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Bendamustine-based conditioning for non-Hodgkin lymphoma autologous transplantation: an increasing risk of renal toxicity

Les biopuces a ADN font partie des avancees technologiques importantes des toutes dernieres annees qui permettent d'ouvrir la voie a une comprehension plus globale et plus approfondie de la biologie tumorale. Par l'hybridation de l'ADN complementaire synthetise a partir d'un echantillon tumoral sur une surface contenant des sequences de genes differents, cette technique va permettre en une seule etape d'evaluer de facon quantitative l'expression de plusieurs milliers de genes au sein de cette tumeur. Les lymphomes malins, de par leur heterogeneite tant biologique que clinique, constituent un modele de choix pour l'utilisation de ce nouvel outil. Appuyee sur des analyses bio-informatiques complexes, l'analyse de plusieurs series d'echantillons a recemment permis la description des voies de signalisation activees dans ces tumeurs lymphoides, aboutissant a la reconnaissance de la proximite ou au contraire a l'individualisation de certains sous-types histologiques, a l'identification de predicteurs biologiques de diagnostic ou de pronostic, et enfin a la caracterisation de nouvelles cibles pour des agents pharmacologiques specifiques. A travers les publications realisees a ce jour, nous allons discuter dans cette revue l'impact de cette technique sur la comprehension de la biologie et la clinique des lymphomes malins, de type Hodgkinien et non Hodgkinien.

It is now established that a subgroup of non-Hodgkin's lymphoma (NHL) patients probably benefit from high-dose therapy (HDT). We therefore retrospectively analyzed survival of 126 consecutive patients with large cell lymphoma (LCL) and high-intermediate (HI) or high-risk (H) age-adjusted international prognostic index (Aa-IPI). They received either standard chemotherapy (CT) (66 patients), or HDT (60 patients). Distribution of the Aa-IPI scores showed no statistical significant difference between the two treatment groups. Complete response (CR) rate was 51% for the whole series, with 41% and 62% for the standard CT group and HDT group, respectively. With a median follow-up of 63 months (range, 16 to 159), the 5-year overall survival (OS) and event-free survival (EFS) for all patients was 52% and 43%, respectively. There was a statistical significant difference in terms of survival towards the HDT group: OS at 76% vs 31%, EFS at 64% vs 24%. Patients who achieved CR with front-line therapy had a 5-year OS at 70%, while it was 34% for patients who were not in CR. These results are comparable to those reported in the literature, and strongly suggest that both initial CR achievement and HDT as front-line treatment are predictive factors for prolonged survival of patients with poor-risk LCL. Bone Marrow Transplantation (2000) 25, 35-40.

Aims The oesophageal mucosa is a frequent target of opportunistic infections in human immunodeficiency virus (HIV) infection. Langerhans cells (LC) are known as a target and reservoir of HIV in the skin. The aim of this study was to characterize oesophageal LC in HIV-infected patients. Methods and results Thirty oesophageal biopsies were obtained from 29 patients (median age 35.5), all in stage IV of the HIV Center of Disease Control Classification. We performed histological assessment of the oesophageal mucosa and immunohistochemical detection of oesophageal LC using an anti-CD1a antibody, followed by morphometric analysis. Biopsies from 17 noninfected patients were studied using the same procedure. LC in oesophageal mucosa of the HIV positive patients showed a significantly and dramatically decreased number (LC(N) median = 5.85/mm2) and surface/epithelial surface (LC (S) ratio = 0.09) when compared with HIV-negative controls (LC(N) median = 29.7/mm2, LC(S) ratio = 1.83) with P = 0.003 for LC(N) and P

