Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

Prognostic significance of interferon regulating factor 4 (IRF4) in node-negative breast cancer.

PURPOSE: The transcription factor IRF4 regulates immunoglobulin class switch recombination as well as plasma cell differentiation. We examined the prognostic significance of IRF4 expression in node-negative breast cancer (BC). METHODS: IRF4 expression was evaluated by immunostaining in a cohort of 197 node-negative BC patients not treated in adjuvant setting, referred to as Mainz cohort. The prognostic significance of immunohistochemically determined IRF4 expression for metastasis-free survival (MFS) was examined by Kaplan-Meier survival analysis as well as univariate and multivariate Cox analysis adjusted for age, pT stage, histological grade, ER, and HER2 status. For verification of immunohistochemical results, IRF4 mRNA expression was evaluated using microarray-based gene expression profiling in four previously published cohorts (Mainz, Rotterdam, Transbig, Yu) consisting of 824 node-negative breast cancer patients in total, who were not treated with adjuvant therapy. The prognostic significance of IRF4 mRNA expression on metastasis-free survival (MFS) was examined by univariate and multivariate Cox analysis in the Mainz cohort and by a meta-analysis of all node-negative BC patients and different molecular subtypes. IRF4 mRNA levels were compared to immunohistochemically determined IRF4 expression in 140 patients of the Mainz cohort using Spearman correlation. RESULTS: Immunohistochemically determined high IRF4 expression was associated with higher MFS in univariate Cox regression (HR 0.178, 95% CI 0.070-0.453, p < 0.001). IRF4 maintained its significance independently of established clinical factors for MFS (HR 0.088, 95% CI 0.033-0.232, p < 0.001). Immunohistochemically, determined IRF4 correlated moderately with IRF4 mRNA expression (ρ = 0.589). Higher expression of IRF4 was associated with better MFS in a meta-analysis of the total cohort (HR 0.438, 95% CI 0.307-0.623, p < 0.001). Prognostic significance was more pronounced in the HER2+ molecular subtype (HR 0.215, 95% CI 0.090-0.515, p = 0.001) as compared to the luminal A (HR 0.549, 95% CI 0.248-1.215, p = 0.139), luminal B (HR 0.444, 95% CI 0.215-0.916, p = 0.028), and basal-like subtypes (HR 0.487, 95% CI 0.269-0.883, p = 0.018). Further, IRF4 expression showed independent prognostic significance in a multivariate analysis of the Mainz cohort (HR 0.236, 95% CI 0.105-0.527, p < 0.001). CONCLUSIONS: IRF4 had independent prognostic significance in node-negative BC. Higher expression of IRF4 was associated with improved outcome. The prognostic impact differed between diverse molecular subtypes and was most pronounced in HER2+ breast cancer.

[Liaison psychiatry nurse: the development of supervision in somatic medicine]. / Infirmier en psychiatrie de liaison : développement de la supervision en milieu somatique.

