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Follicular lymphoma is an incurable B cell malignancy characterized by the t(14;18) translocation and mutations affecting the epigenome. Although frequent gene mutations in key signaling pathways, including JAK-STAT, NOTCH and NF-κB, have also been defined, the spectrum of these mutations typically overlaps with that in the closely related diffuse large B cell lymphoma (DLBCL). Using a combination of discovery exome and extended targeted sequencing, we identified recurrent somatic mutations in RRAGC uniquely enriched in patients with follicular lymphoma (17%). More than half of the mutations preferentially co-occurred with mutations in ATP6V1B2 and ATP6AP1, which encode components of the vacuolar H(+)-ATP ATPase (V-ATPase) known to be necessary for amino acid-induced activation of mTORC1. The RagC variants increased raptor binding while rendering mTORC1 signaling resistant to amino acid deprivation. The activating nature of the RRAGC mutations, their existence in the dominant clone and their stability during disease progression support their potential as an excellent candidate for therapeutic targeting.

The purpose of this work was to modernize recommendations for evaluation, staging, and response assessment of patients with Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). A workshop was held at the 11th International Conference on Malignant Lymphoma in Lugano, Switzerland, in June 2011, that included leading hematologists, oncologists, radiation oncologists, pathologists, radiologists, and nuclear medicine physicians, representing major international lymphoma clinical trials groups and cancer centers. Clinical and imaging subcommittees presented their conclusions at a subsequent workshop at the 12th International Conference on Malignant Lymphoma, leading to revised criteria for staging and of the International Working Group Guidelines of 2007 for response. As a result, fluorodeoxyglucose (FDG) positron emission tomography (PET)–computed tomography (CT) was formally incorporated into standard staging for FDG-avid lymphomas. A modification of the Ann Arbor descriptive terminology will be used for anatomic distribution of disease extent, but the suffixes A or B for symptoms will only be included for HL. A bone marrow biopsy is no longer indicated for the routine staging of HL and most diffuse large B-cell lymphomas. However, regardless of stage, general practice is to treat patients based on limited (stages I and II, nonbulky) or advanced (stage III or IV) disease, with stage II bulky disease considered as limited or advanced disease based on histology and a number of prognostic factors. PET-CT will be used to assess response in FDG-avid histologies using the 5-point scale. The product of the perpendicular diameters of a single node can be used to identify progressive disease. Routine surveillance scans are discouraged. These recommendations should improve evaluation of patients with lymphoma and enhance the ability to compare outcomes of clinical trials.

Problems of sexual function and fertility in long-term survivors (≥5 years) of haematological malignancy are often neglected in clinic. Our centre carried out a questionnaire study in this population addressing patient-perceived fertility and sexual function. 718 patients responded (56% of those invited; 39% Hodgkin, 45% non-Hodgkin lymphoma, 16% acute leukaemia). Respondent women were more likely to remain childless than a normal control population. Self-reported infertility was more likely in men than women [odds ratio (OR) 1·77, P = 0·001]. Myeloablative therapy increased the likelihood of childlessness (OR 2·48, P = 0·004). Few attended fertility support services (12%). 24% of men banked sperm and 29% of these used the sample, of which 46% resulted in successful pregnancy. Fertility clinic attendance and sperm storage was more likely post-1990 (OR 4·05, P < 0·001; OR 5·05, P < 0·001 respectively). Reporting a negative impact of cancer on sexual function was more common in women than men (OR 2·20, P < 0·001), and increased with current age and age at diagnosis (by 3-4% per year, P ≤ 0·001) but decreased with longer follow-up (by 2%/year, P = 0·005). Patients on anti-depressants and those reporting cancer-related body change/appearance concerns more frequently reported a negative impact (P < 0·04 and P < 0·03 respectively). These self-reported outcomes confirm literature findings, suggest improvement over time, but highlight a need for involvement of support services., (© 2013 John Wiley & Sons Ltd.)

Follicular lymphoma is an incurable malignancy, with transformation to an aggressive subtype representing a critical event during disease progression. Here we performed whole-genome or whole-exome sequencing on 10 follicular lymphoma-transformed follicular lymphoma pairs followed by deep sequencing of 28 genes in an extension cohort, and we report the key events and evolutionary processes governing tumor initiation and transformation. Tumor evolution occurred through either a 'rich' or 'sparse' ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histone, JAK-STAT signaling, NF-κB signaling and B cell developmental genes. Longitudinal analyses identified early driver mutations in chromatin regulator genes (CREBBP, EZH2 and KMT2D (MLL2)), whereas mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides new insights into the genetic basis of follicular lymphoma and the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations in the CPC represents an attractive therapeutic strategy.

Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.

Defining the role of high-dose therapy with autologous stem cell rescue in the therapeutic algorithm of follicular lymphoma remains a major challenge. In contrast to the acknowledged poor outcome associated with cyclophosphamide/total body irradiation conditioning in heavily pretreated patients, the prognostic impact of the number of previous therapy lines in patients treated with the chemotherapy-only containing regimen, BEAM, is unknown. From 1997 to 2008 80 patients (41 males, 39 females; median age, 51 years; range, 31-67) received high-dose therapy with autologous stem cell rescue with BEAM for relapsed follicular lymphoma at our center. Overall survival and time-to-progression were analyzed according to the number of prior treatment lines. The median number of previous treatment lines was three, with 61% of the patients having received more than three lines (including rituximab in 47%). After a median follow-up of 76 months (range, 14-160), three patients developed secondary myelodysplastic syndrome. The 5-year overall survival rate was 71% and 5-year time-to-progression was 44%. There were no differences in time-to-progression or overall survival according to the number of previous treatment lines or episodes of disease. In conclusion, high-dose therapy with autologous stem cell rescue with BEAM appears to be equally effective in second or third remission of follicular lymphoma.

