Preliminary results on a proposed histopathological assessment of predictive factors for basal cell carcinoma recurrence after primary free margin excision.

Background: Basal cell carcinoma (BCC) incidence is steadily increasing but therapeutic solutions remain limited and present a public health challenge. Aims: To identify predictive factors of BCC recurrence after primary free margin excision, with automated methods, by evaluating cell proliferation, the Hedgehog pathway activation and primary cilia. Materials and Methods: This case-control study included 32 patients (16 with recurrence occurring at least 6 months after complete resection, and 16 without recurrence) who underwent surgery for BCC. Formalin-fixed paraffin-embedded cutaneous resections were processed for immunohistochemistry or immunostaining with the following primary antibodies: mouse anti-MCM6, rabbit anti-ARL13B and rabbit anti-GLI1. Results: BCC recurrence after free margin excision was significantly linked to a higher proliferative index (p < 0.001) and a lower cilia count (p = 0.041) in the primary lesion. No significant differences were observed regarding cilia length (p = 0.39) or GLI1-positive nuclei. Discussion: The complex interplay between essential signaling pathways, cell proliferation and cilia requires further experimental investigations in the context of BCC recurrence. Conclusion: A higher proliferative index evaluated with MCM6 antibody could be a useful prognosis marker of BCC risk of recurrence. The lower cilia count in the primary lesion unveiled novel perspectives to understand BCC recurrence molecular mechanisms.

[Vulvar invasive squamous cell carcinoma and hidradenoma papilliferum. Case report]. / Carcinome épidermoïde et hidradénome papillifère vulvaire. A propos d'un cas.

We report a case of a vulvar invasive squamous cell carcinoma associated to a benign tumor with apocrine differenciation, the hidradenoma papilliferum, infiltrated by the carcinoma. Diagnosis was established by clinical and histopathological examination, first on biopsy and then on local vulvar excision. Classical association between hidradenoma papilliferum and Paget's disease is described, but to the best of our knowledge, there have been no previous reports of such case in published literature. A common physiopathological etiology cannot be completely excluded.

Usefulness of HPV testing in the follow-up of untreated cervical low grade lesions.

The aim of the present work was to evaluate the usefulness of high-risk human papillomavirus (HR-HPV) testing for the follow-up of women with untreated low grade cervical squamous cell lesions (LSIL). For that, 412 women with a cytological diagnosis of LSIL at entry were monitored by cytology, HR-HPV testing with the Hybrid Capture II assay (HC-II) and colposcopy. Our primary endpoint was clinical progression defined by the presence of a high grade cervical intraepithelial neoplasia (CIN2 and CIN3) at the biopsy. At baseline, histological control revealed 10 CIN2 and 11 CIN3 only in the cohort of women HR-HPV+. In the follow-up, 4 CIN2 and 8 CIN3 were detected, always in the women initially HR-HPV+. Thus, the recurrence of a HR-HPV+ infection clearly selects a population at high-risk for CIN2-3. The semi-quantitative appreciation of the viral load with HC-II could not be used as a good prognostic factor for the follow-up of women with LSIL. HR-HPV testing reduces the number of cytology and colposcopy examinations in the follow-up of women aged >35 years when HPV testing is initially negative. Thus HR-HPV testing should be reserved for the follow-up of this population of women initially HR-HPV+ and proposed 6 to 12 months after the cytological diagnosis of LSIL.

Comparison of liquid based cytology and histology for the evaluation of HER-2 status using immunostaining and CISH in breast carcinoma.

