Frontline low-dose alemtuzumab with fludarabine and cyclophosphamide prolongs progression-free survival in high-risk CLL.

The randomized Haemato Oncology Foundation for Adults in The Netherlands 68 phase 3 trial compared front-line chemotherapy with chemotherapy plus the CD52 monoclonal antibody alemtuzumab for high-risk chronic lymphocytic leukemia, defined as at least 1 of the following: unmutated immunoglobulin heavy chain genes, deletion 17p or 11q, or trisomy 12. Fit patients were randomized to receive either 6 28-day cycles of oral FC chemotherapy (days 1 through 3: fludarabine 40 mg/m(2) per day and cyclophosphamide 250 mg/m(2) per day: n = 139) or FC plus subcutaneous alemtuzumab 30 mg day 1 (FCA, n = 133). FCA prolonged the primary end point, progression-free survival (3-year progression-free survival 53 vs 37%, P = .01), but not the secondary end point, overall survival (OS). However, a post hoc analysis showed that FCA increased OS in patients younger than 65 years (3-year OS 85% vs 76%, P = .035). FCA also increased the overall response rate (88 vs 78%, P = .036), and the bone marrow minimal residual disease-negative complete remission rate (64% vs 43%, P = .016). Opportunistic infections were more frequent following FCA, but without an increase in treatment related mortality (FCA: 3.8%, FC: 4.3%). FCA improves progression-free survival in high-risk chronic lymphocytic leukemia. As anticipated, FCA is more immunosuppressive than FC, but with due vigilance, does not lead to a higher treatment-related mortality. This study was registered at www.trialregister.nl as trial no. NTR529.

Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies.

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.

Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells.

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies.

LPL is the strongest prognostic factor in a comparative analysis of RNA-based markers in early chronic lymphocytic leukemia.

BACKGROUND: The expression levels of LPL, ZAP70, TCL1A, CLLU1 and MCL1 have recently been proposed as prognostic factors in chronic lymphocytic leukemia. However, few studies have systematically compared these different RNA-based markers. DESIGN AND METHODS: Using real-time quantitative PCR, we measured the mRNA expression levels of these genes in unsorted samples from 252 newly diagnosed chronic lymphocytic leukemia patients and correlated our data with established prognostic markers (for example Binet stage, CD38, IGHV gene mutational status and genomic aberrations) and clinical outcome. RESULTS: High expression levels of all RNA-based markers, except MCL1, predicted shorter overall survival and time to treatment, with LPL being the most significant. In multivariate analysis including the RNA-based markers, LPL expression was the only independent prognostic marker for overall survival and time to treatment. When studying LPL expression and the established markers, LPL expression retained its independent prognostic strength for overall survival. All of the RNA-based markers, albeit with varying ability, added prognostic information to established markers, with LPL expression giving the most significant results. Notably, high LPL expression predicted a worse outcome in good-prognosis subgroups, such as patients with mutated IGHV genes, Binet stage A, CD38 negativity or favorable cytogenetics. In particular, the combination of LPL expression and CD38 could further stratify Binet stage A patients. CONCLUSIONS: LPL expression is the strongest RNA-based prognostic marker in chronic lymphocytic leukemia that could potentially be applied to predict outcome in the clinical setting, particularly in the large group of patients with favorable prognosis.

Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.

BACKGROUND: High-resolution genomic microarrays enable simultaneous detection of copy-number aberrations such as the known recurrent aberrations in chronic lymphocytic leukemia [del(11q), del(13q), del(17p) and trisomy 12], and copy-number neutral loss of heterozygosity. Moreover, comparison of genomic profiles from sequential patients' samples allows detection of clonal evolution. DESIGN AND METHODS: We screened samples from 369 patients with newly diagnosed chronic lymphocytic leukemia from a population-based cohort using 250K single nucleotide polymorphism-arrays. Clonal evolution was evaluated in 59 follow-up samples obtained after 5-9 years. RESULTS: At diagnosis, copy-number aberrations were identified in 90% of patients; 70% carried known recurrent alterations, including del(13q) (55%), trisomy 12 (10.5%), del(11q) (10%), and del(17p) (4%). Additional recurrent aberrations were detected on chromosomes 2 (1.9%), 4 (1.4%), 8 (1.6%) and 14 (1.6%). Thirteen patients (3.5%) displayed recurrent copy-number neutral loss of heterozygosity on 13q, of whom 11 had concurrent homozygous del(13q). Genomic complexity and large 13q deletions correlated with inferior outcome, while the former was linked to poor-prognostic aberrations. In the follow-up study, clonal evolution developed in 8/24 (33%) patients with unmutated IGHV, and in 4/25 (16%) IGHV-mutated and treated patients. In contrast, untreated patients with mutated IGHV (n=10) did not acquire additional aberrations. The most common secondary event, del(13q), was detected in 6/12 (50%) of all patients with acquired alterations. Interestingly, aberrations on, for example, chromosome 6q, 8p, 9p and 10q developed exclusively in patients with unmutated IGHV. CONCLUSIONS: Whole-genome screening revealed a high frequency of genomic aberrations in newly diagnosed chronic lymphocytic leukemia. Clonal evolution was associated with other markers of aggressive disease and commonly included the known recurrent aberrations.

