B-cell Lymphoid Neoplasms Research Articles

LGL in MGUS or MGUS in LGL? Lessons of Nature and Their Clinical Implications

Associations between seemingly unrelated entities often reveal important lessons of nature, instructive for the understanding of mutual pathogenic roots. While investigating the clinical features of T-cell large granular lymphocytic leukemia (LGL), we noted the frequent presence of plasma cell dyscrasias/monoclonal gammopathies (MGs), chiefly MGUS. There are several potential explanations for this co-occurrence: 1) similar demographics (both conditions mostly presenting in the elderly); 2) the possibility that the two precursors originate from an initial polyclonal immune response to a common target; and 3) as alternative, that monoclonal plasma cells or the Ig product may be the targets of cytotoxic T-LGL cells in a context of tumor surveillance reaction. For instance, clonal TCR may recognize the M protein idiotype or vice versacausing aberrant activations. In addition, while MGUS should not be associated with cytopenias, responses to anti-myeloma therapies have been observed in LGL with MGUS refractory to traditional immunosuppressive therapies. We hypothesized that the study of clinical and epidemiologic features of MGs and LGL may be helpful to elucidate some of these fundamental questions. To address this, a dual approach was applied: 1) MG-forward strategy: screening of LGL in 2348 individuals with documented diagnosis of MGs and available bone marrow exam; 2) LGL-forward strategy: search of MGs in 235 LGL cases with M protein assessment. Overall, among 2348 cases with MGs, we identified 42 cases (1.8%) with concurrent evidence of LGL. We then selected an exploratory cohort of 787 MG patients with clinical annotations for further phenotypic dissection and comparative analyses. Within 533 patients with non-active MGs (i.e., MGUS or smoldering myeloma -SM), 144 (27%) had cytopenias of any type and grade, which could not be attributable to plasma cell infiltration. Screening for comorbid conditions explaining cytopenias left 32 cases (22%) presumed to be idiopathic. LGL was overrepresented in MGUS-SM with cytopenias (6 vs 0.3% in cases without counts alterations, p< .001). This frequency was higher when considering only cases with idiopathic cytopenias (22 vs 1.8% in cases with cytopenias of other causes, p< .001) ( FigA). Reverse analysis of the LGL cohort demonstrated that 71 (30%) LGL cases had M proteins. Overall, the MG/LGL dyad was more frequently associated with concurrent autoimmune conditions (54%), as well as monoclonal B-cell lymphocytosis and other B-cell lymphoid neoplasms (19%). Next, we compared cases with MG/LGL vs those with MGs (N = 775) or LGL (N = 157) exclusively. When compared to the MGs cohort, MG/LGL cases presented a younger age (median 64 vs 70 years, p < .001), had higher frequency of M protein of the IgM type (21 vs 10%, p= .002), and oftentimes exhibited small/transient paraproteinemia (42 vs 2%, p< .001). No specific features were enriched in the MG/LGL cases when compared to the LGL cohort, except for a higher proportion of proliferations of NK-cell subtype (12 vs 4%, p= .02). Finally, to confirm the observed association, we focused on the subgroup of non-active MGs by excluding cases with initial diagnosis of MM or lymphoma. Multivariable analysis (adjusting by sex, age and MG risk) revealed a lower risk of immunoparesis (aOR: 0.4, 95%CI: 0.2 - 0.7, p= .002) in MG/LGL as opposed to MG without LGL, whereas no effect was found in progression to MM/lymphoma (aHR: 0.9, 95%CI: 0.4 - 2.1, p= .81 in a competitive-risk setting). Moreover, when opposed to its MG and LGL counterparts, MG/LGL more frequently presented with severe cytopenias (aOR vsMGs: 28.9, 95%CI: 14.0 - 59.0, p< .001; aOR vsLGL: 2.7, 95%CI: 1.7 - 5.6, p= .008), and with immune-mediated cytopenias (aOR vsMGs: 10.5, 95%CI: 4.9 - 22.2, p< .001; aOR vsLGL: 2.8, 95%CI: 1.0 - 4.8, p= .04) ( FigB). In conclusion, our study reveals that: 1) the association between MGUS and LGL is more than an age-related epidemiological coincidence; 2) distinctive nosologic features for the MG/LGL dyad; and 3) that even “non-active” MGs associated with LGL might be of hematological clinical significance through humoral or cellular immune mechanisms. These findings point towards alternative treatment strategies of cytopenias in MGUS and LGL (immunosuppression vs anti-myeloma therapies, respectively), and provide impetus for the investigation of tumor surveillance in MGUS vs common etiologic factors intertwining these clonopathies.

ERBB4-Mediated Signaling Is a Mediator of Resistance to PI3K and BTK Inhibitors in B-cell Lymphoid Neoplasms.

