Background/Objectives: The aim of this study was to evaluate the cycle threshold (Ct) values of self-collected vaginal samples as a triage method to colposcopy for high-risk (hr) HPV-positive women. Methods: We analyzed data from GRECOSELF, a nationwide observational cross-sectional study on HPV primary cervical cancer screening in Greece. Self-collected vaginal samples were tested with the cobas® HPV test (Roche® Molecular Systems, Pleasanton, CA, USA). The Ct value, i.e., the number of cycles needed until DNA amplification occurs exponentially in a PCR, reflects the viral load, and it was evaluated as a triage method to colposcopy for hrHPV-positive women. Results: For CIN2 and more advanced lesions, the Ct value, as a dichotomous variable at a cut-off of 29.7, had 54.8% (95%CI: 38.7–70.2) sensitivity, 35.4% (23.9–48.2) Positive Predictive Value (PPV), 74.2% (66.8–80.8) specificity, and 86.4% (73.6–91.6) Negative Predictive Value (NPV) for HPV16/18, while for other hrHPV types, sensitivity was 26.7% (12.3–45.9), PPV 6.7% (2.9–12.8), specificity 78.8% (75.1–82.2), and NPV 95.0% (92.5–96.8). For CIN3 and more advanced lesions, the NPV for non-HPV16/18 was 97.9 (96.1–99.1). Conclusions: For self-collected vaginal samples of hrHPV-positive women, the Ct value may be used as a triage method to colposcopy. As Ct values inversely reflect the viral loads, they are lower in high-grade CIN and/or carcinoma.

Background: CLL subset #4 is the largest stereotyped subset in IGHV-mutated CLL (M-CLL). The clonotypic B cell receptor immunoglobulin (BcR IG) in subset #4, encoded by the IGHV4-34/IGKV3-20 gene pair, displays long heavy complementarity determining region 3 (VH CDR3), enriched in positively charged residues; ubiquitous expression of gamma heavy chain isotype; distinctive imprint of somatic hypermutation (SHM), characterized by the frequent introduction of acidic residues and pronounced intraclonal diversification. These features are reminiscent of edited autoantibodies, implicating ongoing (auto)antigen interactions in the natural history of CLL subset #4. Aims: Here, we sought to explore the immune trajectory and clonal dynamics of CLL subset #4, particularly focused on the role of SHM. Methods: We studied longitudinal samples from 6 subset #4 and 6 non-subset #4 IGHV4-34-expressing M-CLL cases. Clonotypic IG heavy (HC) and light chain (LC) gene rearrangements were profiled by next generation sequencing (NGS). The clonotype accounting for the majority of NGS reads in a given sample was defined as dominant; related clonotypes expressing the same IGHV/IGKV gene and VH CDR3 amino acid (aa) sequence yet differing in the aa sequence of the VH and/or VK domains were defined as subclonotypes. The dominant clonotypes of all cases as well as 32 subclonotypes of subset #4 cases were expressed as recombinant monoclonal antibodies (rmAbs) and screened for antigen reactivity using ELISA and flow cytometry. Results: NGS revealed two distinct patterns of subclonal architecture in the IGH repertoire of subset #4. The first was characterized by the presence of a single dominant clonotype (4 cases) whereas the second by the co-existence of ≥2 significantly expanded clonotypes with comparable frequencies (2 cases). Turning to the LC, 5/6 cases carried a single dominant clonotype, while 1 case carried ≥1 expanded clonotypes. In contrast, a single dominant clonotype was detected in all non-subset #4 cases for both HC and LC. Longitudinal analysis revealed significantly (p<0.05) more pronounced subclonal drift (changing relative frequencies) of the expanded clonotypes in subset #4 vs non-subset #4 cases, particularly for the HC. Surprisingly, in all subset #4 cases we identified clonotypic IgG transcripts (same IGH gene and VH CDR3 length, high VH CDR3 aa sequence identity) with 100% germline IGHV identity (‘truly unmutated’), present at low frequency (median: 0.003%, range: 0.0003-0.03%). Prompted by this, we produced rmAbs for both the dominant, somatically hypermutated BcR IG clonotype of all 6 subset #4 cases, as well as 32 truly unmutated subclonotypes (from all studied subset #4 