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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. [ABSTRACT FROM AUTHOR]

Self-sampling for human papillomavirus (HPV) testing is an alternative to physician sampling particularly for cervical cancer screening nonattenders. The GRECOSELF study is a nationwide observational cross-sectional study aiming to suggest a way to implement HPV-DNA testing in conjunction with self-sampling for cervical cancer screening in Greece, utilizing a midwifery network. Women residing in remote areas of Greece were approached by midwives, of a nationwide network, and were provided with a self-collection kit (dry swab) for cervicovaginal sampling and asked to answer a questionnaire about their cervical cancer screening history. Each sample was tested for high-risk (hr) HPV with the Cobas HPV test. HrHPV-Positive women were referred to undergo colposcopy and, if needed, treatment according to colposcopy/biopsy results. Between May 2016 and November 2018, 13,111 women were recruited. Of these, 12,787 women gave valid answers in the study questionnaire and had valid HPV-DNA results; hrHPV prevalence was 8.3%; high-grade cervical/vaginal disease or cancer prevalence was 0.6%. HrHPV positivity rate decreased with age from 20.7% for women aged 25-29 years to 5.1% for women aged 50-60 years. Positive predictive value for hrHPV testing and for HPV16/18 genotyping ranged from 5.0% to 11.6% and from 11.8% to 27.0%, respectively, in different age groups. Compliance to colposcopy referral rate ranged from 68.6% (for women 25-29) to 76.3% (for women 40-49). For women residing in remote areas of Greece, the detection of hrHPV DNA with the Cobas HPV test, on self-collected cervicovaginal samples using dry cotton swabs, which are provided by visiting midwives, is a promising method for cervical cancer secondary prevention., (©2019 American Association for Cancer Research.)

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness., (© 2018 UICC.)

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., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

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., (Copyright © 2019 Ferrata Storti Foundation.)

The formation of B-cell receptor immunoglobulin (BcR IG) is the result of a multi-step process that starts at the pro-B cell stage with the VDJ gene recombination of IG genes of the heavy chain, followed by VJ recombination of the light chain genes at the pre-B II cell stage. As a result, a fully functional BcR IG is expressed on the surface of any given naive B cell. After antigen encounter, somatic hypermutation (SHM) and class-switch recombination (CSR) act on the rearranged IG genes within the context of affinity maturation, leading to the expression of a BcR IG with unique immunogenetic and functional characteristics. Since B-cell neoplasms arise from the transformation of a single B cell, this renders IG gene rearrangements ideal clonal markers as they will be identical in all neoplastic cells of each individual clone. Furthermore, the rearranged IG sequence can also serve as a cell development/maturation marker, given that its configuration is tightly linked to specific B-cell developmental stages. Finally, in certain instances, as in the case of chronic lymphocytic leukemia (CLL), the clonotypic IG sequence and, more specifically, the load of somatic hypermutations within the rearranged IG heavy variable (IGHV) gene, holds prognostic and potentially predictive value. However, in order to take full advantage of the information provided from the analysis of the clonotypic IG gene rearrangement sequences, robust methods and tools need to be applied. Here, we provide details regarding the methodologies necessary to ensure reliable IG sequence analysis based on the recognized expertise of the European Research initiative on CLL (ERIC). All methodological and analytical steps are described below, starting from the isolation of blood mononuclear cells (PBMC), moving to the identification of the clonotypic IG rearrangement and ending with the accurate interpretation of the SHM status.

High-throughput, next-generation sequencing (NGS) offers a unique opportunity for in-depth characterization of adaptive immune receptor repertoires. Nevertheless, limitations and pitfalls exist in every step of both the experimental and the analytical procedure, leading to discrepancies in the literature and incomprehensive and/or altogether misleading results. Thus, standardization of protocols in NGS immunogenetics is urgently needed.Here, we describe the experimental protocol that we developed for T-cell receptor beta chain (TRB) gene repertoire analysis in chronic lymphocytic leukemia, aiming to provide a reproducible and biologically meaningful output. Although optimized for TRBV-TRBD-TRBJ gene rearrangements, this protocol may be customized for other adaptive immune receptor sequences, as well.

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a 'molecular timeline' since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

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)., (Copyright© Ferrata Storti Foundation.)

Patients with chronic lymphocytic leukemia (CLL) assigned to stereotyped subset #4 express highly homologous B-cell receptor immunoglobulin (BcR IG) sequences with intense intraclonal diversification (ID) in the context of ongoing somatic hypermutation (SHM). Their remarkable biological and clinical similarities strongly support derivation from a common ancestor. We here revisited ID in subset #4 CLL to reconstruct their evolutionary history as a community of related clones. To this end, using specialized bioinformatics tools we assessed both IGHV-IGHD-IGHJ rearrangements (n = 511) and IGKV-IGKJ rearrangements (n = 397) derived from eight subset #4 cases. Due to high sequence relatedness, a number of subclonal clusters from different cases lay very close to one another, forming a core from which clusters exhibiting greater variation stemmed. Minor subclones from individual cases were mutated to such an extent that they now resembled the sequences of another patient. Viewing the entire subset #4 data set as a single entity branching through diversification enabled inference of a common sequence representing the putative ancestral BcR IG expressed by their still elusive common progenitor. These results have implications for improved understanding of the ontogeny of CLL subset #4, as well as the design of studies concerning the antigenic specificity of the clonotypic BcR IGs.

