Cytogenetic complexity in chronic lymphocytic leukemia: definitions, associations, and clinical impact.

Recent evidence suggests that complex karyotype (CK) defined by the presence of ≥3 chromosomal aberrations (structural and/or numerical) identified by using chromosome-banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges toward the routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with ≥5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcomes, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and/or TP53 mutations [TP53abs]), and the expression of somatically hypermutated (M-CLL) or unmutated immunoglobulin heavy variable genes. Thus, they contrasted with CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs, and immunoglobulin heavy variable gene somatic hypermutation status, we propose a novel hierarchical model in which patients with high-CK exhibit the worst prognosis, whereas those with mutated CLL lacking CK or TP53abs, as well as CK with +12,+19, show the longest overall survival. Thus, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with ≥5 chromosomal aberrations emerges as prognostically adverse, independent of other biomarkers. Prospective clinical validation is warranted before ultimately incorporating high-CK in risk stratification of CLL.

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tailored approaches grounded on immunogenetic features for refined prognostication in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL.

IRProfiler - a software toolbox for high throughput immune receptor profiling.

BACKGROUND: The 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. RESULTS: This 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. CONCLUSION: IRProfiler 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.

GLASS: assisted and standardized assessment of gene variations from Sanger sequence trace data.

MOTIVATION: Sanger sequencing is still being employed for sequence variant detection by many laboratories, especially in a clinical setting. However, chromatogram interpretation often requires manual inspection and in some cases, considerable expertise. RESULTS: We present GLASS, a web-based Sanger sequence trace viewer, editor, aligner and variant caller, built to assist with the assessment of variations in 'curated' or user-provided genes. Critically, it produces a standardized variant output as recommended by the Human Genome Variation Society. AVAILABILITY AND IMPLEMENTATION: GLASS is freely available at http://bat.infspire.org/genomepd/glass/ with source code at https://github.com/infspiredBAT/GLASS. CONTACT: nikos.darzentas@gmail.com or malcikova.jitka@fnbrno.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Numerous Ontogenetic Roads to Mantle Cell Lymphoma: Immunogenetic and Immunohistochemical Evidence.

To obtain insight into the ontogeny of mantle cell lymphoma (MCL), we assessed 206 patients from a morphological, immunohistochemical, and immunogenetic perspective. Our series included nodal (n = 151), extranodal (n = 28), and primary splenic (n = 27) MCL cases. Skewing of the immunoglobulin heavy variable (IGHV) gene repertoire was noted, with only four IGHV genes accounting for 46% of cases and approximately 70% of cases (107/154) bearing an imprint of somatic hypermutation (SHM) ranging from minimal to pronounced. Interestingly, a distinctive immunophenotypic and immunogenetic profile was identified for primary splenic MCL, which was enriched for DBA.44-positive cases (P < 0.001) and used the IGHV1-8 gene more frequently (P = 0.02) compared to nodal or extranodal cases, alluding to distinct immunopathogenetic and antigen selection processes. Expression of CD27 (considered a marker of activated B cells) was generally dissociated from SHM and was more prevalent in cases with no or minimal/borderline SHM. These findings support the idea that antigen drive is relevant for most MCL cases, although the specific antigens and the precise location of affinity maturation remain to be elucidated. Moreover, they raise the intriguing hypothesis of multiple cellular origins for MCL.

Molecular evidence for antigen drive in the natural history of mantle cell lymphoma.

To further our understanding about antigen involvement in mantle cell lymphoma (MCL), we analyzed the expression levels of activation-induced cytidine deaminase (AID), a key player in B-cell responses to antigen triggering, in 133 MCL cases; assessed the functionality of AID by evaluating in vivo class switch recombination in 52 MCL cases; and sought for indications of ongoing antigen interactions by exploring intraclonal diversification within 14 MCL cases. The AID full-length transcript and the most frequent splice variants (AID-ΔE4a, AID-ΔE) were detected in 128 (96.2%), 96 (72.2%), and 130 cases (97.7%), respectively. Higher AID full-length transcript levels were significantly associated (P < 0.001) with lack of somatic hypermutation within the clonotypic immunoglobulin heavy variable (IGHV) genes. Median AID transcript levels were higher in lymph node material compared to cases in which peripheral blood was analyzed, implying that clonal behavior is influenced by the microenvironment. Switched tumor-derived IGHV-IGHD-IGHJ transcripts were identified in 5 of 52 cases (9.6%), all of which displayed somatic hypermutation and AID-mRNA expression. Finally, although most cases exhibited low levels of intraclonal diversification, analysis of the mutational activity revealed a precise targeting of somatic hypermutation indicative of an active, ongoing interaction with antigen(s). Collectively, these findings strongly allude to antigen involvement in the natural history of MCL, further challenging the notion of antigen naivety.

