Malignant Lymphoid Tissue Research Articles

Spatially-resolved transcriptomics reveal macrophage heterogeneity and prognostic significance in diffuse large B-cell lymphoma

Macrophages are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL). Macrophage estimation by immunohistochemistry shows varying prognostic significance across studies in DLBCL, and does not provide a comprehensive analysis of macrophage subtypes. Here, using digital spatial profiling with whole transcriptome analysis of CD68+ cells, we characterize macrophages in distinct spatial niches of reactive lymphoid tissues (RLTs) and DLBCL. We reveal transcriptomic differences between macrophages within RLTs (light zone /dark zone, germinal center/ interfollicular), and between disease states (RLTs/ DLBCL), which we then use to generate six spatially-derived macrophage signatures (MacroSigs). We proceed to interrogate these MacroSigs in macrophage and DLBCL single-cell RNA-sequencing datasets, and in gene-expression data from multiple DLBCL cohorts. We show that specific MacroSigs are associated with cell-of-origin subtypes and overall survival in DLBCL. This study provides a spatially-resolved whole-transcriptome atlas of macrophages in reactive and malignant lymphoid tissues, showing biological and clinical significance.

Deep learning analysis of mid-infrared microscopic imaging data for the diagnosis and classification of human lymphomas.

The present study presents an alternative analytical workflow that combines mid-infrared (MIR) microscopic imaging and deep learning to diagnose human lymphoma and differentiate between small and large cell lymphoma. We could show that using a deep learning approach to analyze MIR hyperspectral data obtained from benign and malignant lymph node pathology results in high accuracy for correct classification, learning the distinct region of 3900 cm-1 to 850 cm-1 . The accuracy is above 95% for every pair of malignant lymphoid tissue and still above 90% for the distinction between benign and malignant lymphoid tissue for binary classification. These results demonstrate that a preliminary diagnosis and subtyping of human lymphoma could be streamlined by applying a deep learning approach to analyze MIR spectroscopic data. This article is protected by copyright. All rights reserved.

SPATIALLY‐RESOLVED TRANSCRIPTOMICS DEFINE CLINICALLY RELEVANT SUBSETS OF MACROPHAGES IN DIFFUSE LARGE B‐CELL LYMPHOMA

Introduction: Tumor-associated macrophages (TAMs) are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL) and are implicated in tumor progression and therapy resistance. Conventional immunohistochemistry-based studies have relied on the oversimplified M1/M2 classification of TAMs which does not fully capture the functional diversity of macrophage biology, thus potentially contributing to their inconsistent prognostic significance in DLBCL. Spatial whole-transcriptomic analysis allows in-depth investigation of specific cell types in different spatial regions within normal and malignant lymphoid tissues, enabling detailed macrophage phenotyping. Here, we employed spatial whole-transcriptomic analysis in the characterization of CD68+ cells of DLBCL and reactive lymphoid tissues (RLTs) to identify novel macrophage subsets with biological and clinical significance. Methods: Digital spatial profiling with whole-transcriptomic analysis of CD68+ cells was performed in 47 DLBCL and 17 RLTs, to define macrophage signatures (termed “MacroSigs”) of distinct lymphoid spatial niches and clinical scenarios. Eight independent DLBCL datasets (4594 patients) with complete transcriptomic and survival information were used for validation of these spatial-derived MacroSigs. Results: Digital spatial profiling revealed previously unrecognized transcriptomic differences between macrophages populating distinct spatial compartments in RLTs (light zone (LZ)/dark zone (DZ), germinal center (GC)/interfollicular (IF) regions), and in between disease states (RLTs and DLBCL with or without relapsed disease). This transcriptomic diversity of macrophages was categorized into eight MacroSigs. Spatial-derived MacroSigs associate with specific cell-of-origin (COO) subtypes of DLBCL, of particular interest being the IF-MacroSig enriched in the unclassified COO (p < 0.005, 6/8 datasets). MacroSigs of relapsed-DLBCL and DZ were prognostic for shorter overall survival in multiple datasets (p < 0.05 in 5/8 datasets; p < 0.05 in 8/8 datasets, respectively). Projection onto a macrophage single-cell RNA-sequencing atlas reveals the Non-relapse-DLBCL MacroSig to depict HES1/FOLR2-like macrophages, while relapse-DLBCL-MacroSig represents IL1B-like monocytes, with unique therapeutic vulnerabilities for each. The research was funded by: Work in ADJ’s laboratory is funded by a core grant from the Cancer Science Institute of Singapore, National University of Singapore through the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative. CT was supported by the Italian Foundation for Cancer Research (AIRC) Investigator Grant IG ID.22145; 5 × 1000 Grant ID.22759. Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Immunohistochemical expression of transcription factors PAX5, OCT2, BCL6 and transcription regulator P53 in Non-Hodgkin lymphomas: A diagnostic cross-sectional study