Νext generation sequencing studies in Homo sapiens have identified novel immunoglobulin heavy variable (IGHV) genes and alleles necessitating changes in the international ImMunoGeneTics information system (IMGT) GENE-DB and reference directories of IMGT/V-QUEST. In chronic lymphocytic leukaemia (CLL), the somatic hypermutation (SHM) status of the clonotypic rearranged IGHV gene is strongly associated with patient outcome. Correct determination of this parameter strictly depends on the comparison of the nucleotide sequence of the clonotypic rearranged IGHV gene with that of the closest germline counterpart. Consequently, changes in the reference directories could, in principle, affect the correct interpretation of the IGHV mutational status in CLL. To this end, we analyzed 8066 productive IG heavy chain (IGH) rearrangement sequences from our consortium both before and after the latest update of the IMGT/V-QUEST reference directory. Differences were identified in 405 cases (5 % of the cohort). In 291/405 sequences (71.9 %), changes concerned only the IGHV gene or allele name, whereas a change in the percent germline identity (%GI) was noted in 114/405 (28.1 %) sequences; in 50/114 (43.8 %) sequences, changes in the %GI led to a change in the mutational set. In conclusion, recent changes in the IMGT reference directories affected the interpretation of SHM in a sizeable number of IGH rearrangement sequences from CLL patients. This indicates that both physicians and researchers should consider a re-evaluation of IG sequence data, especially for those IGH rearrangement sequences that, up to date, have a GI close to 98 %, where caution is warranted.

Immunoglobulin gene sequence analysis is widely utilized for prognostication in chronic lymphocytic leukemia (CLL) and the definition of standardized procedures has allowed reliable and reproducible results. Occasionally, a straightforward interpretation of the sequences is not possible because of the so-called 'problematic sequences' that do not fit the 'classic' interpretation and pose scientific questions at the cross-road between hematology and immunology. Thanks to a dedicated effort within the European Research Initiative on CLL (ERIC), we have now the possibility to present such cases, offer a scientific explanation and propose recommendations in terms of prognostication.

Chronic lymphocytic leukemia (CLL) is uniquely characterized by the existence of subsets of cases with quasi-identical, 'stereotyped' B-cell receptors (BCRs). Herein we investigate this stereotypy in 2662 patients with CLL, the largest series yet, using purpose-built bioinformatics methods based on sequence pattern discovery. Besides improving the identification of 'stereotyped' cases, we demonstrate that CLL actually consists of two different categories, based on the BCR repertoire, with important biological and ontogenetic differences. The first ( approximately 30% of cases) shows a very restricted repertoire and is characterized by BCR stereotypy (clustered cases), whereas the second includes cases with heterogeneous BCRs (nonclustered cases). Eleven major CLL clusters were identified with antigen-binding sites defined by just a few critically positioned residues, regardless of the actual immunoglobulin (IG) variable gene used. This situation is closely reminiscent of the receptors expressed by cells participating in innate immune responses. On these grounds, we argue that whereas CLL cases with heterogeneous BCRs likely derive from the conventional B-cell pool, cases with stereotyped BCRs could derive from progenitor cells evolutionarily adapted to particular antigenic challenges, perhaps intermediate between a true innate immune system and the conventional adaptive B-cell immune system, functionally similar to what has been suggested previously for mouse B1 cells.

Background and Objectives: The available data on the immunoglobulin gene (IG) repertoire in multiple myeloma (MM) derive mainly from heavy chains; considerably less is known about light chains. We assessed in parallel IGH and IGK/IGL rearrangements in 101 MM patients so as to gain insight into: (i) IG repertoires; (ii) antigen impact; (iii) the role of receptor editing., Design and Methods: Bone marrow aspirates were collected from all cases. IGHV-(D)-J and IGLV-J rearrangements were amplified by reverse transcriptase polymerase chain reaction (PCR). In all cases, IGKV-J rearrangements were analyzed in parallel on cDNA/genomic DNA. IGKV-KDE and IGKJ-C-INTRON-KDE were also amplified by DNA-PCR. RT-PCR products were directly sequenced., Results: IGHV3 genes predominated; the IGHV4-34 gene was used in only one case. Five IGKV and five IGLV genes accounted for the majority of in-frame, transcribed IGKV-J or IGLV-J rearrangements. Taking IGKV-J, IGKV-KDE and IGKJ-C-INTRON-KDE rearrangements together, biallelic IGK locus rearrangements were detected in 22/43 k-MM cases. In l-MM, 36/42 cases had at least one rearranged IGK allele; 8/19 IGKV-J rearrangements in l-MM were in-frame. All in-frame, transcribed IGH/IGK/IGL sequences were mutated; parallel heavy/light chain analysis demonstrated a comparable impact of somatic hypermutation., Interpretation and Conclusions: Biases in IG repertoire did not seem disease-related but followed a similar pattern to that of the normal repertoire. The under-representation of the IGHV4-34 gene provides an explanation for the paucity of autoimmune phenomena in MM. Somatic mutation patterns indicate the complementary role of MM IGH/IGK/IGL sequences in antigen recognition. Finally, the frequent inactivation of productive IGKV-J joints by secondary rearrangements in MM suggests active receptor editing.