To examine the association between early HIV viremia and mortality after HIV-associated lymphoma.
Multicenter observational cohort study.
Center for AIDS Research Network of Integrated Clinical Systems cohort.
HIV-infected patients with lymphoma diagnosed between 1996 and 2011, who were alive 6 months after lymphoma diagnosis and with ≥2 HIV RNA values during the 6 months after lymphoma diagnosis.
Cumulative HIV viremia during the 6 months after lymphoma diagnosis, expressed as viremia copy-6-months.
Main outcome measure
All-cause mortality between 6 months and 5 years after lymphoma diagnosis.
Of 224 included patients, 183 (82%) had non-Hodgkin lymphoma (NHL) and 41 (18%) had Hodgkin lymphoma (HL). At lymphoma diagnosis, 105 (47%) patients were on antiretroviral therapy (ART), median CD4 count was 148 cells/µlL (IQR 54– 322), and 33% had suppressed HIV RNA (<400 copies/mL). In adjusted analyses, mortality was associated with older age [adjusted hazard ratio (AHR) 1.37 per decade increase, 95% CI 1.03–1.83], lymphoma occurrence on ART (AHR 1.63, 95% CI 1.02– 2.63), lower CD4 count (AHR 0.75 per 100 cell/µL increase, 95% CI 0.64–0.89), and higher early cumulative viremia (AHR 1.35 per log10copies × 6-months/mL, 95% CI 1.11–1.65). The detrimental effect of early cumulative viremia was consistent across patient groups defined by ART status, CD4 count, and histology.
Exposure to each additional 1-unit log10 in HIV RNA throughout the 6 months after lymphoma diagnosis, was associated with a 35% increase in subsequent mortality. These results suggest that early and effective ART during chemotherapy may improve survival.
AIDS; Burkitt lymphoma; diffuse large B-cell lymphoma; HIV; Hodgkin lymphoma; lymphoma; non-Hodgkin lymphoma
HIV increases risk of non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). The effect of HIV on presentation, treatment, and outcomes of NHL and HL in routine care in the combination antiretroviral therapy (cART) merits further characterization. We performed a retrospective analysis of HIV-infected patients with NHL and HL receiving care at the University of North Carolina at Chapel Hill from January 1, 2000 until December 31, 2010. Statistical analyses were conducted using SAS, version 9.2 (SAS Institute Inc). Sixty-five HIV-infected patients with NHL and HL were identified. Patients with non-CNS NHL and HL presented with advanced disease (85% stage III or IV) and adverse prognostic features. Patients completed 87% of planned chemotherapy cycles, and 68% of patients completed stage-appropriate therapy. Dose reduction, interruption, and/or delay occurred during more than 25% of administered cycles in 64% of patients. Infectious complications, febrile neutropenia, and myelosuppression accounted for 78% of deviations from planned cumulative dose and dose intensity. Primary CNS lymphoma (PCNSL) was associated with poor prognosis, but 2-year overall survival was 66% for all non-CNS lymphoma. Among patients surviving at least 2 years, 75% had CD4 count >200 cells/μl and 79% had HIV viral load <400 copies/ml at last follow-up. Despite advanced disease and difficulty tolerating chemotherapy with optimal cumulative dose and dose intensity, most patients with non-CNS HIV-associated lymphoma survived more than 2 years after diagnosis, the majority with suppressed HIV RNA.
Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.
The anti-CD20 monoclonal antibody, rituximab, provides a significant therapeutic benefit for patients with B-cell disorders. However, response to therapy varies and relapses are common, so an understanding of both inherited and acquired rituximab resistance is needed. In order to identify mechanisms of inherited resistance, sensitive versus resistant individuals were selected from a survey of 92 immortalized lymphoblastoid B-cell lines from normal individuals. Levels of CD20 protein and surface expression were lower in the resistant group. In contrast, CD20 mRNA levels were not correlated with susceptibility, suggesting regulation at a post-transcriptional level. To examine acquired resistance, resistant sublines were selected from both lymphoblastoid as well as lymphoma cell lines. Confirming previous findings, there was significant down-regulation of CD20 protein expression in all the resistant sublines. CD20 mRNA splice variants are reported to be associated with development of resistance. Three splice variants were observed in our cell lines, each lacking the binding epitope for rituximab, but none were associated with rituximab resistance. The second generation anti-CD20 mAb, ofatumumab, was more active compared with rituximab in vitro in the survey of all B-cell lines, mirroring results that have been reported previously with malignant B-cells. These studies show that normal B-lymphoblastoid cell lines can be used to model both innate and acquired mechanisms of resistance. They validate the important role of CD20 expression and enable future genetic studies to identify additional mediators of anti-CD20 mAb resistance.