Clonal B-cell lymphocytosis exhibiting immunophenotypic features consistent with a marginal-zone origin: is this a distinct entity?

The biological and clinical significance of a clonal B-cell lymphocytosis with an immunophenotype consistent with marginal-zone origin (CBL-MZ) is poorly understood. We retrospectively evaluated 102 such cases with no clinical evidence to suggest a concurrent MZ lymphoma. Immunophenotyping revealed a clonal B-cell population with Matutes score ≤2 in all cases; 19/102 were weakly CD5 positive and all 35 cases tested expressed CD49d. Bone marrow biopsy exhibited mostly mixed patterns of small B-lymphocytic infiltration. A total of 48/66 (72.7%) cases had an abnormal karyotype. Immunogenetics revealed overusage of the IGHV4-34 gene and somatic hypermutation in 71/79 (89.8%) IGHV-IGHD-IGHJ gene rearrangements. With a median follow-up of 5 years, 85 cases remain stable (group A), whereas 17 cases (group B) progressed, of whom 15 developed splenomegaly. The clonal B-cell count, degree of marrow infiltration, immunophenotypic, or immunogenetic findings at diagnosis did not distinguish between the 2 groups. However, deletions of chromosome 7q were confined to group A and complex karyotypes were more frequent in group B. Although CBL-MZ may antedate SMZL/SLLU, most cases remain stable over time. These cases, not readily classifiable within the World Heath Organization classification, raise the possibility that CBL-MZ should be considered as a new provisional entity within the spectrum of clonal MZ disorders.

Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors.

We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s).

Cancer care pathways across seven countries in Europe: What are the current obstacles? And how can artificial intelligence help?

BACKGROUND: Cancer poses significant challenges for healthcare professionals across the disease pathway including cancer imaging. This study constitutes part of the user requirement definition of INCISIVE EU project. The project has been designed to explore the full potential of artificial intelligence (AI)-based technologies in cancer imaging to streamline diagnosis and management. The study aimed to map cancer care pathways (breast, prostate, colorectal and lung cancers) across INCISIVE partner countries, and identify bottle necks within these pathways. METHODS: Email interviews were conducted with ten oncology specialised healthcare professionals representing INCISIVE partner countries: Greece, Cyprus, Spain, Italy, Finland, the United Kingdom (UK) and Serbia. A purposive sampling strategy was employed for recruitment and data was collected between December 2020 and April 2021. Data was entered into Microsoft Excel spreadsheet to allow content examination and comparative analysis. RESULTS: The analysed pathways all shared a common characteristic: inequalities in relation to delays in cancer diagnosis and treatment. All the studied countries, except the UK, lacked official national data about diagnostic and therapeutic delays. Furthermore, a considerable variation was noted regarding the availability of imaging and diagnostic services across the seven countries. Several concerns were also noted for inefficiencies/inequalities with regards to national screening for the four investigated cancer types. CONCLUSIONS: Delays in cancer diagnosis and treatment are an ongoing challenge and a source for inequalities. It is important to have systematic reporting of diagnostic and therapeutic delays in all countries to allow the proper estimation of its magnitude and support needed to address it. Our findings also support the orientation of the current policies towards early detection and wide scale adoption and implementation of cancer screening, through research, innovation, and technology. Technologies involving AI can have a great potential to revolutionise cancer care delivery. POLICY SUMMARY: This study highlights the widespread delay in cancer diagnosis across Europe and supports the need for, systematic reporting of delays, improved availability of imaging services, and optimised national screening programs. The goal is to enhance cancer care delivery, encourage early detection, and implement research, innovation, and AI-based technologies for improved cancer imaging.

