KI67 and DLX2 predict increased risk of metastasis formation in prostate cancer-a targeted molecular approach.

BACKGROUND: There remains a need to identify and validate biomarkers for predicting prostate cancer (CaP) outcomes using robust and routinely available pathology techniques to identify men at most risk of premature death due to prostate cancer. Previous immunohistochemical studies suggest the proliferation marker Ki67 might be a predictor of survival, independently of PSA and Gleason score. We performed a validation study of Ki67 as a marker of survival and disease progression and compared its performance against another candidate biomarker, DLX2, selected using artificial neural network analysis. METHODS: A tissue microarray (TMA) was constructed from transurethral resected prostatectomy histology samples (n=192). Artificial neural network analysis was used to identify candidate markers conferring increased risk of death and metastasis in a public cDNA array. Immunohistochemical analysis of the TMA was carried out and univariate and multivariate tests performed to explore the association of tumour protein levels of Ki67 and DLX2 with time to death and metastasis. RESULTS: Univariate analysis demonstrated Ki67 as predictive of CaP-specific survival (DSS; P=0.022), and both Ki67 (P=0.025) and DLX2 (P=0.001) as predictive of future metastases. Multivariate analysis demonstrated Ki67 as independent of PSA, Gleason score and D'Amico risk category for DSS (HR=2.436, P=0.029) and both Ki67 (HR=3.296, P=0.023) and DLX2 (HR=3.051, P=0.003) as independent for future metastases. CONCLUSIONS: High Ki67 expression is only present in 6.8% of CaP patients and is predictive of reduced survival and increased risk of metastasis, independent of PSA, Gleason score and D'Amico risk category. DLX2 is a novel marker of increased metastasis risk found in 73% patients and 8.2% showed co-expression with a high Ki67 score. Two cancer cell proliferation markers, Ki67 and DLX2, may be able to inform clinical decision-making when identifying patients for active surveillance.

Norepinephrine inhibits the migratory activity of pancreatic cancer cells.

We have shown previously that norepinephrine induces migratory activity of tumour cells from breast, colon and prostate tissue via activation of beta-2 adrenergic receptors. Consequently, this effect can be inhibited pharmacologically by clinically established beta-blockers. Tumour cell migration is a prerequisite for metastasis formation, and accordingly we and others have shown that breast cancer patients, which take beta-blockers due to hypertension, have reduced metastasis formation and increased survival probability as compared to patients without hypertension or using other anti-hypertensive medication. Unlike the aforementioned tumour cells, pancreatic cancer cells show a reduced migratory activity upon norepinephrine treatment. By means of our three-dimensional, collagen-based cell migration assay, we have investigated the signal transduction pathways involved in this phenomenon. We have found that this conflicting effect of norepinephrine on pancreatic cancer cells is due to an imbalanced activation of the two pathways that usually mediate a pro-migratory effect of norepinephrine in other tumour cell types. Firstly, the inhibitory effect results from activation of a pathway which causes a strong increase of the secondary cell signalling molecule, cAMP. In addition, activation of phospholipase C gamma and the downstream protein kinase C alpha were shown to be already activated in pancreatic cancer cells and cannot be further activated by norepinephrine. We hypothesize that this constitutive activation of the phospholipase C gamma pathway is due to a cross-talk with receptor tyrosine kinase signalling, and this might also deliver an explanation for the unusual high spontaneous migratory activity of pancreatic cancer cells.

The oestrogen receptor coactivator CARM1 has an oncogenic effect and is associated with poor prognosis in breast cancer.

