05.11.2015

Ki 67 cancer test vinegar

Human Papilloma Virus (HPV) infection is thought to play a role in laryngeal carcinogenesis; particularly, p16 expression has been reported as a surrogate marker for high-risk HPV (HR-HPV) infection, associated with a subgroup of laryngeal cancers showing favourable prognosis. Among head and neck Squamous cell carcinomas (HNSCCs), the larynx is one of the sites most involved, being traditionally associated with tobacco and alcohol use, especially when in combination [1].
With the present study, using Immunohistochemistry assay in a series of 73 laryngeal biopsies with and without dysplasia, we aimed to identify the staining pattern of p16INK4a using a more objective immunohistochemical method for assessing HR-HPV. Seventy-three T1 N0 or carcinoma in situ (CIS) laryngeal biopsies were selected from the archives of the Fatebenefratelli Hospital from May 1992 through September 2004. Five, 10-Aµm sections of formalin-fixed, paraffina€“embedded tissue from the same sixty-eight specimen blocks were transferred to eppendorf vials.
A cross-tabulation analysis of p16INK4a immunohistochemical distribution with clinical and Histopathological findings was performed by the Chi-square test for trend or Fishera€™s exact test. HPV genotyping detection was conducted on all 73 cases (Table 3): 18 samples (25%) were classified as HPV+ and 55 samples (75%) were classified as HPV-. Table 2: Correlation between p16INK4 (dual-staining) immunohistochemical distribution and clinical data. Table 3: Correlation between HPV-DNA (INNO LiPA) and p16INK4 (dual-staining) immunohistochemical status. P16 expression was therefore considered as an indicator of HR-HPV presence and correlated to clinic pathological data.
There was no significant correlation regarding tobacco consumption, as p16 expression was found in 29% and 24% samples from non smoking patients and former (defined as having quit smoking 5 years prior to diagnosis of laryngeal cancer) and current smokers, respectively.
Figure 1: Detection of p16 expression (brown cytoplasmic staining) and the additional staining for the Ki-67 proliferation marker (nuclear red staining) within the same cell was regarded as a positive test result (immunoperoxidase, x400, original magnification).
The clinical significance of tumor HPV status in HNSCC is now well established; these tumors have been defined by HPV detection and belong to a subgroup which responds far better to radiotherapy than the smoking and alcohol related HNSCC [5]. The prognostic impact of p16 in laryngeal carcinoma was evaluated to consider whether such a biological marker can be used as a potential diagnostic tool. Le QT, Giaccia AJ (2003) Therapeutic exploitation of the physiological and molecular genetic alterations in head and neck cancer. Gillison ML, Lowy DR (2004) A causal role for human papillomavirus in head and neck cancer.
Agudelo D, Quer M, Leon X, Diez S, Burgues J (1997) Laryngeal carcinoma in patients without a history of tobacco and alcohol use. Koscielny S, Dahse R, Ernst G, von Eggeling F (2007) The prognostic relevance of p16 Inactivation in head and neck cancer. Vinokurova S, Wentzensen N, von Knebel Doeberitz M (2005) Analysis of p16INK4a and integrated HPV genomes as progression markers. Yuen PW, Man M, Lam KY, Kwong YL (2002) Clinicopathological significance of p16 gene expression in the surgical treatment of head and neck squamous cell carcinomas.
Bradley KT, Budnick SD, Logani S (2006) Immunohistochemical detection of p16INK4a in dysplastic lesions of the oral cavity. Gologan O, Barnes EL, Hunt JL (2005) Potential diagnostic use of p16INK4A, a new marker that correlates with dysplasia in oral squamoproliferative lesions.
Samarawardana P, Singh M, Shroyer KR (2011) Dual stain immunohistochemical localization of p16INK4 and Ki-67: a synergistic approach to identify clinically significant cervical mucosal lesions. Shanmugaratnam K, Sobin LH (1991) Histological typing of tumors of the upper respiratory tract and ear (2nd edn), Springer-Verlag, Newyork, USA. Aberrant activation of oncogenes or loss of tumour suppressor genes opposes malignant transformation by triggering a stable arrest in cell growth, which is termed cellular senescence1, 2, 3.