cases) and compared their antigen reactivity profiles. We report consistent, significantly (p<0.05) stronger reactivity of the truly unmutated, subclonotypic BcR IG compared to the mutated BcR IG expressed by the dominant CLL #4 clone. With few exceptions, all subclonotypic truly unmutated BcR IG displayed more intense recognition against dsDNA and lipopolysaccharides, antigenic elements from CMV, Influenza A and Mycoplasma pneumoniae as well as viable HEK293 and Jurkat T cells. Summary/Conclusion: Our findings support that SHM in CLL subset #4 is functionally driven by persistent selection by (auto)antigens, ultimately leading to a significant redemption from autoreactivity. The identification of truly unmutated clonotypic IgG gene transcripts likely reflects a complex trajectory of clonal evolution, offering hints about the precise timing of SHM in relation to class switch recombination in the natural history of CLL subset #4. Keywords: Antigen presentation, B cell chronic lymphocytic leukemia, B cell lymphoma, Antibody

BackgroundMicroenvironmental interactions of the malignant clone with T cells are critical throughout the natural history of chronic lymphocytic leukemia (CLL). Indeed, clonal expansions of T cells and shared clonotypes exist between different CLL patients, strongly implying clonal selection by antigens. Moreover, immunogenic neoepitopes have been isolated from the clonotypic B cell receptor immunoglobulin sequences, offering a rationale for immunotherapeutic approaches. Here, we interrogated the T cell receptor (TR) gene repertoire of CLL patients with different genomic aberration profiles aiming to identify unique signatures that would point towards an additional source of immunogenic neoepitopes for T cells.Experimental designTR gene repertoire profiling using next generation sequencing in groups of patients with CLL carrying one of the following copy-number aberrations (CNAs): del(11q), del(17p), del(13q), trisomy 12, or gene mutations in TP53 or NOTCH1.ResultsOligoclonal expansions were found in all patients with distinct recurrent genomic aberrations; these were more pronounced in cases bearing CNAs, particularly trisomy 12, rather than gene mutations. Shared clonotypes were found both within and across groups, which appeared to be CLL-biased based on extensive comparisons against TR databases from various entities. Moreover, in silico analysis identified TR clonotypes with high binding affinity to neoepitopes predicted to arise from TP53 and NOTCH1 mutations.ConclusionsDistinct TR repertoire profiles were identified in groups of patients with CLL bearing different genomic aberrations, alluding to distinct selection processes. Abnormal protein expression and gene dosage effects associated with recurrent genomic aberrations likely represent a relevant source of CLL-specific selecting antigens.

Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.

Increasing evidence supports a role for the vaginal microbiome (VM) in the severity of HPV infection and its potential link to cervical intraepithelial neoplasia. However, a lot remains unclear regarding the precise role of certain bacteria in the context of HPV positivity and persistence of infection. Here, using next generation sequencing (NGS), we comprehensively profiled the VM in a series of 877 women who tested positive for at least one high risk HPV (hrHPV) type with the COBAS® 4,800 assay, after self-collection of a cervico-vaginal sample. Starting from gDNA, we PCR amplified the V3-V4 region of the bacterial 16S rRNA gene and applied a paired-end NGS protocol (Illumina). We report significant differences in the abundance of certain bacteria compared among different HPV-types, more particularly concerning species assigned to Lacticaseibacillus, Megasphaera and Sneathia genera. Especially for Lacticaseibacillus, we observed significant depletion in the case of HPV16, HPV18 versus hrHPVother. Overall, our results suggest that the presence or absence of specific cervicovaginal microbial genera may be linked to the observed severity in hrHPV infection, particularly in the case of HPV16, 18 types.

