Lack of extensive mutations in the VH5 genes used in common B cell chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) is a malignancy of the CD5+ B cells. Prior studies indicated that CLL B cells generally express immunoglobulin (Ig) VH and VL genes with little or no somatic mutations. However, a recent report indicated that VH251, one of three VH genes belonging to the VH5 subgroup (e.g., VH251, VH32, and VH15), not only is frequently rearranged in this disease, but also has extensive and selective mutations when expressed by CLL B cells. The extent and nature of these mutations contrasts markedly from the low level of mutations noted in VH5 genes used by normal B cells or other Ig V genes found expressed in CLL. To determine whether this difference reflects a unique property of VH251 or a previously unrecognized subgroup of CLL, we examined for VH5 Ig gene rearrangements in leukemia cells from 68 patients that satisfied clinical and diagnostic criteria for CD5+ B cell CLL. Southern blot hybridization studies with probes for VH251 and the JH locus revealed that only 7 (10%) of the 68 monoclonal CLL cell populations had undergone Ig gene rearrangement involving VH5 genes. Two (3%) were found to have functionally rearranged VH5 genes that shared > or = 98% sequence homology with 5-2R1, a VH251 gene isolated from a pre-B cell acute lymphocytic leukemia. The other five CLL (7%) had functionally rearranged VH5 genes that each shared > or = 99% nucleic acid sequence homology with a germline VH32 isolated from human sperm DNA. These data indicate that VH251 or VH32 also may be expressed by CD5+ CLL B cells with little or no somatic mutation. These findings contrast with a recently published study on VH5 gene expression in B CLL and contest the hypothesis that extensive somatic mutation is a common property of the VH5 genes used in this disease. Further work to define the clinical and/or phenotypic characteristics of patients with leukemia cells that express mutated versus nonmutated Ig V genes may reveal subsets of CLL that possibly differ in their cytogenesis, etiopathogenesis, and/or clinical behavior.

Lack of allelic exclusion in B cell chronic lymphocytic leukemia.

We determined the immunoglobulin (Ig) V(H) subgroup expressed by the leukemia cells of 108 patients with B cell chronic lymphocytic leukemia (CLL). Surprisingly, we found that six samples (5%) each expressed Ig of more than one V(H) subgroup. Southern blot analysis demonstrated that these samples each had rearrangements involving both Ig heavy chain alleles. Nucleic acid sequence analyses of the Ig cDNA revealed each to express two functional Ig V(H) genes: V(H)3-33 and V(H)4-39; V(H)3-7 and V(H)4-39; V(H)3-23 and V(H)4-61; V(H)2-70 and V(H)3-30.3; or V(H)3-30 and V(H)4-b (DP67). One sample expressed three Ig V(H) genes: V(H)2-70, V(H)3-7, and V(H)4-59. Despite having more than one Ig heavy chain transcript, each sample was found to express only one functional Ig light chain. From the primary sequence, we deduced that the Ig of some of these CLL samples should react with Lc1, a monoclonal antibody (mAb) reactive with a supratypic cross-reactive idiotype present on Ig encoded by a subgroup of Ig V(H)4 genes (namely, V(H)4-39, V(H)4-b [DP-67], V(H)4-59, or V(H)4-61), and B6, an mAb that reacts with Ig encoded by certain Ig V(H)3 genes (namely, V(H)3-23, V(H)3-30, or V(H)3-30.3), and/or modified staphylococcal protein A (SpA), a 45-kilodalton bacterial "superantigen" that reacts with most Ig of the V(H)3 subgroup. Flow cytometric analyses revealed that such samples did in fact react with Lc1 and B6 and/or SpA, but not with control mAbs of irrelevant specificity. This study demonstrates that a subset of CLL patients have leukemic B cells that express more than one functional Ig heavy chain.

Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression "signature," irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Relative seroprevalence of human herpes viruses in patients with chronic lymphocytic leukaemia.

