ALBERT

Description: Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Description: The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with 〉90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p

Description: The LRF CLL4 trial randomised 777 previously untreated patients with Binet stage progressive A, B or C disease between January 1999 and October 2004 to receive either Chlorambucil, Fludarabine or Fludarabine and Cyclophosphamide. Interphase FISH for deletions of chromosome 6q, 11q, 13q, 17p and trisomy 12, IgVH gene mutational status (98% cut off), CD38 (7% cut off) and ZAP70 (10% cut off) expression were measured at randomisation on 579, 523, 535 and 478 patients respectively. Leukemic cells from 39 patients utilised the VH3-21 gene of whom 33 had homologous CDR3’s. Among the biological markers, log rank analysis showed that 〉20% p53 loss, del 11q, unmutated VH genes, high CD38 and high ZAP 70 correlated with disease progression or death (Table 1) but not deletion of chromosome 6q, 13q and trisomy 12 (p=0.7, 0.3 and 0.2 respectively). There was no difference in PFS or response duration between the 52 patients with 5–20% p53 loss and the 494 patients with no p53 loss. Multivariate Cox regression analysis showed that 〉20% p53 loss (p

Description: Abstract 3113 The natural history of patients with monoclonal B-cell lymphocytosis (MBL) who present with a lymphocytosis of 98% homology to germline (unmutated) 7/37 (19%) 22/57 (38.5%) P=0.0666 V gene usage: . . . 1–2 1/37 (2.7%) 19/57 (33%) P=0.0002 4–34 9/37 (24%) 4/57 (7%) P=0.0297 3–23 4/37 (11%) 4/57 (7%) P=0.7077 All 10 MBL cases for whom a CT or abdominal ultrasound scan was performed at or close to the time of presentation, showed no abnormality. None of the 47 cases has evolved into a chronic lymphoproliferative disorder other than SMZL. Lymphocyte morphology is often difficult to evaluate in cases with a slight lymphocytosis, but only 12 MBL cases did not show morphological features of SMZL in 〉10% of cells. MBL cases typically expressed moderate or strong surface IG and were CD5-, CD23-, CD79d+ and FMC7+, however, 9 expressed weak CD5. All had a Matutes CLL score of