Temporal dynamics of clonal evolution in chronic lymphocytic leukemia with stereotyped IGHV4-34/IGKV2-30 antigen receptors: longitudinal immunogenetic evidence.

Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset 4 possess distinctive patterns of intraclonal diversification (ID) within their immunoglobulin (IG) genes. Although highly indicative of an ongoing response to antigen(s), the critical question concerning the precise timing of antigen involvement is unresolved. Hence, we conducted a large-scale longitudinal study of eight subset 4 cases totaling 511 and 398 subcloned IG heavy and kappa sequences. Importantly, we could establish a hierarchical pattern of subclonal evolution, thus revealing which somatic hypermutations were negatively or positively selected. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially; however, at later time points, the minor cluster had often disappeared and hence not been selected. Despite the high intensity of ID, it was remarkable that certain residues remained essentially unaltered. These novel findings strongly support a role for persistent antigen stimulation in the clonal evolution of CLL subset 4.

Cancer care at the time of the fourth industrial revolution: an insight to healthcare professionals' perspectives on cancer care and artificial intelligence.

BACKGROUND: The integration of Artificial Intelligence (AI) technology in cancer care has gained unprecedented global attention over the past few decades. This has impacted the way that cancer care is practiced and delivered across settings. The purpose of this study was to explore the perspectives and experiences of healthcare professionals (HCPs) on cancer treatment and the need for AI. This study is a part of the INCISIVE European Union H2020 project's development of user requirements, which aims to fully explore the potential of AI-based cancer imaging technologies. METHODS: A mixed-methods research design was employed. HCPs participating in cancer care in the UK, Greece, Italy, Spain, Cyprus, and Serbia were first surveyed anonymously online. Twenty-seven HCPs then participated in semi-structured interviews. Appropriate statistical method was adopted to report the survey results by using SPSS. The interviews were audio recorded, verbatim transcribed, and then thematically analysed supported by NVIVO. RESULTS: The survey drew responses from 95 HCPs. The occurrence of diagnostic delay was reported by 56% (n = 28/50) for breast cancer, 64% (n = 27/42) for lung cancer, 76% (n = 34/45) for colorectal cancer and 42% (n = 16/38) for prostate cancer. A proportion of participants reported the occurrence of false positives in the accuracy of the current imaging techniques used: 64% (n = 32/50) reported this for breast cancer, 60% (n = 25/42) for lung cancer, 51% (n = 23/45) for colorectal cancer and 45% (n = 17/38) for prostate cancer. All participants agreed that the use of technology would enhance the care pathway for cancer patients. Despite the positive perspectives toward AI, certain limitations were also recorded. The majority (73%) of respondents (n = 69/95) reported they had never utilised technology in the care pathway which necessitates the need for education and training in the qualitative finding; compared to 27% (n = 26/95) who had and were still using it. Most, 89% of respondents (n = 85/95) said they would be opened to providing AI-based services in the future to improve medical imaging for cancer care. Interviews with HCPs revealed lack of widespread preparedness for AI in oncology, several barriers to introducing AI, and a need for education and training. Provision of AI training, increasing public awareness of AI, using evidence-based technology, and developing AI based interventions that will not replace HCPs were some of the recommendations. CONCLUSION: HCPs reported favourable opinions of AI-based cancer imaging technologies and noted a number of care pathway concerns where AI can be useful. For the future design and execution of the INCISIVE project and other comparable AI-based projects, the characteristics and recommendations offered in the current research can serve as a reference.

The Clinical Utility of ABO and RHD Systems as Potential Indicators of Health Status, a Preliminary Study in Greek Population.

