Image analysis of immunohistochemistry is superior to visual scoring as shown for patient outcome of esophageal adenocarcinoma.

Quantification of protein expression based on immunohistochemistry (IHC) is an important step in clinical diagnoses and translational tissue-based research. Manual scoring systems are used in order to evaluate protein expression based on staining intensities and distribution patterns. However, visual scoring remains an inherently subjective approach. The aim of our study was to explore whether digital image analysis proves to be an alternative or even superior tool to quantify expression of membrane-bound proteins. We analyzed five membrane-binding biomarkers (HER2, EGFR, pEGFR, ß-catenin, and E-cadherin) and performed IHC on tumor tissue microarrays from 153 esophageal adenocarcinomas patients from a single center study. The tissue cores were scored visually applying an established routine scoring system as well as by using digital image analysis obtaining a continuous spectrum of average staining intensity. Subsequently, we compared both assessments by survival analysis as an end point. There were no significant correlations with patient survival using visual scoring of ß-catenin, E-cadherin, pEGFR, or HER2. In contrast, the results for digital image analysis approach indicated that there were significant associations with disease-free survival for ß-catenin, E-cadherin, pEGFR, and HER2 (P = 0.0125, P = 0.0014, P = 0.0299, and P = 0.0096, respectively). For EGFR, there was a greater association with patient survival when digital image analysis was used compared to when visual scoring was (visual: P = 0.0045, image analysis: P < 0.0001). The results of this study indicated that digital image analysis was superior to visual scoring. Digital image analysis is more sensitive and, therefore, better able to detect biological differences within the tissues with greater accuracy. This increased sensitivity improves the quality of quantification.

Epidermal growth factor receptor (EGFR) is an independent adverse prognostic factor in esophageal adenocarcinoma patients treated with cisplatin-based neoadjuvant chemotherapy.

Neoadjuvant platin-based therapy is accepted as a standard therapy for advanced esophageal adenocarcinoma (EAC). Patients who respond have a better survival prognosis, but still a significant number of responder patients die from tumor recurrence. Molecular markers for prognosis in neoadjuvantly treated EAC patients have not been identified yet. We investigated the epidermal growth factor receptor (EGFR) in prognosis and chemotherapy resistance in these patients. Two EAC patient cohorts, either treated by neoadjuvant cisplatin-based chemotherapy followed by surgery (n=86) or by surgical resection (n=46) were analyzed for EGFR protein expression and gene copy number. Data were correlated with clinical and histopathological response, disease-free and overall survival. In case of EGFR overexpression, the prognosis for neoadjuvant chemotherapy responders was poor as in non-responders. Responders had a significantly better disease-free survival than non-responders only if EGFR expression level (p=0.0152) or copy number (p=0.0050) was low. Comparing neoadjuvantly treated patients and primary resection patients, tumors of non-responder patients more frequently exhibited EGFR overexpression, providing evidence that EGFR is a factor for indicating chemotherapy resistance. EGFR overexpression and gene copy number are independent adverse prognostic factors for neoadjuvant chemotherapy-treated EAC patients, particularly for responders. Furthermore, EGFR overexpression is involved in resistance to cisplatin-based neoadjuvant chemotherapy.

MALDI imaging mass spectrometry reveals COX7A2, TAGLN2 and S100-A10 as novel prognostic markers in Barrett's adenocarcinoma.

To characterize proteomic changes found in Barrett's adenocarcinoma and its premalignant stages, the proteomic profiles of histologically defined precursor and invasive carcinoma lesions were analyzed by MALDI imaging MS. For a primary proteomic screening, a discovery cohort of 38 fresh frozen Barrett's adenocarcinoma patient tissue samples was used. The goal was to find proteins that might be used as markers for monitoring cancer development as well as for predicting regional lymph node metastasis and disease outcome. Using mass spectrometry for protein identification and validating the results by immunohistochemistry on an independent validation set, we could identify two of 60 differentially expressed m/z species between Barrett's adenocarcinoma and the precursor lesion: COX7A2 and S100-A10. Furthermore, among 22 m/z species that are differentially expressed in Barrett's adenocarcinoma cases with and without regional lymph node metastasis, one was identified as TAGLN2. In the validation set, we found a correlation of the expression levels of COX7A2 and TAGLN2 with a poor prognosis while S100-A10 was confirmed by multivariate analysis as a novel independent prognostic factor in Barrett's adenocarcinoma. Our results underscore the high potential of MALDI imaging for revealing new biologically significant molecular details from cancer tissues which might have potential for clinical application. This article is part of a Special Issue entitled: Translational Proteomics.