Rituximab; Ofatumumab; CD20 expression; mRNA splice variants; Complement-dependent cytotoxicity; Drug resistance; Lymphoblastoid cell line
The prognosis for patients with Hodgkin lymphoma (HL) has improved in recent decades. On the other hand, not all patients can be cured with the currently established therapy regimes and this therapy is associated with several adverse late effects. Therefore it is necessary to develop new therapy strategies. After treatment of L-540 HL cells with 5′-azacytidine (5AC), we observed increased expression of the preferentially expressed antigen in melanoma (PRAME). In addition, we detected an increased resistance of 5AC-treated cells against cytotoxic drugs. We analyzed the influence of PRAME on cell survival of HL cells by knocking down PRAME in the chemotherapy resistant cell line L-428, a cell line that express PRAME at a high level. After knock-down of PRAME using vector based RNA interference we observed increased sensitivity for cisplatin, etoposide and retinoic acid. DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors. Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling. Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.
Individualization of cancer chemotherapy based on the patient’s genetic makeup holds promise for reducing side effects and improving efficacy. However, the relative contribution of genetics to drug response is unknown.
Materials & methods
In this study, we investigated the cytotoxic effect of 29 commonly prescribed chemotherapeutic agents from diverse drug classes on 125 lymphoblastoid cell lines derived from 14 extended families.
The results of this systematic study highlight the variable role that genetics plays in response to cytotoxic drugs, ranging from a heritability of <0.15 for gemcitabine to >0.60 for epirubicin.
Putative quantitative trait loci for cytotoxic response were identified, as well as drug class-specific signatures, which could indicate possible shared genetic mechanisms. In addition to the identification of putative quantitative trait locis, the results of this study inform the prioritization of chemotherapeutic drugs with a sizable genetic response component for future investigation.
cancer; cell line; chemotherapy; cytotoxicity; pharmacogenetics; pharmacogenomic; QTL
Kaposi sarcoma (KS) is the most frequent AIDS-defining cancer worldwide. KS-associated herpesvirus (KSHV) is the etiological agent of KS, and the virus is also associated with two lymphoproliferative diseases. Both KS and KSHV-associated lymphomas, are cancers of unique molecular composition. They represent a challenge for cancer treatment and an opportunity to identify new mechanisms of transformation. Here, we review the current clinical insights into KSHV-associated cancers and discuss scientific insights into the pathobiology of KS, primary effusion lymphoma, and multicentric Castleman’s disease.
KSHV; lymphoma; sarcoma; viral cancer; therapy
The transcription factor TCF21 is involved in mesenchymal-to-epithelial differentiation and was shown to be aberrantly hypermethylated in lung and head and neck cancers. Because of its reported high frequency of hypermethylation in lung cancer, we sought to characterize the stages and types of non-small cell lung cancer (NSCLC) that are hypermethylated and to define the frequency of hypermethylation and associated “second hits”.
We determined TCF21 promoter hypermethylation in 105 NSCLC including various stages and histologies in smokers and nonsmokers. Additionally, we examined TCF21 loss-of-heterozygosity and mutational status. We also assayed 22 cancer cell lines from varied tissue origins. We validated and expanded our NSCLC results by examining TCF21 immunohistochemical expression on a tissue microarray containing 300 NSCLC cases.
Overall, 81% of NSCLC samples showed TCF21 promoter hypermethylation and 84% showed decreased TCF21 protein expression. Multivariate analysis showed that TCF21 expression, although below normal in both histologies, was lower in adenocarcinoma than squamous cell carcinoma, and was not independently correlated with gender, smoking and EGFR mutation status, or clinical outcome. Cell lines from other cancer types also showed frequent TCF21 promoter hypermethylation.
Hypermethylation and decreased expression of TCF21 were tumor-specific and very frequent in all NSCLC, even early-stage disease, thus making TCF21 a potential candidate methylation biomarker for early-stage NSCLC screening. TCF21 hypermethylation in a variety of tumor cell lines suggests it may also be a valuable methylation biomarker in other tumor types.