BTK and PI3K inhibitors are among the drugs approved for the treatment of patients with lymphoid neoplasms. Although active, their ability to lead to long-lasting complete remission is rather limited, especially in the lymphoma setting. This indicates that tumor cells often develop resistance to the drugs. We started from a marginal zone lymphoma cell line, Karpas-1718, kept under prolonged exposure to the PI3Kδ inhibitor idelalisib until acquisition of resistance, or with no drug. Cells underwent transcriptome, miRNA and methylation profiling, whole-exome sequencing, and pharmacologic screening, which led to the identification of the overexpression of ERBB4 and its ligands HBEGF and NRG2 in the resistant cells. Cellular and genetic experiments demonstrated the involvement of this axis in blocking the antitumor activity of various BTK/PI3K inhibitors, currently used in the clinical setting. Addition of recombinant HBEGF induced resistance to BTK/PI3K inhibitors in parental cells and in additional lymphoma models. Combination with the ERBB inhibitor lapatinib was beneficial in resistant cells and in other lymphoma models already expressing the identified resistance factors. An epigenetic reprogramming sustained the expression of the resistance-related factors, and pretreatment with demethylating agents or EZH2 inhibitors overcame the resistance. Resistance factors were also shown to be expressed in clinical specimens. In conclusion, we showed that the overexpression of ERBB4 and its ligands represents a novel mechanism of resistance for lymphoma cells to bypass the antitumor activity of BTK and PI3K inhibitors and that targeted pharmacologic interventions can restore sensitivity to the small molecules.

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Combining cell-free RNA with cell-free DNA in liquid biopsy for hematologic and solid tumors

Current use of liquid biopsy is based on cell-free DNA (cfDNA) and the evaluation of mutations or methylation pattern. However, expressed RNA can capture mutations, changes in expression levels due to methylation, and provide information on cell of origin, growth, and proliferation status. We developed an approach to isolate cell-free total nucleic acid (cfDNA) and used targeted next generation sequencing to sequence cell-free RNA (cfRNA) and cfDNA as new approach in liquid biopsy. We demonstrate that cfRNA is overall more sensitive than cfDNA in detecting mutations. We show that cfRNA is reliable in detecting fusion genes and cfDNA is reliable in detecting chromosomal gains and losses. cfRNA levels of various solid tumor biomarkers were significantly higher (P < 0.0001) in samples from solid tumors as compared with normal control. Similarly, cfRNA lymphoid markers and cfRNA myeloid markers were all higher in lymphoid and myeloid neoplasms, respectively as compared with control (P < 0.0001). Using machine learning we demonstrate cfRNA was highly predictive of diagnosis (AUC >0.98) of solid tumors, B-cell lymphoid neoplasms, T-cell lymphoid neoplasms, and myeloid neoplasms. In evaluating the host immune system, cfRNA CD4:CD8B and CD3D:CD19 ratios in normal controls were as expected (median: 5.92 and 6.87, respectively) and were significantly lower in solid tumors (P < 0.0002). This data suggests that liquid biopsy combining analysis of cfRNA with cfDNA is practical and may provide helpful information in predicting genomic abnormalities, diagnosis of neoplasms and evaluating both the tumor biology and the host response.

Combined Reverse-Transcriptase Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing Analyses to Assign Unclassified BCL2−/BCL6− Nonrearranged Small B-Cell Lymphoid Neoplasms as Follicular or Nodal Marginal Zone Lymphoma

Distinguishing between follicular lymphoma (FL) and nodal marginal zone lymphoma (NMZL) can be difficult when morphologic and phenotypic features are unusual and characteristic cytogenetic rearrangements are absent. We evaluated the diagnostic contribution of ancillary techniques—including fluorescence in situ hybridization (FISH)-detected 1p36 deletion; reverse-transcriptase, multiplex, ligation-dependent probe amplification (RT-MLPA); and next-generation sequencing (NGS)—for tumors that remain unclassified according to standard criteria. After review, 50 CD5-negative small B-cell lymphoid neoplasms without BCL2 and BCL6 FISH rearrangements were diagnosed as FLs (n = 27), NMZLs (n = 5), or unclassified (n = 18) based on the 2016 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. FISH helped identify the 1p36 deletion in 3 FLs and 1 unclassified tumor. Most classified FLs had an RT-MLPA germinal center B-cell (GCB) signature (93%) or were noncontributive (7%). Classified NMZLs had an RT-MLPA activated B-cell signature (20%), had an unassigned signature (40%), or were noncontributive (40%). Among unclassified tumors, the RT-MLPA GCB signature was associated with mutations most commonly found in FLs (CREBBP, EZH2, STAT6, and/or TNFRSF14) (90%). An RT-MLPA-detected GCB signature and/or NGS-detected gene mutations were considered as FL identifiers for 13 tumors. An activated B-cell signature or NOTCH2 mutation supported NMZL diagnosis in 3 tumors. Combining the RT-MLPA and NGS findings successfully discriminated 89% of unclassified tumors in favor of one or the other diagnosis. NGS-detected mutations may be of therapeutic interest. Herein, we detected 3 EZH2 and 8 CREBBP mutations that might be eligible for targeted therapies.

