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European LeukemiaNet (ELN) acute myeloid leukemia (AML) genetic risk classification systems were based on response to intensive chemotherapy; their ability to discriminate outcomes in older patients treated with venetoclax-azacitidine may be suboptimal. Here, pooled analysis of patients in the phase 3 VIALE-A trial (NCT02993523) and phase 1b study (NCT02203773) examined prognostic stratification according to 2017 and 2022 ELN risk classifications. A bioinformatic algorithm derived new molecular signatures differentiating venetoclax-azacitidine-treated patients based on median overall survival (OS). 279 patients treated with venetoclax-azacitidine and 113 patients treated with placebo-azacitidine were analyzed. When classified by ELN 2017 or 2022 prognostic criteria, most patients had adverse-risk AML (60.2% and 72.8% for venetoclax-azacitidine and 65.5% and 75.2% for placebo-azacitidine, respectively). While outcomes with venetoclax-azacitidine were improved across all ELN risk groups compared with placebo-azacitidine, ELN classification systems poorly discriminated venetoclax-azacitidine outcomes. By applying a bioinformatic algorithm, new molecular signatures were derived differentiating OS outcomes with venetoclax-azacitidine; the mutational status of TP53, FLT3-ITD, NRAS, and KRAS categorized patients into higher-, intermediate-, and lower-benefit groups (52%, 25%, and 23% of patients, respectively), each associated with a distinct median OS (26.5 months [95% CI, 20.2 to 32.7], 12.1 months [95% CI, 7.3 to 15.2], and 5.5 months [95% CI, 2.8 to 7.6], respectively). ELN prognostic classifiers do not provide clinically meaningful risk stratification of OS outcomes for patients with AML treated with venetoclax-azacitidine. TP53, FLT3-ITD, NRAS, and KRAS mutation status allows classification of these patients into three risk groups with distinct differences in median OS., (Copyright © 2024 American Society of Hematology.)

Historically, older adults with B-cell acute lymphoblastic leukemia (ALL) have done poorly with chemotherapy-based treatment. Therefore, new innovative approaches are urgently needed to improve outcomes for this population. CD19-targeted immunotherapies such as blinatumomab and chimeric antigen receptor (CAR) T cell therapy have produced remarkable responses in relapsed/refractory (r/r) B-cell ALL, including clearance of minimal residual disease (MRD). Available data support the efficacy and safety of blinatumomab in older adults with advanced B-cell ALL. Therefore, its application is being extended to frontline regimens for B-cell ALL, particularly in older adults. There are several studies actively examining the role of blinatumomab alone or in combination with attenuated dosing of conventional chemotherapy or novel agents in older adults with newly diagnosed ALL and early data are encouraging. While CD19-targeted CAR (CD19CAR) T cell therapy is active in children and young adults with r/r B-cell ALL, data supporting its efficacy and safety in older adults with ALL is scarce. Furthermore, the commercially FDA approved CD19CAR T cell therapy product for r/r ALL is restricted only to patients ≤25 years of age. Although there are concerns about older adults tolerating the expected toxicities associated with CAR T cell therapy, which may be life threatening, tailored approaches for prophylactic and pre-emptive interventions combined with utilization of safer CAR T cell platforms may improve tolerability and further extend the use of this promising treatment to older patients with ALL. In this review, we will discuss the progress in immunotherapies for older adults with B-cell ALL and their potential for transforming frontline therapy for newly diagnosed patients., (Published by Elsevier Ltd.)