Hairy cell leukemia (HCL) is a rare indolent B-cell malignancy thought to derive from a BRAF-mutant antigen-experienced B cell. Supporting evidence includes a transcriptional profile that is most similar to post-germinal center (GC) memory B cells; the presence of somatic hypermutation (SHM) in the majority of HCL cases; and, the preferential usage of the IGHV4-34 gene in the minority of classic HCL (HCLc) cases with low or no SHM. IGHV4-34-expressing HCLc is clinically more aggressive than other HCLc; moreover, it is enriched for BRAF-wildtype cases while it displays frequent MAP2K1 mutations. Overall, this constellation of features raises questions regarding the precise nature and ontogeny of IGHV4-34-expressing HCLc. Here we addressed this issue through investigating the subclonal architecture of the B cell receptor immunoglobulin (BcR IG) gene repertoire in a series of 17 cases with a typical phenotype of HCLc expressing the IGHV4-34 gene. Previous Sanger analysis had documented 10 cases carrying IGHV genes with 100% germline identity (GI), 2 cases with minimal SHM (GI>99%) and 5 with a more significant SHM load (GI<98%). Total RNA was isolated from peripheral blood mononuclear cells and IGHV-IGHD-IGHJ rearrangements were RT-PCR amplified and subjected to NGS sequencing using a paired-end protocol. Quality filtering of NGS raw reads was performed with a purpose-built bioinformatics pipeline and downstream analysis was performed with the IMGT/HighV-QUEST, tripr and IgIDivA softwares. Clonotypes were defined as clusters of rearrangement sequences expressing the IGHV4-34 gene and bearing identical variable heavy complementarity-determining region 3 (VH CDR3) amino acid (aa) sequences. Variants of a given clonotype i.e. gene rearrangement sequences with a VH CDR3 of identical length differing in up to 2 aa positions were defined as sub-clonotypes; these are likely to have differentiated from the main clonotype due to ongoing SHM. For the analysis of intraclonal diversification (ID) within the clonotypic rarranged IGHV genes, we used the metrics 'convergence score', describing the tendency of the BcR IG to acquire more mutations, and 'maximal pathway length', showing whether ongoing SHM acquisition takes place. Overall, we obtained 3,238,715 raw reads (median 178,446 reads/sample). Of these, 2,226,444 reads corresponded to productive IGHV4-34 gene rearrangements (median 127,647 sequences/sample) that were assigned to a total of 10,532 clonotypes (median 593 clonotypes/sample). The median frequency of the dominant clonotype was 64.4%, indicating a significant subclonal branching due to ID. Indeed, overall, we detected 5,267 subclonotypes (median 213 subclonotypes/sample) corresponding to 242,271 unique nucleotide (nt) variants (median 8098 nt variants/sample). ID was observed in both IG-mutated (<100% GI) and IG-unmutated (100% GI) cases, albeit was significantly (p=0.01) more extensive in the former (median number of nucleotide variants/sample: 21,306 vs 7,099, respectively). Next, we compared the present HCLc dataset against a corresponding dataset produced following the same approach from 11 IG-mutated CLL (M-CLL) cases expressing the IGHV4-34 gene, of which 5 belonged to stereotyped subset #4, notable for a high degree of ID. No statistically significant differences were identified in convergence scores between HCLc vs CLL, indicating that the former also displays considerable ID despite an overall lower number of clonal SHMs (underlying the higher GI at the clonal level i.e. by Sanger sequencing analysis). Moreover, HCLc exhibited significantly (p=0.002) higher maximal pathway lengths compared to CLL, suggesting that acquisition of subclonal SHM in IGHV4-34 expressing HCLc is an ongoing process that takes place progressively in many steps. Also relevant to mention, different recurrent replacement SHMs were seen in IGHV4-34 expressing HCLc vs CLL: indicatively, G36S in VH CDR1 was present in subclones of 11/17 HCLc cases whereas it was totally absent in CLL. In conclusion, we report a complex immunogenetic architecture for IGHV4-34 expressing HCLc with distinctive, disease-biased features and extensive ID, likely mediated by ongoing antigen interactions. This finding questions a post-GC origin for at least a subgroup of HCLc, while also highlighting the need to reappraise the ontogenetic relationship of this particular HCLc subgroup vs all other HCLc.

Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a 'truly unmutated' CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3'IGHV, IGHD, 5'IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.

