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Objective Several lines of evidence indicate that B cells may be involved in the immunopathology of giant cell arteritis (GCA) and polymyalgia rheumatica (PMR). This study was undertaken to examine the distribution of defined B cell subsets, including effector B (Beff) cells and regulatory B (Breg) cells, in patients with GCA and patients with PMR before and after corticosteroid treatment. Methods Circulating B cells were analyzed in 34 newly diagnosed, untreated patients with GCA or PMR, and in 44 followup samples from patients with GCA or PMR who received corticosteroids for 2 weeks or 3 months. For comparison, 40 age-matched healthy controls and 11 rheumatoid arthritis (RA) patients were included. Serum BAFF levels were determined, and temporal arteries were studied by immunohistochemistry. Results Patients newly diagnosed as having GCA or PMR, but not patients with RA, had decreased numbers of circulating B cells compared to healthy controls. B cell numbers recovered rapidly in treated patients with GCA and PMR in remission. This recovery was not achieved by compensatory hyperproliferation or enhanced bone marrow production. B cell numbers inversely correlated with erythrocyte sedimentation rates, C-reactive protein levels, and serum BAFF levels. Tumor necrosis factor α–positive Beff cells, but not interleukin-10 (IL-10)–positive Breg cells, were decreased in patients newly diagnosed as having GCA or PMR. Following treatment, circulating numbers of Beff cells normalized. The returning Beff cells demonstrated an enhanced capacity to produce IL-6. Few B cells were found in temporal artery biopsy specimens from GCA patients. Conclusion We show for the first time that the distribution of B cells is highly disturbed in GCA and PMR and that B cells likely contribute to the enhanced IL-6 response in both diseases.

AimsThe Fas-associated death domain gene (FADD) is often overexpressed in squamous cell carcinoma of the head and neck (HNSCC), and is considered to be a driver gene in amplification of the chromosomal 11q13.3 region. Amplification of 11q13.3 is associated with increased metastasis in HNSCC and breast cancer. The aim of this study was to investigate the association between FADD protein expression in advanced-stage HNSCC and clinicopathological features and outcome.Methods and resultsTumour tissues of 177 HNSCC patients uniformly treated with primary surgery and postoperative radiotherapy were collected. FADD expression was assessed on pretreatment tumour biopsies using immunohistochemistry. High FADD expression was detected in 44% of the HNSCC patients. High expression was associated with an increased rate of lymph node metastasis (P=0.001) and with a shorter distant metastasis-free interval (DMFI) (HR 2.6, 95% CI 1.0-6.7, P=0.046) when lymph node metastases were present.ConclusionsOur data show that an increase in FADD expression is associated with a higher incidence of lymph node metastasis at presentation, and with shorter DMFI when lymph node metastases are present. High FADD expression in the primary tumour could be a useful marker to select patients for systemic treatment strategies that reduce the risk of distant metastases.

For the past 20 years most malignant lymphomas have been classified as clinicopathological entities, each with its own combination of clinical, morphological, immunophenotypic and molecular genetic characteristics. Molecular and cytogenetic abnormalities can be detected by a wide range of techniques, ranging from conventional karyotyping to single nucleotide polymorphism analysis. In this review, we consider the common genetic abnormalities found in lymphoma and discuss the advantages and disadvantages of individual techniques used in their detection. Finally, we discuss briefly possible novel developments in the field of lymphoma diagnostics.

Amplification of the 11q13 region is one of the most frequent aberrations in squamous cell carcinomas of the head and neck region (HNSCC). Amplification of 11q13 has been shown to correlate with the presence of lymph node metastases and decreased survival. The 11q13.3 amplicon carries numerous genes including cyclin D1 and cortactin. Recently, we reported that FADD becomes overexpressed upon amplification and that FADD protein expression predicts for lymph node positivity and disease-specific mortality. However, the gene within the 11q13.3 amplicon responsible for this correlation is yet to be identified. In this paper, we compared, using immunohistochemical analysis for cyclin D1, FADD and cortactin in a series of 106 laryngeal carcinomas which gene correlates best with lymph node metastases and increased disease-specific mortality. Univariate Cox regression analysis revealed that high expression of cyclin D1 (P = 0.016), FADD (P = 0.003) and cortactin (P = 0.0006) predict for increased risk to disease- specific mortality. Multivariate Cox analysis revealed that only high cortactin expression correlates with disease- specific mortality independent of cyclin D1 and/or FADD. Of genes located in the 11q13 amplicon, cortactin expression is the best predictor for shorter disease-specific survival in late stage laryngeal carcinomas.

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.

Objective. To assess the value of the parotid biopsy as a diagnostic tool for primary Sjogren's syndrome ( pSS), and to compare the parotid biopsy and the labial biopsy with regard to diagnostic value and biopsy-related morbidity.Methods. In 15 consecutive patients with pSS and 20 controls, the parotid biopsy was assessed as a diagnostic tool based on the presence of lymphocytic foci, benign lymphoepithelial lesions and lymphoid follicles. These new histological criteria were compared with established diagnostic criteria for the labial biopsy in 35 consecutive patients suspected for pSS who underwent simultaneous biopsies from both sites. In addition, both biopsies were compared for morbidity.Results. The first analysis revealed a focus score of >= 1 or lymphocytic infiltrates ( not fulfilling the criterion of a focus score of 1) combined with benign lymphoepithelial lesions as diagnostic criteria for pSS. When comparing the parotid biopsy with the labial biopsy sensitivity and specificity were comparable (sensitivity 78%, specificity 86%). Level of pain was comparable and no loss of motor function was observed. No permanent sensory loss was observed after parotid biopsy, while labial biopsy led to permanent sensory loss in 6% of the patients. Malignant lymphoma was detected in one parotid biopsy by chance, without involvement of the labial salivary gland.Conclusion. A parotid biopsy has a diagnostic potential comparable with that of a labial biopsy in the diagnosis of pSS, and may be associated with less morbidity.

Although Burkitt's lymphoma (BL) is classified as one entity in the World Health Organisation (WHO) classification, we wondered whether BL should not be considered as a different disease in children compared with adults. Netherlands Cancer Registry (NCR) data were obtained from 1994 to 1998 (n=203). Detailed clinical data from two treatment protocols were compared: one for adults up to the age of 65 years (n=27) and one for children (n=80). All slides of the two clinical studies were centrally reviewed which included immunophenotyping and when necessary breakpoint analysis of MYC/8q24. Only cases with an unambiguous diagnosis of BL (classical and atypical BL) were accepted. The age distribution of BL-patients showed a bimodal distribution with a peak at the paediatric age and a steady increase after approximately 60 years of age. Most of the patients were males (89% for children and 78% for adults) and only male patients showed this bimodality. Children more often had extranodal disease (81% vs. 59%), whereas adults more often had nodal disease (89% vs. 53%). Based on epidemiology and clinical presentation, the concept that BL is one disease should be re-challenged.

Background: The definition of primary extranodal non-Hodgkin's lymphoma (NHL) is a controversial issue, especially in patients where both nodal and extranodal sites are involved.Patients and methods: The impact of different definitions of primary extranodal NHL on incidence and prognosis is explored using data from a population-based NHL registry.Results: Using liberal criteria, 389 (34%) cases were classified as primary extranodal NHL. Overall survival (OS) rates of nodal and extranodal NHL patients defined this way were comparable; however, extranodal NHL patients had a better disease-free survival (DFS). When strict criteria were applied, 231 cases (20%) were classified as primary extranodal NHL, OS and DFS rates of extranodal NHL patients defined this way were superior to nodal NHL patients; however, the difference in OS was reversed after correction for differences in International Prognostic Index and malignancy grade.Conclusion: This study illustrates the selection bias that is introduced when a strict definition of primary extranodal NHL, that excludes cases with disseminated disease, is used. Patients with primary extranodal NHL were found to have a superior DFS, irrespective of which definition of primary extranodal NHL was used.

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n =18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with `malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n =26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n =17) designated LCT; and cytologically malignant (n =9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n =13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n =4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated whether oncogenic tyrosine kinase activation also occurs in other categories of lymphoma by staining 145 cases of lymphoma covering those tumours with a range of different subtypes including those with morphological similarity to ALK-positive anaplastic large cell lymphoma (ALCL). Twelve cases of the borderline malignant disorder lymphomatoid papulosis were also studied. Twenty seven of the 28 cases of ALK-positive ALCL showed the extensive cytoplasmic labelling for phosphotyrosine in the neoplastic cells. The remaining case containing moesin-ALK exhibited membrane-associated phosphotyrosine expression. There was no nuclear phosphotyrosine labelling in any of the ALK-positive ALCL, even though ALK was present within the cell nuclei in 23 of the tumours. Variable degrees of phosphotyrosine labelling, usually membrane-restricted, were observed in 7/40 cases of ALK-negative ALCL, 9/29 cases of diffuse large B-cell lymphoma, 3/6 cases of mediastinal B-cell lymphoma, 2/7 cases of Hodgkin's lymphoma, 3/6 cases of peripheral T-cell lymphomas unspecified, 4/6 cases of B-cell chronic lymphocytic leukaemia, 2/6 cases of follicular lymphomas and 2/12 cases of lymphomatoid papulosis studied. However none of these phosphotyrosine-positive cases showed the strong cytoplasmic labelling comparable to that seen in ALK-positive lymphoma. We conclude that activation of a tyrosine kinase is probably not a major oncogenic event in lymphomas other than ALK-positive ALCL.

Follicular lymphoma (FL) is defined as a neoplasm of germinal center B cells, usually with a follicular growth pattern. The neoplastic population typically consists predominantly of centrocytes, the resting cells present in the light zone of normal germinal centers, and relatively few centroblasts

Diffuse large B cell lymphomas relapsing in the CNS lack oncogenic MYD88 and CD79B mutations

The relationship between Epstein-Barr virus (EBV) and the germinal centre (GC) of the asymptomatic host remains an enigma. The occasional appearance of EBV-positive germinal centres in some patients, particularly those with a history of immunosuppression, suggests that EBV numbers in the GC are subject to immune control. The relationship, if any, between lymphoid hyperplasia with EBV-positive germinal centres and subsequent or concurrent lymphomagenesis remains to be clarified. As far as the development of EBV-associated Hodgkin's lymphoma is concerned, the suppression of virus replication, mediated by LMP1 on the one hand, and the loss of B-cell receptor signalling on the other, appears to be an important pathogenic mechanism. A further important emerging concept is that alterations in the microenvironment of the EBV-infected B-cell may be important for lymphomagenesis.

A retrospective analysis of patients presenting with primary lymphoma of bone (PLB) was performed to determine clinical factors affecting prognosis in relation to histological subtype and treatment outcome. Data from 106 patients, presenting with a PLB between 1943 and 1996, were retrieved from the files of the Netherlands Committee on Bone Tumours and Leiden University Medical Centre. The lymphomas were reclassified according to the REAL and updated Kiel classification. The clinical presentation, survival and prognostic factors were investigated. Sixty patients had sufficient clinical information and adequate follow-up to be included in the study. All 33 PLB that could be immunophenotyped were of B cell origin. According to the REAL classification, most PLB were large (B) cell lymphomas (92%) and according to the Kiel classification 45% of the tumours were centroblastic multilobated. PLB presented most often in the long bones (48%), with Ann Arbor stage I (46%), II (16%), IV (16%) and unknown (20%). Stage IV disease was exclusively caused by the presence of multiple bone lesions. Notwithstanding the heterogeneous treatment, the 5-year overall survival was 61%; 46% of patients were progression free at 5 years. Patients at presentation older than 60 had a worse overall survival (76% vs 37%, P = 0.0002) and a worse progression-free period (58% vs 28%, P = 0.0073). Patients with the immunoblastic subtype had a worse survival than the centroblastic mono/polymorphic subtype or the centroblastic multilobated subtype (P = 0.015). Primary lymphoma of bone represents an uncommon bone tumour with a relatively homogeneous morphology and clinical behaviour. Compared to other aggressive lymphomas, PLB have a favourable prognosis.

