Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.

To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.

An impending crisis in the provision of histopathology expertise for mouse functional genomics.

The generation of new mouse models of human disease is accelerating rapidly, due to the completion of whole-genome sequencing efforts and technological advances in the manipulation of the mouse genome. We sought to investigate manpower issues in the provision of histopathology expertise for mouse functional genomics and compared this to the perceived demand from principal investigators (PIs). Through the European Commission (EC)-funded PRIME pathology training initiative, two questionnaires were devised to collect information from pathologists and EC-funded PIs on the current provision of mouse histopathology expertise in Europe and the demands for this service. We find that pathological analysis is being performed almost exclusively by professionally qualified pathologists, generally employed in clinical diagnostic posts, where the work is undertaken as collaboration outside of their contractual commitments but without previous training in veterinary or comparative pathology. The results indicate that there is a lack of both trainees and provision of specialist training in this field. Unsurprisingly, the availability of diagnostic expertise and advice falls far short of the number of genetically engineered mice (GEM) being generated for analysis. We analyse these results with reference to previous studies and discuss solutions for the future recruitment, training and funding for pathologists in mouse functional genomics in Europe.

Lack of beta-catenin in early life induces abnormal glucose homeostasis in mice.

AIMS/HYPOTHESIS: Wingless and iNT-1 (WNT) pathway members are critical for pancreatic development and exocrine tissue formation. Recently, much attention has focused on delineating the roles of beta-catenin in pancreatic organogenesis. However, little is known about the involvement of beta-catenin in the endocrine or exocrine function of the mature pancreas. We report for the first time the impact of beta-catenin deletion in the pancreatic beta cells. METHODS: We targeted the deletion of the beta-catenin gene in pancreatic beta cells by crossing a floxed beta-catenin mouse strain with a RIP-Cre mouse strain. RESULTS: Surprisingly, the majority of the mutant mice died shortly after birth and had deregulated glucose and insulin levels. The newborn mutant pancreases demonstrated increased insulin content, reflecting a defect in insulin release confirmed in vitro. Moreover, there was a reduction in total endocrine tissue at birth, while cellularity in islets was greater, suggesting that lack of beta-catenin affects beta cell size. Some newborns survived beta-catenin deletion and showed a milder phenotype during adulthood. CONCLUSIONS/INTERPRETATION: The deletion of beta-catenin in the maturing beta cells negatively impacts on islet morphology and function. This work reveals that lack of beta-catenin in early life is related to severe deregulation of glucose homeostasis.

Progress in updating the European Radiobiology Archives.

PURPOSE: The European Radiobiology Archives (ERA), together with corresponding Japanese and American databases, hold data from nearly all experimental animal radiation biology studies carried out between 1960 and 1998, involving more than 300,000 animals. The Federal Office for Radiation Protection, together with the University of Cambridge have undertaken to transfer the existing ERA archive to a web-based database to maximize its usefulness to the scientific community and bring data coding and structure of this legacy database into congruence with currently accepted semantic standards for anatomy and pathology. METHODS: The accuracy of the primary data input was assessed and improved. The original rodent pathology nomenclature was recoded to replace the local 'DIS-ROD' (Disease Rodent) formalism with Mouse Pathology (MPATH) and Mouse Anatomy (MA) ontology terms. A pathology panel sampled histopathological slide material and compared the original diagnoses with currently accepted diagnostic criteria. RESULTS: The overall non-systematic error rate varied among the studies between 0.26% and 4.41%, the mean error being 1.71%. The errors found have been corrected and the studies thus controlled have been annotated. The majority of the original pathology terms have been successfully translated into a combination of MPATH and MA ontology terms. CONCLUSIONS: ERA has the potential of becoming a world-wide radiobiological research tool for numerous applications, such as the re-analysis of existing data with new approaches in the light of new hypotheses and techniques, and using the database as an information resource for planning future animal studies. When the database is opened for new data it may be possible to offer long-term storage of data from recent and future animal studies.

Splitting bone marrow trephines into frozen and fixed fragments allows parallel histological and molecular detection of B cell malignant infiltrates.