Venetoclax (ven), an inhibitor of the BCL2 anti-apoptotic protein, has been proven remarkably effective in the treatment of patients with chronic lymphocytic leukemia (CLL), where the malignant B cells typically overexpress BCL2. BCL2 is also expressed by various bystander cells in the CLL microenvironment, including T cells. Hence, investigating the effects of ven treatment on T cells is highly relevant, especially considering that distinct T cell subpopulations exhibit different sensitivity to anti-apoptotic signaling blockade. Herein we interrogated the molecular and phenotypic profiles of the T cell compartment in 1 treatment-naïve and 15 relapsed/refractory CLL patients who received ven either as monotherapy (n=12) or in combination with an anti-CD20 antibody (obinutuzumab, n=1; rituximab, n=3). We studied peripheral blood samples collected prior to ven initiation and after 5 months of treatment, from which we isolated T cell subpopulations (CD3+ T cells n=8; CD4+ and CD8+ T cells, n=8). Longitudinal analysis of pre/post-treatment samples by flow cytometry (n=7) revealed a significant (p=0.01) decrease in the numbers of central memory (CD45RO+/CCR7+) CD4+ (21.8% vs 8.8%) and CD8+ cells (21.4% vs 9.7%) under ven; in contrast, a significant (p=0.01) increase of CD8+ effector memory cells (CD45RO+/CCR7-) was observed (27.8% vs 32.9%). Profiling of the T cell receptor beta (TRB) chain gene repertoire overtime (n=15) was performed by next generation sequencing (NGS). Clonotypes (i.e. TRBV-TRBD-TRBJ gene rearrangements with unique pairs of TRBV genes and identical CDR3 amino acid sequences) were calculated and clonality was estimated as the average cumulative frequency of the 10 most frequent clonotypes per sample (ACF-10). A significantly (p=0.01) more diverse repertoire was identified in CD4+ vs CD8+ cells at both pre- and post-treatment timepoints (average numbers of clonotypes pre-/post-treatment: CD4+ cells, 16,908/13,768; CD8+ cells, 4,437/3,633). Expanded clonotypes corresponding to oligoclonal expansions were documented in all examined subpopulations at both timepoints. However, significantly (p=0.01) more pronounced skewing was observed in CD8+ vs CD4+ cells (pre-/post-treatment ACF-10 values: 64.4%/70.6% vs 31.4%/42.2%, respectively). Repertoire comparisons overtime highlighted a fraction of T clonotypes that were retained after treatment in all examined subpopulations. Of note, repertoire conservation was significantly (p=0.01) greater in CD8+ vs CD4+ T cells [retained pre-treatment clonotypes: 7,102/31,063 (28%) vs 11,525/118,359 (12%), respectively]. Transcriptome profiling of CD4+ and CD8+ cells pre-/post-treatment was performed using the Oncomine Immune Response Research Assay (395 genes) in 5 cases attaining a complete response after treatment. While the transcriptomes of CD4+ cells remained essentially unaltered, significant differences were identified in CD8+ cells, where 19 genes were found to be downregulated (adj.p<0.05, fold change>2). These genes are implicated in functions such as immune response (e.g. TLR7, TLR9, CD40), regulation of immune system (e.g. HLA genes, CIITA, TNFAIP8) and lymphocyte activation pathways (e.g. ICOSLG, CEACAM1, CXCR5), with at least some of these having immunosuppressive functions (e.g. BTLA and CTLA4 encoding inhibitory receptors associated with T cell exhaustion). In conclusion, ven treatment led to increased numbers of CD8+ effector subpopulations along with prominent clonal expansions and transcriptional rewiring that could conceivably contribute to clinical response. Downregulation of immune-related genes implicated in core regulatory/activation pathways supports immune recovery by ven, offering a rationale for future combination strategies aiming to increase the depth of clinical response.

Chronic graft-versus-host disease (cGVHD) is the main cause of morbidity and transplant-related mortality following allogeneic hematopoietic stem cell transplantation (alloHSCT), however effective treatment options are limited. Lack of objective surrogates (biomarkers) for treatment response has hindered progress in this respect. T cells are considered the major effectors of cGVHD, yet the respective repertoires are insufficiently charted. Here, we investigated the dynamic architecture of T-cell repertoires in cGVHD by exploiting next-generation sequencing (NGS), aiming to uncover immunogenetic signatures linked with cGHVD occurrence and response to treatment.

The localization of bacterial components and/or metabolites in the central nervous system may elicit neuroinflammation and/or neurodegeneration. Helicobacter pylori (a non-commensal symbiotic gastrointestinal pathogen) infection and its related metabolic syndrome have been implicated in the pathogenesis of gastrointestinal tract and central nervous system disorders, thus medications affecting the nervous system - gastrointestinal tract may shape the potential of Helicobacter pylori infection to trigger these pathologies. Helicobacter pylori associated metabolic syndrome, by impairing gut motility and promoting bacterial overgrowth and translocation, might lead to brain pathologies. Trimebutine maleate is a prokinetic drug that hastens gastric emptying, by inducing the release of gastrointestinal agents such as motilin and gastrin. Likewise, it appears to protect against inflammatory signal pathways, involved in inflammatory disorders including brain pathologies. Trimebutine maleate also acts as an antimicrobial agent and exerts opioid agonist effect. This study aimed to investigate a hypothesis regarding the recent advances in exploring the potential role of gastrointestinal tract microbiota dysbiosis-related metabolic syndrome and Helicobacter pylori in the pathogenesis of gastrointestinal tract and brain diseases. We hereby proposed a possible neuroprotective role for trimebutine maleate by altering the dynamics of the gut-brain axis interaction, thus suggesting an additional effect of trimebutine maleate on Helicobacter pylori eradication regimens against these pathologies.

