Tertiary lymphoid structural heterogeneity determines tumour immunity and prospects for clinical application.

Tertiary lymphoid structures (TLS) are clusters of immune cells that resemble and function similarly to secondary lymphoid organs (SLOs). While TLS is generally associated with an anti-tumour immune response in most cancer types, it has also been observed to act as a pro-tumour immune response. The heterogeneity of TLS function is largely determined by the composition of tumour-infiltrating lymphocytes (TILs) and the balance of cell subsets within the tumour-associated TLS (TA-TLS). TA-TLS of varying maturity, density, and location may have opposing effects on tumour immunity. Higher maturity and/or higher density TLS are often associated with favorable clinical outcomes and immunotherapeutic response, mainly due to crosstalk between different proportions of immune cell subpopulations in TA-TLS. Therefore, TLS can be used as a marker to predict the efficacy of immunotherapy in immune checkpoint blockade (ICB). Developing efficient imaging and induction methods to study TA-TLS is crucial for enhancing anti-tumour immunity. The integration of imaging techniques with biological materials, including nanoprobes and hydrogels, alongside artificial intelligence (AI), enables non-invasive in vivo visualization of TLS. In this review, we explore the dynamic interactions among T and B cell subpopulations of varying phenotypes that contribute to the structural and functional diversity of TLS, examining both existing and emerging techniques for TLS imaging and induction, focusing on cancer immunotherapies and biomaterials. We also highlight novel therapeutic approaches of TLS that are being explored with the aim of increasing ICB treatment efficacy and predicting prognosis.

Single cell sequencing data identify distinct B cell and fibroblast populations in stricturing Crohn's disease.

Single cell RNA sequencing of human full thickness Crohn's disease (CD) small bowel resection specimens was used to identify potential therapeutic targets for stricturing (S) CD. Using an unbiased approach, 16 cell lineages were assigned within 14,539 sequenced cells from patient-matched SCD and non-stricturing (NSCD) preparations. SCD and NSCD contained identical cell types. Amongst immune cells, B cells and plasma cells were selectively increased in SCD samples. B cell subsets suggested formation of tertiary lymphoid tissue in SCD and compared with NSCD there was an increase in IgG, and a decrease in IgA plasma cells, consistent with their potential role in CD fibrosis. Two Lumican-positive fibroblast subtypes were identified and subclassified based on expression of selectively enriched genes as fibroblast clusters (C) 12 and C9. Cells within these clusters expressed the profibrotic genes Decorin (C12) and JUN (C9). C9 cells expressed ACTA2; ECM genes COL4A1, COL4A2, COL15A1, COL6A3, COL18A1 and ADAMDEC1; LAMB1 and GREM1. GO and KEGG Biological terms showed extracellular matrix and stricture organization associated with C12 and C9, and regulation of WNT pathway genes with C9. Trajectory and differential gene analysis of C12 and C9 identified four sub-clusters. Intra sub-cluster gene analysis detected 13 co-regulated gene modules that aligned along predicted pseudotime trajectories. CXCL14 and ADAMDEC1 were key markers in module 1. Our findings support further investigation of fibroblast heterogeneity and interactions with local and circulating immune cells at earlier time points in fibrosis progression. Breaking these interactions by targeting one or other population may improve therapeutic management for SCD.

Role of IL-27 in Epstein-Barr virus infection revealed by IL-27RA deficiency.

