Allogeneic hematopoietic stem cell transplantation (HSCT) has been considered as the treatment of choice for patients with high-risk chronic lymphocytic leukemia (CLL) (i.e., refractory to purine analogues, short response (< 24 months) to chemoimmunotherapy, and/or presence of 17p-/TP53 mutations). Currently, treatment algorithms for HR-CLL are being challenged by the introduction of novel classes of drugs. Among them, BCR-signal inhibitors (BCRi) and BCL2 antagonists (BCL2a) appear particularly promising. As a result of the growing body of favourable outcome data reported for BCRi/BCL2a, uncertainty is emerging on how to advise patients with high-risk CLL about indication and timing of HSCT. This position paper provides an overview of currently available evidence and theoretical considerations to guide this difficult decision process. As long as the risks and benefits of different treatment strategies are not settled, all patients with high-risk CLL should be considered for treatment with BCRi/BCL2a. For those patients responding to these agents there are two treatment possibilities: (1) performing an HSCT, and (2) to continue on the novel drug. Individual disease-specific and transplant-related risk factors, along with patient's preferences, should be taken into account when advising one of these treatments over the other.
Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2- lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2- Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease.
Discovery of the enzymatic activity that catalyses oxidation of 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) mediated by the MLL fusion partner TET1 has sparked intense research to understand the role this new DNA modification has in cancer. An unambiguous picture has emerged where tumours are depleted of 5hmC compared to corresponding normal tissue, but it is not known whether lack of 5hmC is a cause or a consequence of tumourigenesis. Experimental data reveals a dual tumour-suppressive and oncogenic role for TET proteins. Tet2 is a driver in haematological malignancies but Tet1 had an oncogenic role in MLL-rearranged leukaemia, where Tet1 is overexpressed. Overexpression of Tet2 in melanoma cells re-established the 5hmC landscape and suppressed cancer progression but inhibiting Tet1 in non-transformed cells did not initiate cellular transformation. In this review we summarise recent findings that have shaped the current understanding on the role 5hmC plays in cancer.
Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-?B activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.
ZAP-70 methylation 223 nucleotides downstream of transcription start (CpG+223) predicts outcome in chronic lymphocytic leukemia (CLL), but its impact relative to CD38 and ZAP-70 expression or immunoglobulin heavy chain variable region (IGHV) status is uncertain. Additionally, standardizing ZAP-70 expression analysis has been unsuccessful. CpG+223 methylation was quantitatively determined in 295 untreated CLL cases using MassARRAY. Impact on clinical outcome vs CD38 and ZAP-70 expression and IGHV status was evaluated. Cases with low methylation (<20%) had significantly shortened time to first treatment (TT) and overall survival (OS) (P < .0001). For TT, low methylation defined a large subset of ZAP-70 protein-negative cases with significantly shortened TT (median, 8.0 vs 3.9 years for high vs low methylation; hazard ratio [HR] = 0.43; 95% confidence interval [CI], 0.25-0.74). Conversely, 16 ZAP-70 protein-positive cases with high methylation had poor outcome (median, 1.1 vs 2.3 years for high vs low methylation; HR = 1.62; 95% CI, 0.87-3.03). For OS, ZAP-70 methylation was the strongest risk factor; CD38 and ZAP-70 expression or IGHV status did not significantly improve OS prediction. A pyrosequencing assay was established that reproduced the MassARRAY data (? coefficient > 0.90). Thus, ZAP-70 CpG+223 methylation represents a superior biomarker for TT and OS that can be feasibly measured, supporting its use in risk-stratifying CLL.
Over the past decade, there have been significant advances in our understanding of the pathogenesis of chronic lymphocytic leukemia (CLL), which has been accompanied by an explosion in treatment options. Although the combination of fludarabine, cyclophosphamide, and rituximab is the current frontline treatment of choice for fit patients, targeted therapies such ibrutinib, idelalisib, and ABT-199 are showing great promise in clinical trials. However, none of these drugs seems curative, and allogeneic hematopoietic stem cell transplantation remains the only strategy that produces durable clinical remissions in otherwise poor-risk disease. Immune reconstitution remains an enticing prospect in CLL, as malignant B cells should be particularly susceptible to a T cell-mediated attack. It has recently been demonstrated that the T-cell defect in CLL can be effectively overcome by both lenalidomide treatment and by adoptive transfer of chimeric antigen receptor T cells. A variety of other immunotherapies are in development, including CLL vaccines, CD40 ligand therapies, and monoclonal antibody immune checkpoint blockade. This review explores the nature of the immune defect in CLL and summarizes the recent developments in the immunotherapeutic field.
The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with ?2-integrin expression being modulated by NOTCH1 mutation status.
Most patients with chronic lymphocytic leukemia (CLL) are elderly and/or have comorbidities that may make them ineligible for fludarabine-based treatment. For this population, chlorambucil monotherapy is an appropriate therapeutic option; however, response rates with chlorambucil are low, and more effective treatments are needed. This trial was designed to assess how the addition of rituximab to chlorambucil (R-chlorambucil) would affect safety and efficacy in patients with CLL.
The cellular microenvironment in follicular lymphoma is of biological and clinical importance. Studies on the clinical significance of non-malignant cell populations have generated conflicting results, which may partly be influenced by poor reproducibility in immunohistochemical marker quantification. In this study, the reproducibility of manual scoring and automated microscopy based on a tissue microarray of 25 follicular lymphomas as compared to flow cytometry is evaluated. The agreement between manual scoring and flow cytometry was moderate for CD3, low for CD4, and moderate to high for CD8, with some laboratories scoring closer to the flow cytometry results. Agreement in manual quantification across the 7 laboratories was low to moderate for CD3, CD4, CD8 and FOXP3 frequencies, moderate for CD21, low for MIB1 and CD68, and high for CD10. Manual scoring of the architectural distribution resulted in moderate agreement for CD3, CD4 and CD8, and low agreement for FOXP3 and CD68. Comparing manual scoring to automated microscopy demonstrated that manual scoring increased the variability in the low and high frequency interval with some laboratories showing a better agreement with automated scores. Manual scoring reliably identified rare architectural patterns of T-cell infiltrates. Automated microscopy analyses for T-cell markers by two different instruments were highly reproducible and provided acceptable agreement with flow cytometry. These validation results provide explanations for the heterogeneous findings on the prognostic value of the microenvironment in follicular lymphoma. We recommend a more objective measurement, such as computer-assisted scoring, in future studies of the prognostic impact of microenvironment in follicular lymphoma patients.