The finding that (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins IGs) are expressed in a significant fraction of chronic lymphocytic leukemia (CLL) highlighted the importance of antigen selection in disease pathogenesis. Subsets of patients sharing the same stereotyped BcR IG display consistent biological features and, at least for certain subsets, clinical presentation and outcome, including the response to particular treatment. On these grounds, BcR IG stereotypy emerges as a useful tool for dissecting the pronounced heterogeneity of CLL toward refining risk stratification and therapeutic management aligned with the principles of precision medicine.

Chronic lymphocytic leukemia (CLL) B cells engage in multifaceted bi-directional interactions with bystander cells, including T cells. Immunogenetic studies in CLL revealed clonal expansions of T cells and shared T cell clonotypes between different patients, strongly implying clonal selection by antigens. Although the exact nature of these antigens remains largely elusive, evidence exists that the clonotypic B cell receptor immunoglobulin (BcR IG) may serve as a source of antigenic epitopes for T cells. That said, recurrent genomic aberrations associated with distinct abnormal expression profiles could represent an alternative, non mutually exclusive, source of potent immunogenic onco-antigens that might shape the T cell repertoire in CLL. On these grounds, here we interrogated the T cell receptor (TR) gene repertoire of CLL patients with different genomic aberration profiles with the aim to identify unique signatures that would allude to distinct antigen selection pressures. The study group included 46 patients with CLL, sampled before treatment initiation, who were categorized in 5 subgroups defined by a unique genomic aberration, as follows: +12, n=18; del(11q), n=10; del(13q), n=7; del(17p)/TP53mut, n=6; NOTCH1mut, n=5. Confounding effects of multiple aberrations have been minimized, as we previously established through comprehensive characterization (including FISH, SNP arrays and gene panels) that the analyzed patients carried only one of the above aberrations. Starting material was RNA extracted from blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end next generation sequencing (NGS). Raw NGS reads (n=13,213,563| median: 294,757/sample) were processed through a purpose-built bioinformatics pipeline. Only productive rearrangements (n=9,249,546 | median=199,184/sample) were taken into consideration for the computation of clonotypes i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 (CDR3) sequence. Overall, 513,984 distinct clonotypes (median=10,304 clonotypes/sample) were assessed. The main measure of clonality employed in this study was the median cumulative frequency of the 10 most expanded T cell clonotypes/sample (MCF-10). For comparisons of the clonality profiles, a group of 17 aged-matched healthy individuals were used as controls. All patients displayed oligoclonal T cell expansions with the following MCF-10 values: del(11q): 21.6%, +12: 25%, del(13q): 20.6%, NOTCH1mut: 9.1%, del(17p)/TP53mut: 12.9%; the difference between the del(11q) and +12 groups versus the NOTCH1mut group was statistically significant (p<0.05). The MCF-10 value of the control group was estimated at 17.5%, supporting the notion of age-related decrease in TR repertoire diversity. However, the del(11q), +12 and del(13q) CLL groups displayed elevated clonality, reaching statistical significance (p<0.002) in the case of +12. TRBV gene repertoire analysis revealed that the TRBV12-3 gene predominated in most groups, except for the del(17p)/TP53mut, where the predominant gene was TRBV10-3. Clonotype comparisons disclosed the presence of shared TR clonotypes both within a particular group but also between groups. Overall, 446/513,984 clonotypes were found to be shared by at least two patients across all groups; the vast majority (392/446, 88%) of shared clonotypes appeared to be CLL-biased since they did not match entries in public databases of TR clonotypes from various contexts. Subgroup-specific clonotypes were identified for all aberrations examined; these emerged as unique to the particular subgroups, as revealed by extensive comparisons against both public databases but also a large TR clonotype database from CLL available to us from our previous studies. In conclusion, recurrent genomic aberrations, especially large chromosomal abnormalities, display an oligoclonal TR gene repertoire. The distinct immunogenetic profile of each group examined here and, most importantly, the existence of subgroup-specific clonotypes, suggest that abnormal protein expression and gene dosage effects likely represent a relevant source of CLL-specific selecting antigens.