Purpose: The immune microenvironment is key to the pathophysiology of classical Hodgkin lymphoma (CHL). Twenty percent of patients experience failure of their initial treatment, and others receive excessively toxic treatment. Prognostic scores and biomarkers have yet to influence outcomes significantly. Previous biomarker studies have been limited by the extent of tissue analyzed, statistical inconsistencies, and failure to validate findings. We aimed to overcome these limitations by validating recently identified microenvironment biomarkers (CD68, FOXP3, and CD20) in a new patient cohort with a greater extent of tissue and by using rigorous statistical methodology., Patients and Methods: Diagnostic tissue from 122 patients with CHL was microarrayed and stained, and positive cells were counted across 10 to 20 high-powered fields per patient by using an automated system. Two statistical analyses were performed: a categorical analysis with test/validation set-defined cut points and Kaplan-Meier estimated outcome measures of 5-year overall survival (OS), disease-specific survival (DSS), and freedom from first-line treatment failure (FFTF) and an independent multivariate analysis of absolute uncategorized counts., Results: Increased CD20 expression confers superior OS. Increased FOXP3 expression confers superior OS, and increased CD68 confers inferior FFTF and OS. FOXP3 varies independently of CD68 expression and retains significance when analyzed as a continuous variable in multivariate analysis. A simple score combining FOXP3 and CD68 discriminates three groups: FFTF 93%, 62%, and 47% (P < .001), DSS 93%, 82%, and 63% (P = .03), and OS 93%, 82%, and 59% (P = .002)., Conclusion: We have independently validated CD68, FOXP3, and CD20 as prognostic biomarkers in CHL, and we demonstrate, to the best of our knowledge for the first time, that combining FOXP3 and CD68 may further improve prognostic stratification.

Purpose: To investigate breast cancer risk after supradiaphragmatic radiotherapy administered to young women with Hodgkin's lymphoma (HL) in a much larger cohort than previously to provide data for patient follow-up and screening individualized according to treatment type, age, and time point during follow-up., Patients and Methods: Breast cancer risk was assessed in 5,002 women in England and Wales treated for HL with supradiaphragmatic radiotherapy at age < 36 years from 1956 to 2003, who underwent follow-up with 97% completeness until December 31, 2008., Results: Breast cancer or ductal carcinoma in situ developed in 373 patients, with a standardized incidence ratio (SIR) of 5.0 (95% CI, 4.5 to 5.5). SIRs were greatest for those treated at age 14 years (47.2; 95% CI, 28.0 to 79.8) and continued to remain high for at least 40 years. The maximum absolute excess risk was at attained ages 50 to 59 years. Alkylating chemotherapy or pelvic radiotherapy diminished the risk, but only for women treated at age ≥ 20 years, not for those treated when younger. Cumulative risks were tabulated in detail; for 40-year follow-up, the risk for patients receiving ≥ 40 Gy mantle radiotherapy at young ages was 48%., Conclusion: This article provides individualized risk estimates based on large numbers for patients with HL undergoing follow-up after radiotherapy at young ages. Follow-up of such women needs to continue for 40 years or longer and may require more-intensive screening regimens than those in national general population programs. Special consideration is needed of potential measures to reduce breast cancer risk for girls treated with supradiaphragmatic radiotherapy at pubertal ages.

The relative merits of reduced-intensity allogeneic stem cell transplantation (RISCT) for high-risk indolent lymphoid malignancies are emerging, although the preferred conditioning regimen to manage the risks of graft-versus-host disease (GVHD) is not clearly defined. Here we report the outcome of 73 patients with lymphoid malignancies who received RISCT with a fludarabine/cyclophosphosphamide conditioning regimen and a median follow-up of 3 years. Median age was 54 years. Forty-eight per cent of patients had previously undergone autologous stem cell transplantation with a median of three prior therapies. Non-relapse mortality at 3 years was 19% but only 5% for patients with multiple myeloma (MM). Three-year overall survival and current progression-free survival was 67% and 63% respectively. Grade 2-4 acute GVHD occurred in 14% of patients while 49% had chronic GVHD requiring systemic immunosuppression. The preparatory regimen in this study has the advantage of reduced acute GVHD and low mortality, notably in patients with MM. In addition, this strategy provides long-term disease control in a significant proportion of patients with particular benefit in those with high-risk follicular lymphoma., (© 2012 Blackwell Publishing Ltd.)

An intention-to-treat (ITT) analysis was performed in 103 unselected patients with relapsed/refractory classical Hodgkin lymphoma (CHL) comparing early relapse (<12 months) or failure of first-line therapy (ER/FTF) with late relapses (LR). Seventy one percentage proceeded to high-dose therapy/autologous stem cell rescue (HDT/ASCR) following salvage treatment. By ITT, 5-year overall survival (OS) was 50% for ER/FTF compared to 73% for LR patients (P = 0·012). However OS was equivalent for both groups if salvage treatment response was adequate to proceed to HDT/ASCR. ER/FTF patients remain a high-risk group largely due to a failure of salvage therapy: a point at which novel interventions could impact survival., (© 2012 Blackwell Publishing Ltd.)

