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.

Round-robin testing for LMO2 and MYC as immunohistochemical markers to screen MYC rearrangements in aggressive large B-cell lymphoma.

Aggressive large B-cell lymphomas (aLBCL) include a heterogeneous group of lymphomas with diverse biological features. One of the approaches to the diagnosis of aLBCL is based on the identification of MYC rearrangements (MYC-R), in addition to BCL2 and BCL6 rearrangements by genetic techniques, mainly fluorescent in situ hybridization (FISH). Because of the low incidence of MYC-R, the identification of useful immunohistochemistry markers to select cases for MYC FISH testing may be useful in daily practice. In a previous work, we identified a strong association between the profile CD10 positive/LMO2 negative expression and the presence of MYC-R in aLBCL and obtained good intralaboratory reproducibility. In this study, we wanted to evaluate external reproducibility. To evaluate whether LMO2 can be a reproducible marker between observers 50 aLBCL cases were circulated among 7 hematopathologists of 5 hospitals. Fleiss' kappa index for LMO2 and MYC were 0.87 and 0.70, respectively, indicating high agreement between observers. In addition, during 2021-2022, the enrolled centers included LMO2 in their diagnostic panels to evaluate prospectively the utility of the marker, and 213 cases were analyzed. Comparing LMO2 with MYC, the group of CD10 positive cases showed higher specificity (86% vs 79%), positive predictive value (66% vs 58%), likelihood positive value (5.47 vs 3.78), and accuracy (83% vs 79%), whereas the negative predictive values remained similar (90% vs 91%). These findings place LMO2 as a useful and reproducible marker to screen MYC-R in aLBCL.

Pre- and Post-Neoadjuvant Clinicopathological Parameters Can Help in the Prognosis and the Prediction of Response in HER2+ and Triple Negative Breast Cancer.

Neoadjuvant treatment (NAT) is one of the most widely used options for HER2+ and triple negative (TN) early breast cancer (BC). Since around half of the patients treated with NAT do not achieve a pathologically complete response (pCR), biomarkers to predict resistance are urgently needed. The correlation of clinicopathological factors with pCR was studied in 150 patients (HER2 = 81; TN = 69) and pre- and post-NAT differences in tumour biomarkers were compared. Low estrogen receptor (ER) expression, high tumour-infiltrating lymphocytes (TILs) and low cT-stage were associated with pCR in HER2+ tumours (p = 0.022; p = 0.032 and p = 0.005, respectively). Furthermore, ER expression was also associated with residual cancer burden (RCB; p = 0.046) in the HER2+ subtype. Similarly, pre-NAT, low progesterone receptor expression (PR; 1-10%) was associated with higher RCB (p < 0.001) in TN tumours. Only clinical and pathological T-stage (cpT-stage) had prognostic capacity in HER2+ tumours, whereas pre-NAT cpT-stage and post-NAT TILs had this capacity for the prognosis of TN tumours. We conclude that ER and PR expression may help predict response to NAT in HER2 and TN BC and should be taken into account in residual tumours. Also, changes observed in the phenotype after NAT suggest the need to reevaluate biomarkers in surviving residual tumour cells.

Diffuse Large B-Cell Lymphoma in the HIV Setting.

Despite the widespread use of combined antiretroviral therapy (cART) and the subsequent decrease in AIDS-defining cancers, HIV-related lymphomas remain a leading cause of morbidity and mortality in people with HIV (PWH). Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) subtype in PWH. This lymphoma is a heterogeneous disease including morphological variants and molecular subtypes according to the cell of origin or the mutation profile. In the pre-cART era, treatment with standard-dose chemotherapy induced high rates of toxicity and outcomes were very poor. The introduction of cART and the incorporation of infection prophylaxis allowed the use of conventional intensive chemotherapy regimens used in the general population, such as R-CHOP or R-EPOCH. The use of cART during chemotherapy treatment was initially controversial due to the potential risk of adverse drug-drug interactions. However, the availability of current cART regimens with less potential to cause drug interactions and evidence that cART improves survival rates in NHL strongly support the use of cART in PWH with DLBCL. Consequently, interdisciplinary collaboration between HIV specialists and hemato-oncologists for the management of potential interactions and overlapping toxicities between antiretroviral and antineoplastic drugs is crucial for the optimal treatment of PWH with NHL.