BACKGROUND: HER-2 amplification is an important prognostic biomarker and treatment determinant in breast carcinoma. AIMS: To correlate immunocytochemical (ICC) expression of HER-2 and gene amplification determined by chromogenic in situ hybridisation (CISH) using liquid based cytology (LBC) with immunohistochemistry (IHC) and CISH using histological samples of the same breast carcinomas. METHODS: Frozen sections and cytobrushings of 103 breast carcinomas were analysed. Four techniques were performed on each tumour: two on LBC samples (ICC, and CISH, both graded as positive, indeterminate, or negative) and two on histological samples (IHC and CISH). Two cell lines (MCF-7, negative; BT 474, positive) were used as controls for cytological analysis. A complementary fluorescence in situ hybridisation technique was carried out in histological samples with low amplification (4-10 dots/nucleus). RESULTS: Interobserver agreement for the four techniques calculated by the kappa coefficient indicated a substantial agreement. Nine cases failed in cytology because of poor cellularity. Among 94 cases, 19 were amplified; 73, 12, and 9 tumours were scored 0 or 1+, 2+, and 3+, respectively by IHC and 75, 13, and 6, respectively, by ICC. CISH found no amplification in 72 tumours. Correlations between the IHC and CISH results in the histological and cytological samples were always significant. CONCLUSIONS: Her-2 status could be determined in LBC samples and correlated well with reference histological methods using in situ hybridisation. ICC was less reliable because of the presence of the cytoplasmic membrane. However, these results should be confirmed by a large multicentre study.

[Gestational trophoblastic diseases: a retrospective study from 1997 to 2003]. / Maladies trophoblastiques gestationnelles: étude rétrospective de 1997 a 2003.

BACKGROUND: Trophoblastic diseases correspond to a very heterogeneous group of rare pathology in young women which fertility should be preserved. PATIENT AND METHODS: We conducted a retrospective study from 1997 to 2003, including all patients with molar pregnancy or trophoblatic tumor in our department of Obstetrics and Gynecology. RESULTS: Fifteen patients were identified with 9 molar pregnancies, 5 trophoblastic tumors and 1 placental tumor of implantation site. The outcome was favorable for 14, and one patient died from her metastatic disease. For 4 patients we asked our university colleague for the optimal approach. DISCUSSION: Management of molar pregnancies is well established. Persistent gestational disease is more rare and problematic with potential metastatic dissemination. Multidisciplinary care is often needed. CONCLUSION: Persistent gestational disease should be managed in a highly specialized centre, as developed in the Lyon University Hospital.

Quantitative computer image analysis of chondroitin sulfate A expression in placentas infected with Plasmodium falciparum.

Most pathological conditions resulting from infection with the human malaria parasite Plasmodium falciparum occur as a consequence of the sequestration by several adhesion molecules of parasite-infected red blood cells (IRBCs). Recent reports have provided evidence that placental vascular endothelial ligands for IRBCs were mostly restricted to chondroitin sulfate A (CSA). The expression of CSA in malaria-infected placentas was investigated in a prospective case-control study in a hypoendemic area (Dakar, Senegal). The tissue distribution of CSA was measured in the terminal villi by immunostaining combined with image processing in 20 infected and 20 noninfected frozen sections of placenta. The villous surface immunostained by anti-CSA antibody was higher in infected than in noninfected placentas (p<0.03), in placentas with active infection than in those with past chronic infection (p<0.05), and in infected placentas with positive imprints than in those with negative imprints (not significant; p=0.06). Labeling was found in the extracellular matrix and in endothelial and stromal cells of all the placentas. Syncytiotrophoblast immunostaining was detected in all placentas associated with active or active chronic infection (n=7) but in only 4/13 placentas with past chronic infection (p<0.01). The presence of P. falciparum in the imprint was significantly correlated with immunostaining of CSA in syncytiotrophoblasts (p=0.003). These results suggest that CSA can play an important role in the sequestration of P. falciparum in human placentas during the acute phase of infection.

Tumor collagenase stimulatory factor (TCSF) expression and localization in human lung and breast cancers.

Tumor cell-derived collagenase stimulatory factor (TCSF) stimulates in vitro the biosynthesis of various matrix metalloproteinases involved in tumor invasion, such as interstitial collagenase, gelatinase A, and stromelysin 1. The expression of TCSF mRNAs was studied in vivo, using in situ hybridization and Northern blotting analysis, in seven normal tissues and in 22 squamous cell carcinomas of the lung, and in seven benign proliferations and in 22 ductal carcinomas of the mammary gland. By in situ hybridization, TCSF mRNAs were detected in 40 of 44 carcinomas, in pre-invasive and invasive cancer cells of both lung and breast cancers. TCSF mRNAs and gelatinase A mRNAs were both visualized in the same areas in serial sections in breast cancers, and were expressed by different cells, tumor cells, and fibroblasts. The histological results were confirmed by Northern blot analysis, which showed a higher expression of TCSF mRNAs in cancers than in benign and normal tissues. These observations support the hypothesis that TCSF is an important factor in lung and breast tumor progression.