Acute myeloid leukemia-initiating cells (LICs) are responsible for the emergence of leukemia and relapse after chemotherapy. Despite their identification more than 15 years ago, our understanding of the mechanisms responsible for their self-renewal activity and their chemoresistance remains poor. The slow progress in this area is partly due to the difficulty of studying these cells ex vivo. Indeed, current studies are reliant on xenotransplantation assays in immunodeficient mice. In this paper, we report that by modeling key elements of the bone marrow niche using different stromal feeder layers and hypoxic culture conditions, we can maintain LICs over at least 3 weeks and support their self-renewal properties demonstrated through primary and secondary successful xenograft. We provide a proof of principle that this niche-like culture system can be used to study LIC chemoresistance following in vitro cytarabine treatment similarly to the xenograft chemotherapy model. We found that although LICs are believed to be more chemoresistant than non-LICs, functionally defined LICs are not enriched after cytarabine treatment, and heterogeneity in their resistance to treatment can be seen between patients and even within the same patient. We present a culture system that can be used as an in vitro surrogate for xenotransplantation and that has the potential to dramatically increase the throughput of the investigation of LICs. This would further provide the means by which to identify and target the functionality of the different signaling pathways involved in the maintenance and resistance of LICs to improve acute myeloid leukemia treatments.
Distinct patterns of DNA methylation characterize the epigenetic landscape of promyelocytic leukemia/retinoic acid receptor-? (PML-RAR?)-associated acute promyelocytic leukemia (APL). We previously reported that the microRNAs (miRNAs) clustered on chromosome 14q32 are overexpressed only in APL. Here, using high-throughput bisulfite sequencing, we identified an APL-associated hypermethylation at the upstream differentially methylated region (DMR), which also included the site motifs for the enhancer blocking protein CCCTC-binding factor (CTCF). Comparing the profiles of diagnostic/remission paired patient samples, we show that hypermethylation was acquired in APL in a monoallelic manner. The cytosine guanine dinucleotide status of the DMR correlated with expression of the miRNAs following a characteristic position-dependent pattern. Moreover, a signature of hypermethylation was also detected in leukemic cells from an established transgenic PML-RARA APL mouse model at the orthologous region on chromosome 12, including the CTCF binding site located upstream from the mouse miRNA cluster. These results, together with the demonstration that the region does not show DNA methylation changes during myeloid differentiation, provide evidence that 14q32 hypermethylation is implicated in the pathogenesis of APL. We propose a model in which loss of imprinting at the 14q32 domain leads to overexpression of the miRNAs in APL.
Chronic lymphocytic leukemia (CLL) is a disease of an accumulation of mature B cells that are highly dependent on the microenvironment for maintenance and expansion. However, little is known regarding the mechanisms whereby CLL cells create their favorable microenvironment for survival. High-mobility group protein B-1 (HMGB1) is a highly conserved nuclear protein that can be actively secreted by innate immune cells and passively released by injured or dying cells. We found significantly increased HMGB1 levels in the plasma of CLL patients compared with healthy controls, and HMGB1 concentration is associated with absolute lymphocyte count. We therefore sought to determine potential roles of HMGB1 in modulating the CLL microenvironment. CLL cells passively released HMGB1, and the timing and concentrations of HMGB1 in the medium were associated with differentiation of nurse-like cells (NLCs). Higher CD68 expression in CLL lymph nodes, one of the markers for NLCs, was associated with shorter overall survival of CLL patients. HMGB1-mediated NLC differentiation involved internalization of both receptor for advanced glycation end products (RAGE) and Toll-like receptor-9 (TLR9). Differentiation of NLCs can be prevented by blocking the HMGB1-RAGE-TLR9 pathway. In conclusion, this study demonstrates for the first time that CLL cells might modulate their microenvironment by releasing HMGB1.
Pretherapy patients with chronic lymphocytic leukemia (CLL) from US Intergroup trial E2997 were analyzed with single nucleotide polymorphism microarrays to detect acquired chromosomal anomalies. The four CLL-typical anomalies (11q-, +12, 13q-, and 17p-) were found at expected frequencies. Acquired anomalies in other regions account for 70% of the total detected anomalies, and their number per participant has a significant effect on progression-free survival after adjusting for the effects of 17p- (and other covariates). These results were compared with those from a previous study of more than 50,000 participants from the GENEVA consortium of genome-wide association studies, which analyzed individuals with a variety of medical conditions and healthy controls. The percentage of individuals with acquired anomalies is vastly different between the two studies (GENEVA 0.8%; E2997 80%). The composition of the anomalies also differs, with GENEVA having a higher percentage of acquired uniparental disomies and a lower percentage of deletions. The four common CLL anomalies are among the most frequent in GENEVA participants, some of whom may have CLL-precursor conditions or early stages of CLL. However, the patients from E2997 (and other studies of symptomatic CLL) have recurrent acquired anomalies that were not found in GENEVA participants, thus identifying genomic changes that may be unique to symptomatic stages of CLL.
Despite the recent advances with targeted therapies in chronic lymphocytic leukemia (CLL), allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option. However, this procedure is associated with significant morbidity and mortality due to high rates of infection and the toxicity of graft versus host disease (GVHD). One of the principle aims of cellular immunotherapy is to target the malignant cells without damaging the other tissues of the body. T lymphocytes offer the opportunity to do this, due to the exquisite specificity that they exhibit as part of the adaptive immune response. Chimeric antigen receptor (CAR) T cells are lymphocytes that have been genetically modified to express the antigen binding component of an immunoglobulin molecule coupled to T-cell signaling domains. The use of an immunoglobulin molecule eliminates MHC restriction, enabling the same CAR to be used for several different patients and increasing the feasibility of widespread clinical use. They can be constructed to target a huge range of antigens, allowing the targeting of cancer cells with unprecedented levels of specificity. The addition of co-stimulatory domains to the CAR construct has enhanced the efficacy and durability of these T cells, which are under investigation in several clinical trials. The early results from these trials have been very encouraging with dramatic responses being observed in heavily pre-treated patients with otherwise poor risk disease.
Although there have been recent advances with targeted therapies in chronic lymphocytic leukemia (CLL), chemoimmunotherapy remains the treatment of choice; however, this approach is not curative. A key feature of CLL is that it induces a state of immunosuppression, causing increased susceptibility to infections and failure of an antitumor immune response, often worsened by the immunosuppressive effect of treatment. Because of its improved specificity, immunotherapy potentially offers a way out of this dilemma. Allogeneic stem cell transplantation remains the only curative option, but is hampered by the toxicity of GVHD. After many years of promise but little reward, many other immunotherapeutic approaches are now in transition to the clinical setting. Clinical trials including CLL vaccines, CXCR4 antagonists, and adoptive cellular immunotherapies such as chimeric antigen receptor-modified T cells, CD40 ligand gene therapy, and the immunomodulatory drug lenalidomide are ongoing. Results to date suggest that immunotherapeutic approaches for the treatment of CLL might finally be fulfilling their promise.
The opportunity to improve therapeutic choices on the basis of molecular features of the tumor cells is on the horizon in diffuse large B-cell lymphoma (DLBCL). Agents such as bortezomib exhibit selective activity against the poor outcome activated B-cell type (ABC) DLBCL. In order for targeted therapies to succeed in this disease, robust strategies that segregate patients into molecular groups with high reliability are needed. Although molecular studies are considered gold standard, several immunohistochemistry (IHC) algorithms have been published that claim to be able to stratify patients according to their cell-of-origin and to be relevant for patient outcome. However, results are poorly reproducible by independent groups.
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.
CD4(+) T-helper cells (THs) dominate the classical Hodgkin lymphoma (CHL) microenvironment, but their role is poorly understood. Advances in flow cytometry and immunohistochemistry permit more detailed investigation of this aspect of CHL pathophysiology. To address the hypothesis that the TH-infiltrate, rather than being TH2-enriched, senescent and hypofunctional, is TH1 and activation marker-rich, cytokine-secretory and proliferative, we applied comprehensive flow cytometric immunophenotyping and functional assays of cytokine secretion/proliferation to TH cells from 18 CHL-derived single-cell suspensions (SCSs) compared to reactive lymph nodes (RLNs). CHL-derived TH cells express TH1-associated CXCR3/CCR5 and TNF?/IFN?/interleukin-2 (IL-2) and less TH2-associated CCR3/CCR4, with no IL-4/IL-13. They lack exhaustion-/suppression-associated PD1, CD57 and terminally differentiated effector memory cells, with more central memory cells, activation-associated partners of Hodgkin Reed Sternberg (HRS) cell-expressed CD30/OX40-L/ICOS-L, and other activation markers. TH cell lines established from CHL and RLN-derived SCSs remain cytokine-secretory. We confirmed and extended these studies using tissue microarray immunohistochemistry (TMA-IHC) from a large CHL tissue bank (n = 122) and demonstrate TH1-associated TBET is abundant in CHL, and TH2-associated CMAF/GATA3 and exhaustion-associated PD1 expressed at significantly lower levels. These molecular insights into the CHL-associated TH offer potential diagnostic, prognostic and pharmacologically modifiable therapeutic targets and do not support the established view of a TH2-enriched, senescent/exhausted, hypofunctional, hypoproliferative infiltrate.
In this issue of Blood, Morin et al report their findings on complete genome sequence analysis of primary diffuse large B-cell lymphoma (DLBCL) primary tumors and cell lines and reveal novel somatic point mutations, rearrangements, and fusions. Importantly, they also demonstrate the temporal acquisition of mutations, providing insight into the evolution of mutations occurring in DLBCL.
To assess the impact of cancer (IOC) on subsequent quality of life (QOL), 718 long-term haematological cancer survivors completed validated psychosocial, functional and QOL scales, including IOC. Fifteen percent reported significant psychological distress, 18% high levels of fatigue and 10% moderate to severe functional impairment. These groups of participants also showed poorer QOL. There were no significant differences in psychological distress (P = 0·76), fatigue (P = 0·23) or functional impairment (P = 0·74) across different cancer subtypes. Two separate hierarchical regression analyses examined the combined association of disease-type, psychosocial and other factors on negative and positive IOC scores respectively. Higher negative IOC scores were significantly associated (P ? 0·001) with medical comorbidity, psychological distress, lower social support, high fatigue levels and functional impairment. Paediatric patients (diagnosed at <17 years) were significantly more negative than adult patients (P = 0·001); greater years since diagnosis was significantly (P < 0·001) associated with less negative IOC. Higher positive IOC was associated with acute leukaemia (P = 0·01); lower positive IOC with paediatric patients (P < 0·001), white ethnicity (P < 0·001), higher education (P = 0·003), no partner (P = 0·01) and lower social support (P = 0·01). Screening for medical comorbidity, psychological distress and fatigue identifies those needing most support and should allow earlier interventions to address negative and positive IOC to improve the well-being of cancer survivors.
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-?B 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-?B 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.
Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC-progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34(+)CD38(-) stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34(+)CD38(+) progenitors were reduced. Residual normal CD34(+) cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC-progenitor transition.
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.
Over the last decade, the active role of the microenvironment in the pathogenesis of B cell lymphomas has been recognized, delivering signals that favor clonal expansion and drug resistance. We are only beginning to understand the complex cross talk between neoplastic B cells and the tissue microenvironment, for example in secondary lymphoid organs, but some key cellular and molecular players have emerged. Mesenchymal stromal cells, nurselike cells (NLC) and lymphoma-associated macrophages (LAM), in concert with T cells, natural killer cells and extracellular matrix components participate in the dialog with the neoplastic B cells. B cell receptor signaling, activation via TNF family members (i.e. BAFF, APRIL), and tissue homing chemokine receptors and adhesion molecules are important in the interaction between malignant B cells and their microenvironment. Disrupting this cross talk is an attractive novel strategy for treating patients with B cell malignancies. Here, we summarize the cellular and molecular interactions between B cell lymphoma/leukemia cells and their microenvironment, and the therapeutic targets that are emerging, focusing on small molecule inhibitors that are targeting B cell receptor-associated kinases SYK, BTK, and PI3Ks, as well as on immunomodulatory agents and T cell mediated therapies. Clinically relevant aspects of new targeted therapeutics will be discussed, along with an outlook into future therapeutic strategies.
Hematopoietic stem and progenitor cells (HSPCs) are exposed to low levels of oxygen in the bone marrow niche, and hypoxia-inducible factors (HIFs) are the main regulators of cellular responses to oxygen variation. Recent studies using conditional knockout mouse models have unveiled a major role for HIF-1? in the maintenance of murine HSCs; however, the role of HIF-2? is still unclear. Here, we show that knockdown of HIF-2?, and to a much lesser extent HIF-1?, impedes the long-term repopulating ability of human CD34(+) umbilical cord blood cells. HIF-2?-deficient HSPCs display increased production of reactive oxygen species (ROS), which subsequently stimulates endoplasmic reticulum (ER) stress and triggers apoptosis by activation of the unfolded-protein-response (UPR) pathway. HIF-2? deregulation also significantly decreased engraftment ability of human acute myeloid leukemia (AML) cells. Overall, our data demonstrate a key role for HIF-2? in the maintenance of human HSPCs and in the survival of primary AML cells.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells.
Previous studies have demonstrated the prognostic importance of the immune microenvironment in follicular lymphoma (FL). To investigate the molecular mechanisms during which tumor-infiltrating T cells (TILs) are altered in the FL microenvironment, we studied highly purified CD4 and CD8 TILs from lymph node biopsies at diagnosis in treatment-naive patients with FL compared with reactive tonsils and the peripheral blood of healthy donors.
Despite the use of highly active antiretroviral therapy (HAART), AIDS-related lymphoma remains common. We investigated the tumor, microenvironment, and viral components in 41 AIDS-related diffuse large B-cell lymphomas (AR-DLBCLs) in the pre- and post-HAART era. The outcome has improved and the frequency of the prognostically unfavorable immunoblastic histology has decreased after HAART. Compared with sporadic cases, AR-DLBCL demonstrated increased hyperproliferation (P < .001) and c-Myc rearrangements, reduced CD4(+) (P < .001) and FOXP3(+) T cells (P < .001), increased activated cytotoxic cells (P < .001), but no difference in tumor-associated macrophages. Our analysis showed that AR-DLBCL is highly angiogenic with higher blood-vessel density than sporadic cases (P < .001) and highlighted the role of Epstein-Barr virus in angiogenesis. We recognized viral profiles and as a second step examined the reactive cytotoxic cell infiltrates. Our observation of markedly higher numbers of cytotoxic cells in AR-DLBCL with LMP1 and/or p24 compared with cases lacking viral antigens (P < .001) has important clinical implications, implicitly linked to the immunosurveillance theory. Whereas early initiation of HAART should improve immunosurveillance and reduce the incidence of LMP1-positive AR-DLBCL, cases without viral antigens appear able to avoid immunologic reaction and likely require additional strategies to improve surveillance.
Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Although significant advances have been made in the treatment of CLL in the last decade, it remains incurable. Treatments may be too toxic for some elderly patients, who constitute most of the individuals with this disease, and there remain subgroups of patients for which this therapy has minimal activity. This article summarizes the current understanding of the immune defects in CLL. It also examines the potential clinical implications of these findings.
Professor John Gribben is Chair of the International Workshop on non-Hodgkin Lymphoma and the Gordon Hamilton Fairley Chair of Medical Oncology at St. Bartholomews Hospital, Barts Cancer Institute, London, UK, a Cancer Research UK Centre of Excellence. His doctoral studies were performed at University College London, UK as the recipient of a Wellcome Trust Fellowship Award and he continued post-doctoral training with Professor Lee Nadler at the Dana-Farber Cancer Institute (Harvard Medical School, MA, USA). In 1992, Gribben was appointed to the Faculty at Harvard Medical School, where he remained as Associate Professor of Medicine and an Attending Physician at the Dana-Farber Cancer Institute and Brigham and Womens Hospital (MA, USA), until returning to London in 2005. Gribben is a founding member of the CLL Research Consortium, Associate Editor of Blood and was elected a Fellow of the Academy of Medical Science. His primary research interests include the immunotherapy of cancer (including stem cell transplantation), the identification of B-cell-tumor antigens and the detection and treatment of minimal residual disease in leukemia and lymphoma.
The aim of this study was to analyse the immunophenotypic and molecular features of a large series of follicular lymphomas, focusing in particular on atypical cases that fail to express CD10 and/or bcl-2. Such cases present diagnostic pitfalls, especially with regard to the differential diagnosis from follicular hyperplasia and marginal zone B-cell lymphoma. Therefore, we also included an immunohistochemical evaluation of stathmin, which is strongly expressed by germinal centre B cells, as a putative new marker for follicular lymphomas, particularly those with an atypical phenotype.
The toxicity burden and long-term anti-leukaemic effect of non-myeloablative (NMA) allogeneic haematopoietic stem-cell transplantation (AHSCT) for acute myeloid leukaemia (AML) and myelodysplasia (MDS) remains undefined. We report the outcome of 56 patients with AML/MDS transplanted from human leucocyte antigen-matched donors using NMA conditioning without T-cell depletion. With a median follow-up of 5 years, treatment-related mortality was 9% and current disease-free survival (CDFS) was 45% (overall) and 55% (patients transplanted in remission). Development of graft-versus-host disease upon withdrawal of post-transplant immunosuppression was associated with less relapse and better CDFS. These data confirm that NMA AHSCT without T-cell depletion is safe and can result in sustained remissions of AML/MDS.
Phosphoinositide-3 kinase (PI3K) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis, but early-phase studies of the PI3K p110? inhibitor GS-1101 have reported inferior responses in MCL compared with other non-Hodgkin lymphomas. Because the relative importance of the class IA PI3K isoforms p110?, p110?, and p110? in MCL is not clear, we studied expression of these isoforms and assessed their contribution to PI3K signaling in this disease. We found that although p110? was highly expressed in MCL, p110? showed wide variation and expression increased significantly with relapse. Loss of phosphatase and tensin homolog expression was found in 16% (22/138) of cases, whereas PIK3CA and PIK3R1 mutations were absent. Although p110? inhibition was sufficient to block B-cell receptor-mediated PI3K activation, combined p110? and p110? inhibition was necessary to abolish constitutive PI3K activation. In addition, GDC-0941, a predominantly p110?/? inhibitor, was significantly more active compared with GS-1101 against MCL cell lines and primary samples. We found that a high PIK3CA/PIK3CD ratio identified a subset of primary MCLs resistant to GS-1101 and this ratio increased significantly with relapse. These findings support the use of dual p110?/p110? inhibitors in MCL and suggest a role for p110? in disease progression.
T lymphocytes have an essential role in adaptive immunity and rely on the activation of integrin lymphocyte function-associated antigen-1 (LFA-1) to mediate cell arrest and migration. In cancer, malignant cells modify the immune microenvironment to block effective host antitumor responses. We show for the first time that CD4 and CD8 T cells from patients with chronic lymphocytic leukemia (CLL) exhibit globally impaired LFA-1-mediated migration and that this defect is mediated by direct tumor cell contact. We show that following the coculture of previously healthy T cells with CLL cells, subsequent LFA-1 engagement leads to altered Rho GTPase activation signaling by downregulating RhoA and Rac1, while upregulating Cdc42. Of clinical relevance, repair of this T-cell defect was demonstrated using the immunomodulatory drug lenalidomide, which completely rescued adhesion and motility function by restoring normal Rho GTPase activation signaling. Our report identifies a novel cancer immune evasion mechanism whereby tumor cells induce Rho GTPase signaling defects in T cells that prevent appropriate LFA-1 activation and motility. We believe these findings identify important biomarkers and highlight the clinical utility of immunotherapy to rescue normal T-cell function in CLLs that are likely to have relevance in other cancers.
In this issue of Blood, Dovedi et al demonstrate convincing evidence for the therapeutic efficacy of a new systemic immunostimulatory agent, the Toll-like receptor-7 (TLR7) agonist R848, to augment radiotherapy-induced anticancer immunity.
Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56(+)/CD3(-)) and less than 1% CD3(+) cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.
In this issue of Blood, Laurent et al use 3-D confocal imaging to visualize CD8+ T-cell lytic immune synapses in follicular lymphoma and report that this activity may influence progression-free survival after rituximab-chemotherapy.
Based on clinical activity in phase 2 studies, lenalidomide was evaluated in a phase 2/3 study in patients with relapsed/refractory chronic lymphocytic leukemia (CLL). Following tumor lysis syndrome (TLS) complications, the protocol was amended to a phase 1 study to identify the maximum tolerated dose-escalation level (MTDEL). Fifty-two heavily pretreated patients, 69% with bulky disease and 48% with high-risk genomic abnormalities, initiated lenalidomide at 2.5 mg/day, with dose escalation until the MTDEL or the maximum assigned dose was attained. Lenalidomide was safely titrated to 20 mg/day; the MTDEL was not reached. Most common grade 3-4 adverse events were neutropenia and thrombocytopenia; TLS was mild and rare. The low starting dose and conservative dose escalation strategy resulted in six partial responders and 30 patients obtaining stable disease. In summary, lenalidomide 2.5 mg/day is a safe starting dose that can be titrated up to 20 mg/day in patients with CLL.
Combination chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) has emerged as the current standard of care in the treatment of chronic lymphocytic leukemia (CLL). Despite very high response rates, this treatment is too toxic for many patients, and it remains unclear as how to manage patients who do not respond to these agents or who relapse early after treatment. An increase in our understanding of the biology of CLL has led to the development of a wide range of therapies aimed at specific defects in this disease. B-cell receptor signaling is aberrantly increased in CLL, and so many of these drugs target key steps in these pathways. Antitumor immunity is also impaired, and a number of strategies are being developed to repair this acquired immune dysfunction. This review highlights some of the emerging agents and describes the biological rationale for their use in CLL.
