Evaluation of MYC status in oral lichen planus in patients with progression to oral squamous cell carcinoma.

BACKGROUND: Malignant transformation of oral lichen planus (OLP) to oral squamous cell carcinoma (OSCC) is controversial. C-MYC is a proto-oncogene involved in various solid tumours, including OSCC. OBJECTIVES: To determine MYC status using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in OLP lesions from 10 patients with progression to OSCC (group I) and to compare this with OLP lesions from patients without progression to OSCC (group II). METHODS: We constructed two tissue microarrays with 11 OSCC samples (group IA), 17 OLP samples from the same patients (group IB) and 13 OLP specimens from 12 control patients (group II). FISH evaluation of the MYC gains was determined in 100 nonoverlapping nuclei per sample. IHC evaluation was determined by calculating the percentage C-MYC expression in the epithelial cells. RESULTS: OSCC samples showed MYC copy number gains and C-MYC overexpression in 91% and 73% of cases, respectively. MYC gains were detected in 47% of samples from group IB and were absent from all samples from group II. C-MYC was overexpressed in 87% of cases from group IB and in only 44% of control specimens (group II). The differences in MYC status between groups IB and II were statistically significant. CONCLUSIONS: OLP lesions in patients with progression to OSCC show MYC gains and C-MYC overexpression. In patients with severe OLP, determining MYC status may predict a subgroup of subjects with a higher risk of progression to OSCC.

Multiple genetic copy number alterations in oral squamous cell carcinoma: study of MYC, TP53, CCDN1, EGFR and ERBB2 status in primary and metastatic tumours.

BACKGROUND: Oncogenesis in the oral cavity is believed to result from genetic alterations that cause a stepwise transformation of the mucosa to invasive carcinoma. In oral squamous cell carcinoma (OSCC) multiple cytogenetic abnormalities have been reported, but their practical significance remains uncertain. OBJECTIVE: To evaluate the usefulness of the assessment of CCND1, MYC, EGFR, ERBB2 and TP53 in OSCC and lymph node metastases. METHODS: Fifty-one consecutive samples of OSCC, nine lymph node biopsies showing metastatic spread from OSCC, 16 biopsies diagnosed as oral leucoplakia (OLK), 13 samples corresponding to oral lichen planus (OLP) and 14 samples from normal oral mucosa were included in the study. Clinical and histopathological characteristics were reviewed. The genetic and protein status of the CCND1, MYC, EGFR, ERBB2 oncogenes and the TP53 tumour suppressor gene were assessed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). The obtained results were compared with the clinical characteristics and the outcome of the OSCCs. RESULTS: TP53 gene losses and MYC, ERBB2, CCND1 and EGFR copy number gains and amplifications were detected in a higher proportion in OSCC and lymph node samples than in OLK and OLP samples (P < 0·005). Overexpression of p53, Myc, Cyclin D1, c-erbB-2 and epidermal growth factor receptor (EGFR) was more prevalent in malignant samples than benign samples (P < 0·05). Correlation between FISH and IHC results was demonstrated in MYC, EGFR and CCND1 studies. The presence of two or more genetic abnormalities in the studied loci was exclusively detected in primary and metastatic OSCC. CONCLUSIONS: In our series, genetic abnormalities in TP53, MYC, CCND1, ERBB2 and EGFR detected by FISH were absent in inflammatory lesions, infrequent in precursor lesions and common in tumoral lesions. Evaluation of the genetic status of TP53, MYC, CCND1, ERBB2 and EGFR may be an additional diagnostic tool in distinguishing benign from malignant oral lesions in histopathologically challenging cases.

MYC gene numerical aberrations in actinic keratosis and cutaneous squamous cell carcinoma.

