p53, Ki-ras, and DNA ploidy in human pancreatic ductal adenocarcinomas.

Ki-ras mutations and DNA aneuploidy are common findings in human pancreatic ductal adenocarcinomas. An altered p53 tumor-suppressor gene has been suggested to cooperate with activated Ki-ras in malignant cellular transformation and could enhance genomic instability. We have investigated a panel of well-documented pancreatic carcinomas with defined ploidy and Ki-ras mutations for the presence and pattern of genetic alterations of the p53 gene, their coincidence with Ki-ras point mutations, and their correlation with DNA ploidy, tumor pathology, and clinical course. DNA was isolated from formalin-fixed and paraffin-embedded tumor tissue and polymerase-chain-reaction-amplified fragments of the p53 gene exons 5 to 9 were screened by the single-strand conformation polymorphism method. The positive cases were further examined for mutations by direct sequencing. Twenty-nine of seventy-one (41%) tumors showed mutations of the p53 gene, however, five tumors carried two mutations resulting in a total of 34/71 (48%) genetic alterations of the p53 gene. The majority were missense point mutations and distributed primarily within the evolutionary conserved domains (62%). Ten of Thirty-four (29%) affected the hotspot codons 248, 273, and 282, respectively, and 21/34 (62%) of the p53 gene mutations clustered on exons 7 and 8. Transitions (71%) predominated over transversions (15%), deletions were identified in 7/34 (21%) tumors. One third of the carcinomas showed both Ki-ras codon 12 and p53 gene mutations. p53 mutations correlated with distant metastasis (p < 0.05) and survival (p < 0.05). DNA triploidy was associated with a mutated Ki-ras gene (p < 0.05) as well as with double mutations of c-Ki-ras and p53 (p < 0.05). Unlike most other malignant tumors pancreatic ductal adenocarcinomas exhibit a significantly higher incidence of c-Ki-ras than p53 gene mutations. However, like other neoplasms p53 gene mutations seem to be associated with a metastatic phenotype possibly acquired during tumor progression.

Expression of c-fos correlates with IFN-alpha responsiveness in Philadelphia chromosome positive chronic myelogenous leukemia.

This study evaluates (i) constitutive levels of oncogene and p53 transcripts in chronic phase CML patients and (ii) their modulations subsequent to in vivo therapy with rIFN-alpha 2c. Peripheral blood mononuclear cells (pbmc) and bone marrow cells of 26 patients were examined for c-fos, c-myc, p53 and the hybrid bcr/abl mRNA levels. Results indicated that (i) constitutive c-fos transcript levels are significantly higher in patients subsequently responding to IFN-alpha therapy (p < 0.01) and positively correlated with the proportion of lymphocytes (r = 0.6895, p < 0.01) and negatively with the proportion of immature cells (r = -0.568, p < 0.01) contained in the pbmc preparations tested, (ii) constitutive mRNA levels of the hybrid bcr/abl, c-myc and p53 are positively correlated with each other, but failed to relate to disease parameters, and (iii) acute and chronic in vivo exposure to IFN-alpha is accompanied by upregulation of c-fos and downregulation of c-myc mRNA levels in responder patients.

Syringotropic cutaneous T-cell lymphoma: a variant of mycosis fungoides?

We report two patients with the typical clinical (patches/plaques studded with brownish-red papules) and histopathological (hyperplastic eccrine ducts and glands surrounded and infiltrated by lymphocytes) features of syringotropic cutaneous T-cell lymphoma. Immunohistochemical studies confirmed the T-cell character of the infiltrate, and gene rearrangement studies its monoclonality (in one case). Our patients did not present with visceral involvement.

Early diagnosis of gastric lymphoma: gene rearrangement analysis of endoscopic biopsy samples.

The diagnosis of gastric lymphoma in endoscopic biopsy specimens remains difficult despite the emergence of accepted criteria for the histologic diagnosis of lymphomas originating from mucosa-associated lymphoid tissue (MALT). The sensitivity and validity of immunoglobulin (Ig) gene rearrangement analysis of mucosal biopsies for the diagnosis of malignant B-cell lymphoma were investigated in comparison with conventional histology and immunohistology. Biopsy specimens from 34 different endoscopies of 20 patients with a previous history, or tentative diagnosis of gastric lymphoma, and 12 control samples were analyzed for the presence of clonal Ig gene rearrangements. A clonal B-cell population was detected by Southern blot analysis in all patients with a definitive histologic diagnosis of lymphoma. In addition, in two patients the detection of clonal rearrangements in biopsy specimens preceded by several months the histologic diagnosis of lymphoma, and clonality was confirmed in three further patients where histology remained inconclusive. In some cases of low-grade MALT-lymphoma, discrete spreading of malignant cells within chronically inflamed mucosa was suggested by the presence of identical clonal rearrangements in all simultaneously obtained biopsies, with or without histologically detectable involvement by lymphoma. Our results show that immunoglobulin gene rearrangement studies of endoscopic biopsy samples are an additional powerful tool for the diagnosis of gastric lymphoma, especially for detecting early recurrence, and improve the preoperative assessment of the extent of mucosal involvement.