Clonality analysis of the immunoglobulin heavy chain (IgH) gene is helpful in identifying malignant B cell infiltrates in the bone marrow and is usually carried out on separate aspirates or on the same formalin-fixed decalcified bone marrow specimen. To determine whether the removal of the decalcification step would improve the molecular analysis, we first studied 12 bone marrow specimens with lymphoma infiltration split into a fixed and a small frozen fragment. Both the detection rate of IgH gene monoclonality and DNA quality were found to be superior in the frozen part than in the fixed part. Conversely, to evaluate whether the split would compromise histological analysis, we selected a series of 134 bone marrow specimens obtained from patients with small B cell lymphoma and showing IgH monoclonality on the frozen part. The histological detection rate of infiltrated or suspicious infiltrates (95%) on the fixed part was not altered by saving a frozen part.

An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress.

Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21(Cip1) and p27(Kip1) proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21(Cip1) and p27(Kip1) in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development.

All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth.

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-trans-retinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.

Helicobacter pylori infection and stem cells at the origin of gastric cancer.

Helicobacter pylori infection is now recognized as the main and specific infectious cause of cancer in the world. It is responsible for gastric adenocarcinomas of both intestinal and diffuse types, which are the long-term consequences of the chronic infection of the gastric mucosa. Case-control studies have shown an association between the two, recognized as early as 1994 and further substantiated by interventional studies in which H. pylori eradication has led to the prevention of at least part of the gastric cancers. Experimental studies have highlighted the role of bone marrow-derived cells (BMDCs) and particularly mesenchymal stem cells, in the neoplastic process in about a quarter of the cases and possibly an epithelial-mesenchymal transition (EMT) in the other cases. Different studies have confirmed that chronic infection with H. pylori induces a chronic inflammation and subsequent damage of the gastric epithelial mucosa, leading to BMDC recruitment. Once recruited, these cells home and differentiate by cell-cell fusion with local gastric epithelial cells, bearing local stem cell failure and participating in tissue regeneration. The context of chronic infection and inflammation leads to an EMT and altered tissue regeneration and differentiation from both local epithelial stem cells and BMDC. EMT induces the emergence of CD44+ cells possessing mesenchymal and stem cell properties, resulting in metaplastic and dysplastic lesions to give rise, after additional epigenetic and mutational events, to the emergence of cancer stem cells (CSCs) and adenocarcinoma.

Evidence that an identical T cell clone in skin and peripheral blood lymphocytes is an independent prognostic factor in primary cutaneous T cell lymphomas.

The monoclonality of the T cell receptor gamma-chain gene was analyzed by polymerase chain reaction in skin and blood specimens of 85 patients with cutaneous T cell lymphomas including 67 mycosis fungoides, seven Sézary syndromes, and 11 CD30- nonepidermotropic cutaneous T cell lymphomas. A cutaneous T cell clone was detected in 69% of mycosis fungoides and 100% of Sézary syndromes. This frequency varied according to the clinical stage: 57% in early stages (Ia-IIa) to 96% in advanced stages (IIb-IV, Sézary syndrome). A peripheral blood T cell clone was detected in 42% of early stages and in 74% of late stages but was identical to the cutaneous one in 15% and in 63%, respectively. A significant association between initial clinical stage and T cell monoclonality was observed. In nonepidermotropic cutaneous T cell lymphomas, T cell monoclonality was detected in 55% of skin and 36% of blood samples. Univariate and multivariate analyses showed that, besides the initial clinical stage, an identical cutaneous and blood T cell clone was an independent prognostic factor for disease progression of mycosis fungoides/Sézary syndrome (hazard ratio 3.4, 95% confidence interval 1.4-9.9). Parallel polymerase chain reaction study of skin and blood specimens may therefore provide an initial prognostic marker that could help to monitor therapeutic strategies. A fully prospective study, with simultaneous therapeutic trials, needs to be done to confirm our findings and to include treatment variables in the statistical analysis.

Identification of novel trkA variants with deletions in leucine-rich motifs of the extracellular domain.

The peripheral expression of trkA encoding for NGF receptor was investigated by RNase protection assay. A thymus-specific protected fragment was identified. Using 5' rapid amplification of cDNA ends, three different trkA fragments were characterized. The longer fragment corresponded to the classical trkA L3 transcripts while the two shorter fragments lacked sequences encoding for leucine-rich motifs of the extracellular domain of TrkA, similarly to the trkB L1 and L0 variants. RT-PCR analysis of adult rat tissues showed the expression of trkA L1 transcripts in the thymus, testis, lung and kidney but not in the central nervous system. Their combined expression with trkA L3 transcripts suggests that specific peripheral TrkA oligomers may modulate NGF binding and function in non-neuronal cells.

Expression of NGF receptors in normal and pathological human thymus.