Epstein-Barr virus (EBV) infection can engender severe B cell lymphoproliferative diseases1,2. The primary infection is often asymptomatic or causes infectious mononucleosis (IM), a self-limiting lymphoproliferative disorder3. Selective vulnerability to EBV has been reported in association with inherited mutations impairing T cell immunity to EBV4. Here we report biallelic loss-of-function variants in IL27RA that underlie an acute and severe primary EBV infection with a nevertheless favourable outcome requiring a minimal treatment. One mutant allele (rs201107107) was enriched in the Finnish population (minor allele frequency = 0.0068) and carried a high risk of severe infectious mononucleosis when homozygous. IL27RA encodes the IL-27 receptor alpha subunit5,6. In the absence of IL-27RA, phosphorylation of STAT1 and STAT3 by IL-27 is abolished in T cells. In in vitro studies, IL-27 exerts a synergistic effect on T-cell-receptor-dependent T cell proliferation7 that is deficient in cells from the patients, leading to impaired expansion of potent anti-EBV effector cytotoxic CD8+ T cells. IL-27 is produced by EBV-infected B lymphocytes and an IL-27RA-IL-27 autocrine loop is required for the maintenance of EBV-transformed B cells. This potentially explains the eventual favourable outcome of the EBV-induced viral disease in patients with IL-27RA deficiency. Furthermore, we identified neutralizing anti-IL-27 autoantibodies in most individuals who developed sporadic infectious mononucleosis and chronic EBV infection. These results demonstrate the critical role of IL-27RA-IL-27 in immunity to EBV, but also the hijacking of this defence by EBV to promote the expansion of infected transformed B cells.

[Persistent Polyclonal B-Cell Lymphocytosis (PPBL): An Entity That Is Not What it Seems]. / Linfocitose Policlonal Persistente de Células B (LPPB): Uma Entidade que Não É o que Parece.

Persistent polyclonal B-cell lymphocytosis is a rare disease with chronic lymphocytosis of polyclonal origin, which is more frequent in mostly asymptomatic middle-aged female smokers. The hallmark of this entity is the presence of bilobed/binucleated B lymphocytes, which are polyclonal as demonstrated by immunophenotyping; an elevated IgM level is common. This disease shows, in most cases, an indolent course over many years and, although controversial, it may rarely convert to malignant lymphoma. In addition to smoking, a genetic predisposition for persistent polyclonal B-cell lymphocytosis is likely. Recurrent genetic aberrations have been described. The differential diagnosis includes non-Hodgkin's lymphoma and a clear distinction between both entities is of the utmost importance because treatment is generally not indicated in the former: instead, regular follow-up is recommended. The authors describe the case of a 46-year-old female smoker, who presented with chronic lymphocytosis, elevated IgM and circulating binucleated lymphocytes. Excluding lymphoma was important considering the unusual presentation with constitutional symptoms and splenomegaly.

A linfocitose policlonal persistente de células B é uma doença rara, caracterizada por linfocitose crónica policlonal, que ocorre mais frequentemente em mulheres fumadoras de meia-idade, que se apresentam assintomáticas ou com sintomas inespecíficos. A presença de linfócitos B binucleados é considerada a assinatura citomorfológica desta entidade. A imunofenotipagem comprova a sua origem policlonal, observando-se muitas vezes uma elevação da IgM sérica. É controverso se existe um risco aumentado de desenvolvimento de linfoma. A predisposição genética é também um fator de risco, além do tabagismo. Apesar da sua natureza policlonal, alterações genéticas recorrentes estão descritas. Na linfocitose policlonal persistente de células B a abordagem terapêutica consiste habitualmente numa vigilância regular, o que reforça a importância do seu reconhecimento. Os autores descrevem o caso de uma mulher de 46 anos, fumadora, com linfocitose crónica, IgM elevada e linfócitos binucleados. O diagnóstico diferencial com linfoma assumiu particular importância, considerando os sintomas constitucionais e esplenomegalia que apresentava.

Prognostic significance of lymphocytic foci composition in minor salivary gland biopsies for severe disease flare and severity in Sjögren's syndrome: a 3-year follow-up cohort study.