To evaluate HPV status, different detection molecular tests have been used and a large variation of HPV prevalence has been reported. As squamous carcinoma develops throughout a step progression in various stages of dysplasia, many molecular changes have been described [2]. Such a biological marker for laryngeal carcinoma and its prognostic role were investigated whether they can be used as potential prognostic biomarkers in routine clinical practice.
The analysis of time to recurrence rates for p16INK4a positivity was calculated using the Kaplan-Meier method and compared using the log-rank test. Sixteen out of 18 HPV+ samples were associated to p16 expression and the genotyping assay demonstrated the presence of HR-HPV: 8 cases were typed as HPV-16 (50%), 5 were typed as HPV-18 (31%), 2 were typed as HPV 31 (13%) and 1 was typed as HPV 52 (6%).
However, in this analysis the overa€“expression of p16 in 29% samples from non smokers, suggested the evidence of a causal role for HPV in carcinogenesis in patients without a history of tobacco use. Log-rank (Mantel-Cox) curve showed that p16 negative expression was found to have a significant effect on recurrence whereas a positive effect on outcome was evidenced in p16 positive patients. This morphology independent biomarker approach, which recently has been assessed [14-15] allowed us to found a 25% of p16 positivity in a retrospective study of T1 N0 or CIS laryngeal biopsies. We postulate that p16 positivity might be regarded as a protective factor: log-rank (Mantel-Cox) curve showed that freedom from recurrence was worse among p16-negative cases and in multivariate Cox proportional hazards analysis for the time-to-recurrence evidenced such an association.
Prior studies identified ?TopBP1 as a key mediator for the oncogenic gain-of-function activities of mutant ?p53 (mut?p53) in cancer. The normalized activities of ?E2F1 were shown as fold induction relative to that of the empty vector control.


This process is finely tuned by both cell-autonomous and non-cell-autonomous mechanisms that regulate the entry of tumour cells to senescence4, 5, 6.
3: Gr-1+ myeloid cells oppose senescence in both Pten-loss-induced cellular senescence and oncogene-induced senescence. Briefly, LNCaP prostate cancer cells were cultured in the absence or presence of docetaxel, with or without human recombinant IL-1RA. 7: Efficiency of magnetic-activated cell sorting (MACS) purification and bone marrow transplantation.
The implication of Human Papilloma Virus (HPV) in HNSCC development [3,4], particularly, the infection with high-risk HPV types (HR-HPV) has also been etiologically linked with a subset of laryngeal carcinoma [5] thus suggesting a different carcinogenetic pathway. Mean age of the patients at diagnosis was 62 years (range, 42 to 77 years); sex, smoking history, histology and tumor location are summarized in (Table 1).
Visualization reagents comprising a polymer reagent conjugated to horseradish peroxidases and goat anti-mouse antibody (15 minutes) and a polymer reagent conjugated to alkaline phosphatase and goat anti-rabbit antibody (15 minutes) were used.