The coactivator-associated arginine methyltransferase-1 (CARM1) is implicated in regulation of oestrogen receptor (ER) α-mediated gene pathways in response to ER activation. It plays an important role in breast cancer growth by regulating the E2F1 expression suggesting that CARM1 could be a target in the subclassification of oestrogen-dependent breast cancer. This study aims to investigate the clinical and biological importance of CARM1 protein expression in a large (1,130 patients), well-characterised and annotated series of invasive breast cancers using tissue microarrays and immunohistochemistry. In the whole series, increased CARM1 expression is correlated with features associated with aggressive behaviour such as young age, premenopausal status, large tumour size and high tumour grade. There is a positive correlation between CARM1 expression and biomarkers associated with non-luminal phenotype and poor prognosis such as HER2, basal cytokeratins, EGFR, p53 and the proliferation markers Ki67, TK1, CD71 and Cyclin E. Negative associations with the luminal-associated markers including steroid receptors and luminal cytokeratins are found. Similar associations are identified in the ER-positive/luminal subgroup (n = 767). Outcome analyses indicate that CARM1 expression is an independent predictor of shorter breast cancer-specific survival and disease-free interval in the whole series and in the ER-positive subgroup. CARM1 shows an oncogenic effect in breast cancer and its expression is associated with poor prognosis. CARM1 could be a potential marker of luminal class subclassification and for target therapy, particularly in the ER-positive luminal-like subgroup.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies.

BACKGROUND: The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. METHODS: Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at -80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. RESULTS: The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 µm2 in LEC to 392,887 µm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/µl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 µl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 µm2 to 130,0000 µm2. RNA concentration of these samples ranged from 10.88 ng/12 µl to 25.8 ng/12 µl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. CONCLUSIONS: The optimised protocol for sample collection and laser microdissection improved the RNA yield of the in situ ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.

Local allergic rhinitis: concept, pathophysiology, and management.

Local allergic rhinitis (LAR) is a localized nasal allergic response in the absence of systemic atopy characterized by local production of specific IgE (sIgE) antibodies, a T(H)2 pattern of mucosal cell infiltration during natural exposure to aeroallergens, and a positive nasal allergen provocation test response with release of inflammatory mediators (tryptase and eosinophil cationic protein). Although the prevalence remains to be established, a number of patients previously given a diagnosis of nonallergic rhinitis or idiopathic rhinitis are now being classified as having LAR. Culprit allergens responsible include house dust mite, grass and olive pollens, and many others. For the diagnosis of LAR, neither skin prick testing nor determination of the presence of serum sIgE antibodies is useful, and a nasal allergen provocation test is needed to identify the culprit allergen or allergens. In a certain proportion of cases, local sIgE can be detected, and conjunctivitis, asthma, or both can be associated. Whether patients with LAR will have systemic atopy in the future is a matter of debate. Further studies are needed for examine the prevalence of this phenomenon in different areas, to improve the diagnostic methods to better identify these patients, and to develop therapeutic approaches, including the use of immunotherapy.

EpCAM expression is an indicator of recurrence in basal-like breast cancer.

Advances in the understanding of the molecular basis of breast cancer have necessitated a definition of more sensitive and specific indicators of prognosis that are central to the underlying cancer biology and that reflect the complicated and heterogeneous nature of the disease. This study investigates the expression of epithelial cell adhesion molecule (EpCAM) in breast cancer particularly basal-like phenotype group which remains unclear. EpCAM expression was assessed using immunohistochemistry in a large well-characterised series of breast carcinomas prepared as tissue microarrays. Relationships between EpCAM expression with molecular subtypes, clinicopathological variables and patients' outcome were examined. EpCAM expression was associated with higher tumour grade (P < 0.001), larger tumour size (P < 0.001) and basal phenotype (P = 0.03). Importantly, within the basal-like tumours those positive for EpCAM showed a significantly shorter DFI (LR = 7.97, P = 0.005) and MFS (LR 4.01, P = 0.045). EpCAM may play a role in breast cancer progression and its expression is associated with poor patient outcome in basal-like breast cancer, independent of other prognostic factors.

A review of the biological and clinical characteristics of luminal-like oestrogen receptor-positive breast cancer.

