Role of nicotinic acetylcholine receptors in cell proliferation and tumour invasion in broncho-pulmonary carcinomas.

OBJECTIVES: Nicotine and its associated nicotinic acetylcholine receptors (nAChRs) are believed to be involved in the progression of lung carcinomas. This study aimed at examining the localization of nAChRs in human lung tumours and, by using primary cultures of tumour cells derived from these tumours, determining the nAChR roles in cell proliferation and tumour invasion. MATERIALS AND METHODS: Immunohistochemistry was used to assess nAChR expression in non-small cell lung carcinomas (NSCLC). Primary cultures of tumour cells were established from NSCLC tissue samples and the effects of nicotine and nAChR antagonists on cell proliferation and invasion were assessed. RESULTS: α5, α7, ß2 and ß4 nAChR subunits were expressed in all adenocarcinomas (AC) and squamous cell carcinomas (SCC) tissue samples. In AC, all subunits were identified in glandular structures. In SCC, α5, ß2 and ß4 subunits were essentially identified in tumour cells at invasive fronts, whereas α7 subunit was mainly present in the most differentiated tumour cells and less frequently at invasive fronts. In AC and SCC, there was an inverse distribution of cell proliferation marker Ki-67 and α7 nAChR. Both α7 nAChR and heteromeric nAChRs positively regulated in vitro tumour invasion in NSCLC. Heteromeric nAChRs had a limited activity in regulating tumour cell proliferation in vitro. In contrast, α7 nAChR was a repressor of proliferation in tumour cells isolated from well differentiated NSCLC but mediated the pro-proliferative activity of nicotine in cells isolated from poorly differentiated NSCLC. CONCLUSION: α7 nAChR and heteromeric α5*ß2*ß4* nAChRs play a role in ex vivo tumour progression by stimulating invasion and, depending on the differentiation status of the tumour, by regulating proliferation. Our results suggest that the use of α7 nAChR antagonists to prevent lung cancer progression should be restricted to poorly differentiated tumours.

The elastin peptide (VGVAPG)3 induces the 3D reorganisation of PML-NBs and SC35 speckles architecture, and accelerates proliferation of fibroblasts and melanoma cells.

During melanoma tumour growth, cancerous cells are exposed to the immediate surrounding the micro- and macro environment, which is largely modified through the degradation of the extracellular matrix by fibroblast-derived metalloproteinases. Among the degradation products, (VGVAPG)3, an elastin peptide is known to stimulate the proliferation of both fibroblasts and cancerous cells by binding to the elastin-binding receptor and activating the MEK/ERK signal transduction pathway. As this process strongly modifies mRNA synthesis, we investigated its effect on the relative three-dimensional organisation of the major partners of the mRNA splicing machinery: promyelocytic nuclear bodies (PML-NBs ) and splicing component 35 speckles (SC35) of normal fibroblasts and melanoma SK-MEL-28 cells. SC35 and PML-NBs proteins were immunolabeled and imaged by confocal microscopy within these cells cultured with (VGVAPG)3. Three-dimensional reconstruction was performed to elucidate the organisation of PML-NBs and SC35 speckles and their spatial relationship. In G0 cells, SC35 speckles were sequestered in PML-NBs. Shortly after (VGVAPG)3 stimulation, the three-dimensional organisation of PML-NBs and SC35 speckles changed markedly. In particular, SC35 speckles gradually enlarged and adopted a heterogeneous organisation, intermingled with PML-NBs. Conversely, inhibition of the elastin-binding protein or MEK/ERK pathway induced a remarkable early sequestration of condensed SC35 speckles in PML-NBs, the hallmark of splicing inhibition. The 3D architecture of speckles/PML-NBs highlights the modulation in their spatial relationship, the multiple roles of PML-NBs in activation, inhibition and sequestration, and provides the first demonstration of the dependence of PML-NBs and SC35 speckles on the elastin peptide for these functions.

Endothelin-1: a predictor of extracapsular extension in clinically localized prostate cancer?