B-CELL LYMPHOID NEOPLASMS IN THE 5TH EDITION OF THE WHO CLASSIFICATION OF HEMATOLYMPHOID TUMORS (2022). 1. General principles of classification. Precursor B-cell neoplasms

Tumors of lymphoid and hematopoietic tissues are among the most prevalent malignancies. The tumors originating from lymphoid cells are of the utmost heterogeneity. The development of the generally accepted classifi ation sys- tem for this group of the malignancies became a priority task and its clinical implementation has been already advantageous. The in-depth insight into the pathogenesis of lymphoid neoplasms requires the continuous revision of the classification of these diseases. In 2022, World Health Organization specialists prepared the regular 5th issue of the Classification of Hematolymphoid Tumors representing a revision of the previous one (2017). The basic provisions of the new classification concerning B-cell lymphoid neoplasms are disclosed in this review composed of two parts. The review focuses on the principal changes as compared with the previous version of the classification. The first part of the review relates to two major categories such as tumor-like lesions with B-cell predominance and precursor B-cell neoplasms. The laboratory criteria important for the final diagnosis of the various nosological forms are discussed. Mature B-cell neoplasms, plasma cell neoplasms and other diseases with paraproteins will be presented in the next issue. the classification of these diseases. In 2022, World Health Organization specialists prepared the regular 5th issue of the Classification of Hematolymphoid Tumors representing a revision of the previous one (2017). The basic provisions of the new classification concerning B-cell lymphoid neoplasms are disclosed in this review composed of two parts. The review focuses on the principal changes as compared with the previous version of the classification. The first part of the review relates to two major categories such as tumor-like lesions with B-cell predominance and precursor B-cell neoplasms. The laboratory criteria important for the final diagnosis of the various nosological forms are discussed. Mature B-cell neoplasms, plasma cell neoplasms and other diseases with paraproteins will be presented in the next issue.

A rare case of mantle cell lymphoma of nasopharynx and larynx with synchronous presentation treated with volumetric modulated arc technique radiotherapy

Mantle cell lymphoma (MCL) or mature peripheral B-cell lymphoid neoplasm is a type of non-Hodgkin's lymphoma. It is more common in elderly males. It is very rare, accounting for less than 1% of head and neck malignancies. MCL is usually positive for CD20, CD5, CD43, cyclin D1 and negative for CD10, CD23, and BCL6. It is a very aggressive neoplasm, and hence, patients often present in advanced stage. Due to its rare incidence and short clinical course, there are limited data on standard treatment protocols. Even with the present treatment protocols, only 30% of patients achieve complete response. Here, we present a case of synchronous presentation of MCL of the nasopharynx and larynx in a 69-year-old male. We aim to discuss the investigations and treatment done with volumetric modulated arc technique radiotherapy that led to complete response.

Co-Transduced CD19/BCMA Dual-Targeting CAR-T Cells for the Treatment of Non-Hodgkin Lymphoma

CAR-T cells targeting CD19 have accomplished impressive responses in relapsed/refractory non-hodgkin lymphoma (NHL) patients, but disease recurrence is a concerning hurdle to overcome. This complication is mainly due to a selective pressure on tumor cells expressing CD19, which leads to loss of expression or downregulation to levels that cannot be detected by CAR-T cells. Simultaneous targeting of more than one antigen has been proposed as a compelling strategy to prevent tumor escape. CD19 is expressed along B-cell differentiation, and BCMA is expressed not only on plasmatic cells but also on mature B-cells. Thus, most NHL subtypes express both CD19 and BCMA. Here, we hypothesized that dual CD19 and BCMA targeting could reduce tumor antigen escape and enhance the antitumor efficacy of CAR-T cells. CAR constructs targeting CD19 (ARI0001) or BCMA (ARI0002h) were engineered in our institution and both have demonstrated safety and efficacy for treating patients with B-cell lymphoid neoplasms and multiple myeloma, respectively. Both CARs contain the 41BB co-stimulatory domain in tandem with the CD3z. We tested the antitumoral efficacy of three different dual-targeting CD19/BCMA strategies: 1) a bicistronic vector encoding both CAR constructs separated by a T2A cleavage peptide (ARI0001-T2A-ARI0002h); 2) co-transduced T cells with ARI0001 and ARI0002h lentiviral vectors to obtain a product with three CAR populations comprising both single-CARs and a double positive population; 3) pooled ARI0001 and ARI0002. Conventional functional assays such as cytokine secretion and specific lysis after short-term co-culture of CAR-T cells with NHL cell lines co-expressing CD19 and BCMA at constitutive levels revealed that all strategies were active but, did not discriminate between single- and dual-CARs groups. In order to mimic a potential relapse due to CD19 downregulation, we developed a CD19low density cell line of Burkitt lymphoma through its expression under a weak promoter. After 7-days of co-culture of CAR-T cells with RAMOS-CD19Low, only the co-transduction product maintained tumor elimination. Next, we analyzed the capacity of single versus dual CAR-T cells to kill malignant B cells and release cytokines in co-culture with NHL Patient Derived Lymphoma Spheroids. We tested samples from patients with 1) follicular lymphoma, 2) diffuse large B cell lymphoma (DLBCL), and 3) DLBCL patients who relapsed or had refractory disease post-CD19 CAR-T cell treatment. Under these more challenging conditions, differences among effector groups were maximized. ARI0001 and bicistronic CAR-T cells displayed similar effector functions in terms of killing capacity and cytokine production, especially when CD19 was expressed at high densities. ARI0002h presented antitumor efficacy but not as high as ARI0001. Co-transduction and pool products showed increased cytokine production and killing, being capable of B-cell elimination in all cases. A high disease burden of Burkitt lymphoma in vivo model revealed that co-transduction product significantly increased mice survival compared to ARI0001. This increase in survival was even more remarkable when the model was CD19low. No significant differences were observed between the co-transduction and pool groups. In summary, we propose a relatively simple preparation of a dual-CAR-T cells targeting CD19 and BCMA, which is particularly effective against NHL cell lines expressing low CD19 densities. Co-transduction is advantageous with respect to the pool since it only requires one manufacturing process, decreasing the cost of the therapy and increasing patients' access to these novel therapies. Our studies provide a strong rational to test CD19/BCMA dual-CAR-T cells in clinical trials for the treatment of NHL patients.

