Paraffin-embedded Lymph Node Samples Research Articles

Slug and Vimentin downregulation at the metastatic site is associated with Skip-N2 metastasis of lung adenocarcinoma

ObjectiveLung cancer displays heterogeneity both in the tumor itself and in its metastatic regions. One interesting behavior of the tumor is known as Skip N2 metastasis, which N2 lymph nodes contain tumor cells while N1 are clean. In this study, mRNA levels of epithelial mesenchymal transition (EMT) related genes in skip N2 and normal N2 involvements of non-small cell lung cancer tissues were investigated to evaluate the possible molecular background that may contribute to the pathogenesis of Skip N2 metastasis.Materials and methodsEighty-three surgically resected and paraffin embedded lymph node samples of lung cancer patients were analyzed in this study, which 40 of them were Skip N2. N2 tissues were sampled from 50% tumor containing areas and total RNA was extracted. mRNA levels for 18S, E-cadherin, Vimentin, ZEB1 and SLUG were analyzed via qPCR and E-cadherin and vimentin protein levels via immunohistochemistry (IHC). Bioinformatic analysis were adopted using online datasets to evaluate significantly co-expressed genes with SLUG in lung cancer tissue samples.ResultsSkip-N2 patients who had adenocarcinoma subtype had better survival rates. Comparative analysis of PCR results indicated that Skip N2 tumor tissues had increased E-Cadherin/Vimentin ratio and ZEB1 mRNA expression, and significantly decreased levels of SLUG. E-cadherin IHC staining were higher in Skip N2 and Vimentin were in Non-Skip N2. TP63 had a strong correlation with SLUG expression in the bioinformatics analyses.ConclusionThe results indicate that, at molecular level, Skip N2 pathogenesis has different molecular background and regulation of SLUG expression may orchestrate the process.

Clinical Utility of Chromosomal Microarray in Establishing Clonality and High Risk Features in Patients with Richter Transformation

Background: Richter Transformation (RT) is defined as the transformation of chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) into an aggressive lymphoma. Diffuse large B-cell lymphoma (DLBCL) makes up the vast majority of RT and these patients have a poor prognosis compared to de novo DLBCL (Rossi D, et al. Blood, 2011; 117(12):3391-3401). Prognostic factors include therapy for CLL/SLL prior to transformation, TP53/CDKN2A abnormalities, as well as clonal relationship between CLL/SLL and RT. There are no randomized controlled trials to direct treatment of RT and many centers use clonal relationship to direct treatment decisions including whether or not patients should receive consolidation with autologous or allogeneic stem cell transplantation (Parikh SA, et al. Blood; 123(11): 1647-1657). One method for establishing clonality involves genetic analysis of the immunoglobulin heavy variable chain (IgHV) gene in both the CLL/SLL portion as well as the RT portion by means of IGHV V-D-J sequencing. Whole genome Chromosomal microarray (CMA) provides a method of tracking duplications or deletions of chromosomal segments sometimes referred to as copy number variants (CNVs). CMA is used at our institution to establish chromosomal aberrations at diagnosis for all CLL/SLL patients. This provides prognostic information at diagnosis as well as the ability to track aberrations in the event of progression or transformation of disease. Use of CMA at diagnosis of CLL/SLL and upon transformation to DLBCL (RT) provides a method to determine clonal relationship. It also identifies aberrations involving TP53 and CDKN2A, which are known to be markers of poor prognosis. Methods: We performed a single center retrospective analysis of all patients at MUSC with pathologically confirmed CLL or SLL and Richter transformation between 1/01/2010 to 02/14/2019. We collected baseline demographic, clinical, laboratory, pathology, and outcomes data from the electronic medical record. Chromosomal Microarray Analysis (CMA) was performed on genomic DNA extracted from peripheral blood and fresh or formalin fixed paraffin-embedded lymph node samples using the Infinium HD Human Omni1 BeadChip or the CytoSNP-850K v1.1 BeadChip Array (Illumina, Inc., San Diego, CA). Copy number and genotype data were analyzed using NxClinical (BioDiscovery, Inc) software. Aberrations that were in 100% of cells were considered constitutional and were not included in the data analysis; whereas regions noted to be in less than 100% of cells were considered clonal changes. Results: We identified a total of 24 patients with a diagnosis of Richter transformation to DLBCL with prior history of CLL/SLL. Of these patients, 7 had CMA performed on both the CLL/SLL and RT samples. Clinical characteristics were collected for each patient and are included in table 1. CMA data including deletions, duplications, LOH, loss of TP53 and/or CDKN2A is displayed for each patient in table 2. Six of the 7 patients (85%) had common CMA aberrations identified in the CLL/SLL and RT samples providing evidence of a common clonality. Five of the 7 patients (71%) were identified to have TP53 loss and/or CDKN2A loss by CMA at the time of RT diagnosis. Discussion: Chromosomal microarray was able to provide proof of common clonality in 6 of 7 patients with Richter transformation to DLBCL, which is a poor prognostic feature of RT. It also identified a loss of TP53 and/or CDKN2A in 5 of 7 patients which is also a poor prognostic feature of RT. This information can be used to counsel patients on prognosis and could effect clinical recommendations such as treatment with Allogeneic stem cell transplantation. Institutions with the ability to run CMA should utilize this modality in patients Richter transformation to DLBCL. Disclosures No relevant conflicts of interest to declare.

Suppression of CUL4B Induces G1 Cell Cycle Arrest through the Modulation of AKT Signaling Pathway

IntroductionT-cell lymphomas (TCL) are a heterogeneous group and one of the more aggressive of non-Hodgkin lymphomas. Previous studies have illustrated the poor prognosis in major propotion of patients due to refractory and recurrent TCL. CUL4B, a member of the CUL4B-RING E3 ubiquitin ligase complex, has been demonstrated to be involved in tumorigenesis and correlated to poor survival, contributing to tumor progression via several mechanisms, including epigenetic silencing of tumor suppressors. Although CUL4B was recently identified as a novel oncogene in several solid malignancies, the potential functional role of CUL4B in TCL are poorly understood to data. Hence, the aim of this study was to determine the pro-tumor function and mechanism of CUL4B in TCL.MethodsThe paraffin-embedded lymph node samples were collected from 30 newly diagnosed cases of T-cell lymphoma and 20 normal lymph nodes with approval in our hospital. TCL samples and purified normal T cells (including CD4+, CD8+, HLA-DR+, and HLA-DR-cells) (Pier Paolo Piccaluga et al., 2007) were evaluated for CUL4B expression. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. Expression levels of CUL4B mRNA and protein in TCL cells was detected by RT-PCR and western blot assay. Lentiviral vectors were used to transfected TCL cells to stably silence CULB. To evaluate the effects of CULB on cell proliferation and cycle, cell counting kit-8 and Propidium iodide staining were assessed respectively.ResultsWe found that the expression levels of CUL4B were elevated in TCL samples compared with purified normal T cells via analyzing previously published gene expression data (Pier Paolo Piccaluga et al., 2007) (Fig. 1A). IHC assay indicated that CUL4B was higher expressed compared to normal lymph nodes (Fig. 1B). Moreover, RT-PCR and western blotting assays confirmed the elevated expression level of CUL4B in TCL cells compared with PBMCs in agreement with ICH assay (Fig. 1C-D). The function of cell viability maintenance of CUL4B as well as correlation with poor outcome has been reported previously in solid tumor. In consistent with previous studies in solid malignancies, knockdown CUL4B in TCL cells inhibited cell proliferation, induced G1/S phase arrest (Fig. 1E). Additionally, loss-of-function assay showed that the expression of phosphorylated AKT were reduced in TCL cells. Besides, p21 and p27 were observed in TCL cells (Fig. 1F).ConclusionOur investigations firstly identified the elevated expression levels and the oncogenic role of CUL4B in TCL tumorigenesis. Expression of CUL4B was upregulated in TCL. Targeting CUL4B with shRNA interference exerted therapeutic potential in abrogating cell survival, inducing cell cycle arrest through regulating the AKT signaling pathway. Taken together, targeting the expression of CUL4B may provide a novel approach to TCL therapy. DisclosuresNo relevant conflicts of interest to declare.

Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis

BackgroundMolecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples.ResultsIn lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE.ConclusionsReal-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.

Bcl-2/IgH expression in minimal bone marrow infiltration by follicular lymphoma cells

The purpose of this study was to investigate the roles of bcl-2 chromosomal translocation and Bcl-2 protein expression in follicular lymphoma (FL) minimal bone marrow (BM) infiltration. We identified the same bcl-2/IgH fusion gene in paraffin-embedded lymph node (LN) samples and BM samples using immunohistochemistry (IHC), immunocytochemistry (ICC), cytologic morphology and fluorescence in situ hybridization (FISH). The presence of the Bcl-2/IgH fusion gene in the BM samples and paraffin-embedded LN samples from 56 patients with follicular lymphomas was detected using FISH. The Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using ICC and IHC, respectively. Approximately 78.6% (44/56) of the paraffin‑embedded LN tissue sections that underwent FISH analysis had a bcl-2/IgH translocation. The primary lesion was also positive for the bcl-2/IgH fusion gene, as were the BM minimal infiltrates. The bcl-2/IgH rearrangement occurred in 88.6% (39/44) of the BM specimens. The bcl-2/IgH recombination rate in stage III/IV cancers was significantly different to that observed in stage I/II cancers (p=0.041). In 59% (23/39) of the cases with t(14;18), Bcl-2 was found to be present as assessed by ICC. Positive Bcl-2 ICC staining and the t(14;18) translocation (as detected using FISH) were positively correlated (p=0.028). We then applied the FISH method to slides that had previously been morphologically evaluated using Wright-Giemsa staining; any slides with at least one abnormal cell were subjected to FISH analysis following staining. The assessment of bcl-2/IgH translocation status may contribute to the better detection of minimal BM infiltration by FL cells. Utilizing FISH and cytologic morphology techniques allows for earlier and more accessible BM examination.

Detection of Epstein-Barr virus and human T-cell lymphotropic virus type 1 in malignant nodal lymphoma, studied in Okinawa, a subtropical area in Japan.

Formalin-fixed paraffin-embedded lymph node samples were collected from 100 cases of malignant nodal lymphoma documented in Okinawa in the period from 1973 through 1998. According to the new World Health Organization classification, 12 cases were Hodgkin's lymphoma (HL). Eighty-eight cases of non-Hodgkin's lymphoma (NHL) included 54 cases of T-cell type and 34 cases of B-cell type. Using polymerase chain reaction (PCR), Epstein-Barr virus (EBV) was detected in 11 cases (91.7%) of HL and in 57 cases (64.8%) of NHL, and human T-cell lymphotropic virus type 1 (HTLV-1) was detected in 23 cases (26.1%) of NHL. Clonal integration of HTLV-1 was detected in 10 (43.5%) of 23 HTLV-1 PCR-positive cases by the inverse PCR technique. The EBV-infected cells were detected by EBER-1 in situ hybridization in 11 (91.7%) of 12 HL cases and in 64 (72.7%) of 88 NHL cases. Irrespective of phenotype and tissue type of the malignant lymphoma, the rate of EBV-positive infection in Okinawa was higher than that in any other districts reported in Japan. This characteristic high rate of EBV-positive infection in Okinawa can be ascribed to various factors, such as racial and geographical differences.