BACKGROUND: Herpes virus infections may have a significant role in chronic lymphocytic leukaemia (CLL) due to their ability to modulate the host's immune system. MATERIALS AND METHODS: We examined the seroprevalence of four herpes viruses [Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), human herpes virus (HHV)-6 and -7] in a cohort of European CLL patients (cohort 1, n = 100) in relation to the immunoglobulin variable heavy (IGHV) chain gene use and compared serological results with those obtained from age- and gender-matched healthy adults (n = 100). RESULTS: CMV-seroprevalence was significantly higher in CLL cohort 1 (79%) than in the control cohort (57%, P = 0.001); the seroprevalence of EBV (89% vs. 94%), HHV-6 (73% vs. 60%), or HHV-7 (35% vs. 35%) was not. In CLL cohort 1, use of IGHV3-30 was more prevalent among CMV-seropositive and of IGHV3-21 among HHV-7-seronegative cases. To investigate the generalizability of these findings, we investigated the herpes virus seroprevalence in a second cohort of age-matched CLL patients from a different geographical area (USA, n = 100, cohort 2). In cohort 2, CMV-seroprevalence was comparable with that of the control cohort (53%). Seroprevalence of EBV, HHV-6 and HHV-7 were 85%, 88% and 73% respectively. In CLL cohort 2, use of IGHV3-30 or IGHV3-21 was not associated with any of the herpes viruses investigated. CONCLUSIONS: CMV-seropositivity is associated with CLL in selected patient cohorts. However, the considerable variation in herpes virus-specific seropositivity between geographically distinct CLL cohorts indicates that seropositivity for any of the four human herpes viruses investigated is not generally associated with CLL.

Elevation of soluble CD307 (IRTA2/FcRH5) protein in the blood and expression on malignant cells of patients with multiple myeloma, chronic lymphocytic leukemia, and mantle cell lymphoma.

CD307 is a differentiation antigen expressed in B-lineage cells. One soluble and two membrane-bound forms have been predicted and an enzyme-linked immunosorbent assay (ELISA) for soluble CD307 established. Our goal was to determine if CD307 is expressed on the surface of cells from patients with multiple myeloma (MM), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and other B-cell malignancies and if soluble CD307 levels are elevated in the blood of patients with these B-cell malignancies. Cells and blood were collected from patients. Expression of CD307 was measured by flow cytometry and blood levels of soluble CD307 by ELISA. High soluble CD307 levels were detected in 21/43 (49%) of patients with MM, 36/46 (78%) with CLL and 9/24 (38%) with MCL. Soluble CD307 levels correlated with plasma cell percentages in bone marrow aspirates in MM and total white blood cells in CLL. CD307 on the cell membrane was detected by flow cytometry in 8/8 MM, 23/29 CLL and 4/5 MCL samples. Because CD307 is present on malignant cells from patients with MM, CLL and MCL, CD307 may be a useful therapeutic target for the treatment of these diseases.

International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

Susceptibility to multiple sclerosis associated with an immunoglobulin gamma 3 restriction fragment length polymorphism.

Susceptibility to multiple sclerosis (MS) has been linked to the immunoglobulin G (Gm) markers as well as HLA-DR genes. We have used a genomic Ig gamma 1 probe which detects polymorphisms in the gamma 1, gamma 2, gamma 3 and pseudogamma genes to identify restriction fragment length polymorphisms associated with MS. A negative association was found between a 5.9-kilobase (kb) Bst EII gamma 3 fragment and MS. Southern blot analysis of genomic DNA revealed the presence of this fragment in 84 of 140 (60.0%) controls, but in only 17 of 59 (28.8%) MS patients. The frequency of the fragment in 47 myasthenia gravis and 16 Graves' disease patients was similar to that in controls, 60.0 and 62.5%, respectively.

The human immunoglobulin V(H) gene repertoire is genetically controlled and unaltered by chronic autoimmune stimulation.