Global gene expression profiling (GEP) studies of breast cancer have identified distinct biological classes with different clinical and therapeutic implications. Oestrogen receptor (ER) has been found to be a central marker of the molecular signature. GEP studies have consistently recognized a molecularly distinct class of tumours that is characterized by high-level expression of ER and other biomarkers recognized to be characteristic of normal luminal cells of the breast. This class is the largest of the GEP-defined molecular subclasses, comprising 60-70% of breast cancer cases. Moreover, it has been proposed that this group of tumours is composed of at least two subclasses distinguished by differing GEP profiles. At present, there is no consensus on the definition of the luminal subclasses and, in clinical practice, luminal-like tumours and ER-positive tumours are frequently considered to be the same. A better understanding of the biological features of luminal tumours could lead to their improved characterization and consistent identification. In this review, we explore the concept and definitions of the luminal-like class of breast carcinoma and their contribution to our understanding of their molecular features, clinical significance and therapeutic implications.

FOXO3a nuclear localisation is associated with good prognosis in luminal-like breast cancer.

Oestrogen receptor (ER)-positive breast cancer (BC) constitutes a heterogeneous group of tumours with regard to outcome and response to therapy. Accurate stratification of ER-positive BC according to risk of relapse and response to therapy will be achieved through an improved understanding of ER and ER-related biological pathways. Recent studies have identified Forkhead box O3a (FOXO3a) transcription factor as an intracellular mediator of ERα expression and as an important downstream target of the Akt/PI3K pathway indicating a biological and potential clinical role for FOXO3a in ER-positive BC. In this study, we investigated the clinical relevance and biological associations of FOXO3a protein expression, using tissue microarrays and immunohistochemistry, in a large series of patients with invasive breast cancer. FOXO3a protein expression showed both nuclear and/or cytoplasmic staining patterns. FOXO3a predominant nuclear expression was positively associated with biomarkers of good prognosis including PgR, FOXA1 and p27 expression. There was an inverse association with mitotic counts, MIB1 growth fraction, C-MYC and PIK3CA expression. With respect to patient outcome, FOXO3a nuclear localisation was associated with longer BC specific survival (P < 0.001) and longer distant metastasis free interval (P = 0.001), independently of the well-established breast cancer prognostic factors. In conclusion, our results demonstrate the biological and prognostic role of FOXO3a protein expression and its subcellular localisation in ER-positive/luminal-like BC possibly through its involvement in controlling cell proliferation.

Clinicopathologic and molecular significance of phospho-Akt expression in early invasive breast cancer.

Akt/PKB serine/threonine kinase is a leading signalling modulator for several cellular processes including metabolism, growth, proliferation and survival. However, complexity and diversity in the upstream/downstream arms of Akt pathway, as recent genetic studies reported, challenge considerably the evolvement of effective targeted therapies. The aim was to study the expression of phospho-Akt1 (pAkt) in breast cancer (BC), with respect to different component proteins upstream/downstream of Akt pathway activation, clinicopathologic parameters and patients' outcome. pAkt (Ser473) was evaluated by immunohistochemistry, on tissue microarrays containing 1,202 early invasive BC with long-term clinical follow-up. Seventy-six percent of the studied tumours overexpressed pAkt, where it was associated with expression of oestrogen and androgen receptors, PIK3CA, cytokeratin (CK)18, CK19 and PTEN. Loss of pAkt was correlated with high grade, CK5/6, p53 and high Ki67 labelling index. Higher proportions of luminal tumours were pAkt positive relative to triple negative/basal subtypes. However, pAkt overexpression was not associated with breast cancer specific (BCSS) or metastasis-free survival (MFS). Four tumour phenotypes were identified based on PIK3CA and pAkt expression, with substantial proportions being PIK3CA⁻/pAkt⁺ or PIK3CA⁺/pAkt⁻. These four combinatorial phenotypes were significantly associated with BCSS (p = 0.001) and MFS (p = 0.002). Although pAKT is an oncogene correlated with poor prognostic variables, it was not a prognostic marker. Combinatorial phenotypic groups of PIK3CA/pAkt denoted functional complexity, at translational level, within the upstream and downstream arms of Akt activation with significant impact on patients' outcome. These findings may help development more adequate therapeutic regimens for specific subgroups of this key cancer pathway.

CTEN (C-terminal tensin-like), a novel oncogene overexpressed in invasive breast carcinoma of poor prognosis.