The Fas receptor (APO-1/CD95) is capable of inducing apoptosis of lymphoid cells and is expressed in some non-Hodgkin's lymphomas (NHLs). Fas expression is up-regulated at the surface of normal B cells upon triggering of the CD40 receptor. In this report, we investigated the sensitivity of NHLs to Fas-mediated apoptosis induced by anti-Fas monoclonal antibodies (MAbs) and its possible modulation by CD40 ligation in 18 NHL biopsy samples of various histological subtypes. Flow cytometric analysis showed that the fraction of Fas-expressing lymphoma cells was highly variable from sample to sample (from 1% to 93%, mean value 46%). The frequency of apoptotic cells was not significantly increased upon treatment with an anti-Fas MAb compared with control MAb in the 18 NHL cases analysed. The sensitivity of lymphoma cells to Fas-mediated apoptosis was correlated neither with the histological subtypes nor with the level of Fas expression. Activation of neoplastic B cells by CD40 ligation resulted in significant increases in Fas expression and Fas-induced apoptosis among the five B-NHL cases tested. The overall increase in apoptotic rates was moderate and remained lower in tumour samples than in control CD40-activated normal tonsil B cells. Altogether, our results indicate that the sensitivity to Fas-induced apoptosis is null or weak in NHL cells, irrespective of their histological subtype, and that it can be increased to a moderate and variable degree by CD40 ligation on neoplastic B cells. This may be an impediment to the development of Fas-based therapies for NHLs.

AIMS: To confirm the recent data obtained in mice, showing that the Fas ligand (FasL) is involved in the phenomenon of "immune privilege" (the apparent defect of the immune system in specific anatomical sites) and to extend this finding to humans. METHODS: The expression of FasL was analysed in a panel of histologically normal human tissues by reverse transcriptase polymerase chain reaction and Western blotting. The tissues sampled were brain, breast, bone marrow, oesophagus, kidney, liver, lung, lymph node, ovary, pancreas, pituitary gland, prostate, spleen, stomach (antrum and fundus), striated muscle, testis, thyroid, and uterus. These were obtained from patients with various neoplastic and non-neoplastic disorders; placental tissue was obtained after normal obstetric delivery, and spontaneous or voluntary abortion. RESULTS: Strong FasL expression was detected in testis and placenta. FasL expression was also detectable, although it was seen to a lesser extent, in oesophagus, prostate, lung, and uterus, which also coexpressed variable amounts of Fas mRNA or protein or both. The other organs tested for FasL expression were all negative. CONCLUSIONS: FasL in humans is expressed predominantly in immune "sanctuaries" such as testis and placenta, suggesting that, similar to mice, this expression may contribute to the immune privileged status of these organs, by preventing dangerous inflammatory responses. The coexpression of FasL and Fas in particular epithelia suggests that the physiological cell turnover of some tissues may be regulated by the Fas-FasL apoptotic pathway.

FAS/APO-1 (CD95) is a membrane glycoprotein belonging to the tumour necrosis factor/nerve growth factor receptor family, and which can trigger apoptosis in some lymphoid cell lines. Immunohistochemistry combined with Northern blotting allowed determination of the pattern of FAS/APO-1 expression in a series of Ki-1 [CD30] positive lymphoid malignancies, including 27 Hodgkin's disease and eight anaplastic large cell lymphomas. CD30 negative tumours used as controls included 27 B-cell non-Hodgkin's lymphomas. 14 T-cell non-Hodgkin's lymphomas, four reactive lymphadenitis, and non-lymphoid tissues. Immunohistochemistry, performed on frozen sections, revealed a strong FAS/APO-1 expression in 25 out of 27 (92%) Hodgkin's disease cases, predominantly in Reed Sternberg cells; 50 to 100% of the neoplastic cells in eight out of (100%) anaplastic large cell lymphoma cases were positive. In contrast, positive FAS/APO-1 immunostaining was observed only in 22 out of 41 (53%) CD30 negative non-Hodgkin's lymphomas. Northern blot analysis detected variable amounts of the FAS/APO-1 transcript in the immunohistochemistry-positive samples. These results suggest possible hyper-expression of FAS/APO-1 (CD95) in Hodgkin's disease and anaplastic large cell lymphomas.

