Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

[Incidental finding lymphocytosis. From normal variants up to leukemia]. / Zufallsbefund Lymphozytose. Von der Normvariante bis zur Leukämie.

A relative or absolute increase of lymphocytes in peripheral blood is a frequent incidental finding. The differential diagnosis of such a finding includes a broad spectrum ranging from normal variations to neoplastic diseases. As for other laboratory parameters lymphocytosis must be interpreted within a given clinical situation. Further diagnostics aim to reliably differentiate between reactive and neoplastic conditions. If a lymphoma or leukemia is suspected this should lead to a rapid hemato-oncological investigation.

Prophylaxis of infectious complications with colony-stimulating factors in adult cancer patients undergoing chemotherapy-evidence-based guidelines from the Infectious Diseases Working Party AGIHO of the German Society for Haematology and Medical Oncology (DGHO).

BACKGROUND: Current evidence on myelopoietic growth factors is difficult to overview for the practicing haematologist/oncologist. International guidelines are sometimes conflicting, exclude certain patient groups, or cannot directly be applied to the German health system. This guideline by the Infectious Diseases Working Party (AGIHO) of the German Society of Haematology and Medical Oncology (DGHO) gives evidence-based recommendations for the use of G-CSF, pegylated G-CSF, and biosimilars to prevent infectious complications in cancer patients undergoing chemotherapy, including those with haematological malignancies. METHODS: We systematically searched and evaluated current evidence. An expert panel discussed the results and recommendations. We then compared our recommendations to current international guidelines. RESULTS: We summarised the data from eligible studies in evidence tables, developed recommendations for different entities and risk groups. CONCLUSION: Comprehensive literature search and expert panel consensus confirmed many key recommendations given by international guidelines. Evidence for growth factors during acute myeloid leukaemia induction chemotherapy and pegfilgrastim use in haematological malignancies was rated lower compared with other guidelines.

ASXL1 exon 12 mutations are frequent in AML with intermediate risk karyotype and are independently associated with an adverse outcome.

We aimed at evaluating ASXL1mut in 740 AML with intermediate risk karyotype for frequency, association with other mutations and impact on outcome. Five hundred fifty-three cases had a normal karyotype (NK) and 187 had intermediate risk aberrant cytogenetics. Overall, ASXL1mut were detected in 127/740 patients (17.2%). ASXL1mut were more frequent in males than in females (23.5% vs 9.9%, P<0.001). They were associated with higher age (median: 71.8 vs 61.8, P<0.001), a history of preceding myelodysplastic syndromes, and with a more immature immunophenotype compared with patients with wild-type ASXL1 (ASXL1wt). ASXL1mut were more frequent in patients with aberrant karyotype (58/187; 31.0%), especially in cases with trisomy 8 (39/74; 52.7%), than in those with NK (69/553; 12.5%; P<0.001). ASXL1mut were observed more frequent in RUNX1mut (P<0.001), and less frequent in NPM1mut (P<0.001), FLT3-internal tandem duplication (ITD) (P<0.001), FLT3-TKD (P=0.001) and DNMT3Amut (P<0.001). Patients with ASXL1mut had a shorter overall survival (OS) (P<0.001) and event free survival (P=0.012) compared with ASXL1wt. In multivariable analysis, ASXL1mut was an independent adverse factor for OS (P=0.032, relative risk: 1.70). In conclusion, ASXL1mut belong to the most frequent mutations in intermediate risk group AML. Their strong and independent dismal prognostic impact suggests the inclusion into the diagnostic work-up of AML.

Survival, prognostic factors and rates of leukemic transformation in 381 untreated patients with MDS and del(5q): a multicenter study.

Myelodysplastic syndromes (MDS) with del(5q) are considered to have a benign course of the disease. In order to address the issue of the propensity of those patients to progress to acute myeloid leukemia (AML), data on 381 untreated patients with MDS and del(5q) characterized by low or intermediate I International Prognostic Scoring System (IPSS) risk score were collected from nine centers and registries. Median survival of the entire group was 74 months. Transfusion-dependent patients had a median survival of 44 months vs 97 months for transfusion-independent patients (P<0.0001). Transfusion need at diagnosis was the most important patient characteristic for survival. Of the 381 patients, 48 (12.6%) progressed to AML. The cumulative progression rate calculated using the Kaplan-Meier method was 4.9% at 2 years and 17.6% at 5 years. Factors associated with the risk of AML transformation were high-risk World Health Organization adapted Prognostic Scoring System (WPSS) score, marrow blast count >5% and red-cell transfusion dependency at diagnosis. In conclusion, patients with MDS and del(5q) are facing a considerable risk of AML transformation. More detailed cytogenetic and molecular studies may help to identify the patients at risk of progression.