MALDI imaging identifies prognostic seven-protein signature of novel tissue markers in intestinal-type gastric cancer.

Proteomics-based approaches allow us to investigate the biology of cancer beyond genomic initiatives. We used histology-based matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that predict disease outcome in gastric cancer after surgical resection. A total of 181 intestinal-type primary resected gastric cancer tissues from two independent patient cohorts were analyzed. Protein profiles of the discovery cohort (n = 63) were directly obtained from tumor tissue sections by MALDI imaging. A seven-protein signature was associated with an unfavorable overall survival independent of major clinical covariates. The prognostic significance of three individual proteins identified (CRIP1, HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of an independent validation cohort (n = 118). Whereas HNP-1 and S100-A6 were found to further subdivide early-stage (Union Internationale Contre le Cancer [UICC]-I) and late-stage (UICC II and III) cancer patients into different prognostic groups, CRIP1, a protein previously unknown in gastric cancer, was confirmed as a novel and independent prognostic factor for all patients in the validation cohort. The protein pattern described here serves as a new independent indicator of patient survival complementing the previously known clinical parameters in terms of prognostic relevance. These results show that this tissue-based proteomic approach may provide clinically relevant information that might be beneficial in improving risk stratification for gastric cancer patients.

High number of CD45RO+ tumor infiltrating lymphocytes is an independent prognostic factor in non-metastasized (stage I-IIA) esophageal adenocarcinoma.

BACKGROUND: The validation of novel prognostic indicators is of greatest interest for the management of esophageal adenocarcinoma (Barrett's cancer), particularly for non-metastasized (stage I-IIA) disease. The prognostic role of tumor infiltrating T-lymphocytes (TILs) in Barrett's cancer has not been reported so far. Here we evaluated the impact of TILs on survival, recurrence, and metastasis in Barrett's cancer, particularly in stage I-IIA patients. METHODS: The levels of the adaptive immune markers CD3, CD8, and CD45RO were analyzed by immunohistochemistry and image analysis in tissue microarrays consisting of tumor tissues of 118 patients with primary resected Barrett's cancer. The findings were correlated with clinicopathological parameters including patient outcome. RESULTS: In multivariate analysis, a low density of intratumoral CD45RO+ immune cells was an independent unfavorable factor for disease-free survival in stages I-IIA patients (P = 0.004, RR = 4.7, 95% CI = 1.6-13.5) as well in the entire cohort (P = 0.048, RR = 2.0, 95% CI = 1.0-4.0). High CD3+ and CD45RO+ levels were associated with prolonged disease-free survival and overall survival as well with low recurrence rates of disease (P = 0.005 and P = 0.018, respectively). In addition, low CD3+ levels were correlated with a higher frequency of lymph node metastasis (P = 0.025). CONCLUSIONS: This study demonstrates that the density of CD45RO+ TILs is an independent prognostic factor in non-metastasized (stage I-IIA) Barrett's cancer patients and indicates an important role for the adaptive immunologic microenvironment. The inclusion of CD45RO+ density may help to improve the management of stage I-IIA Barrett's cancer.

Centrosome abnormalities in head and neck squamous cell carcinoma (HNSCC).

CONCLUSIONS: Numerical and structural centrosome abnormalities play a critical role in the tumor progression of in head and neck squamous cell carcinoma (HNSCC) and may provide useful information as a prognostic factor for these patients. OBJECTIVES: Centrosome alterations are often linked with aneuploidy, cell transformation, and tumor progress. We investigated centrosome abnormalities in HNSCC and correlated these variables to clinicopathological parameters and clinical follow up data of the patients. METHODS: Retrospective analysis of numerical and structural alterations of centrosomes in tumor tissues and corresponding normal epithelium (n=50 and 31, respectively). Immunohistochemistry was performed using an anti-gamma-tubulin antibody. Image acquisition was done by an Orthoplan microscope, centrosomes were segmented interactively, and area as well as mean optical density was measured. Aneuploidy was evaluated by fluorescence in situ hybridization in a subset of cases (n=29). RESULTS: Numerical and structural centrosome abnormalities differed significantly between normal squamous epithelium and tumor cells (both P<0.0001). Especially numerical centrosome abnormalities were significantly associated with T category and tumor stage (both P<0.0001) and the occurrence of distant metastasis (P=0.002 and P=0.019, respectively). Numerical centrosome abnormalities correlated also with disease free survival of the patients (P=0.032) as well as shorter overall survival (P=0.003).