CD160 is a human natural killer (NK)-cell-activating receptor that is also expressed on T-cell subsets. In the present study, we examined 811 consecutive cases of B-cell lymphoproliferative disorders (B-LPDs), and demonstrated CD160 expression in 98% (590 of 600) of chronic lymphocytic leukemia (CLL) cases, 100% (32 of 32) of hairy cell leukemia (HCL) cases, 15% (5 of 34) of mantle cell lymphoma (MCL) in the leukemic phase, and 16% (23 of 145) of other B-LPD cases. CD160 transcript and protein were absent in the normal B-cell hierarchy, from stem cells, B-cell precursors, maturing B cells in the germinal center, and circulating B cells, including CD5(+)CD19(+) B1 cells in umbilical cord. CD160 positivity was significantly higher in CLL and HCL in terms of percentage (65.9% and 67.8%, respectively, P < .0001) and median fluorescence intensity (552 and 857, respectively, P < .0001) compared with all other B-LPD cases. Lymph node CLL samples were also CD160(+). Using the disease-specific expression of CD5, CD23, and CD160, a score of 3 characterized CLL (diagnostic odds ratio, 1430); a score of 0 excluded CLL, MCL, and HCL; and the CD23/CD5 ratio differentiated CLL from leukemic CD23(+) MCL. In the B-cell lineage, CD160 is a tumor-specific antigen known to mediate cellular activation signals in CLL, and is a novel target for therapeutic manipulation and monitoring of minimal residual disease.
Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70(-) and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70(+) and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70(-)) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease.
The addition of rituximab to fludarabine-based regimens in chronic lymphocytic leukemia (CLL) has been shown to produce high response rates with extended remissions. The long-term follow-up of these regimens with respect to progression, survival, risk of secondary leukemia, and impact of genomic risk factors has been limited.
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??) 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.
There have been tremendous advances in the treatment of chronic lymphocytic leukemia (CLL) over the past decade, with the goal of therapy no longer being just to palliate symptoms but now to achieve complete remission, eradicate minimal residual disease, and improve survival. During this period, there have also been major advances in identification of molecular factors associated with increased risk of progression. The clinical utility of these factors is being explored to determine whether we can identify groups of patients who should be treated earlier in their disease course and whether we can tailor therapy for groups of patients with specific molecular markers of disease. First-line chemoimmunotherapy approaches now offer prolonged survival, and there is a need to identify patients who are suitable candidates for allogeneic stem-cell transplantation that uses reduced-intensity conditioning regimens. The vast majority of CLL patients are either too old or do not have sufficiently high-risk disease to warrant these approaches, and effective therapies that can be tolerated by the more frail elderly patients with this disease are urgently needed. Numerous novel agents are being developed, and their role in the first-line treatment of frail patients or those who relapse after previous treatment is being explored in clinical trials.
The indolent lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) remain incurable with standard therapy. Autologous hematopoietic stem cell transplantation (HSCT) is feasible and has low treatment-related mortality in follicular lymphoma, but there are questions relating to optimal timing of the procedure, conditioning regimen, and late effects. Myeloablative allogeneic HSCT is associated with high treatment-related morbidity and mortality, few late relapses, but is applicable to only a small number of patients. The major focus of HSCT in these lymphomas has been with reduced-intensity conditioning (RIC) allogeneic HSCT, which is applicable to the age distribution of these diseases and which exploits the graft-versus-lymphoma effect in these diseases. Steps to further decrease the morbidity and mortality of the RIC HSCT and in particular to reduce the incidence of chronic extensive graft-versus-host disease (GVHD) while maintaining tumor control remain the major focus. Many potential treatments are available for indolent lymphomas and CLL, and appropriate patient selection and the timing of HSCT remain controversial. The use of HSCT must always be weighed against the risk of the underlying disease, particularly in a setting where improvements in treatment are leading to improved outcome.
Chronic lymphocytic leukemia (CLL) remains incurable, but over the past decade there have been major advances in the understanding of the pathophysiology of CLL and in the treatment of this disease. This has led to greatly increased response rates and durations of response, as well as improved survival. CLL is a disease of the elderly and not all patients are eligible for the aggressive upfront chemoimmunotherapy regimens that are resulting in improved response rates and survival, so what is the optimal treatment approach for more frail elderly patients? It is highly likely that our treatment approaches will continue to evolve as the results of ongoing clinical trials are released. The age range of patients involved in clinical trials is not representative of this disease, and more research is required in patients who are representative of the majority of CLL patients seen in practice before we will see outcome improvements in these more elderly and often more frail patient populations.
The microenvironment in which cancer arises plays a critical role in tumorigenesis. Although previously regarded as an innocent bystander, evidence has accumulated over the past 10 years that the microenvironment contributes to tumor growth and progression by providing nutrients and survival signals, and protecting the tumor from normal immune responses and anticancer drugs. Exactly how normal stromal cells, whose function should be to suppress malignant growth, become co-opted into facilitating tumor development is only just beginning to be understood, but a complex story is emerging wherein tumor and stromal cells appear to co-evolve. A better understanding of tumor-stromal interactions and the molecular alterations that result in stromal dysfunction may help to identify patients who will benefit from either more aggressive or risk-adapted therapy regimens, and/or novel compounds that disrupt the tumor microenvironment and re-establishing normal control mechanisms.
Direct contact with stromal cells protects chronic lymphocytic leukaemia (CLL) B cells from chemotherapy-induced apoptosis in vitro. Blockade of CXCR4 signalling antagonizes stroma-mediated interactions and restores CLL chemosensitivity. In vivo, administration of CXCR4 antagonists effectively mobilizes haematopoietic progenitor cells. Therefore, combinations of CXCR4 blockade and cytoreductive treatment with selective activity on CLL cells may avoid potential haematotoxicity. Hence, we tested CXCR4 antagonists in the context of passive and active immunotherapeutic approaches. We evaluated how efficiently rituximab, alemtuzumab and cytotoxic T cells killed CLL cells cocultured with stromal cells in the presence and absence of a CXCR4 antagonist. Stromal cell contact attenuated rituximab- and alemtuzumab-induced complement-dependent cytotoxicity of CLL cells. Addition of CXCR4 antagonists abrogated the protective effect of stroma. In contrast, stromal cells did not impair antibody-dependent cell-mediated cytotoxicity and cytotoxicity induced by activated T cells. Destruction of microtubules in CLL target cells restored the protective effect of stroma coculture for CLL cells during Natural Killer cell attack by preventing mitochondrial relocalization towards the immunological synapse. Our data identify the combination of CXCR4 antagonists with passive - but not active - immunotherapy as a promising potential treatment concept in CLL.