OBJECTIVE: To assess the value of endothelin-1 (ET-1) expression in predicting extracapsular extension (ECE) in clinically localized prostate cancer (PCa). PATIENTS AND METHODS: ET-1 expression was determined by immunohistochemistry on archival needle biopsies (NBs) from 94 patients (49 pT2 and 45 pT3a) who underwent radical prostatectomy (RP) for clinical T1-T2 PCa. Each sample was analysed independently by two pathologists blinded to the clinical data. RESULTS: In univariate analysis, high ET-1 expression in NBs, pre-operative prostate-specific antigen (PSA) level >10 ng/ml, percentage of positive biopsy cores and NB Gleason score ≥7 were significantly associated with ECE as determined on subsequent RP. No significant association was found between clinical stage and ECE. In multivariate analysis, there was a significant association with high ET-1 expression in NBs (p = 0.006), pre-operative PSA level >10 ng/ml (p = 0.049), and NB Gleason score ≥7 (p = 0.002). These three pre-operative factors combined provided the best model for predicting ECE with 93.3% sensitivity, 49% specificity, 62.5% positive predictive value, 88.9% negative predictive value. The combination yielded a higher concordance index (0.760 vs 0.720) and offered a higher log partial likelihood than the same model without ET1 (112.8 vs 105.7, p = 0.01). CONCLUSIONS: ET-1 expression was strongly associated with ECE and, when combined with pre-operative PSA level and Gleason score, improved the predictive accuracy of pre-operative NBs. Its assessment in patients with localized PCa might be useful when making treatment decisions. Further studies with standardisation of immunohistochemical staining and multi-institutional validation are now needed to establish the appropriate use of ET-1 staining in PCa staging and to evaluate inter-observer reproducibility.

Contribution of DNA ploidy image cytometry to the management of ASC cervical lesions.

BACKGROUND: The Bethesda system classifies smears that suggest an underlying cervical intraepithelial neoplasia (CIN) as ASC (atypical squamous cell) smears. ASC smears are subdivided into ASCUS (of undetermined significance) and ASCH (cannot exclude a high-grade lesion). Today the management of ASCUS is a triage with HR-HPV testing and colposcopy is recommended for ASCH. The aim was to conduct a study on ASC smears to determine DNA ploidy measurement for the detection of CIN2+. METHODS: The link between a suspect DNA ploidy assessed by image cytometry and/or a positive HR-HPV testing was analyzed on 69 ASCUS and 82 ASCH smears, and the presence of CIN2+ within 12 months after ASC diagnosis. The ploidy was suspect in case of aneuploidy, multiploidy, or in the presence of cells with a DNA content >5c or >9c. RESULTS: Every woman who had a CIN2+ had a suspect DNA profile in the ASCUS smears and every woman except 1 was HR-HPV-positive. The link between a positive HR-HPV test or a suspect DNA profile or both and a CIN2+ was high (P = .019, .023, and .008, respectively). The presence of >9c cells was particularly linked to CIN2+ (P = .0031). In all, 90.9% and 87.9% of the ASCH smears with CIN2+ were, respectively, HR-HPV positive or had a suspect ploidy (P = .0000 and P = .0043), and the presence of >9c cells was also linked to CIN2+ (P = .003). CONCLUSIONS: HR-HPV testing and determination of the ploidy profile with special attention to 9c-exceeding cells could be accurate for a better management of ASC smears.

Aneuploidy, but not Ki-67 or EGFR expression, is associated with recurrences in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC), the most common skin cancer, has an overall excellent prognosis, but recurrences are frequent. The value of classical clinical and histological prognostic factors to predict recurrences remain limited. METHODS: In order to evaluate the prognostic value of Epidermal Growth Factor Receptor (EGFR) expression, Ki-67 antigen expression and DNA ploidy, we compared primary tumors in 20 patients who had subsequent local recurrences and 20 matched controls without recurrences. DNA ploidy was determined by image cytometry, and EGFR and Ki-67 expression were studied by immunohistochemistry. RESULTS: Statistical analysis of the intensity and the percentage of EGFR expression and Ki-67 antigen expression did not show any significant difference between the two groups. In contrast, we found that 78% of primary BCC in patients who experienced recurrences vs. 32% in the control group were aneuploid (p = 0.005). CONCLUSIONS: Aneuploidy is a risk factor for recurrences. This factor should be useful in clinical practice and require evaluation in further studies.

Prognostic value of a proliferation index including MIB1 and argyrophilic nucleolar organizer regions proteins in node-negative breast cancer.