OBJECTIVE: The objective of this study is to further highlight the differences between different ABO blood groups and Rhesus types with health biomarkers. METHODS: In total 150 active healthy blood donors participated in our study comprising of 80 males from 19-61 years and 70 females aged from 21 to 64. Participants carrying blood group A were 55 individuals, blood group B 32, blood group O 51, and blood group AB 12, RHD+ 132, and RHD- 18. All the volunteer regular blood donors were selected recognizing them as a healthy population excluding drug and supplements intake. Their blood samples were analyzed just before blood donation for biochemical, hematological, and antioxidant markers. Statistical computations were performed using the SPSS tool, specifically, the one-way ANOVA test, Chi-square statistics, and logistic regression were used as statistical models. RESULTS: O blood donors presented better iron absorption and the worst lipid profile. Indeed, a significant trend of high atheromatic index values revealed an increased risk for hyperlipidemia, in contrast with blood group A presenting a better lipid profile with lower atheromatic index values. There was also a gender related association for blood group A compared with O that was further highlighted using binary logistic regression. CONCLUSION: In this study, a significant difference was observed among the ABO blood groups in several of the examined biochemical and hematological biomarkers. O blood group appeared different behavior in comparison to all the tested blood groups and furthermore the RHD-group presented a better lipid profile in comparison to the RHD+ group. In order to obtain a more comprehensive view of the correlation between the ABO blood group and biochemical markers, further studies are required.

Experiences of cancer survivors in Europe: Has anything changed? Can artificial intelligence offer a solution?

Introduction: Cancer is a major global health issue. Despite technological advancements in oncology, challenges remain in many aspects related to cancer management. This study constitutes one part of the user requirement definition of INCISIVE EU H2020 project, which has been designed to explore the full potential of artificial intelligence (AI) based technologies in cancer imaging. The study aimed to explore cancer survivors' experiences of cancer care in five European countries. Methods: A qualitative study employing semi-structured interviews was conducted. A purposive sampling strategy was used to recruit participants across the five validation countries of INCISIVE project: Greece, Cyprus, Spain, Italy, and Serbia. Forty cancer survivors were interviewed between November 2020 and March 2021. Data was analysed thematically using the framework approach and coded using NVivo12 software. Results: The analysis yielded several gaps within the cancer care pathway which reflected on the participants experiences. Five key themes were revealed; (1) perceived challenges during the cancer journey, (2) the importance of accurate and prompt diagnosis, (3) perceived need for improving cancer diagnosis, (4) absence of well-established/designated support services within the pathway and (5) suggestions to improve cancer care pathway. Conclusion: Cancer survivors experienced significant burdens pertaining to cancer diagnosis and treatment. Our findings underscored some main gaps within the cancer care pathway which contributed to the challenges articulated by the participants including lack of resources and delays in diagnostic and treatment intervals. Additionally, several suggestions were provided by the cancer survivors which could be considered towards the improvement of the current state of care, some of which can be optimised using new technologies involving AI such as the one proposed by INCISIVE.

The Potential Impact of Blood System on Dietary Habits and Smoking.

The 'Blood-Type' diet advises individuals to eat according to their ABO blood group to improve their health and decrease the risk of chronic diseases. However, the food preferences of individuals with different blood groups have not been examined. The aim of our study was to investigate, in healthy regular blood donors (rBDs), the associations of smoke, alcohol, caffeine, vitamin and fat intake with their different blood groups and if ABO groups could be a potential predictor tool for disease prevention. A total of 329 volunteers were divided into four groups according to their ABO types: Group 1 (A) comprised 141 rBDs; Group 2 (B), 65 rBDs; Group 3 (O), 96 rBDs; and Group 4, 27 rBDs. Additionally, they were divided into two groups according to their rhesus types and their preferences for smoke, too. Dietary intake was assessed using 3-day food recall and the Food Processor computer program for nutrient analysis. Alcohol, caffeine, sugar and Vitamin D consumption were significantly (p < 0.05) higher in the O group. The A group presented statistically significantly (p < 0.05) greater preferences for cholesterol intake and a higher trend for smoking (25%) habits compared with all the other groups, whereas Group B preferred more fatty foods. The blood group AB appeared to be the most controlled food intake group. Regarding the rhesus comparisons, alcohol; caffeine; and Vitamin C, D, E and K consumptions were significantly (p < 0.05) higher in rhesus-positive individuals than their rhesus-negative counterparts. For the non-smoker group, compared with the smokers, a higher consumption of Vitamin D and fibers was found. In conclusion, in the present study, statistically significant correlations of the ABO and rhesus system with some dietary parameters were found, indicating a consequent influence of these preferences on the progression of different diseases.