Cell-free RNA in liquid biopsy and biomarkers profiling of hematologic and solid tumors.

3047 Background: Expressed RNA can capture mutations, changes in expression levels due to methylation, and provide information on cell of origin, growth, and proliferation status. We developed an approach to isolate fragmented RNA from peripheral blood plasma and explored its potential to be used in liquid biopsy. Methods: Peripheral blood cfRNA was extracted from patients with neoplasms in B-cell (#105), T-cell (#16), Myeloid (#73), and from solid tumors (#44), Normal individuals (#51), and reactive post-transplant (#137). RNA was sequenced using a 1459-gene panel. Expression profile was generated using Cufflinks. Results: cfRNA levels of various solid tumor biomarkers (CA-125, CA-15-3, CEA 8, Keratin19, Keratin6A...) were significantly higher (P &lt; 0.0001) in samples from solid tumors as compared with normal control. Similarly, cfRNA lymphoid markers (CD19, CD22, CD79A, and CD79B...) and cfRNA myeloid markers (CD33, CD14, CD117, CD56...) were all higher in B-cell lymphoid neoplasms and myeloid neoplasms, respectively (P &lt; 0.0001), as compared with control. In evaluating the host immune system, cfRNA CD4:CD8B and CD3D:CD19 ratios in normal controls were as expected (median: 5.92 and 6.87, respectively) and were significantly lower in solid tumors (median 3.40 and 2.23, respectively, P &lt; 0.0002). Solid tumor cfRNA showed CTLA4:CD8B ratio significantly higher in tumors than in normal (median 0.74 vs 0.19, P = 0.0001), while there was no difference in cfRNA PD-L1:CD8B ratio (median 1.45 vs 1.77, P = 0.96). Similar distinct patterns are noted for various cytokine and chemokines. cfRNA was highly predictive of diagnosis (AUC &gt; 0.98) of solid tumors, B-cell lymphoid neoplasms, T-cell lymphoid neoplasms, and myeloid neoplasms as compared with normal control. When a specific neoplastic disease was considered against all cases including control and other neoplasms, the AUC varied between 0.77 and 0.949. Conclusions: This data shows that liquid biopsy using targeted sequencing of cfRNA in patients with various types of cancer provides comprehensive and reliable information on the neoplastic disease as well as the host. [Table: see text]

Combining cell-free RNA (cfRNA) with cell-free total nucleic acid (cfTNA) as a new paradigm for liquid biopsy.

3048 Background: Expressed RNA can capture mutations, gene fusions, and biomarker profiles. In principle, each abnormal cell has one copy of mutated gene, but numerous copies of mutated RNA. Cell-free RNA (cfRNA) is not used due to the assumption that it is degraded. Next Generation Sequencing (NGS) by design is particularly adaptable for fragmented DNA and RNA. We developed an approach to isolate cell-free total nucleic acid (cfTNA) and cell-free RNA (cfRNA) from peripheral blood. Using targeted sequencing, we explored the potential of this approach to detect mutations, fusion mRNA, and copy number variation (CNV) in solid tumors and hematologic neoplasms. Methods: Peripheral blood cfTNA and cfRNA were extracted from B-cell lymphoid neoplasms (#105), T-cell neoplasms (#16), Myeloid neoplasms (#73), solid tumors (#44), and Normal individuals (#51), and sequenced using a targeted panel of 1459 genes. Results: Numbers of mutations detected in solid tumors and hematologic neoplasms were significantly (P &gt; 0.0001) higher in cfRNA (No. = 1229) than in cfTNA (No. = 1004). Overall variant allele frequency (VAF) was significantly higher in cfRNA than in cfTNA (P &lt; 0.0001). However, numerous mutations detected by RNA were not detected by cfTNA and vice versa. In general, nonsense mutations were more likely to be detected by cfTNA than by cfRNA and at higher VAF. Low-level mutations (VAF &lt; 10%) were more likely to be detected by cfRNA than by cfTNA. For example, 136 mutations in TP53 gene were detected using cfRNA and only 70 mutations were detected in cfTNA. KRAS mutations were also higher in cfRNA (#33) as compared with cfTNA (#21). In contrast, when most of the mutations were nonsense, as in ASXL1 gene, more mutations were detected by cfTNA (24 vs 23). When mutations were detected in both cfRNA and cfTNA, mutation load (level of mutant copies) was overall slightly higher in cfTNA (P = 0.06), likely due to higher degradation of RNA, but varied significantly dependent on the type of mutated gene and type of mutation. cfRNA was reliable in detecting fusion transcripts in solid tumors and in hematologic neoplasms (SLC34A2-ROS1, DDX5-BCL6, ETV6-RUNX1, RUNX1T1-RUNX1, PML-RARA, RUNX1-ZFPM2, DEK-NUP214, EP300-ZNF384) irrespective of the breakpoint or partner gene. The cfTNA detected various CNVs expected by cytogenetic analysis when tumor fraction was adequate (VAF &gt; 10%). Conclusions: This data demonstrates that using cfRNA and cfTNA provides complementary comprehensive information for evaluating mutations, fusion genes, and CNV. This approach increased sensitivity and reliability of liquid biopsy. Furthermore, the cfRNA provides critical information on relative expression of various genes that can be used as biomarkers in characterizing the neoplastic process (see ASCO abstract, Liquid Biopsy Based on Cell-Free RNA and Biomarkers profiling of hematologic and solid tumors).