HHV8 and EBV-negative primary effusion-based lymphoma: A case report of a new provisional entity and review of literature.

Key Clinical Message: HHV8- and EBV-negative primary effusion lymphoma is an extremely rare neoplasm involving body cavities without detectable tumor mass. It usually presents in elderly patients without known immunodeficiency. Compared to primary effusion lymphoma, it has a better prognosis.Primary effusion lymphoma (PEL) is a rare non-Hodgkin lymphoma confined exclusively to body cavities without detectable tumor masses. The term PEL-like is an entity similar to PEL in clinical presentation but without relation to human herpesvirus 8 (HHV8). We report a case of HHV8- and EBV-negative primary effusion-based lymphoma.

Macrophage CD5L is a target for cancer immunotherapy.

BACKGROUND: Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. METHODS: Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. FINDINGS: Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p = 0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages in vitro. Its administration in vivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. INTERPRETATION: CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. FUNDING: For a full list of funding bodies, please see the Acknowledgements.

Flow cytometry to detect bone marrow involvement by follicular lymphoma.

BACKGROUND: High-quality data on bone marrow involvement (BMI) assessed by flow cytometry (FC) in follicular lymphoma (FL) is lacking. AIMS: We set up a prospective protocol with a 10-color tube and acquisition of 500.000 leukocytes on a Nav flow cytometer for evaluation of BMI in FL by FC. MATERIALS AND METHODS: FC was compared with a combination of histopathology and IGH gene rearrangement, which were considered the gold standard. We also compared BMI by FC with PET. RESULTS: Fifty-two patients were included (median 67 years, 54% female). BMI by FC was seen in 35 (67%), with a median involvement of 1.2% (interquartile range: 0.3%-7%) of leukocytes. Comparison with the gold standard revealed two false negatives and two false positives (potentially true involvement undetected by the gold standard). BMI by PET was seen in 14/46 (30%). Immunophenotype of FL in the bone marrow was highly heterogeneous. The most common phenotypic abnormality was dim expression of CD19 (>0.5 log loss in 30% of patients). CD10 was negative in 13 (37%) and incompletely positive (overlap with the negative population) in a further 8 (28%) while entirely positive only in 14 (48%). Other abnormalities (loss of CD20, gain or loss of CD79b, expression of CD43, and substantial loss of CD45) were rare. Computational analysis by means of FlowSOM confirmed the heterogeneous phenotype, with FL from different patients clustering in unrelated metaclusters. CONCLUSION: BMI by FL was frequent and immunophenotype was heterogeneous. However, this protocol enabled detection of FL in bone marrow in the vast majority of patients with bone marrow involvement by the gold standard.

Genetic and phenotypic characterisation of HIV-associated aggressive B-cell non-Hodgkin lymphomas, which do not occur specifically in this population: diagnostic and prognostic implications.

The frequency of aggressive subtypes of B-cell non-Hodgkin lymphoma (B-NHL), such as high-grade B-cell lymphomas (HGBL) with MYC and BCL2 and/or BCL6 rearrangement (HGBL-DH/TH) or Burkitt-like lymphoma (BL) with 11q aberration, is not well known in the HIV setting. We aimed to characterise HIV-associated aggressive B-NHL according to the 2017 WHO criteria, and to identify genotypic and phenotypic features with prognostic impact. Seventy-five HIV-associated aggressive B-NHL were studied by immunohistochemistry (CD10, BCL2, BCL6, MUM1, MYC, and CD30), EBV-encoded RNAs (EBERs), and fluorescence in situ hybridisation (FISH) to evaluate the status of the MYC, BCL2, and BCL6 genes and chromosome 11q. The 2017 WHO classification criteria and the Hans algorithm, for the cell-of-origin classification of diffuse large B-cell lymphomas (DLBCL), were applied. In DLBCL cases, the frequencies of MYC and BCL6 rearrangements (14.9 and 27.7%, respectively) were similar to those described in HIV-negative patients, but BCL2 rearrangements were infrequent (4.3%). MYC expression was identified in 23.4% of DLBCL cases, and coexpression of MYC and BCL2 in 13.0%, which was associated with a worse prognosis. As for BL cases, the expression of MUM1 (30.4%) conferred a worse prognosis. Finally, the prevalence of HGBL-DH/TH and BL-like with 11q aberration are reported in the HIV setting. The phenotypic and genotypic characteristics of HIV-associated aggressive B-NHL are similar to those of the general population, except for the low frequency of BCL2 rearrangements in DLBCL. MYC and BCL2 coexpression in DLBCL, and MUM-1 expression in BL, have a negative prognostic impact on HIV-infected individuals.