OBJECTIVE: This study was designed to evaluate a cell proliferation marker, including the percentage of cycling cells (MIB1), and the duration of the cell cycle (assessed by argyrophilic nucleolar organizer regions proteins [AgNORs] measurement). STUDY DESIGN: We included 90 patients with invasive node-negative breast cancer. None received chemotherapy. With the help of a double-staining technique, a proliferation index (PI) was determined by multiplying the percentage of MIB1-positive cells by the mean area of the AgNORs present in those MIB1-positive cells. PI was evaluated for its impact on overall survival (OS) and disease-free survival (DFS). RESULTS: We demonstrated that PI was correlated to OS. For DFS, it conserved its high prognostic value only in univariate analysis. The global amount of AgNORs was more discriminative for DFS. CONCLUSION: PI and AgNOR quantification supplied additional prognosis information in node-negative patients, and we propose to integrate them in further studies.

Fluorescence in situ hybridization and immunohistochemical analysis of p53 expression in endometrial cancer: prognostic value and relation to ploidy.

BACKGROUND: Endometrial carcinoma is the most common gynecological malignancy. Several molecular biological characteristics have been studied for their potential value in patient management. OBJECTIVES: Our objectives were to compare p53 immunohistochemical expression with P53 gene status determined by fluorescence in situ hybridization (FISH) and to compare these characteristics with ploidy and with classical clinical and histological prognostic factors. MATERIALS AND METHODS: We reviewed stored specimens from 43 patients with endometrial cancer diagnosed in 1999-2004. P53 FISH and immunohistochemistry were performed, together with imaging cytometry to calculate DNA ploidy. RESULTS: Thirteen of the 43 endometrial carcinomas (30.2%) showed P53 loss of heterozygosity (LOH). P53 LOH correlated with the histological type (P = .03) and the histological grade (P = .004). Quantitative immunohistochemical expression of p53 protein correlated with the histological type (P = .0001). With a cutoff of 10% of p53-positive cells, p53 overexpression correlated with the histological type (P = .003) and grade (P = .0008). No relation was found between P53 LOH or immunohistochemical expression and the disease stage, the depth of myometrial invasion, lymph node status, lymphovascular space involvement, recurrence, or death from cancer. Nondiploid carcinomas showed deeper myometrial invasion than diploid carcinomas (P = .01). No relation was observed between ploidy and qualitative or semiquantitative p53 expression or P53 LOH. CONCLUSION: In endometrial cancer, FISH analysis of P53 status adds no significant prognostic information compared with immunohistochemical p53 analysis.

[Cell ploidy: predictive factor of locally advanced prostate cancer]. / La ploïdie cellulaire: facteur prédictif de cancer de prostate localement avancé.

OBJECTIVE: This study was designed to demonstrate that the study of cell ploidy on biopsies of clinically localized prostate cancers can contribute to the diagnosis of a tumour extending beyond the prostatic capsule and can complete imaging for local staging. METHODS: Analysis of the histological results of 140 patients operated for clinically localized prostate cancer distinguished two groups of patients in whom the initial tumour was Gleason score 6 or 7. The first group was composed of 33 patients whose tumour was classified as pT3 and the second group was composed of 24 patients whose tumour was classified as pT2. The cell ploidy study was performed on biopsies and operative specimens in the two groups. RESULTS: In the pT3N0M0 group, 72% of tumours presented an aneuploid contingent versus 16% of tumours of the pT2N0M0 group. A strong correlation was demonstrated between cell ploidy and tumour stage (p = 0.0002) and a highly significant correlation was observed between tumour stage and the presence of a tumour contingent with ploidy greater than 5C (p = 0.0009). CONCLUSION: The presence of an aneuploid contingent on biopsies of clinically localized prostate cancer significantly increases the risk of a more advanced tumour. This technique could therefore constitute a simple complementary tool in the staging of prostate cancer in combination with transrectal MRI, but this needs to be confirmed by other studies.

P16 is overexpressed in cutaneous carcinomas located on sun-exposed areas.

