Baliakas, Panagiotis

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin 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 immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. 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.

Abstract 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<0.0001). Regarding other genomic aberrations, they were either absent (NOTCH1 mutations) or rare (SF3B1 mutations, trisomy 12, del(11q), TP53 aberrations due to either del(17p) and/or TP53 mutations). The sole exception concerned a high frequency (14%) of TP53 aberrations in subset #29 (p<0.05 compared with the other subsets), which is notable for M-CLL cases in general. Time to first treatment (TTFT) could be analyzed in 228 cases. IgG subsets #4 and #16 had significantly (p=0.036) longer TTFT (median TTFT: not yet reached) compared to the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). In conclusion, we have identified distinct clinicobiological profiles for different stereotyped IGHV4-34 M-CLL subsets, highlighting subsets #4 and #16 as particularly indolent, which is important for both medical and social reasons, especially considering that a significant proportion of patients in these subsets are diagnosed at younger ages. Our findings support the notion that BcR IG stereotypy refines prognostication in CLL, superseding the crude immunogenetic distinction based on SHM load only. Additionally, the observed heterogeneity suggests that not all M-CLL are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Disclosures Tausch: Gilead: Other: Travel support. Shanafelt:Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Polyphenon E Int'l: Research Funding; Hospira: Research Funding; Janssen: Research Funding; Pharmactckucs: Research Funding; Cephalon: Research Funding. Niemann:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy; Novartis: Other: Travel grant. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:Janssen Pharmaceuticals: Research Funding.

Key Points The ClinGen MM-VCEP has specified RUNX1-specific curation rules to address gene function, gene-specific domains, and phenotypic criteria. RUNX1-specific criteria resulted in a reduction in CONF and VUS variants by 33%, emphasizing the need for expert variant curation.

Abstract Abstract 2638 We recently demonstrated that over 30% of cases with splenic marginal-zone lymphoma (SMZL) express distinctive immunoglobulin (IG) receptors that utilize a single polymorphic variant of the IGHV1-2 gene (IGHV1-2*04) and also exhibit restricted antigen-binding site motifs and precise targeting of somatic hypermutation (SHM). On these grounds, we proposed the existence of molecular subtypes of SMZL defined by immunogenetic analysis of the IG receptors with implications for selection by specific (super) antigenic element(s) in the development of at least a major subset of SMZL. In order to gain insight as to whether antigen involvement is relevant only prior to the malignant transformation or if it continues to affect the SMZL clone itself leading to intraclonal diversification (ID) through ongoing SHM, we conducted a large-scale subcloning study of rearranged IG heavy variable genes, in a total of 471 subcloned sequences from 22 SMZL cases. The analysis was intentionally biased towards cases expressing IGHV1-2*04 receptors that exhibit a series of distinctive immunogenetic features, including biased usage of the IGHD3-3 and IGHD3-10 genes, unusually long heavy complementarity-determining region 3 (VH CDR3) and minimally/borderline mutated status (identity to the germline in the range of 97–99.6%). Hence, the study group included 16 IGHV1-2*04 cases and 6 cases utilizing other IGHV genes. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and a median of 21 (9–46) colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences from the same sample. Overall, 15/22 cases (68%) carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, of which 12 utilized the IGHV1-2*04 gene whereas the remaining 3 utilized other IGHV genes. The high frequency of ID within the IGHV1-2*04 group, ranging from limited to (often) pronounced, is noteworthy in view of the generally low level of SHM among IGHV1-2*04 receptors. Detailed analysis of the distribution and molecular features of CMs revealed: (i) restricted ID patterns, in the sense of identical mutations in certain VH positions among subclones of different cases; (ii) “hotspots” of ID, i.e. particular codons exhibiting intense ongoing mutational activity (especially notable in this respect was codon 39 in VH FR2); (iii) a predominance of conservative amino acid changes, characterized by somatically introduced amino acid belonging to the same biochemical category as the mutating amino acid; and (iv) limited diversification within VH CDR3. Additionally, in 7/12 IGHV1-2*04 cases with CMs, identified ID patterns delineate distinct “clusters” of subcloned sequences with unique as well as shared mutations and closely similar if not identical VH CDR3s (including identical VH CDR3 length), pointing to “branching” of the malignant clone into distinct subclones, perhaps able to evolve along related yet distinct pathways. In conclusion, our study indicates that the SHM mechanism may continuously operate in certain subsets of SMZL, especially those expressing IGHV1-2*04 receptors. Although the precise timing of interactions with antigen(s) and their functional implications for SMZL evolution will likely remain difficult to define accurately, the results reported here suggest a role for persistent antigenic stimulation, at least for certain immunogenetically defined subsets of SMZL. Disclosures: Mollejo: Red Temática de Investigación Cooperativa en Cancer (RETICC): Research Funding; Asociación Española contra el Cancer: Research Funding.