Diffuse large B-cell lymphoma (DLCL) is characterized by a marked degree of morphologic and clinical heterogeneity. We studied 156 patients with de novo DLCL for rearrangements of the BCL2, BCL6, and MYC oncogenes by Southern blot analysis and BCL2 protein expression. We related these data to the primary site of presentation, disease stage, and other clinical risk factors. Structural alterations of BCL2, BCL6, and MYC were detected in 25 of 156, 36 of 116, and 10 of 151 patients, respectively. Three cases showed a combination of BCL2 and BCL6 rearrangements, and two cases had a combination of BCL6 and MYC rearrangements. BCL2 rearrangement was found more often in extensive (39%) and primary nodal (17%) lymphomas than in extranodal cases (4%) (P = .003). BCL2 rearrangement was present in none of 40 patients with stage I disease, but in 22% of patients with stage II to IV (P = .006). The presence of BCL2 rearrangements did not significantly affect overall survival (OS) or disease-free survival (DFS). In contrast, high BCL2 protein expression adversely affected both OS (P = .008) and DFS (P = .01). BCL2 protein expression was poorly correlated with BCL2 rearrangement: only 52% of BCL2-rearranged lymphomas and 37% of BCL2-unrearranged cases had high BCL2 protein expression. Rearrangement of BCL6 was found more often in patients with extranodal (36%) and extensive (39%) presentation versus primary nodal disease (28%). No significant correlation was found with disease stage, lymphadenopathy, or bone marrow involvement. DFS and OS were not influenced by BCL6 rearrangements. MYC rearrangements were found in 16% of primary extranodal lymphomas, versus 2% of primary nodal cases (P = .02). In particular, gastrointestinal (GI) lymphomas (5 of 18 cases, 28%) were affected by MYC rearrangements. The distinct biologic behavior of these extranodal lymphomas was reflected by a high complete remission (CR) rate: 7 of 10 patients with MYC rearrangement attained complete remission and 6 responders remained alive for more than 4 years, resulting in a trend for better DFS (P = .07). These data show the complex nature of molecular events in DLCL, which is a reflection of the morphologic and clinical heterogeneity of these lymphomas. However, thus far, these genetic rearrangements fail as prognostic markers. © 1998 by The American Society of Hematology.

Patients with monoclonal gammopathies comprise a heterogenous group. The few studies on incidence and follow-up are single-centre-based and may reflect referral bias. To avoid this, all patients (n = 1275) in mid-western Netherlands with a newly discovered paraproteinaemia in 1991, 1992 and 1993 were included in a population-based registry and divided into four major diagnostic groups: multiple myeloma and plasmacytoma (n = 230, 18%), other haematological diseases (n = 141, 11%), paraprotein-related internal diseases (n = 191, 15%) and monoclonal gammopathy of undetermined significance (MGUS, n = 713, 56%). To avoid a possibly erroneous diagnosis, patients who were classified as having MGUS but who did not undergo confirmatory bone marrow examination were included in a separate group ‘provisional MGUS’ (n = 524, 41%), whereas patients who did were classified as having ‘definite MGUS’ (n = 189, 15%). The ‘provisional MGUS’ patients were relatively older and had more often a poor performance status, but differences between this and the ‘definite MGUS’ group were otherwise small. Patients complaining of general malaise more often had a full work-up of their paraproteinaemia. Bone pain, hypercalcaemia, high total protein, and high ESR occurred predominantly in the myeloma group, whereas fever or infection was less often seen in these patients. This registry of patients with paraproteinaemias provided valuable data related to all different diseases associated with paraproteinaemia.

Although the results of treatment of Hodgkin's disease (HD) have improved considerably in the last decades, the disease remains fatal in a minority of patients. We have recently shown that numbers of activated cytotoxic T cells (CTLs), present in tumor biopsy specimens, differ considerably among individual HD patients. Because CTLs are the major effector cells in elimination of neoplastic cells, we investigated whether the number of activated CTLs is related to the clinical outcome of the individual patient with HD. Activated CTLs present in tumor biopsy specimens of patients with nodular sclerosis or mixed cellularity HD were identified by immunohistochemistry using an antibody directed against granzyme B (GrB), a major constituent of the cytotoxic granules of activated CTLs and natural killer cells, and an antibody directed against CD8. The presence of a high percentage of GrB+ lymphocytes was found to be an unfavorable prognostic marker. The large majority of GrB+ cells were also CD8+, indicating that these cells are activated CTLs. Prognosis was found to decrease with increasing percentages of GrB+ lymphocytes. Optimal discrimination between patients with good and poor prognosis was obtained when the threshold was set at 15% GrB+ cells; 6 of 10 patients with ≥15% GrB+ lymphocytes died as a result of the disease, as compared with 6 of 70 patients with less than 15% GrB+ lymphocytes (P < .0001). In stage-2 patients, the percentage of GrB+ lymphocytes retained its predictive value in a multivariate analysis including histology, sex, age, erythrocyte sedimentation rate, and the presence of B symptoms as covariables. In addition, patients with ≥15% GrB+ lymphocytes had a shortened progression-free survival time (P = .002). We conclude that a high percentage of activated CTLs present in biopsy material of HD patients is a strong indicator for an unfavorable clinical outcome.

Objective. To define variations in the cellular infiltrate and in the expression of monokines in synovial tissue (ST) from rheumatoid arthritis (RA) patients with different durations of disease and different levels of disease activity. Methods. The immunohistologic features of synovial biopsy specimens from 31 patients with early RA ( 5 years) were compared. The possible associations between these features and local disease activity, as measured by the score for pain in the biopsied knee joint, were also evaluated. Results. The immunohistologic features were not dependent on disease duration. We found a positive correlation between the scores for knee pain and the semiquantitative scores for the number of macrophages, as well as the expression of interleukin-6 and tumor necrosis factor α, whereas the correlation with the scores for CD4+ T cells was negative. Multivariate analysis showed that these correlations were highly statistically significant (P < 0.003). Conclusion. The results do not support the view that inflammatory mechanisms in the synovial tissues of RA patients differ between early and late stages of the disease. The findings presented here are consistent with the concept that early RA is the result of a synovitis process of longer duration and that macrophage-derived cytokines play an important role in maintaining the clinical signs of inflammation.