BCL2 translocation in high grade B cell lymphoma (NOS, DH/TH) is associated with reduced progression free survival.

High Grade B Cell Lymphoma, NOS, and High Grade B Cell Lymphoma with Dual Hit or Triple Hit have been recently recategorized in the 2016 revision of the WHO classification of lymphoid neoplasms. In this study we have characterized the genetic, histopathological, and clinical features of a series of this type of lymphoid neoplasia (17 HGBCL NOS and 53 HGBCL DH/TH).HGBCL NOS showed better response to first line treatment than HGBCL with DH/TH but no significant differences in PFS or OS were found between the two categories. Survival analysis in the whole cohort of cases found that only the presence of BCL2 translocation was significantly associated with PFS. Other clinical features such as IPI, LDH or stage were equivalent in both categories. Furthermore, both high grade and DLBCL morphological patterns showed equivalent PFS and OS in this set of High grade BCL NOS/DH/TH.Key pointsBCL2 translocation in High Grade B Cell Lymphoma NOS and High Grade B Cell Lymphoma with DH/TH is associated with reduced progression free survival.Both high grade and DLBCL morphological patterns showed equivalent outcome regarding PFS and OS in HGBCL.

Clinical and Therapeutic Implications of Epstein-Barr Virus in HIV-Related Lymphomas.

The incidence of lymphomas is increased in people living with HIV (PLWH). Aggressive B-cell non-Hodgkin lymphomas (NHLs) are the most common and are considered an AIDS-defining cancer (ADC). Although Hodgkin lymphoma (HL) is not considered an ADC, its incidence is also increased in PLWH. Among all HIV-related lymphomas (HRL), the prevalence of Epstein-Barr virus (EBV) is high. It has been shown that EBV is involved in different lymphomagenic mechanisms mediated by some of its proteins, contributing to the development of different lymphoma subtypes. Additionally, cooperation between both HIV and EBV can lead to the proliferation of aberrant B-cells, thereby being an additional lymphomagenic mechanism in EBV-associated HRL. Despite the close relationship between EBV and HRL, the impact of EBV on clinical aspects has not been extensively studied. These lymphomas are treated with the same therapeutic regimens as the general population in combination with cART. Nevertheless, new therapeutic strategies targeting EBV are promising for these lymphomas. In this article, the different types of HRL are extensively reviewed, focusing on the influence of EBV on the epidemiology, pathogenesis, clinical presentation, and pathological characteristics of each lymphoma subtype. Moreover, novel therapies targeting EBV and future strategies to treat HRL harboring EBV are discussed.

Molecular and functional profiling identifies therapeutically targetable vulnerabilities in plasmablastic lymphoma.

Plasmablastic lymphoma (PBL) represents a rare and aggressive lymphoma subtype frequently associated with immunosuppression. Clinically, patients with PBL are characterized by poor outcome. The current understanding of the molecular pathogenesis is limited. A hallmark of PBL represents its plasmacytic differentiation with loss of B-cell markers and, in 60% of cases, its association with Epstein-Barr virus (EBV). Roughly 50% of PBLs harbor a MYC translocation. Here, we provide a comprehensive integrated genomic analysis using whole exome sequencing (WES) and genome-wide copy number determination in a large cohort of 96 primary PBL samples. We identify alterations activating the RAS-RAF, JAK-STAT, and NOTCH pathways as well as frequent high-level amplifications in MCL1 and IRF4. The functional impact of these alterations is assessed using an unbiased shRNA screen in a PBL model. These analyses identify the IRF4 and JAK-STAT pathways as promising molecular targets to improve outcome of PBL patients.

Hodgkin Lymphoma in People Living with HIV.

