Rapidly progressive amyotrophic lateral sclerosis is associated with microglial reactivity and small heat shock protein expression in reactive astrocytes.

AIMS: Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive loss of motor neurons, muscle weakness, spasticity, paralysis and death usually within 2-5 years of onset. Neuroinflammation is a hallmark of ALS pathology characterized by activation of glial cells, which respond by upregulating small heat shock proteins (HSPBs), but the exact underlying pathological mechanisms are still largely unknown. Here, we investigated the association between ALS disease duration, lower motor neuron loss, TARDNA-binding protein 43 (TDP-43) pathology, neuroinflammation and HSPB expression. METHODS: With immunohistochemistry, we examined HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 expression in cervical, thoracic and sacral spinal cord regions in 12 ALS cases, seven with short disease duration (SDD), five with moderate disease duration (MDD), and ten age-matched controls. Expression was quantified using ImageJ to examine HSP expression, motor neuron numbers, microglial and astrocyte density and phosphorylated TDP-43 (pTDP-43+) inclusions. RESULTS: SDD was associated with elevated HSPB5 and 8 expression in lateral tract astrocytes, while HSP16.2 expression was increased in astrocytes in MDD cases. SDD cases had higher numbers of motor neurons and microglial activation than MDD cases, but similar levels of motor neurons with pTDP-43+ inclusions. CONCLUSIONS: Increased expression of several HSPBs in lateral column astrocytes suggests that astrocytes play a role in the pathogenesis of ALS. SDD is associated with increased microgliosis, HSPB5 and 8 expression in astrocytes, and only minor changes in motor neuron loss. This suggests that the interaction between motor neurons, microglia and astrocytes determines neuronal fate and functional decline in ALS.

Angiogenesis-Related Markers and Prognosis After Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy for Metastatic Colorectal Cancer.

BACKGROUND: Patients presenting with peritoneal metastases (PM) of colorectal cancer (CRC) can be curatively treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Angiogenesis is under control of multiple molecules of which HIF1a, SDF1, CXCR4, and VEGF are key players. We investigated these angiogenesis-related markers and their prognostic value in patients with PM arising from CRC treated with CRS and HIPEC. PATIENTS AND METHODS: Clinicopathological data and tissue specimens were collected in 2 tertiary referral centers from 52 patients who underwent treatment for isolated PM of CRC. Whole tissue specimens were subsequently analyzed for protein expression of HIF1a, SDF1, CXCR4, and VEGF by immunohistochemistry. Microvessel density (MVD) was analyzed by CD31 immunohistochemistry. The relationship between overall survival (OS) and protein expression as well as other clinicopathological characteristics was analyzed. RESULTS: Univariate analysis showed that high peritoneal cancer index (PCI), resection with residual disease and high expression of VEGF were negatively correlated with OS after treatment with CRS and HIPEC (P < 0.01, P < 0.01, and P = 0.02, respectively). However, no association was found between the other markers and OS (P > 0.05). Multivariate analysis showed an independent association between OS and PCI, resection outcome and VEGF expression (multivariate HR: 6.1, 7.8 and 3.8, respectively, P ≤ 0.05). CONCLUSIONS: An independent association was found between high VEGF expression levels and worse OS after CRS and HIPEC. The addition of VEGF expression to the routine clinicopathological workup could help to identify patients at risk for early treatment failure. Furthermore, VEGF may be a potential target for adjuvant treatment in these patients.

Epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are prognostic biomarkers for patients with resected colorectal cancer liver metastases.