Background: Thrombopoietin receptor agonists (TPO-RAs) are used to treat primary immune thrombocytopenia (ITP). Some patients have discontinued treatment while maintaining a hemostatic platelet count., Objectives: To develop expert consensus on when it is appropriate to consider tapering TPO-RAs in ITP, how to taper patients off therapy, how to monitor patients after discontinuation, and how to restart therapy., Methods: We used a RAND/UCLA modified Delphi panel method. Ratings were completed independently by each expert before and after a meeting. Second-round ratings were used to develop the panel's guidance. The panel was double-blinded: The sponsor and nonchair experts did not know each other's identities., Results: Guidance on when it is appropriate to taper TPO-RAs in children and adults was developed based on patient platelet count, history of bleeding, intensification of treatment, trauma risk, and use of anticoagulants/platelet inhibitors. For example, it is appropriate to taper TPO-RAs in patients who have normal/above-normal platelet counts, have no history of major bleeding, and have not required an intensification of treatment in the past 6 months; it is inappropriate to taper TPO-RAs in patients with low platelet counts. Duration of ITP, months on TPO-RA, or timing of platelet response to TPO-RA did not have an impact on the panel's guidance on appropriateness to taper. Guidance on how to taper patients off therapy, how to monitor patients after discontinuation, and how to restart therapy is also provided., Conclusion: This guidance could support clinical decision making and the development of clinical trials that prospectively test the safety of tapering TPO-RAs., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Management of acute lymphoblastic leukemia (ALL) in a socioeconomically vulnerable population without ready access to a hematopoietic stem cell transplant (HCT) center and clinical trials is challenging. Data regarding the outcomes of such patients are sparse., Patients and Methods: This retrospective analysis included 90 consecutive patients with ALL who presented to Harbor-UCLA between 2003 and 2018. The primary objective was overall survival (OS), whereas secondary objectives included leukemia-free survival, toxicities of therapy, and referral for HCT and incidence of successful HCT., Results: Most patients were male (56.7%) and Hispanic (72.2%). The median age of diagnosis was 36 years (range, 18-63 years). The median OS was 26.8 months (95% confidence interval [CI], 17.4-59.0 months). In patients who achieved complete remission with therapy, the median leukemia-free survival was 16.4 months. Fifty percent of patients experienced at least 1 episode of bacteremia, and nearly 25% of patients developed an invasive fungal infection. Thirty-six percent (n = 32) of patients were referred for HCT. The referral rate increased over time, which led to improved OS in patients who underwent evaluation at a tertiary cancer center (hazard ratio, 0.44; 95% CI, 0.21-0.89; P = .02). Patients who underwent HCT had significantly better OS compared with those who did not (OS not reached vs. 21.9 months; hazard ratio, 0.16; 95% CI, 0.04-0.68; P = .01)., Conclusion: Risk stratification and evidence-based treatment approaches are important for patients with ALL treated in a resource-limited setting. Most patients can be induced successfully and achieve complete remission with therapy. Partnership with a cancer center with early referral for HCT can facilitate curative HCT to be performed., (Published by Elsevier Inc.)

Acute lymphoblastic leukemia (ALL) arising in patients with prior malignancy is increasingly being reported. Although terms such as 'secondary' and 'therapy-related' have been interchangeably used to characterize these cases of ALL in a similar fashion to acute myeloid leukemia (AML), it must be noted that some reported cases have not had exposure to cytotoxic therapy and hence a causal relationship between the prior malignancy and subsequent ALL is difficult to establish. Therefore, the use of the term secondary ALL to describe such cases without exposure to cytotoxic therapy is preferably avoided and will not be discussed here. ALL related to prior cytotoxic therapy (t-ALL) appears to be a distinct entity when compared to the de novo ALL, with characteristics including older age at the time of onset, female predominance and leukemia genetics enriched with KMT2A (MLL) gene rearrangement and chromosomes 5/7 abnormalities. Outcome of t-ALL appears inferior to de novo ALL when treated with conventional combination chemotherapy and this adverse outcome may be related to unfavorable patient factors as well as high risk genetic features of the disease itself. Additional genomic and molecular studies are needed to better characterize pathologic features of ALL arising after exposure to cytotoxic therapy in patients with prior malignancies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Mutations in isocitrate dehydrogenase (IDH)1/2 genes result in nicotinamide adenine dinucleotide phosphate-dependent reduction of α-ketoglutarate and formation of 2-hydroxyglutarate, which blocks normal cellular differentiation and promotes leukemogenesis. Nearly 20% of acute myeloid leukemia (AML) patients carry IDH1/2 mutations. Although multiple investigators have described the prognostic implications of IDH mutations in AML patients receiving chemotherapy, the effect of these mutations on outcomes after allogeneic (allo) hematopoietic cell transplantation (HCT) is unknown., Patients and Methods: We report on the clinical outcome of a cohort of AML patients, who were tested for IDH mutations and underwent alloHCT at City of Hope (2015-2017). Of a total of 317 screened patients, 99 (31%) underwent alloHCT, of whom 23 carried and 76 did not carry IDH mutations (control)., Results: No statistical significance was detected in patient's overall survival (P = .84). With a median follow-up of 7.8 months, 1-year relapse rate of 29% and 13% was seen in the IDH-mutated and control group, respectively (P = .033). IDH1/2 mutation status remained significantly associated with relapse (hazard ratio, 2.8; P = .046) after inclusion of pre-HCT disease status in a multivariable model., Conclusion: Our results, despite low patient numbers, indicate that IDH mutations are associated with higher relapse rate after alloHCT. Further prospective studies on post transplantation IDH inhibition is required to improve outcomes in AML patients who carry IDH mutations., (Copyright © 2019. Published by Elsevier Inc.)