DNA image cytometry and Ag-NORs-staining application in biocompatibility studies on human osteoblast cells in vitro.

We report here the study of the biocompatibility of a bone graft material, the Pyrost, using a previously established in vitro model of human osteoblasts. The effect of this material on cell proliferation was evaluated by the MTS assay. Results indicated the absolute absence of cytotoxic or cytostatic effect of Pyrost on cultured osteoblasts. Viability rate was more than 90% in cells cultured with the material compared to the control. Morphological analysis, undertaken by scanning electron microscopy showed a good adhesion and a spreading of osteoblasts in contact with the material that was colonized by cultured cells. In the second part of this work, we have introduced two methods as complementary biocompatibility tests: DNA image cytometry and interphase Ag-NORs quantification. DNA content was measured in cells cultured with or without Pyrost for 3, 9, 15 and 30 days. The determination of DNA indicated that the majority of osteoblasts population was diploid without aneuploidy. The DNA index and cell distribution profile in DNA histograms were similar in all cell populations. The Ag-NORs amount was used as a parameter for cell kinetic evaluation. We have measured the Ag-NORs index like DNA quantification. The proliferation rate, evaluated by Ag-NORs counts in osteoblasts cultured with or without the material, was identical. However, a decrease in Ag-NORs index was observed from day 3 to day 15 of incubation. These results showed a satisfactory biocompatibility of the Pyrost in human osteoblasts culture. The material did not alter cell viability and had no inducing effect either on proliferation rate or on cell ploidy as demonstrated by DNA image cytometry and Ag-NORs proteins staining.

Human papillomaviruses and DNA ploidy in anal condylomata acuminata.

Previous studies have emphasized the usefulness of DNA ploidy measurement and Human Papillomavirus (HPV) detection as prognostic markers in low grade cervical lesions. We addressed the eventual relationship between HPV type, DNA profile, and p53 tumor suppressor protein expression in anal condylomata acuminata to eventually determine parameters which may be considered as predictive risk factors for the development of cancer. DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of 45 anal condylomata acuminata without atypia containing HPV detected by in situ hybridization and Polymerase Chain Reaction (PCR). p53 expression was detected by immunohistochemistry. DNA aneuploidy was found in 53.3% of these lesions, 48.9% containing non oncogenic HPV types 6 and/or 11 and 4.4% harbouring HPV types 11 and 18. The DNA diploid lesions were all associated with non oncogenic HPV types 6 and/or 11 and one case also contained HPV type 33. There was no significant correlation between the detection of DNA aneuploidy and the presence of immuno-detected p53. DNA aneuploidy was not related to the presence of oncogenic HPV in anal condylomata acuminata. The DNA aneuploid profile frequently observed, especially in lesions associated with non oncogenic HPV types, is not yet well explained and cannot be considered as a prognostic factor. In contrast, a more intensive clinical follow-up should be proposed in patients with oncogenic HPV associated to DNA aneuploidy.

Oncogenic human papillomaviruses and ploidy in cervical lesions.

AIM: To compare ploidy measurements obtained on tissue sections of selected low and high grade squamous intraepithelial lesions containing oncogenic HPV (types 16, 18 or 33) detected by in situ hybridisation (ISH) or PCR. METHODS: DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of eight lesions exhibiting atypical squamous cells or squamous atypia and 53 low and 63 high grade squamous intraepithelial lesions in which HPV had been detected by ISH or PCR. RESULTS: Aneuploidy was strongly associated with the presence of oncogenic HPV, being detected in 50% of lesions with squamous atypia and 75.5% of the low and 95.2% of the high grade squamous intraepithelial lesions. The multiploid profile was highly associated with high grade lesions and with the pattern of HPV DNA integration. CONCLUSIONS: The presence of aneuploidy is strongly suggestive of the presence of oncogenic HPV types. Combining the detection of HPV by ISH and PCR with DNA image cytometry may provide the pathologist and the physician with important prognostic information about low grade lesions, especially when these lesions have a multiploid DNA profile and contain oncogenic HPV.