High prevalence of oncogenic MYD88 and CD79B mutations in primary testicular diffuse large B-cell lymphoma

In follicular lymphoma, somatic hypermutation of the immunoglobulin heavy chain genes facilitates the identification of different lymphoma cell clones, and the construction of genealogical trees. To investigate the dissemination of lymphoma cells, and the role of bone marrow in disease progression, we simultaneously analyzed the somatic hypermutation patterns of lymph node and bone marrow specimens taken from three patients at onset and relapse of their disease. Immunoglobulin heavy chain genes were amplified by polymerase chain reaction, cloned and sequenced. Mutational pedigrees were constructed in a hierarchical order. When direct transition of one mutation pattern into that of a successor clones was not feasible, hypothetical predecessor clones were created, and a probability measurement calculation was introduced. Eighty-five sequenced clones were generated. The average mutation rates were 13.45% for the lymph node specimens, and 9.78% for the bone marrow ones. Forty-two hypothetical predecessor clones were introduced into inter-compartment pedigrees. The genealogical trees showed that early lymphoma clones with a low mutational load quickly migrate from lymph nodes into the bone marrow. Bi-directional lymphoma cell migration was detectable between the two compartments. In one case of follicular lymphoma, a clone identical to the initial lymph node clone was detected 2 years later in the bone marrow. The newly introduced algorithm allows the evaluation of both time and direction of follicular lymphoma cell migration. We found evidence that follicular lymphoma originates in the lymph node, and infiltrates the bone marrow early in the course of the disease. Moreover, inter-compartment migration between lymph nodes and bone marrow occurs in both directions.

Previous results suggested increased mRNA expression of CCND1 in hairy cell leukemia (HCL). The CCND1 gene is involved in the t(11;14)(q13;q32) chromosomal rearrangement, a characteristic abnormality in mantle cell lymphoma (MCL). We and others reported that, in contrast to other B-cell lymphomas, almost all MCL have over-expression of the CCND1 gene with a good correlation between RNA and protein analysis. Recent studies showed that overexpression of the cyclin D1 protein can be easily detected by immunohistochemistry (IHC) on formalin-fixed, paraffin embedded tissues.To investigate whether the CCND1 gene is involved in HCL, we performed IHC on a series of 22 cases using formalin-fixed paraffin embedded splenectomy specimens. For IHC the sections were boiled in citrate buffer. The presence of rearrangements within the BCL-1 locus and the CCND1 gene was analyzed in 13 of 22 cases by Southern blot analysis using all available break-point probes. Expression of CCND1 was analyzed at the mRNA level (Northern blot) and protein level (IHC).Overexpression of the cyclin D1 protein using IHC was observed in all cases, with strong expression in 5 cases. Pre-existing B- and T-cell areas of the spleen did not express significant levels of the cyclin D1 protein. Seven of 9 cases analyzed by both IHC and Northern blotting showed overexpression of the CCND1 gene with both methods. No genomic abnormalities were observed in any of the 13 cases studied by Southern blot analysis. Additionally, no 11q13 abnormalities were detected by banding analysis of 19 of 22 cases.The elevated levels of CCND1 mRNA and protein in conjunction with the absence of overt rearrangements within the BCL-1 locus distinguish HCL from MCL and other B-cell malignancies. This suggests that activation of the CCND1 gene in HCL is due to mechanisms other than chromosomal rearrangement.

An International Prognostic Index (IPI) for patients with aggressive non-Hodgkin's lymphoma (NHL) has recently been published. The IPI is based on pretreatment clinical characteristics and developed on clinical trial patients, classified as intermediate grade according to the Working Formulation (WF). We applied this IPI in a population-based registry of NHL patients. This registry does not have the restrictions that usually hold for patients in clinical trials, eg, with respect to age and performance status. Moreover, it covers all the three WF classes (low, intermediate, and high). The IPI turned out to be of prognostic value for response rate and survival in our unselected cohort of 744 patients, as well. In each of the three WF classes separately, the four IPI classes showed going from low to high substantially decreasing response rates and survival percentages. For our cohort of WF intermediate grade patients 5-year survival levels were lower in all four IPI classes (59%, 34%, 14%, and 10%, respectively), probably reflecting the selection of clinical trial patients in the original study (73%, 51%, 43%, and 26%).

Successive oncogenic steps are necessary to generate cancer. In many B-cell lymphomas, chromosomal translocations are considered to be an early oncogenic hit. We investigated whether the lymphoma-associated t(14;18) involving the BCL2 oncogene can occur outside the context of malignancy. To this end, we extensively screened blood cells from healthy blood donors by a very sensitive seminested polymerase chain reaction (PCR) for breakpoint junctions at JH1–5 on 14q32 and the major breakpoint region of BCL2 on 18q21. In each individual, mononuclear cells, granulocytes, flow-sorted B cells, and T cells were separately tested in five to seven independently performed PCRs (in total, 0.5 x 10(6) to 1.0 x 10(6) cells per fraction per individual). Amplification products that hybridized with an internal BCL2 probe and a JH probe were sequenced. Six of nine individuals harbored t(14;18) breakpoints. Translocations were restricted to B cells, with an estimated frequency of 1 in 10(5) or less circulating B cells. In total, 23 of 51 experiments on B cells were positive in contrast to 1 of 48 on T cells and 2 of 47 experiments on granulocytes. Consistent with the presence of 4.7% to 13.0% B cells in the mononuclear cell fractions, only very few (4 of 47) tests were positive in these fractions. Sequence analysis showed that four of six individuals harbored two to five unrelated t(14;18)-carrying B-cell clones. All breakpoints had a structure similar to that in follicular lymphoma. We propose that B cells with the t(14;18) translocation are regularly generated in normal individuals, but that only very few cells with the translocation will acquire the additional oncogenic hits necessary to establish the malignant phenotype.