BACKGROUND: The genetic alterations that drive the transition from actinic keratoses (AKs) to cutaneous squamous cell carcinomas (SCCs) have not been defined precisely. Amplification and/or overexpression of the MYC proto-oncogene have been demonstrated in several human, malignant tumours including head and neck SCCs. OBJECTIVES: To evaluate the presence of MYC genomic aberrations in both AKs and SCCs. METHODS: Skin biopsy specimens corresponding to AKs, SCCs and control samples were included in two paraffin-embedded tissue microarrays. MYC cytogenetic profile was evaluated by fluorescence in situ hybridization (FISH). The results obtained were compared with MYC immunohistochemical expression. RESULTS: Twenty-three AKs and 30 SCCs were evaluated. MYC numerical aberrations were observed in eight of 23 (35%) AKs and 19 of 30 (63%) SCCs (P = 0.05). MYC numerical aberrations were more frequent in moderately to poorly differentiated SCCs (77%) when compared with well-differentiated SCCs (25%; P = 0.027). A significant association between copy number gains of MYC by FISH analysis and MYC protein expression was demonstrated. CONCLUSIONS: MYC gains and amplifications are frequent cytogenetic abnormalities in SCCs and may play a relevant role in promoting SCC undifferentiation and tumoral progression.

Role of Leishmania spp. infestation in nondiagnostic cutaneous granulomatous lesions: report of a series of patients from a Western Mediterranean area.

BACKGROUND: Leishmaniasis is a parasitic disease prevalent in countries of the Mediterranean area. OBJECTIVES: The potential role of Leishmania as the aetiological factor for cutaneous granulomatous lesions in a series of patients from a Western Mediterranean area was evaluated. The practical usefulness of Leishmania-specific polymerase chain reaction (PCR) amplification and immunohistochemical techniques in skin biopsy specimens was assessed. METHODS: Twenty-five skin biopsies diagnosed as nonspecific granulomatous dermatoses were included in the study. A panel of histopathological features was blindly evaluated by two independent observers. Only those cases showing nondiagnostic clinicopathological features and lacking demonstrable microorganisms after bacteriological, mycological or mycobacteriological cultures and specific stains (Ziehl-Neelsen, Giemsa, Gram, periodic acid-Schiff stains) were finally selected. Quantitative real-time PCR was performed in all selected samples. In available samples, immunohistochemical detection of specific Leishmania spp. antigens was also performed. RESULTS: From the selected 25 biopsies, Leishmania spp. DNA was detected by real-time PCR in 13 cases. In seven of eight PCR-positive cases the presence of a varying density of amastigotes could also be demonstrated immunohistochemically. CONCLUSIONS: Leishmania infection seems to be an important aetiological factor in cutaneous granulomatous lesions showing nondiagnostic features in endemic areas. In such areas, Leishmania-specific PCR amplification and/or immunohistochemical studies may be useful diagnostic tools. These techniques may be specifically indicated in the evaluation of patients showing nonspecific granulomatous inflammatory infiltrates of unknown aetiology lacking the histopathological evidence of parasites.

A study of Ki-67, c-erbB2 and cyclin D-1 expression in CIN-I, CIN-III and squamous cell carcinoma of the cervix.

The histological criteria for cervical intraepithelial neoplastic lesions and their follow-ups have been established, but their reproducibility, specificity and sensibility are not certain. Immunohistochemical markers provide more information on each specific case, in order to facilitate its classification and, eventually, its prognosis. Using immunohistochemical techniques, this study analyzes the prognostic value of three markers (Ki-67, c-erbB2 and Cyclin D1) in cases of low grade squamous intraepithelial neoplasia (CIN-I), high grade squamous intraepithelial neoplasia (CIN-III), and infiltrating squamous cell carcinoma (SCC) taken from a group of cervical samples. In situ hybridization was performed in order to detect high-risk HPV. High risk HPV was demonstrated in 82%, 89% and 100% of the LGSIL, HGSIL and SCC cases, respectively. C-erbB2 expression was detected in 9%, 33% and 50% of the LSIL, HGSIL and SCC cases, respectively. The Ki-67 LI was 25%, 68% and 65.5% in the LGSIL, HGSIL and SCC cases, respectively. Nuclear Cyclin D1 expression was seen in 82%, 11% and 30% of the CIN-I,CIN-III and SCC cases, respectively. We observed that the cytoplasmic cyclin D1 expression increased with the severity of the lesion instead of the nuclear expression decreasing with the progression of the pathology. Nuclear and cytoplasmic Cyclin D1 expression seemed to be related to HPV high risk infection. We concluded that Cyclin D1, cerbB2 and The Ki-67 LI expression changed in relation to the severity of the lesion and that they could be helpful in making a differential diagnosis.