SBA-15 Mesoporous Silica as Delivery Vehicle for rhBMP-2 Bone Morphogenic Protein for Dental Applications.

(1) Background: A proposed approach to promote periodontal tissue regeneration in cases of peri-implantitis is the local administration of growth factors at the implant site. Recombinant human bone morphogenetic protein-2 (rh-BMP-2) can effectively promote bone regeneration and osseointegration and the development of appropriate carriers for its delivery is of paramount importance. The aim of the present study was to develop SBA-15 mesoporous nanoparticles (MSNs) with varying porosity, evaluate their biocompatibility with human Periodontal Ligament Cells (hPDLCs) and to investigate their effectiveness as carriers of rh-BMP-2. (2) Methods: SBA-15 type mesoporous silicas were synthesized via sol-gel reaction. The calcined SBA-15 samples were characterized by N2 porosimetry, Fourier transform-infrared spectrometry (FTIR), Scanning (SEM) and Transmission Electron Microscopy (TEM). Rh-BMP-2 loading and release kinetics were evaluated by UV spectroscopy. (3) Results: MSNs presented hexagonally arranged, tubular pores of varying length and diameter. Slightly higher loading capacity was achieved for SBA-15 with large pores that presented good hemocompatibility. MTT assay revealed no cytotoxic effects for all the tested materials, while SBA-15 with large pores induced a significant upregulation of cell viability at day 5. (4) Conclusions: SBA-15 MSNs may prove a valuable delivery platform towards the effective release of bone-inducing proteins.

Activation-induced cytidine deaminase splicing patterns in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) is critically implicated in somatic hypermutation (SHM) and class switch recombination (CSR). AID is expressed as a native transcript and as several splice variants, with as yet undefined roles. Chronic lymphocytic leukemia (CLL) leukemic B cells have also been shown to express AID transcripts, especially in cases with unmutated immunoglobulin (IG) genes. Therefore, AID expression in CLL might potentially be relevant to the disease. The available data on AID-mRNA splicing patterns in CLL are limited and conflicting. Here, we investigated AID-mRNA isoform expression in a series of 195 CLL patients and explored associations with IG gene mutational status and surface immunoglobulin (sIg) isotype expression. Full-length AID transcripts and two splice variants were detected in 110/91/95 cases, respectively. Co-expression of all three AID-mRNA isoforms was significantly more frequent (p<0.001) in cases with unmutated IGHV genes. No significant differences were identified between sIgG vs. sIgMD cases regarding the frequency of AID-mRNA expression. However, expression of at least one AID-mRNA isoform predominated among mutated IgG vs. mutated IgMD cases (p=0.05). These results attest to the biological heterogeneity of CLL and also indicate that AID splice variants may inhibit SHM in CLL cells of the unmutated subtype.

Skewing of the T-cell receptor repertoire in patients receiving rituximab after allogeneic hematopoietic cell transplantation: what lies beneath?

Rituximab is known to affect T cell immune responses. We and others have reported expansions of T large granular lymphocytes (T-LGLs) in lymphoma patients after Rituximab. We report here the immunogenetic profiling of the T cell receptor (TR) gene repertoire in 14 patients who received Rituximab post allo-HCT and explore clinicobiological correlations. All experienced antigenic triggers, CMV, EBV re-activation and chronic GvHD and had been treated with Rituximab. Skewing of TRBV genes was observed: 3 TRBV genes accounted for half of the repertoire. Oligoclonal pattern with expanded clonotypes was common. Patients with oligoclonality exhibited frequently cGvHD. Longitudinal samples in one revealed distinct clonotypes, suggesting clonal drift. T-LGL leukemia of donor origin with mixed chimerism eventually developed. In conclusion, we report development of oligoclonal T-LGLs after Rituximab post allo-HCT, alluding to antigen selection. Persistence of this phenomenon likely reflects strong antigenic stimulation by viruses and/or cGVHD aggravated by Rituximab.

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

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 ß 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.