Description: Chronic lymphocytic leukemia (CLL) is a paradigmatic malignancy where the interplay of cell-extrinsic and cell-intrinsic factors has a major impact on disease evolution. Indeed, extrinsic triggering, e.g. antigenic stimulation through the B-cell receptor (BcR), together with intrinsic aberrations, e.g. accumulation of genetic defects, play a major role throughout the natural history of CLL. The importance of antigen involvement is underscored by the existence of 'stereotyped' BcR in up to 30% of CLL patients. Notably, CLL patients with stereotyped BcR can be grouped into different subsets, each with a subset-biased biological and clinical profile. For instance, while the clinically aggressive subset #2 (IGHV3-21/IGLV3-21, comprising both mutated (M-CLL) and unmutated (U-CLL) IGHV genes) displays a remarkably high frequency of SF3B1 mutations, subset #8, a subset with the highest risk of Richter transformation, shows a strong association with trisomy 12 and NOTCH1 mutations. ATM defects are implicated in the evolution of CLL and are associated with a dismal prognosis, however the extent to which they contribute to the genetic landscape in stereotyped subsets remains unexplored. To gain insight into this issue, we assessed the frequency of ATM mutations in 249 well-characterized CLL patients assigned to major subsets #1-8. The entire coding region of ATM (62 exons) was investigated with two different targeted deep-sequencing approaches, i.e. Haloplex technology (HiSeq, coverage ~1500X) or the Nextera XT kit (MiSeq, coverage ~4000X). A conservative variant allele frequency cut-off of 10% was selected, and mutations were validated by Sanger sequencing. Altogether, we identified 61 ATM mutations in 47/249 (19%) patients across all major subsets (Fig. 1). As expected, the majority of identified ATM mutations (n=43, 70%) have not yet been reported while the remaining 30% were listed in the HGMD or COSMIC mutation databases. The spectrum of ATM mutations included missense (n=31), nonsense (n=9), splicing (n=6), and frame-shift (n=14) mutations, and one in-frame deletion. Missense substitutions were distributed along the entire gene without any 'hotspot' region or preferred domain. The highest mutation frequency was detected in subset #2 (26%), with a significant enrichment in U-CLL vs. M-CLL cases, (13/33 vs. 8/48 subset #2 cases, respectively; p=0.021). Within poor-prognostic U-CLL subsets, ATM mutations were also frequent in subsets #6 (25%) and #7 (23%), while subsets #3, #5, #1, and #8 showed lower frequencies (17%, 17%, 13%, and 7%, respectively). The favorable prognostic M-CLL subset #4 exhibited a low frequency (7%) of ATM mutations. Notably, when comparing the two most populated subsets, i.e. #1 and #2, ATM mutations were overrepresented in the latter with a borderline significance value (p=0.086); when restricting the analysis to U-CLL #2 cases a significantly higher frequency was observed compared to #1 (13/33 vs 9/68; p=0.0045). Regarding the clinical impact of ATM defects in subset #2, we divided patients into subgroups with biallelic inactivation (def-ATM), sole 11q-, sole ATM -mutation, TP53 -aberrations and WT. While both groups with mono-allelic ATM disruption showed a significantly reduced overall survival compared to WT (median survival sole ATM -mutation, 71 months, sole 11q-, 40 months, vs. 123 months in the WT group; p=0.002 and 0.02, respectively), a non-significant reduction of overall survival was observed for patients with bi-allelic ATM aberrations (70 months, p=0.29) (Fig. 2). The few subset #2 patients with TP53 defects showed a similar survival as WT group, underscoring previous observations that TP53 dysfunction per se plays a minor role in this subset. In summary, we demonstrate that ATM mutations can be added to the list of genetic defects with a biased distribution in stereotyped subsets. The enrichment of ATM defects in subset #2 was associated with a negative impact on overall survival, suggesting a role for ATM inactivation in shaping the aggressive phenotype of this subset. This study further supports the recent suggestion that CLL development is driven by antigenic selection, coupled with preferential acquisition of specific genetic defects. The work was supported by the projects MSMT CR CZ.1.05/1.1.00/02.0068, IGA NT13493-4/2012 and TACR TE02000058. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. . Strefford:Roche: Research Funding. Stamatopoulos:Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding.