Primary extramedullary plasmacytoma: similarities with and differences from multiple myeloma revealed by interphase cytogenetics.

Primary extramedullary plasmacytoma is an indolent neoplasm that infrequently converts to multiple myeloma. Since cytogenetic data on extramedullary plasmacytoma are lacking, we studied 38 cases of this type of neoplasm by fluorescence in situ hybridization. Fourteen cases (37%) contained IGH breaks, including six with a t(4;14) translocation. No translocations t(11;14), t(14;16), t(8;14), nor breaks involving MALT1, BCL6 or FOXP1 were found. Loss of 13q (40%), as well as chromosomal gains (82%) were common. There was no correlation between chromosomal alterations and clinical features or local relapse. Cytogenetically, extramedullary plasmacytoma and multiple myeloma are closely related. However, the distribution of IGH translocation partners, with the notable absence of t(11;14), is different. Key words: extramedullary plasmacytoma, multiple myeloma, cytogenetics, IGH translocation, fluorescence in situ hybridization.

Electrophysiologic assessment of sciatic nerve regeneration in the rat: surrounding limb muscles feature strongly in recordings from the gastrocnemius muscle.

Striking inconsistencies between the results of morphometric and electrophysiologic examinations of the regenerating nerve were observed in a previous study featuring the bridging of a 14 mm gap in the rat sciatic nerve. To shed light on this dichotomy, seven further rats were subjected to permanent sciatic nerve transection and assessed electrophysiologically, histologically and by retrograde axonal tracing at various postoperative intervals (1 h to 8 weeks). The results of the histological examinations and retrograde tracing revealed that in spite of the fact that compound muscle action potentials could be recorded in the gastrocnemius muscle, no reinnervation of the gastrocnemius muscle, either physiological or aberrant, had actually taken place. Furthermore, it was established that the electrical activity recorded in the gastrocnemius muscle after stimulation of the proximal or distal stump is generated by surrounding hind limb muscles unaffected by denervation. These are stimulated either directly, or indirectly due to spreading of the impulse. It is therefore strongly recommended that caution should be exercised when interpreting recordings from the gastrocnemius muscle after stimulation of a regenerating sciatic nerve in laboratory rodents.

Predictive value of Aurora-A/STK15 expression for late stage epithelial ovarian cancer patients treated by adjuvant chemotherapy.

PURPOSE: To investigate the expression and regulation of the centrosomal kinase Aurora-A/STK15 (AURKA) in epithelial ovarian cancers and to determine the prognostic and predictive value of this marker for patients with late stage epithelial ovarian cancer treated by distinct adjuvant chemotherapies. EXPERIMENTAL DESIGN: Archival resection specimens of epithelial ovarian cancers (n=115) and nonneoplastic ovaries (n=28) were analyzed for AURKA mRNA and protein expression by microdissection and quantitative reverse transcriptase-PCR and immunohistochemistry. AURKA DNA copy numbers were measured by fluorescence in situ hybridization in 37 cases. Statistical evaluation was done with respect to clinicopathologic variables, disease-free survival, and overall survival. RESULTS: AURKA mRNA expression was significantly elevated in cancers (P<0.001) and correlated with AURKA protein expression (P=0.0134). Overexpression of AURKA protein was detected in 68 of 107 (63.5%) cases and was linked with increased AURKA DNA copy numbers (P=0.0141) and centromere 20 aneusomy (P=0.0137). Moreover, AURKA overexpression was associated with improved overall survival in optimal debulked patients receiving taxol/carboplatin therapy (n=43, P=0.018). Finally, in an exploratory approach, patients receiving non-taxane-based therapy, AURKA overexpression was predictive for worse overall survival (n=30, P=0.049). CONCLUSIONS: AURKA overexpression is seen in the majority of late stage epithelial ovarian cancers, most likely due to increased AURKA DNA copy numbers and/or chromosome 20 aneusomy. Importantly, AURKA overexpression may differentially affect taxane and non-taxane-based adjuvant therapy responses. The study sheds new light on AURKA expression and regulation in epithelial cancers in vivo and specifically shows its value as a clinically relevant marker and as a potential therapeutic target per se.