The management of patients presenting with metastatic malignant melanoma (MM) is hampered by the substantial variability in survival of these patients and the lack of prognostic markers. In the search for a reliable predictive parameter, we have investigated the expression of the nm23 gene, considered to be a major regulator of the metastatic process. We have analysed by Northern blot the nm23 mRNA level in tumour tissue obtained from metastases of 20 stage II and ten stage III patients with MM. Normal human tissues and benign naevi were simultaneously examined. The level of nm23 expression was highly heterogeneous in MM metastases, with a mean value which was higher than the mean level in normal tissues and naevi. Correlative study was focused on the overall survival following resection of the metastasis in which nm23 Northern blot analysis was performed. Patients displaying higher nm23 expression in metastatic tissue (above the mean level) tended to have a longer survival than others (P = 0.08), and this difference was significant for patients presenting with isolated regional lymph node involvement (P = 0.035). The time from biopsy of the primary MM to the appearance of the first lymph node metastasis also showed a positive correlation with the nm23 mRNA level in this metastasis. The present study is not only in accordance with previous reports showing that the nm23 gene may be implicated in MM progression, but also suggests the reliable value of nm23 expression as a prognostic marker for patients presenting with metastatic MM. Images Figure 1

The influence of the mode of intravenous (i.v.) administration (bolus injection or continuous infusion) on the tissue penetration of ceftazidime was studied in the rat. The antibiotic concentration was monitored in serum, pleural exudate, vitreous humor, kidney, liver, lung, testicles and epididymal fat tissue. Administration as a bolus resulted in a significantly higher AUC in pleural exudate and in higher peak levels in serum, liver and lung than continuous infusion, which produced a higher peak concentration in kidney than a bolus. No differences in AUC and peak concentrations between the two methods of administration were observed in the other tissues or fluids. With either method of administration the highest antibiotic accumulation was observed in kidney.

Summary Primary central nervous system lymphomas (CNSL) are uncommon neoplasms accounting for about 1% of primary brain tumors. Patients with congenital or acquired immunodeficiencies including AIDS patients and transplant recipients represent the main high-risk population for CNSL occurrence. An important point emerging from the literature is that CNSL incidence has dramatically increased during the last years not only in HIV infected patients by virtue of the AIDS epidemic spread, but also for unclear reasons in immunologically normal persons. Although c-myc oncogene activation and Epstein-Barr virus infection are considered to playa role in CNSL development, the peculiar tendency of these lymphomas to occur and remain inside the CNS is not well understood and may involve putative CNS binding molecules carried by lymphocytes. The clinical presentation is characterized by a great variety of neurological disorders. Radiological features consist of hyperdense homogeneous deposits within the subcortical white matter with a pattern of marked enhancement after injection of contrast material. The tumor masses are usually ill-defined and multicentric. Although all cytological types can be observed, the most common types belong to the high-grade category of non-Hodgkin's lymphoma. Monoclonal antibodies reactive with formalin-fixed, paraffin-embedded sections can be used in conjunction with stereotactic needle biopsy to provide accurate immunological characterization of CNSL. The large majority of CNSL is of B-cell origin but T-cell lymphomas seem at the present time less exceptional than previously thought. Although radiotherapy and chemotherapy can increase length of survival, the prognosis of CNS remains dramatically poor, the shortest survival being observed in AIDS patients.

*Correspondence to: Dr T. Rudiger, Department of Pathology, University of Wurzburg, Josef-Schneider-Str. 2, D-97080 Wurzburg, Germany. Tel: +49-931-201-1783; Fax: +49-931-888-7518; E-mail: thomas.ruediger@mail.uni-wuerzburg.de University of Wurzburg, Wurzburg, Germany; British Columbia Cancer Agency, Vancouver, Canada; Department of Health & Human Services, NIH-NCI, Bethesda, USA; The Netherlands Cancer Institute, Amsterdam, The Netherlands; The Norwegian Radium Hospital and Institute for Cancer Research, Oslo, Norway; Universitaire Ziekenhuizen Leuven, Leuven, Belgium; University Hospital Groningen, The Netherlands; Institut Paoli-Calmettes, Marseille; Hopital Saint-Louis, Paris, France; Clinic of Internal Medicine I, University of Cologne, Cologne, Germany