Introduction: This was an ambispective cohort study evaluating the prognostic significance of lymphocytic foci and its lymphoid composition in minor salivary gland biopsy (MSGB) for short-term disease flare and severity in Sjögren's syndrome (SS). Methods: The inclusion criteria comprised individuals meeting the ACR/EULAR 2016 criteria who underwent MSGB with an infiltration of more than 50 lymphocytes and received clinical diagnosis between September 2017 and December 2018. Patients with inadequate biopsy samples were excluded. The number of lymphocytic foci and their lymphoid composition in MSGB were assessed using immunofluorescence staining. Major organ damage and improvements in the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) were measured. Statistical analyses, including Cox and linear regressions, were conducted. Results: A total of 78 patients with at least one lymphocytic focus were included in the study. The presence of higher T-cell counts in lymphocytic foci in MSGB was associated with severe disease flare, and a logarithmic transformation of T-cell count indicated increased risk (HR 1.96, 95% CI 0.91-4.21). Improvements in the ESSDAI were associated with higher total lymphocyte count and T- and B-cell numbers in the lymphoid composition of the lymphocytic foci. Seropositive patients exhibited higher T CD4+ cell numbers. Correlation analysis showed negative associations between age and lymphocytic foci and the T-cell count. Positive correlations were observed between antinuclear antibody (ANA) titers and total lymphocyte numbers. Discussion: Patients with a higher number of T cells in the lymphocytic infiltrates of lymphocytic foci may have a two-fold risk of severe disease flare. The number of B cells and T CD4+ cells in the lymphocytic infiltrates of lymphocytic foci showed a weak but positive relation with the ESSDAI improvement during follow-up. Age and seropositivity appeared to influence the lymphoid composition of the lymphocytic foci.

Non-sclerosing (T-cell) and sclerosing (B-cell) lymphocytic lobulitis in diagnostic breast biopsies: Clinical, imaging, and pathologic features.

Lymphocytic lobulitis (LL) is characterized by prominent lymphocytic infiltrates centered on lobules. Sclerosing lymphocytic lobulitis (SCLL) associated with diabetes mellitus (DM) or autoimmune disease (AI) was the first type to be described. Subsequently, non-sclerosing LL (NSCLL) was reported as an incidental finding in prophylactic mastectomies due to high risk germline mutations or a family history of breast cancer. The two types of LL were distinguished by stromal features and a predominant population of B-cells in the former and T-cells in the latter. In this study, 8 cases of NSCLL detected clinically or by screening were compared to 44 cases of SCLL. One case of NSCLL presented as a palpable mass, 2 as masses on screening, and 5 as MRI enhancement. In contrast, 80% of SCLL cases presented as palpable masses. Half the cases of NSCLL were associated with a BRCA1 or 2 mutation compared to 1 case of SCLL (2%). Three additional cases of NSCLL were associated with a strong family and/or personal history of breast cancer. Almost half (52%) of SCLL cases were associated with DM or AI, but only 25% of NSCLL. Immunoperoxidase studies confirmed a predominance of T-cells in NSCLL and B-cells in SCLL associated with DM or AI. It is important for pathologists to be aware of this new observation that NSCLL can be detected as a palpable mass or an imaging finding in diagnostic biopsies, as its presence can be indicative of a significant risk for breast cancer.

Patterns of B-cell lymphocyte expression changes in pre- and post-malignant prostate tissue are associated with prostate cancer progression.

BACKROUND: Inflammation characterized by the presence of T and B cells is often observed in prostate cancer, but it is unclear how T- and B-cell levels change during carcinogenesis and whether such changes influence disease progression. METHODS: The study used a retrospective sample of 73 prostate cancer cases (45 whites and 28 African Americans) that underwent surgery as their primary treatment and had a benign prostate biopsy at least 1 year before diagnosis. CD3+, CD4+, and CD20+ lymphocytes were quantified by immunohistochemistry in paired pre- and post-diagnostic benign prostate biopsy and tumor surgical specimens, respectively. Clusters of similar trends of expression across two different timepoints and three distinct prostate regions-benign biopsy glands (BBG), tumor-adjacent benign glands (TAG), and malignant tumor glandular (MTG) regions-were identified using Time-series Anytime Density Peaks Clustering (TADPole). A Cox proportional hazards model was used to estimate the hazard ratio (HR) of time to biochemical recurrence associated with region-specific lymphocyte counts and regional trends. RESULTS: The risk of biochemical recurrence was significantly reduced in men with an elevated CD20+ count in TAG (HR = 0.81, p = 0.01) after adjusting for covariates. Four distinct patterns of expression change across the BBG-TAG-MTG regions were identified for each marker. For CD20+, men with low expression in BBG and higher expression in TAG compared to MTG had an adjusted HR of 3.06 (p = 0.03) compared to the reference group that had nominal differences in CD20+ expression across all three regions. The two CD3+ expression patterns that featured lower CD3+ expression in the BBG compared to the TAG and MTG regions had elevated HRs ranging from 3.03 to 4.82 but did not reach statistical significance. CONCLUSIONS: Longitudinal and spatial expression patterns of both CD3+ and CD20+ suggest that increased expression in benign glands during prostate carcinogenesis is associated with an aggressive disease course.

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication.

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.

Current Insights into Tissue Injury of Giant Cell Arteritis: From Acute Inflammatory Responses towards Inappropriate Tissue Remodeling.

Giant cell arteritis (GCA) is an autoimmune disease affecting large vessels in patients over 50 years old. It is an exemplary model of a classic inflammatory disorder with IL-6 playing the leading role. The main comorbidities that may appear acutely or chronically are vascular occlusion leading to blindness and thoracic aorta aneurysm formation, respectively. The tissue inflammatory bulk is expressed as acute or chronic delayed-type hypersensitivity reactions, the latter being apparent by giant cell formation. The activated monocytes/macrophages are associated with pronounced Th1 and Th17 responses. B-cells and neutrophils also participate in the inflammatory lesion. However, the exact order of appearance and mechanistic interactions between cells are hindered by the lack of cellular and molecular information from early disease stages and accurate experimental models. Recently, senescent cells and neutrophil extracellular traps have been described in tissue lesions. These structures can remain in tissues for a prolonged period, potentially favoring inflammatory responses and tissue remodeling. In this review, current advances in GCA pathogenesis are discussed in different inflammatory phases. Through the description of these-often overlapping-phases, cells, molecules, and small lipid mediators with pathogenetic potential are described.

Tumor mutational load is prognostic for progression to therapy among high-count monoclonal B-cell lymphocytosis.

ABSTRACT: High-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.

Participation of B cell in immunotherapy of cancer.

Even though their effector roles extend beyond conventional humoral immunity, B and plasma cells may exhibit antitumor effects through antibody-dependent cell cytotoxicity (ADCC) and activation of the complement cascade. Depending on whether they are positioned in immature or mature compartments termed tertiary lymphoid structures (TLS), which include T cells, B cells are believed to play numerous functions in modulating the immune system's capacity to destroy cancer cells. These formations represent a process of lymphoid neogenesis that takes place in peripheral tissues in response to prolonged exposure to inflammatory signals. Activated in the germinal centres of tertiary lymphoid structures, B cells may directly present tumor-associated antigens to T cells, make antibodies that enhance antigen presentation to T cells, or kill tumour cells, resulting in a favourable therapeutic effect. Immune complexes may also enhance inflammation, angiogenesis, and immunosuppression via the activation of macrophages and complement, resulting in detrimental effects. The functional variety of B-cell subsets includes professional antigen-presenting cells, regulatory cells, memory populations, and plasma cells that produce antibodies. Importantly, antibodies may independently generate innate immune responses and the cancer immunity cycle. B cells and B-cell-mediated antibody responses constitute the largely underestimated second arm of the adaptive immune system and unquestionably need more consideration in cancer. This article reviews the known roles of B lymphocytes in the tumour microenvironment, their contribution to anticancer activity of immunotherapies, and their significance in overall survival of cancer patients. In addition to producing antibodies, B cells regulate the immune system and serve as effective antigen-presenting cells.

A tower of babel of acronyms? The shadowlands of MGUS/MBL/CHIP/TCUS.

With the advent of outperforming and massive laboratory tools, such as multiparameter flow cytometry and next-generation sequencing, hematopoietic cell clones with putative abnormalities for a variety of blood malignancies have been appreciated in otherwise healthy individuals. These conditions do not fulfill the criteria of their presumed cancer counterparts, and thus have been recognized as their precursor states. This is the case of monoclonal gammopathy of unknown significance (MGUS), the first blood premalignancy state described, preceding multiple myeloma (MM) or Waldenström macroglobulinemia (WM). However, in the last 2 decades, an increasing list of clonopathies has been recognized, including monoclonal B cell lymphocytosis (MBL), which antecedes chronic lymphocytic leukemia (CLL), clonal hematopoiesis of indeterminate potential (CHIP) for myeloid neoplasms (MN), and T-cell clones of uncertain significance (TCUS) for T-cell large chronic lymphocytic leukemia (LGLL). While for some of these entities diagnostic boundaries are precisely set, for others these are yet to be fully defined. Moreover, despite mostly considered of "uncertain significance," they have not only appeared to predispose to malignancy, but also to be capable of provoking set of immunological and cardiovascular complications that may require specialized management. The clinical implications of the aberrant clones, together with the extensive knowledge generated on the pathogenetic events driving their evolution, raises the question whether earlier interventions may alter the natural history of the disease. Herein, we review this Tower of Babel of acronyms pinpointing diagnostic definitions, differential diagnosis, and the role of genomic profiling of these precursor states, as well as potential interventional strategies.

Multimodal and spatially resolved profiling identifies distinct patterns of T cell infiltration in nodal B cell lymphoma entities.

The redirection of T cells has emerged as an attractive therapeutic principle in B cell non-Hodgkin lymphoma (B-NHL). However, a detailed characterization of lymphoma-infiltrating T cells across B-NHL entities is missing. Here we present an in-depth T cell reference map of nodal B-NHL, based on cellular indexing of transcriptomes and epitopes, T cell receptor sequencing, flow cytometry and multiplexed immunofluorescence applied to 101 lymph nodes from patients with diffuse large B cell, mantle cell, follicular or marginal zone lymphoma, and from healthy controls. This multimodal resource revealed quantitative and spatial aberrations of the T cell microenvironment across and within B-NHL entities. Quantitative differences in PD1+ TCF7- cytotoxic T cells, T follicular helper cells or IKZF3+ regulatory T cells were linked to their clonal expansion. The abundance of PD1+ TCF7- cytotoxic T cells was associated with poor survival. Our study portrays lymphoma-infiltrating T cells with unprecedented comprehensiveness and provides a unique resource for the investigation of lymphoma biology and prognosis.

Dual T-cell constant ß chain (TRBC)1 and TRBC2 staining for the identification of T-cell neoplasms by flow cytometry.

The diagnosis of leukemic T-cell malignancies is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. Recently developed therapeutic antibodies specific for the mutually exclusive T-cell receptor constant ß chain (TRBC)1 and TRBC2 isoforms provide a unique opportunity to assess for TRBC-restriction as a surrogate of clonality in the flow cytometric analysis of T-cell neoplasms. To demonstrate the diagnostic utility of this approach, we studied 164 clinical specimens with (60) or without (104) T-cell neoplasia, in addition to 39 blood samples from healthy donors. Dual TRBC1 and TRBC2 expression was studied within a comprehensive T-cell panel, in a fashion similar to the routine evaluation of kappa and lambda immunoglobulin light chains for the detection of clonal B-cells. Polytypic TRBC expression was demonstrated on total, CD4+ and CD8+ T-cells from all healthy donors; and by intracellular staining on benign T-cell precursors. All neoplastic T-cells were TRBC-restricted, except for 8 cases (13%) lacking TRBC expression. T-cell clones of uncertain significance were identified in 17 samples without T-cell malignancy (13%) and accounted for smaller subsets than neoplastic clones (median: 4.7 vs. 69% of lymphocytes, p < 0.0001). Single staining for TRBC1 produced spurious TRBC1-dim subsets in 24 clinical specimens (15%), all of which resolved with dual TRBC1/2 staining. Assessment of TRBC restriction by flow cytometry provides a rapid diagnostic method to detect clonal T-cells, and to accurately determine the targetable TRBC isoform expressed by T-cell malignancies.