The expression of NGF receptors was investigated in normal human thymus and in thymic hyperplasias, thymomas and thymic carcinomas. By RT-PCR, we detected TrkAI transcripts encoding for the high-affinity NGF receptor. Western blot analysis showed the presence of both TrkA and p75NGFR proteins. In normal thymuses, epithelial subcapsular and medullar cells were TrkA immunoreactive. Interdigitated medullar cells were stained for both TrkA and p75NGFR. While epithelial cells of normal thymuses or benign thymomas exhibited a TrkA positive-p75NGFR negative phenotype, a switch to a TrkA negative-p75NGFR positive phenotype was observed in malignant epithelial cell tumours and was associated with cell proliferation-associated MIB1 expression. Our results argue for a local role of NGF and its receptors on thymic stromal cells both in normal and neoplastic conditions.

Low prevalence of monoclonal B cells in Helicobacter pylori gastritis patients with duodenal ulcer.

We have studied the prevalence of B-cell clonality among a large group of 320 patients with Helicobacter pylori gastritis and duodenal ulcer. These patients underwent endoscopic examination with multiple gastric biopsies at diagnosis and were followed 2 and 12 months after therapy. Histopathologic examination of 809 sets of biopsy specimens showed lymphoid gastritis with lymphoid aggregates or follicles, but without lymphoepithelial lesion, in 302 samples corresponding to initial biopsy specimens (n=130) or to posttreatment biopsy specimens (n=172). DNA extracted from fresh antral specimens allowed the amplification of Helicobacter pylori DNA in all cases before therapy. The arrangement of the immunoglobulin heavy chain gene was studied by polymerase chain reaction (PCR) in the 302 selected lymphoid gastritis samples. Single or dominant bands were seen only in four specimens from three patients (1.3%), whereas a polyclonal pattern was seen in the other 298 samples. The detection threshold of our PCR technique was approximately 3% of clonal B cells diluted in a polyclonal population. This threshold appeared to be a reliable cutoff between polyclonal gastritis and clonal MALT lymphoma. In our experience, Helicobacter pylori lymphoid gastritis appeared mainly as a benign polyclonal condition.

CD30-positive cutaneous large cell lymphomas. A comparative study of clinicopathologic and molecular features of 16 cases.

The authors have analyzed and compared the clinicopathologic and molecular features of 16 cases of large cell cutaneous lymphomas expressing CD30 antigen. Three main clinical groups were defined: (1) a group of localized skin disease (7 cases); (2) a group of multicentric skin disease (5 cases); and (3) a group of concomitant skin and extracutaneous disease. Good prognosis was associated with localized skin disease and no history of lymphoma. Interestingly, a majority of Reed Sternberg-like cells was only observed in this group (5 of 6 cases). The two other groups did not show distinctive evolutive nor morphologic features. Southern blot and/or polymerase chain reaction (PCR) technique showed clonality and a T-cell genotype in respectively 13 of 14 and 12 of 12 analyzed cases. Viral infection of tumoral cells was investigated by PCR, in situ hybridization (ISH) or electron microscopy. Epstein-Barr virus (EBV) sequences were detected by PCR and ISH in tumoral cells of cutaneous lesions in one case of skin lymphoma with extracutaneous spreading. No EBV sequence was detected by ISH in the localized lymphomas, whereas HIV particles were visible in tumoral cells in one of these cases. No human T-cell lymphotropic virus (HTLV) tax sequence was amplified by PCR in any case of our series. Our results confirm that CD30-positive cutaneous large cell lymphomas are different clinical and molecular entities. However, a combined clinical and morphologic analysis may help to identify a subset of CD30 cutaneous lymphomas with favorable prognosis.

Intrasinusoidal bone marrow infiltration revealing intravascular lymphomatosis.

Intravascular lymphoma is a rare, often fatal disease characterized by a widespread intravascular proliferation of neoplastic lymphoid cells. Dermatological and bizarre neurological manifestations usually predominate. We report a case of intravascular lymphomatosis with an exceptional clinical presentation showing splenomegaly combined with early bone marrow involvement. The diagnosis was made on bone marrow biopsy examination using both immunohistochemistry and molecular biology analysis. We stress the histopathological features of bone marrow involvement by intravascular lymphoma which allow the prompt recognition of this disease. Early systemic chemotherapy, which represents the only chance of remission in such an aggressive disease, can then be initiated.

Primary cardiac lymphoma in an immunocompetent woman.