BACKGROUND: Resection of colorectal cancer liver metastasis (CRCLM) with curative intent has long-term benefit in ~40% of cases. Prognostic biomarkers are needed to improve clinical management and reduce futile surgeries. Expression of epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase-2) has been associated with carcinogenesis and survival. We investigated the prognostic value of EGFR and PTGS2 expression in patients with resected CRCLM. METHODS: Formalin-fixed paraffin-embedded CRCLM tissue and corresponding primary tumour specimens from a multi-institutional cohort of patients who underwent liver resection between 1990 and 2010 were incorporated into tissue microarrays (TMAs). TMAs were stained for EGFR and PTGS2 by immunohistochemistry. The hazard rate ratio (HRR) for the association between expression in CRCLM and overall survival was calculated using a 500-fold cross-validation procedure. RESULTS: EGFR and PTGS2 expression could be evaluated in 323 and 351 patients, respectively. EGFR expression in CRCLM was associated with poor prognosis (HRR 1.54; P<0.01) with a cross-validated HRR of 1.47 (P=0.03). PTGS2 expression was also associated with poor prognosis (HRR 1.60; P<0.01) with a cross-validated HRR of 1.63 (P<0.01). Expression of EGFR and PTGS2 remained prognostic after multivariate analysis with standard clinicopathological variables (cross-validated HRR 1.51; P=0.02 and cross-validated HRR 1.59; P=0.01, respectively). Stratification for the commonly applied systemic therapy regimens demonstrated prognostic value for EGFR and PTGS2 only in the subgroup of patients who were not treated with systemic therapy (HRR 1.78; P<0.01 and HRR 1.64; P=0.04, respectively), with worst prognosis when both EGFR and PTGS2 were highly expressed (HRR 3.08; P<0.01). Expression of PTGS2 in CRCLM was correlated to expression in patient-matched primary tumours (P=0.02, 69.2% concordance). CONCLUSIONS: EGFR and PTGS2 expressions are prognostic molecular biomarkers with added value to standard clinicopathological variables for patients with resectable CRCLM.

Aurora kinase A (AURKA) expression in colorectal cancer liver metastasis is associated with poor prognosis.

BACKGROUND: Five-year survival after resection of colorectal cancer liver metastasis (CRLCM) is <30%. We recently found that aurora kinase A (AURKA) drives 20q gain-associated tumour progression and is associated with disease recurrence. This study evaluates the prognostic value of AURKA expression in CRCLM of patients who underwent liver resection. METHODS: Tissue microarrays (TMAs) were generated using formalin-fixed paraffin-embedded CRCLM and matched primary tumour from a multi-institutional cohort of patients with CRCLM who underwent liver resection between 1990 and 2010. Tissue microarrays were stained for AURKA using immunohistochemistry, and a hazard rate ratio (HRR) for the association between overall survival (OS) and nuclear AURKA expression in CRCLM was calculated. Results were validated by 500-fold cross-validation. RESULTS: The expression of AURKA was evaluated in CRCLM of 343 patients. High AURKA expression was associated with poor OS (HRR 1.55, P<0.01), with a cross-validated average HRR of 1.57 (P=0.02). Average HRR was adjusted for the established prognostic clinicopathological variables in a multivariate analysis (average HRR 1.66; P=0.02). The expression of AURKA in CRCLM was correlated to its expression in corresponding primary tumour (P<0.01). CONCLUSION: The expression of AURKA protein is a molecular biomarker with prognostic value for patients with CRCLM, independent of established clinicopathological variables.

There is no increased risk for colorectal cancer and adenomas in patients with diverticulitis: a retrospective longitudinal study.

AIM: This study was designed to assess the relationship between diverticulitis and the development of colorectal cancer (CRC) and colonic adenomas. METHOD: A retrospective study was longitudinally conducted. Patients who had been admitted to the hospital between 1990 and 2000 with diverticulitis were retrieved and the incidence of CRC and prevalence of colonic adenomas in these patients was determined. Data were collected from the electronic clinical and pathology records. The incidence of CRC and prevalence of adenomas in this patient cohort was compared with the general population. The patients were followed until 2008. RESULTS: A total of 288 patients with diverticulitis were included (167 of whom were female patients [58%]). The mean age of patients at admittance for diverticulitis was 66 years (range: 27-92). CRC was detected in five patients (1.7%) (95% CI 0.8-3.5) with a mean age of 77 years; colonic adenomas were found in 18 patients (6.3%) (95% CI 4.3-9.0) with a mean age of 62 years. The lifetime risks of developing CRC and adenomas were presumed to be 4% and 20% respectively. Expected rates for CRC and adenomas in our patients were calculated as 17 (95% CI 4.0-8.6) and 69 patients (95% CI 20.1-28.3) respectively. CONCLUSION: This study showed a lower prevalence of CRC and colonic adenomas in patients with diverticulitis compared with the lifetime risk which means that diverticulitis is not a risk factor for development of CRC and adenomas. Long-term colonic screening after a negative colonoscopy for diverticulitis (generally performed several weeks after recovery) does not seem to be justified.

Gross genomic damage measured by DNA image cytometry independently predicts gastric cancer patient survival.

BACKGROUND: DNA aneuploidy reflects gross genomic changes. It can be measured by flow cytometry (FCM-DNA) or image cytometry (ICM-DNA). In gastric cancer, the prevalence of DNA aneuploidy has been reported to range from 27 to 100%, with conflicting associations with clinicopathological variables. The aim of our study was to compare the DNA ploidy status measured using FCM-DNA and ICM-DNA in gastric cancer and to evaluate its association with clinicopathological variables. METHODS: Cell nuclei were isolated from 221 formalin-fixed, paraffin-embedded gastric cancer samples. DNA ploidy was assessed using FCM-DNA and ICM-DNA. RESULTS: A total of 178 (80.5%) gastric cancer samples were classified as DNA aneuploid using FCM-DNA, compared with 172 (77.8%) gastric cancer samples when using ICM-DNA. Results obtained from both methods were concordant in 183 (82.8%) cases (kappa=0.48). Patients with ICM-DNA diploid gastric cancer survived significantly longer than those with ICM-DNA aneuploid gastric cancer (log rank 10.1, P=0.001). For FCM-DNA data, this difference did not reach statistical significance. The multivariate Cox model showed that ICM-DNA ploidy status predicted patient survival independently of tumour-node-metastasis status. CONCLUSION: ICM-DNA ploidy status is an independent predictor of survival in gastric cancer patients and may therefore be a more clinically relevant read out of gross genomic damage than FCM-DNA.

Combined assessment of EGFR pathway-related molecular markers and prognosis of NSCLC patients.

The purpose of this study is to evaluate the prognostic value of the combined assessment of multiple molecular markers related to the epidermal growth factor receptor (EGFR) pathway in resected non-small cell lung cancer (NSCLC) patients. Tumour specimens of 178 NSCLC patients were collected and analysed for EGFR and KRAS mutation status by DNA sequencing, and for EGFR copy number by fluorescent in situ hybridisation. Tissue microarrays were generated and used to determine the expression of multiple EGFR pathway-related proteins by immunohistochemistry. We analysed the association between each marker and patient prognosis. Univariate analyses for each clinical variable and each molecular marker were performed using Kaplan-Meier curves and log-rank tests. From these results, we selected the variables KRAS mutations and expression of cytoplasmic EGFR, granular pERK, nuclear pSTAT3, cytoplasmic E-cadherin and cytoplasmic pCMET to enter into a Cox proportional hazards model, along with stage as the strongest clinical variable related with prognosis. Of the EGFR-related markers evaluated here, the markers EGFR, pERK, pSTAT3, E-cadherin, pCMET and mutations in KRAS were associated with survival when analysed in combination in our patient cohort, with P=0.00015 as the P-value for a test of the additional impact of markers on prognosis, after taking stage into consideration. Confirmation of the impact of these markers in independent studies will be necessary.

Contacts between medial and lateral perforant pathway fibers and parvalbumin expressing neurons in the subiculum of the rat.

The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects ('boutons') of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones.

Drug resistance-associated marker Lrp for prediction of response to chemotherapy and prognoses in advanced ovarian carcinoma.