Detection of enteroviral ribonucleic acid in myocardial biopsies from patients with idiopathic dilated cardiomyopathy by polymerase chain reaction.

Infection by enteroviruses, especially by Coxsackie B viruses, has been incriminated in pathogenesis of dilated cardiomyopathy. We developed polymerase chain reaction tests for the detection of enteroviral and Coxsackie B3 genomes, respectively, in myocardial biopsies obtained from a homogeneous group of 19 patients with idiopathic dilated cardiomyopathy. To determine unambiguously the incidence of enteroviruses and Coxsackie B3 viruses in these patients, we used two primer pairs, one common to all enteroviruses and the other specific for Coxsackie B3 viruses. In six patients of the dilated cardiomyopathy group, enteroviral ribonucleic acid (RNA) could be detected; only one was subspecified as Coxsackie B3 RNA. In contrast, no enteroviral RNA could be detected in a contrast group of 21 patients with other cardiac disorders. These results suggest that enteroviruses other than Coxsackie B3 are causally linked to the pathogenesis of dilated cardiomyopathy.

Immunoglobulin gene rearrangement in plasma cell dyscrasias: detection of small clonal cell populations in peripheral blood and bone marrow.

The bone marrow (BM) and peripheral blood (PB) samples of 71 patients with plasma cell dyscrasias were analysed by the Southern blot technique for the presence of clonal immunoglobulin (Ig) gene rearrangements. 53% of BM samples examined were archival material such as air dried BM slides or frozen trephine biopsies. The results were related to bone marrow plasmacytosis as determined by cytology and flow cytometry, and other clinical parameters. Clonal Ig gene rearrangements were found in BM samples of 45 (83%) of 54 MM patients and in 3 of 6 patients with monoclonal gammopathy of unknown significance (MGUS). Clonal cell populations in the PB were detected in 11 (30%) of 37 examined MM patients, but in none of the patients with MGUS or solitary plasmacytoma of bone. PB involvement was associated with progressive disease. Circulating monoclonal cells were significantly associated with higher M-protein levels (p < 0.05). Thus, circulating clonal precursor cells are encountered more frequently in active MM.

Long-term follow-up of patients with hairy cell leukaemia treated with pentostatin: lymphocyte subpopulations and residual bone marrow infiltration.

Peripheral blood lymphocyte (PBL) subsets and bone marrow biopsies were analysed in six patients with hairy cell leukaemia (HCL) treated with 2'-deoxycoformycin (pentostatin, DCF) according to a phase II trial of the EORTC Leukemia Cooperative Group. All patients responded to DCF with four complete and two partial remissions according to conventional criteria. Within the PBL subsets, major changes concerned the CD4+ T cells, which during DCF therapy were distinctly suppressed to nadir values of 0.038-0.18 (median 0.126) x 10(9)/l. In five patients these cells returned to normal 3.0-49.5 (median 14.5) months after the last DCF injection. CD8+ cells were decreased to a lesser extent, and NK cell numbers improved during treatment. Bone marrow immunohistology applying the MoAb B-ly7 demonstrated residual hairy cells (HCs) in all of the six patients following DCF treatment with nadir HC numbers of 0.2-3.0% of bone marrow cells. Immunoglobulin gene rearrangement analysis of DNA obtained from these biopsies showed only germline bands, whereas rearranged bands had been present on the pretreatment specimens. Within the observation period of 15.5-54.0 (median 47.0) months after discontinuation of DCF therapy, immunohistology demonstrated a continuous increase in HC numbers in five of the six patients with clonal rearrangement detectable in bone marrow specimens from three of these patients at last follow-up date. Although established on the basis of a small number of patients, these data suggest that DCF treatment as currently employed in HCL is unable to eradicate the malignant B cell clone.

[Proliferative activity and p53 expression in transitional cell carcinoma of the urinary bladder]. / Proliferative Aktivität und Expression des p53-Proteins in Ubergangszellkarzinomen der Harnblase.