Preliminary evidence for T cell receptor (TR) repertoire renewal and increased TR clonality has been reported by our group (Vlachonikola et al., ASH 2019) in multiple myeloma (MM) patients (pts) receiving daratumumab monotherapy within the context of the REBUILD study, an ongoing prospective, multicenter, non-comparative, open-label, phase II study in pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Herein, we report the results from the longitudinal analysis of the TR repertoire employing next generation sequencing (NGS) and multi-color flow cytometry in 24 pts who completed 3 cycles (n=24) and 6 cycles (n=11/24) of daratumumab monotherapy, in order to assess the immunomodulatory effects of daratumumab. We assessed 59 peripheral blood samples collected at screening (SCR, n=24), on Day 1 of Cycle 4 (C4, n=24) and Day 1 of Cycle 7 (C7, n=11). Patients were grouped based on best responses at C4 into responders (i.e. pts with partial response [PR, n=7] and very good PR [VGPR, n=8]), and non-responders (i.e. pts with minimal response [MR, n=2], stable disease [SD, n=5], or progressive disease [PD, n=2]). TRBV-TRBD-TRBJ gene rearrangements were subjected to paired-end NGS and raw reads (n=13,886,646 | median 239,969/sample) were processed through a purpose-built bioinformatics pipeline. Productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=6,324,986 | median 100,738/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 325,789 distinct clonotypes (median 4,535 clonotypes/sample) were analyzed. The TR repertoire displayed clonal T cell expansions in both groups (responders/non-responders) in all pre/post-treatment timepoints. Clonality increased after treatment for both responders and non-responders in all assessed timepoints, with statistical significance at C4 in both groups (median cumulative frequency of the 10 most expanded T cell clonotypes/sample in responders: 31.6% pre-treatment vs 43% C4 post-treatment, p=0.009; and, in non-responders: 19.8% pre-treatment vs 39.6% C4 post-treatment, p=0.009). In both groups, the clonotype repertoire appeared to be renewed. Interestingly, in the responders' group a significant shift was noticed in the major clonotype repertoire at screening vs C4. In particular, the 10 most expanded clonotypes/sample at C4 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment. Additionally, although the major post-treatment clonotype at C4 also dominated at C7 in most cases, certain lower frequency clonotypes at C4 emerged among the top-10 at C7. Thirteen shared clonotypes were identified amongst the post-treatment repertoires of different patients but not in other entities in public databases, raising the possibility that they may be "MM-specific" and selected by common MM-associated antigens. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at SCR 63% vs 78.7% at C4 | p=0.0045 and 87.4% at C7 | p=0.0009), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at SCR 31.4% vs 45.3% at C4 | p=0.0045 and 53.8% at C7 | p=0.001) in both groups. In conclusion, we document T cell clonal expansions and clonal drift after daratumumab treatment in MM. Our results suggest that daratumumab acts through renewing the greatest part of the pre-treatment TR clonotype repertoire, suggesting dynamic changes of the T cell compartment under treatment, a claim also supported by the significant increase in CD8+ cytotoxic T cell numbers overtime. The significant post-treatment expansion of certain low-frequency pre-treatment clones in responders raises the intriguing hypothesis that daratumumab treatment may have led to the outgrowth of anti-MM T cell clones, arguably contributing to clinical response. Disclosures Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Hatjiharissi:Abbvie: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria. Katodritou:Theagenion Cancer Hospital: Current Employment; Takeda: Honoraria, Other: Expenses, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genesis Pharma: Honoraria, Other: Expenses, Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gavriatopoulou:Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genesis Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Delimpasi:GENESIS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Symeonidis:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Research Funding; Astellas: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding. Stamatopoulos:AstraZeneca: Honoraria; Janssen, Gilead, Abbvie: Honoraria, Research Funding. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, Research Funding, Speakers Bureau. Terpos:Amgen: Honoraria, Research Funding; Genesis pharma SA: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Sanofi: Honoraria; BMS: Honoraria. Chatzidimitriou:Janssen: Research Funding.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic condition of monoclonal B-cell expansions in the blood of healthy, mostly elderly, individuals. MBL is classified into three distinct subtypes: (i) “chronic lymphocytic leukemia (CLL)-like” MBL (CD5CD23), which accounts for the vast majority of cases; (ii) “atypical CLLlike” MBL (CD5CD23-CD20; and (iii) “non CLL-like” MBL (CD5). “CLL-like” MBL is subdivided into two different categories based on clonal size; cases with 0.55x10 cells/L are categorized as “high-count MBL” (HCMBL), whereas those with <0.5x10 cells/L as ”low-count MBL” (LC-MBL). HC-MBL progresses to CLL requiring treatment at a rate of 1-2% per year, whereas the risk of progression for “CLL-like” LC-MBL is negligible despite persisting over time. Recently, we reported that the genomic profiles of LCMBL, HC-MBL and ‘ultra-stable’ CLL (no disease progression for ≥10 years) are very similar. On these grounds, we proposed that cross-talk between CLL progenitor cells and the microenvironment might represent a major driver in early stages of the disease. Relevant microenvironmental triggers might be provided by T cells, considering ample evidence for their implication in CLL pathogenesis and the existence of T-cell expansions. Such clonal expansions were also evident in HCMBL and LC-MBL, however, the relevant studies had important limitations, such as limited coverage of the expressed repertoire and sequencing depth, thus precluding firm conclusions from being drawn. In order to overcome these limitations, we characterized the T-cell receptor beta (TRB) chain gene repertoire using a high-throughput sequencing approach. Blood samples were collected from individuals from Val Borbera, Italy, where a LC-MBL cohort is regularly followed up (Online Supplementary Material). Samples from aged-matched, healthy individuals without MBL from the same region were analyzed as controls; comparisons to CLL were also performed. The research protocol was approved by the Ethics Committee of the San Raffaele Institute and all participants gave written informed consent in accordance with the Declaration of Helsinki. Samples of blood (5 mL) were obtained from all individuals and processed within 24 h. LC-MBL was diagnosed following a standardized flow cytometry approach (Online Supplementary Methods). We analyzed 48 samples from individuals with LC-MBL (“CLL -like” LC-MBL, n=41; “other” LC-MBL subtypes, n=7), and 17 samples from healthy controls (Online Supplementary Table S1). TRBV-TRBD-TRBJ gene rearrangements were amplified by polymerase chain reaction, sequenced on a MiSeq Sequencer and bioinformatically processed, as previously described. Overall, 2,357,648 distinct TRB clonotypes were identified. Of these, 1,006,126 (42.7%) were expanded (>1 read), whereas the remainder (1,351,522, 57.3%) concerned singletons (=1 read). The relevance of age in shaping the TRB repertoire, reported in a recent next-generation sequencing study, was also evident in our cohort; clonal expansions were present in all sample categories, including healthy donors. However, significantly different mean relative frequencies of expanded clonotypes were evident between sample categories [analysis of variance (ANOVA), P<0.05]. In more detail, expanded clonotypes were larger in “CLL-like” LC-MBL (0.018%; range, 0.0030.14%) than in “other” LC-MBL (0.007%; range, 0.0010.04%) or in the “healthy” category (0.01%; range, 0.002-0.012%) (Table 1, Online Supplementary Figure S1). Two different approaches were followed to assess clonality in each sample: (i) the ten “major” (most expanded) clonotypes; and (ii) all expanded clonotypes with an individual frequency of >1%. The first approach led to the identification of slightly higher clonality levels in LC-MBL than in healthy individuals, yet lower than those reported in CLL (ANOVA, P=0.3) (Figure 1, Online Supplementary Table S2). When considering clonotypes with a relative frequency >1%, the average number of expanded clonotypes for the “CLL-like” LC-MBL, “other” LC-MBL and healthy groups was five (range, 0-27), four (range, 2-6) and four (range, 1-11), respectively. The average sum of relative frequencies was 20% in “CLL-like” LC-MBL, 17% in “other” LC-MBL and 15% in the

Epigenetic changes, including altered small non-coding RNAs, appear to be implicated in the pathogenesis of sporadic parathyroid adenomas (PAs). In this study, we investigated the circular RNAs (circRNAs) expression profile in sporadic PAs. Sixteen tissue samples of sporadic PAs, and four samples of normal parathyroid tissue (NPT) were investigated. Sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols, and circRNAs sequences were predicted by bioinformatics tools. We identified 35 circRNAs that were differentially expressed in sporadic PAs compared to NPT; 22 were upregulated, and 13 were downregulated, according to the pre-defined thresholds of fold-change > 2.0 and p < 0.05. In the subgroup analysis of PAs from male patients (n = 7) compared to PAs from female patients (n = 9), we also find a different expression profile. In particular, 19 circRNAs were significantly upregulated, and four circRNAs were significantly downregulated in male patients, compared to female counterparts. We show here for the first time a differential circRNA expression pattern in sporadic PAs compared to NPT, and a different expression profile in PA samples from male compared to female patients, suggesting an epigenetic role in the PA pathogenesis, and also an effect of gender in the epigenetic regulation of PAs.