Factors influencing the pharmacokinetics of prophylactic posaconazole oral suspension in patients with acute myeloid leukemia or myelodysplastic syndrome.

OBJECTIVES: To estimate the pharmacokinetic (PK) properties of posaconazole in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) undergoing chemotherapy in a clinical setting. METHODS: Posaconazole concentrations in patients with AML/MDS receiving prophylactic posaconazole were determined by high-performance liquid chromatography. A population PK model with nonlinear mixed effect modeling was developed. The list of tested covariates included age, weight, height, gender, posaconazole dose, ethnicity, co-administration of antineoplastic chemotherapy, ranitidine or pantoprazole, coincident fever, diarrhea, leukocyte counts, and γ-glutamyltransterase plasma activity. RESULTS: A total of 643 serum concentrations of posaconazole from 84 patients were obtained. A one-compartment model with first order absorption and elimination as the basic structural model appropriately described the data, with an apparent clearance of 56.8 L/h [95% confidence interval (CI) 52.8­60.8 L/h] and an apparent volume of distribution of 2,130 L (95% CI 1,646­2,614 L). Significant effects on apparent clearance (CL/F) were found for presence of diarrhea and for co-medication with proton-pump inhibitors (1.5- and 1.6-fold increase in CL/F, respectively), weight (33.4 L larger apparent volume of distribution per kilogram), and co-administration of chemotherapy (0.6-fold lower apparent volume of distribution). CONCLUSION: We developed a prediction basis for mean posaconazole concentrations in AML/MDS patients. Patient weight, presence of diarrhea, and concomitant medication (chemotherapy and pantoprazole) showed significant effects on posaconazole exposure. Corresponding adjustments of the starting dose according to the presence of diarrhea and during the co-administration of chemotherapy or proton-pump inhibitors appear justified before therapeutic drug monitoring results are available. Further investigation of the interaction between different chemotherapeutic regimens and posaconazole is warranted.

Wnt/ß-catenin/LEF-1 signaling in chronic lymphocytic leukemia (CLL): a target for current and potential therapeutic options.

There is a growing body of evidence that Wnt signaling, which is already known to play a critical role in various types of cancer, also has a vital function in B cell neoplasias, particularly in chronic lymphocytic leukemia (CLL). It is known that Wnt proteins are overexpressed in primary CLL cells and several physiological inhibitors are partly inactivated in this disease. Furthermore, ß-catenin is upregulated upon Wnt stimulation and cooperates with the transcription factor lymphoid enhancer binding factor-1 (LEF-1). LEF-1 is excessively overexpressed in CLL cells by more than 3,000-fold compared to normal B cells. Moreover, LEF-1 could be identified as an important regulator of pathophysiologically relevant genes in CLL, and several Wnt/ß-catenin signaling components substantially influence CLL cell survival. In this review we summarize the current state of knowledge about Wnt/ß-catenin/LEF-1 signaling in CLL. Following a short overview of current treatment concepts in CLL, we briefly describe Wnt signaling in human cancers. We then discuss recent progress in understanding regulation of the Wnt/ß-catenin/LEF-1 signaling pathway in this disease. Based on the present scientific evidence we highlight which components of this important signaling pathway could serve as therapeutic targets in CLL. We then present previous results gained from experimental approaches to target different parts of the Wnt/ß-catenin/LEF-1 cascade. Together with potentially promising approaches we also critically reflect on the kind of difficulties and problems that may arise using such strategies.

Clinical effectiveness of posaconazole prophylaxis in patients with acute myelogenous leukaemia (AML): a 6 year experience of the Cologne AML cohort.