Mutations of the p53 gene are important mechanisms in malignant transformation and are associated with dysregulation of normal cell growth. In the present study the expression of mutated p53-protein and proliferating cell nuclear antigen (PCNA) was investigated in a series of 31 human transitional cell carcinomas (TCC) by immunohistochemistry (IHC). The number of PCNA-positive cells and the pattern of expression was distinct in normal urothelium being confined to the basal cell layer. In dysplastic urothelium and in Carcinoma in situ (CIS) PCNA-immunoreactive nuclei were irregularly distributed throughout all layers. In tumor cell complexes the pattern of PCNA-immunoreactivity was different in papillary and primary infiltrating TCCs. Densitometric quantification of the intensity of the PCNA-reactivity using image analysis revealed an increase from normal to dysplastic urothelium and from dysplastic urothelium to invasive tumors. 21/31 (68%) of the tumors and tumor-associated CIS showed overexpression of p53 varying in percentage, pattern and reaction intensity. The percentage of PCNA-positive cells was higher in tumors overexpressing p53. Double IHC showed colocalization of both molecules in a significant proportion of tumor cells suggesting a link of p53 overexpression and the abnormal proliferative activity. The present results show that p53 over-expression is found in a significant percentage of TCCs and indicate a close association with a defective growth regulation resulting in increased PCNA-levels and enhanced cellular proliferation.

A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired immunodeficiency syndrome.

High-grade malignant nonHodgkin's lymphomas--five lymphoblastic, three pleomorphic, and two immunoblastic--developed in 10/25 cynomolgus monkeys (Macaca fascicularis) followed for up to 746 d after infection with simian immunodeficiency virus, strain SIVsm. These lymphomas were shown to be associated with an Epstein-Barr (EB)-like cynomolgus B-lymphotropic herpesvirus (CBLV) by electron microscopy, by Southern blot hybridization with probes against human EBV, and by the expression of antigens corresponding to EBV-associated nuclear antigens (EBNAs) involved in human B cells transformation. Southern blot demonstration of immunoglobulin gene rearrangements and homogeneous EBV episomes indicated that all the lymphomas were CBLV-associated monoclonal B cell proliferations. Our findings suggest that these tumors correspond to the EBV-associated malignant lymphomas in acquired immunodeficiency syndrome with respect to clinical, morphological, phenotypic, and genotypic characteristics. The particular susceptibility of SIVsm immunodeficient cynomolgus monkeys for CBLV-associated lymphomagenesis appears therefore a useful model for EBV-associated lymphomas in humans.

[Opportunistic malignant lymphomas in SIV infected primates--a model for Epstein-Barr virus associated lymphomas in AIDS]. / Opportunistische maligne Lymphome bei SIV-infizierten Primaten--Ein Modell für Epstein-Barr Virus assoziierte Lymphome bei AIDS.

In a series of 33 cynomolgus monkeys (Macaca fascicularis) experimentally infected with Simian Immunodeficiency virus (SIV), strain smm3, 13 animals developed malignant Non-Hodgkin lymphomas. These lymphomas presented with unusual primary manifestations like in the orbita, testes, and brain. The morphological features and immunophenotyping identified the tumors as high malignant B-cell lymphomas. In all tumors as well as in tumor-derived cell lines a cynomolgus B-lymphotropic herpes virus (CBLV) with structural homogeneity to the Epstein-Barr virus (EBV) could be demonstrated by Southern blotting with EBV-specific probes. The lymphoma cells also expressed CBLV-associated nuclear antigens involved in B-cell transformation crossreacting with EBNA-specific human sera and monoclonal antibodies. Ig-gene rearrangement studies revealed clonal populations, however, no translocations of the c-myc oncogene could be detected. The lymphomas developing with high frequency in SIV-induced immunodeficiency resemble a major subtype of human EBV-associated AIDS lymphomas. This animal model can therefore be used to further elucidate interactions of HIV and EBV in AIDS-related lymphomagenesis.

Gene rearrangement studies in the diagnosis of primary systemic and nodular primary localized cutaneous amyloidosis.

Difficulties may arise in the diagnosis of patients with clinical features suggestive of plasma cell dyscrasia-related amyloidosis (amyloidosis L), but without evidence of a paraprotein. We have employed gene rearrangement methodology to demonstrate the clonality of bone marrow cells not only in a patient with myeloma-associated systemic amyloidosis, but also in a patient with "primary" systemic amyloidosis without overt myeloma or a detectable paraprotein. Furthermore, we have shown the clonality of the amyloid-producing plasma cells within a skin nodule of a patient with primary localized cutaneous amyloidosis; by contrast, clonal rearrangement was not detected in bone marrow cells from this patient. This finding provides definitive proof that organ-limited nodular primary localized cutaneous amyloid deposits arise in relation to cutaneous plasmacytomas. Gene rearrangement studies may enable early diagnosis and initiation of treatment in patients with systemic amyloidosis L, as well as their differentiation from patients with organ-limited nodular cutaneous amyloidosis, who do not require aggressive therapy.