We report a fatal primary cardiac non-Hodgkin's lymphoma in a 62 years old immunocompetent woman presenting with tamponade and complete atrioventricular block. CT-scan, echocardiography and autopsy examination showed a tumor largely infiltrating the heart without extracardiac involvement. A surgical biopsy revealed high grade B-cell non-Hodgkin's lymphoma with a misleading myelomonocytic CD68 (KPI) expression. Polymerase Chain Reaction analysis revealed a clonal rearrangement of the immunoglobulin heavy chain gene and confirmed the B-cell origin of the lymphoma. Our report also emphasizes the role of immunohistochemical and molecular techniques in the diagnosis.

Differential expression of NGF receptors in human thymic epithelial tumors.

NGF receptor (TrkA and p75NGFR) expression was investigated in human thymuses, including normal thymuses, thymic hyperplasias, thymomas and thymic carcinomas. TrkAI but not TrkAII transcripts were demonstrated by RT-PCR. In normal thymuses, immunohistochemistry revealed a restricted TrkA-immunoreactivity to epithelial and interdigitated reticular cells, while only interdigitaded reticular cells were immunoreactive for p75NGFR. Thymocytes were negative for both receptors. A switch from the normal TrkA positive-p75NGFR negative phenotype to a TrkA negative-p75NGFR positive phenotype was found in histologically aggressive epithelial cell tumors, suggesting that NGF and its receptors are potentially involved in thymus stroma organogenesis and proliferation.

Leukemic dissemination within a glioblastoma in a patient with chronic lymphoid leukemia.

Metastasis from an extracranial tumor to a primary central nervous system tumor is a rare event, and most reported cases concern metastases to meningiomas. The authors describe the first case of leukemic cell dissemination within a glioblastoma. The patient likely presented a genetic predisposition to multiple neoplasms, and the unusual localization of leukemic cells might be partly related to the characteristic microvascular proliferation in glioblastoma.

[Littoral cell angioma: a rare vascular splenic tumor]. / L'angiome à cellules littorales: une tumeur vasculaire rare de la rate.

Littoral cell angioma is a rare primitive vascular tumor of the spleen considered as benign. Its clinical presentation is non specific. The diagnostic is based on histological and immunohistological analysis. The lesion is composed of anastomosing vascular channels often featuring papillary projections. They are lined by tumoral cells which exhibit an immunoreactivity for vascular and histiomonocytic markers. We report one case of littoral cell angioma and we discuss the diagnosis and the histogenesis of this tumor.

[Hepatic MALT lymphoma disclosing a nodal extension]. / Lymphome du MALT hépatique avec extension ganglionnaire révélatrice.

We report a case of a 50-year-old man with chronic viral hepatitis B presenting with a primary hepatic lymphoma of mucosa-associated lymphoid tissue, revealed clinically by a pedicular nodal mass. The liver biopsy showed an active chronic hepatitis and a dense portal lymphoid infiltrate with centrocyte-like cells inducing typical biliary lympho-epithelial lesions. The lymph-node biopsy revealed a marginal zone lymphoma pattern. A monoclonal rearrangement of the immunoglobulin heavy chain gene was detected in the lymph node by polymerase chain reaction. This case of primary hepatic mucosa-associated lymphoid tissue lymphoma confirms that the liver also contains mucosa-associated lymphoid tissue, in which low grade lymphoma can arise.

[Study of B-lymphocyte clonality using in vitro gene amplification (PCR) in paraffin embedded samples]. / Etude de la clonalité B lymphocytaire par la technique d'amplification de gène in vitro (PCR) à partir de prélèvements fixés et inclus en paraffine.

Family-specific primers were used in polymerase chain reaction (PCR) to analyse clonality of immunoglobulin heavy chain (IgH) gene rearrangement. DNA templates were extracted from formalin-fixed paraffin-embedded biopsies from B-cell lymphomas (n = 19), T-cell lymphomas (n = 3), reactive lymphadenopathies (n = 20) and negative controls (n = 2). PCR was also performed on DNA extracted from fragments of the same biopsies that were either fixed in Bouin's solution (n = 24) or snap-frozen (n = 22). The latter were also studied for IgH gene rearrangement by the Southern blot technique. Polyclonal or clonal fragments were obtained from all frozen biopsies. No amplification was observed in 3 out of 44 formalin fixed specimens and 18 out of 24 Bouin fixed specimens. A clonal amplification was only observed in 16 out of 19 formalin-fixed B-cell lymphoma specimens. Therefore this technique allows to study B-cell clonality from formalin-fixed, embedded material.