BACKGROUND: Large randomized controlled trials have shown significant decreases in morbidity and mortality in leukaemia patients with posaconazole prophylaxis. However, the value of prophylaxis has been questioned in centres with a low incidence of invasive fungal diseases (IFDs) and pre-emptive treatment strategies. METHODS: We prospectively evaluated the epidemiology of IFDs in acute myelogenous leukaemia (AML) patients undergoing first remission-induction chemotherapy before and after posaconazole prophylaxis had been introduced as a standard of care. Patients admitted from January 2003 to December 2005 received topical polyenes as antifungal prophylaxis (first group), while those admitted between January 2006 and December 2008 received 200 mg of oral posaconazole three times daily (second group). Other diagnostic and therapeutic standard operating procedures remained unchanged. RESULTS: A total of 82 patients in the polyene prophylaxis group and 77 in the posaconazole prophylaxis group were included in the final analysis. Baseline characteristics were well matched between groups. Patients receiving topical polyene prophylaxis were more likely to experience breakthrough IFDs (19.5% and 3.9%; P = 0.003) or breakthrough aspergillosis (13.4% and 2.6%; P = 0.018) than patients receiving systemic posaconazole prophylaxis. They also had more febrile days (mean 10.7 +/- 9.66 and 7.3 +/- 5.73; P = 0.007), longer need for inpatient treatment (mean 53.0 +/- 24.16 and 46.0 +/- 14.39; P = 0.026) and a shorter fungal-free survival (78.7 and 90.4 days; P = 0.024). No significant differences were observed for persistent fever, pneumonia, lung infiltrates indicative of invasive pulmonary aspergillosis, or attributable and overall mortality. CONCLUSIONS: After introduction of posaconazole prophylaxis for patients with AML, the number of febrile days, the incidence rate of IFDs and aspergillosis and the duration of hospitalization decreased significantly.

Patients with haematological malignancies show a restricted body image focusing on function and emotion.

The diagnosis of cancer threatens the psychological and bodily integrity. Based on this assumption, we aimed to explore how newly diagnosed patients cope with special regard to the body image (BI). In total, 40 patients (32 haematological malignancies) were assessed by questionnaires on mood, complaints, self-regulation and quality of life (QOL). The BI was assessed by the 'Body Grid' which reveals the constructs patients choose to characterize the body. The constructs were categorized using a model of six predefined categories comprising: emotion, control, activity, strength, function and appearance. Tinnitus sufferers and medical students served as comparison groups. Cancer patients showed significantly more anxious depression and a significantly lower QOL than controls. Their BI was restricted, focusing the functional status of body organs (e.g. opposing healthy vs. ill organs) as well as emotional aspects (e.g. trust vs. fear). The data convey fundamental psychological distress in newly diagnosed cancer patients. Restriction of BI and use of functional constructs may help to buffer the threat to body integrity. The emotional constructs reflect the existential impact. The data give a clear indication for the need for early psychosocial support which should aim at stabilizing the psychological and bodily integrity of the patient.

Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique.

Recently, various mutations within the Abl sequence have been described that negatively affect imatinib binding to Bcr/Abl resulting in cellular resistance of chronic myeloid leukemia (CML) cells. So far, little is known as to whether these mutations are preexisting or develop under imatinib therapy as current mutation analyses are limited by a low sensitivity of approximately 1:2 (50%) to 1:5 (20%). By combining peptide nucleic acid (PNA)-based DNA clamping with a fluorescence hybridization probe assay, we developed a new and highly sensitive technique for the detection of known mutations within the Bcr/Abl kinase domain. With this approach we investigated 19 cases of CML refractory to imatinib treatment before and during therapy. By clamping of wild-type Abl through PNA we could effectively enhance the detection sensitivity for the Bcr/Abl mutations Thr315Ile, Glu255Lys, and Tyr253His such that 1 mutant cDNA molecule could be detected in 500 negatives (0.2%). We observed in one case that a Gly255Lys mutation was detectable before treatment. By DNA analysis of buccal swaps, a genetic polymorphism could be excluded. In two cases clonal evolution of known mutations developed gradually under treatment. In another case an initially detectable Tyr253His mutation disappeared after therapy onset but was again observed after 6 weeks of imatinib treatment. Preexisting and evolving Bcr/Abl mutations associated with an unfavorable prognosis could be safely detected by the presented technique. This may facilitate risk stratification in CML and may serve as a model for individualized molecular monitoring and therapeutic strategies in other malignant diseases.

Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.

Quantitation of target mRNAs using the reverse-transcription polymerase chain reaction found a widespread field of application in diverse biomedical diagnostic assays. However, the problem of varying sample quality has to be solved by correcting target molecule amounts through detection of an endogenous control template. The choice of an appropriate reference gene is still object of debate as pseudogene co-amplification and expression level variations may limit the usefulness of some currently used reference reactions. We compared quantitative expression levels of the commonly used endogenous reference genes beta-actin (beta-actin), beta-2-microglobulin (beta2-MG) and porphobilinogen deaminase (PBDG) using the TaqMan chemistry. With these assays we investigated the respective expression patterns in K562 cells and leucocytes of normal individuals as well as of malignoma patients. In K562 cells 1544+246 beta-actin, 65+30 beta2-MG and 22+/-8 PBDG copies/cell were detected. In normal leucocytes 491+/-97 beta-actin, 40+/-17 beta2-MG and <1 PBDG copies/cell were quantified. Leucocytes of various malignancies exhibited 84+/-51 beta-actin, 106+/-8 beta2-MG and <1 PBDG copies/cell. We conclude that beta2-MG is the most suitable reference gene tested as its variation between different sample origins and within distinct cell types was acceptable low.

Selection of B-cell chronic lymphocytic leukemia cell variants by therapy with anti-CD20 monoclonal antibody rituximab.

OBJECTIVE: Anti-CD20 chimeric monoclonal antibody rituximab (Mabthera; IDEC-C2B8) is currently tested in several clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). In the present study, we investigated whether rituximab therapy may select for CD20(-) subclones. MATERIALS AND METHODS: Leukemic B-CLL cells were isolated from patients with B-CLL and sensitivity to rituximab-induced cell death was examined. Levels of CD20 protein and mRNA were determined using flow cytometry and real-time PCR, respectively. Clonality analyses of leukemic cells throughout rituximab therapy were performed by GeneScan analysis of patient clone specific rearrangements of the complementarity determining region III of the heavy chain immunoglobulin. RESULTS: Cytotoxicity of rituximab in vitro did not depend on the protein levels of CD20. During therapy with rituximab CD20(+) B-CLL cells were depleted and CD20(-) leukemic cells emerged. After treatment, the initial CD20(+) B-CLL cell clone reexpanded. CD20(-) B-CLL cells retained their capacity to synthesize the CD20 molecule. CONCLUSIONS: These data support the concept that in B-CLL rituximab treatment may not lead to the emergence of CD20(-) leukemic variants. Our findings support clinical studies investigating the benefit of prolonged period of rituximab therapy in B-CLL disease.

Fluorescent 5'-exonuclease assay for the absolute quantification of Wilms' tumour gene (WT1) mRNA: implications for monitoring human leukaemias.

The Wilms' tumour gene (WT1) has been suggested as a powerful parameter for molecular monitoring of minimal residual disease (MRD) in leukaemias. However, molecular monitoring via WT1 RNA levels is far from being routinely performed, which is possibly owing to the complex and inaccurate quantitative reverse transcription polymerase chain reaction (RT-PCR) procedures. Using a newly-developed quantitative real time RT-PCR, we measured WT1 transcripts in peripheral blood leucocytes of patients with acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid leukaemia (CML). While healthy blood donors did not show measurable amounts of WT1 transcripts, WT1 RNA levels were detectable in all types of leukaemia. Furthermore, intraindividual WT1 transcript kinetics were exclusively dependent on disease progression, treatment and subsequent disease outcome. Using this approach, we could distinguish between treatment response and failure within the first days of therapeutic intervention. Moreover, gradually rising WT1 levels over a period of weeks and months paralleled long-term disease progression and appeared to be a prognostic indicator for subsequent clinical relapse. A linear correlation between quantities of WT1 and bcr/abl fusion transcripts could be seen in CML. We conclude that quantitative assessment of WT1 transcripts using real-time PCR is an appropriate method for molecular monitoring of AML, ALL and CML, and can be used independently for both short- and long-term monitoring of leukaemia patients.

Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia.

The p73 protein shares structural and functional similarities with the tumour-suppressor p53, but its role in neoplastic transformation is unknown. Alternative splicing leads to the expression of at least nine p73 C-terminal mRNA splice variants (alpha, beta, gamma, delta, epsilon, zeta, eta, eta1, theta). In this survey, we analyse the expression of p73 by real-time quantitative RT-PCR, its known C-terminal variants with an RT-PCR-Southern technique and by Western blot in samples of 51 patients with B-CLL, normal B lymphocytes from eight individuals, and five haematopoetic cell lines. p73alpha protein expression positively correlated with higher risk B-CLL stages (P = 0.046). Total p73 mRNA expression was higher (P = 0.01) and p73alpha protein more frequently detected (P = 0.008) in B-CLL compared with normal CD19+-B-lymphocytes. p73 C-terminal mRNA variants were expressed both in B-CLL and in normal B-lymphocytes, but their expression was biased since the gamma (P = 0.041), the theta (P < 0.001), and the eta variant (P = 0.033) prevailed in normal B-lymphocytes. In summary, we conclude that the accumulation of p73, the expression pattern of particular p73 variants and its link to progression may play a distinct role in the molecular pathology B-CLL.

The tight linkage between DNA replication and double-strand break repair in bacteriophage T4.

Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.