Primitive neuroectodermal tumor of the central nervous system with glial differentiation: a FISH study of an adult case.

Primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS), a rare occurrence in adults, may show glial differentiation and can be misinterpreted as pure astrocytic neoplasms. Few fluorescence in situ hybridization (FISH) studies have been carried out on these tumors; isochromosome 17q was found to be the major chromosomal abnormality. We present the case of an adult in which we performed a FISH study of both the glial and neuronal components. A complex array of FISH changes, not including an isochromosome 17q were identified.

Expression of androgen, oestrogen alpha and beta, and progesterone receptors in the canine prostate: differences between normal, inflamed, hyperplastic and neoplastic glands.

The expression of receptor for androgen (AR), oestrogen alpha and beta (ERalpha and ERbeta) and progesterone (PR) was examined immunohistochemically in canine prostate specimens (normal, hyperplastic, inflamed [prostatitis] or neoplastic). AR immunolabelling was seen in 100% of epithelial cells of normal and hyperplastic tissue, the corresponding figures for inflamed and carcinomatous tissue being 74% and 65%, respectively. ERalpha labelling was seen in 85% of epithelial cells in normal prostate glands, the corresponding figures for hyperplastic, inflamed and neoplastic glands being 35%, 22% and 12%, respectively. ERbeta labelling was seen in 85% of epithelial cells of normal glands and in about 70% of such cells in glands showing pathological changes. On the other hand, PR expression (weak) in normal glands was observed in fewer epithelial cells (44%) than in hyperplastic (70%), inflamed (62%) or neoplastic (64%) glands.

Expression of Snail protein in tumor-stroma interface.

The product of Snail gene is a repressor of E-cadherin transcription and an inductor of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. In order to examine Snail expression in animal and human tissues, we have raised a monoclonal antibody (MAb) that reacts with the regulatory domain of this protein. Analysis of murine embryos shows that Snail is expressed in extraembryonic tissues and embryonic mesoderm, in mesenchymal cells of lungs and dermis as well as in cartilage. Little reactivity was detected in adult tissues as Snail was not constitutively expressed in most mesenchymal cells. However, Snail expression was observed in activated fibroblasts involved in wound healing in mice skin. Moreover, Snail was detected in pathological conditions causing hyperstimulation of fibroblasts, such as fibromatosis. Analysis of Snail expression in tumors revealed that it was highly expressed in sarcomas and fibrosarcomas. In epithelial tumors, it presented a more limited distribution, restricted to stromal cells placed in the vicinity of the tumor and to tumoral cells in the same areas. These results demonstrate that Snail is present in activated mesenchymal cells, indicate its relevance in the communication between tumor and stroma and suggest that it can promote the conversion of carcinoma cells to stromal cells.

Molecular pathology of endometrial carcinoma: transcriptional signature in endometrioid tumors.

A dualistic model, which has been established on a morphological basis and that differentiates type I endometrioid from type II non-endometrioid endometrial cancer, is widely accepted. Molecular genetics have provided us with data supporting the dualistic model of endometrial tumorigenesis and with some clues to speculate about the sequence of the molecular alterations defining the tumorigenesis pathways. In type I endometrioid endometrial cancer, PTEN gene silencing, microsatellite instability associated with defects in DNA mismatch repair genes, or mutations in the K-ras gene are the known major alterations defining the progression from normal endometrium to hyperplasia and then on to carcinoma. Recently, cDNA microarray technology for identifying the differences in gene expression patterns between the histological types of endometrial cancer have permitted the identification of differentially expressed genes that could help us to understand differences in the biology and the clinical outcome between histiotypes. Genes involved in the mitotic checkpoint as a major mechanism of carcinogenesis in non-endometrioid endometrial cancer, or altered genes associated with the initial steps of myometrial infiltration in endometrioid endometrial cancer, represent examples of how useful large genetic screenings can be for understanding the tumorigenesis process and the future directions in the molecular pathogenesis of endometrial cancer.

The Ki-67 labeling index is not a useful predictor for the follow-up of cervical intraepithelial neoplasia 1.