C-terminal tensin-like (CTEN) gene is a member of the TENSIN gene family, involved in cell migration and localised at focal adhesion sites. This study was designed to explore the prevalence and clinical significance of CTEN expression in a large and well-characterised series (n = 1,409) invasive breast cancer (BC) cases with long term follow-up (median 11 years), using immuno-histochemistry and tissue microarray. Moderate to strong cytoplasmic immunoreactivity for CTEN was observed in 90% of the studied cases. CTEN expression was significantly associated with poor prognostic variables including larger tumour size (P = 0.044), higher histological grade (P = 0.019), axillary nodal involvement (P = 0.035) and poor Nottingham Prognostic Index (P = 0.016). Significant associations were observed between increased CTEN expression and up-regulation of phosphorylated-Akt (P-Akt), PIK3 and N-cadherin proteins (P\0.001). Kaplan-Meier survival analysis demonstrated that patients with high CTEN expression had significantly shorter Breast Cancer Specific Survival (P = 0.004) and Metastasis-Free Survival (P = 0.041) than those with low-CTEN expression. Multivariate analysis showed that CTEN was not an independent prognostic marker in BC. In conclusion, our results demonstrated the oncogenetic role of increased CTEN expression and its association with poor prognostic parameters. These data could help in prognostic assessment in BC patients.

MIB1/Ki-67 labelling index can classify grade 2 breast cancer into two clinically distinct subgroups.

Histological grade is recognized as one of the strongest prognostic factors in operable breast cancer (BC). Although grade 1 and grade 3 tumours are biologically and clinically distinct, grade 2 tumours bear considerable difficulty in outcome prediction and planning therapies. Several attempts such as genomic grade index have been performed to subclassify grade 2 into two subgroups with clinical relevance. Here, we present evidence that the routinely evaluable immunohistochemical MIB1/Ki67 labelling index (MIB-LI) can classify grade 2 tumours into two clinically distinct subgroups. In this study, growth fractions of 1,550 primary operable invasive breast carcinomas were immunohistochemically assayed on full-face tissue sections using the MIB1 clone of Ki-67. Growth fractions were assessed as number of MIB1 positive nuclei in 1,000 tumour nuclei at high-power magnification and expressed as MIB1-LI. Using a 10% cut-point of MIB1-LI, grade 2 BCs were classified into low (49.8%) and high (50.2%) proliferative subgroups. Univariate and multivariate survival analysis revealed statistically significant differences between these subgroups regarding patients' BC specific survival (P < 0.001), and metastasis free survival (P < 0.001) which was independent of the well-established prognostic factors (HR = 2.944, 95% CI = 1.634-5.303, P < 0.001). In conclusion, our results further demonstrate that grade 2 BCs may represent at least two biological or behaviourally different entities. Assay of growth fraction in BC using MIB1/Ki67 immunohistochemistry is a robust cost-effective diagnostic tool that subdivides grade 2 tumours into low and high risk populations providing additional prognostic information in planning therapies and outcome prediction.

RERG (Ras-like, oestrogen-regulated, growth-inhibitor) expression in breast cancer: a marker of ER-positive luminal-like subtype.

Global gene expression profiling studies have classified breast cancer into a number of distinct biological and molecular classes with clinical relevance. The heterogeneous luminal group, which is largely characterised by oestrogen receptor (ER) expression, appears to contain distinct subgroups with differing behaviour. In this study, we analysed 47,293 gene transcripts in 128 invasive breast carcinomas (BC) using Artificial Neural Networks and a cross-validation analysis in combination with an ensemble sample classification to identify genes that can be used to subclassify ER+ luminal tumours. The results were validated using immunohistochemistry on TMAs containing 1,140 invasive breast cancers. Our results showed that the RERG gene is one of the highest ranked genes to differentiate between ER+ luminal-like and ER- non-luminal cancers based on a 10-fold external cross-validation analysis with an average classification accuracy of 89%. This was confirmed in our protein expression studies that showed RERG positive associations with markers of luminal differentiation including ER, luminal cytokeratins (CK19, CK18 and CK7/8) and FOXA1 (P = 0.004) and other markers of good prognosis in BC including small size, lower histologic grade and positive expression of androgen receptor, nuclear BRCA1, FHIT and cell cycle inhibitors p27 and p21. RERG expression was inversely associated with the proliferation marker MIB1 (P = 0.005) and p53. Strong RERG expression showed an association with longer breast cancer specific survival and distant metastasis free interval in the whole series as well as in the ER+ luminal group and these associations were independent of other prognostic variables. In conclusion, we used novel bioinformatics methods to identify candidate genes to characterise ER+ luminal-like breast cancer. RERG gene is a key marker of the luminal BC class and can be used to separate distinct prognostic subgroups.