Purpose: We investigated the long-term risk of second primary malignancy after chemotherapy for Hodgkin's lymphoma (HL) in a much larger cohort than any yet published, to our knowledge., Patients and Methods: We followed 5,798 patients with HL treated with chemotherapy in Britain from 1963 to 2001--of whom 3,432 also received radiotherapy--to assess second primary malignancy risks compared with general population-based expectations., Results: Second malignancies occurred in 459 cohort members. Relative risk (RR) of second cancer was raised after chemotherapy alone (RR, 2.0; 95% CI, 1.7 to 2.4) but was much lower than after combined modalities (RR, 3.9; 95% CI, 3.5 to 4.4). After chemotherapy alone, there were significantly raised risks of lung cancer, non-HL, and leukemia, each contributing approximately equal absolute excess risk. After combined modalities, there were raised risks of these and several other cancers. Second cancer risk peaked 5 to 9 years after chemotherapy alone, but it remained raised for 25 years and longer after combined modalities. Risk was raised after each common chemotherapy regimen except, based on limited numbers and follow-up, adriamycin, bleomycin, vinblastine, and dacarbazine. The age and time-course relations of lung cancer differed between chemotherapy alone and combined modalities., Conclusion: Although chemotherapy alone leads to raised risk of second malignancy, this risk is lower and affects fewer anatomic sites than that after combined modalities, and it is slight if at all after 15 years follow-up. The mechanism of lung cancer etiology may differ between chemotherapy and radiotherapy.

Dysregulation of the Wnt/β-catenin pathway has been observed in various malignancies, including acute myeloid leukemia (AML), where the overexpression of β-catenin is an independent adverse prognostic factor. β-catenin was found upregulated in the vast majority of AML samples and more frequently localized in the nucleus of leukemic stem cells compared with normal bone marrow CD34(+) cells. The knockdown of β-catenin, using a short hairpin RNA (shRNA) lentiviral approach, accelerates all-trans retinoic acid-induced differentiation and impairs the proliferation of HL60 leukemic cell line. Using in vivo quantitative tracking of these cells, we observed a reduced engraftment potential after xenotransplantation when β-catenin was silenced. However, when studying primary AML cells, despite effective downregulation of β-catenin we did not observe any impairment of their in vitro long-term maintenance on MS-5 stroma nor of their engraftment potential in vivo. Altogether, these results show that despite a frequent β-catenin upregulation in AML, leukemia-initiating cells might not be 'addicted' to this pathway and thus targeted therapy against β-catenin might not be successful in all patients.

Inherited risk determinants for follicular lymphoma (FL) have recently been described in the immune gene-rich human leukocyte antigen region on chromosome 6p. The known importance of host immune response to FL survival led us to evaluate these germline factors in FL outcome. We confirm the association of single nucleotide polymorphisms rs10484561 (P = 3.5 × 10⁻⁹) and rs6457327 (P = .008) with risk of FL and demonstrate that rs6457327 predicts both time to (P = .02) and risk of (P < .01) FL transformation independently of clinical variables, including the Follicular Lymphoma International Prognostic Index.

Background: The impact of active surveillance, comprising annual computed tomography scanning and bone marrow biopsies, in the follow-up of patients after high-dose therapy with autologous stem cell rescue for recurrent follicular lymphoma was analyzed., Design and Methods: Seventy-one of 99 patients who received high-dose therapy commenced the surveillance program. Response duration, time to next treatment and overall survival were compared according to whether disease progression had been diagnosed on the basis of surveillance investigations or on clinical grounds., Results: After a median follow-up of 16 years, progression was documented by surveillance in 16 patients and clinically in 18, the median response duration being 2.4 and 2.3 years, respectively (P=NS). Ten patients with a relapse detected clinically started treatment immediately, contrasting with one patient whose relapse was detected by surveillance investigations. Five patients with relapses detected by surveillance investigations have not required treatment after a median follow-up of 18 years, whereas all but two patients with a relapse detected clinically have been treated. The median time to next treatment was 7 years for patients with a relapse identified by surveillance investigations and 4 years for those whose relapse was manifested clinically (P=0.03). Overall survival was not significantly different between the two groups., Conclusions: Surveillance investigations, consisting of annual computed tomography scanning and bone marrow biopsies, have no impact on the management of patients with recurrent follicular lymphoma and do not improve the outcome of these patients.

Follicular lymphoma has considerable clinical heterogeneity, and there is a need for easily quantifiable prognostic biomarkers. Microvessel density has been shown to be a useful prognostic factor based on numerical assessment of vessel numbers within histologic sections in some studies, but assessment of tumor neovascularization through angiogenic sprouting may be more relevant. We therefore examined the smallest vessels, single-staining structures measuring less than 30 microm(2) in area, seen within histologic sections, and confirmed that they were neovascular angiogenic sprouts using extended focal imaging. Tissue microarrays composing diagnostic biopsies from patients at the extremes of survival of follicular lymphoma were analyzed with respect to numbers of these sprouts. This analysis revealed higher angiogenic activity in the poor prognostic group and demonstrated an association between increased sprouting and elevated numbers of infiltrating CD163(+) macrophages within the immediate microenvironment surrounding the neovascular sprout.