Description: 80% of patients with CLL present with a lymphocytosis detected on a routine blood count. Many studies have identified risk factors for disease progression but there remains uncertainty about the magnitude of risk and which factors are most useful in determining this risk. To address this we have studied a cohort of 122 stage A0 patients who presented with a lymphocytosis and typical CLL immunophenotype before 1996 and whose CLL has either progressed or remained stable over a 10 year period. All patients were reviewed at least annually. Disease progression was defined as the need for anti-leukemic therapy, an increase in stage or a downward trend in haemoglobin or platelet count, progressive lymphadenopathy or splenomegaly or constitutional symptoms. Karyotypic data, from analysis of G banded metaphases derived fromTPA stimulated lymphocyte cultures, were available on all cases at diagnosis. CD38 and ZAP 70 expression were measured on DMSO frozen cells. VH gene usage and mutational status, and telomere length were assessed on frozen DNA samples, while interphase FISH for del 13q14, del 11q and 17p (ATM and p53 loss) and trisomy 12, was undertaken on fixed cell suspensions. All samples had been collected and stored at, or close to the time of diagnosis. 50 patients had progressive disease. Of the 72 patients with stable disease, the lymphocyte count remained below 20x109/l in 53 and rose with a lymphocyte doubling time of 〉 1 year in the remainding 19 (termed slowly progressive). In univariate analysis, a lymphocyte count of 〉 20x109/l, unmutated VH genes, high expression of CD38 (cut off 30%) and ZAP70, del 11q, abnormal presenting karyotype and short telomere length all predicted for disease progression. There was no difference in VH gene usage between the progressive and stable groups. Among the 107 patients for whom lymphocyte count, VH gene mutational status, CD38 and ZAP 70 expression are all currently available, the number of poor risk factors in the progressive and stable groups is shown in Table 1. There was no difference in the number of risk factors between the stable and slowly progressive groups. Del 11q was found in 9 progressive cases all of whom had 2 or more risk factors. P53 loss was found in a single patient with stable disease and no risk factors. 20/122 patients presented with a lymphocyte count 〈 5.0x109/l, below which patients have been considered to have monoclonal B cell lymphocytosis. Interestingly, 5 of these developed progressive disease. Using only the lymphocyte count, CD38 and ZAP 70 expression, Table 2 shows the risk of disease progression according to the risk factors present. In summary, once ZAP 70 measurement by flow cytometry is standardized, readily available prognostic tests can provide a quantitative estimate of the risk of disease progression. Performing interphase FISH for 11q and p53 loss in poorer risk cases only may provide additional prognostic information. Table 1 No. of adverse factors at presentation. (%) 0 1 2 3 4 Group Progressive 11(25) 10(23) 11(25) 9(20) 3(7) Stable/Slowly progressive 44(70) 16(25) 3(5) 0 0 Table 2 Odds of Progression % of Patients Progressing No risk factors 0.2 16.9 ZAP70 only 0.74 42.7 Lymphs〉20 0.89 47.0 CD38 only 1.57 61.1 ZAP70+lymphs 3.24 76.4 ZAP70+CD38 5.75 85.2 CD38+lymphs 6.85 87.3 All 25.1 96.2

Description: Whilst the spectrum and clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies is well established, their incidence, biological and clinical importance in splenic marginal zone lymphoma (SMZL) remains uncertain. Accordingly we screened 175 well-characterised SMZL cases for mutations in 768 genes (Haloplex Target Enrichment System) with a known or postulated role in B-cell physiology, B-cell malignancies in general and SMZL pathophysiology in particular. After sequencing (HiSeq 2000) we achieved a mean depth across our gene panel of 297-fold (range 128-702x), with more than 85% of all bases covered at 〉50-fold. After comprehensive filtering, 1,374 single nucleotide variants and insertions/deletions were identified. 221 genes were recurrently mutated at a gene frequency of 2-16% [n=2-28]. Sanger sequencing confirmed 86/86 selected variants in our recurrent genes, and showed 99% concordance between our Haloplex and Sanger sequencing of NOTCH2 exon 34, which was performed in all patients. Comprehensive validation of both germ-line Haloplex [n=18 patients] and Sanger sequencing established the sensitivity and specificity of our approach, and confirmed the biological importance and somatic origin of the genes described herein. To identify biologically relevant genes, we employed MutSigCV analysis, an algorithm that identifies significantly mutated genes by accounting for background mutation rate, DNA replication time and the gene size. 18 mutated genes were identified with TP53 [n=28], KLF2 [n=21], MYD88 [n=12], NOTCH2 [n=17], TNFAIP3 [n=13] and CCND3 [n=15] being the most significant; genes that encode components of pathways important in the regulation and differentiation of mature B-cells were also identified, including CREBBP [n=9], MAP3K6 [n=5], KDM2B [n=7], SETD1B [n=6], TRAF3 [n=9], ARID1A [n=10], BIRC3 [n=3], BCL10 [n=5], BTG1 [n=3], ATM [n=10], NFKBIE [n=4] and DDI1 [n=4]. Then, we searched for significant pairwise gene correlations and mutually exclusive relationships between our mutated genes demonstrating the following: (1) independent events, such as MYD88, where a mutation is invariably observed as an isolated event; (2) cancer drivers that have a similar proportion of co-occurring and mutually-exclusive relationships, such as NOTCH2, TP53, TNFAIP3 and CREBBP, and (3) genes such as KLF2, CCND3 and ARID1A that have proportionally more co-occurring relationships, thus suggesting a synergistic function to promote tumorigenesis. Finally, we studied clonal evolution, by differentiating between early, clonal events, and later, subclonal mutations (ABSOLUTE algorithm), and we were able to classify clonal or subclonal mutation in 6/18 of our MutSigCV genes. Paradigmatically, we observed that all the CREBBPmutations were fully clonal. Amongst our most novel findings was KLF2, or Krüppel-like factor 2, mutations that were distributed across the entire protein, with a cluster in the C2H2 domain and were all somatically acquired. All mutations tested were clonal, significantly associated with del(7q) (P=0.001), IGHV1-2*04 gene usage (P