TCF21; methylation; biomarker; lung cancer; screening
FMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit FLT3 ITD mutations.
We molecularly characterized FLT3 mutations in two dogs and one cell line, by DNA sequencing, gene expression analysis via quantitative real-time PCR, and sensitivity to the FLT3 inhibitor lestaurtinib via in vitro proliferation assays. FLT 3 and downstream mediators of FLT3 activation were assessed by Western blotting.
The canine B-cell leukemia cell line, GL-1, and neoplastic cells from 2/7 dogs diagnosed cytologically with ALL were found to have FLT3 ITD mutations and FLT3 mRNA up-regulation. Lestaurtinib, a small molecule FLT3 inhibitor, significantly inhibited the growth of GL-1 cells, while not affecting the growth of two other canine lymphoid cell lines without the FLT3 mutation. Finally, western blots were used to confirm the conserved downstream mediators of FLT3 activating mutations.
These results show that ALL and FLT3 biology is conserved between canine and human patients, supporting the notion that canine ALL, in conjunction with the GL-1 cell line, will be useful in the development of a relevant large animal model to aid in the study of human FLT3 mutant leukemias.
Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.
A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major α-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitive tub1 alleles were synthetically lethal in combination with tub3Δ, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of α-tubulin. The systematic tub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast α–β-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of β-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of α-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the α–β interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201 allele.
Non-Hodgkin lymphomas (NHL) are a heterogeneous group of solid tumours of lymphoid cell origin. Three important aspects of lymphocyte development include immunity and inflammation, DNA repair, and programmed cell death. We have used a previously established case-control study of NHL to ask whether genetic variation in genes involved in these three important processes influences risk of this cancer. 118 genes in these three categories were tagged with single nucleotide polymorphisms (SNPs), which were tested for association with NHL and its subtypes. The main analysis used logistic regression (additive model) to estimate odds ratios in European-ancestry cases and controls. 599 SNPs and 1116 samples (569 cases and 547 controls) passed quality control measures and were included in analyses. Following multiple-testing correction, one SNP in MSH3, a mismatch repair gene, showed an association with diffuse large B-cell lymphoma (OR: 1.91; 95% CI: 1.41–2.59; uncorrected p = 0.00003; corrected p = 0.010). This association was not replicated in an independent European-ancestry sample set of 251 diffuse large B-cell lymphoma cases and 737 controls, indicating this result was likely a false positive. It is likely that moderate sample size, inter-subtype and other genetic heterogeneity, and small true effect sizes account for the lack of replicable findings.
CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-κB and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-κB p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1α were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2α levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies.
Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.
Multiple myeloma (MM) is characterized by complex genetic abnormalities whose complexity signifies varying degree of chromosomal instability (CIN). In this study, we introduced a novel CIN measure, chromosome instability genome event count (CINGEC), which considered both copy number aberrations and interstitial breakpoints from high-resolution genome-wide assays. When assessed in two aCGH MM datasets, higher CINGEC was associated with poor survival. We then derived a CINGEC-associated gene expression profile (GEP) signature, CINGECS, using a dataset that has both aCGH and GEP. Genes in CINGECS were mainly involved in DNA damage responses besides in aneuploidy and other generic oncogenic processes contrary to other CIN associated GEP signatures. Finally, we confirmed its survival association in three GEP datasets that encompassed newly diagnosed patients treated with transplant-based protocol with or without novel agents for induction as well as relapsed patients treated with bortezomib. Furthermore, CINGECS was independent of many GEP-based prognostic signatures. In conclusion, our novel CIN measure has definite biological and clinical significance in myeloma.
This retrospective study was undertaken to evaluate the outcome of patients with stage I or II (limited stage), grade I–II follicular non-Hodgkin’s lymphoma (FL) treated with radiation therapy (RT) alone as initial management.
Patients with stage I or II and pathologically confirmed WHO grade I or II FL treated initially with RT alone between 1982 and 2008 were identified from a population based cancer registry.