A variety of genetic lesions, including chromosomal translocations, internal tandem duplications, and mutations, have been described in acute myeloid leukemia (AML). Expression profiling has shown that chromosomal translocations, in particular, are associated with distinctive patterns of gene expression. AML exhibiting the translocation t(8;21), which fuses the AML1 and ETO genes, has such a characteristic expression profile. One gene whose expression is highly correlated with the presence of the AML1/ETO fusion is POU4F1, which encodes the POU homeodomain transcription factor BRN3A. Here we show using specific siRNA in t(8;21) cells and overexpression studies in progenitor cells that AML1/ETO promotes expression of POU4F1/BRN3A. This effect requires DNA-binding function of AML1/ETO, and accordingly, AML1/ETO is bound to the POU4F1 locus in t(8;21) cells. Functionally, whereas overexpression of Brn3a in murine hematopoietic progenitor cells induces terminal myeloid differentiation, coexpression of AML1/ETO or AML1/ETO9a blocks this effect. Furthermore, Brn3a reduction by shRNA impairs AML1/ETO-induced immortalization of murine progenitors. In summary, we identify POU4F1/BRN3A as a novel potential upregulated AML1/ETO target gene whose dramatically high expression may cooperate with AML1/ETO in t(8;21) cells., ((c)2010 AACR.)

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

PURPOSE: There is a need for new treatments for Hodgkin and T-cell lymphoma due to the development of drug resistance in a proportion of patients. This phase I study of radioimmunotherapy used CHT-25, a chimeric antibody to the alpha-chain of the interleukin-2 receptor, CD25, conjugated to iodine-131 ((131)I) in patients with refractory CD25-positive lymphomas. EXPERIMENTAL DESIGN: Fifteen patients were treated (Hodgkin lymphoma, 12; angioimmunoblastic T-cell lymphoma, 1; adult T-cell leukemia/lymphoma, 2). Tumor was monitored by computed tomography and in all but two patients by (18)F-fluorodeoxyglucose positron emission tomography. RESULTS: There were no grade 3 or 4 infusion reactions. At the maximum tolerated dose of 1,200 MBq/m(2), the major side effect was delayed myelotoxicity with the nadir for platelets at 38 days and for neutrophils at 53 days. One patient treated with 2,960 MBq/m(2) developed prolonged grade 4 neutropenia and thrombocytopenia and died of Pneumocystis jiroveci pneumonia. Nonhematologic toxicity was mild. Single photon emission computer tomography imaging showed tumor-specific uptake and retention of (131)I and no excessive retention in normal organs. Of nine patients receiving >/=1,200 MBq/m(2), six responded (three complete response and three partial response); one of six patients with administered radioactivity of

An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.

Understanding how the immune system in patients with cancer interacts with malignant cells is critical for the development of successful immunotherapeutic strategies. We studied peripheral blood from newly diagnosed patients with acute myeloid leukemia (AML) to assess the impact of this disease on the patients' T cells. The absolute number of peripheral blood T cells is increased in AML compared with healthy controls. An increase in the absolute number of CD3+56+ cells was also noted. Gene expression profiling on T cells from AML patients compared with healthy donors demonstrated global differences in transcription suggesting aberrant T-cell activation patterns. These gene expression changes differ from those observed in chronic lymphocytic leukemia (CLL), indicating the heterogeneous means by which different tumors evade the host immune response. However, in common with CLL, differentially regulated genes involved in actin cytoskeletal formation were identified, and therefore the ability of T cells from AML patients to form immunologic synapses was assessed. Although AML T cells could form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signaling molecules to the synapse was significantly impaired. These findings identify T-cell dysfunction in AML that may contribute to the failure of a host immune response against leukemic blasts.

To date, studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation, block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens, we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique, unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia, was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33, CD34, CD123, CD11c and CD36, to deplete myeloblasts.

To investigate the cell of origin linking follicular (FL) and transformed (t-FL) lymphomas, we analyzed the somatic hypermutation (SHM) pattern of the variable region of the immunoglobulin heavy gene (IgH-VH) in 18 sequential FL/t-FL samples and a father (donor) and son (recipient), who developed FL and t-FL, after transplantation. Genealogic trees showed a pattern compatible with a common progenitor cell (CPC) origin in 13 cases. The identification of the t-FL clonotype in the previous FL sample and of the putative CPC sequence in both the FL/t-FL biopsies showed that the intraclonal diversity of FL and t-FL germinal centers (GCs) is more intricate than previously described, and all 3 clonotypes (CPC, FL, t-FL) may occur simultaneously within the same lymph node. On the basis of the father/son model, this CPC must be long-lived, providing a possible explanation for the incurable nature of this disease.

Acquired homozygosity in the form of segmental acquired uniparental disomy (aUPD) has been described in follicular lymphoma (FL) and is usually due to mitotic recombination. SNP array analysis was performed with the use of the Affymetrix 10K 2.0 Gene-chip array on DNA from 185 diagnostic FL patients to assess the prognostic relevance of aUPD. Genetic abnormalities were detected in 118 (65%) of 182 patients. Number of abnormalities was predictive of outcome; more than 3 abnormalities was associated with inferior overall survival (OS; P < .03). Sites of recurrent aUPD were detected on 6p (n = 25), 16p (n = 22), 12q (n = 17), 1p36 (n = 14), 10q (n = 8), and 6q (n = 8). On multivariate analysis aUPD on 1p36 correlated with shorter OS (P = .05). aUPD on 16p was predictive of transformation (P = .03) and correlated with poorer progression-free survival (P = .02). aUPD is frequent at diagnosis of FL and affects probability of disease transformation and clinical outcome.

The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein overexpression in most follicular lymphomas. However the expression of BCL2 is not always homogeneous and may demonstrate a variable degree of heterogeneity. This study analysed BCL2 protein expression pattern in 33 cases of t(14;18)-positive follicular lymphomas using antibodies against two different epitopes (i.e. the widely used antibody BCL2/124 and an alternative antibody E17). 16/33 (49%) cases demonstrated strong BCL2 expression. In 10/33 (30%) cases, BCL2 expression was heterogeneous and in some of these, its loss appeared to be correlated with cell proliferation, as indicated by Ki67 expression. Double immunofluorescence labelling confirmed an inverse BCL2/Ki67 relationship, where in 24/28 (86%) cases cellular expression of BCL2 and Ki67 was mutually exclusive. In addition, seven BCL2 'pseudo-negative' cases were identified in which immunostaining was negative with antibody BCL2/124, but positive with antibody E17. Genomic DNA sequencing of these 'pseudo-negative' cases demonstrated eleven mutations in four cases and nine of these were missense mutations. It can be concluded that in follicular lymphomas, despite carrying the t(14;18) translocations, BCL2 protein expression may be heterogeneous and loss of BCL2 could be related to cell proliferation. Secondly, mutations in translocated BCL2 genes appear to be common and may cause BCL2 pseudo-negative immunostaining.

Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is an autosomal dominant syndrome characterized by platelet abnormalities and a predisposition to myelodysplasia (MDS) and/or acute myeloid leukemia (AML). The disorder, caused by inherited mutations in RUNX1, is uncommon with only 14 pedigrees reported. We screened 10 families with a history of more than one first degree relative with MDS/AML for inherited mutations in RUNX1. Germ- line RUNX1 mutations were identified in 5 pedigrees with a 3:2 predominance of N-terminal mutations. Several affected members had normal platelet counts or platelet function, features not previously reported in FPD/AML. The median incidence of MDS/AML among carriers of RUNX1 mutation was 35%. Individual treatments varied but included hematopoietic stem cell transplantation from siblings before recognition of the inherited leukemogenic mutation. Transplantation was associated with a high incidence of complications including early relapse, failure of engraftment, and posttransplantation lymphoproliferative disorder. Given the small size of modern families and the clinical heterogeneity of this syndrome, the diagnosis of FPD/AML could be easily overlooked and may be more prevalent than previously recognized. Therefore, it would appear prudent to screen young patients with MDS/AML for RUNX1 mutation, before consideration of sibling hematopoietic stem cell transplantation.

Purpose: To determine the clinical relevance of Wilms' tumor 1 (WT1) gene mutations in acute myeloid leukemia (AML) with normal karyotype (NK)., Patients and Methods: Exons 7 and 9 of WT1 were screened in samples from 470 young adult NK AMLs using a combination of direct sequencing and high-resolution capillary electrophoresis., Results: Overall, 51 mutations were detected in 47 cases (10%): 46 frameshift mutations with insertion/deletion of one to 28 base pairs in exon 7 (n = 45) or exon 9 (n = 1), with a median mutant level of 45% (range, 8% to 86%), and five substitutions in exon 9: D396N (n = 3), H397Y (n = 1) and H397Q (n = 1). Patients with WT1 mutations had an inferior response to induction chemotherapy compared with wild-type cases (complete remission rate, 79% v 90%, odds ratio [OR] = 3.02; 95% CI, 1.17 to 7.82; P = .02), a higher rate of resistant disease (15% v 4%; OR = 9.33; 95% CI, 2.38 to 36.6; P = .001), an increased cumulative incidence of relapse (67% v 43%, hazard ratio [HR] = 3.02; 95% CI, 1.69 to 5.38; P = .0008), with a reduction in both relapse-free survival (22% v 44%; HR = 2.16; 95% CI, 1.32 to 3.55; P = .005) and overall survival (26% v 47%; HR = 1.91; 95% CI, 1.23 to 2.95; P = .007) at 5 years. In multivariate analysis, which included FLT3 internal tandem duplication and NPM1 mutation status, the presence of a WT1 mutation remained an independent adverse prognostic factor., Conclusion: WT1 mutations are a negative prognostic indicator in NK AML and may be suitable for the development of targeted therapy.

The International Prognostic Index and the Follicular Lymphoma International Prognostic Index are widely used for the risk assessment of follicular lymphoma (FL). Although molecular studies have provided insight into the biology of FL, no molecular marker has impacted on treatment stratification. Because TP53 mutations are associated with poor prognosis in hematologic malignancies, we investigated the prognostic value of TP53 mutation at diagnosis in FL. Heterozygous TP53 mutation was detected in 12 of 185 (6%) analyzed cases. Mutation was associated with older age (P = .02) and higher International Prognostic Index score (P = .04). On multivariate analysis, TP53 mutation correlated with shorter progression-free survival (P < .001) and overall survival (P = .009). TP53 mutation was associated with low expression of the immune-response 1 gene expression signature (P = .016) and with an unfavorable gene expression-based survival predictor score (P < .001), demonstrating for the first time that molecular features of the malignant cell may correlate with the nature of the immune response in FL.

Despite advances in the curative treatment of acute myeloid leukemia (AML), recurrence will occur in the majority of cases. At diagnosis, acquisition of segmental uniparental disomy (UPD) by mitotic recombination has been reported in 15% to 20% of AML cases, associated with homozygous mutations in the region of loss of heterozygosity. This study aimed to discover if clonal evolution from heterozygous to homozygous mutations by mitotic recombination provides a mechanism for relapse. DNA from 27 paired diagnostic and relapsed AML samples were analyzed using genotyping arrays. Newly acquired segmental UPDs were observed at relapse in 11 AML samples (40%). Six were segmental UPDs of chromosome 13q, which were shown to lead to a change from heterozygosity to homozygosity for internal tandem duplication mutation of FLT3 (FLT3 ITD). Three further AML samples had evidence of acquired segmental UPD of 13q in a subclone of the relapsed leukemia. One patient acquired segmental UPD of 19q that led to homozygosity for a CEBPA mutation 207C>T. Finally, a single patient with AML acquired segmental UPD of chromosome 4q, for which the candidate gene is unknown. We conclude that acquisition of segmental UPD and the resulting homozygous mutation is a common event associated with relapse of AML.

Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukemia-initiating cells. One method commonly used to isolate these rare cells is to sort cells stained with fluorochrome-conjugated antibodies into fractions, then transplant the different fractions into immunodeficient mice to test their repopulating ability. The antibodies are generally treated as being neutral in terms of their effects on the experiment. Human repopulating cells are thought to express CD34 and lack CD38. Here we present evidence that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukemia cells. We show that this effect is Fc-mediated and can be overcome by treating mice with immunosuppressive antibodies. When this inhibitory effect is prevented, we demonstrate that the CD34(+)CD38(+) fraction of certain acute myeloid leukemia samples contains all, or at least most, leukemia-initiating cell capacity. This study highlights the potential pitfall of antibody-mediated clearance of repopulating cells and is important for any groups working with this model. More importantly, the work suggests that there is greater variation in the phenotypes of leukemia-initiating cells than previously suggested.

We present here a genome-wide map of abnormalities found in diagnostic samples from 45 adults and adolescents with acute lymphoblastic leukemia (ALL). A 500K SNP array analysis uncovered frequent genetic abnormalities, with cryptic deletions constituting half of the detected changes, implying that microdeletions are a characteristic feature of this malignancy. Importantly, the pattern of deletions resembled that recently reported in pediatric ALL, suggesting that adult, adolescent, and childhood cases may be more similar on the genetic level than previously thought. Thus, 70% of the cases displayed deletion of one or more of the CDKN2A, PAX5, IKZF1, ETV6, RB1, and EBF1 genes. Furthermore, several genes not previously implicated in the pathogenesis of ALL were identified as possible recurrent targets of deletion. In total, the SNP array analysis identified 367 genetic abnormalities not corresponding to known copy number polymorphisms, with all but two cases (96%) displaying at least one cryptic change. The resolution level of this SNP array study is the highest used to date to investigate a malignant hematologic disorder. Our findings provide insights into the leukemogenic process and may be clinically important in adult and adolescent ALL. Most importantly, we report that microdeletions of key genes appear to be a common, characteristic feature of ALL that is shared among different clinical, morphological, and cytogenetic subgroups.

Although recent reports suggest that selenium can modulate the activity of cytotoxic drugs, the mechanism underlying this activity remains unclear. This has been investigated using a panel of human B-cell lymphoma cell lines. The cytotoxic effects of chemotherapeutic agents (e.g., doxorubicin, etoposide, 4-hydroperoxycyclophosphamide, melphalan, and 1-beta-d-arabinofuranosylcytosine) were increased by up to 2.5-fold when combined with minimally toxic concentrations (EC(5-10)) of the organic selenium compound, methylseleninic acid (MSA). DNA strand breaks were identified using comet assays, but the measured genotoxic activity of the combinations did not explain the observed synergistic effects in cell death. However, minimally toxic (EC(10)) concentrations of MSA induced a 50% decrease in nuclear factor-kappaB (NF-kappaB) activity after an exposure of 5 h, similar to that obtained with the specific NF-kappaB inhibitor, BAY 11-7082. Combinations of BAY 11-7082 with these cytotoxic drugs also resulted in synergism, suggesting that the chemosensitizing activity of MSA is mediated, at least in part, by its effects on NF-kappaB. Basal intracellular selenium concentration was higher in a MSA-sensitive cell line. After exposure to MSA, methylselenocysteine and selenomethionine were identified as the main intracellular species generated. Volatile selenium species, trapped using solid-phase microextraction fibers, were identified as dimethylselenide and dimethyldiselenide. These volatile species are thought to be the most biologically active forms of selenium. Taken together, these results show that the NF-kappaB pathway is one target for MSA underlying the interaction between MSA and chemotherapy. These data encourage the further clinical development of selenium as a potential modulator of cytotoxic drug activity in B-cell lymphomas.

Purpose: The aim of this retrospective analysis was to determine the outcome of patients with follicular lymphoma who received myeloablative therapy supported by autologous bone marrow transplantation as consolidation of second or subsequent remission, with a minimum follow-up of 12 years., Patients and Methods: One hundred twenty-one adults received cyclophosphamide (CY) and total-body irradiation (TBI) supported by autologous bone marrow transplantation, with the marrow mononuclear cell fraction having been treated with monoclonal antibodies and complement. Data from St Bartholomew's Hospital and Dana-Farber Cancer Institute were combined for the purpose of this analysis because the patients were treated in an identical manner., Results: Fifty-seven patients are alive, 41 without progression between 9 and 19 years; 64 patients have died, 20 without progression. With a median follow-up of 13.5 years, 60 patients have developed recurrent lymphoma. There is an apparent plateau on the remission duration curve at 48% at 12 years. Survival of patients treated in second remission was significantly longer than the survival of patients treated later in the course of the illness. Both remission duration and overall survival were also significantly longer for patients treated in second remission compared with an age-matched, remission-matched group of patients treated at St Bartholomew's Hospital before the introduction of this treatment. However, use of CY+TBI was associated with a significant risk of secondary myelodysplasia and secondary acute myeloblastic leukemia, resulting in 15 patient deaths., Conclusion: These mature data confirm that prolonged freedom from recurrence may be achieved with myeloablative therapy and that a plateau on the curve seems to emerge with long follow-up.