Downregulation of MEIS1 impairs long-term expansion of CD34 + NPM1-mutated acute myeloid leukemia cells

During the past 10 years, MDS has become a topic of considerable interest due to the development of new cytogenetic and molecular techniques and the introduction of the clinical administration of hematopoietic growth factors. The cytological and clinical classifications of the diseases comprising the syndrome are based on two main subtypes, those with a low risk of acquiring acute leukemia and those with a high risk. Which one will prove to be the simplest and most reliable classification system for the individual patient is not yet clear.

In this issue of Blood , Tellier et al report on extremely rare t(14;18)-positive cells within reactive lymph nodes of normal individuals. They convincingly show that these cells preferentially reside as nonproliferating cells in germinal centers.[1][1] ![Figure][2] Tellier et al[1][1]

A 70-yr-old woman presented with a severe haemorrhagic diathesis due to an acquired factor X deficiency. A plasma infusion study showed that exogenous factor X was eliminated very effectively from the patient's circulation. A bone marrow biopsy was consistent with plasma cell dyscrasia. Neither an abdominal fat biopsy nor the bone marrow biopsy confirmed an amyloidosis, although clinically no other diagnosis seemed possible. Treatment with intermittent chemotherapy, consisting of vincristine, cytoxan and prednisone, yielded definite clinical and laboratory improvement.

Clonality of myeloid and lymphoid cell fractions obtained from peripheral blood (PB) or bone marrow (BM) of five patients with a myelodysplastic syndrome (MDS), was studied by combined immunophenotypic analysis and DNA in situ hybridization. This novel technique enables quantitative and direct analysis of cytogenetic alterations in nondividing cells of distinct cell lineages. Four patients with a trisomy 8 and one patient with a translocation (1;7) were studied. For cell lineage determination, antibodies specific for progenitor cells (CD34), myeloid cells (CD15), monocytes (63D3), T cells (CD3), and B cells (CD19,20,22) were used. In one patient with a trisomy 8, BM cells were available and the erythroid lineage could be studied. For detection of cytogenetic aberrations, we used chromosome- specific repetitive DNA probes. In three patients, all nonlymphoid cells carried the cytogenetic abnormality; in two patients, mosaicism within these lineages was suggested by the relative low numbers (35% to 55%) of aberrant cells. None of the T or B cells of the five patients carried the chromosomal aberrations. We conclude that combined immunophenotyping and in situ hybridization is a feasible technique to study lineage involvement. Our data suggest that the chromosomal aberrations studied in MDS are restricted to the myeloid lineages.

Mantle cell lymphoma (MCL) is characterized by genetic instability and a poor prognosis. Many blastoid variants are (hypo)tetraploid and have ail even worse prognosis. We investigated the role of signalling by mitogen-activated protein kinases (MAPKs) in MCL. As compared to normal tonsil B cells, MCL, cells showed higher activation of the JNK MAPK in both an MAPK array and a sandwich ELISA assay. Immunohistochemistry showed overexpression of phospho (p)-JNK (Thr183/Tyr185) in 30 of 37 MCL cases. Inhibition of p-JNK with SP600125 resulted in growth arrest in all four MCL cell lines (Jeko-1, HBL-2, UPN-1, Granta-519), which could be partly reversed by the addition of CD40L and IL-4. Furthermore, SP600125 led to G2/M phase arrest on day I and a striking increase in endoreduplication on day 2 and day 3, which was confirmed by karyotype analysis. G2/M arrest was associated with down-regulation of EGR1 and p21 protein expression. SP600125 induced polyploidy could be blocked by the BCL-2 inhibitor YC137. These data suggest that constitutive JNK activity is necessary to promote proliferation and maintain diploidy in MCL. JNK inhibition leads to cell cycle deregulation and endoreduplication, mimicking the tetraploid state seen in a subset of MCL cases. Thus, our data also provide an experimental model to study polyploid MCL. cells. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Forty-four B-chronic lymphocytic leukemias (CLL) were studied by Southern blot analysis using probes for the Ig genes and bcl-1, bcl-2 (major, minor and 5′ breakpoint region), bcl-3, c-myc, and retinoblastoma (Rb) loci. Eight cases had three or more rearranged JH bands, indicating oligoclonality, clonal evolution, or chromosomal translocation. One case had a rearrangement of the bcl-1 locus and three of the bcl-2 locus. In the first case, comigration of the rearranged bcl-1 and JH sequences indicated a t(11;14)(q13;q32) translocation, which, in contrast to previously described cases, seems to be completely reciprocal. One case with a bcl-2 rearrangement showed comigration of the bcl-2 major breakpoint region and a rearranged JH band. This indicates a t(14;18) (q32;q21). The two other cases showed rearrangements of the bcl-2 5′ breakpoint region without apparent comigration. No rearrangements were detected of c-myc and bcl-3, located at chromosome 19, nor was a deletion of Rb found. All but three cases had CD5 expression. The exceptions included the t(11;14) and the t(14;18) cases. Our results confirm recent data on rearrangements at the 5′ site of bcl-2 in CLL. Additionally, they corroborate the presumption that CD5-negative chronic B-cell leukemias should be considered apart from classical CLL.

