Yields correlated with pre-mobilization CD34+ cells x 10(6)/l (r = 0.37, P-value0.0001); correlation was stronger in allogeneic donors (r = 0.56, P-value = 0.0004) and males (r = 0.46, P-value0.0001). Based on classification and regression tree multivariate analysis, the predictive value of pre-mobilization blood CD34+ cell count was confounded by other variables, including age, gender, mobilization regimen and malignancy type. We generated an algorithm to predict a minimum PBPC yield of 1 x 10(6) CD34+ cells/kg/leukapheresis procedure after mobilization. A threshold pre-mobilization blood CD34+ cell count of 2.65 cells x 10(6)/l was the most important decision point in predicting successful mobilization. Only 2% of subjects with pre-mobilization blood CD34+ cell counts2.65 cells x 10(6)/l did not achieve the minimum per apheresis, whereas 24% with pre-mobilization values below threshold had inadequate mobilization. Prospectively identifying individuals at risk for mobilization failure would allow for improved treatment planning, resource utilization and time saving.

Background: Lower incidence and severity of acute graft versus host disease (GVHD) has been observed in leukemia patients receiving HLA-mismatched umbilical cord (UCB) transplants. However, despite the increased use of UCB in stem cell transplantation, the mechanisms underlying these favorable outcomes are not well delineated.Methods: We analyzed antigen specific lymphocyte responses after transplant to determine whether the decreased allogeneic responsiveness of UCB lymphocytes is attributable to pan-unresponsiveness, lymphocyte repressive or recipient-specific tolerance.Results: Circulating lymphocytes collected early (3 months) after UCB transplant demonstrate a less naive phenotype compared with that in the infused graft. Additionally, after transplant, circulating peripheral blood UCB-derived lymphocytes produced normal levels of interferon-[gamma] and proliferated normally when stimulated with mitogen or third party alloantigen. In contrast, when stimulated with recipient antigen, circulating lymphocytes emerging posttransplant did not proliferate nor produce interferon-[gamma]. Moreover, analysis of interleukin-4 production revealed a Th2 response to recipient antigens. These data indicate early induction of immune tolerance of naive UCB graft lymphocytes with skewing of transplant recipient-specific immune response towards Th2 cytokine profile.Conclusions: UCB graft lymphocyte immune naivety and observed early tolerance induction may contribute to the observed favorable GVHD incidence, despite infusion of HLA mismatch grafts in the unrelated allogeneic setting.

Purpose: A novel regimen designed to maximize antileukemia activity of carboplatin through inhibiting repair of platinum-DNA adducts was conducted in poor prognosis, acute leukemia patients. Experimental Design: Patients received fludarabine (10 to 15 mg/m2 × 5 days), carboplatin (area under the curve 10 to 12 by continuous infusion over 5 days), followed by escalated doses of topotecan infused over 72 hours (fludarabine, carboplatin, topotecan regimen). Twenty-eight patients had acute myelogenous leukemia (7 untreated secondary acute myelogenous leukemia, 11 in first relapse, and 10 in second relapse or refractory), 1 patient had refractory/relapsed acute lymphoblastic leukemia, and 2 patients had untreated chronic myelogenous leukemia blast crisis. Six patients had failed an autologous stem cell transplant. Patients ranged from 19 to 76 (median 54) years. Measurement of platinum-DNA adducts were done in serial bone marrow specimens. Results: Fifteen of 31 patients achieved bone marrow aplasia. Clinical responses included 2 complete response, 4 complete response with persistent thrombocytopenia, and 2 partial response. Prolonged myelosuppression was observed with median time to blood neutrophils ≥200/μl of 28 (0 to 43) days and time to platelets ≥20,000/μl (untransfused) of 40 (24 to 120) days. Grade 3 or greater infections occurred in all of the patients, and there were 2 infection-related deaths. The nonhematologic toxicity profile was acceptable. Five patients subsequently received allografts without early transplant-related mortality. Maximum tolerated dose of fludarabine, carboplatin, topotecan regimen was fludarabine 15 mg/m2 × 5, carboplatin area under the curve 12, and topotecan 2.55 mg/m2 over 72 hours. An increase in bone marrow, platinum-DNA adduct formation between the end of carboplatin infusion and 48 hours after the infusion correlated with bone marrow response. Conclusions: Fludarabine, carboplatin, topotecan regimen is a promising treatment based on potential pharmacodynamic interactions, which merits additional study in poor prognosis, acute leukemia patients.

