Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature

Stacchini A, Barreca A, Demurtas A, Aliberti S, di Celle P F & Novero D
(2012) Histopathology  60, 452–459
Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature Aim: To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). Methods and results: CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl‐6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. Conclusions: CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.     

Immunohistochemical demonstration of leucocyte differentiation antigens on paraffin sections using a modified AMeX (ModAMeX) method

The AMeX method (cold Acetone fixation with subsequent Methyl benzoate and Xylene treatment and routine paraffin embedding) has been recently revived for simultaneous preservation of morphology of cells and their antigens. We propose a modification of this method (ModAMeX), with the use of proteolytic enzyme inhibitors and low temperature paraffin wax embedding, which results in better preservation of a large number of leucocyte differentiation antigens and diagnostic morphologic detail. T-cell antigens (CD1, CD2, CD3, CD7 & CD8), B-cell antigens (CD22), macrophage associated antigens (CD11c, CD14 and others), activation antigens (CD25 and others), as well as some other antigens of diagnostic interest (CD10) were found to be preserved with a staining intensity equal to that of sections of fresh frozen tissue. Although the staining intensity of other T-cell antigens (CD4 & CD5), B-cell antigens (CD19, CD21 & CD37), activation antigens (Ki-1) and nuclear proliferation antigen (Ki-67) was slightly weaker as compared with frozen sections, this could be corrected by increasing the monoclonal antibody concentration. Staining for heavy and light chains of immunoglobulins was minor, sometimes compromised due to persistence of background staining as a result of extracellular immunoglobulins. The ModAMeX method has the advantages of simplicity, low cost and the possibility of exchange of tissue material between laboratories.     

Expression of the neural cell adhesion molecule (CD56) by human myeloma cells.

Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T,B, myelomonocytic, and natural killer (NK)-cell antigens. Leu-19/NKH1 (CD56), a molecule identical to N-CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual-parameter flow cytometry was used to correlate N-CAM positivity with DNA aneuploidy or cytoplasmic immunoglobulin expression as markers of myeloma cells. When N-CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N-CAM frequently exhibited pre-B cell markers, myeloid antigen, and HLA-DR, respectively (seven out of eight cases, 88%). Six out of eight N-CAM-negative myelomas were of the IgG lambda isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N-CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N-CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained.     

The bi-specific CD3 x NCAM antibody: a model to preactivate T cells prior to tumour cell lysis

To target the neural cell adhesion molecule (NCAM, CD56) on neuroblastoma by T cell-based immunotherapy we have generated a bi-specific CD3 x NCAM antibody (OE-1). This antibody can be used to redirect T cells to NCAM+ cells. Expectedly, the antibody binds specifically to NCAM+ neuroblastoma cells and CD3+ T cells. OE-1 induces T cell activation, expansion and effector function in peripheral blood mononuclear cell (PBMC)-derived CD4+ and CD8+ T cells. T cell activation was shown to depend on the presence of normal natural killer (NK) cells in the culture. Interestingly, while PBMC- derived T cells were activated by OE-1, NK cells were almost completely depleted, suggesting that T cells activated by OE-1 deleted the NK cells. Activated CD4+ and CD8+ T cells differentiate into a larger CCR7+ central memory and a smaller CCR7- effector memory cell population. Most importantly, preactivated T cells were highly cytotoxic for neuroblastoma cells. In eight of 11 experiments tumour-directed cytotoxicity was enhanced when NK cells were present during preactivation with OE-1. These data strongly support a bi-phasic therapeutic concept of primarily stimulating T cells with the bi-specific antibody in the presence of normal NCAM+ cells to induce T cell activation, migratory capacity and finally tumour cell lysis.     

Correlation of very late activation integrin and CD44 expression with extrarenal invasion and metastasis of renal cell carcinomas

Cell adhesion molecules mediate cell-cell and cell-matrix interactions, and they are thought to play an important role in tumor invasion and metastasis. Altered expression of integrins and CD44 in renal cell carcinoma has been recently demonstrated, but an association with invasive or metastatic behavior has not been reported. We examined very late activation (VLA) integrin and CD44 expression in 37 renal cell carcinomas and correlated adhesion molecule expression with multiple histological and clinical parameters. Most tumors exhibited positive staining for VLA3 (81%). Approximately one third of the tumors stained positively for VLA6 and CD44, and fewer (27%) were positive for VLA2. Only a few tumors were positive for VLA4 (8%) and VLA5 (14%). Most of the tumors exhibiting positive staining showed a combination of membranous and cytoplasmic staining patterns. Low-grade tumors positive for VLA6 showed a tendency for basilar staining of the tumor cells, whereas high-grade tumors exhibited diffuse cytoplasmic staining. All tumors exhibiting weak or strong positive staining for VLA4 or VLA5 showed extrarenal invasion or were known to have developed metastases at the time of nephrectomy. All tumors strongly positive for VLA2 or CD44 showed invasion beyond the renal capsule or metastases. In contrast to a previous study, no association was observed between positive staining and tumor grade. Nor were tumor size, architectural pattern, cell type, or DNA ploidy found to be associated with particular staining patterns. Although many of the invasive tumors showed no difference in VLA integrin or CD44 expression compared with tumors confined to the kidney, increased expression in some of them suggests that these cell adhesion molecules may contribute to the invasive or metastatic phenotype.     

抗CD34单抗标记血管生成在非小细胞肺癌中的表达及临床意义

目的探讨非小细胞肺癌中 CD34标记的血管生成的表达及临床意义。方法采用免疫组化法检测 81例手术切除的非小细胞肺癌标本中抗内皮细胞表面抗原 CD34单抗标记的血管生成。结果 CD34以其染色的特异性、敏感性及清晰的背景而易于识别。统计学分析显示微血管密度同肺癌临床分期的进展及血行转移密切相关 ,但未发现同淋巴结转移及其它临床病理特征有关。结论肺癌中微血管密度对血行转移的进展有重要作用且同肺癌的发生有关。     

Expression of the embryonal neural cell adhesion molecule N-CAM in lung carcinoma. Diagnostic usefulness of monoclonal antibody 735 for the distinction between small cell lung cancer and non-small cell lung cancer

Paraffin sections of 19 surgically resected small cell lung carcinomas (SCLC), 33 non-small cell lung carcinomas (NSCLC) of various types, and four bronchial carcinoids were immunostained with monoclonal antibodies (MoAbs) 735 and anti-Leu 7, both recognizing some sugar epitopes present on the neural cell adhesion molecule N-CAM. With MoAb 735, all SCLC were stained focally or diffusely, and one carcinoid was stained focally. Only three of the 33 NSCLC were faintly and focally positive with MoAb 735; these three tumours showed relatively small tumour cells and small, oval nuclei. Anti-Leu 7 stained all the carcinoids, only eight SCLC, sometimes focally, and eight NSCLC. MoAb 735 was thus superior to anti-Leu 7 in distinguishing between SCLC and NSCLE. Since MoAb 735 stained all SCLC strongly and is applicable on paraffin sections, it provides a well-needed addition to the immunomarkers used in the diagnostic distinction of SCLC and NSCLC.     

Standard and variant CD44 isoforms are commonly expressed in lung cancer of the non-small cell type but not of the small cell type

Cluster of differentiation 44 (CD44) encompasses a polymorphic family of cell membrane glycoproteins involved in the mechanism of tumour invasion and metastasis. Since non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) display very different rates of progression, a significant discrepancy in their CD44 expression profiles is to be expected. An immunohistochemical study was undertaken on the expression of standard CD44 (CD44s) and the variant isoforms containing the domains encoded by variant exon 3 (CD44v3) or variant exon 6 (CD44v6) in paraffin-embedded bronchial biopsy specimens from 32 NSCLC cases and 11 SCLC cases. An absolute lack of immunoreactivity for CD44s, CD44v3, and CD44v6 was obtained in every case of SCLC, whereas 28 of the 32 NSCLC cases showed a positive immunoreaction for at least one of the three epitopes investigated. In conclusion, the occurrence of standard and variant CD44 isoforms in NSCLC and their absence in SCLC suggest the possibility that CD44 is in some way instrumental in conditioning the biological behaviour of NSCLC, but not of SCLC, whose metastatic cascade would be set in motion by the activation of hitherto unidentified, CD44-independent pathways.     

CD8+, CD56+ (natural killer-like) T-cell lymphoma involving the small intestine with no evidence of enteropathy: clinicopathology and molecular study of five Japanese patients

The present study reports five CD8+, CD56+ (natural killer (NK)-like) T-cell lymphomas involving the small intestine without evidence of enteropathy, from Japan. Three were intestinal T-cell lymphoma. The site of origin of the other two was not definitive. Four of five patients underwent emergency operation because of intestinal perforation. The small intestines of these patients had multiple ulcerative lesions with or without demarcated tumors. Histologically, the lymphoma cells were monomorphic or slightly pleomorphic and displayed epitheliotropism of varying degrees. Lymphoma cells of all patients shared the common phenotype: CD3+, CD4-, CD5-, CD8+, CD56+, CD57-, T-cell intracellular antigen-1+, granzyme B+. In contrast to nasal/nasal type NK-cell lymphomas, they had clonal rearrangement of T-cell receptor(TCR) genes and were negative for EBV-encoded RNA. Immunohistochemistry and genetics suggested that three cases were of alpha beta T-cell origin and two cases were of gamma delta T-cell origin. There was no evidence of enteropathy in any patient. The cases followed a clinically aggressive course with a frequent involvement of lung. According to the classification based on the recent genetic studies of European enteropathy-type intestinal T-cell lymphoma (ETL), the present cases could be classified as type 2 ETL.     

Inconsistent association of Epstein-Barr virus with CD56 (NCAM)-positive angiocentric lymphoma occuring in sites other than the upper and lower respiratory tract

We previously described nine cases of angiocentric lymphoma of a possible natural killer (NK)-cell lineage with a surface CD3− CD56+ phenotype occurring in sites other than the upper and lower respiratory tract. This study was performed to investigate the association of Epstein-Barr virus (EBV) with these lymphomas, using the polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small RNAs (EBERs) and immunohistology for EBV-determined nuclear antigen-2 (EBNA-2) and latent membrane protein-1 (LMP-1) in paraffin sections. PCR and ISH produced almost identical results, and EBERs were identified in the nuclei of the lymphoma cells of three cases, two of which exhibited LMP-1 in the cytoplasm of tumour cells without EBNA-2 expression. Molecular genetic analysis revealed EBV to be incorporated into these three EBER-positive cases either clonally or biclonally. It was revealed by re-evaluation of their morphology with the established EBV status on each case that, in contrast to the rather variable and irregular cellular composition of the EBV- positive tumours, the EBV-negative tumours stood out because of their remarkably uniform 'blastoid' appearance, and could be grouped as blastic NK-cell lymphoma. The relationship of the EBV-positive cases with nasal NK-cell tumours has yet to be clarified.     

CD56 may be a more useful immunohistochemical marker than somatostatin receptor 2A for the diagnosis of phosphaturic mesenchymal tumors

Phosphaturic mesenchymal tumors (PMTs) are the most typical cause of tumor-induced osteomalacia (TIO) associated with mesenchymal neoplasms. Specifically, TIO is attributed to the production of phosphatonins, such as fibroblast growth factor 23 (FGF23), participating in the homeostasis of phosphate. Although immunohistochemistry (IHC) for FGF23 showed characteristic positive staining in PMTs, FGF23 antibodies that can be used for the reliable diagnosis of PMTs are hard to obtain in common pathology laboratories. Somatostatin receptor 2A (SSTR2A) has been previously proposed as an alternatively useful marker for the diagnosis of PMTs. However, SSTR2A is not commonly utilized in pathological laboratories. The CD56 marker is a useful alternative that is comparable to SSTR2A and is similar considering the sensitivity. Even in cases of PMTs originating in the bones, ethylenediaminetetraacetic acid-based decalcification for tissue processing does not seem to affect the IHC of CD56. As CD56 immunopositivity in mesenchymal tumors is limited, it also has some degree of specificity for PMTs. Thus, when PMTs are suspected, the use of CD56 is recommended.     

小鼠抗人C5aR短肽（9—30）单克隆抗体制备及鉴定

目的获得有生物功能的小鼠抗人 C5 a R短肽单克隆抗体。方法对 C5 a受体 (CD88)二级结构和 B细胞表位进行分析研究 ,采用 Fmoc方案固相合成 C5 a R N-端第 9～ 30位氨基酸残基的 2 2 -肽 ,以此为抗原免疫 Balb/ c小鼠。结果建立了 1株小鼠抗人 C5 a R短肽杂交瘤细胞系 E3,其平均染色体数目为 10 2条 ,所分泌抗体为 Ig G1,κ型 ,腹水抗体效价为 1×10 - 4 ～ 1× 10 - 6 ,可识别 U 937、人脐静脉内皮细胞 (VEC)和 PMN等表达 C5 a R细胞。 E3单抗的亲和常数 Ka=2 .5× 10 5 ,其结合表位为 C5 a R第 15～ 2 1位氨基酸基序 D1 5 DKDTL2 0 D。结论 B细胞表位多肽具有免疫原性 ,可制作单克隆抗体 ,为 C5 a-C5 a R相互作用的研究以及 AL I、ARDS等 C5 a相关疾病的研究提供实验材料。     

The p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/CD3/ζ complex

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are thought to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56^lck SH2 domain with ζ-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/ζ complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8αβ and CD8αβ isoforms to the TCR. We demonstrate in vivo in association of CD8αα/p56^lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56^lck specifically impedes the association of ZAP-70 with CD8αα/p56^lck without affecting the ζ/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8αα or CD8αβ isoform with the TCR/CD3/ζ complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56^lck SH2 domain mediates recruitment of CD8/p56^lck to the activated TCR/CD3/ζ complex.     

Immunohistochemical application of an antibody specific for human CD1a to the diagnosis of canine mast cell tumour

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.     

Monoclonal antibodies to denatured human ACE (CD 143), broad species specificity,reactivity on paraffin sections,and detection of subtle conformational changes in the C-terminal domain of ACE

Two new mouse monoclonal antibodies (mAbs) were generated to denatured human angiotensin-converting enzyme (ACE, CD143). The clones 2E2 and 3C5, each of the IgG1 kappa chain isotype, detect ACE with high sensitivity, respectively, at 20 ng and 2 ng of protein per lane in Western blotting. They both recognize different epitopes on the C-domain of ACE located between amino acid residues 740 and 992. In formalin-fixed and paraffin-embedded human tissues, immunohistochemistry revealed all known expression sites of ACE, e.g. the epithelial brush borders of proximal kidney tubules, epithelial cells of epididymis, endothelial cells, activated macrophages as well as germ cells during spermatogenesis. In contrast to other mAbs to denatured human ACE, mAbs 2E2 and 3C5 demonstrate cross-reactivity with a broad spectrum of animal species such as monkey, rat, rabbit, cattle, dog, cat, and guinea pig. In addition, mAb 2E2 recognized mouse ACE in Western blotting and on paraffin sections. Our findings suggest that mAbs 2E2 and 3C5 are useful for identifying even subtle changes in ACE conformation resulting from denaturation. These mAbs are also sensitive tools for the detection of minimal amounts of ACE in biological fluids and tissues using proteomics approaches. Their reactivity in routinely processed tissues of various species may prove useful for correlation of ACE expression in animal models to human diseases.     

Aldo-keto reductase family 1 member C3 (AKR1C3) is expressed in adenocarcinoma and squamous cell carcinoma but not small cell carcinoma

Human aldo-keto reductase family 1 member C3 (AKR1C3) was initially identified as a critical enzyme in reducing 5α-dihydrotestosterone (5α-DHT) to 5α-androstane-3α,17β-diol (3α-diol) and oxidizing 3α-diol to androsterone. Based on these enzymatic activities, AKR1C3 was originally named type 2 3α-hydroxysteroid dehydrogenase (HSD)/type 5 17β-HSD. Additionally, AKR1C3 was demonstrated to be capable of metabolizing other steroids including estrogen and progesterone. Subsequently, AKR1C3 was shown to possess 11-ketoprostaglandin reductase activity in metabolizing prostaglandins and dihydrodiol dehydrogenase x (DDx) activity in metabolizing xenobiotics. Tissue distribution of AKR1C3 has been detected in both sex hormone-dependent organs such as the testis, breast, endometrium, and prostate as well as sex hormone-independent organs including the kidney and urothelium. Although prominent expression of AKR1C isozymes has been reported in human non-small cell lung carcinoma (NSCLC), the expression of AKR1C3 in small cell carcinoma of the lung has not been described. Also, the expression of AKR1C3 in normal lung has not been described. In this study, we demonstrated strong AKR1C3 immunoreactivity in bronchial epithelium but not in bronchial glands or alveolar pneumocytes. Strong AKR1C3 immunoreactivity was also demonstrated in columnar epithelium but only weak immunoreactivity in squamous epithelium of the gastrointestinal junction. Although AKR1C3 immunoreactivity was absent in small cell carcinoma of the lung, positive AKR1C3 immunoreactivity was extensively present in both adenocarcinoma and squamous cell carcinoma arising from the lung and the gastroesophageal junction. AKR1C3 may serve as an adjunct marker for differentiating small cell carcinoma from NSCLC. However, roles of AKR1C3 in adenocarcinoma, squamous cell carcinoma, and small cell carcinoma pathogenesis require further studies.