The factors controlling immunoglobulin (Ig) gene repertoire formation are poorly understood. Studies on monozygotic twins have helped discern the contributions of genetic versus environmental factors on expressed traits. In the present experiments, we applied a novel anchored PCR-ELISA system to compare the heavy chain V gene (V(H)) subgroup repertoires of mu and gamma expressing B lymphocytes from ten pairs of adult monozygotic twins, including eight pairs who are concordant or discordant for rheumatoid arthritis. The results disclosed that the relative expression of each Ig V(H) gene subgroup is not precisely proportional to its relative genomic size. The monozygotic twins had more similar IgM V(H) gene repertoires than did unrelated subjects. Moreover, monozygotic twins who are discordant for RA also use highly similar IgM V(H) gene-subgroup repertoires. Finally, the V(H) gene repertoire remained stable over time. Collectively, these data reveal that genetic factors predominantly control V(H) gene repertoire formation.

The soluble CD40 ligand sCD154 in systemic lupus erythematosus.

We found that the plasma of patients with active systemic lupus erythematosus (SLE) could induce a human B-cell line (Ramos) to express high levels of immune accessory molecules that are commonly found on blood B cells of patients with active SLE. The ability of SLE plasma to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for the CD40 ligand (CD154) but not by antibodies to TNF-alpha. Immunoprecipitation studies with anti-CD154 identified a 20-kDa protein in the plasma of SLE patients with active disease, but not in plasma of normal donors, indicating that such plasma contained soluble CD154 (sCD154). Using a quantitative ELISA method, we found that the plasma of patients with active disease had levels of sCD154 that were significantly higher than those found in plasma of normal donors. Levels of CD154 transcripts in SLE blood lymphocytes correlated with the relative concentrations of sCD154 found in SLE plasma. Furthermore, plasma levels of sCD154 correlated with the titers of anti-double-stranded DNA autoantibody and with clinical disease activity. These studies indicate that sCD154 of patients with SLE may act as a functional ligand for CD40 that is associated with SLE disease activity.

Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors.

To better understand the stage(s) of differentiation reached by B-type chronic lymphocytic leukemia (B-CLL) cells and to gain insight into the potential role of antigenic stimulation in the development and diversification of these cells, we analyzed the rearranged VH genes expressed by 83 B-CLL cells (64 IgM+ and 19 non-IgM+). Our results confirm and extend the observations of a bias in the use of certain VH, D, and JH genes among B-CLL cells. In addition, they indicate that the VH genes of approximately 50% of the IgM+ B-CLL cells and approximately 75% of the non-IgM+ B-CLL cells can exhibit somatic mutations. The presence of mutation varies according to the VH family expressed by the B-CLL cell (VH3 expressers displaying more mutation than VH1 and VH4 expressers). In addition, the extent of mutation can be sizeable with approximately 32% of the IgM+ cases and approximately 68% of the non-IgM+ cases differing by > 5% from the most similar germline gene. Approximately 20% of the mutated VH genes display replacement mutations in a pattern consistent with antigen selection. However, CDR3 characteristics (D and JH gene use and association and HCDR3 length, composition, and charge) suggest that selection for distinct B cell receptors (BCR) occurs in many more B-CLL cells. Based on these data, we suggest three prototypic BCR, representing the VH genes most frequently encountered in our study. These data suggest that many B-CLL cells have been previously stimulated, placing them in the "experienced" or "memory" CD5(+) B cell subset.

Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration.

Cirmtuzumab inhibits Wnt5a-induced Rac1 activation in chronic lymphocytic leukemia treated with ibrutinib.

Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK) that can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting alternative pathways also contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. In this study, we found that CLL cells of patients treated with ibrutinib had activated Rac1. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated with ibrutinib at concentrations that were effective in completely inhibiting BTK and BCR-signaling. Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab (UC-961), an anti-ROR1 mAb. We found that treatment with cirmtuzumab and ibrutinib was significantly more effective than treatment with either agent alone in clearing leukemia cells in vivo. This study indicates that cirmtuzumab may enhance the activity of ibrutinib in the treatment of patients with CLL or other ROR1+ B-cell malignancies.

Wnt5a induces ROR1 to associate with 14-3-3ζ for enhanced chemotaxis and proliferation of chronic lymphocytic leukemia cells.

