Primary cutaneous follicle center lymphoma with Hodgkin and Reed-Sternberg-like cells: a new histopathologic variant.

Primary cutaneous follicle center lymphoma (PCFCL) is the most frequent cutaneous B-cell lymphoma. A 62-year-old man presented with a solitary indolent subcutaneous nodule for 3 years duration, without other abnormalities. Histological examination showed lymphoproliferation with a nodular growth pattern characterized by fibrous collagen bands surrounding nodules. The nodules were composed of medium-sized centrocytes admixed with many large multilobulated and lacunar cells without eosinophils or granulomatous aspect. Hodgkin-like cells were CD30+, CD15+, PAX5+, OCT2+, BOB1+, MUM1+, Ki67+, Bcl6+ and focally CD20+ and EMA-, CD79a-, Bcl2- and CD10-. The medium-sized cells were CD20+, CD79a+, Bcl2+, Bcl6+ and CD10+, enmeshed in a network of CD21-positive follicular dendritic cells. Epstein-Barr virus detection was negative. Interphase fluorescence in situ hybridization showed the absence of BCL2 or BCL6 rearrangement. In such a case, the presence of Hodgkin-like cells intermixed with the tumor population may result in a pitfall diagnosis of classical Hodgkin lymphoma (CHL). Differential diagnoses to be ruled out are secondary or primary skin localization of rather CHL, or systemic follicular lymphoma. Several clinical, radiological, histological, immunohistochemical and molecular arguments indicated the diagnosis of PCFCL. To our knowledge, this is the first report of PCFCL with Hodgkin-like cells.

Protrusion-guided extracellular vesicles mediate CD30 trans-signalling in the microenvironment of Hodgkin's lymphoma.

Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.

Differences in nuclear DNA organization between lymphocytes, Hodgkin and Reed-Sternberg cells revealed by structured illumination microscopy.

Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first time, the organization of nuclear DNA and that of DNA-free space in control lymphocytes, Hodgkin cells and Reed-Sternberg cells using 3D structured illumination microscopy (SIM). We have observed detail in these SIM images that was not observed in conventional widefield images. We have measured the size distribution of the DNA structure using granulometry and noted a significant, progressive increase in the amount of sub-micron structures from control lymphocytes to Hodgkin cells to Reed-Sternberg cells. The DNA-free space changes as well; "holes" in the DNA distribution start to appear in the malignant cells. We have studied whether these "holes" are nucleoli by staining for upstream binding factor (UBF), a protein associated with the nucleolus. We have found that the relative UBF content progressively and significantly decreases-or is absent-in the DNA-free space when measured as either the Pearson correlation coefficient with the DNA-free space or as the number of "holes" that contain UBF. Similar differences exist within the population of Reed-Sternberg cells between binucleated and multinucleated cells with four or more subnuclei. To our knowledge, this is the first study that investigates the changes of the nuclear DNA structure in any disease with superresolution light microscopy.

3D nuclear organization of telomeres in the Hodgkin cell lines U-HO1 and U-HO1-PTPN1: PTPN1 expression prevents the formation of very short telomeres including "t-stumps".

BACKGROUND: In cancer cells the three-dimensional (3D) telomere organization of interphase nuclei into a telomeric disk is heavily distorted and aggregates are found. In Hodgkin's lymphoma quantitative FISH (3D Q-FISH) reveals a major impact of nuclear telomere dynamics during the transition form mononuclear Hodgkin (H) to diagnostic multinuclear Reed-Sternberg (RS) cells. In vitro and in vivo formation of RS-cells is associated with the increase of very short telomeres including "t-stumps", telomere loss, telomeric aggregate formation and the generation of "ghost nuclei". RESULTS: Here we analyze the 3D telomere dynamics by Q-FISH in the novel Hodgkin cell line U-HO1 and its non-receptor protein-tyrosine phosphatase N1 (PTPN1) stable transfectant U-HO1-PTPN1, derived from a primary refractory Hodgkin's lymphoma. Both cell lines show equally high telomerase activity but U-HO1-PTPN differs from U-HO1 by a three times longer doubling time, low STAT5A expression, accumulation of RS-cells (p < 0.0001) and a fourfold increased number of apoptotic cells.As expected, multinuclear U-HO1-RS-cells and multinuclear U-HO1-PTPN1-RS-cells differ from their mononuclear H-precursors by their nuclear volume (p < 0.0001), the number of telomeres (p < 0.0001) and the increase in telomere aggregates (p < 0.003). Surprisingly, U-HO1-RS cells differ from U-HO1-PTPN1-RS-cells by a highly significant increase of very short telomeres including "t-stumps" (p < 0.0001). CONCLUSION: Abundant RS-cells without additional very short telomeres including "t-stumps", high rate of apoptosis, but low STAT5A expression, are hallmarks of the U-HO1-PTPN1 cell line. These characteristics are independent of telomerase activity. Thus, PTPN1 induced dephosphorylation of STAT5 with consecutive lack of Akt/PKB activation and cellular arrest in G2, promoting induction of apoptosis, appears as a possible pathogenetic mechanism deserving further experimental investigation.

3D structural and functional characterization of the transition from Hodgkin to Reed-Sternberg cells.

Recent research using an innovative 3D quantitative FISH approach of nuclear remodelling associated with the transition from mononuclear Hodgkin to diagnostic multinuclear Reed-Sternberg cells revealed profound changes in the 3D nuclear organization of telomeres. Analogous 3D telomere dynamics were identified in Hodgkin's lymphoma derived cell-lines and diagnostic patient biopsies. These changes were observed in both, EBV positive and EBV-negative Hodgkin's lymphoma and independent of the age of the patients at presentation. Compared to mononuclear Hodgkin cells, multinuclear Reed-Sternberg cells are characterized by a highly significant increase of telomere aggregates, often composed of very short telomeres, telomere shortening and loss. RS-cells with telomere free "ghost" nuclei are regularly observed. The telomere protecting shelterin complex appears to be disrupted and deregulation of DNA-repair mechanisms is observed. Our findings are consistent with the hypothesis that distinct 3D telomere changes and shelterin disruption represent a common pathogenetic denominator in the generation of Reed-Sternberg cells.

The 3D nuclear organization of telomeres marks the transition from Hodgkin to Reed-Sternberg cells.

To get an insight into the transition from mononuclear Hodgkin cells (H cells) to diagnostic multinuclear Reed-Sternberg cells (RS cells), we performed an analysis of the three-dimensional (3D) structure of the telomeres in the nuclei of the Hodgkin cell lines HDLM-2, L-428, L-1236 and lymph node biopsies of patients with Hodgkin's disease. Cellular localization of key proteins of the telomere-localized shelterin complex, the mitotic spindle and double-stranded DNA breaks was also analyzed. RS cells show significantly shorter and significantly fewer telomeres in relation to the total nuclear volume when compared with H cells; in particular, telomere-poor 'ghost' nuclei are often adjacent to one or two nuclei displaying huge telomeric aggregates. Shelterin proteins are mainly cytoplasmic in both H and RS cells, whereas double-stranded DNA breaks accumulate in the nuclei of RS cells. In RS cells, multipolar spindles prevent proper chromosome segregation. In conclusion, a process of nuclear disorganization seems to initiate in H cells and further progresses when the cells turn into RS cells and become end-stage tumor cells, unable to divide further because of telomere loss, shortening and aggregate formation, extensive DNA damage and aberrant mitotic spindles that may no longer sustain chromosome segregation. Our findings allow a mechanistic 3D understanding of the transition of H to RS cells.

Hodgkin lymphoma presenting with exclusive or preponderant pulmonary involvement: a clinicopathologic study of 5 new cases.

A review of the pathological features of Hodgkin lymphoma manifesting with exclusive or preponderant lung involvement is given for 5 patients. Three patients were men and 2 were women, with an age range 17 to 48 years (median, 42 years). They presented with nonspecific symptoms including dry cough, fever, or chest pain. Initial clinical assessment suggested a lung tumor. Pathological evaluation was carried out on lung biopsy, wedge resection, lobectomy, or pneumonectomy specimens. All the cases showed diagnostic Reed Sternberg cells within the proper background. Immunopositivity for CD15 and CD30 was documented as well. Nodular sclerosing and mixed cellularity were the documented subtypes. Additional histologic features were a pronounced nodular growth pattern with or without necrosis, a diffuse hypersensitivity pneumonia-like picture, or acute pneumonia-like changes. Our study confirms that the recognition of Hodgkin lymphoma in lung, although based on well-established morphologic criteria, may represent a source of interpretative problems because of the unusual clinical presentation as well as the peculiar histologic changes induced within the pulmonary microenvironment.

Hodgkin's disease variant of Richter's syndrome in chronic lymphocytic leukaemia patients previously treated with fludarabine.

The transformation of chronic lymphocytic leukaemia (CLL) into large-cell lymphoma (Richter's syndrome, RS) is a well-documented phenomenon. Only rarely does CLL transform into Hodgkin's lymphoma (HL). To further analyse the clinico-pathological and genetic findings in the HL variant of RS, we performed a single-institution study in four patients, who developed HL within a mean of 107 months after diagnosis of CLL. All were treated with fludarabine. Three cases were Epstein-Barr virus (EBV)-associated mixed cellularity (MC) HL, the fourth was nodular sclerosis (NS) HL without EBV association. The sites involved by HL included supra- and infradiaphragmal lymph nodes and the tonsils; stage IV disease was also documented. All patients presented with CLL treatment-resistant lymphadenopathies and B-symptoms. In two of the MC cases, molecular analysis performed on CLL samples and microdissected Hodgkin and Reed-Sternberg cells (HRSC) suggested a clonal relationship, while in NS no indication of a clonal relationship was detected. In summary, HL can occur in CLL patients at any site, up to 17 years after initial diagnosis, especially after treatment with fludarabine. The majority present with B-symptoms and CLL treatment-resistant lymphadenopathy, are of the MC type, clonally related to CLL and might be triggered by an EBV infection.

Allelic losses on chromosome 6q25 in Hodgkin and Reed Sternberg cells.

We established a molecular cytogenetic approach to identify consistent genetic aberrations in classical Hodgkin lymphoma. Single laser-micromanipulated Hodgkin and Reed Sternberg (H-RS) cells and the respective germ line tissue were PCR-amplified using highly polymorphic microsatellite probes. Loss of heterozygosity and genomic imbalances of the fluorochrome-labeled microsatellites were determined by fragment length analysis. Eleven cases of in classical Hodgkin lymphoma (cHL) were initially screened with 21 microsatellite markers scattered over the entire genome. Loss of heterozygosity was detected in >40% of informative loci in most cases indicating a deletion of a substantial part of the genome of H-RS cells. Allelic losses and imbalances on chromosome 6q were detected in most of these cases. A deletion mapping of 6q was performed in 16 cases of cHL. This detailed analysis of 6q led to the identification of a 3.3-Mbp region around D6S311 flanked by D6S978 and D6S1564 that was altered in 11 of 14 cases of cHL analyzed. In conclusion, allelotyping of single H-RS cells revealed monoallelic chromosomal deletions and genomic imbalances on 6q that might affect genes critically involved in the pathogenesis of H-RS cells.

Chromosome 16q deletion and loss of E-cadherin expression in Hodgkin and Reed-Sternberg cells.

Hodgkin and Reed-Sternberg (H&RS) cells are generally considered to be the neoplastic cells of Hodgkin's disease (HD); however, such H&RS cells are a few in number due to the numerous reactive cells. Very few data have so far been published on the cytogenetic abnormalities in HD. We have previously used the analysis of comparative genomic hybridization (CGH), employing sorted H&RS cells. The most commonly observed genetic aberrations were a loss on 16q11/21, a gain on 1p13 and a gain on 7q35/36. To confirm the loss of 16q, we analyzed the loss of heterozygosity (LOH) using the regions D16S3075 (16p13), D16S3068 (16q11), D16S3136 (16q12), D16S503 (16q13), D16S515 (16q21), D16S3091 (16q23) and D16S520 (16q24). A total of 100 sorted H&RS cells were compared with a similar number of sorted reactive T cells in 15 cases with HD, including 5 cases with nodular sclerosis (NS) type and 10 cases with mixed cellularity (MC) type. LOHs of 16q, especially 16q21-23, were frequently detected, but 16p deletions were infrequent. Analysis of 16q21 showed LOH in 12 of 15 cases with HD (80%), including 9 cases with MC type (90%) and 3 cases with NS type (60%). 16q23 showed LOH in 9 of 15 cases with HD (60%), including 5 cases with MC type (50%) and 4 cases with NS (80%). On the other hand, 16p13 showed LOH in 3 of 15 cases with HD (20%). Immunohistochemical staining showed that H&RS cells rarely expressed E-cadherin, which is located on 16q. Our findings suggest that 16q deletion, especially 16q21-23, is probably involved in H&RS giant cell formation and tumorigenesis.

Chromosomal abnormalities in Hodgkin's disease are not restricted to Hodgkin/Reed-Sternberg cells.

Hodgkin and Reed-Sternberg cells are considered to represent the malignant fraction in Hodgkin's disease. Several studies have shown that the Hodgkin and Reed-Sternberg cells are chromosomally abnormal, but genetic data about the morphologically normal cell population in Hodgkin's disease are very limited. This latter cell population has therefore been examined for chromosomal aberrations, using the in situ hybridization (ISH) procedure, making use of DNA probes for chromosomes 1, 7, 8, 9, 11, 12, 15, 17, and 18. Nuclei were isolated from freshly frozen (10 cases) and paraffin-embedded (16 cases) biopsy samples and 1000 nuclei per case were evaluated. The cases of Hodgkin's disease were compared with reactive lymph nodes, which show aberrant chromosome copy numbers in less than 1 per cent of the cells. Using strict scoring criteria, nuclei in the tumour were found to show an abnormal genotype, in the range of 1-12 per cent, with trisomies occurring most frequently. No characteristic numerical chromosome abnormality was observed. ISH on 4 microns thick paraffin sections of six cases of Hodgkin's disease revealed numerical aberrations for chromosome 1 in cells which appeared to be morphologically normal. The genomically abnormal nuclei did not differ in morphology or size from the nuclei of morphologically normal cells, but differed considerably in size when compared with the nuclei of Hodgkin/Reed-Sternberg cells after the ISH procedure. Three of these six cases revealed a population of apparently normal cells with an aberrant copy number which differed notably from the fraction observed in reactive lymph nodes. It is concluded, therefore, that a subset of morphologically normal cells, next to the Hodgkin/Reed-Sternberg cells, are chromosomally aberrant and may participate in the malignant cell fraction of Hodgkin's disease.

Epstein-Barr virus-associated Hodgkin's disease in HTLV-I seropositive patients: a report of two cases.

Diagnosis of Hodgkin's disease (HD) is quite difficult in the patient with seropositivity for human T cell lymphotropic virus I (HTLV-I). Herein, two cases of Epstein-Barr virus (EBV)-associated HD, which occurred in males with seropositivity for anti-HTLV-I, are reported. One patient is alive and was diagnosed as having interfollicular HD with CD20+CD15-CD30-CD3-CD4-CD8-CD45RO-Read-Sternberg (R-S) cells. Positivity for EBV-encoded RNA 1 (EBER-1) and latent membrane protein 1 (LMP-1) was shown on follicular germinal center cells and R-S cells. In that case, neither T cell receptor (TCR) beta chain rearrangement nor integration of the HTLV-I provirus was demonstrated in the lymph nodes, although atyical lymphocytes (2%) were found in the peripheral blood. The other case pursued an aggressive clinical course and the patient was diagnosed as having an adult T cell leukemia/lymphoma (ATLL) because of the presence of anti-HTLV-I antibody, lymph node swelling, and the appearance of flower-like cells in the peripheral blood. However, an autopsy revealed no obvious ATLL cell infiltration in any of the organs examined. Multiple granulomatous lesions were found in the bone marrow, liver, kidneys, spleen, and lymph nodes. Reassessment of lymph node lesions in biopsies and granulomatous lesions in autopsy samples demonstrated that both lesions contained CD15+CD30+CD3-CD4-CD8-CD20-CD45RO-EBER-1+L MP-1+R-S cells, and they were considered to be a composite lymphoma of HD and ATLL. These two cases therefore suggest that EBV-associated HD can develop in patients with seropositivity for HTLV-I.

Nucleoli and AgNORs in Hodgkin's disease.

AIM: To evaluate the morphofunctional characteristics of lymph node cells from patients with Hodgkin's disease by measuring silver stained nucleolar organiser regions (AgNORs). METHODS: Nucleoli in Hodgkin's and Reed-Sternberg (HRS) cells, lymphocytes and prolymphocytes were investigated in cytological smears and histological sections of lymph nodes from 32 patients with Hodgkin's disease, and from 34 patients with reactive lymphadenopathy. According to the Rye histological classification of Hodgkin's disease, three cases were the lymphocyte predominant (LP) type, 14 the nodular sclerosing (NS) type, and 15 the mixed cellularity (MC) type. The investigation was done before treatment, by means of a one step silver staining method. In each case, 50 to 100 HRS cells, lymphocytes, and prolymphocytes were evaluated to determine the mean numbers of nucleoli and AgNORs per nucleus. The nonparametric Wilcoxon Mann-Whitney test was used to compare the groups. RESULTS: The mean numbers of nucleoli and AgNORs were higher in lymphocytes and prolymphocytes compared with those from reactive lymph nodes used as controls. Numbers of nucleoli and AgNORs were higher (not significant) in the NS type of Hodgkin's disease than in the MC type. There was a significant increase in numbers of nucleoli in HRS cells, and their AgNOR counts were increased. The greatest number of nucleoli in HRS cells was found in the NS type. Furthermore, the nucleolar activity of HRS cells was greater in the NS type compared with the MC type (50.2 (SEM 3.9) v 37.7 (2.9) AgNORs per nucleus (p = 0.025)). Comparative analysis of cytological and histological samples showed that the AgNOR score was significantly higher in touch imprints than in tissue sections with tumours of the same histological type. CONCLUSIONS: Assessment of cell activity in Hodgkin's disease patients by silver staining is more convenient and informative in lymph node imprints than in histological sections. The highest expression of interphase ribosomal RNA cistrons found in NS HRS cells is probably explained by their high proliferative activity and elevated production of transforming growth factor 1 which is known to be the most potent cytokine present in the NS subtype of Hodgkin's disease.

Molecular single cell studies of normal and transformed lymphocytes.

The polymerase chain reaction allows the characterization of RNA and DNA sequences from single cells. Methods were established to analyse single cells isolated from suspension or by multicolour flow cytometry. We established a method to isolate single immunostained cells from frozen tissue sections and to analyse those cells for immunoglobulin gene rearrangements. This method was first used to study B cell differentiation within human germinal centres. In another series of experiments, Hodgkin and Reed-Sternberg (HRS) cells from a total of 14 cases of HD were analysed for B lineage derivation and clonality. In 13 of the 14 cases, clonal V gene rearrangements were identified. This shows that HRS cells generally represent the outgrowth of a clonal population of B cells. The detection of somatic mutations in all VH gene rearrangements amplified from HRS cells and the nature of those mutations identifies a GC B cell as the HRS precursor.

Molecular biology of Hodgkin's disease.

The mist surrounding the origin and genesis of HRS cells of classical HD is beginning to dissipate. Molecular biological studies of classical HD at the single cell level strongly suggest that the HRS cells in the majority of cases represent a monoclonal outgrowth of late germinal centre B cells that have lost their capacity to express IG through crippling mutations introduced during the germinal centre reaction. Because of the expression of T cell antigens and/or cytotoxic molecules, the HRS cells of a minority of classical HD cases appear to originate from T cells. Under physiological conditions, B cells that are unable to express IG are eliminated by apoptosis. In most B cell derived classical HD cases, the HRS cells have lost their IG gene coding capacity through mutation and should therefore die of apoptosis. Since this usually does not happen, blockade of the apoptotic pathway may be a major event in the pathogenesis of B cell related classical HD. It is tempting to assume that viruses such as EBV, as well as regulator genes that normally monitor the human genome for damaged DNA, such as TP53, might be involved in the postulated hindrance of the apoptotic pathway, leading to the genesis of classical HRS cells.

A comparison of distinct modes of tumor cell death in Hodgkin's disease using morphology and in situ DNA fragmentation.

The study examined the morphology and frequency of cell death occurring spontaneously in lymph nodes from patients with Hodgkin's disease. In addition to necrosis, which was infrequent and usually in patches, we document two cell types showing features of individual cell death: mummy cells end apoptotic cells. Mummy cells present no evidence of DNA fragmentation, but show electron microscopic features of "dark cells." Apoptotic Hodgkin-Reed-Sternberg cells are found frequently and are easier to demonstrate by in situ and labeling of fragmented DNA than by light microscopy only. In many cases phagocytosis of apoptotic cells is also documented. The significance of these findings to the limited number of Hodgkin-Reed-Sternberg cells in most cases of Hodgkin's disease is discussed.

Detection of numerical karyotype changes in the giant cells of Hodgkin's lymphomas by a combination of FISH and immunohistochemistry applied to paraffin sections.

Conventional karyotyping of Hodgkin's disease (HD) has until now yielded only limited insight into karyotypic characteristics of this disease. For this reason, fluorescence in situ hybridization (FISH) using alpha-satellite chromosome-specific probes was applied to paraffin sections of HD tumors in order to verify numerical aberrations suggested to be specific for HD in the literature. The FISH technique was combined with immunohistochemical detection of the CD30 antigen to allow easier identification of the Reed-Sternberg and Hodgkin (RS&H) cells. The number of specific FISH signals per nucleus was determined both in CD30-positive RS&H cells as well as in non-malignant "bystander" cells in order to assess differences in the signal distribution. Contrasted with normal lymphoid cells, the tumor cells in HD were found to be polysomic for at least one of the chromosomes analyzed (1,2,4, and 8). The technique described is a reliable method for confirmation of results obtained from conventional cytogenetics, which is especially suited for archival material or samples not containing dividing cells.

Detection of immunoglobulin heavy-chain gene rearrangement at the single-cell level in malignant lymphomas: no rearrangement is found in Hodgkin and Reed-Sternberg cells.

Hodgkin and Reed-Sternberg (HRS) cells, the neoplastic cells of Hodgkin's disease (HD), represent only a minority of the cellular infiltrate in affected tissue. Therefore, rearrangements of the immunoglobulin heavy-chain (IgH) gene detected in DNA extracted from an entire Hodgkin's lymph node cannot be attributed to the HRS cells and cannot be used as an argument for the B-cell origin of HRS cells. We developed a new method for the amplification of rearranged DNA of the IgH gene from single cells. Using 6 "forward primers" which were constructed corresponding to consensus sequences of the 6 known families of the IgH variable (V) region (framework region I) and a mix of 2 "reverse primers" corresponding to consensus sequences of the different joining (J) segments, rearrangements of all 6 V-families were detected in human peripheral blood lymphocytes. Rearranged IgH DNA could be amplified from single cells of B-cell lymphoma-cell lines and from 13 patients with B-cell non-Hodgkin's lymphomas. However, analysis of HRS cells isolated from lymph nodes of 13 patients with Hodgkin's disease did not show any rearrangement of the IgH gene locus. These findings, obtained by polymerase chain reaction (PCR) on isolated single HRS cells, contrast with previous studies that used Southern-blot analysis of entire tissues affected by Hodgkin's disease. We conclude that the neoplastic HRS cells in Hodgkin's disease--with the possible exception of the nodular paragranuloma subtype--are probably not derived from B cells.

Localization of anti-Leu-M1 (CD15) binding sites in Hodgkin's disease by immunoelectron microscopic examination.

Anti-Leu-M1 (CD15) is a monoclonal antibody used in surgical pathology to diagnoses Hodgkin's disease. By light microscopic immunohistochemistry, anti-Leu-M1 reacts with Reed-Sternberg cells and their variants, notably lacunar cells in nodular sclerosing Hodgkin's disease, as well as granulocytes in Hodgkin's disease. The immunostaining of Reed-Sternberg cells has been characteristically described as a diffuse cytoplasmic pattern with a prominent perinuclear globular component. In addition, irregular plasma membrane reactivity has been observed. To define the intracellular localization of Leu-M1 precisely, the authors performed postembedding immunoelectron microscopy with the protein A-gold technique on sections embedded in Lowicryl K4M from a patient with nodular-sclerosing-type Hodgkin's disease. At the electron microscopic level, gold particle staining indicative of Leu-M1 binding was found within cytoplasmic granules and the Golgi apparatus, as well as focally at the plasma membrane. The cytoplasmic granules were located in a perinuclear region and in the cell periphery. Although the morphology of the granules was suggested of lysosomal structures, immunolabel was not detected on serial sections of these granules with three different antibodies directed against lysosomal antigens.