BACKGROUND: Drug resistance is a major impediment to the successful treatment of ovarian carcinoma. None of the earlier-identified resistance mechanisms, such as overexpression of the MDR1 gene product, P-glycoprotein (Pgp), has been shown to be a major determinant of clinical response to chemotherapy and survival for ovarian cancer patients. The multidrug resistance-associated protein (Mrp) and the lung resistance protein (Lrp, also called the p110 major vault protein), are newly described proteins associated with multidrug resistance in vitro. PURPOSE: The aim of this retrospective study was to investigate the expression of Mrp and Lrp, in addition to Pgp, in advanced ovarian carcinoma and to determine whether such expression was predictive of response to chemotherapy and survival. METHODS: Fifty-seven banked frozen specimens, previously collected and frozen at the time of diagnosis from an equal number of patients with International Federation of Gynecology and Obstetrics (FIGO) stage III or IV ovarian carcinoma, were immunostained for Pgp (with monoclonal antibodies [MAbs] MRK-16 and JSB-1), Mrp (with MAb MRPrl), and Lrp (with MAb LRP-56). All patients had received platinum- or alkylating-based chemotherapy after debulking surgery. Clinicopathologic parameters determined at diagnosis were retrospectively assessed for their relationship with Pgp, Mrp, and Lrp expression. Response to treatment and survival were compared between Pgp, Mrp, and Lrp expression groups. Qualitative variables were analyzed using Fisher's exact test or the chi-squared test. All reported P values are two-tailed. RESULTS: Nine (16%), 39 (68%), and 44 (77%) of the 57 tumor specimens examined showed positive immunostaining for Pgp, Mrp, and Lrp, respectively. Positive immunostaining for these proteins was not associated with any other prognostic factor examined. No association was found between Pgp and Mrp expression and response to chemotherapy and survival. In contrast, patients with Lrp-positive tumors had poorer response to chemotherapy (P = .004) and shorter progression-free (P = .003) and overall (P = .007) survival than Lrp-negative patients. Multivariate analysis of Lrp expression, FIGO stage, residual tumor after initial surgery, tumor grade, and presence or absence of ascites showed that only Lrp status was independently related to both progression-free survival and overall survival. CONCLUSIONS: Positive Lrp immunostaining in advanced ovarian carcinoma appears to be an indicator of poor response to standard chemotherapy (platinum or alkylating agents) and of adverse prognoses. IMPLICATIONS: The functional characterization of Lrp and related proteins may reveal new approaches to modulate Lrp-associated drug resistance. A large prospective study is warranted to confirm the prognostic value of Lrp.

Versican and vascular endothelial growth factor expression levels in peritoneal metastases from colorectal cancer are associated with survival after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) can increase survival of colorectal cancer (CRC) patients with peritoneal metastases (PM). This treatment is associated with high morbidity and mortality rates. Therefore, improvement of patient selection is necessary. Assuming that the clinical phenotype is dictated by biological mechanisms, biomarkers could play a crucial role in this process. Since it is unknown whether and to what extent angiogenesis influences the course of disease in patients with PM, we investigated the expression of two angiogenesis-related markers and their relation to overall survival (OS) in CRC patients after CRS and HIPEC. Clinicopathological data and tissue samples were collected from 65 CRC patients with isolated metastases to the peritoneum that underwent CRS and HIPEC. Whole tissue specimens from PM were evaluated for versican (VCAN) expression, VEGF expression and microvessel density (MVD) by immunohistochemistry. The relation between these markers and OS was assessed using univariate and multivariate analysis. Associations between VEGF expression, VCAN expression, MVD and clinicopathological data were tested. High stromal VCAN expression was associated with high MVD (p = 0.001), better resection outcome (p = 0.003) and high T-stage (p = 0.027). High epithelial VCAN expression was associated with MVD (p = 0.007) and a more complete resection (p < 0.001). In multivariate analysis, simplified peritoneal cancer index (p = 0.001), VEGF expression levels (p = 0.012), age (p = 0.030), epithelial VCAN expression levels (p = 0.042) and lymph node status (p = 0.053) were associated with OS. Concluding, VCAN and VEGF were associated with survival in CRC patients with PM after CRS and HIPEC. Independent validation in a well-defined patient cohort is required to confirm the putative prognostic role of these candidate biomarkers.

Fully automated microvessel counting and hot spot selection by image processing of whole tumour sections in invasive breast cancer.

BACKGROUND: Manual counting of microvessels is subjective and may lead to unacceptable interobserver variability, which may explain conflicting results. AIMS: To develop and test an automated method for microvessel counting and objective selection of the hot spot, based on image processing of whole sections, and to compare this with manual selection of a hot spot and counting of microvessels. METHODS: Microvessels were stained by CD31 immunohistochemistry in 10 cases of invasive breast cancer. The number of microvessels was counted manually in a subjectively selected hot spot, and also in the same complete tumour sections by interactive and automated image processing methods. An algorithm identified the hot spots from microvessel maps of the whole tumour section. RESULTS: No significant difference in manual microvessel counts was found between two observers within the same hot spot, and counts were significantly correlated. However, when the hot spot was reselected, significantly different results were found between repeated counts by the same observer. Counting all microvessels manually within the entire tumour section resulted in significantly different hot spots than manual counts in selected hot spots by the same observer. Within the entire tumour section no significant differences were found between the hot spots of the manual and automated methods using an automated microscope. The hot spot was found using an eight connective path search algorithm, was located at or near the border of the tumour, and (depending on the size of the hot spot) did not always contain the field with the largest number of microvessels. CONCLUSIONS: The automated counting of microvessels is preferable to the manual method because of the reduction in measurement time when the complete tumour is scanned, the greater accuracy and objectivity of hot spot selection, and the possibility of visual inspection and relocation of each measurement field afterwards.