BackgroundNon-Hodgkin lymphoma represents a heterogeneous group of tumors that constitute the seventh most common malignancy. Immunohistochemistry plays a major role in the detection of specific cell receptors. Transcription factors are a heterogeneous group of genes that play a critical role in the commitment, differentiation, and proliferation of specific cell types. MethodsParaffin-embedded tissue sections of non-Hodgkin lymphoma cases were selected, classified, and evaluated before staining with immunohistochemical markers (PAX5, OCT2, BCL6, and P53). Expression of the aforementioned markers was compared with histological subtypes and grades of lymphoma cases. Means of expression were also compared among histological subtypes. ResultsA total of 55 cases of NHL including 26 cases of low-grade lymphomas and 29 cases of high-grade lymphomas were included in the study. DLBCL and FL were the most common subtypes of high-grade and low-grade lymphomas respectively. Both PAX5 and OCT2 were positive in 44 cases of NHL (80%) including all cases of B-cell lymphomas. BCL6 and P53 demonstrated positive expression in 29.1% and 67.3% respectively. Interestingly, we found a significant association between the histological subtypes and the aforementioned markers (P-value<0.05). DiscussionExpression of PAX5, OCT2, BCL, and P53 played a major role in the diagnosis and grading of non-Hodgkin lymphomas in our study. Both PAX5 and OCT2 provided more accuracy and specificity in the diagnosis of B-cell neoplasms compared to the classical B-cell markers. BCL6 expression reflected its role in germinal center formation in normal and malignant lymphoid tissues, and expression of P53 mirrored the accumulation of gene mutations in more aggressive lymphoma subtypes. ConclusionIn this manuscript, we aimed to present a unique study that highlights the immunohistochemical expression of all the aforementioned factors among various histological subtypes of non-Hodgkin lymphomas with disparities in histological aggressiveness, highlighting a promising diagnostic and prognostic panel for non-Hodgkin lymphomas.

Stem cell-derived CAR T cells traffic to HIV reservoirs in macaques.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5– donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell–mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.

A case of Barrett\u2019s esophageal cancer with gastric mucosa-associated lymphoma

BackgroundAlthough the first-line therapy for early-stage gastric mucosa-associated lymphoid tissue lymphoma is the eradication of Helicobacter pylori, the effect of eradication in Helicobacter pylori-negative cases is unclear. In this case report, we describe a surgical option for a case of Barrett’s esophageal cancer with concurrent gastric mucosa-associated lymphoid tissue lymphoma.Case presentationA 79-year-old man was admitted to our hospital with Barrett’s esophageal cancer and gastric mucosa-associated lymphoid tissue lymphoma. Initially, we performed endoscopic submucosal dissection for Barrett’s esophageal cancer. Since residual tumor was observed after the endoscopic submucosal dissection, we performed an esophagectomy with two-field lymph node dissection, which was followed by placement of a gastric conduit via the posterior mediastinal route. He was discharged 14 days after surgery. Although no additional treatment exists for mucosa-associated lymphoid tissue lymphoma, no recurrent disease has been detected to date.ConclusionAn option to use a portion of the stomach with low-grade malignant mucosa-associated lymphoid tissue lymphoma as a conduit after esophagectomy was suggested.

3D image analysis reveals differences of CD30 positive cells and network formation in reactive and malignant human lymphoid tissue (classical Hodgkin Lymphoma).

AimsThe examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues.Materials and resultsUsing immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL.ConclusionIn contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.

Treatment of primary gastric lymphoma

Stomach is the most common location of lymphoma in digestive tract, accounting for 50%-60% of gastrointestinal lymphomas. The most common histological types are low malignant mucosa-associated lymphoid tissue (MALT) lymphoma from non-Hodgkin lymphoma (NHL) and diffuse large B-cell and marginal zone B-cell lymphoma (DLBCL) from NHL. Chronic gastritis secondary to Helicobacter pylori(HP) infection has been considered a major predisposing factor for MALT lymphoma. At present, the most widely accepted initial therapy for localized disease is aimed at the eradication of HP using regimens combining antibiotics and proton-pump inhibitors. The irradiation has become the therapy of choice for patients with early stage MALT lymphoma without HP infection or with persistent lymphoma following antibiotic therapy. In all the patients with advanced disease, treatment options include chemotherapy and the use of monoclonal antibodies. Treatment of DLBCL in stomach is based on aggressive poly-chemotherapy that is usually combined with rituximab. The same guidelines followed for nodal aggressive lymphoma can also be applied to gastric lymphoma with aggressive histology as DLBCL. For localized stages (stages I( and II(), these guidelines suggest front-line therapy with 3 to 4 cycles of standard R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) followed by radiotherapy. Advanced stage patients (stage IIII() usually undergo only 6 to 8 cycles of R-CHOP in order to obtain a complete remission rate. Nowadays surgery is limited to rare cases and radiotherapy combined or not with chemotherapy represents an effective therapeutic option ensuring long-term, organ-salvage benefits mainly in aggressive histological types.

Investigation of optical coherence micro-elastography as a method to visualize micro-architecture in human axillary lymph nodes.

BackgroundEvaluation of lymph node involvement is an important factor in detecting metastasis and deciding whether to perform axillary lymph node dissection (ALND) in breast cancer surgery. As ALND is associated with potentially severe long term morbidity, the accuracy of lymph node assessment is imperative in avoiding unnecessary ALND. The mechanical properties of malignant lymph nodes are often distinct from those of normal nodes. A method to image the micro-scale mechanical properties of lymph nodes could, thus, provide diagnostic information to aid in the assessment of lymph node involvement in metastatic cancer. In this study, we scan axillary lymph nodes, freshly excised from breast cancer patients, with optical coherence micro-elastography (OCME), a method of imaging micro-scale mechanical strain, to assess its potential for the intraoperative assessment of lymph node involvement.MethodsTwenty-six fresh, unstained lymph nodes were imaged from 15 patients undergoing mastectomy or breast-conserving surgery with axillary clearance. Lymph node specimens were bisected to allow imaging of the internal face of each node. Co-located OCME and optical coherence tomography (OCT) scans were taken of each sample, and the results compared to standard post-operative hematoxylin-and-eosin-stained histology.ResultsThe optical backscattering signal provided by OCT alone may not provide reliable differentiation by inspection between benign and malignant lymphoid tissue. Alternatively, OCME highlights local changes in tissue strain that correspond to malignancy and are distinct from strain patterns in benign lymphoid tissue. The mechanical contrast provided by OCME complements the optical contrast provided by OCT and aids in the differentiation of malignant tumor from uninvolved lymphoid tissue.ConclusionThe combination of OCME and OCT images represents a promising method for the identification of malignant lymphoid tissue. This method shows potential to provide intraoperative assessment of lymph node involvement, thus, preventing unnecessary removal of uninvolved tissues and improving patient outcomes.

The relationship between RUVBL1 (Pontin, TIP49, NMP238) and BCL6 in benign and malignant human lymphoid tissues

The human BCL6 gene, which is involved in the pathogenesis of certain human lymphomas, encodes a transcriptional repressor that is needed for germinal center B cell development and T follicular helper cell differentiation. Our goal was to identify BCL6 target genes using a cell system in which BCL6 repressive effects are inhibited followed by subtractive hybridization, and we detected the RUVBL1 (Pontin, TIP49) gene as a potential target of BCL6 repression. Here we show that the BCL6 protein significantly represses RUVBL1 transcription (6.8-fold). Knockdown of endogenous BCL6 in a human B cell lymphoma line leads to significant upregulation of RUVBL1, and there is an inverse expression pattern between the BCL6 and RUVBL1 proteins in certain human lymphomas. RUVBL1 is part of the AAA+ superfamily and participates in multiple processes, including gene transcription regulation, chromatin remodeling, and DNA repair, which, if dysregulated, may promote lymphoma development. A further understanding of the relationship between RUVBL1 and BCL6 should improve our understanding of the pathogenesis of human lymphomas.

Increased IL-6 detection in adult and pediatric lymphoid tissue harboring Parvovirus B19

BackgroundParvovirus B19 (B19V) is a common pathogenic virus infecting humans. Previous studies have shown evidence of B19V infection in patients with non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma using ELISA and PCR on serum specimens. B19V nonstructural protein is known to alter the expression of cellular factors including interleukin-6 (IL-6), which can increase the risk for and worsen the prognosis of lymphomas. ObjectiveThe objective of this study was to detect B19V capsid protein and IL-6 expression in normal and malignant lymphoid tissue, as well as any correlation between the two. Study designIHCs for B19V capsid protein, IL-6, and B19V co-receptors P-antigen and α5β1 integrin were performed on a tissue array containing 70 duplicated pediatric and adult lymphoma tissues and 5 duplicated benign lymph node sections. Cases were identified as normal, B-cell NHL, diffuse large B-cell NHL, Hodgkin's lymphoma, extranodal NK/T cell lymphoma, anaplastic large cell lymphoma, or mantle cell lymphoma. IL-6 and B19V capsid staining were quantified using a positive pixel count algorithm, and P-antigen and α5β1 staining using a membrane quantification algorithm. ResultsB19V capsid protein was detected in both benign and malignant lymphoid tissue. The Spearman rank correlation coefficient analysis was performed to determine the relationship between the level of positivity for B19V and IL-6 staining, yielding an overall correlation coefficient of 0.679 (p-value<0.0001). ConclusionsOur results show a moderate correlation between the levels of positive B19V and IL-6 staining by IHC, indicating a possible role for B19V in the pathogenesis of lymphomas.

BCL7A protein expression in normal and malignant lymphoid tissues

BCL7A protein expression in normal and malignant lymphoid tissues

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.

Effect of inactivation of argininosuccinate synthetase on sensitivity of lymphomas to caspase-dependent apoptosis following treatment with arginine deiminase.

8093 Background: Arginine is a semi-essential amino acid, critical to the growth of human cancers. Downregulation of the enzyme argininosuccinate synthetase (ASS1), a key rate-limiting enzyme involved in arginine metabolism, results in an intrinsic dependence on extracellular arginine with arginine deprivation offering a potential treatment option for patients with arginine auxotrophic tumors. Several early phase clinical trials of the arginine lowering drug, pegylated arginine deiminase (ADI-PEG20), have demonstrated clinical efficacy in patients with solid tumors. Methods: We assessed ASS1 expression (ASS1 promoter methylation, ASS1 mRNA and protein levels) in normal and malignant lymphoid cell lines and tissues, and tested the effect of ADI-PEG20. Results: The ASS1 promoter is hypermethylated in lymphomas using the Illumina Infinium methylation platform and provided a discriminator between lymphoma (cell lines and primary tumor samples, n=20) and B cell controls (benign lymph nodes and lymphoblastoid cell lines, n=6). A good correlation was observed between ASS1 mRNA and protein levels in tumor (n=12) and normal control cell lines (n=2), with most malignant, but not normal, cell lines showing absence of ASS1 expression. Notably, ASS1 protein was absent in >99% of lymphoma cases (n=300), as assessed by immunohistochemical staining. The ASS1-negative t(14;18) follicular lymphoma cell line, Karpas-422, and the cutaneous T cell lymphoma cell line, MyLa, were sensitive to ADI-PEG 20 with activation of caspases 3 and 7, PARP cleavage and downregulation of mTOR signalling. In contrast, the normal lymphoblastoid cell lines, NcNC and HRC57 and the Ramos Burkitt's lymphoma cell line, expressed constitutive and inducible levels of ASS1 protein, and were resistant to the apoptotic effects of ADI-PEG20. Conclusions: ASS1 is epigenetically inactivated in human lymphomas resulting in a potential dependency on extracellular arginine for cell growth. ASS1-negative lymphomas are sensitive to arginine depletion suggesting that this approach is worth exploring as a potential novel therapeutic approach in the management of patients with lymphoma. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Polaris Pharmaceuticals, Inc. Polaris Pharmaceuticals, Inc.

Absence of chlamydial infection in Japanese patients with orbital adnexal MALT lymphoma

18523 Background: The extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a distinct B-cell lymphoma that develops in extranodal sites and usually has an indolent clinical course as a localized disease. Recent clinicopathological studies suggest a strong relation between MALT lymphoma and inflammatory diseases of the epithelium such as autoimmune diseases and infections (Sjogren’s syndrome, Hashimoto’s thyroiditis, Helicobacter pylori infection, and etc.), because chronic antigen stimulation has been causally linked to the development of B-cells lymphoproliferative diseases. MALT lymphoma grows in extranodal lymphoid tissue and exists in epithelium tissues, and it is often difficult to distinguish malignant lymphoid tissue from benign inflammation. This is one cause since recent positive or negative correlations between ocular adnexal MALT lymphomas and chlamydial infections have been reported from different researchers, indicate several possible explanations. Methods: Twenty-three cases of ocular adnexal MALT lymphoma in southern regions of Japan, whose monoclonality was confirmed by immunoglobulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors using ELISA analysis to detect antibodies and Southern blot analysis followed by specific PCR gene amplification. Results: Our series of patients did not show any serological relations to Epstein-Barr virus, Hepatitis C virus, and Chlamydophila psittaci. Two cases had positive serum antibodies for autoimmunity, and another two cases had positive antibodies against Chlamydia trachomatis. None of our cases showed the presence of chlamydial 16S rRNA and 16S-23S spacer rRNA genes (C. psittac, C trachomatis, C. pneumoniae, and C. felis) after Southern blot analysis followed by PCR amplification. Conclusions: Inflammatory agents are still unknown in our series of orbital adnexal MALT lymphomas, and some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan. No significant financial relationships to disclose.

Absence of Chlamydial Infection in Japanese Patients with Ocular Adnexal MALT Lymphoma.

The extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a distinct type of B-cell lymphoma that develops in extranodal sites and usually has an indolent clinical course as a localized disease. Recent clinicopathological studies suggest a strong relationship between MALT lymphoma and inflammatory diseases of the epithelium such as autoimmune diseases and infections (Sjogren's syndrome, Hashimoto's thyroiditis, Helicobacter pylori infection, and etc.), because chronic antigen stimulation has been causally linked to the development of lymphoproliferative diseases in B cells. MALT lymphoma grows in extranodal lymphoid tissue and exists in epithelium tissues, and it is often difficult to distinguish malignant lymphoid tissue from benign inflammation. However, this is only one of the possible causes; recent positive and negative correlations between ocular adnexal MALT lymphomas and chlamydial infections reported by different researchers indicate several different possible explanations. Here, we analyze twenty-three cases of ocular adnexal MALT lymphoma whose monoclonality was confirmed by immunoglobulin heavy chain gene rearrangement and/or the analysis of cell surface antigens for evidence of expected causative factors, using both ELISA analysis to detect antibodies and specific PCR gene amplification followed by Southern blot analysis. Our series of patients did not show any serological relations to the Epstein-Barr virus, the Hepatitis C virus, or to Chlamydophila psittaci. Two cases had positive serum antibodies for autoimmunity, another two cases had positive antibodies against Chlamydia trachomatis. None of our cases showed the presence of chlamydial 16S rRNA or 16S-23S spacer rRNA genes (C. psittac, C trachomatis, C. pneumoniae, and C. felis) after PCR amplification. These results indicate that the inflammatory agents are still unknown in our series of ocular adnexal MALT lymphomas, and that some types of chlamydial infections are not associated with ocular adnexal MALT lymphoma in southern regions of Japan.

MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by α4β1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration

AbstractB-cell chronic lymphocytic leukemia (B-CLL) progression is determined by malignant cell extravasation and lymphoid tissue infiltration. We have studied the role and regulation of matrix metalloproteinase-9 (MMP-9) in B-CLL cell migration and invasion. Adhesion of B-CLL cells to the fibronectin fragment FN-H89, VCAM-1, or TNF-α–activated human umbilical vein endothelial cells (HUVECs) up-regulated MMP-9 production, measured by gelatin zymography. This effect was mediated by α4β1 integrin and required PI3-K/Akt signaling. The chemokine CXCL12 also up-regulated MMP-9, independently of α4β1 and involving ERK1/2 but not Akt activity. Accordingly, α4β1 engagement activated the PI3-K/Akt/NF-κB pathway, while CXCL12/CXCR4 interaction activated ERK1/2/c-Fos signaling. Anti–MMP-9 antibodies, the MMP-9 inhibitor TIMP-1, or transfection with 3 different MMP-9 siRNAs significantly blocked migration through Matrigel or HUVECs. Cell-associated MMP-9 was mainly at the membrane and contained the proactive and mature forms. Moreover, B-CLL cells formed podosomes upon adhesion to FN-H89, VCAM-1, or fibronectin; MMP-9 localized to podosomes in a PI3-K–dependent manner and degraded a fibronectin/gelatin matrix. Our results are the first to show that MMP-9 is physiologically regulated by α4β1 integrin and CXCL12 and plays a key role in cell invasion and transendothelial migration, thus contributing to B-CLL progression. MMP-9 could therefore constitute a target for treatment of this malignancy.

Telomerase activity and telomerase reverse transcriptase catalytic subunit expression in canine lymphoma: correlation with Ki67 immunoreactivity

Increased telomerase activity (TA) has been found in human and canine solid tumours, stem cells and somatic tissues with enhanced proliferative potential. The relationship between TA in normal and malignant lymphoid tissues remains unclear. The TA and the expression of canine telomerase reverse transcriptase catalytic subunit (dogTERT) messenger RNA (mRNA) were analyzed in malignant lymph nodes from 30 dogs with lymphoma, from two dogs with non-neoplastic illness and from two clinically normal dogs, demonstrating a statistically significant difference between TA in lymphoma lymph nodes (n = 30) and normal nodes (n = 4) but no significant difference in dogTERT mRNA expression. In addition, the expression of telomerase reverse transcriptase catalytic subunit (TERT) protein and Ki67 was analyzed in malignant lymph nodes from 10 dogs with lymphoma and from two clinically normal dogs by immunohistochemistry. TERT expression was associated with Ki67 in all lymphoma nodes (n = 10), and differences were illustrated between TERT and Ki67 expression between lymphoma (n = 10) and non-lymphoma (n = 2) nodes. This data support further investigation of telomerase in canine haematopoietic neoplasia through large-scale prospective studies.

Expression of the Rho‐family GTPase gene RHOF in lymphocyte subsets and malignant lymphomas

We have studied the expression of RHOF, a member of the Rho-GTPase family, in an array of lymphoid cells and tissues. Previous microarray studies demonstrated RHOF upregulation in a subset of transformed follicular lymphomas. Real-time quantitative polymerase chain reaction evaluated RHOF expression in lymphocyte subpopulations, and normal and malignant lymphoid tissue. Cells and tissues of B-cell origin expressed higher RHOF levels than their T-cell counterparts. Neoplastic cells and tissues of B-cell origin expressed higher levels of RHOF than their benign cellular counterparts. Relatively elevated levels of RHOF were seen in lymphomas derived from germinal centre origin.

ETO protein of t(8;21) AML is a corepressor for Bcl-6 B-cell lymphoma oncoprotein

AbstractThe multiplicity of transcription factors involved in hematologic malignancies suggests a complicated scenario in which many different molecular mechanisms lead to malignant transformation. We hypothesized that some of these proteins might physically and functionally interact and thus mechanistically link different diseases. The ETO protein of t(8;21) acute myeloid leukemia (AML) is an excellent candidate as a common factor because it is normally expressed in human hematopoietic cells, it binds to histone deacetylases (HDACs), and it interacts with the PLZF protein of t(11;17) acute promyelocytic leukemia. To determine whether ETO functionally links a broader range of disease entities, we asked whether ETO forms a complex with the Bcl-6 oncoprotein of B-cell lymphomas. We found that ETO and Bcl-6 are coexpressed in normal and malignant lymphoid tissue, where they interact and colocalize in nuclear speckles. ETO binds to the fourth zinc finger of Bcl-6, enhances Bcl-6 repression of artificial and endogenous genes in an HDAC-dependent manner, and forms a complex with Bcl-6 on the promoters of its endogenous target genes in B-cell lymphoma cells. Therefore, ETO is a bona fide corepressor that links the transcriptional pathogenesis of acute leukemias and B-cell lymphomas and offers a compelling target for transcriptional therapy of hematologic malignancies.