Despite widespread use of combined antiretroviral therapy (cART) and increased life expectancy in people living with HIV (PLWH), HIV-related lymphomas (HRL) remain a leading cause of cancer morbidity and mortality for PLWH, even in patients optimally treated with cART. While the incidence of aggressive forms of non-Hodgkin lymphoma decreased after the advent of cART, incidence of Hodgkin lymphoma (HL) has increased among PLWH in recent decades. The coinfection of Epstein-Barr virus plays a crucial role in the pathogenesis of HL in the HIV setting. Currently, PLWH with HRL, including HL, are treated similarly to HIV-negative patients and, importantly, the prognosis of HL in PLWH is approaching that of the general population. In this regard, effective cART during chemotherapy is strongly recommended since it has been shown to improve survival rates in all lymphoma subtypes, including HL. As a consequence, interdisciplinary collaboration between HIV specialists and hemato-oncologists for the management of potential drug-drug interactions and overlapping toxicities between antiretroviral and antineoplastic drugs is crucial for the optimal treatment of PLWH with HL. In this article the authors review and update the epidemiological, clinical and biological aspects of HL presenting in PLWH with special emphasis on advances in prognosis and the factors that have contributed to it.

Flow cytometric expression of CD71, CD81, CD44 and CD39 in B cell lymphoma.

Flow cytometry is a useful ancillary tool for the diagnosis of nodal B cell lymphomas. Well-established antigens have diagnostic limitations. This study aimed to assess the expression of CD71, CD81, CD44 and CD39 by flow cytometry in B cell lymphomas. Expression of these 4 antigens was queried in 185 samples with a diagnosis of a B cell lymphoma according to a histological examination of the lymph node and the World Health Organization (WHO) classification (follicular lymphoma [FL, n = 96], diffuse large B cell lymphoma/High grade B cell lymphoma [DLBCL/HGBH, n = 48], marginal zone lymphoma/lymphoplasmacytic lymphoma [MZL/LPL, n = 14], chronic lymphocytic leukemia/small lymphocytic lymphoma [CLL, n = 10], mantle cell lymphoma [MCL, n = 11], Burkitt lymphoma [BL, n = 4] and other [n = 2]). CD81 was bright and CD44 was dim in germinal center-derived malignancies, particularly aggressive lymphomas (BL and CD10-positive DLBCL/HGBL). CD81 was very dim in CLL. CD71 was bright in aggressive lymphomas (DLBCL/HGBL and BL). CD39 was bright in CD10-negative DLBCL. CD71 appeared valuable in the differential diagnosis between indolent and aggressive lymphomas, CD39 between CD10-negative DLBCL and MZL/LPL and CD81 between MCL and CLL. To conclude, we report the expression of CD71, CD81, CD44 and CD39 by FC in B cell lymphomas. Further studies will have to determine the value they add to specific FC panels.

Lack of expression of LMO2 clone SP51 identifies MYC rearrangements in aggressive large B-cell lymphomas.

MYC rearrangements (MYC-R) confer unfavorable prognosis to large B-cell lymphomas (LBCL). Because of the low incidence of such genetic alteration, surrogates to screen MYC-R may be useful in daily practice. Previous studies suggested that clone 1A9-1 of LMO2 loss may be a good predictor for the presence of MYC-R in LBCL. The present study examines the utility of LMO2 clone SP51. For this purpose, we have analyzed 20 Burkitt lymphomas and 325 LBCL. Among them, 245 cases were studied prospectively using whole tissue sections, and 100 retrospectively by tissue microarrays. The cohort of CD10-positive prospective cases achieved the best results. Lack of LMO2 SP51 expression predicted the presence of MYC-R with high specificity, accuracy, positive and negative predictive value (PPV/NPV), and positive and negative likelihood ratios (PLR/NLR). Compared with MYC protein expression, LMO2 SP51 obtained significantly higher specificity, accuracy, PPV, and PLR (94%, 91%, 85%, and 14.33 vs 73%, 77%, 56%, and 3.26, respectively), and similar NPV and NLR (92% and 0.22 vs 95% and 0.12). Compared with LMO2 clone 1A9-1, the sensitivity of LMO2 SP51 was lower (79% vs 89%). We conclude that LMO2 SP51 may be a useful marker to screen MYC-R in CD10-positive LBCL.

Dissemination of Mycobacterium tuberculosis is associated to a SIGLEC1 null variant that limits antigen exchange via trafficking extracellular vesicles.

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non-functional variant in SIGLEC1, which encodes the myeloid-cell receptor Siglec-1/CD169 implicated in HIV-1 cell-to-cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb-infected Siglec-1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec-1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec-1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination.