Non-angiogenic tumours unveil a new chapter in cancer biology.

The term 'angiogenesis' was coined in 1787 and the role of vessels in cancer has been studied ever since. In 1971 Folkman introduced the hypothesis, until now widely accepted, that tumour growth is strictly dependent on angiogenesis. However, the discovery that tumours can also grow without angiogenesis by exploiting pre-existing vessels, both in humans and more recently in mice, has demonstrated that this is not always the case. These observations highlight a new aspect of the interaction between vessels and tumours and demonstrate the existence of a previously unrecognized group of tumours that grow without angiogenesis and whose biology is, so far, largely unknown.

Incidence, management, and outcome of high-grade transformation of nodular lymphocyte predominant Hodgkin lymphoma: long-term outcomes from a 30-year experience.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a rare form of Hodgkin lymphoma that typically presents as early stage, indolent disease in young adult males. The relationship between NLPHL and DLBCL is incompletely understood, and there remains a paucity of data with regard the incidence and management of high-grade transformation. We report the largest study to date describing the incidence, management and long-term outcome of 26 cases of high-grade transformation of NLPHL over a 30-year period. We report a transformation incidence of 17.0%. Bone marrow, splenic, and liver infiltration with DLBCL was frequent. Patients with an aa-IPI 2-3 have poorer OS and PFS (P = 0.034 and P = 0.009, respectively). Although the approach to treatment was somewhat variable, typically young, otherwise fit patients received anthracycline-based induction, platinum-based consolidation with stem cell harvesting, followed by autologous SCT with BEAM conditioning. Long-term (5 year) PFS was over 60% with this approach, and comparable to our de novo DLBCL historical age and time period-matched cohort largely treated with CHOP-like chemotherapy alone. The transformation rate of 17.0% highlights the importance of accurate initial diagnosis, long-term follow-up, and re-biopsy at relapse.

High DLL4 expression in tumour-associated vessels predicts for favorable radiotherapy outcome in locally advanced squamous cell head-neck cancer (HNSCC).

INTRODUCTION: Expression of the DLL4 (a notch pathway ligand) by tumor-associated endothelium is a postulated marker of vascular maturity and functionality. As vascular functionality is an important parameter defining chemotherapy and oxygen intra-tumoral distribution, we investigated the role of DLL4 expression in tumour vasculature in the efficacy of radio-chemotherapy for HNSCC patients. MATERIALS AND METHODS: Sixty-five biopsy specimens from HNSCC patients with inoperable disease were immunohistochemically examined using anti-CD31 (pan-endothelial cell marker) and anti-DLL4 antibodies and the vascular density (VD) was recorded. Patients were treated with platinum based hypofractionated accelerated conformal radiotherapy. The median follow-up period was 24 months (4-80 months). RESULTS: Using the 33rd and 66th percentiles cases were grouped in three categories of low, medium and high CD31+ or DLL4+ VD. The percentage of vessels expressing DLL4 (DLL4-ratio) ranged from 17 to 100 % (mean 71 %), showing substantial variation among cases. In accordance with previous published studies, a biphasic pattern of association of CD31+ VD with poor outcome was noted. Cases with a medium VD had a significantly better local relapse free survival (LRFS) compared to cases of high VD (p = 0.0005, HR 0.15) and of low VD (p = 0.02, HR 0.28). High DLL4/CD31 ratio defined improved LRFS in both these subgroups of poor prognosis. CONCLUSIONS: The expression of DLL4 is associated with reduced radio-resistance, presumably by reducing hypoxia and improving chemotherapy accessibility. Using the combination of CD31 and DLL4 staining, a classification is suggested so that HNSCCs are categorized in sub-groups to be targeted by different anti-angiogenic and hypoxia targeting agents.

CD31 angiogenesis and combined expression of HIF-1α and HIF-2α are prognostic in primary clear-cell renal cell carcinoma (CC-RCC), but HIFα transcriptional products are not: implications for antiangiogenic trials and HIFα biomarker studies in primary CC-RCC.