Human papillomavirus genotyping was performed by deparaffinization of the tissue sections and DNA extraction (Norgena€™s FFPE DNA Purification Kit), followed by HPV genotype detection using the INNO-LiPA HPV Genotyping Kit (Innogenetics). Cox proportional hazards regression was performed to analyze the effect of all variables on recurrence times. Positive dual-staining immunoreactivity was found only in carcinomatous cells and was mainly observed in neoplastic nests (Figure 1). No significant correlation was found between p16 expression and anatomical sites as p16 was expressed in 26% of Glottic carcinomas and 22% of Supraglottic cases, respectively. Previous studies demonstrated that over-expression of p16 assessed has been consistently related to HR-HPV infection it is easy to carry in paraffin embedded tissues and in finea€“needle aspiration (FNA) of cytological specimens of cervical metastasis. Cells were then lysed and immunoprecipitated with anti-FLAG beads followed by immunoblotting as described10. Most of the discrepancy was found in cells with low pT308-AKT levels except HOP-92 cells (dotted box). ShRNAs were then induced with ?doxycycline (?DOX) on day 0 (pink lines), and cells were counted using a Beckman Coulter counter. The cells were then washed and stained with anti-PE magnetic beads and collected for magnetic separation. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Studies regarding HPV induced laryngeal cancers indicate that they are associated with improved outcomes [6], moreover they have more favorable prognosis when treated with surgery alone [7]. The patients had not received any preoperative therapy and surgery was the main mode of treatment given; clinical follow-up, ranging from 24 to 48 months, was conducted with a median follow-up of 36 months. The samples were then developed with two different chromogenic reactions based on horseradish peroxidases-mediated conversion of a DAB chromogenic (10 minutes), and alkaline phosphatase-mediated conversion of Fast Red chromogenic (15 minutes) to visible reaction products at the respective antigen sites. This assay permits specific detection of at least 25 HPV genotypes (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, .54, 56, 58, 59, 66, 70, 73 and 74). Recently, as a result from the expression of the HR-HPV related protein E7, p16INK4a positivity can be considered as a surrogate of HR-HPV infection [10]; it has been currently established in HNSCC by using different monoclonal antibody clones [12,13,18,19], particularly, E6H4 monoclonal antibody has been employed and its reactivity has been assessed by different cut-off regarding the intensity of the immunoreaction, the staining pattern and the percentage of p16 positive atypical cells [5,8,19-23]. We utilized a highly validated assay that screens from a broad panel of HPV types (INNO-LiPA HPV Genotyping Kit) and found a correlation between HR-HPV and p16 expression status, thus we believe that p16 expression is a reliable biomarker of HPV infection in HNSCC. Our findings confirm that p16 is of considerable diagnostic and prognostic value; moreover, the additional staining for Ki-67 might improve the reproducibility of the assay. The immunoblot showed the ?TopBP1 expression levels in these four cell lines (upper panel). Here we show that at the onset of senescence, PTEN null prostate tumours in mice2, 7 are massively infiltrated by a population of CD11b+Gr-1+ myeloid cells that protect a fraction of proliferating tumour cells from senescence, thus sustaining tumour growth. Normal-like prostate areas were compared with PIN and prostate cancer (PCA) areas in the same section. Patients with p16-negative tumours were more likely to relapse (83%) than p16-positive cancers (17%). At present, the causal role of HR-HPV in laryngeal carcinogenesis is controversial, mainly because no unanimously accepted detection method exists. A Squamous cell carcinoma of the cervix with known positive staining for p16INK4A was used as the positive control; an immunoglobulin class-matched no immune antibody was substituted for the primary antibody in the negative control. After amplification of the HPV genome (L1 region), the PCR products were hybridized with specific oligonucleotide probes immobilized on a strip and detected using a chromogenic procedure following the manufacturera€™s protocol. As a consequence, the reported p16 positivity in HNSCC various significantly: Lassen et al. Further evaluations of this biomarker could better identify the biological behavior and the clinical outcome of HR-HPV related HNSCC and could be taken into account for future therapeutic strategies. Mechanistically, we found that Gr-1+ cells antagonize senescence in a paracrine manner by interfering with the senescence-associated secretory phenotype of the tumour through the secretion of interleukin-1 receptor antagonist (IL-1RA). The arrow heads point to positions where UBC-GFP+ cells colocalize in close proximity to Ki-67+ cells. Commonly used HPV detection methods for formalin fixed paraffin embedded tissue include: HPV consensus PCR method, type specific HPV detection by fluorescence in situ hybridization (FISH) and real-time PCR assays.


After counterstaining, a two-step mounting protocol was applied: in a first step an aqueous mounting medium, subsequently a permanent mounting medium were used. DNA from the Caski cell line was used as a positive PCR control; PCR reagents lacking DNA served in each PCR amplification as a negative control. Strikingly, Pten-loss-induced cellular senescence was enhanced in vivo when Il1ra knockout myeloid cells were adoptively transferred to PTEN null mice.