Hepatosplenic T-cell lymphoma is an uncommon neoplasm characterized by a lymphoid infiltrate within the sinusoids of the liver, spleen, and bone marrow, without significant nodal involvement. The majority of cases express the γδ T-cell receptor and are associated with an isochromosome 7q cytogenetic abnormality. Recently, a small number of cases have been reported that express the αβ T-cell receptor. Here, we report our findings of a case of an S100-positive hepatosplenic αβ T-cell lymphoma in a 20-year-old woman who presented with pancytopenia and hepatosplenomegaly. The case adds to the growing literature of hepatosplenic αβ T-cell lymphomas.

Purpose: Primary follicular lymphoma of the testis in childhood is rare with only 6 cases previously reported. We present 3 additional cases.Materials and Methods: We extensively analyzed primary follicular lymphoma of the testis in 3 boys. Clinical data were obtained by reviewing patient charts.Results: The patients were 4, 5 and 11 years old, respectively. Two patients presented with painless unilateral testicular enlargement and 1 presented with unilateral hydrocele. Laboratory findings were within normal limits in all patients. Radical orchiectomy was done in all cases. The excised testes were partially or completely replaced by tumor. In all cases the features were those of follicular, large cell-type malignant lymphoma. Tumor cells in all cases were CD20 and CDw75 positive, focally CD23 positive and bcl-2 negative, while in 2 they were CD10 positive and bcl-6 positive. Surface Ig was absent in the 2 cases studied. Karyotyping in 1 case showed a normal karyotype. Staging revealed no evidence of extra...

As the threshold nucleated cell dose for single unit umbilical cord blood (UCB) in adults has not to date been firmly established, we prospectively compared single vs. 2-unit UCB transplantation after reduced intensity conditioning (RIC) in adult patients with hematologic malignancies. Study design specified one UCB unit if the cryopreserved total nucleated cell (TNC) dose was ≥2.5×107/kg recipient weight, otherwise 2-units matched at minimum 4/6 HLA loci to the patient and 3/6 to each other were infused. Twenty-seven patients received 1 unit; 23 patients received 2 units. Median time to absolute neutrophil count (ANC) >500/μL was 24 days (95% CI 22–28 days), 25 days for 1-unit and 23 days for 2-units (p=0.99). At day 100, ANC >500/μL was 88.4% and 91.3% in the 1 and 2-unit groups (p=0.99), respectively. Three-year event free survival (EFS) was 28.6% and 39.1% in the 1 and 2-unit groups (p=0.71), respectively. Infusion of 2 units was associated with significantly lower relapse risk, 30.4% vs. 59.3% (p=0.045). Infused cell doses (TNC, CD3+, CD34+, CD56+CD3neg) did not impact engraftment, overall survival (OS), or EFS. Taken together, single unit UCB transplantation with threshold cell dose ≥2.5×107/kg recipient weight after RIC is a viable option for adults, although infusion of 2 units confers a lower relapse incidence.

Here we report a 3-year-old boy with myelomastocytic leukemia. The patient presented with fatigue and right eye proptosis. Bone marrow revealed acute myeloid leukemia with t(8;21) and trisomy 8. Induction therapy produced marked reduction in marrow myeloblasts with the emergence of 13% atypical mast cells. These cells were subsequently identified in retrospect in the diagnostic marrow consistent with myelomastocytic leukemia. His clinical course was notable for the difficulty in the eradication of the leukemic process and resembled that of adults with systemic mastocytosis with associated hematologic non-mast cell lineage disease. To the best of our knowledge, this is the youngest individual reported. The implications of mast cell lineage involvement in acute myeloid leukemia are reviewed.

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis. We report a fatal case of ECD with extensive cardiac involvement associated with a marginal zone lymphoma and monoclonal proteinemia in a young man. This is the first reported association of ECD with a monoclonal gammopathy or a lymphoma.

The flow cytometric analysis of T-cell malignancies is difficult due to the heterogeneity of T-cells and the lack of convenient methods to detect T-cell clonality. Neoplastic T-cells are most often detected by their altered level of surface antigen expression, and detection requires an extensive knowledge of the phenotype of normal T-lymphocytes. This review focuses on the methods to distinguish malignant T-cells from their normal counterparts and the phenotypic features of the T-cell lymphoproliferative disorders.