Description: Several lines of evidence implicate antigenic stimulation in the pathogenesis of chronic B-lymphoproliferative disorders. This is illustrated by B-cell chronic lymphocytic leukaemia (B-CLL) in which there is non-random use of immunoglobulin (Ig) variable heavy chain (Vh) genes and a gene expression profile and immunophenotype in keeping with antigen exposure and activation. Sequence analysis of IgVh genes reveals that within B-CLL there is a subset of patients whose tumors utilize Vh3–21 and show such homology between antigen binding sites that the Ig must recognize the same antigen. In splenic marginal zone lymphoma (SMZL) overuse of the Vh1–02 gene is well documented and there is similarly some restriction of antigen binding sites. Engagement of the B cell receptor by a relatively restricted range of antigens might thus select cells that subsequently form the malignant clone. T lymphocytes may also be involved in this process since IgVh genes in about 50% of B-CLL patients show evidence of somatic hypermutation and CD40L expressing T lymphocytes surround proliferating tumor cells in pseudofollicles in the bone marrow and lymph node. Although both Vh3–21 B-CLL and Vh1–02 SMZL have a relatively low Vh gene mutational rate, it is thought that somatic hypermutation has taken place indicating possible interaction with T cells. To investigate whether such T cell/tumor interactions might be antigen specific we looked for evidence of MHC restriction in homologous cases of Vh3–21 B-CLL and Vh1–02 SMZL. Medium resolution HLA typing was performed by PCR-SSO and the phenotypes compared with normal controls and other disease groups by Fishers exact test with Bonferroni correction (shown following the uncorrected values). Groups included: Vh3–21 B-CLL with/without homologous CDR3 (n=33/7), B-CLL cases with random Vh gene use (without Vh1–02 or Vh3–21, n=22), SMZL with (n=18) and without (n=18) Vh1–02, B-CLL with Vh1–02 (n=17) and 1667 normal controls drawn from the same population. Of 18 patients with Vh1–02 SMZL, 11 (61%) had an HLA-DR15 phenotype compared to only 441/1667 (26%) controls (p=0.0022, 0.029) and 2/18 (11%) of non Vh1–02 SMZL (p=0.0045, 0.058). Of the 7 Vh1–02 SMZL that did not express HLA-DR15, 5 expressed HLA-DR4. Non Vh1–02 SMZL was associated with HLA-DR4 (p=0.0023, 0.023) but not HLA-DR15. A number of other associations between IgVh homologous subgroups and MHC phenotype were noted including Vh3–21 B-CLL and HLA-B15 and non Vh3–21/Vh1–02 B-CLL and HLA-B58 however once corrected for the number of variables, statistical significance was lost. In general the Vh genes of both normal and neoplastic human marginal zone B cells show evidence of somatic hypermutation although whether this occurs through a T-dependent or independent pathway is currently unknown. The finding of MHC restriction in Vh subsets of SMZL however suggest that this disorder derives from a marginal zone B cell that has responded to a T-dependent antigen. Whether similar mechanisms apply in B-CLL is unclear from the current data and further study of a larger number of cases is therefore warranted.