Diet-induced obesity reduces bone marrow T and B cells and promotes tumor progression in a transplantable Vk*MYC model of multiple myeloma.

Obesity is associated with an increased risk of developing multiple myeloma (MM). The molecular mechanisms causing this association is complex and incompletely understood. Whether obesity affects bone marrow immune cell composition in multiple myeloma is not characterized. Here, we examined the effect of diet-induced obesity on bone marrow immune cell composition and tumor growth in a Vk*MYC (Vk12653) transplant model of multiple myeloma. We find that diet-induced obesity promoted tumor growth in the bone marrow and spleen and reduced the relative number of T and B cells in the bone marrow. Our results suggest that obesity may reduce MM immune surveillance and thus may contribute to increased risk of developing MM.

Distribution of multi-level B cell subsets in thymoma and thymoma-associated myasthenia gravis.

B-cell subsets in peripheral blood (PB) and tumor microenvironment (TME) were evaluated to determine myasthenia gravis (MG) severity in patients with thymoma-associated MG (TMG) and the distribution of B cells in type B TMG. The distribution of mature B cells, including Bm1-Bm5, CD19+ and CD20+ B cells and non-switched (NSMBCs) and switched (SMBCs) memory B cells, were determined in 79 patients with thymoma or TMG. Quantitative relationships between the T and TMG groups and the TMG-low and TMG-high subgroups were determined. NSMBCs and SMBCs were compared in TME and PB. Type B thymoma was more likely to develop into MG, with types B2 and B3 being especially associated with MG worsening. The percentage of CD19+ B cells in PB gradually increased, whereas the percentage of CD20+ B cells and the CD19/CD20 ratio were not altered. The (Bm2 + Bm2')/(eBm5 + Bm5) index was significantly higher in the TMG-high than in thymoma group. The difference between SMBC/CD19+ and NSMBC/CD19+ B cell ratios was significantly lower in the thymoma than TMG group. NSMBCs assembled around tertiary lymphoid tissue in thymomas of patients with TMG. Few NSMBCs were observed in patients with thymoma alone, with these cells being diffusely distributed. MG severity in patients with TMG can be determined by measuring CD19+ B cells and Bm1-Bm5 in PB. The CD19/CD20 ratio is a marker of disease severity in TMG patients. Differences between NSMBCs and SMBCs in PB and TME of thymomas can synergistically determine MG severity in patients with TMG.

Clinicopathologic features of relapsed CD19(-) B-ALL in CD19-targeted immunotherapy: Biological insights into relapse and LILRB1 as a novel B-cell marker for CD19(-) B lymphoblasts.

INTRODUCTION: The mechanism of relapsed CD19(-) B-ALL after anti-CD19 immunotherapy (Kymriah [CART-19] and blinatumomab) is under active investigation. Our study aims to assess LILRB1 as a novel B-cell marker for detecting CD19(-) B-lymphoblasts and to analyze the clinicopathologic/genetic features of such disease to provide biological insight into relapse. METHODS: Six patients (3 males/3 females, median age of 14 years) with relapsed CD19(-) B-ALL were analyzed for cytogenetic/genetic profile and immunophenotype. RESULTS: CD19(-) B-ALL emerged after an interval of 5.8 months following anti-CD19 therapy. Five of six patients had B-cell aplasia, indicative of a persistent effect of CART or blinatumomab at relapse. Importantly, LILRB1 was variably expressed on CD19(-) and CD19(+) B lymphoblasts, strong on CD34(+) lymphoblasts and dim/partial on CD34(-) lymphoblasts. Three of six patients with paired B-ALL samples (pre- and post-anti-CD19 therapy) carried complex and different cytogenetic abnormalities, either as completely different or sharing a subset of cytogenetic abnormalities. CONCLUSION: LILRB1 can be used as a novel B-cell marker to identify CD19(-) B lymphoblasts. The emergence of CD19(-) B-ALL appears to be associated with complex cytogenetic evolutions. The mechanism of CD19(-) B-ALL relapse under anti-CD19 immune pressure remains to be explored by comprehensive molecular studies.