Temporal progression and extent of the return of sensation in the foot provided by the saphenous nerve after sciatic nerve transection and repair in the rat - implications for nociceptive assessments.

Sensory testing, by providing stimuli for nociceptors of the foot, is a popular method of evaluating sensory regeneration after damage to the sciatic nerve in the rat. In the following study, 20 rats were submitted to double transection of the sciatic nerve. The subsequent 14 mm gap was repaired through guidance interponation. In order to evaluate nerve regeneration, sensory testing was performed additionally to other methods, which included motor testing, morphometry, and electron microscopic assessments of nerves. Somatosensory testing revealed that all animals exhibited next to the same amount of sensory reinnervation on their foot regardless of their experimental group. In motor tests, however, two out of the three experimental groups did not improve at all. These groups also failed to show neural regrowth in morphometric and electron microscopic assessments of the associated nerve. Retrograde tracing was able to prove the saphenous nerve as an alternative source of sensory reinnervation in animals with failed sciatic regeneration. This means that results of sensory testing in the rat should be treated with caution, taking into account the areas tested and the likelihood that in these areas saphenous sprouting could have taken place. Furthermore, it is strongly advised that somatosensory testing should be conducted only on toe 5.

STAT3 mRNA and protein expression in colorectal cancer: effects on STAT3-inducible targets linked to cell survival and proliferation.

AIMS: To evaluate mRNA and protein expression of signal transducers and activators of transcription (STAT)3 in colorectal carcinomas (CRCs) and to define the association of STAT3 activity with the STAT3-inducible targets cyclin D1, survivin, Bcl-xl and Mcl-1. MATERIALS AND METHODS: Matching serial sections of normal colonic epithelium and invasive CRCs (n = 32) were subjected to quantitative reverse transcriptase polymerase chain reaction specific to STAT3, cyclin D1, survivin, Bcl-xl and Mcl-1, as well as immunohistochemistry. For STAT3 immunohistochemistry, two antibodies, recognising unphosphorylated (UP-) and phosphorylated (tyr705, P-) STAT3 were used. Ki-67 (MIB-1) staining was included as a proliferation marker. RESULTS: Compared with normal colonic epithelium, UP-STAT3 and P-STAT3 (p = 0.023 and 0.006) protein expression and expression of its associated targets cyclin D1, survivin and Bcl-xl were significantly (all p<0.001) increased in carcinoma. In carcinomas, STAT3 (p = 0.019) and Bcl-xl (p = 0.001) mRNAs were correlated with lymph node status. Moreover, nuclear P-STAT3 protein expression (active state) was associated with the expression of its target genes Bcl-xl (p = 0.038) and survivin (p = 0.01) as well as with Ki-67 (p = 0.017). By contrast, cytoplasmic UP-STAT was significantly linked to Bcl-xl mRNA (p = 0.024) and protein (p = 0.001) as well as to cytoplasmic survivin protein expression (p = 0.019). CONCLUSION: Both inactive (UP-STAT3) and active (P-STAT3) STAT3 proteins are markedly increased in invasive CRCs. This is associated with Bcl-xl and survivin induction, increased proliferation and lymph node metastasis. This study therefore provides the basis for further examination of the prognostic or predictive value of these molecular markers in CRC.

Aurora kinase A messenger RNA overexpression is correlated with tumor progression and shortened survival in head and neck squamous cell carcinoma.

PURPOSE: Aurora kinase A (AURKA/STK15/BTAK) encodes a serine/threonine kinase associated with chromosomal distribution and its up-regulation induces chromosomal instability, thereby leading to aneuploidy and cell transformation in several types of cancer. In this study, we investigated the role of AURKA in head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: The mRNA expression levels of AURKA were compared in tumor tissues of 66 HNSCC patients with those in corresponding normal squamous epithelium by real-time quantitative reverse transcriptase-PCR. In addition, the association between AURKA mRNA and protein expression, centrosome abnormalities, and aneuploidy was studied in a subset of cases (n=34). All molecular variables were correlated to histomorphologic findings and clinical follow-up data of the patients. RESULTS: AURKA mRNA up-regulation was significantly associated with tumor stage and the occurrence of regional lymph node, as well as distant metastasis (P<0.0001 for all). Similarly, a correlation was found for protein expression and the occurrence of regional lymph node (P=0.0183) and distant metastasis (P=0.03). The mRNA was positively associated with protein expression (P=0.003) and centrosome abnormalities (P=0.03). Cox regression analysis revealed that AURKA mRNA up-regulation correlated with disease-free survival of the patients (P=0.03) as well as shorter overall survival (P<0.001). CONCLUSIONS: We conclude that the up-regulation of AURKA mRNA may play a critical role in the tumor progression of HNSCC and provides useful information as a prognostic factor for HNSCC patients.