Abstract The existence of stereotyped B cell receptor immunoglobulins (BcR IG) in chronic lymphocytic leukemia (CLL) strongly implicated antigen selection in disease ontogeny. We have previously shown that the stereotyped fraction encompasses ~30% of all CLL and includes multiple subsets with distinct BcR IG configuration and variable size. Eventually, certain major subsets emerged as distinct clinical entities, exemplified by subset #2 (IGHV3-21/IGLV3-21, ~2.5-3% of all CLL, mixed somatic hypermutation (SHM) status) of a particularly aggressive clinical course, thus, sharply contrasting subset #4 (IGHV4-34/IGKV2-30, ~1% of all CLL, mutated IGHV genes, M-CLL), a prototype for indolent disease. Here, taking advantage of a multi-institutional cohort of 21,123 CLL IG rearrangements, almost three times the size of the largest previous study, and the availability of validated, purpose-built immunoinformatics methods, we reappraised BcR IG stereotypy especially focusing on major subsets and the degree of their sequence similarity to related minor subsets. Stereotypy discovery was performed with ARResT/Teiresias, while stereotypy assignment to existing subsets previously deemed as major was performed with ARResT/AssignSubsets (http://bat.infspire.org/arrest/). In the present study, a subset was characterized as major if representing > 0.2% of the cohort (i.e. at least 50 cases). Minor subsets closely related to major ones (termed satellite) were identified applying the following criteria: (i) usage of IGHV genes from the same phylogenetic clan; (ii) VH CDR3 length difference ranging from -2 to +2 compared to the respective major subset; (iii) shared VH CDR3 sequence motif; and, (iv) -2 to +2 difference in the offset of the VH CDR3 motif compared to the respective major subset. In total, 7378/21123 (34.9%) IG sequences were grouped into subsets with stereotyped VH CDR3, with the previously characterized 19 major subsets accounting collectively for 2594 sequences (12.3%) of the cohort: of these, 12 included cases with unmutated IGHV genes (U-CLL), 6 concerned M-CLL and 1 (subset #2) included cases with mixed SHM status. Four additional subsets exceeded 50 cases, and, thus, were also considered as 'major'. These results reinforce the notion that not all CLL will end up being stereotyped but rather that a plateau for stereotypy exists at ~1/3 of the cohort. Subset #2 was the largest subset (n=572, 2.7%), while subset #1 (IGHV clan I (IGHV1,5,7 subgroups)/IGKV1(D)-39) was the most frequent subset within U-CLL (n=515, 2.4%) and subset #4 the most common M-CLL subset (n=192, 0.9%), hence displaying remarkable consistency regarding their frequency in all cohorts published since the pioneering studies. Altogether, Teiresias and AssignSubsets gave concordant results for previously identified major subsets, illustrating the validity of our approach. Satellite subsets were sought for individually for each major subset. In general, few satellite subsets were identified, most of which concerned U-CLL major subsets. That notwithstanding, notable cases of satellite subsets were exemplified by major subset #1 and its satellite subset #99 from which it differed only in VH CDR3 length (13 aminoacids in subset #1 versus 14 in subset #99); interestingly, both subsets displayed equally aggressive clinical course. Another example concerned subset #8 (IGHV4-39/IGKV1(D)-39, U-CLL), an aggressive subset with very high risk for Richter's transformation, that, except for a one-aminoacid difference in VH CDR3 length, was otherwise identical to satellite subset #215, also displaying clinical aggressiveness. Overall, our results confirm that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease subgroups amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Most major subsets display unique sequence motifs, however satellite subsets exist, especially within U-CLL. Considering ever-increasing evidence that major stereotyped subsets may represent distinct disease subgroups, the existence of satellite subsets reveals a novel aspect of repertoire restriction and has implications for refined molecular classification of CLL. Disclosures Shanafelt: Genentech: Research Funding; GlaxoSmithkKine: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding. Gaidano:Roche: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Niemann:Janssen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Gilead: Consultancy. Langerak:F. Hofmann-LaRoche, Genentech: Research Funding; InVivoScribe Technologies: Patents & Royalties: Royalties are provided to European Network (EuroClonality). Jaeger:Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Kater:Celgene: Research Funding; Gilead: Research Funding; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Stilgenbauer:Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding. Hallek:Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Rosenquist:Gilead Sciences: Speakers Bureau. Ghia:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding; Adaptive: Consultancy; Abbvie: Consultancy, Honoraria. Stamatopoulos:Novartis: Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding.

Abstract In chronic lymphocytic leukemia (CLL), the molecular features of the clonotypic B-cell receptor immunoglobulins (BcR IG) are set from the birth of the clone and in contrast to genetic aberrations, remain stable overtime, rendering the BcR IG a reference biomarker that is usually not significantly affected by clonal evolution. Approximately 30% of CLL cases carry quasi-identical BcR IGs and can be assigned to distinct stereotyped subsets. While preliminary evidence alludes to BcR IG stereotypy being relevant from a clinical viewpoint, this aspect has never been explored systematically or in a cohort of adequate size to enable meaningful conclusions. In order to assess the clinical implications of BcR IG stereotypy, we evaluated clinicobiological data from 8593 CLL patients, particularly focusing on 14 major stereotyped subsets of cases with unmutated (U-CLL) or mutated IGHV genes (M-CLL). The largest subset was #2 (n=254, 3%), within which 156 and 98 cases were M-CLL and U-CLL, respectively, followed by U-CLL subsets #1 (n=173, 2%) and #7 (n=123, 1.4%). Amongst M-CLL, the largest subsets were #4 (n=94, 1.1%) and #148 (n=92, 1%). Stereotyped subsets, even of the same mutational category i.e. U-CLL or M-CLL, exhibited significant clinicobiological differences regarding: age at diagnosis (median age ranging from 53-68 years, p<0.001); gender distribution (male/female ratios ranging from 0.85-4.4, p<0.001); the frequency of advanced stage disease (Binet C) at diagnosis (ranging from 9-21%, p<0.001); CD38 positivity (median percentage of positive cells ranging from 2-73%, p<0.05). Moving to cytogenetic findings, we detected subset-biased spectra of FISH-detected aberrations which can be summarized as follows: (i) isolated del(13q) varied from 10% in subset #8 (U-CLL), to 29% in subsets #59 (U-CLL) and #16 (M-CLL), and >75% in M-CLL subsets #77 and #148 (p<0.001); (ii) trisomy 12 was absent in subsets #31 (U-CLL) and #77 (M-CLL), in 20% of subset #201 (M-CLL) and, 60% in subset #8 (U-CLL) (p<0.001); (iii) del(11q) ranged from 69% in subset #31 (U-CLL) to 12% in subset #8 (U-CLL) and 0% in M-CLL subsets #16 and #201 (p<0.001); (iv) del(17p): absent from all M-CLL subsets except #148 (7.5%); absent from U-CLL subsets #5 and #59, and present in 19% and 17% of stereotyped subset #8 and #6 cases, respectively (p<0.05). We considered subset #2 separately since it included both M-CLL and U-CLL and identified a distinctive clinicobiological profile characterized by a constellation of features, including high incidence of Binet stage C disease (21%), CD38 positivity (41%) and certain cytogenetic abnormalities i.e. del(13q) (56%) and del(11q) (23%) and, in contrast, low frequency of del(17p) (4.5%). Notably, no differences were found between U-CLL versus M-CLL subset #2 cases regarding the incidence of these parameters. Survival analysis for time-to-first-treatment (TTFT) revealed that each stereotyped subset exhibited a distinct clinical course, different from that of other subsets even with similar IGHV gene mutational status and/or expressing the same IGHV gene. Interestingly, subsets #1 and #2 demonstrated a similar TTFT to that of del(17p) positive cases, while within subset #2 the clinical outcome was independent of IGHV mutational status. By integrating BcR IG stereotypy into the Döhner cytogenetic prognostication model, we found that subsets #1, #2 and #4, collectively accounting for ~7% of our series, and representing both U-CLL and M-CLL, constituted distinct clinical entities, with the latter exhibiting a particularly indolent disease even when compared to isolated del(13q) cases or cases with no cytogenetic aberrations. Finally, in multivariate analysis for TTFT, assignment to subset #2 emerged as a new independent unfavorable factor (p=0.002); furthermore, when restricting the analysis to Binet A cases, subset #2 exhibited the second highest hazard ratio after U-CLL. In conclusion, the molecular classification of CLL based on BcR IG stereotypy refines prognostication beyond IG mutational status and improves the Döhner hierarchical model. Hence, we suggest that a compartmentalized approach focusing on and comparing different subsets sheds light on CLL clinical behavior and biology and that BcR IG stereotypy represents a companion molecular diagnostic for personalized medicine in CLL. Disclosures Montillo: Janssen: Honoraria.

Abstract Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication. Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes. Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P &lt; 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

Key Points CLL stereotyped subset #2 (IGHV3-21/IGLV3-21) is uniformly aggressive independently of somatic hypermutation status. The prognosis for non–subset #2/IGHV3-21 CLL resembles that of the remaining CLL cases with similar somatic hypermutation status.