Wnt5a can activate Rho GTPases in chronic lymphocytic leukemia (CLL) cells by inducing the recruitment of ARHGEF2 to ROR1. Mass spectrometry on immune precipitates of Wnt5a-activated ROR1 identified 14-3-3ζ, which was confirmed by co-immunoprecipitation. The capacity of Wnt5a to induce ROR1 to complex with 14-3-3ζ could be blocked in CLL cells by treatment with cirmtuzumab, a humanized mAb targeting ROR1. Silencing 14-3-3ζ via small interfering RNA impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance in vitro exchange activity of ARHGEF2 and (3) induce activation of RhoA and Rac1 in CLL cells. Furthermore, CRISPR/Cas9 deletion of 14-3-3ζ in ROR1-negative CLL cell-line MEC1, and in MEC1 cells transfected to express ROR1 (MEC1-ROR1), demonstrated that 14-3-3ζ was necessary for the growth/engraftment advantage of MEC1-ROR1 over MEC1 cells. We identified a binding motif (RSPS857SAS) in ROR1 for 14-3-3ζ. Site-directed mutagenesis of ROR1 demonstrated that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2 to ROR1, and activation of RhoA and Rac1. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1 signaling, leading to enhanced CLL migration and proliferation.

Leukemia-cell proliferation and disease progression in patients with early stage chronic lymphocytic leukemia.

The clinical course of patients with recently diagnosed early stage chronic lymphocytic leukemia (CLL) is highly variable. We examined the relationship between CLL-cell birth rate and treatment-free survival (TFS) in 97 patients with recently diagnosed, Rai stage 0-II CLL in a blinded, prospective study, using in vivo 2H2O labeling. Birth rates ranged from 0.07 to 1.31% new cells per day. With median follow-up of 4.0 years, 33 subjects (34%) required treatment by NCI criteria. High-birth rate was observed in 44% of subjects and was significantly associated with shorter TFS, unmutated IGHV status and expression of ZAP70 and of CD38. In multivariable modeling considering age, gender, Rai stage, expression of ZAP70 or CD38, IGHV mutation status and FISH cytogenetics, only CLL-cell birth rate and IGHV mutation status met criteria for inclusion. Hazard ratios were 3.51 (P=0.002) for high-birth rate and 4.93 (P<0.001) for unmutated IGHV. The association between elevated birth rate and shorter TFS was observed in subjects with either mutated or unmutated IGHVs, and the use of both markers was a better predictor of TFS than either parameter alone. Thus, an increased CLL birth rate in early stage disease is a strong predictor of disease progression and earlier treatment.

EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.

Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

Increased expression of CD152 (CTLA-4) by normal T lymphocytes in untreated patients with B-cell chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) have defects in both cellular and humoral immunity. Since CD152 (CTLA-4) plays a critical role in downregulating T-cell responses, we studied the expression of surface and cytoplasmic CD152 (sCD152 and cCD152, respectively) in freshly isolated T cells from treatment-naïve patients with CLL. CD4+ and CD8+ T cells from these patients demonstrated significantly increased sCD152 and cCD152 compared to normal donors. Furthermore, these patients had an increased proportion of the regulatory CD4(+)/CD25(+)/CD152+ subset that correlated with advanced Rai stage, unfavorable cytogenetics and low serum IgG and IgA levels. The expression of sCD152 by T cells also correlated with ZAP-70 expression by CLL B cells. The proportion of CD4(+)/CD25+ cells was also correlated with unmutated immunoglobulin heavy chain variable gene status. Blockade of CD152 with monoclonal antibody (mAb) in proliferation assays was associated with potent T-cell proliferation in response to autologous and allogeneic CD40-activated CLL B cells. In summary, T cells from patients with CLL may be primed for anergy by expressing increased amounts of CD152; anti-CD152 mAb may represent a therapeutic opportunity to enhance an immune response against autologous leukemia cells.

Cloning and characterization of CLLD6, CLLD7, and CLLD8, novel candidate genes for leukemogenesis at chromosome 13q14, a region commonly deleted in B-cell chronic lymphocytic leukemia.