Older patients with acute myeloid leukemia (AML) respond poorly to standard induction therapy. B-cell lymphoma 2 (BCL-2) overexpression is implicated in survival of AML cells and treatment resistance. We report safety and efficacy of venetoclax with decitabine or azacitidine from a large, multicenter, phase 1b dose-escalation and expansion study. Patients (N = 145) were at least 65 years old with treatment-naive AML and were ineligible for intensive chemotherapy. During dose escalation, oral venetoclax was administered at 400, 800, or 1200 mg daily in combination with either decitabine (20 mg/m 2 , days 1-5, intravenously [IV]) or azacitidine (75 mg/m 2 , days 1-7, IV or subcutaneously). In the expansion, 400 or 800 mg venetoclax with either hypomethylating agent (HMA) was given. Median age was 74 years, with poor-risk cytogenetics in 49% of patients. Common adverse events (>30%) included nausea, diarrhea, constipation, febrile neutropenia, fatigue, hypokalemia, decreased appetite, and decreased white blood cell count. No tumor lysis syndrome was observed. With a median time on study of 8.9 months, 67% of patients (all doses) achieved complete remission (CR) + CR with incomplete count recovery (CRi), with a CR + CRi rate of 73% in the venetoclax 400 mg + HMA cohort. Patients with poor-risk cytogenetics and those at least 75 years old had CR + CRi rates of 60% and 65%, respectively. The median duration of CR + CRi (all patients) was 11.3 months, and median overall survival (mOS) was 17.5 months; mOS has not been reached for the 400-mg venetoclax cohort. The novel combination of venetoclax with decitabine or azacitidine was effective and well tolerated in elderly patients with AML (This trial was registered at www.clinicaltrials.gov as #NCT02203773)., (© 2019 by The American Society of Hematology.)

Higher incidence rates and poor outcomes have been reported among Latin American patients (Latinos) with acute lymphoblastic leukemia (ALL). Distinct patterns in recent genomic studies allude to a predisposing genetic component. In this review, we critically examine the increasing amount of empirical information on the epidemiology, outcomes and genomics of Latinos with ALL. We discuss the immense diversity within the Latino community and varying definitions of what is considered Latino, which pose an epidemiological challenge. Environmental factors have been evaluated as possible predisposing factors in the development of ALL but studies have produced conflicting results. In this review we describe chromosomal abnormalities and the specific genomic landscape in Latinos with ALL and their association with unfavorable prognosis, focusing on the higher frequency of Philadelphia chromosome-Like ALL. Recent data suggest an association between polymorphisms that are commonly found in indigenous Americans and high rates of ALL. The review compares the distribution of ALL throughout the various countries in Latin America in an attempt to shed some epidemiological light on the genetic ancestry of ALL. We additionally identify areas where research is warranted in efforts toward the advent of novel targeted agents that are relevant in improving outcomes within the Latino community., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The outcome of patients with acute lymphoblastic leukemia (ALL) relapsing after allogeneic hematopoietic cell transplantation (AlloHCT) is poor. Although morphologic remission can sometimes be achieved, such remissions are usually transient if not consolidated by a second AlloHCT (AlloHCT2)., Materials and Methods: We retrospectively analyzed the outcomes of 27 patients with ALL who had undergone AlloHCT2 for relapsed disease at our center during a 12-year period., Results: With a median follow-up of 50.9 months for living patients, the 2-year overall and event-free survival were 40.7% and 29.6%, respectively. Patients with either a disease-free interval or interval between transplants of > 1 year had better overall survival (P = .02 and P = .0005) after AlloHCT2., Conclusion: AlloHCT2 remains a potential curative option in a subset of patients with relapsed ALL after the first AlloHCT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8(+) T cells predominantly recognize a nonameric HLA-A2-restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8(+) T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8(+) T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