A novel diagnostic approach for the classification of small B-cell lymphoid neoplasms based on the NanoString platform

Small B-cell lymphoid neoplasms (SBCLNs) are a heterogeneous group of diseases characterized by malignant clonal proliferation of mature B-cells. However, the classification of SBCLNs remains a challenge, especially in cases where histopathological analysis is unavailable or those with atypical laboratory findings or equivocal pathologic data. In this study, gene expression profiling of 1039 samples from 27 gene expression omnibus (GEO) datasets was first investigated to select highly and differentially expressed genes among SBCLNs. Samples from 57 SBCLN cases and 102 nonmalignant control samples were used to train a classifier using the NanoString platform. The classifier was built by employing a cascade binary classification method based on the random forest algorithm with 35 refined gene signatures. Cases were successively classified as chronic lymphocytic leukemia/small lymphocytic lymphoma, conventional mantle cell lymphoma, follicular lymphoma, leukemic non-nodal mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia, and other undetermined. The classifier algorithm was then validated using an independent cohort of 197 patients with SBCLNs. Under the distribution of our validation cohort, the overall sensitivity and specificity of proposed algorithm model were >95%, respectively, for all the cases with tumor cell content greater than 0.72. Combined with additional genetic aberrations including IGH-BCL2 translocation, MYD88 L265P mutation, and BRAF V600E mutation, the optimal sensitivity and specificity were respectively found at 0.88 and 0.98. In conclusion, the established algorithm demonstrated to be an effective and valuable ancillary diagnostic approach for the sub-classification and pathologic investigation of SBCLN in daily practice.

Resistance to PI3Kδ inhibitors in marginal zone lymphoma can be reverted by targeting the IL-6/PDGFRA axis.

PI3Kδ inhibitors are active in patients with lymphoid neoplasms and a first series of them have been approved for the treatment of multiple types of B-cell lymphoid tumors, including marginal zone lymphoma (MZL). The identification of the mechanisms underlying either primary or secondary resistance is fundamental to optimize the use of novel drugs. Here we present a model of secondary resistance to PI3Kδ inhibitors obtained by prolonged exposure of a splenic MZL cell line to idelalisib. The VL51 cell line was kept under continuous exposure to idelalisib. The study included detailed characterization of the model, pharmacological screens, silencing experiments, and validation experiments on multiple cell lines and on clinical specimens. VL51 developed resistance to idelalisib, copanlisib, duvelisib, and umbralisib. An integrative analysis of transcriptome and methylation data highlighted an enrichment of upregulated transcripts and low-methylated promoters in resistant cells, including IL-6/STAT3- and PDGFRA-related genes and surface CD19 expression, alongside the repression of the let-7 family of miRNA, and miR-125, miR-130, miR-193 and miR-20. The IL-6R blocking antibody tocilizumab, the STAT3 inhibitor stattic, the LIN28 inhibitor LIN1632, the PDGFR inhibitor masitinib and the anti-CD19 antibody drug conjugate loncastuximab tesirine were active compounds in the resistant cells as single agents and/or in combination with PI3Kδ inhibition. Findings were validated on additional in vitro lymphoma models and on clinical specimens. A novel model of resistance obtained from splenic MZL allowed the identification of therapeutic approaches able to improve the antitumor activity of PI3Kδ inhibitors in B-cell lymphoid tumors.

CD200 as a Valuable Marker for Differentiating between CLL and Other B-cell Lymph Proliferative Disorders

Chronic lymphocytic leukaemia (CLL) being the commonest subtype of mature B-cell lymphoid neoplasms need to be differentiated correctly from other B-chronic lymphoproliferative disorders (BCLPD). CD200 is being an essential marker for prognosis and diagnosis of CLL. Sixty patients diagnosed with BCLPD were evaluated for CD200 expression with eight-colour flow cytometry. An integration of modified Matutes score and CD200 were applied for CLL diagnosis. The overall immunophenotype of CLL patients in this study showed significant, frequent expression of CD5, CD22, CD23, CD43 and CD200. Atypical versus typical CLL patients’ phenotype comparison revealed significant lower expression of CD5 and CD43 with higher expression of CD79b in atypical CLL patients. CD200 was expressed significantly higher in CLL patients (typical and atypical) than in MCL patients who showed dim CD200 expression in only twenty-five percent of patients. The inclusion of CD200 within the diagnostic markers can empower the modified Matutes score accuracy for CLL.

Spectrum of B-cell neoplasms associated with immunoglobulin G4-related disease.

Immunoglobulin G4-related disease (IgG4-RD) has rarely been associated with lymphoid neoplasms, the spectrum of which remains unclear. B-cell lymphoid neoplasms (LN) associated with IgG4-RD diagnosed in a 4-year period were analysed. There were five men and three women at a median age of 76.5 (52-90) years; three with synchronous IgG4-RD and LN; three with IgG4-RD preceding LN by 2, 3, and 22years; and two with LN preceding IgG4-RD by 2.5 and 7years. All patients presented with disseminated lymphadenopathy. Monoclonal gammopathy of undetermined significance (MGUS)/smouldering multiple myeloma (SMM) was found in three patients, all with an IgGκ paraprotein. Levels of IgGκ and IgG4 correlated. Diffuse large B-cell lymphoma (DLBCL) was found in three patients, with one case showing co-existing lymphoma and IgG4-RD in the same lymph node biopsy. The remaining two cases were marginal zone lymphoma (MZL) developing in a lacrimal gland previously involved by IgG4-RD; and nodular lymphocyte predominant Hodgkin lymphoma (NLP-HL) diagnosed in a lymph node with concomitant IgG4-RD. Low-dose continuous prednisolone was given for MGUS/SMM, with both monoclonal IgGκ and IgG4 responding. Combination chemotherapy was given for DLBCL, with two patients achieving complete response and one patient dying from refractory lymphoma. The patient with MZL refused treatment, whereas the case of NLP-HL responded completely to chemotherapy. Our findings together with previous observations suggest that IgG4-RD has an increased risk of B-cell neoplasms. Patients with IgG4-RD presenting with lymphadenopathy require vigorous investigations to exclude lymphoid neoplasms.

CLL-393: Knockdown of WEE1 Gene Induces Cytotoxic Effects in Hairy Cell Leukemia

Background: Hairy cell leukemia (HCL) is a rare B-cell lymphoid neoplasm with morphological variations that can be difficult to distinguish. A BRAFV600E mutation is found in 80% of patients. Despite the fact that purine analogs (PNA) with or without anti-CD20 antibodies remain the first-line treatment, resistance is still a major issue that has a severe impact on patient outcomes. The successful therapeutic application of short interfering RNA (siRNA) in tumor suppression is being paved by gene silencing. In the cell cycle, the WEE1 gene is the master inhibitor of cyclin-dependent kinase 1. Cumulative data suggests that the WEE1 gene plays a definite function in cell biological processes and that silencing the WEE1 gene could have a variety of implications in cancer cells. Objectives: The purpose of this study was to examine how ex vivo knockdown of the WEE1 gene affects the viability of hairy cell leukemia (HCL) cells in order to see if it has therapeutic potential. Methods: The HCL cell line [Mo T] was used in an experimental in vi’ro investigation. To silence the WEE1 gene ex vivo, cells were transfected using siRNA. Furthermore, the effect of gene knockdown on cell viability was assessed using the MTT test, as well as cell cycle and apoptosis assays performed by flow cytometry. Furthermore, the WEE1 gene expression level in silenced cells was measured and correlated with cellular protein expression. Results: Silencing the WEE1 gene resulted in a significant decrease in cell viability, increased cell apoptosis, and arrested the cell cycle transition from the G1 to the S phase of the cell cycle. In addition, transfected cells showed a significant reduction in WEE1 gene expression, demonstrating the efficiency of gene silencing. Conclusion: In primary hairy cells, silencing the WEE1 gene resulted in significant cytotoxic effects, suggesting that knockdown of the WEE1 gene could be a promising therapeutic target in HCL. Hairy cell leukemia (HCL) is a rare B-cell lymphoid neoplasm with morphological variations that can be difficult to distinguish. A BRAFV600E mutation is found in 80% of patients. Despite the fact that purine analogs (PNA) with or without anti-CD20 antibodies remain the first-line treatment, resistance is still a major issue that has a severe impact on patient outcomes. The successful therapeutic application of short interfering RNA (siRNA) in tumor suppression is being paved by gene silencing. In the cell cycle, the WEE1 gene is the master inhibitor of cyclin-dependent kinase 1. Cumulative data suggests that the WEE1 gene plays a definite function in cell biological processes and that silencing the WEE1 gene could have a variety of implications in cancer cells. The purpose of this study was to examine how ex vivo knockdown of the WEE1 gene affects the viability of hairy cell leukemia (HCL) cells in order to see if it has therapeutic potential. The HCL cell line [Mo T] was used in an experimental in vi’ro investigation. To silence the WEE1 gene ex vivo, cells were transfected using siRNA. Furthermore, the effect of gene knockdown on cell viability was assessed using the MTT test, as well as cell cycle and apoptosis assays performed by flow cytometry. Furthermore, the WEE1 gene expression level in silenced cells was measured and correlated with cellular protein expression. Silencing the WEE1 gene resulted in a significant decrease in cell viability, increased cell apoptosis, and arrested the cell cycle transition from the G1 to the S phase of the cell cycle. In addition, transfected cells showed a significant reduction in WEE1 gene expression, demonstrating the efficiency of gene silencing. In primary hairy cells, silencing the WEE1 gene resulted in significant cytotoxic effects, suggesting that knockdown of the WEE1 gene could be a promising therapeutic target in HCL.

Variable Distribution of DOCK-D Proteins between Cytosol and Nucleoplasm in Cell Lines, Effect of Interleukin-4 on DOCK10 in B-Cell Lymphoid Neoplasms, and Validation of a New DOCK10 Antiserum for Immunofluorescence Studies.

Dedicator-of-cytokinesis (DOCK), a family of guanine-nucleotide exchange factors (GEFs), comprises four subfamilies, named from A to D. DOCK-D comprises DOCK9, DOCK10, and DOCK11. The GEF activity involves translocation from the cytoplasm to the plasma membrane (PM), as assessed by the transfection of tagged proteins. However, the cellular localization of endogenous DOCK proteins is poorly understood. In this paper, to gain a better understanding of the role of the DOCK-D proteins, we studied their distribution between cytosol and nucleoplasm in 11 cell lines. DOCK-D proteins were distributed with variable cytosolic or nuclear predominance, although the latter was common for DOCK9 and DOCK11. These results suggest that the DOCK-D proteins may perform new nuclear functions, which remain to be discovered. Furthermore, we found that DOCK10 levels are increased by interleukin-4 (IL-4) in B-cell lymphoid neoplasms other than chronic lymphocytic leukemia (CLL) such as mantle cell lymphoma and diffuse large B-cell lymphoma. We also found evidence for an induction of the cytosolic levels of DOCK10 by IL-4 in CLL. Finally, we obtained a valid DOCK10 antiserum for immunofluorescence (IF) microscopy that, as an antibody against the hemagglutinin (HA) tag, marked PM ruffles and filopodia in HeLa cells with inducible expression of HA-DOCK10.

Waldenström's Macroglobulinemia: An Exploration into the Pathology and Diagnosis of a Complex B-Cell Malignancy.

After 77 years since the initial description, Waldenström macroglobulinemia (WM) remains as a bone marrow neoplastic disorder with lymphoplasmacytic differentiation oversecreting a monoclonal immunoglobulin M (IgM). However, many biological and genetic aspects of this entity have been unraveled and it is now easy to correctly diagnose patients with this illness. The diagnosis requires the presence of a monoclonal IgM component and bone marrow lymphoid infiltration must be demonstrated. In addition, other small B-cell lymphoid neoplasms with plasma cell differentiation must be discarded. Although the clinical picture is highly heterogeneous, the diagnosis is much easier today compared to the past, since now we can demonstrate the presence of somatic mutations, especially the L265P mutation in the MYD88 gene, highly characteristic of WM (>90% of the patients), followed by the WHIM-like mutations in the CXCR4 gene (~35%). The identification of these mutations is very important, because they can modulate the response to new treatments with Bruton’s tyrosine kinase (BTK) inhibitors. Thus, the conventional prognostic factors that predict the outcome of these patients (anemia, thrombopenia, high M component, high B2M, and advanced age), must be complemented with the genetic evaluation of the patient, that can help us in the prediction of the risk of transformation from asymptomatic to symptomatic forms (Del6q) and/or from indolent forms of the disease to aggressive lymphomas (CD79b mutations).

ENDOG Impacts on Tumor Cell Proliferation and Tumor Prognosis in the Context of PI3K/PTEN Pathway Status.

Simple SummaryThe PI3K/AKT pathway is involved in cell survival and proliferation. Molecular aberrations and/or hyperactivation of the PI3K-PTEN-AKT axis are frequent in distinct cancer types such as endometrial carcinoma, the most common type of cancer of the female genital tract, and chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm depending on B-cell receptor (BCR) activity, which induces chronical activation of this pathway. The mitochondrial nuclease ENDOG was found to influence PI3K/AKT activity in somatic cells. Our aim was to assess the value of ENDOG gene silencing to block cancer cell proliferation and to evaluate the relevance of ENDOG as prognostic marker. In vivo, in vitro and in silico experiments show that Endog/ENDOG silencing blunts proliferation of tumor cells dependent on high p-AKT/low PTEN activity and that ENDOG has prognostic value in specific cancer types.EndoG influences mitochondrial DNA replication and is involved in somatic cell proliferation. Here, we investigated the effect of ENDOG/Endog expression on proliferation in different tumor models. Noteworthy, ENDOG deficiency reduced proliferation of endometrial tumor cells expressing low PTEN/high p-AKT levels, and Endog deletion blunted the growth of PTEN-deficient 3D endometrial cultures. Furthermore, ENDOG silencing reduced proliferation of follicular thyroid carcinoma and glioblastoma cell lines with high p-AKT expression. High ENDOG expression was associated with a short time to treatment in a cohort of patients with chronic lymphocytic leukemia (CLL), a B-cell lymphoid neoplasm with activation of PI3K/AKT. This clinical impact was observed in the less aggressive CLL subtype with mutated IGHV in which high ENDOG and low PTEN levels were associated with worse outcome. In summary, our results show that reducing ENDOG expression hinders growth of some tumors characterized by low PTEN activity and high p-AKT expression and that ENDOG has prognostic value for some cancer types.