Propionic acid secreted from propionibacteria induces NKG2D ligand expression on human-activated T lymphocytes and cancer cells.

We found that propionic acid secreted from propionibacteria induces expression of the NKG2D ligands MICA/B on activated T lymphocytes and different cancer cells, without affecting MICA/B expression on resting peripheral blood cells. Growth supernatant from propionibacteria or propionate alone could directly stimulate functional MICA/B surface expression and MICA promoter activity by a mechanism dependent on intracellular calcium. Deletion and point mutations further demonstrated that a GC-box motif around -110 from the MICA transcription start site is essential for propionate-mediated MICA promoter activity. Other short-chain fatty acids such as lactate, acetate, and butyrate could also induce MICA/B expression. We observed a striking difference in the molecular signaling pathways that regulate MICA/B. A functional glycolytic pathway was essential for MICA/B expression after exposure to propionate and CMV. In contrast, compounds with histone deacetylase-inhibitory activity such as butyrate and FR901228 stimulated MICA/B expression through a pathway that was not affected by inhibition of glycolysis, clearly suggesting that MICA/B is regulated through different molecular mechanisms. We propose that propionate, produced either by bacteria or during cellular metabolism, has significant immunoregulatory function and may be cancer prophylactic.

Verification that common variation at 2q37.1, 6p25.3, 11q24.1, 15q23, and 19q13.32 influences chronic lymphocytic leukaemia risk.

A recent genome wide association study of chronic lymphocytic leukaemia (CLL) provided evidence that common variation at 2q13 (rs17483466), 2q37.1 (rs13397985), 6p25.3 (rs872071), 11q24.1 (rs735665), 15q23 (rs7176508) and 19q13.32 (rs11083846) affects CLL risk. To verify and further explore the relationship between these variants and CLL risk we genotyped case-control datasets from Spain and Sweden (824 cases, 850 controls). Combined data provided statistically significant support for an association between genotypes at rs13397985, rs872071, rs735665, rs7176508 and rs11083846 and CLL risk. CLL risk increased with increasing numbers of risk alleles (P(trend) = 1.40 x 10(-15)), consistent with a polygenic model of disease susceptibility. These data validate the relationship between common variation and risk of CLL.

Distinct gene expression profiles in subsets of chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors.

BACKGROUND: Numerous subsets of patients with chronic lymphocytic leukemia display similar immunoglobulin gene usage with almost identical complementarity determining region 3 sequences. Among IGHV4-34 cases, two such subsets with "stereotyped" B-cell receptors were recently identified, i.e. subset #4 (IGHV4-34/IGKV2-30) and subset #16 (IGHV4-34/IGKV3-20). Subset #4 patients appear to share biological and clinical features, e.g. young age at diagnosis and indolent disease, whereas little is known about subset #16 at a clinical level. DESIGN AND METHODS: We investigated the global gene expression pattern in sorted chronic lymphocytic leukemia cells from 25 subset/non-subset IGHV4-34 patients using Affymetrix gene expression arrays. RESULTS: Although generally few differences were found when comparing subset to non-subset 4/16 IGHV4-34 cases, distinct gene expression profiles were revealed for subset #4 versus subset #16. The differentially expressed genes, predominantly with lower expression in subset #4 patients, are involved in important cell regulatory pathways including cell-cycle control, proliferation and immune response, which may partly explain the low-proliferative disease observed in subset #4 patients. CONCLUSIONS: Our novel data demonstrate distinct gene expression profiles among patients with stereotyped IGHV4-34 B-cell receptors, providing further evidence for biological differences in the pathogenesis of these subsets and underscoring the functional relevance of subset assignment based on B-cell receptor sequence features.

High-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients with 'stereotyped' IGHV3-21 and IGHV4-34 B-cell receptors.

BACKGROUND: The existence of multiple subsets of chronic lymphocytic leukemia expressing 'stereotyped' B-cell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that 'stereotypy' may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets. DESIGN AND METHODS: We applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 IGHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients. RESULTS: Over 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75-76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and non-subset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy. CONCLUSIONS: Genomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.

Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia--a comparative study of four differently designed, high resolution microarray platforms.

Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K), oligonucleotide arrays (185K, Agilent), and two SNP arrays (250K, Affymetrix and 317K, Illumina). Cross-platform comparison revealed 29 concordantly detected CNAs, including known recurrent alterations, which confirmed that all platforms are powerful tools when screening for large aberrations. However, detection of 32 additional regions present in 2-3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy-number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping detection. Evaluation of baseline variation and copy-number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform-specific CNAs. The SNP arrays displayed higher technical variation, although this was compensated by high density of elements. Affymetrix detected a higher degree of CNAs compared to Illumina, while the latter showed a lower noise level and higher detection rate in the LOH analysis. Large-scale studies of genomic aberrations are now feasible, but new tools for LOH analysis are requested.

Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.

Plasma alemtuzumab levels in patients with chronic lymphocytic leukemia treated with alemtuzumab combined with chemotherapy reflect the efficacy of the treatment: a hypothesis.

In the HOVON68 trial comparing subcutaneous low-dose alemtuzumab (LD-A) used together with fludarabine (F) and cyclophosphamide (C) with FC alone in high-risk chronic lymphocytic leukemia (CLL), LD-AFC resulted in significantly more clinical and molecular responses than FC, but also in more opportunistic infections. In a subgroup analysis of alemtuzumab trough levels during treatment by a sensitive enzyme-linked immunosorbent assay (ELISA) method, detectable levels were found in 4/6 complete and 0/3 partial responders. A relationship between alemtuzumab plasma levels, response and duration of lymphocytopenia was evident. We hypothesize that following combination therapy, the response may not be a function of the alemtuzumab levels, but the opposite, that plasma alemtuzumab levels are a function of the efficacy of the entire treatment, and the fewer leukemic target cells that are remaining, the higher are the levels of plasma alemtuzumab. This concept may well provide a guide for alemtuzumab dosage in future trials.

Quality of life in patients with diffuse large B-cell lymphoma treated with dose-dense chemotherapy is only affected temporarily.

(R)-CHOP-14 has substantially improved outcome in DLBCL, but may have increased morbidity and reduced quality of life (QoL). Our aim was to evaluate QoL during (R)-CHOP-14-based chemotherapy. Twenty-six patients participated (small single-center study). EORTC QLQ-C30 was completed pre-treatment, mid-treatment, 14 days post-treatment, and 3 months post-treatment. Scores were compared to a reference population, and analyzed separately. Pre-treatment, global health status (p = 0.004), physical functioning (p = 0.036), role functioning (p = 0.017), and emotional functioning (p = 0.040) were reduced, and fatigue (p = 0.009) and appetite loss (p = 0.007) increased compared to the reference population. During treatment, physical functioning and role functioning decreased significantly, whereas emotional functioning, fatigue, and diarrhea increased. Three months post-treatment, scores were generally equivalent to those of the reference population, and lower for nausea/vomiting (p < 0.001) and constipation (p < 0.001). Disease-related symptoms were frequent in high-risk DLBCL. Treatment-related symptoms were normalized 3 months post-treatment. In conclusion, QoL is only temporarily affected during (R)-CHOP-14-based chemotherapy, and the treatment regimen is therefore feasible.

TP53 Mutations are infrequent in newly diagnosed chronic lymphocytic leukemia.

TP53 mutations in the absence of 17p-deletion correlate with rapid disease progression and poor survival in chronic lymphocytic leukemia (CLL). Herein, we determined the TP53 mutation frequency in 268 newly diagnosed CLL patients from a population-based material. Overall, we detected TP53 mutations in 3.7% of patients (n = 10), where 7/10 cases showed a concomitant 17p-deletion, confirming the high prevalence of TP53 mutation in 17p-deleted patients. Only 3 (1.1%) of the newly diagnosed patients in our cohort thereby carried TP53 mutations without 17p-deletion, a frequency that is much lower than previous reports on referral cohorts (3-6%). Our findings imply that TP53 mutations are rare at CLL onset and instead may arise during disease progression.

Lack of association between the MDM2 promoter polymorphism SNP309 and clinical outcome in chronic lymphocytic leukemia.

The 309T>G polymorphism in the promoter region of the MDM2 gene, known as SNP309, has recently been suggested as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL) although this has been questioned. To investigate this further, we analyzed the MDM2 SNP309 genotypes in 418 CLL patients and correlated the results with established CLL prognostic factors, time to treatment and overall survival. In this Swedish cohort, no association existed between any particular MDM2 SNP309 genotype, overall survival and time to treatment. Furthermore, no correlation was shown between the MDM2 SNP309 genotypes and Binet stage, IGHV mutational status and recurrent genomic aberrations. In summary, this study argues against the use of the MDM2 SNP309 as a prognostic marker in CLL.

Common variants at 2q37.3, 8q24.21, 15q21.3 and 16q24.1 influence chronic lymphocytic leukemia risk.

To identify new risk variants for chronic lymphocytic leukemia (CLL), we conducted a genome-wide association study of 299,983 tagging SNPs, with validation in four additional series totaling 2,503 cases and 5,789 controls. We identified four new risk loci for CLL at 2q37.3 (rs757978, FARP2; odds ratio (OR) = 1.39; P = 2.11 x 10(-9)), 8q24.21 (rs2456449; OR = 1.26; P = 7.84 x 10(-10)), 15q21.3 (rs7169431; OR = 1.36; P = 4.74 x 10(-7)) and 16q24.1 (rs305061; OR = 1.22; P = 3.60 x 10(-7)). We also found evidence for risk loci at 15q25.2 (rs783540, CPEB1; OR = 1.18; P = 3.67 x 10(-6)) and 18q21.1 (rs1036935; OR = 1.22; P = 2.28 x 10(-6)). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy.