Anna Vardi

Immunogenetics shows that not all MBL are equal: the larger the clone, the more similar to CLL.

Chronic lymphocytic leukemia (CLL) -like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormalities with CLL, from which it is discriminated by a cutoff value of 5 × 10(9)/L circulating clonal B cells. However, the clonal size in MBL is extremely variable and allows discrimination of two distinct entities (high-count [HC] and low-count [LC]-MBL) based on a cutoff value of 0.5 × 10(9)/L clonal B cells. HC-MBL is associated with lymphocytosis and progresses to CLL requiring treatment at a rate of 1.1% per year, whereas LC-MBL is found in the general population only through high-sensitivity techniques and carries limited, if any, risk of progression. We performed an immunogenetic profiling of 333 cases with CLL-like MBL supplemented by detailed comparisons with CLL, focusing especially on CLL Rai stage 0 (CLL-0). LC- and HC-MBL had similar somatic hypermutation status, yet different IGHV gene repertoires and frequencies of B-cell receptor (BcR) stereotypy. In particular, stereotyped BcRs were infrequent in LC-MBL and were often not CLL specific. In contrast, HC-MBL exhibited clear immunogenetic similarities to CLL-0. These findings indicate that LC-MBL may not represent a true preleukemic condition, thus differing from HC-MBL/CLL-0 in which the identification of factors endowing malignant potential is strongly warranted.

Distinct innate immunity pathways to activation and tolerance in subgroups of chronic lymphocytic leukemia with distinct immunoglobulin receptors.

Subgroups of patients with chronic lymphocytic leukemia (CLL) have distinct expression profiles of Toll-like receptor (TLR) pathway-associated genes. To test the hypothesis that signaling through innate immunity receptors may influence the behavior of the malignant clone, we investigated the functional response triggered by the stimulation of TLRs and NOD2 in 67 CLL cases assigned to different subgroups on the basis of immunoglobulin heavy variable (IGHV) gene usage, IGHV gene mutational status or B-cell receptor (BcR) stereotypy. Differences in the induction of costimulatory molecules and/or apoptosis were observed in mutated versus unmutated CLL. Different responses were also identified in subsets with stereotyped BcRs, underscoring the idea that “subset-biased” innate immunity responses may occur independently of mutational status. Additionally, differential modulation of kinase activities was induced by TLR stimulation of different CLL subgroups, revealing a TLR7-tolerant state for cases belonging to stereotyped subset #4. The distinct patterns of TLR/NOD2 functional activity in cells from CLL subgroups defined by the molecular features of the clonotypic BcRs might prove relevant for elucidating the immune mechanisms underlying CLL natural history and for defining subgroups of patients who might benefit from treatment with specific TLR ligands.

Antigen Selection Shapes the T-cell Repertoire in Chronic Lymphocytic Leukemia

Purpose: The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia, although relevant for understanding malignant cell interactions with cognate T cells, is largely unexplored. Experimental Design: Here we profiled the T-cell receptor β chain gene repertoire in 58 chronic lymphocytic leukemia patients, focusing on cases assigned to well-characterized subsets with stereotyped clonotypic B-cell receptor immunoglobulins, therefore those cases most evidently selected by antigen (subsets #1, #2, and #4). Results: Remarkable repertoire skewing and oligoclonality were observed, and differences between subsets were noted regarding both T-cell receptor β chain gene usage and the extent of clonality, with subset #2 being the least oligoclonal. Longitudinal analysis of subset #4 cases revealed that although the repertoire may fluctuate over time, certain clonotypes persist, thus alluding to persistent antigenic stimulation. Shared (“stereotyped”) clonotypes were found between different patients, reflecting selection by common antigenic elements. Cross-comparison of our dataset with public databases showed that some T-cell clonotypes may have expanded secondary to common viral infections; however, the majority of clonotypes proved to be disease-specific. Conclusions: Overall, the T-cell receptor β chain repertoire in chronic lymphocytic leukemia is likely shaped by antigen selection and the implicated antigenic elements may concern epitopes that also select the malignant B-cell progenitors or, more intriguingly, chronic lymphocytic leukemia–derived epitopes. Clin Cancer Res; 22(1); 167–74. ©2015 AACR.

IgG-SWITCHED CLL HAS A DISTINCT IMMUNOGENETIC SIGNATURE FROM THE COMMON MD VARIANT: ONTOGENETIC IMPLICATIONS

Purpose: Immunoglobulin G–switched chronic lymphocytic leukemia (G-CLL) is a rare variant of CLL, whose origin and ontogenetic relationship to the common IgM/IgD (MD-CLL) variant remains undefined. Here, we sought for clues about the ontogeny of G-CLL versus MD-CLL by profiling the relevant IG gene repertoires. Experimental Design: Using purpose-built bioinformatics methods, we performed detailed immunogenetic profiling of a multinational CLL cohort comprising 1,256 cases, of which 1,087 and 169 expressed IG mu/delta and gamma heavy chains, respectively. Results: G-CLL has a highly skewed IG gene repertoire that is distinct from MD-CLL, especially in terms of (i) overuse of the IGHV4-34 and IGHV4-39 genes and (ii) differential somatic hypermutation (SHM) load. Repertoire differences were also found when comparing subgroups with similar SHM status and were mainly attributed to the exclusive representation in G-CLL of two major subsets with quasi-identical (stereotyped) B-cell receptors. These subsets, namely #4 (IGHV4-34/IGKV2-30) and #8 (IGHV4-39/IGKV1(D)-39), were found to display sharply contrasting SHM and clinical behavior. Conclusions: G-CLL exhibits an overall distinct immunogenetic signature from MD-CLL, prompting speculations about distinct ontogenetic derivation and/or immune triggering. The reasons underlying the differential regulation of SHM among G-CLL cases remain to be elucidated. Clin Cancer Res; 20(2); 323–30. ©2013 AACR.

Stereotyped B-cell receptors in chronic lymphocytic leukemia

Abstract Over the last decade, immunogenetic analysis of B-cell receptor immunoglobulins (BcR IGs) has proved to be a particularly fruitful field in chronic lymphocytic leukemia (CLL), not only for understanding disease pathogenesis but also for discriminating clinical subgroups with markedly distinct course and outcome. Of utmost importance was the identification of quasi-identical BcR IGs among unrelated patients with CLL, fittingly coined as “stereotypy,” that set the wheels in motion for unraveling the role of antigen(s) in the selection and expansion of the leukemic clones. The categorization of CLL clones into “subsets” according to shared BcR IG structural characteristics provided a compartmentalized view of this otherwise heterogeneous disease, which eventually led to defining strikingly homogeneous groups of patients in terms of: (i) functional properties of the clonal BcR IGs, e.g. BcR reactivity and signaling; (ii) clonal genetic landscape, e.g. genomic aberrations, gene expression/methylation profiles, microRNA signatures; and (iii) clinical course and outcome. The remarkable restriction of the CLL IG gene repertoire, resulting to a great degree from the high impact of BcR IG stereotypy, may also prompt speculations regarding CLL ontogenesis. Overall, the BcR IG molecule justifiably lies at the heart of CLL clinical research, holding the promise of subset-tailored therapies.

Restrictions in the T-cell repertoire of chronic lymphocytic leukemia: high-throughput immunoprofiling supports selection by shared antigenic elements

Immunoglobulin (IG) gene repertoire restrictions strongly support antigen selection in the pathogenesis of chronic lymphocytic leukemia (CLL). Given the emerging multifarious interactions between CLL and bystander T cells, we sought to determine whether antigen(s) are also selecting T cells in CLL. We performed a large-scale, next-generation sequencing (NGS) study of the T-cell repertoire, focusing on major stereotyped subsets representing CLL subgroups with undisputed antigenic drive, but also included patients carrying non-subset IG rearrangements to seek for T-cell immunogenetic signatures ubiquitous in CLL. Considering the inherent limitations of NGS, we deployed bioinformatics algorithms for qualitative curation of T-cell receptor rearrangements, and included multiple types of controls. Overall, we document the clonal architecture of the T-cell repertoire in CLL. These T-cell clones persist and further expand overtime, and can be shared by different patients, most especially patients belonging to the same stereotyped subset. Notably, these shared clonotypes appear to be disease-specific, as they are found in neither public databases nor healthy controls. Altogether, these findings indicate that antigen drive likely underlies T-cell expansions in CLL and may be acting in a CLL subset-specific context. Whether these are the same antigens interacting with the malignant clone or tumor-derived antigens remains to be elucidated.

Restricted T cell receptor repertoire in CLL-like monoclonal B cell lymphocytosis and early stage CLL

ABSTRACT Analysis of the T cell receptor (TR) repertoire of chronic lymphocytic leukemia-like monoclonal B cell lymphocytosis (CLL-like MBL) and early stage CLL is relevant for understanding the dynamic interaction of expanded B cell clones with bystander T cells. Here we profiled the T cell receptor β chain (TRB) repertoire of the CD4+ and CD8+ T cell fractions from 16 CLL-like MBL and 13 untreated, Binet stage A/Rai stage 0 CLL patients using subcloning analysis followed by Sanger sequencing. The T cell subpopulations of both MBL and early stage CLL harbored restricted TRB gene repertoire, with CD4+ T cell clonal expansions whose frequency followed the numerical increase of clonal B cells. Longitudinal analysis in MBL cases revealed clonal persistence, alluding to persistent antigen stimulation. In addition, the identification of shared clonotypes among different MBL/early stage CLL cases pointed towards selection of the T cell clones by common antigenic elements. T cell clonotypes previously described in viral infections and immune disorders were also detected. Altogether, our findings evidence that antigen-mediated TR restriction occurs early in clonal evolution leading to CLL and may further increase together with B cell clonal expansion, possibly suggesting that the T cell selecting antigens are tumor-related.

Transplant-associated thrombotic microangiopathy: Incidence, prognostic factors, morbidity, and mortality in allogeneic hematopoietic cell transplantation

Renewed interest has emerged in transplant‐associated thrombotic microangiopathy (TA‐TMA) with novel prognostic, diagnostic, and treatment algorithms. We aimed to investigate the incidence, prognostic factors, morbidity, and mortality of TA‐TMA in allogeneic hematopoietic cell transplantation (HCT) recipients. We enrolled consecutive HCT recipients (1990‐2017). Among 758 patients, 116 (15.5%) were diagnosed with TA‐TMA. In the multivariate analysis, TBI‐based conditioning, viral infections, acute and chronic GVHD remained independent predictors of TA‐TMA. With a median follow‐up of 23 (range 0.1‐329) months, TA‐TMA resulted in significantly lower overall survival (OS). In the multivariate analysis, TA‐TMA remained an independent predictor of OS, along with relapse, acute, and chronic GVHD. Among 116 TA‐TMA patients, 70 developed renal (56) and/or neurologic (26) dysfunction that would be necessary for TA‐TMA diagnosis according to the Bone Marrow Transplant Clinical Trials Network criteria. TA‐TMA patients with renal dysfunction showed increased rates of acute GVHD, but no difference in OS compared to patients without renal dysfunction. However, neurologic dysfunction resulted in significantly lower OS. In conclusion, TA‐TMA is associated with increased morbidity and mortality in allogeneic transplant recipients. Successful prevention and treatment strategies of infections and GVHD need to be timely employed to improve survival in this complex setting.

IRProfiler – a software toolbox for high throughput immune receptor profiling

BackgroundThe study of the huge diversity of immune receptors, often referred to as immune repertoire profiling, is a prerequisite for diagnosis, prognostication and monitoring of hematological disorders. In the era of high-throughput sequencing (HTS), the abundance of immunogenetic data has revealed unprecedented opportunities for the thorough profiling of T-cell receptors (TR) and B-cell receptors (BcR). However, the volume of the data to be analyzed mandates for efficient and ease-to-use immune repertoire profiling software applications.ResultsThis work introduces Immune Repertoire Profiler (IRProfiler), a novel software pipeline that delivers a number of core receptor repertoire quantification and comparison functionalities on high-throughput TR and BcR sequencing data. Adopting 5 alternative clonotype definitions, IRProfiler implements a series of algorithms for 1) data filtering, 2) calculation of clonotype diversity and expression, 3) calculation of gene usage for the V and J subgroups, 4) detection of shared and exclusive clonotypes among multiple repertoires, and 5) comparison of gene usage for V and J subgroups among multiple repertoires. IRProfiler has been implemented as a toolbox of the Galaxy bioinformatics platform, comprising 6 tools. Theoretical and experimental evaluation has shown that the tools of IRProfiler are able to scale well with respect to the size of input dataset(s). IRProfiler has been utilized by a number of recently published studies concerning hematological disorders.ConclusionIRProfiler is made freely available via 3 distribution channels, including the Galaxy Tool Shed. Despite being a new entry in a crowded ecosystem of immune repertoire profiling software, IRProfiler founds its added value on its support for alternative clonotype definitions in conjunction with a combination of properties stemming from its user-centric design, namely ease-of-use, ease-of-access, exploitability of the output data, and analysis flexibility.

Monoclonal B-cell lymphocytosis in a hospital-based UK population and a rural Ugandan population : a cross-sectional study

Summary Background Reported incidence of B-cell malignancies shows substantial geographical variation, being more common in the Americas and Europe than in Africa. This variation might reflect differences in diagnostic capability, inherited susceptibility, and infectious exposures. Monoclonal B-cell lymphocytosis (MBL) is a precursor lesion that can be screened for in apparently healthy people, allowing comparison of prevalence across different populations independently of health-care provision. We aimed to compare the prevalence and phenotypic characteristics of MBL in age-and-sex-matched populations from rural Uganda and the UK. Methods In this cross-sectional study, we recruited volunteers aged at least 45 years who were seronegative for HIV-1 from the established Ugandan General Population Cohort and obtained their whole-blood samples. We also obtained blood samples from anonymised waste material of age-and-sex-matched individuals (aged >45 years, with a normal blood count and no history of cancer) in the UK. We used flow cytometry to determine the presence of MBL, defined according to standard diagnostic criteria, in the samples and compared differences in the proportion of cases with chronic lymphocytic leukaemia (CLL)-phenotype MBL and CD5-negative MBL, as well as differences in absolute monoclonal B-cell count between the two cohorts. Findings Between Jan 15 and Dec 18, 2012, we obtained samples from 302 Ugandan volunteers and 302 UK individuals who were matched by age and sex to the Ugandan population. Overall MBL prevalence was higher in the Ugandan participants (42 [14%] individuals) than in the UK cohort (25 [8%]; p=0·038). CLL-phenotype MBL was detected in three (1%) Ugandan participants and 21 (7%) UK participants (p=0·00021); all three Ugandan participants had absolute monoclonal B-cell count below one cell per μL, whereas the 21 UK participants had a median absolute number of circulating neoplastic cells of 4·6 (IQR 2–12) cells per μL. The prevalence of CD5-negative MBL was higher in the Ugandan cohort (41 [14%], of whom two [5%] also had CLL-phenotype MBL) than in the UK cohort (six [2%], of whom two [33%] also had CLL-phenotype MBL; p<0·0001), but the median absolute B-cell count was similar (227 [IQR 152–345] cells per μL in the Ugandan cohort vs 135 [105–177] cells per μL in the UK cohort; p=0·13). Interpretation MBL is common in both Uganda and the UK, but the substantial phenotypic differences might reflect fundamental differences in the pathogenesis of B-cell lymphoproliferative disorders. Funding UK Medical Research Council and UK Department for International Development.