Combination tacrolimus and sirolimus graft-versus-host disease (GVHD) prophylaxis for allogeneic transplant in patients conditioned with a fractionated total body irradiation-based regimen has shown encouraging results. We studied this prophylaxis combination in 85 patients receiving a matched-sibling transplant conditioned with 3 different regimens:fludarabine-melphalan (n = 46); total body irradiation-etoposide (n = 28), and busulfan-cyclophosphamide (n = 11). The conditioning regimens were completed on day -4. Sirolimus and tacrolimus were started on day -3 to avoid overlap with conditioning therapy. All patients engrafted, with a median time to neutrophil engraftment of 15 days. The cumulative incidence of acute GVHD grades II to IV and III to IV was 43% and 19%, respectively, with no significant difference by conditioning regimen. The 2-year cumulative incidence of chronic GVHD was 46%. With a median follow-up of 26 months, disease-free survival was 58% and overall survival, 66%. The day-100 and 2-year nonrelapse mortality was 4.8% and 10.2%, respectively. The overall incidence of thrombotic microangiopathy was 19%, and it was significantly higher with busulfan/cyclophosphamide (55%, P = .005). Tacrolimus plus sirolimus is an effective combination for acute GVHD prophylaxis and is associated with very low nonrelapse mortality. Thrombotic microangiopathy is a significant complication with this regimen, particularly in patients receiving busulfan/cyclophosphamide.

The role of iron chelation therapy in myelodysplastic syndrome (MDS) remains controversial. Averting cardiac dysfunction in low-grade MDS patients who have sufficient longevity to experience deleterious cardiac effects of iron overload has been the major argument in favor of iron chelation. Although there is significant evidence showing the adverse impact of transfusion dependency on survival in MDS, direct evidence linking tissue iron overload to poor survival or in particular to cardiac dysfunction is lacking. Given the heterogeneity of MDS, it is likely that the pathophysiology of iron overload is equally heterogeneous and complex in these patients. In this article, I argue that prevention of cardiac dysfunction in patients with lower grades of MDS may not be the major benefit of iron chelation therapy, and present evidence suggesting a potential benefit of iron chelation on 3 other outcomes, namely (1) lowering infection risk, (2) improving the outcome of allogeneic hematopoietic stem cell transplantation, and (3) delaying leukemic transformation. These outcomes have particular relevance for patients with higher grades of MDS and should be evaluated in future prospective clinical trials that include patients with all grades of MDS to fully evaluate the benefit of iron chelation therapy.

Chronic immune thrombocytopenic purpura (ITP) is characterized by low platelet counts and mucocutaneous bleeding. In previous studies romiplostim (AMG531), a thrombopoiesis-stimulating protein, increased platelet counts in most patients with chronic ITP. This ongoing, long-term open-label, single-arm study investigated safety and efficacy in patients who completed a previous romiplostim study and had platelet counts less than or equal to 50 [corrected] x 10(9)/L. One hundred forty-two patients were treated for up to 156 weeks (mean, 69 weeks). Platelet responses (platelet count > or = 50 x 10(9)/L and double baseline) were observed in 87% of all patients and occurred on average 67% of the time in responding patients. In 77% of patients, the romiplostim dose remained within 2 microg/kg of their most frequent dose at least 90% of the time. Ninety patients (63%) received treatment by self-administration. Treatment-related serious adverse events were reported in 13 patients (9%). Bone marrow reticulin was observed in 8 patients; marrows were not routinely performed in this study, so the true incidence of this event cannot be determined. Severe bleeding events were reported in 12 patients (9%). Thrombotic events occurred in 7 patients (5%). In conclusion, romiplostim increased platelet counts in most patients for up to 156 weeks without tachyphylaxis and had an acceptable safety profile. (ClinicalTrials.gov Identifier NCT00116688).

The anti-cytomegalovirus (CMV) activity and safety of oral maribavir in CMV-seropositive allogeneic stem-cell transplant recipients were evaluated in a randomized, double-blind, placebo-controlled, dose-ranging study. After engraftment, 111 patients were randomized to receive CMV prophylaxis with maribavir (100 mg twice daily, 400 mg once daily, or 400 mg twice daily) or placebo. Within the first 100 days after transplantation, the incidence of CMV infection based on CMV pp65 antigenemia was lower in each of the respective maribavir groups (15%, P = .046; 19%, P = .116; 15%, P = .053) compared with placebo (39%). Similarly, the incidence of CMV infection based on plasma CMV DNA was lower in each of the respective maribavir groups (7%, P = .001; 11%, P = .007; 19%, P = .038) compared with placebo (46%). Anti-CMV therapy was also used less often in patients receiving each respective dose of maribavir (15%, P = .001; 30%, P = .051; 15%, P = .002) compared with placebo (57%). There were 3 cases of CMV disease in placebo patients but none in the maribavir patients. Adverse events, mostly taste disturbance, nausea, and vomiting, were more frequent with maribavir. Maribavir had no adverse effect on neutrophil or platelet counts. These results show that maribavir can reduce the incidence of CMV infection and, unlike ganciclovir, does not cause myelosuppression.

We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph(-)) acute lymphoblastic leukemia (ALL). Two model human Ph(-) ALL cell lines (T-cell MOLT-4 and pre-B-cell Reh) were treated with LBH589 and evaluated for biologic and gene expression responses. Low nanomolar concentrations (IC(50): 5-20 nM) of LBH589 induced cell-cycle arrest, apoptosis, and histone (H3K9 and H4K8) hyperacetylation. LBH589 treatment increased mRNA levels of proapoptosis, growth arrest, and DNA damage repair genes including FANCG, FOXO3A, GADD45A, GADD45B, and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. LBH589 treatment was associated with increased histone acetylation at the GADD45G promoter and phosphorylation of histone H2A.X. Furthermore, treatment with LBH589 was active against cultured primary Ph(-) ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%) and induction of GADD45G mRNA expression (up to 35-fold). Thus, LBH589 possesses potent growth inhibitory activity against including Ph(-) ALL cells associated with up-regulation of genes critical for DNA damage response and growth arrest. These findings provide a rationale for exploring the clinical activity of LBH589 in the treatment of patients with Ph(-) ALL.

We examined the prognostic impact of cytogenetics on the outcome of 200 acute lymphoblastic leukemia (ALL) patients 15 to 65 years of age enrolled in Southwest Oncology Group (SWOG)-9400 study. Evaluable cytogenetics or fluorescence in situ hybridization studies were available in 140 (70%) patients. Four karyotype categories (normal [n = 31, 22%], t(9;22)/BCR/ABL1 [n = 36, 26%], other unfavorable [-7, +8, or 11q23 rearrangement, n = 19, 13%], and miscellaneous [n = 54, 39%]) and the biologically and clinically relevant ALL ploidy subgroups were prospectively defined. Overall survival (OS) decreased significantly with increasing age (P = .009) and varied with karyotype category (P < .001). OS was worst for t(9;22)/BCR/ABL1 followed by other unfavorable karyotypes, with hazard ratios (HR) of 3.45 (95% confidence interval [CI], 1.88-6.31) and 2.14 (95% CI, 1.04-4.04), respectively, compared with normal diploid group. OS of the miscellaneous group was similar to that of the normal diploid group (HR = 0.82; 95% CI, 0.44-1.53). Relapse-free survival (RFS) was not significantly associated with age (P = .30) but was heterogeneous among karyotype categories (P < .001) primarily because of poor RFS in t(9;22)/BCR/ABL1 (HR = 3.49; 95% CI, 1.80-6.75) compared with the normal diploid group. After accounting for the variation among karyotype groups, age was not a significant prognostic factor for OS or RFS, highlighting cytogenetics as the most important prognostic factor in adult ALL. This trial was registered at www.ClinicalTrials.gov as #NCT00002665.