Hypoxia-inducible factors, HIF-1α and HIF-2α, are expressed in the majority of clear-cell renal cell carcinoma (CC-RCC). In vitro, HIFα isoforms regulate a differential set of genes, and their effects in vivo within CC-RCC tumours may affect outcome. The role of angiogenesis and HIFα transcriptional products, including those involved in cell metabolism and morphological dedifferentiation have not been extensively investigated and might have relevance to the development of antiangiogenic or anti-HIFα trials in primary CC-RCC, either before or after radical nephrectomy. We analysed 168 consecutive clear-cell renal tumours from 1983 to 1999 within tissue microarrays and assessed expression of HIF-1α and HIF-2α together with the protein expression of seven of their target genes (BNIP3, CA9, Cyclin D1, GLUT-1, LDH5, Oct-4 and VEGF). The expression of these factors was compared with patient overall survival and CD31 angiogenesis. We found that HIFα antigenicity deteriorated with the age of the paraffin block (P < 0.0001) and in tumours from 1983 to 1992 was deemed not to be reliable. Similar findings were found in aged archival osteosarcoma samples. This might have important implications for retrospective biomarker studies that rely on archival tissue material. HIF-1α(HIGH)/HIF-2α(LOW) tumours had a worse overall survival compared with HIF-1α(LOW)/HIF-2α(LOW) tumours (P = 0.04). Surprisingly, on multivariate analysis, high levels of CD31(+) angiogenesis was shown to be an independent prognostic marker of increased overall survival (P = 0.003). We propose that better differentiation of vascular endothelium may be a reflection of a greater production of vessel stabilization factors versus pro-angiogenic factors, and therefore a less aggressive phenotype.

Lymphoid proliferations of indeterminate malignant potential arising in adults with common variable immunodeficiency disorders: unusual case studies and immunohistological review in the light of possible causative events.

Patients with common variable immunodeficiency disorders (CVIDs) who developed B cell lymphoproliferation of indeterminate malignant potential are described in order to raise a discussion of the relationship between infection and lymphoproliferation in infection prone patients. Those with CVID are at risk of developing either polyclonal or monoclonal lymphoproliferation in part due to the dysregulation of their adaptive immune systems. The aetiologies of the lymphoproliferations are unknown but intriguing; the relevance of infection being particularly problematic. The patients described here demonstrate variability in preceding infection, age at presentation, response to antibiotics and other types of therapy as well as outcome. The question of treatment is also controversial; issues include whether antibiotics or chemotherapy are the first line of therapy in all patients and whether transformation to aggressive B cell malignancy is inevitable or depends on other factors and if so, the length of time for such progression.

LC3A-positive light microscopy detected patterns of autophagy and prognosis in operable breast carcinomas.

Autophagy is a self-degradation mechanism by which cells recycle their own cytoplasmic constituents and dispose of excess or defective organelles after starvation and oxygen deprivation. An antibody to the microtubule-associated protein 1 light chain 3 (LC3A), recognizing both the soluble (LC3A-I) and the membrane-bound form (LC3A-II) of the protein, was used to detect autophagic activity in 102 breast carcinomas. Three distinct patterns were recognized: (1) diffuse cytoplasmic, (2) cytoplasmic/juxta-nuclear, and (3) "stone-like" pattern--dense, rounded, amorphous structures, 5 microm on average, typically enclosed within cytoplasmic vacuoles. The diffuse cytoplasmic pattern showed a direct association with estrogen and progesterone receptor expression. The juxta-nuclear pattern indicated a similar association with hormone receptors, an inverse association with tumor size, and a favorable prognosis. By contrast, an increased number of stone-like structures, probably representing an excessive autophagic response, was related to high-grade tumors and a less favorable outcome. Interestingly, 60 additional epithelial tumors of nonbreast origin disclosed identical autophagic patterns, and so did MDA231 breast cancer xenografts and HCT116 colon tumor spheroids (also analyzed by electron microscopy). Moreover, MCF-7 human breast cancer cell lines confirmed induction of LC3A by anoxia and Thapsigargin. It is concluded that autophagy can be readily recognized in breast carcinomas by light microscopy, after immunohistochemical staining with LC3A, but the significance of the various patterns expressed would need further evaluation.

Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer.

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

Aberrant expression of the neuronal transcription factor FOXP2 in neoplastic plasma cells.

FOXP2 mutation causes a severe inherited speech and language defect, while the related transcription factors FOXP1, FOXP3 and FOXP4 are implicated in cancer. FOXP2 mRNA and protein expression were characterised in normal human tissues, haematological cell lines and multiple myeloma (MM) patients' samples. FOXP2 mRNA and protein were absent in mononuclear cells from different anatomical sites, lineages and stages of differentiation. However, FOXP2 mRNA and protein was detected in several lymphoma (8/20) and all MM-derived cell lines (n = 4). FOXP2 mRNA was expressed in bone marrow samples from 96% of MM patients (24/25), 66.7% of patients with the pre-neoplastic plasma cell proliferation monoclonal gammopathy of undetermined significance (MGUS) (6/9), but not in reactive plasma cells. The frequency of FOXP2 protein expression in CD138(+) plasma cells was significantly higher in MGUS (P = 0.0005; mean 46.4%) and MM patients (P < or = 0.0001; mean 57.3%) than in reactive marrows (mean 2.5%). FOXP2 (>10% nuclear positivity) was detectable in 90.2% of MM (55/61) and 90.9% of MGUS (10/11) patients, showing more frequent expression than CD56 and labelling 75% of CD56-negative MM (9/12). FOXP2 represents the first transcription factor whose expression consistently differentiates normal and abnormal plasma cells and FOXP2 target genes are implicated in MM pathogenesis.

Nuclear and membrane expression of the angiogenesis regulator delta-like ligand 4 (DLL4) in normal and malignant human tissues.

AIMS: Delta-like ligand 4 (DLL4) is one of five known Notch ligands in mammals and interacts predominantly with Notch 1. DLL4 is induced by vascular endothelial growth factor (VEGF) and acts downstream of VEGF as a 'brake' on VEGF-induced vessel growth, forming an autoregulatory negative feedback loop inactivating VEGF. This action was believed to occur only in vascular development, raising hopes that DLL4 could be a specific drug target for controlling vessel growth in tumours and other pathological conditions. Our aim was to pursue this by raising a monoclonal antibody to the internal domain of DLL4 and assess its distribution in normal and malignant tissues in comparison with antibodies against the external domain of DLL4. METHODS AND RESULTS: The anti-DLL4 monoclonal antibody was raised using conventional mouse hybridoma techniques. The antibody has been fully characterized by Western blotting and transfectant immunostaining. It has also been comprehensively compared with other antibodies against both the internal and external domains of DLL4. The antigen is widely expressed on human tissues not only on endothelium but also on epithelium and stromal cells. Indeed, in our comprehensive survey only pulmonary alveoli failed to express DLL4. Of a wide range of malignancies, most also expressed DLL4 on tumour cells with a predominantly cytoplasmic pattern, although a number also displayed nuclear positivity. CONCLUSIONS: Contrary to previous beliefs, DLL4 is widely distributed in tissues other than vessels including many malignancies. Furthermore, the molecule is internalized on binding its receptor and often transported to the nucleus. These findings raise many interesting possibilities for further study of DLL4 and its potential as a therapeutic target.

Overexpression of the oxygen sensors PHD-1, PHD-2, PHD-3, and FIH Is associated with tumor aggressiveness in pancreatic endocrine tumors.