Chromosome 13q14 deletions constitute the most common structural aberration in B-cell chronic lymphocytic leukemia (B-CLL). We constructed a high-resolution physical map covering the critical deleted region in B-CLL at 13q14 and flanking sequences. The order and position of both genomic markers and known genes were determined precisely. Three novel genes, CLLD6, CLLD7, and CLLD8, were isolated and characterized. The predicted protein sequence of CLLD6 revealed no homology with known proteins. However, both CLLD7 and CLLD8 predicted proteins contain known functional domains. CLLD7 has both an RCC1 and a BTB domain, and could thus be involved in cell cycle regulation by chromatin remodeling. CLLD8 contains a methyl-CpG binding, a preSET and a SET domain, suggesting that CLLD8 might be associated with methylation-mediated transcriptional repression. Mutation analysis of hematopoietic tumor cell lines and B-CLL tumor samples revealed no point mutations within the coding region of these three novel genes. The functional domains present within CLLD7 and CLLD8 suggest that the proteins may be involved in critical cellular processes such as cell cycle and transcriptional control and could therefore be directly or indirectly involved in leukemogenesis.

Characterization of the 13q14 tumor suppressor locus in CLL: identification of ALT1, an alternative splice variant of the LEU2 gene.

Chromosome 13q14 deletions constitute the most common genetic abnormality in chronic lymphocytic leukemia (CLL). To identify the putative tumor suppressor gene targeted by 13q14 genomic loss, we completely sequenced and characterized a segment of 790 kb at 13q14 spanning the minimal region of loss in CLL. Transcribed sequences in the region were identified through database homology searches and exon-prediction analysis. Two-hundred kb at the centromeric end of the sequence contain five CpG islands, three previously identified genes LEU5/RFP2, LEU2, and LEU1, seven of seven EST clusters composed of >10 ESTs, and a large number of predicted exons. Homology searches against the mouse EST database have allowed us to identify a highly conserved alternative first exon of the LEU2 gene, giving rise to a novel transcript, ALT1 (GenBank accession no. AF380424), which originates within a G+C region in the vicinity of the D13S272 marker. Two novel 3' exons of LEU2 were also identified and are present in both LEU2 and ALT1 transcripts. However, we have not identified any mutations in leukemia cases, or alterations in expression of mRNAs in the region, that might directly implicate these mRNAs in the pathology of CLL. The centromeric end of the sequence, where all reported genes are located, contains twice the expected amount of ALU repeats, whereas the telomeric end is LINE1 rich and contains four LINE1 elements longer than 4 kb, including two full-length LINE1 sequences. This feature of the sequence may favor the occurrence of chromosomal rearrangements and may confer instability to the region, resulting in deletions that may inactivate an as yet unidentified tumor suppressor.

Lenalidomide treatment and prognostic markers in relapsed or refractory chronic lymphocytic leukemia: data from the prospective, multicenter phase-II CLL-009 trial.

Efficacy of lenalidomide was investigated in 103 patients with relapsed/refractory chronic lymphocytic leukemia (CLL) treated on the prospective, multicenter randomized phase-II CLL-009 trial. Interphase cytogenetic and mutational analyses identified TP53 mutations, unmutated IGHV, or del(17p) in 36/96 (37.5%), 68/88 (77.3%) or 22/92 (23.9%) patients. The overall response rate (ORR) was 40.4% (42/104). ORRs were similar irrespective of TP53 mutation (36.1% (13/36) vs 43.3% (26/60) for patients with vs without mutation) or IGHV mutation status (45.0% (9/20) vs 39.1% (27/68)); however, patients with del(17p) had lower ORRs than those without del(17p) (21.7% (5/22) vs 47.1% (33/70); P=0.049). No significant differences in progression-free survival and overall survival (OS) were observed when comparing subgroups defined by the presence or absence of high-risk genetic characteristics. In multivariate analyses, only multiple prior therapies (⩾3 lines) significantly impacted outcomes (median OS: 21.2 months vs not reached; P=0.019). This analysis indicates that lenalidomide is active in patients with relapsed/refractory CLL with unfavorable genetic profiles, including TP53 inactivation or unmutated IGHV. (ClinicalTrials.gov identifier: NCT00963105).

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.