Forty patients with a mean age 61.3 years at diagnosis were identified. The median follow up was 6.9 years from the end of radiation therapy. Stage was I (n = 26) and II (n = 14). None had B symptoms. The Follicular Lymphoma International Prognostic Index (FLIPI) was low risk in 26 patients and intermediate risk in 5. Doses ranged from 15 Gy to 48 Gy, with a median dose of 35 Gy. All patients achieved a complete clinical response (CR). 5 and 10 year overall survival (OS) was 86% and 59%, progression free survival (PFS) 67% and 54%. Age ≥60 at diagnosis was associated with reduced OS, p = 0.029, but did not affect PFS. No other clinical features including grade or FLIPI were significant for outcomes. Local failure was uncommon occurring in 8% (3/40) although this was 21% (3/14) of all recurrences.
OS and PFS outcomes for radiation alone in limited stage low grade FL patients from this single institution study are consistent with previously published data. No predictors were prognostic for PFS. A dose of ≤35 Gy may be appropriate. In this highly selected homogeneous group the FLIPI loses discriminating ability. Local control is excellent, and a majority of patients are free of disease after 5 years.
Clonal diversity in multiple myeloma (MM) includes both MM-related and MM-unrelated clonal expansions which are subject to dominance exerted by the MM clone. Here we show evidence for the existence of minor but highly expanded unrelated B-cell clones in patients with MM defined by their complementary determining region 3 (CDR3) peak. We further characterize these clones over the disease and subsequent treatment. Second clones were identified by their specific IgH-VDJ sequences that are distinct from those of dominant MM clones. Clonal frequencies were determined through semi-quantitative PCR, quantitative PCR and single-cell polymerase chain reaction of the clone-specific sequence. In 13/74 MM patients, more than one dominant CDR3 peak was identified with 12 patients (16%) being truly biclonal. Second clones had different frequencies, were found in different locations and were found in different cell types from the dominant MM clone. Where analysis was possible, they were shown to have chromosomal characteristic distinct from those of the MM clone. The frequency of the second clone also changed over the course of the disease and often persisted despite treatment. Molecularly-defined second clones are infrequent in monoclonal gammopathy of undetermined significance (MGUS, 1/43 individuals or 2%), suggesting that they may arise at relatively late stages of myelomagenesis. In further support of our findings, biclonal gammopathy and concomitant MM and CLL (chronic lymphocytic leukemia) were confirmed to originate from two unrelated clones. Our data supports the idea that the clone giving rise to symptomatic myeloma exerts clonal dominance to prevent expansion of other clones. MM and second clones may arise from an underlying niche permissive of clonal expansion. The clinical significance of these highly expanded but unrelated clones remains to be confirmed. Overall, our findings add new dimensions to evaluating related and unrelated clonal expansions in MM and the impact of disease evolution and treatment on clonal diversity.
The purpose of the present work was to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo acute myelogenous leukemia by sequencing the entire coding region. EZH2 mutations were identified in 13/714 (1.8%) of AML patients were found to be more common in males (P = 0.033). The presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P<0.0001) and -7/del(7q) (P = 0.025). There were no differences in the incidence of mutation in 13 genes, ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. No difference in complete remission, event-free survival, or overall survival was observed between patients with and without EZH2 mutation (P>0.05). Overall, these results showed EZH2 mutation in de novo acute myeloid leukemia as a recurrent genetic abnormality to be associated with lower blast percentage in BM and -7/del(7q).
Chronic lymphocytic leukemia (CLL) is typically regarded as an indolent B-cell malignancy. However, there is wide variability with regards to need for therapy, time to progressive disease, and treatment response. This clinical variability is due, in part, to biological heterogeneity between individual patients’ leukemias. While much has been learned about this biological variation using genomic approaches, it is unclear whether such efforts have sufficiently evaluated biological and clinical heterogeneity in CLL.
To study the extent of genomic variability in CLL and the biological and clinical attributes of genomic classification in CLL, we evaluated 893 unique CLL samples from fifteen publicly available gene expression profiling datasets. We used unsupervised approaches to divide the data into subgroups, evaluated the biological pathways and genetic aberrations that were associated with the subgroups, and compared prognostic and clinical outcome data between the subgroups.
Using an unsupervised approach, we determined that approximately 600 CLL samples are needed to define the spectrum of diversity in CLL genomic expression. We identified seven genomically-defined CLL subgroups that have distinct biological properties, are associated with specific chromosomal deletions and amplifications, and have marked differences in molecular prognostic markers and clinical outcomes.