CD45− epithelial cells and CD45+CD11b+Gr-1+ myeloid cells were further sorted using a FACSAria Cell Sorter.
In consideration of these findings, investigation of p16 expression by the dual-staining test is sought as technically simple and potentially reliable assay for HR-HPV induced laryngeal cancers thus to be predictive of clinical outcome. Thus, our study provides proof-of-concept evidence for targeting ?TopBP1, a convergent point of multiple pathways, as a cancer therapy. Each treatment was tested in triplicate, and the values were normalized to vehicle control wells. The 490-nm absorbance readings were normalized to the ‘0??M ?Doxorubicin’ controls within each group. Therapeutically, docetaxel-induced senescence and efficacy were higher in PTEN null tumours when the percentage of tumour-infiltrating CD11b+Gr-1+ myeloid cells was reduced using an antagonist of CXC chemokine receptor 2 (CXCR2)8. Total RNA was isolated from the epithelial and myeloid cell populations, and gene expression profiling was carried out using the one-colour labelling method, performing two replicates for each condition. Different monoclonal antibody clones have been used to assess p16 expression, and different cut-off were employed to assess p16 positivity in HNSCC specimens [8,11-13].
Taken together, our findings identify a novel non-cell-autonomous network, established by innate immunity, that controls senescence evasion and chemoresistance.
A heatmap displaying the mRNA expression of 53 secreted factors is shown (n = 2 per group).
Pharmacological interventions aimed at impairing Gr-1+ myeloid cell recruitment (for example, CXCR2a) can enhance senescence, thus improving chemotherapy efficacy. Recently, a novel biomarker concept, based on the combined detection of the p16 and Ki-67 biomarker protein expression has been designed [14]: the simultaneous detection of p16, a cell-cycle regulatory protein, and the expression of a proliferation marker such as Ki-67 within the same epithelial cell may be interpreted as a surrogate marker of cell-cycle deregulation induced by HR-HPV infection [15].
All cases with dual-stain-positive cell (s) were reviewed by two independent pathologists to confirm the result. The histogram shows the frequency of Gr-1+ myeloid cells (n = 5 control group; n = 7 treated groups). Luciferase activity of transfected ?p73 was determined as fold induction relative to that of the empty vector control (means±s.d.
Depletion of ?E2F1 in these samples was confirmed with western blot analysis (right panels). Representative images of H&E, ?Ki-67, TUNEL and cleaved ?caspase 3 staining at ? 40 magnification (lower panels). The cell lysates were then subjected to immunoprecipitation with anti-?p73 antibody or control mouse IgG and then to immunoblotting to detect mut?p53.
A portion of the cell lysates was subjected to immunoblotting with the indicated antibody (upper panel). Total number of living cells was counted with trypan blue exclusion assay and normalized to the vehicle controls. This mechanism is responsible for inhibition of ?E2F1-dependent apoptosis (an oncogenic checkpoint) and inhibition of ATR function (replication checkpoint) in the tumours with high Akt activity. The diameters of 15 mammospheres were measured from three independent culture experiments using a Zeiss digital inverted microscope (Axio Observer). In addition to mutations, cancer cells can have a different mechanism to inactivate ?p53: upregulation of ?p53-negative regulators, such as ?MDM2 (ref.
Adding to the complexity of ?p53 regulation is the presence of 12 ?p53 isoforms with differential expression17, some of which have dominant-negative activities against ?p53 (ref.
Arrows indicate CK18+Ki-67+ cells, which were considered for the analysis, while * indicates CK18−Ki-67+ cells, which were excluded from the analysis.
Cells were then grown in fresh media without ?Calcein AM for eight more days and then stained with ?crystal violet. Representative images are shown in the left panels (a complete set of data are shown in Supplementary Fig. Indeed, ?TopBP1 is frequently overexpressed in breast cancer and its overexpression is associated with a shorter survival12, 19. The number of viable cells was counted by trypan blue exclusion assay and normalized to the vehicle controls.
40, 3493–3502 (2013)." id="ref-link-43">20, T (rs115160714) gene polymorphism and endometrial cancer risk.




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