BACKGROUND: Over the years, somatic care has become increasingly specialized. Furthermore, a rising number of patients requiring somatic care also present with a psychiatric comorbidity. As a consequence, the time and resources needed to care for these patients can interfere with the course of somatic treatment and influence the patient-caregiver relationship. In the light of these observations, the Liaison Psychiatry Unit at the University Hospital in Lausanne (CHUV) has educated its nursing staff in order to strengthen its action within the general care hospital. What has been developed is a reflexive approach through supervision of somatic staff, in order to improve the efficiency of liaison psychiatry interventions with the caregivers in charge of patients. The kind of supervision we have developed is the result of a real partnership with somatic staff. Besides, in order to better understand the complexity of interactions between the two systems involved, the patient's and the caregivers', we use several theoretical references in an integrative manner. PSYCHOANALYTICAL REFERENCE: The psychoanalytical model allows us to better understand the dynamics between the supervisor and the supervised group in order to contain and give meaning to the affects arising in the supervision space. "Containing function" and "transitional phenomena" refer to the experience in which emotions can find a space where they can be taken in and processed in a secure and supportive manner. These concepts, along with that of the "psychic envelope", were initially developed to explain the psychological development of the baby in its early interactions with its mother or its surrogate. In the field of supervision, they allow us to be aware of these complex phenomena and the diverse qualities to which a supervisor needs to resort, such as attention, support and incentive, in order to offer a secure environment. SYSTEMIC REFERENCE: A new perspective of the patient's complexity is revealed by the group's dynamics. The supervisor's attention is mainly focused on the work of affects. However, these are often buried under a defensive shell, serving as a temporary protection, which prevents the caregiver from recognizing his or her own emotions, thereby enhancing the difficulties in the relationship with the patient. Whenever the work of putting emotions into words fail, we use "sculpting", a technique derived from the systemic model. Through the use of this type of analogical language, affects can emerge without constraint or feelings of danger. Through "playing" in that "transitional space", new exchanges appear between group members and allow new behaviors to be conceived. In practice, we ask the supervisee who is presenting a complex situation, to design a spatial representation of his or her understanding of the situation, through the display of characters significant to the situation: the patient, somatic staff members, relatives of the patient, etc. In silence, the supervisee shapes the characters into postures and arranges them in the room. Each sculpted character is identified, named, and positioned, with his or her gaze being set in a specific direction. Finally the sculptor shapes him or herself in his or her own role. When the sculpture is complete and after a few moments of fixation, we ask participants to express themselves about their experience. By means of this physical representation, participants to the sculpture discover perceptions and feelings that were unknown up to then. Hence from this analogical representation a reflection and hypotheses of understanding can arise and be developed within the group. CONCLUSION: Through the use of the concepts of "containing function" and "transitional space" we position ourselves in the scope of the encounter and the dialog. Through the use of the systemic technique of "sculpting" we promote the process of understanding, rather than that of explaining, which would place us in the position of experts. The experience of these encounters has shown us that what we need to focus on is indeed what happens in this transitional space in terms of dynamics and process. The encounter and the sharing of competencies both allow a new understanding of the situation at hand, which has, of course, to be verified in the reality of the patient-caregiver relationship. It is often a source of adjustment for interpersonal skills to recover its containing function in order to enable caregiver to better respond to the patient's needs.

p53 is correlated with low BMI negative progesterone receptor status and recurring disease in patients with endometrial cancer.

OBJECTIVE: P53 tumor suppressor gene plays a role in endometrial carcinogenesis. Former studies described correlations between p53 protein overexpression in endometrial cancer and prognostic factors, measured by immunohistochemistry. But data is still controversial. The aim of this study was to measure p53 and phospho-p53 overexpression by Western blot and evaluate correlations between overexpression and prognostic and clinical factors. Phospho-p53 seems to be the functional p53 protein and was examined for the first time in endometrial cancer. METHODS: 40 patients with endometrial cancer were included in the study. A control group of 20 patients with normal endometrial tissue samples was used. Western blot was performed for detection of p53 and phospho-p53. Clinical and pathological parameters were obtained from medical records. Statistical analysis was performed using the log-rank test, the Mann-Whitney test for two independent groups and the Fisher's exact test for dichotomous groupings. RESULTS: In 17.5% of the patients with endometrial cancer a p53 overexpression could be evaluated. There was a correlation between a p53 overexpression and recurring disease (p: 0.014), a negative progesterone receptor status (p: 0.021) and a low BMI (p: 0.022). Only one of 40 patients had a phospho-p53 expression. CONCLUSION: Western blot is a valid method for the detection of p53 overexpression. As other authors described before, p53 overexpression seems to correlate with negative prognostic factors. The correlation between p53 overexpression and a low BMI may underline the relationship between p53 alterations and biological aggressive endometrial carcinomas.

[Anorexia: somatic assessment and management]. / Anorexie: évaluation et prise en charge somatiques.

Anorexia nervosa, which affects about 2-3% of the general population, is the psychiatric illness with the highest rate of mortality. The management is often complex, requiring multiple stakeholders on the patient's physical and psychiatric. The new specialized centre "abC" (anorexia-bulimia, Centre vaudois) was created with the objective of providing quality services to patients involved and to provide a network facilitating the interaction between physicians and specialized institutions. This is an inter-institutional and interdisciplinary collaboration born of the CHUV and the eHnv (Hospitalized Institutions in Nord Vaudois). The abC includes an outpatient pole (CHUV) and a hospital unit on the site of Saint Loup. At term, it will include a day centre (CHUV).

[Anorexia and bulimia: the canton of Vaud's experience of an interdisciplinary approach]. / Anorexie mentale et boutimie: I'expérience vaudoise d'une prise en charge interdisciplinaire.

As psychiatric disorders attacking the body, anorexia and bulimia may have severe psychological, physical and social consequences, often requiring a long-standing interdisciplinary, coordinated and individualized approach. Recently the canton of Vaud has initiated and developed an interinstitutional structure--between the University Hospital (CHUV) and the hospitals of the Northern region of the canton (eHnv)--for the care of patients suffering from eating disorders. This structure, allowing the above mentioned approach for the treatment of eating disorders, consists of an outpatient facility located in the CHUV and an inpatient unit in the hospital of Saint Loup of the eHnv. Within this structure, the general practitioner plays a crucial role in the prevention of the chronification of these disorders by means of their early detection and management.

Trastuzumab therapy vs tetracycline controlled ERBB2 downregulation: influence on tumour development in an ERBB2-dependent mouse tumour model.

Trastuzumab (Herceptin) has improved therapy of breast cancer. Only patients overexpressing ERBB2 are treated with trastuzumab, whereas its use in tumours without ERBB2 expression is useless. This led to the concept that the subgroup of trastuzumab-sensitive tumours is 'ERBB2-dependent', meaning that ERBB2 signalling is indispensable for growth of these tumours. We used a mouse model that allows anhydrotetracycline (ATc)-controlled downregulation of ERBB2 in tumour tissue. ERBB2 mRNA and protein expression were downregulated below detection limit leading to a macroscopically complete tumour remission within 14 days. Tumour remission was accompanied by a strong decrease in proliferation, a moderate increase in apoptosis, as well as dephosphorylation of ERK1/2 and AKT/PKB. These data clearly indicate ERBB2 dependence. Therefore, a high sensitivity to trastuzumab may be suspected. Surprisingly, trastuzumab caused a much weaker effect compared to ATc-induced ERBB2 downregulation, although a decrease in ERBB2 membrane localisation was induced. Only a slight decrease in proliferation and a weak transient increase in apoptosis were observed. Interestingly, tumours responded to trastuzumab by a sharp fivefold increase in phosphorylated AKT/PKB as well as a 3.5- and 5.3-fold increase in AKT1 and AKT2 mRNA levels, respectively. In conclusion, 'ERBB2 dependence' is not sufficient to define trastuzumab-responsive tumours. The suboptimal effect of trastuzumab compared to the maximally possible effect induced by ATc demonstrates a high potential for improved ERBB2 blocking therapies.

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

Downregulation of beta2-microglobulin in human cord blood somatic stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells?

Adherently growing, non-hematopoietic somatic stem cells isolated from human cord blood were stained with the fluorescent dye PKH26 and transplanted into livers of SCID-mice to examine a possible cell fate transition. Already 7 days after transplantation stem cells were well integrated into the liver tissue. Human albumin that was not expressed by the stem cells before transplantation was detectable in the host's livers after injection of cord blood stem cells. Human alpha1-antitrypsin was detectable in stem cells already before transplantation and remained positive in the mouse liver. The most interesting observation in this study was the downregulation of human beta2-microglobulin (beta2M) in the stem cells after transplantation: beta2M is expressed constitutively in our cord blood stem cells. However, beta2M was no longer detectable by RT-PCR in all tissues where human albumin and alpha1-antitrypsin were expressed after stem cell transplantation. beta2M is known to participate as an integral part of the major histocompatibility complex. Absence of beta2M makes the residual heavy chain inactive as an antigen. Thus, downregulation of beta2M may represent an escape mechanism from killer-T cells and may be a molecular mechanism explaining the recently described "immunological blindness" [37] of stem cells. In contrast to the results obtained after direct injection of stem cells as a suspension, no consistent downregulation of beta2M was observed after transplantation of stem cells encapsulated in alginate beads to generate a compartment where stem cells are protected from the host's natural killer cells. No expression of human genes was observed after transplantation of human cord blood derived mononuclear cells (MNC) that were used as a negative control. In conclusion, we have shown that human cord blood somatic stem cells survive and are reprogrammed after transplantation into mouse livers, although a complete transdifferentiation to hepatocytes did not occur within 7 days, since some marker genes (GATA4 and alpha-fetoprotein) were still negative. Switching off expression of beta2M may be part of an intriguing and novel mechanism explaining why stem cells escape the host's immune system.

Angiogenesis and fibroblast proliferation precede formation of recurrent tumors after radiation therapy in nude mice.

Recently, the combination of ionizing radiation with inhibitors of angiogenesis has been reported to improve tumor eradication compared to treatment with irradiation alone. However, the mechanisms of this effect have not been defined. For this purpose [corrected] we established a non-small cell lung cancer model in nude mice. Tumor vascularization was visualized in vivo by MRI using gadolinium-DTPA as contrast agent. Further, cryosections were produced as close as possible to the MRI slice positions. Since we were interested in examining the formation of a recurrent tumor, irradiation was performed with a single fraction of 4 Gy. This dose caused a partial remission followed by recurrent tumor growth 25 to 35 days after therapy. The process of partial remission as well as formation of the recurrent tumor was examined in 28 nude mice analysing the following parameters: (i) contrast agent enhancement using high-resolution MRI, (ii) proliferation of tumor cells and fibroblasts using Ki-67 immunohistochemistry and (iii) formation of microvessels using CD31 immunohistochemistry. The latter analyses led to differentiation of three stages. Stage 1 (day 1 to day 15 after irradiation) was characterized by increasing areas of dead cell mass in hematoxylin-eosin-stained slides that corresponded to a decrease in tumor cell proliferation as well as contrast agent enhancement in MRI. The percentage of Ki-67-positive tumor cells decreased from initially 45.1% +/- 6.0% (mean +/- standard deviation) to 1.4% +/- 1.2% (mean +/- standard deviation) on day 15. Stage 2 (day 6 to day 20 after irradiation; overlapping with stage 1) was characterized by proliferation of fibroblasts leading to formation of fibrotic septae with abundant microvessels. Already during late stage 2, MRI identified new contrast agent enhancing areas. Stage 3 (day 20 to day 40 after irradiation) was characterized by new tumor cell proliferation. Interestingly, tumor cells almost exclusively proliferated in the direct neighbourhood of the fibrotic septae that had been formed in stage 2. Obviously, proliferation of fibroblasts and blood vessels was a condition prior to formation of recurrent tumor tissue. Thus, our results are in contrast with the view that tumors or recurrent tumors begin as avascular masses that later induce neovascularization. With respect to clinical practice, our results suggest that: (i) adjuvant anti-angiogenic therapy should not be limited to the day of irradiation but should cover a critical period until day 5 to day 20 after radiotherapy, (ii) adjuvant therapy should also include inhibition of fibroblast proliferation and (iii) MRI can identify a recurrent tumor 10 to 15 days before occurrence of new tumor growth.

Malacoplakia simulating organ invasion in a rectosigmoid adenocarcinoma: report of a case.

Malacoplakia is a histiocytic inflammatory response that may be associated with colorectal tumors. We report the case of a 65-year-old male taking steroids for a severe pulmonary disease. He presented with a rectosigmoid tumor that seemed to infiltrate the urinary bladder and the sacrum on the preoperative CT scan and echography and at laparotomy. A low anterior resection en bloc with a partial cystectomy was performed. The pathologic analysis showed a pT3pN0 adenocarcinoma with an extensive malacoplakia infiltrating the bladder and the pericolic and perirectal tissues. This case report emphasizes the overstaging that malacoplakia may induce and underlines a situation the surgeon may possibly confront. Our observation confirms the association of malacoplakia, colorectal carcinoma, and steroid treatment.

Inhibition of phosphofructokinases by copper(II).

The biochemical inhibition by Cu2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant.

c-erbB-2 expression in small-cell lung cancer is associated with poor prognosis.

Small-cell lung cancer (SCLC) carries a bad prognosis despite good initial response to chemotherapy. It is therefore important to identify molecular markers that influence survival as potential new therapeutic targets. In our study, expression of the tyrosine kinase c-erbB-2 (HER2/neu) receptor in tumor tissues of 107 consecutive newly diagnosed patients with primary SCLC was quantified using a monoclonal antibody directed against the c-terminal domain of c-erbB-2. A clear-cut positive expression of c-erbB-2 was observed in 13% of patients. Surprisingly, c-erbB-2 was an independent prognostic factor (RR = 2.16; p = 0.014) when a proportional-hazard model was adjusted to stage (limited vs. extensive disease) and performance status (WHO I-IV), the most relevant clinical parameters. Similarly, a significant association between c-erbB-2 and survival was obtained if a larger number of clinical parameters were included into the analysis, namely response to chemotherapy, TNM stage, lactate dehydrogenase (LDH), neuron-specific enolase (NSE), gender and age (p = 0.033). Interestingly, c-erbB-2 expression was more relevant for patients with advanced tumors. In the subgroup of patients with bad performance status (WHO II-IV), median survival of patients with undetectable c-erbB-2 expression was 274 days compared with only 23 days for patients with clear-cut positive c-erbB-2 immunohistochemistry (p = 0.0031; log-rank test). Similar results were obtained for patients with extensive disease (p = 0.028) and high TNM stages (T>2 or N>1 or M1; p < 0.068, all comparisons). In contrast, c-erbB-2 expression was not associated with survival in patients with limited disease (p = 0.97), low TNM stages (p > 0.56, all comparisons) and good performance status (p = 0.97). In conclusion, c-erbB-2 is expressed in more than 10% of SCLC. Expression of c-erbB-2 is an independent prognostic factor of survival. The effect of c-erbB-2 expression seems to become more important in advanced stages of the disease. Since c-erbB-2 is a therapeutical target in other types of cancer, further studies to identify the role of c-erbB-2 in SCLC are clearly warranted.

Detection of the synovial sarcoma translocation t(X;18) (SYT;SSX) in paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction: a reliable and powerful diagnostic tool for pathologists. A molecular analysis of 221 mesenchymal tumors fixed in different fixatives.

Synovial sarcoma (SS) is a relatively rare sarcoma, which may be confused with several other mesenchymal and nonmesenchymal lesions. It bears the t(X;18) (SYT;SSX) translocation, which seems to be specific for this tumor type and can be detected in paraffin-embedded tissue, using reverse transcriptase-polymerase chain reaction (RT-PCR). However, the specificity and sensitivity of this detection method have rarely been examined in a large series. Using RT-PCR, we examined 250 mesenchymal and nonmesenchymal, benign and malignant, paraffin-embedded lesions for the SS t(X;18) (SYT-SSX) translocation. PCR products were obtained from 221 tumors (88.5%). There were 135 non-SS tumors, 22 biphasic, and 64 monophasic spindle/round cell SS, of which 10 were cytogenetically confirmed as t(X;18)-positive. SYT-SSX gene fusion transcripts were detected in the SS tumor category only (100% specificity), including 100% of the biphasic SS and 86% of monophasic spindle/round cell SS. Nine tumors originally diagnosed as SS were t(X;18) (SYT-SSX)-negative. Following reassessment, only 3 of these tumors showed clinicopathologic, immunohistochemical, and/or ultrastructural features consistent with that diagnosis, thus raising the overall detection sensitivity to 96%. With regard to the potential adverse effect of the fixatives used, PCR products were obtained in 100%, 91.5%, 90.5%, and 0% of tumors fixed with AFA, buffered formalin, Holland Bouin, and conventional Bouin's fluid, respectively. This study shows that the detection of the SS t(X;18) (SYT-SSX) in paraffin-embedded tissue is feasible with a 100% specificity and an overall 96% sensitivity, provided non-Bouin's fluid fixation is used.

Polymerase chain reaction in the diagnosis of T-cell lymphoma in paraffin-embedded bone marrow biopsies: a comparative study.

AIMS: In routine histological analysis of bone marrow biopsies, the distinction between reactive T-cell infiltrates and T-cell lymphoma can be difficult, even with the use of extensive immunohistochemistry. The aim of this study was to evaluate the diagnostic contribution of TCR-gamma gene rearrangement analysed by PCR. METHODS AND RESULTS: The samples studied consisted of 46 paraffin-embedded bone marrow biopsies (diagnosis, staging and follow-up) from 26 patients with T-cell lymphoma. The bone marrow biopsies were categorized into three groups according to the morphological and immunohistochemical results. Group 1, positive for T-cell lymphoma (24 bone marrow biopsies), group 2, suspicion of T-cell lymphoma (15 bone marrow biopsies) and group 3, negative for T-cell lymphoma (seven bone marrow biopsies). DNA could be amplified in 45/46 bone marrow biopsies (98%). Clonal rearrangement was detected in 30/45 bone marrow biopsies tested (67%) including 15/24 bone marrow biopsies (62.5%) of group 1, 11/14 (78.5%) of group 2 and 4/7 (57%) of group 3. In total, PCR analysis supported a diagnosis of T-cell lymphoma in 15/45 bone marrow biopsies (33%), in which histological and/or immunohistochemical examination provided inconclusive evidence of malignancy. CONCLUSIONS: TCR-gamma PCR is a complementary tool for the assessment of T-cell lymphoma in bone marrow biopsies. Optimal evaluation of bone marrow biopsies requires an integrative approach of all available results from morphology, immunohistochemistry, molecular biology and clinical data.

Mutation analysis of the cationic trypsinogen gene in patients with pancreatic cancer.

Recently, an Arg to His mutation at residue 117 of the cationic trypsinogen gene (Arg117His) has been shown to be associated with hereditary pancreatitis (hp). A serious complication of hp is development of pancreatic cancer. Patients suffering from hp have been reported to have a 53-fold increased risk to die from pancreatic cancer. However, the quantitative contribution of mutations in the cationic trypsinogen gene to all pancreatic cancer cases is unknown. A relevant contribution of the Arg117His-mutation to pathogenesis of pancreatic cancer might be possible, since also asymptomatic individuals have been reported to carry this mutation and individuals with only mild symptoms may be undiagnosed as hp. In the present study we analyzed genomic DNA obtained from pancreatic cancer tissue from 34 patients and corresponding normal tissue from 28 of these individuals. The third exon of the cationic trypsinogen gene was amplified by nested PCR and digested with AflIII, since the Arg117His mutation creates an AflIII-restriction site. None of the examined samples carried the Arg117His mutation, whereas the amplification product obtained from a patient with known hp was clearly positive. Sequencing of the complete third exon of the cationic trypsinogen gene in 10 of the pancreatic cancer patients resulted exclusively in the wild-type sequence. In addition DNA obtained from venous blood of 116 further patients with pancreatic cancer did not carry the Arg117His mutation. Our results show that the Arg117His mutation does not contribute to pathogenesis of a substantial fraction of all pancreatic adenocarcinomas. In contrast to most oncogenes or tumor suppressor genes the cationic trypsinogen gene (3rd exon) does not contain mutational hot spots.

Lipomatous hemangiopericytoma: a fat-containing variant of solitary fibrous tumor? Clinicopathologic, immunohistochemical, and ultrastructural analysis of a series in favor of a unifying concept.

The dinicopathologic, immunohistochemical and ultrastructural features of 13 lipomatous hemangiopericytomas are presented. There were 6 male and 7 female patients whose ages at diagnosis ranged from 27 to 75 years (median 48) all presenting with a mass of variable duration. The tumor sizes ranged from 1.7 cm to 19 cm (median 5.5 cm). The locations included the orbit (1), neck (1), mediastinum (1), epicardium (1), retroperitoneum (3), right iliac fossa (1), and upper (1) and lower (4) extremity. Histologically, the lesions were composed of a varying admixture of spindle-shaped to round cells, variably collagenous stroma, adipose tissue, and branched, often thick-walled, hemangiopericytoma-like vessels. For 11 tumors, the mitotic activity ranged from 1 to 3 mitoses per 10 high-power fields (HPF). One tumor which contained hypercellular areas showed 13 mitoses per 10 HPF, and another hypercellular lesion showed up to 43 mitoses per 10 HPF, abnormal mitoses, and necrosis. Immunohistochemically, tumor cells were invariably positive for vimentin and CD99, and mostly for CD34 but negative for desmin, keratin, CD31, CD117 (c-kit), and inhibin. About half of the tumors showed reactivity for bcl-2. Occasionally, focal reactivity was also observed for smooth muscle actin, muscle-specific actin, S100 protein, and epithelial membrane antigen. Ultrastructural examination of seven cases showed features in keeping with fibroblastic, myofibroblastic, or pericytic differentiation. Treatment consisted of simple tumorectomy in 10 cases and wide excision in 3. Follow-up information on 10 patients (range: 6 to 77 months; median: 18 months) showed no recurrence. Lipomatous hemangiopericytoma which share the clinical, pathologic, immunohistochemical, and ultrastructural features of solitary fibrous tumor (SFT) is likely to represent, in most cases, a fat-containing variant of SFT.