BACKGROUND: Recently, an increased expression of P16, a cell cycle regulatory tumor suppressor protein, has been demonstrated in cervical squamous neoplasms as a marker of malignancy. In contrast, studies performed in skin carcinomas led to contradictory results. OBJECTIVES: Our first aim was to evaluate P16 expression in different types of non-melanoma skin cancers compared with normal skin and benign tumors. The second aim was to evaluate the relationship between P16 expression and the location of skin tumors (i.e. exposed versus non exposed sites). Finally, we also studied Ki67 expression in skin carcinomas and control biopsies. METHODS: Skin biopsy specimens with typical histologic features of squamous cell carcinoma (SCC; n = 30), Bowen's disease (BD; n = 17), basal cell carcinoma (BCC; n = 10), seborrheic keratosis (SK; n = 10) and normal human skin (NHS; n = 9) were obtained from 76 patients seen at our institution between 2001 and 2003. In all cases, P16 and Ki67 expression were evaluated by immunohistochemistry and image analysis. RESULTS: P16 overexpression was observed in 58% of cutaneous carcinomas (SCC: 60%; BD: 58%; BCC: 50%) versus 0% of SK or NHS (0%) (p = 0.006). Ki67 expression in over 5% of tumour cells was observed in 69% of cutaneous carcinomas (SCC: 54%; BD: 76%; BCC: 80%) versus 16% in the group including SK (30%) and NHS (0%) (p = 0.04). Overexpression of P16 was associated with a high rate of Ki67 positive tumour cells in 23/57 malignant skin tumors (40%). Both P16 was associated and Ki-67 were negative in 7/57 cases (12%). Sixty-eight percent of tumors located on sun-exposed areas versus 23% of those located on non sun-exposed areas overexpressed P16 (p = 0.02). CONCLUSION: Our study demonstrated that the expression of P16 and Ki67 is associated with skin carcinomas. No difference was observed according to histological types of carcinomas, suggesting that P16 and Ki67 expression did not correlate with the degree of proliferation and malignancy. Within cutaneous carcinoma specimens, P16 overexpression was significantly associated with the location on sun-exposed areas, suggesting a possible induction of P16 overexpression by UV radiation.

Cell cycle and/or proliferation markers: what is the best method to discriminate cervical high-grade lesions?

The aim of this study on a series of biopsies diagnosed as normal, metaplastic, low-grade squamous intraepithelial lesions (LSILs), and high-grade squamous intraepithelial lesions (HSILs) was dual: to determine the chronology of cell cycle and proliferation abnormalities after human papillomavirus infection during the development of squamous intraepithelial lesions and to determine the best diagnostic indicator(s) linked to the appearance of an HSIL. Ninety-nine cervical biopsies, 18 normal, 9 with metaplastic changes, 29 LSIL, and 43 HSIL (23 cervical intraepithelial neoplasia 2 and 20 cervical intraepithelial neoplasia 3), were analyzed by image cytometry for DNA ploidy and p16INK4A determination, AgNOR counting, MIB-1, and ICBP90 immunostaining quantification. The human papillomavirus status had been previously determined on corresponding cytological smears with the Hybrid Capture II test. Suspect DNA profile and p16INK4A staining were the first significant events that preceded the increase of cell proliferation. Indeed, these markers were the best tests for the detection of a lesion, whatever its grade (positive predictive values of 90% and 100%, respectively). The presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium was a very accurate feature to select HSIL (sensitivity, 100% for MIB-1) but with a low specificity. The sensitivity of a suspect DNA profile associated with a positive MIB-1 or ICPB90 immunostaining for the detection of an HSIL was, respectively, 92.8% and 92.7%; their specificities were 54.2% and 44%; their positive predictive values were 78% and 73%; their negative predictive values were 81.2% and 78.6%; and the global values were 78.8% and 74.3%. Thus, the most accurate test to distinguish an LSIL from an HSIL was the association of a suspect DNA profile and the presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium.

Sirolimus-induced thrombotic microangiopathy is associated with decreased expression of vascular endothelial growth factor in kidneys.

The aim of this study was to examine the clinical characteristics, the histological features and the renal expression of vascular endothelial growth factor (VEGF) of five patients with sirolimus-associated thrombotic microangiopathy (TMA). Sirolimus-induced TMA occurs preferentially in kidneys with concomitant endothelial injury: it was observed in three patients with acute cellular rejection on calcineurin inhibitor-free regimen, in one patient with chronic graft rejection on a calcineurin inhibitor-free protocol and in one patient with chronic calcineurin inhibitor nephrotoxicity. We found that renal VEGF expression during sirolimus-induced TMA was significantly lower than VEGF expression in normal transplanted kidneys (p < 0.01). Decreased expression of VEGF seems to be a consequence of sirolimus treatment since (i) analysis of two biopsies performed after the switch of sirolimus to calcineurin inhibitor showed reappearance of VEGF expression, (ii) no decreased expression of VEGF was found in five kidneys with classical TMA and, (iii) an increased expression of VEGF was observed in seven kidneys with acute cellular rejection on a sirolimus-free immunosuppressive regimen (p < 0.01). The potential role of sirolimus-induced downregulation of VEGF as a predisposing factor to the development of TMA is discussed.