An evaluation of the clinical significance of FOXP3+ infiltrating cells in human breast cancer.

Studies in mice have shown that thymic-derived CD4+ CD25+ regulatory T cells (T reg; FOXP3(+) lymphocytes) inhibit an antitumour immune response. Additional studies have also reported that the T reg population increases in peripheral blood and tumour tissues from patients with cancer. However, the relationship between the T reg population and the patient prognosis remains controversial. Our aim was to determine the prognostic value of T reg cell density in breast cancer using immunohistochemical assessment of FOXP3, which has been shown to be the optimal marker for T regs. Tissue microarrays were used, and the density of FOXP3(+) cells was determined in a series of 1445 cases of well-characterised primary invasive breast carcinoma cases with long-term follow up. FOXP3(+) cell numbers were counted in tumour nests, in tumour-adjacent stroma, and in distant stroma. The total number of FOXP3(+) cells significantly correlated with higher tumour grade (r (s) = 0.37, P < 0.001) and ER negativity (Mann-Whitney U test, P < 0.001). In addition, FOXP3 infiltration positively correlated with HER2 expression and basal phenotype subclass. On univariate analysis, FOXP3(+) cells were associated with a worse prognosis (P = 0.012, log rank = 6.36). This association was found for intratumoural FOXP3(+) and for tumour-adjacent stromal FOXP3(+)-cells (tumour-cell associated FOXP3, P = 0.001 and log rank 10.35). However, the number of FOXP3(+) cells was not found to be an independent prognostic factor in multivariate analysis. We therefore conclude that FOXP3(+) infiltrating cells do not have a dominant role in breast cancer prognosis and suggest that other inflammatory cell subsets may be more critical variables.

Luminal and basal-like breast cancer cells show increased migration induced by hypoxia, mediated by an autocrine mechanism.

BACKGROUND: Some breast cancer patients receiving anti-angiogenic treatment show increased metastases, possibly as a result of induced hypoxia. The effect of hypoxia on tumor cell migration was assessed in selected luminal, post-EMT and basal-like breast carcinoma cell lines. METHODS: Migration was assessed in luminal (MCF-7), post-EMT (MDA-MB-231, MDA-MB-435S), and basal-like (MDA-MB-468) human breast carcinoma cell lines under normal and oxygen-deprived conditions, using a collagen-based assay. Cell proliferation was determined, secreted cytokine and chemokine levels were measured using flow-cytometry and a bead-based immunoassay, and the hypoxic genes HIF-1α and CA IX were assessed using PCR. The functional effect of tumor-cell conditioned medium on the migration of neutrophil granulocytes (NG) was tested. RESULTS: Hypoxia caused increased migratory activity but not proliferation in all tumor cell lines, involving the release and autocrine action of soluble mediators. Conditioned medium (CM) from hypoxic cells induced migration in normoxic cells. Hypoxia changed the profile of released inflammatory mediators according to cell type. Interleukin-8 was produced only by post-EMT and basal-like cell lines, regardless of hypoxia. MCP-1 was produced by MDA-MB-435 and -468 cells, whereas IL-6 was present only in MDA-MB-231. IL-2, TNF-α, and NGF production was stimulated by hypoxia in MCF-7 cells. CM from normoxic and hypoxic MDA-MB-231 and MDA-MB-435S cells and hypoxic MCF-7 cells, but not MDA-MB-468, induced NG migration. CONCLUSIONS: Hypoxia increases migration by the autocrine action of released signal substances in selected luminal and basal-like breast carcinoma cell lines which might explain why anti-angiogenic treatment can worsen clinical outcome in some patients.