Although outcomes for patients with follicular lymphoma have improved with chemoimmunotherapy, the disease remains incurable. There is a wide variation in survival, and although the Follicular Lymphoma International Prognostic Index helps to risk-stratify patients, there is a need for robust biomarkers of disease outcome. Most patients will succumb to the emergence of chemoresistance or transformation to diffuse large B-cell lymphoma and there is a need for new treatment approaches in this disease.
According to the immune-surveillance hypothesis, cancer cells evolve strategies to evade or suppress the immune system as part of the development of this disease. The malignant B-cells of chronic lymphocytic leukaemia are prime examples of this premise, having been shown to generate a variety of ways of suppressing T-cell anti-tumour immune responses and these are summarized here. These mechanisms range from impairment of antigen presentation by the tumour cells themselves, to suppression of the immune microenvironment by contact dependent pathways and alterations in the cytokine milieu. By understanding these defects, novel targeted therapies can be developed with the aim of restoring T-cell function. Indeed, some of the recent advances in the treatment of chronic lymphocytic leukaemia have been demonstrated to have profound immunomodulatory effects, repairing these defects in T-cell function.
Peripheral blood natural killer (NK) cell therapy for acute myeloid leukemia has shown promise in clinical trials after allogeneic stem cell transplantation. Cord blood (CB) is another potentially rich source of NK cells for adoptive immune therapy after stem cell transplantation. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. However, despite expressing normal surface activating and inhibitory NK receptors, CB-derived NK cells have poor cytolytic activity. In this study, we investigate the cellular mechanism and demonstrate that unmanipulated CB-NK cells exhibit an impaired ability to form F-actin immunologic synapses with target leukemia cells compared with peripheral blood-derived NK cells. In addition, there was reduced recruitment of the activating receptor CD2, integrin leukocyte function-associated antigen-1, and the cytolytic molecule perforin to the CB-NK synapse site. Exvivo interleukin (IL)-2 expansion of CB-NK cells enhanced lytic synapse formation including CD2 and leukocyte function-associated antigen-1 polarization and activity. Furthermore, the acquired antileukemic function of IL-2-expanded CB-NK cells was validated using a nonobese diabetic severe combined immunodeficient IL-2 receptor common ?-chain null mouse model. We believe our results provide important mechanistic insights for the potential use of IL-2-expanded CB-derived NK cells for adoptive immune therapy in leukemia.
Our understanding of the biology underlying lymphoma is continually increasing and leading to improved treatment strategies for affected patients. However, clinical approaches differ between disease subtypes based on the likelihood of achieving durable remissions. For example, follicular lymphoma (FL) is a common and indolent form of non-Hodgkin lymphoma, in which most patients relapse after treatment and periods of remission become progressively shorter after each course of treatment. Thus, the main focus of research efforts in FL is to develop improved treatment strategies that provide prolonged periods of disease remission. In contrast, Hodgkin lymphoma (HL), although rare, is considered to be highly curable with current treatments and the clinical need is for effective-management strategies, potentially avoiding chemotherapy, that reduce long-term toxicity and improve quality of life for patients. This report summarizes the latest advances in our understanding of FL and HL, and demonstrates the different approaches required when developing novel treatment strategies for the two diseases.
There is no clear consensus regarding the optimal management of chronic lymphocytic leukaemia. Many patients are diagnosed at an advanced age and will die with chronic lymphocytic leukaemia, but of other unrelated causes. A significant minority are diagnosed at an earlier age, or with more aggressive disease, and despite chemotherapy, are likely to die of chronic lymphocytic leukaemia. The infusion of autologous or allogeneic haemopoietic stem cells, following a variety of conditioning regimes, offers the possibility of longer remissions or even cure. We explore the key questions facing clinicians in this field: Who is it best to transplant? When is it best to transplant? How is it best to transplant?
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.
Bendamustine is a novel bifunctional alkylating agent with promising activity in lymphoid malignancies and several solid tumors. Unfortunately, the early development of this agent did not provide sufficient information on which to determine an optimal systematic dose and schedule. As a result, administration of the agent has been inconsistent among studies. The use of this drug has been increasing since it has been approved by the US Food and Drug Administration for chronic lymphocytic leukemia and rituximab-refractory indolent B-cell non-Hodgkin lymphoma, and is expected to increase further following anticipated European regulatory approval. Thus, a consensus meeting was convened to develop recommendations for standardizing the administration of the drug based on the available clinical data. Recommendations were developed including dose and schedule for the various clinical indications, as a single agent and in combination therapy, and to provide guidance for supportive measures. This report, representing the conclusions of that meeting, should provide guidance for the clinician until definitive dose-finding studies have been conducted.