Microarray technology has recently led to the identification of molecular prognostic subgroups in non Hodgkin's lymphomas. In order to determine the usefulness of ready-made macroarrays as routine diagnosis tools in haemato-pathology, we have analysed lymph node biopsies using a cDNA macroarray containing genes involved in apoptosis, including caspases. Nine biopsy specimens were analysed on total frozen tissues: 4 samples of B-cell follicular lymphoma (FL), two of B-cell diffuse large cell lymphoma (DLCL), and three of non-neoplastic lymph nodes from benign lymphadenitis. Eight cell populations were sorted from fresh tissues: malignant B-cells from 2 FL cases and 2 DLCL cases, reactive B-cells from 1 benign lymph nodes, reactive T-cells from 1 benign lymph node, virgin (mantle zone) B-cells and germinal center (GC) B-cells from benign tonsils. Immunohistochemistry (IHC) on paraffin sections was performed for localization of caspases 2, 3, 4, 7, 8, and 9. In the clustered array data, sorted cells from samples sharing common histological lesions grouped together, whereas the array/histology correlation was less satisfactory for tissues. The expression profiles of both array and IHC methods were correlated for most caspases and samples. Variations in array profiles of sorted cell populations can be statistically associated with specific histological features, suggesting a possible diagnostic application of ready-made "Apoptosis macroarrays" in haematopathology.

We report a retrospective study of 11 patients suffering from T-cell non-Hodgkin lymphomas preceded by immunological disorders including two chronic granulomatous diseases, one midline granuloma, four autoimmune hematologic disorders, one hypereosinophilic syndrome, two chronic lymphadenopathies, and one chronic angioedema. In the follow-up of these 11 patients, T-cell non-Hodgkin lymphomas were diagnosed. Even if we cannot exclude a fortuitous association, we feel that these conditions could constitute a 'prelymphomatous' stage.

Gamma-delta (gammadelta) T-cell non-Hodgkin's lymphomas (NHLs) usually present with liver, spleen and marrow infiltration. Lymph node involvement by gammadelta T-cell NHL has been rarely documented so far; its histological pattern needs to be further defined.Two cases of nodal gammadelta T-cell NHL are reported: case 1, a 44-year-old man, presented with cytomegalovirus retinitis and superficial lymphadenopathies. Histological analysis of an inguinal lymph node showed complete destruction by a diffuse pleomorphic lymphoid proliferation, which was positive for CD2, CD3, CD43, CD45, TIA-1 and granzyme B, and displayed a gammadelta phenotype (deltaTCR1+, Vdelta1+, Vdelta2-, Vdelta3-, betaF1-). Bone marrow was normal. Case 2, a male 24-year-old patient with a history of renal transplantation, presented with hepatosplenomegaly and supraclavicular lymph node enlargement. Lymph node architecture was globally preserved. Peripheral sinuses contained scattered nests of medium-sized irregular lymphoid cells. Bone-marrow was infiltrated. Phenotype showed positivity for CD2, CD3, CD45 and TIA1 and expression of gammadelta TCR (deltaTCR1+, deltaV1+, deltaV2-, deltaV3-, betaF1-). Both patients died a short time after diagnosis.These observations suggest that at least two forms of nodal gammadelta T-cell NHL may be encountered: one mimicking classical alphabeta T-cell NHL, with diffuse pleomorphic cell proliferation, and one displaying sinusoidal neoplastic infiltration suggesting a close relationship with hepatosplenic gammadelta T-cell NHL.

Most technical strategies for the analysis of gene expression in tissues are able to study only one protein or RNA product at the same time. A new recent method referred to asDNA arrayorhigh density filteris able to analyze simultaneously several hundreds of different genes. The DNA array is a nylon membrane on which are spotted equal amounts of cDNAs corresponding to different genes. This filter is hybridized with acomplexprobe synthesized with mRNA derived from the tissue analyzed. The result gives a global profile of gene expression within the tissue and allows quantitative and comparative analysis between different tissues or cell types.

We have previously shown that malignant B cells from non-Hodgkin's lymphomas (NHL) are resistant to Fas-mediated apoptosis. To determine the mechanisms underlying this resistance, we analysed by Western blotting the expression of several apoptotic regulators, caspase 3, caspase 8, FADD and poly(ADP-ribose) polymerase (PARP) in fresh lymphoma cells, isolated from 16 B-NHL biopsy samples of different histological subtypes, and displaying variable levels of Fas expression. The profiles of expression of these apoptotic regulators were monitored in cell lysates at different times following Fas with or without CD40 stimulation. Expression of FADD and of the uncleaved forms of PARP, caspase 3 and caspase 8 were detected in all untreated NHL samples. Low levels of PARP cleavage were noted in three untreated samples. Fas stimulation alone induced neither significant apoptosis nor significant changes in the expression profiles of FADD, caspases 3 and 8 and PARP in the 16 samples, except for variations in FADD and caspase 8 expression levels in a minority of samples. Fas/CD40 co-stimulation induced apoptosis and cleavage of caspase 3, caspase 8 and PARP in the five NHLs tested; expression of FADD was not modified. Our results showed (1) that induction of apoptosis in B-NHLs by Fas/CD40 co-stimulation used the same caspase executioner machinery as the normal Fas pathway, and (2) that NHL cells which resisted Fas-mediated apoptosis displayed no defect in either expression or functionality of caspases 3 and 8, nor in FADD expression. The dysfunction underlying NHL resistance to apoptosis must therefore lie upstream of caspase 8, or could alternatively be influenced by anti-apoptotic regulators of the Bcl-2 family.

The oesophageal mucosa is a frequent target of opportunistic infections in human immunodeficiency virus (HIV) infection. Langerhans cells (LC) are known as a target and reservoir of HIV in the skin. The aim of this study was to characterize oesophageal LC in HIV-infected patients.Thirty oesophageal biopsies were obtained from 29 patients (median age 35.5), all in stage IV of the HIV Center of Disease Control Classification. We performed histological assessment of the oesophageal mucosa and immunohistochemical detection of oesophageal LC using an anti-CD1a antibody, followed by morphometric analysis. Biopsies from 17 noninfected patients were studied using the same procedure. LC in oesophageal mucosa of the HIV positive patients showed a significantly and dramatically decreased number (LC(N) median = 5.85/mm2) and surface/epithelial surface (LC (S) ratio = 0.09) when compared with HIV-negative controls (LC(N) median = 29.7/mm2, LC(S) ratio = 1.83) with P = 0.003 for LC(N) and P0.0001 for LC(S).These data suggest that oesophageal LC are, like their epidermal counterparts, a preferential target for HIV infection. Their alterations may provide a clue to the pathogenesis of the decreased local oesophageal immunity and to the occurrence of opportunistic infections.

RON is a receptor protein tyrosine kinase belonging to the hepatocyte growth factor (HGF) receptor family. Using Recepteur d'Origine Nantais (RON) transfected cell lines, Macrophage Stimulating Protein (MSP) was identified as the ligand of RON. RON is synthesized as a single chain precursor, which subsequently is cleaved to yield a disulfide-linked heterodimer, with a 40-kDa alpha chain and a 150-kDa beta chain. Activation of RON by MSP results in cell migration, shape change, and proliferation. The present work centers on the production and characterization of two monoclonal antibodies (MAbs) to RON called ID-1 and ID-2. Antibodies were generated by immunization of mice with Madin-Darby Canine Kidney (MDCK) cells expressing human RON (clone RE7). Both antibodies recognized the mature and precursor form of RON. The specificity of the anti-RON antibodies was confirmed using a hepatocarcinoma cell line HepG2 expressing both task MET and RON receptors. Specific immunoprecipitation with ID-1 and ID-2 or anti-MET antibody followed by Western blotting under reducing conditions with rabbit polyclonal antibodies against RON and MET showed that our anti-RON antibodies recognize specifically the RON receptor. Ligand binding experiments showed that both antibodies are able to block the binding of radiolabeled MSP to RON and showed also that the antibodies recognize two different epitopes in the molecule. The blocking of MSP binding to RON by the anti-RON antibodies was confirmed by inhibition of cell migration induced by MSP in HT-29-D4 cells. Significant immunostaining was not observed in any subpopulation of whole blood with either ID-1 or ID-2. We analyzed the expression of RON receptor in a number of human hematopoietic and nonhematopoietic cells lines by flow cytometry. We found a strong mean of fluorescence intensity (MFI) in colon adenocarcinoma cells SW620 and HT-29-D4, low MFI in SVK14 and HepG2 cells, and no immunostaining in melanoma, lymphoma, and leukemia cells. Immunohistochemistry revealed that RON was expressed in germinal centers of tonsil, in skin, small intestine, and colon. These antibodies defined RON as CDw136 during the last leucocyte typing VI.