Description: Splenic marginal zone lymphoma (SMZL) is a rare indolent B cell malignancy whose diagnosis is based on splenic and/or marrow histology, lymphocyte morphology and immunophenotype. A low level paraprotein and deletion of chromosome 7q are found in approximately 40% of patients. IgVH gene analysis has been performed in several small studies with evidence of biased VH gene usage noted in some studies, and a poor clinical outcome for patients with unmutated VH genes found in a single study. In order to clarify the biological and clinical significance of VH gene usage and mutational state in SMZL we have analysed pooled data from 5 European centres. Of the172 VH sequences analysed 53 (31%) utilised the V1-02 gene compared to a usage of 4% in 939 cases of CLL and 〈 4% in 315 normal B cell rearrangements. 89% of SMZL V1-02’s utilised the V1-02-04 allele compared to only 40% in CLL. D3-3 was used in 43% of the SMZL V1-02 cases compared to only 15% in the non-V1-02 cases. 51% of V1-02 cases had 〉97.9% homology to the germline sequence, most ranged between 96–99%. Constitutional DNA was sequenced from 6 V1-02 cases whose tumor DNA showed 98–99% homology. In each case differences in the tumor VH gene from the germline sequence were due to mutations rather than polymorphisms. The VH gene of 19/22 V1-02’s analysed showed at least 1 of 3 common mutations observed. 13/18 V1-02 cases utilised D3-3 in reading frame 3 and retained 2 aminoacids (GV) in all cases. The mean CDR3 length was 20 aminoacids (range 18–25). 4 additional cases utilising D3-3 in reading frame 1 showed conservation of 3 aminoacids (FLE) and 2 of these cases had virtually identical CDR3’s. These data suggest a role for antigen in the pathogenesis of SMZL, at least in a subset of patients. Clinical outcome data were available in 166 patients presenting with splenomegaly of whom 130 underwent splenectomy. 98 had mutated VH genes and 68 unmutated VH genes using a 98% cut off. There was no difference in overall survival between mutated and unmutated cases nor between the V1-02 and non V1-02 cases. Disease progression after initial therapy occurred in 69 patients overall and in 46 who underwent splenectomy as primary therapy. In neither group of progressive cases was there a correlation between progression and VH gene mutational status. VH gene data was also available on an additional 26 patients who presented with a lymphocytosis and the typical lymphocyte morphology, immunophenotype and incidence of 7q deletions found in SMZL but without splenomegaly. Only 1 case (4%) utilised the V1-02 gene. Further studies are required to determine whether these patients represent a benign subset of SMZL or a separate disorder. In conclusion, VH gene analysis in SMZL confirms biased VH gene usage with evidence of antigen selection but does not support the clinical use of VH genes as a prognostic marker.

Description: Abstract 1587 Monoclonal B-cell lymphocytosis (MBL) with an immunophenotype consistent with marginal zone origin (MBL-MZ), that can be either CD5− or CD5+ but atypical for CLL, and also lacking an IGH/CCND1 translocation, is an increasingly recognised entity with poorly understood biological background and clinical significance. In particular, it is not yet clarified whether it represents a precursor state to one of the distinct lymphoma entities recognized by WHO as deriving from MZ cells or whether it constitutes a novel entity, likely with similar ontogeny. To obtain insight into this issue we retrospectively evaluated a series of 71 patients (male/female: 35/36, median age: 73.3 years) with lymphocytosis (median lymphocyte count: 5.77 × 109/l) detected incidentally on a routine blood test. No case had lymphadenopathy, organomegaly or any clinical features to suggest a concurrent marginal zone lymphoma. Hemoglobin and platelet counts were normal in all cases; 15/57 (26%) cases had paraproteinemia. Peripheral blood immunophenotyping revealed the presence of a clonal B-cell population with Matutes score