Implication of tumstatin in tumor progression of human bronchopulmonary carcinomas.

The NC1 domain of alpha3 chain of type IV collagen, namely tumstatin, has been shown to display specific anti-angiogenic properties by inhibiting endothelial cells' proliferation and inducing their apoptosis via an interaction with alphavbeta3 integrin. Until now, the tumstatin anti-angiogenic effect has only been shown by in vitro studies or mouse xenograft experiments. In the present study, we examined the expression of tumstatin in relationship with tumor vascularization in 34 bronchopulmonary human carcinomas. We observed a clear association between tumstatin expression and tumor vascularization. Indeed, a strong expression of tumstatin in the tumor environment correlated with a mildly developed vascular network. In contrast, tumstatin was absent or poorly detected in highly vascularized tumors. Moreover, alphavbeta3 integrin and tumstatin colocalized in capillary endothelial cells, suggesting a potential interaction between these 2 molecules. Thus, our results plead in favor of an in vivo anti-angiogenic effect of tumstatin. This factor, largely expressed in well-differentiated lung carcinomas, could indeed reduce tumor vascularization and thereby limit tumor progression.

Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

Recurrent human papillomavirus infection detected with the hybrid capture II assay selects women with normal cervical smears at risk for developing high grade cervical lesions: a longitudinal study of 3,091 women.

To test the reliability of the Hybrid Capture II (HC-II) assay detecting 13 high-risk human papillomavirus (HR-HPV) types for the screening of cervical lesions, we monitored by cytology, HR-HPV testing, colposcopy and biopsy, 3,091 women with normal smears at the first entry. Our primary endpoint was clinical progression defined as the presence of a high-grade lesion (HGSIL) at the biopsy. In our population of 659 HR-HPV-infected women, 241 (36.6%) had a positive HR-HPV test at 2 to 4 examinations with a final histological diagnosis of HGSIL in 51 cases (21.2%) within 4 to 36 months, while women with regressive HPV infection did not develop any lesion during the same period. In the cohort of 2,432 women testing negative for HR-HPV infection, only 2 women (0.08%) developed a HGSIL. Both were HR-HPV positive 18 and 24 months after the first entry, at the time of diagnosis of disease. The RR of incident HGSIL when a HR-HPV was detected at enrollment in women with normal smears was 96.7 (95% CI, 95.8-97.7). The RR increased to 237.3 (95% CI, 222.8-251.8) when the HR-HPV test remained positive at 2 controls, and to 314.3 (95% CI, 260.7-367.9) when the HR-HPV test was positive at 3 controls. The evaluation of the viral load of HR-HPV by the HC-II did not represent a sensitive approach to predict the recurrence of HR-HPV infection and/or the apparition of HGSIL. Nevertheless, a recurrent HR-HPV infection detected with HC-II represents a reliable tool to select populations at risk for the development of HGSIL.

Usefulness of DNA ploidy measurement on liquid-based smears showing conflicting results between cytology and high-risk human papillomavirus typing.

To improve the positive predictive value (PPV) for high-risk human papillomavirus (HR-HPV) in primary screening, DNA ploidy was measured on the same liquid-based sample by image cytometry in 984 cases showing discrepancies between cytology and HR-HPV testing. Of the conflicting results, 14.5% corresponded to a cytologic lesion (from atypical squamous cells of undetermined significance to high-grade squamous intraepithelial lesion [HSIL]) without HPV detected, and 85.5% of smears were within normal limits but revealed an HR-HPV infection. A suspect DNA profile was associated significantly with a lesion. In 497 patients who underwent repeated HPV testing, a normal DNA profile at the first smear predicted the clearance of HPV infection (sensitivity, 81.5%; specificity, 45.4%; PPV, 69%; negative predictive value, 62.4%). In persistent HR-HPV infection, a suspect DNA profile at the first smear increased the PPVfrom 10.8% to 22.7% for the detection of a histologically proven HSIL with a sensitivity of 95.2%. DNA ploidy can be used to select smears with high risk of HSIL, especially in cases of persistent HR-HPV infection.