Distribution of Lymphomas in Mexico: A Descriptive Study

IntroductionLymphomas are a heterogeneous group of hematological malignancies of which incidence, prevalence, and epidemiological factors are still not well described in the Mexican population. Most of the recent descriptive studies do not specify details of their analysis and were not based on the WHO classification of tumours. In our study we analyzed 5772 lymphomas diagnosed over a period of 10 years between 2005 and 2014, and classified them according to the 2008 WHO Classification of Tumours of Lymphoid Tissues. The aim of our study is to determine frequencies and subtypes of the main non-Hodgkin's and Hodgkin's lymphomas in a heterogeneous sample of biopsies in the Mexican population.MethodsFiles for tissue samples for lymphomas stored in 5 different hospitals in Mexico City were retrieved for re-analysis. All cases where tissue samples were available were stained with hematoxylin and eosin as well as immunostained. Analysis was performed to differentiate the main mature B-cell, T-cell, Hodgkin's, and precursor lymphoid neoplasms according to the 2008 WHO classification of tumours. Age and sex were obtained and registered from the original submitted request for pathology assessment. All stains were performed in the same laboratory and interpreted by 3 pathologists who provided a diagnosis.Results5772 neoplasms were included in this study. Of these, 4213 were mature B-cell neoplasms (73%; 95%CI 71.83 - 74.12), 888 Hodgkin's lymphomas (HL) (15%; 95%CI 14.48 - 16.34), 496 mature T-cell / NK neoplasms (9%; 95%CI 7.89 - 9.34), and 175 precursor lymphoid neoplasms (3%; 95%CI 2.62 - 3.5). Neoplasms had an even distribution between sexes, with 51% of the cases being male patients.All 4 different categories of neoplasms were further analysed and classified. Six main mature B-cell lymphomas accounted for almost 95% of our cohort. These included diffuse large B-cell lymphoma (DLBCL) (56%; 95%CI 54.39 - 57.39), follicular lymphoma (FL) (20%; 95%CI 18.92 - 21.34), mantle cell lymphoma (6.7%; 95%CI 6.02 - 7.53), marginal zone/MALT lymphoma (5.2%; 95%CI 4.59 - 5.93), chronic lymphocytic leukemia/small lymphocytic lymphoma (4.6%; 95%CI 4.01 - 5.28), and Burkitt's lymphoma (2.5%; 95%CI 2.04 - 2.98). Hodgkin's lymphomas were also classified in 6 main subtypes, with nodular sclerosis (47%; 95%CI 44.14 - 50.7) and mixed cellularity (38%; 95%CI 34.49 - 40.85) being the most common. Several mature T-cell / NK neoplasms were identified, with the majority of cases being peripheral T-cell lymphoma NOS / anaplastic large cell lymphoma ALK negative (44%; 95%CI 39.85 - 48.84) and extranodal NK/T-cell lymphoma, nasal type (30%; 95%CI 25.5 - 33.76). Please refer to Table 1, 2, and 3 for further information.Analysis of cases by group age was also performed for mature B-cell neoplasms and HL. As expected, the majority of cases of DLBCL were seen in the age groups of 41-60 as well as 61-80. The same applied to FL. In the case of HL, the majority of nodular sclerosis subtypes were seen in the group age of 21-40 and mixed cellularity subtype showed a more even distribution in the age groups between 21-80.ConclusionTo our knowledge, this is the first descriptive study with a large sample of confirmed lymphoid neoplasms, using a standardized method of staining, classified according to the 2008 WHO Classification of Tumors representative of the Mexican population. It is a complement to the current epidemiologic registry of neoplasms in Mexico, adding classification details according to global standards set by the WHO. The results obtained are in keeping with the numbers described in other population studies worldwide. [Display omitted] DisclosuresLazo-Langner:Bayer: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Research Funding; Alexion: Research Funding.

Safety and Preliminary Antitumor Activity of the Anti-PD-1 Monoclonal Antibody REGN2810 Alone or in Combination with REGN1979, an Anti-CD20 x Anti-CD3 Bispecific Antibody, in Patients with B-Lymphoid Malignancies

Safety and Preliminary Antitumor Activity of the Anti-PD-1 Monoclonal Antibody REGN2810 Alone or in Combination with REGN1979, an Anti-CD20 x Anti-CD3 Bispecific Antibody, in Patients with B-Lymphoid Malignancies