Progression from colorectal adenoma to carcinoma is associated with non-random chromosomal gains as detected by comparative genomic hybridisation.

AIMS: Chromosomal gains and losses were surveyed by comparative genomic hybridisation (CGH) in a series of colorectal adenomas and carcinomas, in search of high risk genomic changes involved in colorectal carcinogenesis. METHODS: Nine colorectal adenomas and 14 carcinomas were analysed by CGH, and DNA ploidy was assessed with both flow and image cytometry. RESULTS: In the nine adenomas analysed, an average of 6.6 (range 1 to 11) chromosomal aberrations were identified. In the 14 carcinomas an average of 11.9 (range 5 to 17) events were found per tumour. In the adenomas the number of gains and losses was in balance (3.6 v 3.0) while in carcinomas gains occurred more often than losses (8.2 v 3.7). Frequent gains involved 13q, 7p, 8q, and 20q, whereas losses most often occurred at 18q, 4q, and 8p. Gains of 13q, 8q, and 20q, and loss of 18q occurred more often in carcinomas than in adenomas (p = 0.005, p = 0.05, p = 0.05, and p = 0.02, respectively). Aneuploid tumours showed more gains than losses (mean 9.3 v 4.9, p = 0.02), in contrast to diploid tumours where gains and losses were nearly balanced (mean 3.1 v 4.1, p = 0.5). CONCLUSIONS: The most striking difference between chromosomal aberrations in colorectal adenomas and carcinomas, as detected by CGH, is an increased number of chromosomal gains that show a nonrandom distribution. Gains of 13q and also of 20q and 8q seem especially to be involved in the progression of adenomas to carcinomas, possibly owing to low level overexpression of oncogenes at these loci.

An expert system for histological typing and grading of invasive breast cancer. First set up.

This article describes the set up of a rule-based expert system for histologic typing and grading of invasive breast cancer, which is designed to be a user-friendly tool that may be helpful for teaching and to support diagnosis making. The system raises questions and offers fixed choices to the user (usually yes/no) until a histologic diagnosis can be made with reasonable probability or enough data are available to assign a grade. As to histologic typing, the expert system is able to make the following diagnoses: ductal carcinoma, lobular carcinoma, medullary carcinoma, colloidal carcinoma, tubular carcinoma, and invasive cribriform carcinoma. If the diagnosis "ductal carcinoma" is arrived, the system offers the option to assign a histologic grade to the lesion. A first evaluation of the system in 30 cases (five each of the different subtypes) with unequivocal diagnoses by two human experts showed that the system classified 29 of the tumours in the same way as the human experts. The discrepancy case was solved after adding one rule to the system. Ten cases where a discrepancy existed between the original diagnosis of a referring centre and a reviewing human expert were all classified by the expert system in the same way as the human expert. The expert system thus seems to perform well. Further plans for evaluating, modifying and expanding the system are disclosed.

DNA ploidy measurement in oral leukoplakia: different results between flow and image cytometry.

The estimated prevalence of oral leukoplakia is worldwide approximately 2%, with an annual malignant transformation rate of approximately 1%. The aim of the present study was to evaluate the possible contribution of ploidy measurement to the prediction of the clinical course, in a well defined cohort of patients with oral leukoplakia. Ploidy was measured by both flow cytometry (FCM-DNA) and image cytometry (ICM-DNA) and we focussed on the comparison of the two different techniques to determine ploidy. A total of 41 patients have been included, with a mean age of 59 years (range 36-78 years). With FCM-DNA, three lesions were aneuploid, with ICM-DNA, 19 lesions were aneuploid. DNA ploidy was compared with clinicopathological and patients parameters. There were no statistically significant differences between DNA ploidy and any patient factor with both FCM-DNA and ICM-DNA. Using FCM-DNA, DNA aneuploid lesions showed statistically significant more dysplasia (p=0.04) than diploid lesions. Furthermore, DNA aneuploid lesions were more frequently encountered at high-risk locations (p=0.03) as being determined with FCM-DNA. These relations were not found when DNA ploidy was determined with ICM-DNA.