A new 2 beta-Chloromethyl-6 beta-carbamoylmethyl-penam-1,1-dioxide-3-carboxylic acid was synthesised and evaluated for its antimicrobial and beta-lactamase inhibitory activity. The compound was found to be inactive.

Fibroblast growth factor 2 (FGF2) has been implicated in the pathogenesis of malignant melanoma (MM), but the role of other FGFs and their receptors (FGFRs) is not elucidated. To determine whether FGF1 and FGFR1 may be involved in MM growth in vivo, we have studied the expression of the FGF1 and FGFR1 genes in 77 fresh MM biopsy samples, using RT-PCR analysis. Samples of benign nevi, normal skin and carcinoma cell lines were included as controls. Using RT-PCR analysis, expression of FGF1 and FGFR1 was observed in 69/77 and 68/77 cases, respectively. Immunohistochemical detection of the FGFR1 protein was positive in reactive stromal cells and at a much lower level in neoplastic cells. In situ hybridization experiments demonstrated FGFR1 mRNA mainly located in the stromal component. Southern blot analysis of genomic DNA prepared from MM tumors did not show any structural alteration of the FGFR1 gene. There was no correlation between FGF1/FGFR1 expression and the usual clinicopathological parameters of MM. We conclude that FGF1 and FGFR1 are frequently co-expressed in MM, a situation that may contribute to aberrant autocrine and paracrine pathways. Due to the absence of correlation with clinico-pathological parameters, this expression cannot be used as a marker of prognosis in the management of MM patients.

The fusion gene NPM-ALK occurs in a subset of anaplastic large cell lymphomas (ALCLs), as a result of a chromosomal translocation, t(2;5) (p23;q35). It has been suggested that Hodgkin's disease (HD) and ALCL share a common histogenesis because of pathological and phenotypical similarities. In order to check this hypothesis, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the hybrid NPM-ALK gene in 30 tumour samples, including 22 lymph node biopsies from HD and eight ALCL specimens. The threshold level of sensitivity was shown to reach at least 1/10(4) by dilution experiments using cell lines as positive and negative controls. The expected 177 bp product indicative of the NPM-ALK rearrangement was identified in Karpas 299 and SUDHL-1 cell lines and in two out of eight ALCLs. The 22 HD cases were negative, even after two successive tests. Thus, since the ALCL-specific genetic alteration was absent in our series of HD cases, the present study does not support the hypothesis that HD and ALCL are histogenetically related entities.