Proposal for a modified grading system based on mitotic index and Bcl2 provides objective determination of clinical outcome for patients with breast cancer.

We hypothesized that the interaction between mitotic index (M) and Bcl2 could accurately discriminate between low- and high-grade breast cancer (BC) and provide a more objective measure of clinical outcome than histological grade, especially for patients with intermediate histological grade (G2), small size or oestrogen receptor (ER)-negative cancers. A well-characterized series of 1650 BCs with long-term follow-up was subjected to immunohistochemical analysis for Bcl2. Mitotic index (M) was assessed according to Nottingham Grading System (NGS) guidelines: M1: < 10 mitoses; M2: 10-18 mitoses; M3: > 18 mitoses. Results were validated in an independent series of patients (n = 245) uniformly treated with adjuvant anthracycline-based chemotherapy. Subsequently, BCs were classified according to the combined M/Bcl2 profile and compared with NGS. Multivariate Cox regression models using validated prognostic factors demonstrated that the subgroups defined by M/Bcl2 profile remained significantly associated with patients' outcome but also performed better than lymph node status and tumour size. Incorporation of the M/Bcl2 profile into the Nottingham Prognostic Index (NPI) reclassified twice as many patients into the excellent prognosis group, potentially improving decision-making and sparing patients unneeded systemic adjuvant therapy. Patients with M2-3/Bcl2- and M3/Bcl2+ (high risk) had a two- to three-fold increased risk of recurrence when treated with either adjuvant hormone therapy or anthracycline-based chemotherapy compared with those with M1/Bcl2 ± and M2/Bcl2+ (low risk) [HR = 3.4 (2.8-5.6); p < 0.0001 and HR = 2.3 (1.2-4.3); p = 0.0009]. In conclusion, a grading system defined by mitotic counting and Bcl2 expression accurately reclassified patients with NGS-G2, small tumour size or ER-negative cancers into two groups: low risk (NGS-G1-like) versus high risk (NGS-G3-like) of BC mortality and recurrence, improving prognosis and therapeutic planning.

The biological, clinical and prognostic implications of p53 transcriptional pathways in breast cancers.

We hypothesized that the functional status of p53 transcriptional pathways, rather than p53 protein expression alone, could accurately discriminate between low- and high-risk breast carcinoma (BC) and inform about individuals' tumour biological behaviour. To test this, we studied a well-characterized series of 990 BCs with long-term follow-up, immunohistochemically profiled for p53, its main regulators and downstream genes. Results were validated in an independent series of patients (n = 245) uniformly treated with adjuvant anthracycline-based chemotherapy. Eleven p53 transcriptional phenotypes were identified with just two main clinical outcomes. (a) Low risk/good prognosis group (active/partially inactive p53 pathways), defined as p53(+/-)/MDM4(+)/MDM2(+/-)/Bcl2(+/-)/p21(+/-), p53(-)/MDM4(-)/MDM2(+)/Bcl2(+)/p21(+/-) and p53(+/-)/MDM4(-)/MMD2(-)/Bcl2(+)/p21(+/-). These tumours had favourable clinicopathological characteristics, including ER(+) and long survival after systemic adjuvant-therapy (AT). (b) High risk/poor prognosis group (completely inactive p53 pathways), defined as p53(+/-)/MDM4(-) MDM2(-)/Bcl2(-)/p21(-), p53(-)/MDM4(-) MDM2(+)/Bcl2(-)/p21(-) and p53(+/-)/MDM4(-)/MDM2(-)/Bcl2(-)/p21(+). These tumours were characterized by aggressive clinicopathological characteristics and showed shortened survival when treated with AT. Completely inactive p53 pathways but intact p21 axis p53(+/-)/MDM4(-)/MDM2(-)/Bcl2(-)/p21(+) had the worst prognosis, particularly patients who received AT. Multivariate Cox regression models, including validated prognostic factors for both test and validation series, revealed that the functional status of p53 transcriptional pathways was an independent prognosticator for BC-specific survival (HR 2.64 and 4.5, p < 0.001, respectively) and disease-free survival (HR 1.93 and 2.5, p < 0.001, respectively). In conclusion, p53 functional status determined by assessment of p53 regulatory and downstream targets provides independent prognostic value and may help determine more adequate therapeutic regimens for specific subgroups of breast cancer patients.