Forty-two patients with poor prognosis AML were enrolled in a phase II study combining fludarabine, carboplatin, and topotecan (FCT) with thalidomide. Laboratory correlates included serum vascular endothelial growth factor levels (VEGF) and bone marrow microvascular density (MVD). Ten of 42 (24%) patients achieved a complete remission (CR or CRp). Serious thrombotic adverse events were observed in 5 patients suggesting that the combination of cytotoxic chemotherapy and thalidomide may be thrombogenic despite significant thrombocytopenia. VEGF did not correlate with response to therapy, while a trend towards decreased MVD was noted in patients who achieved CR. The addition of thalidomide did not significantly influence angiogenic markers. It is not clear that thalidomide adds any efficacy to the FCT regimen.

Background The pathologic evaluation of mycosis fungoides (MF) is a challenging area in dermatopathology. Objective We sought to determine the usefulness of flow cytometry for the diagnosis of MF from skin biopsy specimens. Methods Skin biopsy specimens from 22 patients with a clinical suggestion for MF were evaluated by 4-color flow cytometry. The results were correlated with the International Society for Cutaneous Lymphoma (ISCL) MF diagnostic score and molecular studies for T-cell receptor gene rearrangement. Results A T-cell abnormality by flow cytometry was identified in all 11 patients with diagnostic ISCL scores whereas the 7 patients with either subdiagnostic ISCL scores or reactive histology showed no phenotypic abnormality by flow cytometry. In all, 10 of 11 patients with diagnostic skin biopsy specimens for MF had T-cell receptor gene rearrangements by polymerase chain reaction. Gene rearrangements were not detected in the subdiagnostic group. Limitations Small study size was a limitation. Conclusion Flow cytometry of skin biopsy specimens is a sensitive method for detecting abnormalities in MF and should be considered part of the routine workup of patients with a clinical suggestion of MF.

While immune hemolysis due to donor isohemagglutinin (IH) production often complicates minor ABO incompatible peripheral blood hematopoietic stem cell transplantation (PBSCT), it is not known if this occurs with umbilical cord blood transplantation (UCBT). We compared IH production and hemolysis following minor ABO allogeneic PBSCT and UCBT. We reviewed 24 ABO minor incompatible allogeneic PBSCTs and 14 ABO minor incompatible UCBTs. Patients were evaluated for donor-derived IH by reverse ABO grouping. Evaluation of hemolysis was based on clinical and laboratory findings of anemia associated with increased RBC transfusion need, concomitant with the appearance of donor-derived IH. Of the 24 ABO minor incompatible allogeneic PBSCTs, 15 produced donor-derived IH from 6 to 88 days following transplantation, with seven of 15 patients exhibiting clinically evident hemolysis. There was no significant difference in days to leukocyte engraftment or infused CD34 cells in patients with or without donor-derived IH. None of the 14 patients receiving ABO incompatible UCBTs showed evidence of donor-derived IH following transplantation with a median follow-up of 60 days. We conclude that donor IHs are not produced in patients undergoing minor ABO incompatible UCBTs suggesting fundamental immunologic differences between allogeneic PBSCT and UCBT.

Summary We investigated the feasibility, safety and efficacy of two B-cell purging methods in patients with CD20+ non-Hodgkin lymphoma (NHL) receiving autologous stsem cell transplantation. Myeloid and immune recoveries between the methods were compared. Twenty-seven patients were randomised to either in vivo purging with rituximab or ex vivo purging by CD34+ cell selection. Both purging methods were efficient at eliminating B-cells in infusates. When compared with in vivo purging, ex vivo purging was associated with CD34+ cell loss and delayed median neutrophil (10 d vs. 11 d) and platelet (12·5 d vs. 17 d) count recoveries. Lymphocyte recovery was similar in both groups, but immunoglobulin recovery was delayed after in vivo purging. Late-infectious complications were few in both arms. At a median follow-up of 27 months, 2-year probabilities of event-free survival (EFS) rates were 81% for in vivo purging and 76% for ex vivo purging (P = 0·66). When compared with 53 unpurged patients, all 27 purged patients had improved 3-year probabilities of overall survival (89% vs. 70%, P = 0·014) and a trend for improved EFS (78% vs. 57%, P = 0·075). In conclusion, although both purging methods were feasible and safe, rituximab purging was superior as it did not impair CD34+ cell mobilisation and was associated with faster myeloid recovery. Further studies are needed to determine whether rituximab purging is more effective than the use of unpurged autografts.