PURPOSE: Tumor hypoxia is associated with poor prognosis and resistance to treatment. Our aim was to assess the expression of proteins that act as cellular oxygen sensors, directly regulating the hypoxia inducible factor (HIF) pathway, i.e., prolyl hydroxylase domain proteins (PHD)-1, PHD-2, PHD-3, and FIH in pancreatic endocrine tumors (PET). EXPERIMENTAL DESIGN: Immunohistochemical expression of these markers was examined in 109 PET included in tissue microarrays and representing various stages of tumorigenesis. The results were correlated with histoprognostic factors including Ki-67 index, presence of a fibrotic focus, and microvascular density (MVD). RESULTS: The cytoplasmic and nuclear expressions of the three PHD isoforms were associated, and their expression was significantly higher in aggressive PETS, malignant, with lymph node metastases or with lower MVD. High nuclear expression of the three isoforms highly correlated with HIF-1alpha nuclear expression (P = 0.02, 0.003, and 0.006, respectively). Moreover, high nuclear PHD-1 or PHD-3 expression was associated with a poorer survival (P = 0.01). Cytoplasmic FIH was significantly higher in malignant PETs (P = 0.05) and in PETs with lymph node metastases (P = 0.02), and its expression correlated positively with those of cytoplasmic PHD isoforms (P < 0001). FIH stromal expression was found in 23% of PETs and correlated with higher FIH nuclear expression (P = 0.0004) and poorer disease-free survival (P = 0.0018). CONCLUSION: HIF regulatory proteins are highly expressed in PET and their expression is correlated with tumor metastases, tumor recurrence, and prognosis. These molecules that play an important role in the control of hypoxia-induced genes may have a function in the regulation of cellular proliferation and differentiation during endocrine tumorigenesis.

The presence of tumor-infiltrating FOXP3+ lymphocytes correlates with intratumoral angiogenesis in endometrial cancer.

OBJECTIVES: CD4(+)CD25(+) regulatory T-cells (Tregs), that express the transcription factor FOXP3, suppress effector T-cell populations and can enable tumour cells to evade the host immune response. In this study, we investigated the numbers of FOXP3(+) Tregs in the normal and malignat endometrium and examined potential links with tumor angiogenesis. METHODS: Paraffin-embedded tissues from 79 patients with stage I endometrial adenocarcinoma and 12 samples from normal endometrium were analyzed using immunohistochemistry for the detection of FOXP3(+) lymphocytes. The presence of FOXP3(+) lymphocytic infiltration was correlated with the tumor vascular density, the hypoxia inducible factors HIF-1alpha and HIF-2alpha, VEGF, estrogen and progesterone receptor expression. Survival analysis was also performed. RESULTS: In normal endometrium, FOXP3 was expressed by stroma infiltrating lymphocytes, with a mean number 8 (range 5-11) lymphocytes per x100 optical field. In tumors, 55/79 (69.6%) cases showed little FOXP3(+) lymphocytic infiltration (0-2 per x100 optical field). In the remaining 24/79 (30.4%) cases that were scored as positive the mean score ranged from 3-8 (median 5). Low numbers of FOXP3(+) lymphocytes significantly correlated with tumoral ER negativity and low vascular density. Survival analysis showed no significant impact of FOXP3 lymphocytic infiltration, although there was a trend towards worse prognosis. CONCLUSIONS: The correlation between the presence of FOXP3(+) Tregs and high vessel density in endometrial adenocarcinomas suggests a link between immunity, intratumoral angiogenesis and poor prognosis. However, further studies are required as significantly fewer Tregs were detected in the tumor microenvironment compared to normal endometrium.

BNIP3 as a progression marker in primary human breast cancer; opposing functions in in situ versus invasive cancer.