Background: Chronic immune thrombocytopenic purpura (ITP) is characterised by accelerated platelet destruction and decreased platelet production. Short-term administration of the thrombopoiesis-stimulating protein, romiplostim, has been shown to increase platelet counts in most patients with chronic ITP. We assessed the long-term administration of romiplostim in splenectomised and non-splenectomised patients with ITP., Methods: In two parallel trials, 63 splenectomised and 62 non-splenectomised patients with ITP and a mean of three platelet counts 30x10(9)/L or less were randomly assigned 2:1 to subcutaneous injections of romiplostim (n=42 in splenectomised study and n=41 in non-splenectomised study) or placebo (n=21 in both studies) every week for 24 weeks. Doses of study drug were adjusted to maintain platelet counts of 50x10(9)/L to 200x10(9)/L. The primary objectives were to assess the efficacy of romiplostim as measured by a durable platelet response (platelet count > or =50x10(9)/L during 6 or more of the last 8 weeks of treatment) and treatment safety. Analysis was per protocol. These studies are registered with ClinicalTrials.gov, numbers NCT00102323 and NCT00102336., Findings: A durable platelet response was achieved by 16 of 42 splenectomised patients given romplostim versus none of 21 given placebo (difference in proportion of patients responding 38% [95% CI 23.4-52.8], p=0.0013), and by 25 of 41 non-splenectomised patients given romplostim versus one of 21 given placebo (56% [38.7-73.7], p<0.0001). The overall platelet response rate (either durable or transient platelet response) was noted in 88% (36/41) of non-splenectomised and 79% (33/42) of splenectomised patients given romiplostim compared with 14% (three of 21) of non-splenectomised and no splenectomised patients given placebo (p<0.0001). Patients given romiplostim achieved platelet counts of 50x10(9)/L or more on a mean of 13.8 (SE 0.9) weeks (mean 12.3 [1.2] weeks in splenectomised group vs 15.2 [1.2] weeks in non-splenectomised group) compared with 0.8 (0.4) weeks for those given placebo (0.2 [0.1] weeks vs 1.3 [0.8] weeks). 87% (20/23) of patients given romiplostim (12/12 splenectomised and eight of 11 non-splenectomised patients) reduced or discontinued concurrent therapy compared with 38% (six of 16) of those given placebo (one of six splenectomised and five of ten non-splenectomised patients). Adverse events were much the same in patients given romiplostim and placebo. No antibodies against romiplostim or thrombopoietin were detected., Interpretation: Romiplostim was well tolerated, and increased and maintained platelet counts in splenectomised and non-splenectomised patients with ITP. Many patients were able to reduce or discontinue other ITP medications. Stimulation of platelet production by romiplostim may provide a new therapeutic option for patients with ITP.

Identification of genetic abnormalities in pathological samples is critical for accurate diagnosis, risk stratification, detection of minimal residual disease, and assessment of response to therapy. Interphase fluorescence in situ hybridization analysis is the standard cytogenetic assay used by many laboratories to detect specific clonal karyotypic aberrations in formalin-fixed, paraffin-embedded tissue. However, direct correlation with immunophenotype or morphology in individual cells is rarely performed because the procedural steps are labor intensive and usually require extensive troubleshooting. In this study, we present a sequential fluorescence in situ hybridization-based technique that uses the identical archived bone marrow smears or paraffin-embedded tissue sections previously evaluated by a pathologist for morphological or immunohistochemical characteristics. This approach is relatively straightforward, using uncomplicated pretreatment and hybridization conditions and basic equipment attached to an automated image analyzer with image capture software to record the location of targeted cells for genotypic/phenotype correlation. Furthermore, the method has proved reliable and reproducible on test samples regardless of specimen age, tissue type, or referring institution.