Mechanisms that protect most high-risk HIV-1 seronegative (HRSN) persons are not well understood. Among hemophiliacs from the Multicenter Hemophilia Cohort Study who remained HIV-1 seronegative despite a high (94%) risk for acquisition of HIV-1 infection, only 7/43 were homozygous for the protective CCR5 Delta32 polymorphism. Among the remainder, neither CCR5 density nor beta-chemokine production, nor in vitro susceptibility to infection with the HIV-1 isolate JR-FL could distinguish HRSN hemophiliacs from healthy controls. When compared to lymphocytes of healthy controls not at risk for HIV-1 infection, diminished spontaneous lymphocyte proliferation was seen in lymphocytes of HRSN hemophiliacs as well as in lymphocytes of hemophiliacs not at risk for HIV-1 infection. Surprisingly sera/plasmas obtained from high-risk HIV-1 seropositve hemophiliacs prior to seroconversion more often contained alloreactive antibodies than date-matched sera/plasmas obtained from HRSN hemophiliacs. Thus alloreactivity may predispose to acquisition of HIV-1 infection after parenteral exposure.

THE FIRST ASSOCIATION OF RUBINSTIEN—Taybi syndrome with immunodeficiency and the successful prevention of infection with intravenous IgG is reported in a 4 year old boy. This case suggests that immunodeficiency maybe a prominent feature of this syndrome and may predispose these patients to recurrent infections.

PURPOSE: Patient response to hematopoietic progenitor-cell mobilizing regimens seems to vary considerably, making comparison between regimens difficult. To eliminate this inter-patient variability, we designed a cross-over trial and prospectively compared the number of progenitors mobilized into blood after granulocyte-macrophage colony-stimulating factor (GM-CSF) days 1 to 12 plus granulocyte colony-stimulating factor (G-CSF) days 7 to 12 (regimen G) with the number of progenitors after cyclophosphamide plus G-CSF days 3 to 14 (regimen C) in the same patient. PATIENTS AND METHODS: Twenty-nine patients were randomized to receive either regimen G or C first (G1 and C1, respectively) and underwent two leukaphereses. After a washout period, patients were then crossed over to the alternate regimen (C2 and G2, respectively) and underwent two additional leukaphereses. The hematopoietic progenitor-cell content of each collection was determined. In addition, toxicity and charges were tracked. RESULTS: Regimen C (n = 50) resulted in mobilization of more CD34+ cells (2.7-fold/kg/apheresis), erythroid burst-forming units (1.8-fold/kg/apheresis), and colony-forming units–granulocyte-macrophage (2.2-fold/kg/apheresis) compared with regimen G given to the same patients (n = 46; paired t test, P < .01 for all comparisons). Compared with regimen G, regimen C resulted in better mobilization, whether it was given first (P = .025) or second (P = .02). The ability to achieve a target collection of ≥ 2 × 106 CD34+ cells/kg using two leukaphereses was 50% after G1 and 90% after C1. Three of the seven patients in whom mobilization was poor after G1 had ≥ 2 × 106 CD34+ cells/kg with two leukaphereses after C2. In contrast, when regimen G was given second (G2), seven out of 10 patients failed to achieve the target CD34+ cell dose despite adequate collections after C1. Thirty percent of the patients (nine of 29) given regimen C were admitted to the hospital because of neutropenic fever for a median duration of 4 days (range, 2 to 10 days). The higher cost of regimen C was balanced by higher CD34+ cell yield, resulting in equivalent charges based on cost per CD34+ cell collected. CONCLUSION: We report the first clinical trial that used a cross-over design showing that high-dose cyclophosphamide plus G-CSF results in mobilization of more progenitors then GM-CSF plus G-CSF when tested in the same patient regardless of sequence of administration, although the regimen is associated with greater morbidity. Patients who fail to achieve adequate mobilization after regimen G can be treated with regimen C as an effective salvage regimen, whereas patients who fail regimen C are unlikely to benefit from subsequent treatment with regimen G. The cross-over design allowed detection of significant differences between regimens in a small cohort of patients and should be considered in design of future comparisons of mobilization regimens.