PURPOSE: BNIP3 is involved in cell death and cell survival via autophagy. Its perinecrotic localization within ductal carcinoma in situ (DCIS) suggests an involvement in neoplastic cellular adaptation to low oxygen tension. This study has investigated the role of BNIP3 in normal and neoplastic breast. EXPERIMENTAL DESIGN: Whole sections from 11 normal breast and microarrayed tissue cores from 81 DCIS and 251 invasive carcinomas were stained for BNIP3 and hypoxia-inducible factor-1alpha. The pattern and level of BNIP3 expression were correlated with clinicopathologic variables and hypoxia-inducible factor-1alpha. RESULTS: BNIP3 expression was significantly up-regulated in the cytoplasm of DCIS and invasive carcinoma compared with normal breast (P = 0.0005 and P < 0.0001, respectively). Nuclear BNIP3 expression was associated with smaller tumor size (P = 0.04), low tumor grade (P = 0.005), and estrogen receptor positivity (P = 0.008) in invasive tumors. Nuclear BNIP3 expression was also associated with a longer disease-free survival among low-grade and estrogen receptor-positive tumors. (P = 0.03 and 0.04, respectively). Conversely, nuclear BNIP3 expression in DCIS was associated with a 3-fold increase in recurrence and a shorter disease-free survival (P = 0.03). CONCLUSIONS: Up-regulation of BNIP3 expression in DCIS and invasive carcinoma suggests a significant role in breast tumor progression. Its association with good survival outcome in invasive carcinoma but with an increased risk of recurrence and shorter disease-free survival in DCIS may suggest a pivotal switch from a cell death to survival function during the transition from preinvasive to invasive breast cancer.

Non-angiogenic tumours and their influence on cancer biology.

Solid tumours need a blood supply, and a large body of evidence has previously suggested that they can grow only if they induce the development of new blood vessels, a process known as tumour angiogenesis. On the basis of this hypothesis, it was proposed that anti-angiogenic drugs should be able to suppress the growth of all solid tumours. However, clinical experience with anti-angiogenic agents has shown that this is not always the case. Reports of tumours growing without the formation of new vessels can be found in the literature dating back to the 1800s, yet no formal recognition, description and demonstration of their special biological status was made until recently. In 1996, we formally recognized and described non-angiogenic tumours in lungs where the only blood vessels present were those originating from normal lung tissue. This is far from an isolated scenario, as non-angiogenic tumour growth has now been observed in tumours of many different organs in both humans and preclinical animal models. In this Opinion article, we summarize how these tumours were discovered and discuss what we know so far about their biology and the potential implications of this knowledge for cancer treatment.

Cytoplasmic location of factor-inhibiting hypoxia-inducible factor is associated with an enhanced hypoxic response and a shorter survival in invasive breast cancer.

INTRODUCTION: Hypoxia-inducible factor (HIF)-1alpha levels in invasive breast carcinoma have been shown to be an adverse prognostic indicator. Cellular HIF-1alpha activity is regulated by factor-inhibiting hypoxia-inducible factor 1 (FIH-1). In hypoxia, FIH-1 hydroxylation of Asn803 within the C-terminal transactivation domain does not occur and HIF-1alpha forms a fully active transcriptional complex. The present study investigates the role of FIH-1 in invasive breast carcinoma and its correlation with hypoxia. METHODS: Microarrayed tissue cores from 295 invasive carcinomas were stained for FIH-1, for HIF-1alpha and for carbonic anhydrase 9. FIH-1 expression was correlated with standard clinicopathological parameters and with the expression of the surrogate hypoxic markers HIF-1alpha and carbonic anhydrase 9. RESULTS: FIH-1 was positive in 239/295 (81%) tumours, 42/295 (14%) exclusively in the nucleus and 54/295 (18%) exclusively in the cytoplasm. Exclusive nuclear FIH-1 expression was significantly inversely associated with tumour grade (P = 0.02) and risk of recurrence (P = 0.04), whereas exclusive cytoplasmic FIH-1 was significantly positively associated with tumour grade (P = 0.004) and carbonic anhydrase 9 expression (P = 0.02). Patients with tumours that excluded FIH-1 from the nucleus had a significantly shorter survival compared with those with exclusive nuclear expression (P = 0.02). Cytoplasmic FIH-1 expression was also an independent poor prognostic factor for disease-free survival. CONCLUSION: FIH-1 is widely expressed in invasive breast carcinoma. As with other HIF regulators, its association between cellular compartmentalization and the hypoxic response and survival suggests that tumour regulation of FIH-1 is an additional important mechanism for HIF pathway activation.

Expression of hypoxia-related tissue factors in astrocytic gliomas. A multivariate survival study with emphasis upon carbonic anhydrase IX.