Our results indicate that investigations focusing on small numbers of patient samples likely provide a biased outlook on CLL biology. These findings may have important implications in identifying patients who should be treated with specific targeted therapies, which could have efficacy against CLL cells that rely on specific biological pathways.
Rituximab is the first line drug to treat non Hodgkin’s lymphoma (B-NHL) alone or in combination with chemotherapy. However, 30–40% of B-NHL patients are unresponsive to rituximab or resistant after therapy. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of PEBP family and functions as an anti-apoptotic molecule. In this study, we found hPEBP4 to be expressed in up to 90% of B-cell lymphoma patients, but in only 16.7% of normal lymph nodes. Interestingly, hPEBP4 overexpression inhibited rituximab-mediated complement dependent cytotoxicity (R-CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in B-NHL cells while downregulation of hPEBP4 augmented the therapeutic efficacy of rituximab both in vitro and in vivo. Furthermore, hPEBP4 silencing sensitized the primary B-acute lymphocytic leukemia (B-ALL) cells to R-CDC. During rituximab-mediated complement dependent cytotoxicity, hPEBP4 was recruited to the cell membrane in a PE-binding domain dependent manner and inhibited R-CDC induced calcium flux and reactive oxygen species (ROS) generation. These events contributed to the decrease of cell death induced by R-CDC in B-cell lymphomas. Meanwhile, hPEBP4 knockdown potentiated the chemosensitization of the rituximab in B-cell lymphoma cells by regulating the expression of Bcl-xl, Cycline E, p21waf/cip1 and p53 and the activation of caspase-3 and caspase-9. Considering that hPEBP4 conferred cellular resistance to rituximab treatment and was preferentially expressed in lymphoma tissue, it could be a potential valuable target for adjuvant therapy for B-cell lymphoma.
Cell of origin classification of diffuse large B-cell lymphoma (DLBCL) identifies subsets with biological and clinical significance. Despite the established nature of the classification existing studies display variability in classifier implementation, and a comparative analysis across multiple data sets is lacking. Here we describe the validation of a cell of origin classifier for DLBCL, based on balanced voting between 4 machine-learning tools: the DLBCL automatic classifier (DAC). This shows superior survival separation for assigned Activated B-cell (ABC) and Germinal Center B-cell (GCB) DLBCL classes relative to a range of other classifiers. DAC is effective on data derived from multiple microarray platforms and formalin fixed paraffin embedded samples and is parsimonious, using 20 classifier genes. We use DAC to perform a comparative analysis of gene expression in 10 data sets (2030 cases). We generate ranked meta-profiles of genes showing consistent class-association using ≥6 data sets as a cut-off: ABC (414 genes) and GCB (415 genes). The transcription factor ZBTB32 emerges as the most consistent and differentially expressed gene in ABC-DLBCL while other transcription factors such as ARID3A, BATF, and TCF4 are also amongst the 24 genes associated with this class in all datasets. Analysis of enrichment of 12323 gene signatures against meta-profiles and all data sets individually confirms consistent associations with signatures of molecular pathways, chromosomal cytobands, and transcription factor binding sites. We provide DAC as an open access Windows application, and the accompanying meta-analyses as a resource.
The clinical impact of aberrant CEBPA promoter methylation (PM) in AML is controversially discussed. The aim of this study was to clarify the significance of aberrant CEBPA PM with regard to clinical features in a cohort of 623 cytogenetically normal (CN) de novo AML. 555 cases had wild-type CEBPA, 68 cases harbored CEBPA mutations. The distal promoter was methylated in 238/623 cases (38.2%), the core promoter in 8 of 326 cases (2.5%), whereas proximal PM was never detected. CEBPA PM and CEBPA mutations were mutually exclusive. CEBPA distal PM positive cases were characterized by reduced CEBPA mRNA expression levels and elevated white blood cell counts. CEBPA distal PM was less frequent in patients with mutations in FLT3, NPM1 and TET2 and more frequent in cases with RUNX1 and IDH2R140 mutations. Overall, no association of methylation to prognosis was seen. However CEBPA distal PM was associated with inferior outcome in cases with low FLT3-ITD ratio or TET2 mutations. A distinct gene expression profile of CEBPA distal PM positive cases compared to CEBPA mutated and CEBPA distal PM negative cases was observed. In conclusion, the presence of aberrant CEBPA PM is associated with distinct biological features but impact on outcome is weak.
Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.