Purpose: To study the clinical significance of transformation to diffuse large B-cell lymphoma (DLBCL) in patients with follicular lymphoma (FL)., Patients and Methods: From 1972 to 1999, 325 patients were diagnosed with FL at St Bartholomew's Hospital (London, United Kingdom). With a median follow-up of 15 years, progression occurred in 186 patients and biopsy-proven transformation in 88 of the 325. The overall repeat biopsy rate was 70%., Results: The risk of histologic transformation (HT) by 10 years was 28%, HT not yet having been observed after 16.2 years. The risk was higher in patients with advanced stage (P = .02), high-risk Follicular Lymphoma International Prognostic Index (FLIPI; P = .01), and International Prognostic Index (IPI; P = .04) scores at diagnosis. Expectant management (as opposed to treatment being initiated at diagnosis) also predicted for a higher risk of HT (P = .008). Older age (P = .005), low hemoglobin level (P = .03), high lactate dehydrogenase (P < .0001), and high-risk FLIPI (P = .01) or IPI (P = .003) score at the time of first recurrence were associated with the diagnosis of HT in a biopsy performed at that time. The median survival from transformation was 1.2 years. Patients with HT had a shorter overall survival (P < .0001) and a shorter survival from progression (P < .0001) than did those in whom it was not diagnosed., Conclusion: Advanced stage and high-risk FLIPI and IPI scores at diagnosis correlate with an increased risk of HT. This event strongly influences the outcome of patients with FL by shortening their survival. There may be a subgroup of patients in whom HT does not occur.

Bortezomib is a proteasome inhibitor with proven efficacy in multiple myeloma and non-Hodgkin's lymphoma. This study reports the effects of bortezomib in B-cell lymphoma cell lines with differing sensitivity to bortezomib to investigate factors that influence sensitivity. Bortezomib induced a time- and concentration-dependent reduction in cell viability in five lymphoma cell lines, with EC(50) values ranging from 6 nmol/L (DHL-7 cells) to 25 nmol/L (DHL-4 cells) after 72 h. Bortezomib cytotoxicity was independent of p53 function, as all cell lines exhibited mutations by sequence analysis. The difference in sensitivity was not explained by proteasome or nuclear factor-kappaB (NF-kappaB) inhibition as these were similar in the most and least sensitive cells. NF-kappaB inhibition was less marked than that of a specific NF-kappaB inhibitor, Bay 11-7082. Cell cycle analysis showed a marked G(2)-arrested population in the least sensitive DHL-4 line only, an effect that was not present with Bay 11-7082 treatment. Conversely, in DHL-7 cells, bortezomib treatment resulted in cells moving into an aberrant mitosis, indicative of mitotic catastrophe that may contribute to increased sensitivity to bortezomib. These studies show that although bortezomib treatment had similar effects on apoptotic and NF-kappaB signaling pathways in these cell lines, different cell cycle effects were observed and induction of a further mechanism of cell death, mitotic catastrophe, was observed in the more sensitive cell line, which may provide some pointers to the difference in sensitivity between cell lines. An improved understanding of how DHL-7 cells abrogate the G(2)-M cell cycle checkpoint may help identify targets to increase the efficacy of bortezomib.

Purpose: Standardized response criteria are needed to interpret and compare clinical trials and for approval of new therapeutic agents by regulatory agencies., Methods: The International Working Group response criteria (Cheson et al, J Clin Oncol 17:1244, 1999) were widely adopted, but required reassessment because of identified limitations and the increased use of [18F]fluorodeoxyglucose-positron emission tomography (PET), immunohistochemistry (IHC), and flow cytometry. The International Harmonization Project was convened to provide updated recommendations., Results: New guidelines are presented incorporating PET, IHC, and flow cytometry for definitions of response in non-Hodgkin's and Hodgkin's lymphoma. Standardized definitions of end points are provided., Conclusion: We hope that these guidelines will be adopted widely by study groups, pharmaceutical and biotechnology companies, and regulatory agencies to facilitate the development of new and more effective therapies to improve the outcome of patients with lymphoma.

Purpose: To examine the immune microenvironment in diagnostic follicular lymphoma (FL) biopsies and evaluate its prognostic significance., Patients and Methods: Immunohistochemistry was used to study numbers and location of cells staining positive for immune cell markers CD4, CD7, CD8, CD25, CD68, forkhead box protein P3 (FOXP3), T-cell intracellular antigen-1, and Granzyme B in tissue microarrays of paraffin-embedded, diagnostic lymph node biopsies taken from 59 FL patients who lived less than 5 years (short-survival group; n = 34) and more than 15 years (long-survival group; n = 25)., Results: CD4 and FOXP3 expression were significantly different between the two groups. Samples from the long-survival group were more likely than those from the short-survival group to have CD4+ staining cells and to have FOXP3-positive cells in a perifollicular location., Conclusion: This study has identified differences in immune cell composition of the diagnostic FL lymph node immune microenvironment and these have the potential for use as prognostic biomarkers in a routine histopathology setting.