Extranodal marginal zone B-cell lymphomas (MZBCLs) arise on a background of chronic inflammation resulting from organ-specific autoimmunity, infection, or by unknown causes. Well-known examples are salivary gland MZBCL in Sjögren's sialadenitis and gastric MZBCL in Helicobacter pylori gastritis. MZBCLs express CXCR3, a receptor for interferon-γ–induced chemokines highly expressed in the chronic inflammatory environment. The immunoglobulin (Ig) variable heavy/light chain (IgVH/IgVL) gene repertoire of salivary gland and gastric MZBCL appears restricted and frequently encodes B-cell receptors with rheumatoid factor reactivity. Primary cutaneous marginal zone B-cell lymphomas (PCMZLs) are regarded as the skin-involving counterparts of extranodal MZBCLs. Although PCMZLs have been associated with Borrelia burgdorferi dermatitis, PCMZLs generally arise because of unknown causes. We studied an extensive panel of PCMZLs and show that PCMZLs do not conform to the general profile of extranodal MZBCL. Whereas most noncutaneous MZBCLs express IgM, PCMZLs in majority express IgG, IgA, and IgE and do not show an obvious immunoglobulin repertoire bias. Furthermore, the isotype-switched PCMZLs lack CXCR3 and seem to arise in a different inflammatory environment, compared with other extranodal MZBCLs.

DNA amplification of the 11q13 region is observed frequently in many carcinomas. Within the amplified region several candidate oncogenes have been mapped, including cyclin D1, TAOS1 and cortactin. Yet, it is unknown which gene(s) is/are responsible for the selective pressure enabling amplicon formation. This is probably due to the use of low-resolution detection methods. Furthermore, the size and structure of the amplified 11q13 region is complex and consists of multiple amplicon cores that differ between different tumor types. We set out to test whether the borders of the 11q13 amplicon are restricted to regions that enable DNA breakage and subsequent amplification. A high-resolution array of the 11q13 region was generated to study the structure of the 11q13 amplicon and analyzed 29 laryngeal and pharyngeal carcinomas and nine cell lines with 11q13 amplification. We found that boundaries of the commonly amplified region were restricted to four segments. Three boundaries coincided with a syntenic breakpoint. Such regions have been suggested to be putatively fragile. Sequence comparisons revealed that the amplicon was flanked by two large low copy repeats known as segmental duplications. These segmental duplications might be responsible for the typical structure and size of the 11q13 amplicon. We hypothesize that the selection for genes through amplification of the 11q13.3 region is determined by the ability to form DNA breaks within specific regions and, consequently, results in large amplicons containing multiple genes.

Several risk factors including immune deficiencies, infections, and autoimmune diseases have been established for non-Hodgkin's lymphoma (NHL). For diffuse large B cell lymphoma (DLBCL), the most common type of lymphoma, no risk factors have been described, which may be due to the intrinsic heterogeneity of this disorder. Previously we reported that, in contrast to nodal DLBCLs, the majority of testicular DLBCLs manifested complete loss of HLA-DR and -DQ expression associated with homozygous deletions of the corresponding genes. To determine the correlation between HLA class II polymorphisms and these lymphomas, we applied DNA typing for HLA-DR81 and HLA-DQB1 on 50 Dutch patients with testicular and 48 with nodal DLBCL and compared the frequencies with a cohort of healthy Dutch controls. Both the patients with nodal and those with testicular DLBCL manifested significantly higher frequencies of HLA-DRB1*15 than the controls (p

Most chromosomal t(8;14) translocations in sporadic Burkitt lymphomas (BL) are mediated by immunoglobulin class switch, recombination (CSR), yet all tumors express IgM, suggesting an incomplete or exclusively monoallelic CSR event. We studied the exact configuration of both the nontranslocated IGH allele and the MYG/IGH breakpoint by applying a combination of low- and high-resolution methods (interphase FISH, DNA fiber FISH, long-distance PCR, and Southern blotting) on 16 BL. IGH class switch events involving the nontranslocated IGH allele were not observed. Thirteen cases had MYG/IGH breakpoints in or nearby IGH switch (S) sites, including five at S mu, three at S gamma and five at S alpha. All eight translocations with a breakpoint at S gamma or S alpha were perfectly reciprocal, without deletion of C mu-C delta or other CH elements. Internal S mu deletions claimed to be a marker for CSR activity and implicated in stabilization of IgM expression were found in BL but did riot correlate with downstream translocation events. This study shows that switch breakpoints in sporadic BL are exclusively resolved by a noncanonical recombination mechanism involving only one switch region. (c) 2006 Wiley-Liss, Inc.

Immunophenotyping of B cell chronic lymphocytic leukaemia (B-CLL) is usually performed by flow cytometry on cell suspensions obtained from peripheral blood, bone marrow or biopsied tissue. Immunohistochemical analysis on routine sections is less commonly performed; however, this approach allows the pathologist and the researcher to appreciate the immuno-architecture of the involved tissues and to gain insight into some of the events that influence the biology of the disease. in this review the authors focus on the following issues: immuno-architecture of the proliferation centres, expression of CD23, MUM1/IRF-4 and cyclin D1, tyrosine phosphorylation and detection of the ZAP-70 kinase. Whenever possible, an attempt is made to interpret the immunohistochemical findings from a functional point of view.

The prognostic significance of age was studied in 372 patients with diffuse large B-cell non-Hodgkin's lymphoma, in relation to the prognostic factors of overexpressed BCL2 and p53 oncoprotein. Overexpression of BCL2 and p53 oncoprotein was defined when more than 50% of the tumor cells showed positive staining. The data were analyzed with respect to the age groups

A patient with follicular lymphoma developed a striking but transient plasmacytosis in blood and bone marrow with paraproteinaemia after treatment by autologous bone marrow transplantation and interleukin-3 (IL-3). By immunophenotyping, cytogenetics and Southern blotting, we proved that the plasma cells were clonally related to the original lymphoma cells. This event was preceded by fever accompanied by high IL-6 concentrations. The patient recovered spontaneously and had a long-lasting remission. We speculate that the bone marrow-derived malignant B cells were stimulated by IL-3 and IL-6 into terminally differentiated plasma cells.