FAS/APO-1 (CD95) is a membrane glycoprotein belonging to the tumour necrosis factor/nerve growth factor receptor family, and which can trigger apoptosis in some lymphoid cell lines. Immunohistochemistry combined with Northern blotting allowed determination of the pattern of FAS/APO-1 expression in a series of Ki-1 [CD30] positive lymphoid malignancies, including 27 Hodgkin's disease and eight anaplastic large cell lymphomas. CD30 negative tumours used as controls included 27 B-cell non-Hodgkin's lymphomas. 14 T-cell non-Hodgkin's lymphomas, four reactive lymphadenitis, and non-lymphoid tissues. Immunohistochemistry, performed on frozen sections, revealed a strong FAS/APO-1 expression in 25 out of 27 (92%) Hodgkin's disease cases, predominantly in Reed Sternberg cells; 50 to 100% of the neoplastic cells in eight out of (100%) anaplastic large cell lymphoma cases were positive. In contrast, positive FAS/APO-1 immunostaining was observed only in 22 out of 41 (53%) CD30 negative non-Hodgkin's lymphomas. Northern blot analysis detected variable amounts of the FAS/APO-1 transcript in the immunohistochemistry-positive samples. These results suggest possible hyper-expression of FAS/APO-1 (CD95) in Hodgkin's disease and anaplastic large cell lymphomas.

The hormone gastrin is mainly produced by the G cells of the antral mucosa and plays a major role in the regulation of digestive mucosal growth. Since it permits identification of cell types containing mRNA, in situ hybridization (ISH) appears to be an interesting method for studying gastrin-producing tissues. In this study, in situ detection of gastrin mRNA has been carried out on frozen sections of four human normal antral mucosa samples and of six colonic carcinomas removed from patients with high levels of plasma gastrin, using a gastrin oligonucleotidic DNA probe. We have compared the results provided respectively by the [35s] labelling and the digoxigenin labelling of the synthetic probe. Positive cells were found in each normal sample analysed with radioactive- as well as digoxigenin-labelled antisense probes. The total number of cells expressing gastrin mRNA appeared slightly higher with the [35s]-labelled probe, while the digoxigenin-labelled probe gave a better definition of positive signals. In contrast, neither radioactive nor cold probes gave positive signals in the six colonic carcinoma samples, although gastrin expression had been demonstrated in these tumours using a reverse transcriptase-PCR method. These results show that, although ISH does not seem sensitive enough to allow the detection of very low levels of gastrin expression, it would appear to be a reliable method for visualizing gastrin mRNA in human antral mucosa.

The histopathologic pattern of tissues involved by Hodgkin's disease (HD) suggests excessive activation of environmental cells by cytokines released by Hodgkin-Reed-Sternberg (HRS) cells, which are considered as the neoplastic component of HD lesions. This hypothesis has been supported by many studies performed in vitro using HD cell lines. In this study, we have tried to demonstrate the cytokine-producing cells in an environment as close as possible to the in vivo conditions, using in situ hybridization onto frozen sections of HD samples. [35S]-labelled single-stranded RNA probes were prepared by transcribing human cDNA fragments of the TNF-alpha and IL-1 alpha genes subcloned into appropriate vectors. A total of 19 specimens of HD lesions, including 7 cases of nodular sclerosing (NS) type and 12 cases of mixed cellularity (MC), were tested with both types of probes. Clinical stages included stage I (6 cases), stage II (4 cases), stage III (6 cases) and stage IV (3 cases). TNF-alpha and/or IL-1 alpha expression was observed in 12 among 19 HD cases. However, neither the histological type nor the clinical status of the patients was correlated with the profile of cytokine secretion. Most of the cytokine-producing cells could be identified as HRS cells due to their morphological appearance. In 3 cases, simultaneous analysis by immunohistochemistry and in situ hybridization showed that IL-1 alpha/TNF-alpha mRNA-producing cells simultaneously expressed the CD30 antigen, thereby confirming the HRS nature of these cells.

A 56-year-old man presented with an inguinal lymph node enlargement. Histologic study of the tumor revealed three intermingled pathologic lesions: a nodular small cell lymphoma, an angiofollicular hyperplasia of vasculohyaline type, and a vascular neoplasia closely resembling Kaposi's sarcoma. The patient was immunocompetent and denied any homosexual relationships, transfusions, or drug use. The serum was negative for the presence of human immunodeficiency virus antibody. Computed tomographic scan and ultrasound examination revealed no other lymphadenopathies. This case shows that both hyperplastic and neoplastic lymphoid proliferations can occur simultaneously with vascular neoplasia. It thereby suggests that the neoplastic populations might interact to favor the tumor growth, the sequence and the nature of the stimulating events remaining unclear.