Evidence for the involvement of free light chain immunoglobulins in allergic and nonallergic rhinitis.

BACKGROUND: Allergic rhinitis is characterized by mast cell degranulation induced by antigen cross-linking of IgE. It has been proposed that some patients with rhinitis show nasal allergy in the absence of systemic markers of atopy, termed entopy. Recent murine studies suggest the existence of an IgE-independent hypersensitivity response involving antigen-induced mast cell activation, mediated by immunoglobulin free light chains (FLCs). OBJECTIVES: To determine whether FLC is associated with mast cell-mediated nasal hypersensitivity and its relationship with eosinophilic activity in allergic and nonatopic rhinitis. METHODS: Patients with allergy and nonallergic rhinitis with eosinophilia syndrome (NARES) had levels of soluble FLC measured in nasal secretions and serum. In addition, levels of the nasal inflammatory mediators mast cell tryptase and eosinophil cationic protein were quantified. Cellular expression of kappa and lambda FLC was characterized in the nasal mucosa of allergic and nonatopic idiopathic rhinitis and control subjects by using immunohistochemistry. Immunopositive cells were phenotyped by using laser microdissection and PCR. RESULTS: Free light chain was significantly increased in nasal secretions of subjects with allergy and NARES, and in serum of patients with NARES. Nonatopic patients with allergy showed significantly increased nasal mast cell tryptase and eosinophil cationic protein. FLC-positive cells were significantly increased in allergic and nonatopic mucosa, and were shown to be mast cells and plasma cells. CONCLUSION: Nasal FLC is significantly increased in allergic and nonatopic rhinitis nasal mucosa, suggesting a role in nasal hypersensitivity. Further studies are needed to identify which allergens trigger FLC-mediated responses in nonatopic rhinitis.

Comparative transcriptional profiling of the limbal epithelial crypt demonstrates its putative stem cell niche characteristics.

BACKGROUND: The Limbal epithelial crypt (LEC) is a solid cord of cells, approximately 120 microns long. It arises from the undersurface of interpalisade rete ridges of the limbal palisades of Vogt and extends deeper into the limbal stroma parallel or perpendicular to the palisade. There are up to 6 or 7 such LEC, variably distributed along the limbus in each human eye. Morphological and immunohistochemical studies on the limbal epithelial crypt (LEC) have demonstrated the presence of limbal stem cells in this region. The purpose of this microarray study was to characterise the transcriptional profile of the LEC and compare with other ocular surface epithelial regions to support our hypothesis that LEC preferentially harbours stem cells (SC). RESULTS: LEC was found to be enriched for SC related Gene Ontology (GO) terms including those identified in quiescent adult SC, however similar to cornea, limbus had significant GO terms related to proliferating SC, transient amplifying cells (TAC) and differentiated cells (DC). LEC and limbus were metabolically dormant with low protein synthesis and downregulated cell cycling. Cornea had upregulated genes for cell cycling and self renewal such as FZD7, BTG1, CCNG, and STAT3 which were identified from other SC populations. Upregulated gene expression for growth factors, cytokines, WNT, Notch, TGF-Beta pathways involved in cell proliferation and differentiation were noted in cornea. LEC had highest number of expressed sequence tags (ESTs), downregulated and unknown genes, compared to other regions. Genes expressed in LEC such as CDH1, SERPINF1, LEF1, FRZB1, KRT19, SOD2, EGR1 are known to be involved in SC maintenance. Genes of interest, in LEC belonging to the category of cell adhesion molecules, WNT and Notch signalling pathway were validated with real-time PCR and immunofluorescence. CONCLUSIONS: Our transcriptional profiling study identifies the LEC as a preferential site for limbal SC with some characteristics suggesting that it could function as a 'SC niche' supporting quiescent SC. It also strengthens the evidence for the presence of "transient cells" in the corneal epithelium. These cells are immediate progeny of SC with self-renewal capacity and could be responsible for maintaining epithelial turn over in normal healthy conditions of the ocular surface (OS). The limbus has mixed population of differentiated and undifferentiated cells.