Human papillomavirus detection by the hybrid capture II assay: a reliable test to select women with normal cervical smears at risk for developing cervical lesions.

The reliability of the Hybrid Capture II (HC-II; Digene, Silver Spring, MD, U.S.A.) assay was tested in detecting 18 human Papillomavirus (HPV) types for the screening of cervical lesions. Cytology, HPV testing, colposcopy, and biopsy were used to monitor 204 women with normal smears at the first entry. The median follow-up was 15 months (range, 4-27 months). The primary endpoint was clinical progression defined as the presence of a cervical intraepithelial lesion at the biopsy. In the patient population of 204 HPV-infected women, 81 (39.7%) had a persistent HPV infection at two or three examinations with a final histologic diagnosis of 14 high-grade and 13 low-grade squamous intraepithelial lesions (SIL) within 4 to 22 months. Women with regressive HPV infection did not develop any lesion during the same period. The evaluation of the viral load of high-risk HPV by the HC-II did not represent a sensitive approach to predict the persistence or the apparition of high-grade lesions. Thus, persistent high-risk HPV infection detected with HC-II represents a reliable tool to select populations at risk for the development of high-grade cervical lesions.

The effects of 5-fluorouracil on contractility and oxygen uptake of the isolated perfused rat heart.

Clinical cardiotoxicity related to 5-fluorouracil (5-FU) simulates either myocardial ischemia or left ventricular dysfunction. In order to characterize the changes occurring in the heart following 5-FU administration, the isovolumic perfused rat heart model according to Langendorff has been used. Particular emphasis was laid on contractility and oxygen uptake. Perfusion of isolated hearts with 1 mg/L 5-FU for 80 minutes failed to show any differences in contractility and oxygen consumption in comparison with the control group. On the contrary, 5-FU pretreatment of Wistar rats (50 mg/kg I.P. for 5 consecutive days) led to a decrease in inotropism without any change in maximum relaxation rate. The most significant finding was the consistent increase in oxygen consumption throughout the 80 minutes of perfusion (p less than 0.05) associated with a decrease in the fractional extraction of oxygen. Mean coronary flow was consistently increased in the 5-FU-pretreated group. Lactate release and CK Leakage did not differ in the two groups. In the 5-FU-pretreated group the ratio of oxygen consumption to rate-pressure product remained significantly elevated throughout 80 minutes of perfusion in comparison with the control group (p less than 0.05). Inappropriately high oxygen uptake could be a reflection of cellular metabolic disturbances responsible for post-ischemic dysfunction.

Efficiency of amifostine as a protection against doxorubicin toxicity in rats during a 12-day treatment.

BACKGROUND: Our purpose was to determine the effects of amifostine, a cytoprotective agent, on doxorubicin tolerance and cardiotoxicity in rats. MATERIALS AND METHODS: Male Wistar rats were treated every other day with an intraperitoneal injection of amifostine or saline 30 minutes before intraperitoneal injection of doxorubicin or saline. Weight change was recorded, and contractile function was evaluated after 11 injections by means of the isolated heart. RESULTS: Weight evolution and cardiac function were significantly improved by 7 and 20 mg/kg amifostine (p < 0.001) but not by 50 mg/kg. The final weight were: controls 349 +/- 16 g; doxorubicin alone 258 +/- 54 g; with amifostine: 7 mg/kg 314 + 28 g; 20 mg/kg 312 +/- 32 g; 50 mg/kg 250 +/- 34 g. Left ventricular developed pressure were: controls 137 +/- 15 mmHg; doxorubicin alone 119 +/- 20 mmHg; with amifostine: 7 mg/kg 140 +/- 20 mmHg; 20 mg/kg 137 +/- 25 mmHg; 50 mg/kg 124 +/- 20 mmHg. CONCLUSION: Seven and 20 mg/kg amifostine protected rats from the toxicity of doxorubicin at the cumulative dose of 18 mg/kg during a 12-day treatment, with regard to weight loss and heart contraction.

Influence of physicochemical reactions of bioactive glass on the behavior and activity of human osteoblasts in vitro.

Bioactive glasses are characterized by a bond to bone with a hydroxyl carbonate apatite layer. They enhance bone tissue formation and for this purpose are used in orthopedic surgery and in dental implantology. In the current work, we studied the biological response of human osteoblasts with a bioactive glass. This bioactive glass is based on 50% Si0(2), 20% Na(2)O, 16% CaO, 6% P(2)O(5), 5% K(2)0, 2% Al(2)O(3) and 1% MgO and designated A9. Cracks and irregularities were observed on the material surface when it was immersed in the culture medium. In addition, energy dispersive X-ray analyses highlighted a selective release of the elements at the surface of the bioactive glass, such as Na(+) and K(+) ions, released from the first day, contrary to the Si, Al, Ca, P, and Mg elements, which were released more slowly. Cell proliferation kinetics, total protein synthesis, and DNA content of the osteoblasts in contact with bioactive glass were similar to control cells. The morphological studies by light and scanning electron microscopy revealed an increasing cellular density in culture with bioactive glass without contact inhibition. The immunohistochemical studies highlighted the expression of types I, III, and V collagens by osteoblasts cultured in the presence of bioactive glass. The pH measurement of the culture medium in the presence of bioactive glass demonstrated a slight alkalinization. We thus conclude that human osteoblasts preserve their properties in the presence of bioactive glass (A9).

Determination of reliable histological features associated with early triploidy using DNA image cytometry.

In order to determine reliable histological features characterizing triploidy, the following features were examined and graded by three pathologists on 46 early abortion specimens: hydropic swelling of the villi, cisterns, villous scalloping, trophoblastic hyperplasia with syncytial vacuolization, single cytotrophoblastic cells in villous stroma, trophoblastic inclusions, microcalcifications and fibrosis. At the same time, the DNA content of the 46 specimens was quantified cytophotometrically using the CAS 200 image analyzer, in order to confirm or not the diagnosis of triploidy. Triploidy was confirmed in 45.7%, and in the triploid group, the grading of three features differed significantly from the non triploid group i.e. cisterns (p < 0.001), trophoblastic hyperplasia (p < 0.05) and trophoblastic inclusions (p < 0.05). Using these three features we were able to give a triploid score per slide which represents the sum of the grades of each of these three features. The minimum score obtained was 0, the maximum was 6. The average of these scores in the triploid group was 3.095, and 1.45 in the non triploid group (p < 0.001). This scoring method on the three significant features (cisterns, trophoblastic hyperplasia and trophoblastic inclusions) appears to be useful to classify an abortion specimen as triploid or not, and to select the specimens for which a DNA quantification may be necessary.

Discrepancies of DNA content of various solid tumours before and after culture measured by image analysis. Comparison of cytogenetical data.

Parallel cytophotometric ploidy studies and cytogenetic analysis were performed on 15 various human solid tumours. The quantification of DNA by image analysis was carried out on cytological imprints of fresh tumours and on smears obtained after cell culture. The results obtained by both sets of calculations were compared with each other and with the cytogenetic results. 6 cases (40%) showed concordance between the 3 techniques. One case was aneuploid for both DNA image analysis measurements but the cytogenetic data showed only a diploid stem line. In 3 cases out of 15 (20%), smears DNA analysis and cytogenetic results were concordant: in 2 tumours, the culture step failed to preserve aneuploid stem lines that were present in the imprint analysis. In the third one, a minority tetraploid peak observed after culture was absent on the imprint slide. Concordance between imprints and cytogenetic data and discordance with smears' analysis was observed in 3 cases (20%). These 3 cases were diploid or near diploid but the DNA analysis on the smears after culture showed an aneuploid stem line in each case. The last 2 cases showed a total disagreement between the 3 techniques. By measuring the DNA content with an image analyser, the observer can ensure that only tumoral cells are taken into account. The present study revealed that cytogenetic data represent only about 60% of the population that is effectively present in the culture dish and that the cultured population represents only 47% of the population present on the fresh tumour imprint.

Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry.