Purpose: A multicenter, single-arm study examining efficacy and toxicity of epratuzumab combined with rituximab was conducted in patients with recurrent or refractory non-Hodgkin's lymphoma., Patients and Methods: Sixty-five patients were enrolled; 34 patients with follicular lymphoma (FL), 15 patients with diffuse large B-cell lymphoma (DLBCL), and 16 patients with other lymphomas. The patients had received a median of two prior therapies (range, 1 to 4); 23% had received rituximab. Epratuzumab was given at 360 mg/m2 intravenously over 60 minutes followed by infusion of 375 mg/m2 rituximab, weekly for 4 consecutive weeks., Results: Combination therapy was well tolerated without greater toxicity than rituximab alone. The objective response (OR) rate was 47% (30 of 64) in assessable patients (46%; 30 of 65 in all patients), being highest in FL (64%; 21 of 33) and DLBCL (47%; seven of 15), and with 24% (eight of 33) and 33% (five of 15) achieving complete response (CR) or complete response unconfirmed (CRu) in these two groups, respectively. Two of six patients with marginal zone lymphoma responded to treatment (one CR). There was a trend for the response rates to be higher in patients with low prognostic index scores (statistically significant with respect to the Follicular Lymphoma International Prognostic Index score in FL patients), with 12 FL patients and three DLBCL patients in groups 0 to 1 having OR (CR/CRu) rates of 83% (33%) and 100% (100%), respectively. The median duration of response was 16 months for FL, with five patients currently progression free for 18 months to 30 months, and 6 months for DLBCL, with two patients currently progression free for 12 months and 18 months., Conclusion: Epratuzumab combined with rituximab was well tolerated, demonstrating promising antilymphoma activity that warrants additional study.

Purpose: To determine the efficacy of bortezomib in patients with lymphoid malignancy, correlating clinical response with effect on plasma cytokines and in vitro activity in primary cultures., Patients and Methods: Patients received bortezomib (1.3 mg/m2) on days 1, 4, 8, and 11 of a 3-week cycle. Plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-6 were measured before each treatment, and bortezomib activity was examined in patient samples grown in primary culture., Results: Fifty-one patients received a total of 193 cycles of treatment. Twenty-four patients had mantle cell lymphoma (MCL), 13 had follicular lymphoma (FL), six had lymphoplasmacytic lymphoma, six had Hodgkin's disease (HD), and one each had diffuse large B-cell lymphoma and adult T-cell leukemia/lymphoma. Patients were heavily pretreated with a median of four previous therapies. Significant grade 3 to 4 toxicities were thrombocytopenia (n = 22), fatigue (n = 10), and peripheral neuropathy (n = 3). Seven patients with MCL responded to treatment (one complete response, six partial responses [PRs]; overall response rate, 29%). Two patients with FL achieved a late PR 3 months after discontinuing therapy. Two patients with Waldenström's macroglobulinemia and one patient with HD achieved a PR. MCL primary cultures demonstrated greater sensitivity to bortezomib than FL (median 50% effective concentration for viability, 209 nmol/L v 1,311 nmol/L, respectively; P = .07), which correlated with clinical response. A median reduction in plasma TNF-alpha of 98% was observed in six patients with MCL who responded to bortezomib compared with a reduction of 38% in six nonresponders (P = .07)., Conclusion: Bortezomib demonstrates encouraging efficacy in MCL in heavily pretreated individuals. Response was associated with a reduction in plasma TNF-alpha and in vitro sensitivity in a small number of patients.

KIT mutation has been implicated in sporadic mastocytosis, yet clusters in only a few sites in the molecule. For those malignancies associated with KIT mutation or over-expression, imatinib offers a specific therapeutic option, yet it has no effect on D816V mutation commonly seen in sporadic mastocytosis. The majority of cases of familial mastocytosis seem to lack KIT mutation. We report a kindred with mastocytosis in whom in vitro and in vivo sensitivity to imatinib was demonstrated. Mutation analysis of the KIT coding region in this family identified a novel A>T mutation at nucleotide 1547 [K509I] in exon 9 in both of the affected patients.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) assay is the current model for assessment of human normal and leukemic stem cells. We explored why 51% of 59 acute myeloid leukemia (AML) patients were unable to initiate leukemia in NOD/SCID mice. Increasing the cell dose, using more permissive recipients, and alternative tissue sources did not cause AML engraftment in most previously nonengrafting AML samples. Homing of AML cells to the marrow was the same between engrafters and nonengrafters. FLT3 internal tandem duplication (ITD) and nucleophosmin mutations occurred at a similar frequency in engrafters and nonengrafters. The only variable that was related to engraftment ability was the karyotypically defined risk stratification of individual AML cases. Of interest, follow-up of younger patients with intermediate-risk AML revealed a significant difference in overall survival between NOD/SCID engrafting and nonengrafting AMLs. Hence, the ability of AML to engraft in the NOD/SCID assay seems to be an inherent property of AML cells, independent of homing, conditioning, or cell frequency/source, which is directly related to prognosis. Our results suggest an important difference between leukemic initiating cells between engrafting and nonengrafting AML cases that correlates with treatment response.

Human hematopoietic stem cells (HSCs) are generally regarded as being devoid of the markers expressed by differentiated blood cells, the lineage-specific antigens. However, recent work suggests that genes associated with the myeloid lineage are transcribed in mouse HSCs. Here, we explore whether myeloid genes are actually translated in human HSCs. We show that CD33, CD13, and CD123, well-established myeloid markers, are expressed on human long-term repopulating cells from cord blood and bone marrow. In addition, we demonstrate that nonobese diabetic/severe combined immunodeficiency (NOD/SCID) leukemia-initiating cells (SL-ICs) are restricted to the CD33+ fraction in 11 of 12 acute myeloid leukemia (AML) samples studied, indicating that leukemic stem cells (LSCs) express this antigen. This study changes our view of HSCs and the process of differentiation. Furthermore, based on the phenotypic similarity of HSCs and LSCs, our data provide support for the hypothesis that AML derives from an HSC. Our findings also provide a challenge to contemporary attempts to improve the outcome of AML using myeloid antigen-targeted therapies, given the potential for HSC killing.