Loss of lamin A/C expression in stage II and III colon cancer is associated with disease recurrence.

AIM OF THE STUDY: Loss of the nuclear lamina protein lamin A/C (LMNA) has been observed in several human malignancies. The present study aimed to investigate associations between LMNA expression and clinical outcome in colon cancer patients. PATIENTS AND METHODS: Clinicopathological data and formalin-fixed paraffin embedded tissues were collected from 370 stage II and III colon cancer patients. Tissue microarrays were constructed, stained for lamin A/C and evaluated microscopically. Microsatellite instability status was determined for 318 tumours. RESULTS: Low levels of LMNA expression were observed in 17.8% of colon tumours, with disease recurrence occurring in 45.5% of stage II and III colon cancer patients with LMNA-low expressing tumours compared to 29.6% of patients with LMNA-high expressing tumours (p=0.01). For stage II patients, disease recurrence was observed for 35.7% of LMNA-low compared to 20.3% of LMNA-high expressing tumours (p=0.03). Microsatellite stable (MSS) tumours exhibited more frequently low LMNA expression than microsatellite instable (MSI) tumours (21% versus 9.8%; p=0.05). Interestingly, disease recurrence among LMNA-low and LMNA-high expressing MSS tumours varied significantly for stage III patients who had not received adjuvant chemotherapy (100% versus 37.8%; p<0.01) while no such difference was observed for patients who received adjuvant chemotherapy (46.7% versus 46.0%; p=0.96). CONCLUSION: These data indicate that low expression of LMNA is associated with an increased disease recurrence in stage II and III colon cancer patients, and suggest that these patients in particular may benefit from adjuvant chemotherapy.

Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1).

The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.

Counting mitoses by image processing in Feulgen stained breast cancer sections: the influence of resolution.

Counting of mitotic cells has been shown to be of prognostic value in breast cancer in different retrospective studies. Up to now the number of mitoses is assessed mainly manually according to a standardized but strict protocol. Although such a manual procedure is reasonably reproducible, automatic counting of mitotic cells offers the potential for greater objectivity and reproducibility. This paper describes the influence of resolution on automatic recognition by image processing of mitotic cells in Feulgen stained breast cancer sections. Using the image recording, correction and segmentation procedure described in a previous study, five specimens were analyzed: one was used to serve as a training set and four were put aside for later use as independent test set. For each slide, objects from a pre-selected area were recorded at increasing resolution. For each object, contour features and optical density measurements were computed and stored in a data file for statistical analysis. The results showed that increased resolution using a 40x objective lowered the number of misclassified mitoses compared with a 20x objective (overall mean percentage of misclassified mitoses over training and all test specimens: 20x, 24.57; 40x, 7.96). The number of misclassifications of non-mitoses was almost stable per specimen but varied between specimens (19-42%) due to differences among tissues. Given the improvement in classifying mitoses and the possibility to evaluate interactively the measurement result, the described semi-automated mitoses pre-screener of histological sections may be suitable for further testing in a clinical setting.

Measurement by confocal laser scanning microscopy of the volume of epidermal nuclei in thick skin sections.

The mean volume and shape of nuclei assessed in standard tissue sections by means of stereologic and morphometric methods are associated with prognosis in tumors of different sites. Thus, accurate quantification of the volume and shape of cell nuclei can be important for cancer patients and also may be useful for a better understanding of basic cellular events, such as growth and differentiation. Confocal laser scan microscopy (CLSM) makes it possible to obtain image sets consisting of very thin serial optical slices from thick tissue sections. These images can be used with digital image processing to construct a three-dimensional (3-D) model of individual nuclei. We used CLSM to study a 50-micron-thick paraffin section of a skin biopsy. Image processing was applied to the CLSM images to precisely segment epidermal nuclei from the background, and serial two-dimensional (2-D) binary images were created. Alignment of the 2-D images that form the 3-D model of the original nuclei was carefully controlled. The series of 2-D binary images was connected with an algorithm to form the 3-D model of each complete nucleus and organized for the 3-D visualization and analysis using a 3-D volume rendering method. In this way we measured the volume and surface area of 22 intact epidermal nuclei. The mean nuclear volume was 174.7 micron3, the standard deviation of the volume 26.47 micron3, the mean surface area 168.0 micron2 and the standard deviation of the surface area 22.00 micron2.(ABSTRACT TRUNCATED AT 250 WORDS)