We conducted a phase 1/2 trial of high-dose 90Y-ibritumomab tiuxetan in combination with high-dose etoposide (VP-16) 40 to 60 mg/kg (day -4) and cyclophosphamide 100 mg/kg (day -2) followed by autologous stem cell transplantation (ASCT) in 31 patients with CD20+ non-Hodgkin lymphoma (NHL). Patients underwent dosimetry (day -21) with 5 mCi (185 MBq) 111In-ibritumomab tiuxetan following 250 mg/m2 rituximab, followed a week later by 90Y-ibritumomab tiuxetan to deliver a target dose of 1000 cGy to highest normal organ. Bone marrow biopsy was done on day -7 to estimate radiation dose and stem cells were reinfused when the radiation dose was estimated to be less than 5 cGy. The median 90Y-ibritumomab tiuxetan dose was 71.6 mCi (2649.2 MBq; range, 36.6-105 mCi; range, 1354.2-3885 MBq). Histology included follicular lymphoma (n = 12), diffuse large B-cell (n = 14), and mantle cell (n = 5). The median number of prior chemo-therapy treatments was 2. The treatment was well tolerated. The median times to reach an absolute neutrophil count greater than 500/microL and platelet count more than 20,000/microL were 10 days and 12 days, respectively. There were 2 deaths and 5 relapses. At a median follow-up of 22 months, the 2-year estimated overall survival and relapse-free survival rates are 92% and 78%, respectively. We conclude that high-dose 90Y-ibritumomab tiuxetan can be combined safely with high-dose etoposide and cyclophosphamide without an increase in transplant-related toxicity or delayed engraftment.

Collagen-mediated platelet activation contributes significantly to coronary and cerebrovascular thrombus formation associated with atherosclerotic plaque destabilization. Recent clinical and laboratory observations support a potential role for the platelet Fc receptor (FcgammaRIIA) in this process. The purpose of this study was to elucidate any association between platelet Fc receptor (FcR) expression levels and both atherosclerosis risk factors (ARFs) along with collagen-dependent platelet activation. Age and gender-independent variation has been described in the expression of this receptor that is stable over time. Platelet Fc surface expression was compared between patients experiencing an acute coronary or cerebrovascular event, healthy patients with two or more ARFs, and healthy patients with fewer than two ARFs. Platelet FcR expression was significantly and stably (6-52 weeks, mean 20 weeks) increased in 101 patients with acute myocardial infarction, unstable angina, or ischemic stroke syndrome (P<0.001) and 38 healthy patients with two or more ARFs (P=0.027) compared with 109 healthy patients with fewer than two ARFs. Patients with diabetes mellitus from all groups had significantly increased platelet FcR expression over those without diabetes (P<0.0001). Platelet aggregation studies suggested a correlation between number of ARFs per patient, platelet Fc expression levels, and relative sensitivity to collagen stimulation. Platelet FcR surface expression is increased in patients with an acute coronary or cerebrovascular event, non-acutely ill patients with two or more ARFs, and in patients with diabetes mellitus. Increased platelet FcR expression may therefore contribute towards risk for atherothrombotic events.

Conventional methods for generating monocyte-derived dendritic cells (DC) for clinical trials utilize the property of plastic adherence to select monocytes from leukapheresis samples. This method is labor-intensive and has the potential for contamination at various steps. We evaluated a large-scale monocyte enrichment procedure using a cell selector (Isolex 300i(R)) followed by culture in a sterile bag system (Stericell(R)) for generation of DC. DC generated in tissue culture flasks after monocyte selection by plastic adherence were compared to those generated in Stericell(R) bags after monocyte enrichment by negative selection with the Isolex(R) 300i. DC were matured with lipopolysaccharide and pulsed with a peptide derived from the melanoma antigen gp100. Peptide-pulsed DC cultured by the two techniques were evaluated for phenotype, viability, ability to induce allogeneic and peptide-specific autologous proliferative responses as well as peptide-specific cytotoxic T-cell responses. The mean monocyte yield from leukapheresis collections was 17+/-2.4%, which increased to 52+/-11% after Isolex(R) selection. The DC yield of plated mononuclear cells from flasks or bags was 2.7+/-0.96% and 4.84+/-2.65%, respectively. DC cultured by both methods expressed high levels of CD86, CD80, CD40, CD83, CD44, CD11c and CD58, and was comparable in their ability to induce allogeneic and peptide-specific autologous proliferative responses as well as gp100 peptide-specific cytotoxic T-cell responses. These results indicate that potent monocyte-derived DC can be generated in a closed culture bag system after monocyte enrichment by immunomagnetic negative selection. Due to the closed nature of the enrichment and culture systems, the potential for contamination is minimized. This protocol is well suited for culturing large numbers of DC for clinical immunotherapy trials.