OBJECTIVE: Our aim was to determine whether the Ki-67 immunostaining pattern, present on diagnosis of cervical intraepithelial neoplasia (CIN), predicts the change from low-grade to high-grade CIN over a 2-year period after diagnosis. MATERIALS AND METHODS: Of 59 cervical biopsy samples from 59 patients diagnosed as having cervical CIN, 35 were diagnosed as CIN 1 and 24 were diagnosed as CIN 2 or CIN 3. The Ki-67 immunostain showed immunopositive cells in the upper two thirds of the epithelium in all specimens. Two hundred nuclei were counted in 25 high-power fields in each specimen, including all of the epithelial layers, to determine the mean number of Ki-67-positive cells. In situ hybridization was used to demonstrate and type human papillomavirus. The chi test, Fisher exact test, Student t test, one-way analysis of variance, and Tukey test were used for statistical analysis, with significance set at p < .05. RESULTS: The mean Ki-67 labeling index for CIN 1, CIN 2, CIN 3, and CIN 2,3 were, respectively, 32.5%, 43.2%, 53.2%, and 47.8%. The statistical study showed significant differences between CIN 1 versus CIN 2, CIN 1 versus CIN 3, and CIN 1 versus CIN 2,3. For CIN 1, the mean Ki-67 labeling index was 32.8% when the lesion disappeared and was 34.6% for persisting lesions. There was no statistically significant difference. CONCLUSIONS: Ki-67 labeling index did not predict persisting CIN 1.

Pathogenicity of Cryptococcus neoformans var. gattii in an immunocompetent mouse model.

The pathogenicity of two different genomic profiles of Cryptococcus neoformans var. gattii serotype B isolated from goats that died from cryptococcal pneumonia was assessed in an experimental model of immunocompetent mice. One strain of each randomly amplified polymorphic DNA (RAPD) profile (GR52 and GR56) and three reference C. neoformans isolates representing serotypes B, D and C were used. BALB/c male mice were inoculated by the intraperitoneal route with each strain. After 4 weeks of follow-up, the animals were sacrificed and autopsy specimens of testes, liver, spleen, kidney, lungs and brain were cultured and stained for histopathology. Although spontaneous mortality was only 2% (one animal), all mice except for those inoculated with serotype C showed positive cultures in almost one organ. The strain GR52 isolated from goat showed the highest rate of positive cultures (80%) followed by serotype D (77%). Serotype B reference strain and second goat strain GR56 were both isolated from 70% of samples. Serotype C was recovered in only 33% of organs, and never from brain or lung specimens. GR52 grew abundantly from all lung cultures, and yeast cells with large capsules were seen in histopathology inside the alveoli, peribronchial vessels and interalveolar spaces. They appeared to elicit no inflammatory response. We conclude that intraperitoneally inoculated C. neoformans var. gattii shows high virulence in this immunocompetent mouse model. Strain GR52 was highest in pathogenicity and had marked lung tropism. In contrast, the serotype C reference strain showed the lowest pathogenicity and seemed not to spread outside the abdominal viscera.

In vitro susceptibility of Cryptococcus neoformans serotypes to GM 237354 derivative of the sordarin class.

In vitro susceptibility to the sordarin derivative GM 237354 and amphotericin B were tested in a total of 190 Cryptococcus neoformans clinical isolates from different geographical areas of Spain and South American countries. Minimal inhibitory concentrations (MICs) were obtained using the NCCLS reference microbroth dilution method and analysed according the serotypes of Cr. neoformans. The MICs for amphotericin B were lower than 1.0 microg ml(-1) (MIC90% 0.5 microg ml(-1) , MIC50% 0.125 microg ml(-1)) but five isolates showed MICs of 2.0 microg ml(-1) to GM 237354 (MIC90% 1.0 microg ml(-1), MIC50% 0.5 microg ml(-1)). Cryptococcus neoformans var. gattii serotype B, was significantly less susceptible than A and AD serotypes (P = 0.047 and P = 0.022, respectively).

Changes in androgenic steroid profile due to urine contamination by microorganisms: a prospective study in the context of doping control.

Urine contamination by microorganisms may affect the interpretation of urinalysis in different areas of clinical diagnosis. This is particularly relevant in doping control. A prospective study was designed to assess the effects of urine contamination by selected pathogens on the endogenous androgenic steroid profile. Pooled urine from a healthy male volunteer with standard steroid profile compared with reference values for the Caucasian population was sterilized by filtration and stored in sterile glass tubes. Aliquots were inoculated with known amounts of 15 different organisms (bacteria, fungi, and moulds) and incubated at 37 degrees C for 2 weeks. Different markers of urine contamination, such as pH, deconjugation of steroids, and metabolic by-products, were determined. Alkalization of urinary pH was not a reliable indicator of urine contamination as several organisms grew in this medium and no alteration of this parameter was found. In uncontaminated urine, less than 10% of steroid glucuronide conjugates were spontaneously hydrolyzed. Higher rates of hydrolysis for sulfate conjugates were found. An unconjugated fraction higher than 10% of the total amount of testosterone was a reliable indicator of urine contamination. However, microbial production of testosterone or epitestosterone was not detected. In contrast, a few organisms were able to synthesize 5alpha-androstanedione, 5beta-androstanedione, and androstenedione using endogenous steroids as substrates.

Serotyping of Cryptococcus neoformans isolates from clinical and environmental sources in Spain.

We determined biovars and serotypes of 154 isolates of Cryptococcus neoformans from clinical and environmental sources from different areas of Spain. All clinical isolates belonged to C. neoformans var. neoformans. Serotypes showed an irregular distribution. C. neoformans var. gattii serotype B was isolated from necropsy specimens from goats with pulmonary disease.

First identification of autochthonous Cryptococcus neoformans var. gattii isloated from goats with predominantly severe pulmonary disease in Spain.

Cryptococcus neoformans var. gattii is associated with Eucalyptus trees growing in various tropical and subtropical regions of the world. The identification of 13 autochthonous strains of C. neoformans var. gattii in Spain is reported. These strains were isolated from lung (10 samples), liver (1 sample), and brain (2 samples) tissue specimens from six goats suffering from predominantly severe pulmonary disease that were autopsied. The animals were members of five different herds of goats grazing in rural areas of the province of Cáceres (Extremadura, Spain). Between 1990 and 1994, there were five outbreaks, in which between 2.5 and 12% of the goats were affected. Although respiratory symptoms (pneumonia) associated with cachexia were the predominant clinical picture in all outbreaks, brain and liver involvement was also documented in three of the five outbreaks. Biotyping was performed by culturing the isolates on L-canavanine-glycine-bromothymol blue medium and testing them for the assimilation of D-proline and D-tryptophan. Serotyping by agglutination tests confirmed the characterization of all strains as C. neoformans var. gattii serotype B. This is the first confirmation of the presence of this variety in Spain, with a peculiar ability to produce severe pulmonary and systemic disease in normal goats, particularly in the form of outbreaks of pneumonia in association with cachexia.

A clinical trial on the prevention of catheter-related sepsis using a new hub model.

BACKGROUND: Catheter hub contamination is being increasingly recognized as a source of catheter-related sepsis. The authors have investigated the efficacy of a new hub design in preventing endoluminal catheter contamination and catheter-related sepsis arising at the hub. METHODS: Adult surgical and intensive care patients requiring a subclavian catheter for at least 1 week were randomly assigned to receive catheters with standard connectors (control group, n=73) or equipped with a new hub model (new hub group, n=78). Skin, catheter tip, and hub cultures were performed at the time the catheter was withdrawn because therapy was terminated or because of suspicion of sepsis, in which case peripheral blood cultures were taken. RESULTS: Of the 151 patients included, 15 (10%) developed catheter-related sepsis. Catheters were more often withdrawn because suspicion of infection in the control group (42 vs. 19%, p<0.005). Catheter sepsis rate was higher in the control group (16 vs. 4%, p<0.01) because of the low rate of catheter sepsis arising at the hub observed in the new hub group (1 vs. 11%, p<0.01). The prevalence of culture-positive catheter hubs without associated bacteremia (colonization) was higher in the control group (18 vs. 5%, P<0.03). CONCLUSIONS: A new catheter hub has proved to be useful in preventing endoluminal bacterial colonization and catheter-related sepsis in subclavian lines inserted for a mean of 2 weeks.