Growth fraction as a predictor of response to chemotherapy in node-negative breast cancer.

Accurate predictive markers of chemotherapeutic response in early breast cancer are still lacking. The role of tumour growth fraction as a predictor of response to chemotherapy was assessed in early breast cancer. In this study, immunohistochemical expression of MIB1 was studied in a well-characterised series of early (Stages I and II) node-negative breast carcinoma cases (n = 100) with long-term follow-up that have received adjuvant chemotherapy (cyclophosphamide/methotrexate/5-fluorouracil regimen). In addition, 728 cases who did not receive adjuvant chemotherapy were used as a control group. Increased tumour growth fraction was associated with a better response to adjuvant chemotherapy in terms of longer breast cancer specific survival and disease-free interval [hazard ratio (HR) = 0.354, 95% CI = 0.177-0.688, p = 0.003 and HR = 0.396, 95% CI = 0.205-0.768, p = 0.006, respectively]. In contrast to the control group, patients with high growth fraction tumour (>70%) showed an excellent outcome with infrequently reported events during the period of follow-up. Importantly, patients with a low growth fraction (< or =10%) showed frequent recurrences and shorter survival time with outcome comparable to those of high growth fraction who did not receive chemotherapy. Therefore, tumour growth fraction can be used to assign patients into distinct groups showing differential response to adjuvant chemotherapy. Patients with a high growth fraction appear to be ideal candidates for adjuvant chemotherapy while those with low growth fraction are less likely to benefit and are prone to the potential serious side effects of adjuvant chemotherapy.

The prognostic significance of PELP1 expression in invasive breast cancer with emphasis on the ER-positive luminal-like subtype.

The transcription functions of oestrogen receptors (ER) are influenced by several coregulators such as PELP1 (proline, glutamate and leucine rich protein 1). The aim of the present study, which uses tissue microarrays and immunohistochemistry, is to explore the clinical and biological relevance of PELP1 protein expression in a large series of consecutive patients (1,162 patients) with invasive breast cancers with particular emphasis on its role in the ER-positive/luminal-like class of tumours. Our results showed that increased PELP1 expression is associated with tumours of larger size, higher histological grade, higher mitotic count, and with positive expression of basal cytokeratins (CK) (CK14; P = 0.018 and CK5/6; P = 0.029), P-cadherin (P = 0.002), p53 and MIB1 (P = 0.018). There was an inverse association between PELP1 expression and ER (P = 0.002), progesterone (PgR) (P = 0.004), androgen (AR) receptor (P < 0.001), and luminal CK (CK18; P = 0.027) expression. A significant association between PELP1 expression and shorter breast cancer specific survival (BCSS) (P = 0.002) and disease-free survival (DFI) (P = 0.006) was found. Multivariate Cox hazard analysis showed that PELP1 expression was an independent predictor of shorter BCSS (Hazard ratio (HR) = 1.349, P = 0.006) and shorter DFI (HR = 1.255, P = 0.011). In the ER-positive/luminal-like group (n = 768), PELP1 expression showed similar association with other clinicopathological variables and was an independent predictor of shorter DFI (HR = 1.256, P = 0.036). In conclusion, PELP1 protein expression is an independent prognostic predictor of shorter BCSS and DFI in breast cancer and its elevated expression is positively associated with markers of poor outcome. PELP1 appears to have a potential application in assessing the clinical outcome of patients with ER-positive breast cancer.