Orbital and extraorbital giant cell angiofibroma: a giant cell-rich variant of solitary fibrous tumor? Clinicopathologic and immunohistochemical analysis of a series in favor of a unifying concept.

The clinicopathologic and immunohistochemical features of one orbital and nine extraorbital soft tissue lesions, the morphology of which overlaps with giant cell angiofibroma and solitary fibrous tumor, are presented. There were 3 male and 7 female patients. Age at diagnosis ranged from 18 to 81 years (median: 45 yrs). Development of a mass was the main presenting symptom. For two patients, the lesion had been evident for several years before excision. Extraorbital tumors were located in the head and neck area (3), back (3), retroperitoneum (1), hip (1), and vulva (1). Tumor size ranged from 1.3 cm to 11 cm (median: 4.5 cm). The lesions presented grossly as well-demarcated, unencapsulated soft tissue masses. Histologically, they were characterized by the presence of alternating cellular and sclerosing areas, keloidal collagen deposition, round- to staghorn-shaped, thick-walled vessels and multinucleated giant stromal cells often lining pseudovascular spaces. Cellular areas were composed of non-atypical spindle to round cells set in a variably collagenous background. Mitotic activity ranged from 1 to 3 mitoses/10 high-power fields. Immunohistochemical studies showed positive staining of the spindle/round cells and multinucleated stromal cells invariably for vimentin, CD34, CD99, and mostly for bcl-2 but negative for muscle specific actin, desmin, CD31, CD117 (c-kit), and inhibin. Occasionally, focal reactivity was observed for smooth muscle actin, S-100 protein, epithelial membrane antigen, and keratin. Treatment consisted of simple tumorectomy in eight patients and wide excision in two. Follow-up information for eight patients (range: 7-32 mos; median: 14 mos), including four with microscopically positive surgical margins, showed no recurrence. These lesions share the clinical, pathologic, and immunohistochemical features of giant cell angiofibroma and solitary fibrous tumor, supporting the view that these tumors are closely related. In addition, it shows that giant cell angiofibroma occurs equally in both sexes and has a wider distribution than initially thought, developing even more often in extraorbital locations than in the orbit.

Metastasizing fibrous histiocytoma of the skin: a clinicopathologic and immunohistochemical analysis of three cases.

The clinicopathologic and immunohistochemical features of three metastasizing fibrous histiocytomas of the skin are presented. The first patient had a 1.3-cm nodule in the right thigh, with right inguinal lymph node metastases 19 years later. The second patient, who had a 3-cm nodule excised from his left thigh and inguinal lymph node metastasis after 4 months, had a favorable outcome 14 years after local radiotherapy and chemotherapy. The third had a 2-cm nodule in his neck, which recurred 16 months later. Four months later, cervical lymph node metastases were found. The patient was alive and well 26 months after initial surgery. All three primary skin tumors involved the dermis and subcutis, appeared well-delineated but nonencapsulated, were associated with some degree of epidermal hyperplasia, and showed features of aneurysmal/atypical or cellular fibrous histiocytoma. The number of mitoses ranged from 6 to 11 per 10 high-power fields. Recurrences and metastases showed morphologic features similar to primary lesions. Tumor cells were positive, at least focally, for CD 68, Ki-M1p, and Factor XIIIa, and occasionally for smooth muscle actin. Desmin, CD 34, S-100 protein, and cytokeratin stainings were negative. Primary neoplasms, recurrences, and metastases showed a Mib-1 labeling index of 10% or less. Cellular, aneurysmal, and atypical (pseudosarcomatous) fibrous histiocytomas of the skin can metastasize, yet they often show a protracted clinical course. Risk factors for metastatic dissemination include large size, high cellularity, aneurysmal changes, marked cellular pleomorphism, high mitotic activity, tumor necrosis, and repeated local recurrences.