Recent evidence suggests immunomodulatory effects of daratumumab in heavily pre-treated Multiple Myeloma (MM) patients (pts); however, the precise effects remain under-characterized, particularly at the molecular level. REBUILD is an ongoing prospective, multicenter, non-comparative, open-label, phase II study that evaluates the effects of daratumumab monotherapy on bone metabolism of pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Secondary endpoint of the study included the evaluation of T cell dynamics by comprehensive analysis of the T cell receptor (TR) repertoire employing next generation sequencing (NGS) and multi-color flow cytometry. Herein we report the results of this secondary endpoint for the first 14 pts who completed 3 cycles of daratumumab monotherapy. In total, we analyzed 28 paired samples collected at screening (n=14) and on Day 1 of Cycle 4 (C4D1, n=14) of treatment in order to assess potential changes in relation to treatment and clinical response. Patients were grouped based on best responses into responders (i.e. patients with partial response [PR, n=1] and very good PR [VGPR, n=6]), and non-responders (i.e. patients with minimal response [MR, n=2], stable disease [SD, n=4], or progressive disease [PD, n=1]). Starting material was peripheral blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw NGS reads (n=6,715,406 | median 221,145/sample) were processed through a previously published, purpose-built bioinformatics pipeline. Only productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=3,097,565 | median 101,670/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 151,153 distinct clonotypes (median 5,084 clonotypes/sample) were assessed. Both groups (responders/non-responders) displayed clonal T cell expansions both pre- and post-treatment. Clonality was found to be increased after treatment for both responders and non-responders, with statistical significance in the former (median cumulative frequency of the 10 most expanded T cell clonotypes/sample: 31% pre-treatment versus 40% post-treatment, respectively | p=0.04). In both groups, the clonotype repertoire appeared to be renewed with only a small fraction of pre-treatment clonotypes remaining after treatment (1% for non-responders; 0.6% for responders). Interestingly, in the responders' group we noticed a significant shift in the major clonotype repertoire at screening vs C4D1. In particular, in the responders' group the 10 most expanded clonotypes/sample at C4D1 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment, suggesting that daratumumab treatment led to the emergence of anti-myeloma T cell clones which contributed to clinical response. On the contrary, the 10 most expanded pre-treatment clonotypes in the non-responders' group tended to dominate also the post-treatment repertoire. Of note, 13 shared clonotypes were identified amongst the post-treatment repertoires of different patients (responders/non-responders); shared clonotypes were not found in other entities in public databases, raising the possibility that they may be "disease-specific" and selected by common tumor-associated antigens. With a single exception, shared clonotypes were detected in cases with relevant HLA restrictions, which is noteworthy given the random HLA background of our cohort. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at screening 60% versus 83% at C4D1 | p=0.003), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at screening 30.8% versus 48.9% at C4D1 | p=0.03) in both groups. In conclusion, TR clonality increases post-treatment through a renewal mechanism; however, pre-treatment clones significantly expanded post-treatment in responders, alluding to the existence of clonotypes with anti-MM properties that may be activated after treatment with daratumumab, arguably contributing to clinical response. Disclosures Kastritis: Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria; Amgen: Honoraria, Research Funding. Hatjiharissi:Janssen: Honoraria. Katodritou:Genesis: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Gavriatopoulou:Genesis: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Amgen: Honoraria. Delimpasi:Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria, Other: Travel grant. Symeonidis:Sanofi: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding. Chatzidimitriou:Janssen: Honoraria.