Lymphomatoid papulosis (LyP) is defined as a recurrent self-healing papulonodular eruption with the histological features of a (CD30+) cutaneous T-cell lymphoma. The atypical cells usually have a CD3+/-, CD4+/-, CD8-, CD30+, CD56- T-cell phenotype. We report an unusual case of LyP, in which the atypical cells expressed a CD3-, CD4-, CD8-, CD30+, CD56+ phenotype. Detailed phenotypic and genotypic analysis confirmed that these cells had a natural killer (NK)-cell phenotype. Lymphomas with an NK-cell phenotype usually have a poor prognosis. However, the waxing and waning of papular lesions for more than 20 years and the excellent response to low-dose oral methotrexate in this patient suggest similar clinical behaviour to LyP cases with a T-cell phenotype.

Gastric MALT lymphoma is a distinct entity related to Helicobacter pylori gastritis. Some studies suggest a role for trisomy 3 in the genesis of these lymphomas, but they mainly focused on low-grade MALT lymphoma. Gastric MALT lymphoma, however, comprises a spectrum from low- to high-grade cases. Furthermore, its exact relation to primary diffuse large B cell lymphoma (DLBCL) of the stomach is not clear. We applied in situ hybridisation (ISH) with centromeric probes on 43 samples of 39 patients with primary gastric lymphoma (13 samples with low-grade MALT lymphoma, 25 with high-grade MALT lymphoma and five with DLBCL) to detect numerical aberrations of 10 chromosomes. ISH was performed immunohistochemically on nuclei isolated from paraffin-embedded resection tissue and on whole paraffin sections using immunofluorescence. In six of 13 low-grade MALT lymphomas trisomy was detected (46%) and mostly involved chromosome 3 (33%). In high-grade MALT lymphomas, trisomies were found in 16 of 25 cases (64%), mainly involving chromosomes 12 and 18. Trisomy 3 was present in only 13% of these cases. Of five DLBCL, only one showed trisomy. Nine of the 16 aberrant high-grade MALT lymphomas (56%) showed trisomy of more than one chromosome per case vs two of six for low-grade cases. In lymphomas with separate low- and high-grade tumour components some trisomies were detected in both components, whereas others occurred only in the high-grade tumour cells. This supports the hypothesis that high-grade MALT lymphomas can develop from a low-grade type and that this progression is accompanied by the acquisition of more genetic aberrations. However, trisomy 3 probably does not play a major role in this progression.

BACKGROUND To compare characteristics, treatment and outcome of patients > or = 70 years with patients < 70 years in a population-based non-Hodgkin's lymphoma (NHL) registry. PATIENTS AND METHODS All new patients with NHL (n = 1168) in a geographically defined region in the western part of The Netherlands were registered during a nearly 10-year period. Patient, tumour and treatment characteristics, response to therapy and survival were analysed for both age groups. An age-adjusted prognostic index was determined for elderly patients with aggressive lymphoma. RESULTS The elderly comprised 41% of the registered patients. There were significantly more females, a preponderance of intermediate-grade histology (diffuse large B-cell lymphoma) and a lower performance status. Incomplete staging in the elderly was mostly due to the omission of a bone marrow biopsy. With respect to WF grading the complete remission rate (except for patients with low-grade/stage I NHL, patients with extranodal NHL and for patients with intermediate grade/extensive NHL) and overall survival at five years (except for patients with low-grade/stage I NHL and for patients with intermediate-grade/extensive NHL) were significantly inferior in the elderly. With respect to the R.E.A.L. Classification the exceptions were in patients with high grade MALT lymphomas (elderly good) and patients with mantle-cell and peripheral T-cell lymphomas (younger group bad too). However, once complete remission was reached, the disease-free survival did not differ significantly between the two age groups, emphasising the importance of achieving complete remission. Although 65% of the classified elderly patients presented with intermediate-grade NHL, only 26% of the elderly patients treated with chemotherapy received anthracycline-based chemotherapy. In the elderly, lymphoma (treatment-related toxicity included) contributed to death in 70% and concomitant disease (other malignancy included) in 30%, versus 78% and 22%, respectively, for the younger group (P = 0.04). The age-adjusted prognostic index, made up of the factors serum LDH, stage and Karnofsky index, showed a clear distinction between the four risk categories low, low/intermediate, intermediate/high and high, with a median survival time of 43, 20, seven and four months, respectively. For the younger group the respective numbers were 144, 45, 19 and 11 months. CONCLUSIONS In a population-based NHL registry the elderly, predominately female patients, formed a larger proportion of the patient group than the one usually reported in the literature. In this population-based cohort inferior remission and overall survival rates were seen in the elderly. However, obtaining complete remission was beneficial for the prognosis of this disease in the elderly. By the application of the R.E.A.L. Classification important subgroups emerge.

Blood cell transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho+/++(dull/bright) and Rho−(negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho+/++cell population contained >99% of committed progenitor cells within vitrocolony-forming ability. The Rho−cell population contained the majority of hematopoietic stem cells within vivomarrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho−stem cells or purified Rho−stem cells in combination with large numbers of Rho+/++committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho−stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.

Osseous involvement is one the predominant features of patients with multiple myeloma (MM). Whole body X-ray (WBX) is the standard method for detecting skeleton abnormalities but has limited value in relapsing disease since it cannot make a distinction between old and new active skeleton lesions. Therefore changes in metabolic activity might be a more useful method to detect new skeleton lesions in MM. This can be assessed by [F-18] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging. Consequently WBX and FDG-PET was studied in relapsing MM prior to treatment. In addition immunohistochemical staining on bone marrow biopsies was performed for glucose transport protein (GLUT) 3 expression, micro vessel density (MVD), and vascular endothelial growth factor (VEGF) to demonstrate whether these parameters might be predictive for the in vivo scanning results. This study included 44 patients (median age 62 yr (range 48-78)) with a median of 3.5 (range 2-12) relapses. Thus far 86% of the patients were treated with autologous stem cell transplantation in combination with bortezomib (78%) or lenalidomide (65%). New lesions on FDG-PET were more frequently demonstrated than new lesions on WBX (p=0.00001). In 78% of the patients FDG-PET demonstrated a median of 4 lesions (range 0-22) per patient with a median standard uptake value (SUV) max of 5.7 (range 0-24). In 42% of the patients new X-ray lesions (median 0 (range 0-7)) were demonstrated and these were concordant with the results of FDG-PET. In 5 % of the patients new X-ray lesions were observed without FDG-PET lesions. For the relapsing patients treatment consisted of dexamethasone in combination with bortezomib (65%) or lenalidomide (50%). CR was obtained in 27% of the patients, PR in 39% and progressive disease in 22%. Average time to next treatment (TTNT) was 11.7 months (2-43). However FDG-PET positivity was not predictive for PFS (p =0.55) and OS (p=0.40) neither if PET positivity was defined as more than 3 lesions nor if the cut off value of SUV max > 4.2 was applied. Immunohistochemical staining on bone marrow biopsies (n=20) with CD34 demonstrated a significant increased MVD of 39 ± 54 (mean ± SD, normal bone marrow MVD 3.5 ± 2.9), in particular when clusters of plasma cells were present. These enhanced MVD was associated with a significant increased expression of VEGF and GLUT-3. However MVD (p = 0.16), VEGF (p = 0.70) or GLUT-3 (p =0.70) expression were not predictive for having a positive or negative FDG-PET. In summary, our results demonstrate that FDG-PET is more informative than WBX for detecting skeleton lesions in relapsing MM patients. However, and in contrast to what was previously shown in patients with newly diagnosed MM, FDG-PET it is not predictive for treatment outcome. This might be due to heterogeneity in studied patients and treatments. Disclosures: No relevant conflicts of interest to declare.

Objective. To study the effect of chimeric anti-CD4 monoclonal antibody (MAb) therapy on synovial inflammation, in order to interpret the clinical experience with anti-CD4 treatment. Methods. The immunohistologic features of synovial biopsy specimens before and 4 weeks after anti-CD4 MAb (cM-T412) therapy were studied in patients with rheumatoid arthritis. The patients received intravenous doses of either placebo (n = 1) or 10 mg (n = 4), 25 mg (n = 2), or 50 mg (n = 1) of cM-T412 daily for 5 consecutive days. Results. Although the patients did not experience clinical improvement, significant decreases in the number of circulating CD4+ cells, the degree of synovial inflammatory infiltration, and the mean scores for expression of adhesion molecules were found in the 7 patients 4 weeks after receiving cM-T412. The scores for infiltration with CD4+ and other inflammatory cells were particularly reduced following treatment with either 25 mg or 50 mg cM-T412. Cytokines, such as interleukin-1β and tumor necrosis factor α, could still be detected in the synovial tissue after treatment. Conclusion. The decline in the numbers of inflammatory cells and adhesion molecules in synovial tissue after CD4+ cell depletion supports the view that CD4+ T cells orchestrate local cellular infiltration. The lack of clinical effect of anti-CD4 therapy might be explained by an insufficient decrease in the number of synovial CD4+ cells and by the persistence of cytokines. Determination of whether more adequate dosing would lead to a clinical improvement must await further study.

We have developed a modified rhodamine (Rho) staining procedure to study uptake and efflux in murine hematopoietic stem cells. Distinct populations of Rho++ (bright), Rho+ (dull), and Rho- (negative) cells could be discriminated. Sorted Rho- cells were subjected to a second Rho staining procedure with the P-glycoprotein blocking agent verapamil (VP). Most cells became Rho positive [Rho-/Rho(VP)+ cells] and some remained Rho negative [Rho-/Rho(VP)- cells]. These cell fractions were characterized by their marrow-repopulating ability in a syngeneic, sex-mismatch transplantation model. Short-term repopulating ability was determined by recipient survival for at least 6 weeks after lethal irradiation and transplantation--i.e., radioprotection. Long-term repopulating ability at 6 months after transplantation was measured by fluorescence in situ hybridization with a Y-chromosome-specific probe, by graft function and recipient survival. Marrow-repopulating cells were mainly present in the small Rho- cell fraction. Transplantation of 30 Rho- cells resulted in 50% radioprotection and > 80% donor repopulation in marrow, spleen, and thymus 6 months after transplantation. Cotransplantation of cells from both fractions in individual mice directly showed that within this Rho- cell fraction, the Rho-/Rho(VP)+ cells exhibited mainly short-term and the Rho-/Rho(VP)- cells exhibited mainly long-term repopulating ability. Our results indicate that hematopoietic stem cells have relatively high P-glycoprotein expression and that the cells responsible for long-term repopulating ability can be separated from cells exhibiting short-term repopulating ability, probably by a reduced mitochondrial Rho-binding capacity.

Adhesion between leukemic cells and the vascular endothelium has been suggested to play a role in the development of leukostasis in myelocytic leukemia. To define the role of adhesion molecules on the surface of endothelial cells in leukostasis, we used immunohistochemistry to study the expression of endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in lung tissue of 4 patients with pulmonary leukostasis. Lung tissue of 2 patients with myelocytic leukemia without leukostasis and 4 patients with irrelevant nonpulmonary disease was used as a negative control. Positive control tissues included a lymph node with angioimmunoblastic lymphadenopathy and a hyperplastic tonsil. Weak positive staining for ELAM-1 was found in 1 patient in vessels, both with and without leukostasis. Expression of VCAM-1 and ICAM-1 in all patients tested was similar to that in the negative controls. The results of this study suggest that activation of endothelium, with increased expression of the endothelial adhesion molecules under study, is not a prerequisite for the development of pulmonary leukostasis in leukemia.

We report the cases of two patients who had infections due to Yersinia enterocolitica. The first patient exhibited chronic recurrent fever, hepatic and splenic granulomas, and bone marrow abnormalities, and the second patient presented with enterocolitis with leukocytoclastic vasculitis of the skin. Cultures and agglutination titers were negative. Indirect immunofluorescence techniques with use of serotype-specific antisera and antisera to Yersinia outer-membrane proteins (Yops) were applied to biopsy specimens, and immunoblotting techniques for determining class-specific circulating antibodies to Yops were used for demonstrating these unusual manifestations of Y. enterocolitica infections.