The expression of the mos proto-oncogene was investigated in frozen sections from 15 ovarian adenocarcinomas, 1 seminoma, 1 immature teratoma and 2 benign stromal tumors by in situ RNA/RNA hybridization with a [35S] labeled mos probe. The presence of mos transcripts was detected in one germ cell tumor and three ovarian carcinomas. The putative role of the mos proto-oncogene activation is discussed.

During the last 10 years, a very large number of molecules with antibacterial activity has been developed by pharmaceutic industry. The principal objectives of pharmacological research are to obtain ever wider spectra of activity and increased antibacterial action in order to combat "emerging" agents and those resistant to traditional treatment, as well as longer acting drugs so as to improve patient compliance. These objectives might be attained by the molecules now in the phase of clinical trial, especially by the new injectable or oral cephalosporins and new fluoroquinolones.

Glucose-6-phosphatase (G6Pase) is a multicomponent system that catalyzes G6P hydrolysis. To determine the specificity of the histochemical reaction of G6Pase, we investigated the inhibitory effect of diethyl pyrocarbonate (DEPC), a specific and very effective inhibitor of the phosphohydrolase component of the G6Pase system, in normal human liver. The inactivation of the histochemical enzymatic activity by DEPC was monitored by determining the mean brightness of the microscopic image and the histogram of light intensity distributions. The results obtained indicate that the histogram is more sensitive than the mean brightness to variations of enzymatic activities, and that the percent of pixels brighter than a convenient level is directly proportional to DEPC concentration. This study indicates that DEPC can be used as an efficient inhibitor of the histochemical reaction of G6Pase.

Immunophenotyping and immunogenotyping were performed in a series of 8 large cell lymphomas exhibiting anaplastic or "histiocytic" morphology and displaying an uncertain phenotype due to a restricted number of differentiation antigens. 6 cases expressed the Ki-1 antigen. 4 cases expressed one or two B-cell markers and contained rearrangements of the immunoglobulin genes. One of them also exhibited a T-cell receptor (TCR) beta gene rearrangement. 3 cases expressed a single T-cell differentiation antigen. Among them, only 1 displayed both gamma and beta TCR gene rearrangement; 1 only contained a gamma TCR gene rearrangement and 1 completely lacked clonal rearrangements. The eight cases expressed an inconclusive immunophenotype due to an abundant population of reactive cells but showed an immunoglobulin gene rearrangement. In conclusion, 5 out of the 8 unusual lymphomas studied here could be characterized by immunogenotyping. This approach was, however, inconclusive in the 3 remaining cases, whose lineage and differentiation stage remain poorly defined.

This report deals with two cases of villous adenoma of the Wirsung duct. The two patients presented with upper abdominal pain, diarrhea, and weight loss. Duodenal intubation showed a complete failure of exocrine pancreatic function. Ultrasound scan, computed tomography, and endoscopic retrograde cholangiopancreatography disclosed marked dilatation of the head portion of the main pancreatic duct. A proximal duodenopancreatectomy was performed. Pathological examination revealed a papillary polyp expanding the pancreatic head and filling the main duct lumen. Histological pattern consisted of a villous adenoma without any feature of malignant change. Previously reported cases exhibit many similarities to our two cases. The putative likelihood of malignant change and the relationship between villous tumors of the Wirsung duct and of other origin remain unclear.

We report a case of granulocytic sarcoma of the bone with pleural involvement diagnosed upon cytologic analysis of the pleural fluid (centrifugation spots stained by May-Grunwald-Giemsa) and confirmed by more complex investigations, i.e., demonstration of granulomonocytic membrane antigens by immunohistochemical monoclonal antibody techniques on frozen sections of the tumor. This case draws attention to the value of cytologic studies in granulocytic sarcomas whose histologic features are suggestive of lymphoma.