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme involved in the reversible metabolism of carbon dioxide to carbonic acid and, hence, in physiological pH regulation. It also participates in cellular differentiation and proliferation, its expression being absent in most normal tissues. It has been recently postulated that the hypoxia-inducible factor (HIF-1) pathway up-regulated by hypoxia accounts for CAIX overexpression in most human tumors. In the present study, we examined the expression of this enzyme in diffuse gliomas of astrocytic origin in relation to vascular endothelial growth factor (VEGF) and HIF-1alpha expression, proliferation rate (as assessed with Ki-67 antigen), microvessel morphology, and survival. Of 84 cases analyzed, 61 cases (72.6%) displayed strong membrane and/or cytoplasmic expression of CAIX and were grouped as positive. Immunoreactivity tended to have a perinecrotic distribution and increased in parallel with the extent of necrosis (P < .001) and histologic grade (P < .001). A positive correlation was also noted with HIF-1alpha and VEGF expression (P < .001), proliferation rate (P = .010), microvessel density (P = .004), and microvessel caliber parameters (P = .014-.038). In univariate survival analysis, increased CAIX expression was associated with shortened survival in the entire cohort (P < .0001), along with VEGF (P = .0205) and HIF-1alpha levels (P = .0190). Multivariate analysis selected the interaction model of CAIX, with grade and age as the only parameters independently affecting survival. CAIX expression was also the only significant parameter for the survival of patients with grades II/III. We conclude that CAIX may be used as a prognostic indicator in diffuse astrocytomas to refine the information provided by grade. Given the role of CAIX in the acidification of tumor environment and its up-regulation by hypoxia, it is thought that CAIX expression may be linked to resistance of tumor cells to radiotherapy by allowing them to acclimatize to a hypoxic and acidic microenvironment.

Whole-genome sequencing identifies homozygous BRCA2 deletion guiding treatment in dedifferentiated prostate cancer.

Whole-genome sequencing (WGS) has transformed the understanding of the genetic drivers of cancer and is increasingly being used in cancer medicine to identify personalized therapies. Here we describe a case in which the application of WGS identified a tumoral BRCA2 deletion in a patient with aggressive dedifferentiated prostate cancer that was repeat-biopsied after disease progression. This would not have been detected by standard BRCA testing, and it led to additional treatment with a maintenance poly ADP ribose polymerase (PARP) inhibitor following platinum-based chemotherapy. This case demonstrates that repeat biopsy upon disease progression and application of WGS to tumor samples has meaningful clinical utility and the potential to transform outcomes in patients with cancer.

Hypoxia-related biological markers as predictors of epirubicin-based treatment responsiveness and resistance in locally advanced breast cancer.

PURPOSE: To identify hypoxia-related biomarkers indicative of response and resistance to epirubicin treatment in patients with locally advanced breast cancer. PATIENTS AND METHODS: One hundred seventy-six women with T2-4 N0-1 breast tumours were randomly assigned to receive epirubicin 120 mg/m2/1-21 (EPI ARM), epirubicin 120 mg/m2/1-21 + erythropoietin 10.000 IU sc three times weekly (EPI-EPO ARM) and epirubicin 40 mg/m2/w-q21 (EPI-W ARM). Sixteen tumour proteins involved in cell survival, hypoxia, angiogenesis and growth factor, were assessed by immunohistochemistry in pre-treatment samples. A multivariate generalized linear regression approach was applied using a penalized least-square minimization to perform variable selection and regularization. RESULTS: VEGF and GLUT-1 expression were significantly positively associated with complete response (CR) to treatment in all leave-one-out iterations. Bcl-2 expression was inversely correlated with pCR, whilst EPO expression was positively correlated with pathological complete response (pCR). Haemaglobin and HIF-1a nuclear expression were inversely correlated with pCR. HB and HIF-1a expression were associated with a higher risk of relapse and overall survival. CONCLUSION: Hypoxic biomarkers determines the epirubicin resistance in breast cancer. Assessment of such biomarkers, may be useful for predicting chemosensitivity and also anthracycline-based treatment outcome.