GCS-100 is a galectin-3 antagonist with an acceptable human safety profile that has been demonstrated to have an antimyeloma effect in the context of bortezomib resistance. In the present study, the mechanisms of action of GCS-100 are elucidated in myeloma cell lines and primary tumor cells. GCS-100 induced inhibition of proliferation, accumulation of cells in sub-G(1) and G(1) phases, and apoptosis with activation of both caspase-8 and -9 pathways. Dose- and time-dependent decreases in MCL-1 and BCL-X(L) levels also occurred, accompanied by a rapid induction of NOXA protein, whereas BCL-2, BAX, BAK, BIM, BAD, BID, and PUMA remained unchanged. The cell-cycle inhibitor p21(Cip1) was up-regulated by GCS-100, whereas the procycling proteins CYCLIN E2, CYCLIN D2, and CDK6 were all reduced. Reduction in signal transduction was associated with lower levels of activated IkappaBalpha, IkappaB kinase, and AKT as well as lack of IkappaBalpha and AKT activation after appropriate cytokine stimulation (insulin-like growth factor-1, tumor necrosis factor-alpha). Primary myeloma cells showed a direct reduction in proliferation and viability. These data demonstrate that the novel therapeutic molecule, GCS-100, is a potent modifier of myeloma cell biology targeting apoptosis, cell cycle, and intracellular signaling and has potential for myeloma therapy.
B-cell chronic lymphocytic leukemia (CLL) expresses CD160, a glycosylphosphatidylinositol-linked receptor found on normal natural killer (NK) and T cells, but not B cells. CD160 is a multifunctional molecule in normal lymphocytes, but its role in CLL biology is unknown. In vitro, CLL cells undergo rapid spontaneous apoptosis, which CD160 activation protected against-mean cell viability increased from 67% to 79% (P < .001). This was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1, but not Bax. As expected from these changes in Bcl-2/Bax and Bcl-xL/Bax ratios, CD160 triggering reduced mitochondrial membrane potential collapse and cytochrome c release. CD160 stimulation also induced DNA synthesis, cell cycle progression, and proliferation. B-cell antigen receptor (BCR)-induced CLL proliferation was generally greater than with CD160, but marked variation was seen. Both BCR and CD160 signaling led to CLL secretion of interleukin-6 (IL-6) and IL-8, although CD160 induced greater increases of IL-6 (51-fold) and IL-8 (15-fold). Survival and activation signals mediated by CD160 showed dose-dependent suppression by phosphoinositide-3 kinase (PI3K) inhibitors. Thus, in vitro, CLL cells can use the CD160 pathway for survival and activation, mimicking CD160 signaling in normal NK and CD8(+) T cells. Establishing the pathophysiologic relevance of these findings may reveal new therapeutic targets.
Alemtuzumab is highly effective at treating chronic lymphocytic leukemia (CLL) in bone marrow, which is the usual site of residual disease after fludarabine-based treatment. Eliminating residual disease potentially is associated with longer remission and overall survival. The authors of this report evaluated the ability of subcutaneous alemtuzumab to treat residual disease.
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.
Although chronic lymphocytic leukemia (CLL) remains incurable, over the past decade there have been major advances in understanding the pathophysiology of CLL and in the treatment of this disease. This has led to greatly increased response rates and durations of response but not yet improved survival. Advances in the use of prognostic factors that identify patients at high risk for progression have led us to the question whether there is still a role for a "watch and wait" approach in asymptomatic high-risk patients or whether they should be treated earlier in their disease course. Questions remain, including, what is the optimal first-line treatment and its timing and is there any role of maintenance therapy or stem cell transplantation in this disease? CLL is a disease of the elderly and not all patients are eligible for aggressive up-front chemoimmunotherapy regimens, so what is the optimal treatment approach for more frail elderly patients? It is highly likely that our treatment approaches will continue to evolve as the results of ongoing clinical trials are released and that further improvements in the outcome of this disease will result from identification of therapies that target the underlying pathophysiology of CLL.
In this issue of Blood, Gentles and colleagues used a computational model to investigate FL and transformed DLBCL, and identified the acquisition of an ESC-like signature as a key feature of transformation.
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.
Stem cell transplantation in chronic lymphocytic leukemia (CLL) is an evolving field. Younger patients with high-risk disease might derive the greatest benefit from this approach and the availability of reduced-intensity conditioning regimens has made allogeneic stem cell transplantation more relevant to patients with CLL. Patient selection, timing of transplantation, and method of conditioning, stem cell delivery and immunosuppression appear to influence outcomes. We collect and review the available data to assist clinical decision-making in this field.
A randomized trial of oblimersen plus fludarabine/cyclophosphamide (OBL-FC; n = 120) versus FC (n = 121) was conducted in patients with relapsed/refractory chronic lymphocytic leukemia (CLL). The primary end point was met: the complete response (CR) rate, defined as complete or nodular partial response, was significantly greater with OBL-FC than with FC (17% v 7%; P = .025). Among patients with CR, response duration was significantly longer with OBL-FC than with FC (median not reached; > 36 months v 22 months; P = .03). Maximum benefit with OBL-FC, including a four-fold increase in CR rate and a survival benefit with 3 years of follow-up (hazard ratio, 0.53; P = .05), was observed in patients with fludarabine-sensitive disease. We evaluated long-term survival and poststudy CLL therapy among all randomly assigned patients.
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.
B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCgamma(2), signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70(+) cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials.
Preclinical animal models have largely ignored the immune-suppressive mechanisms that are important in human cancers. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in nonmalignant cells by cancer, we examined T-cell function in the chronic lymphocytic leukemia (CLL) Emu-TCL1 transgenic mouse model. With development of leukemia, Emu-TCL1 transgenic mice developed functional T-cell defects and alteration of gene and protein expression closely resembling changes seen in CLL human patients. Furthermore, infusion of CLL cells into young Emu-TCL1 mice induced defects comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T-cell defects. Altered pathways involved genes regulating actin remodeling, and T cells exhibited dysfunctional immunological synapse formation and T-cell signaling, which was reversed by the immunomodulatory drug lenalidomide. These results further demonstrate the utility of this animal model of CLL and define a versatile model to investigate both the molecular mechanisms of cancer-induced immune suppression and immunotherapeutic repair strategies.
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