There are two ways of measuring the cell proliferation. The first one consists of quantifying the number of cycling cells with the help of antibodies directed against cells either in G1, S, G2 or M phase. The second way is to assess the cell cycle duration by the quantification of AgNOR proteins. Measuring both the features on the same slide represents an attractive way to tackle the proliferating activity of a cell culture or a tumor. Here, we propose a MIB-1 and AgNOR double staining method especially adapted to image cytometry measurement using MIB-1 antibody coupled to FITC in order to avoid the thresholding problems encountered with such a multilabeling technique. We have applied this new method on a series of 39 breast cancer cases, with at least 4 years follow-up, in order to determine the prognosis significance of this measurement. MIB-1 alone is not linked to prognosis, while the global mean AgNOR area is significantly linked to prognosis in terms of development of visceral metastasis or death. However, the global mean AgNOR area is insufficient to determine the time limit of appearance of metastasis or relapse. Our results clearly demonstrate that a high mean AgNOR area within a cell population having a high MIB-1 index can discern tumors with a high metastatic potential. By multiplying AgNOR area by the percentage of MIB-1 positive cells we calculate the proliferative activity, P, which brings very important information concerning the time limit of relapse.

Comparative study of isoproterenol and 4-deoxypyridoxine effects on the isovolumic rat heart model.

Isovolumic rat hearts were perfused for 20 min (T20) or 40 min (T40) with 10(-6) M isoproterenol (ISO), 10(-5) M 4-Deoxypiridoxine (DOP) or with both drugs (ISO-DOP). ISO increased developed pressure 51% and 31%, coronary flow (C.F.) 37% and 52%, heart rate 50% and 60%, lactate production 160% and 76%, and CK activity in the effluent perfusate 182% and 173% at T20 and T40 respectively, whereas glycogen stores decreased by 47% and 77%. DOP needed at least 40 min to increase heart rate 32%, C.F. 40% and to decrease glycogen reserves by 33% and pyridoxal-5-phosphate 13%. In the ISO-DOP group, the ISO inotropic effect was lowered by 23% (T20) and 25% (T40) and DOP reduced ISO induced glycogenolysis.

Effects of 4-deoxypyridoxine on the isovolumic perfused rat heart: influence of oxygenation and glucose availability.

The study was performed with isolated perfused isovolumic rat hearts. After a 40 min stabilization period, the effect of 4-deoxypyridoxine (DOP) 10(-5) M was studied with 3 Glc concentrations: 0, 3.3 and 11 mM. DOP was perfused during a 20 min normoxic or anoxic period followed by 40 min of normal perfusion. During normoxia with 11 mM Glc, DOP decreased the contracture observed in the control group. With 0 mM Glc, DOP improved developed pressure and dP/dt+ without a decrease in glycogen stores. During anoxia followed by reoxygenation, a partial protection towards CK release was observed with DOP (3.3 mM Glc). Intracellular PLP levels were higher in the 11 mM Glc group than in the other groups with and without DOP, and DOP with 11 mM Glc increased PLP levels (DOP N2 vs. DOP O2). Glycogen stores increased with 11 mM Glc without DOP (O2 vs. N2), whereas they decreased with DOP without Glc (DOP N2 vs. N2). DOP could improve the yield in glycogenolysis in normoxia and might activate mitochondrial anaerobic metabolism during anoxia.

[Survival and infectious processes in pacients with AIDS: analysis based on initial serum vitamin A levels]. / Sobrevida e processos infecciosos em pacientes com AIDS: análise de acordo com os níveis séricos iniciais de vitamina A.

Patients with Aids (n = 39) were followed up for a maximum period of 36 weeks, after which the types and topographies of infectious complications presented and patient survival were analyzed and correlated with the vitamin A levels presented by the patients at the beginning of clinical follow-up. Twenty-one (53,8%) patients presented serum retinol levels below 1.6 micromol/L, 12 (57%) of whom had values lower than 1.05 micromol/L. There was no correlation between low serum vitamin A levels and the types or topographies of the infectious complications that occurred during the follow-up period. Although mean survival at the end of the 36 months follow-up period was similar for the two groups, patients with retinol deficiency presented a lower probability of survival during the first 24 months of follow-up compared to patients without hypovitaminosis A (8.44 x 1.42 months; p = 0.003).