Gene expression signature for angiogenic and nonangiogenic non-small-cell lung cancer.

Angiogenesis is regarded as essential for tumour growth. However, we have demonstrated that some other aggressive non-small-cell lung carcinomas (n-SCLC) do not have angiogenesis. In this study, using cDNA microarray analysis, we demonstrate that angiogenic and nonangiogenic tumour types can be distinguished by their gene expression profiles. Tissue samples from 42 n-SCLC patients were obtained with consent. In all, 12 tumours were nonangiogenic and 30 angiogenic. The two groups were matched by age, sex, smoking and tumour stage. Total RNAs were extracted followed by microarray hybridization and image scan procedure. Data were analysed using GeneSpring 5.1 software. A total of 62 genes were found to be able to separate angiogenic from nonangiogenic tumours. Nonangiogenic tumours have higher levels of genes concerned with mitochondrial metabolism, mRNA transcription, protein synthesis and the cell cycle. Angiogenic tumours have higher levels of genes coding for membrane vesicles, integrins, remodelling, angiogenesis and apoptosis. These results further support our first finding that nonangiogenic lung tumours are fast-growing tumours filling the alveoli in the absence of vascular remodelling. We raise the hypothesis that in nonangiogenic tumours, hypoxia leads to a higher activation of the mitochondrial respiratory chain, which allows tumour growth without triggering angiogenesis.

Phosphorylated KDR can be located in the nucleus of neoplastic cells.

KDR (kinase insert domain receptor) phosphorylation induces several effects which lead eventually to cell proliferation and survival. The precise mechanisms by which KDR, once it is activated, communicates with the nucleus are starting to be understood but have not yet been completely unravelled. Two in vitro studies on animal cell lines reported in the literature have demonstrated that, following stimulation with VEGF, KDR is actually translocated within the nucleus. Our aim was to investigate whether this translocation occurs in human cells both in vitro and in vivo. Using laser scanning confocal microscopy, a variable nuclear localization of phosphorylated and total KDR in cell lines and tumour samples was found. In human neoplastic cell lines, hypoxic stimulation greatly increased the nuclear amount of total KDR but less so that of the phosphorylated form. Only after hypoxia and VEGF stimulation there was a comparably increased expression of phosphorylated and total KDR observed in the nuclei of these cells. We conclude that neoplastic cells show a variable expression of total and phosphorylated KDR in the nucleus. The precise functional meaning of nuclear location remains to be established.

C2028T polymorphism in exon 12 and dinucleotide repeat polymorphism in intron 13 of the HIF-1alpha gene define HIF-1alpha protein expression in non-small cell lung cancer.

OBJECTIVES: In this study, we investigated whether polymorphisms of the HIF-1alpha gene may account for the patterns of HIF-1alpha protein expression in non-small cell lung carcinomas (NSCLC) and the expression of HIF-1alpha down-stream proteins. METHODS: Specific HIF-1alpha polymorphisms were assessed in a series of patients with NSCLC: (a) the C to T transition at nucleotide 1744 (position 2028 according to sequence with accession number , which gives rise to Pro/Ser variation at codon 582), (b) the G to A nucleotide substitution at point 1790 (position 2046 according to sequence with accession number , which gives rise to Ala/Thr variation at codon 588), and (c) the dinucleotide GT repeat polymorphism in intron 13. Immunohistochemistry for HIF-1alpha and down-stream proteins (VEGF, LDH-5, GLUT-1) was also performed in tumor material. RESULTS: A strong association of the P582S polymorphism and of GT repeat polymorphism higher than 14/14 with increased HIF-1alpha expression was noted. HIF-1alpha polymorphism did not relate to the expression of the HIF-1alpha downstream proteins analysed, but significant association of HIF-1alpha expression with LDH-5 was confirmed (p=0.008). CONCLUSIONS: HIF-1alpha polymorphisms may have an important impact on HIF-protein stability and, eventually, function.