Automatic analysis of aqueous specimens for phytoplankton structure recognition and population estimation.

An automatic microscope image acquisition, evaluation, and recognition system was developed for the analysis of Utermöhl plankton chambers in terms of taxonomic algae recognition. The system called PLASA (Plankton Structure Analysis) comprises (1) fully automatic archiving (optical fixation) of aqueous specimens as digital bright field and fluorescence images, (2) phytoplankton analysis and recognition, and (3) training facilities for new taxa. It enables characterization of aqueous specimens by their populations. The system is described in detail with emphasis on image analytical aspects. Plankton chambers are scanned by sizable grids, divers objective(s), and up to four fluorescence spectral bands. Acquisition positions are focused and digitized by a TV camera and archived on disk. The image data sets are evaluated by a large set of quantitative features. Automatic classifications for a number of organisms are developed and embedded in the program. Interactive programs for the design of training sets were additionally implemented. A long-term sampling period of 23 weeks from two ponds at two different locations each was performed to generate a reliable data set for training and testing purposes. These data were used to present this system's results for phytoplankton structure characterization. PLASA represents an automatic system, comprising all steps from specimen processing to algae identification up to species level and quantification.

Reduction of anabolic signals and alteration of osteoblast nuclear morphology in microgravity.

Bone loss has been repeatedly documented in astronauts after flight, yet little is known about the mechanism of bone loss in space flight. Osteoblasts were activated during space flight in microgravity (microg) with and without a 1 gravity (1 g) field and 24 genes were analyzed for early induction. Induction of proliferating cell nuclear antigen (PCNA), transforming growth factor beta (TGFbeta), cyclo-oxygenase-2 (cox-2), cpla2, osteocalcin (OC), c-myc, fibroblast growth factor-2 (fgf-2), bcl2, bax, and fgf-2 message as well as FGF-2 protein were significantly depressed in microg when compared to ground (gr). Artificial onboard gravity normalized the induction of c-myc, cox-2, TGFbeta, bax, bcl2, and fgf-2 message as well as FGF-2 protein synthesis in spaceflight samples. In normal gravity, FGF-2 induces bcl2 expression; we found that bcl2 expression was significantly reduced in microgravity conditions. Since nuclear shape is known to elongate in the absence of mitogens like FGF-2, we used high-resolution image-based morphometry to characterize changes in osteoblast nuclear architecture under microgravity, 1 g flight, and ground conditions. Besides changes in cell shape (roundish/elliptic), other high-resolution analyses show clear influences of gravity on the inner nuclear structure. These changes occur in the texture, arrangement, and contrast of nuclear particles and mathematical modeling defines the single cell classification of the osteoblasts. Changes in nuclear structure were evident as early as 24 h after exposure to microgravity. This documented alteration in nuclear architecture may be a direct result of decreased expression of autocrine and cell cycle genes, suggesting an inhibition of anabolic response in microg. Life on this planet has evolved in a normal gravity field and these data suggest that gravity plays a significant role in regulation of osteoblast transcription.

Vascular endothelial growth factor expression and neovascularization in Barrett's carcinoma.

Angiogenesis is essential for tumor growth and metastasis. An association between microvessel density, a measure of tumor angiogenesis, and conventional prognostic variables has been shown for many tumor entities. For Barrett's carcinoma, the results are controversial. Immature vessels formed in tumors are structurally and functionally different from those in mature vessels. The relation between mature and immature vessels as a prognostic factor for Barrett's carcinoma has not been assessed. Specimens from 45 R0-resected Barrett's carcinomas were immunostained for vascular endothelial growth factor (VEGF), CD 31, and smooth muscle alpha-actin to discriminate between mature and immature vessels. VEGF staining was evaluated quantitatively by measuring optical density with a new computer-based program and expressed as a percentage of the staining (juvenile placental tissue) on control slides. The neovascularization coefficient (i.e., the relation between mature and immature vessels) was estimated with an interactive analytic computer program. The median survival of the study group was 45.7 months. The neovascularization coefficient correlated with the histopathologic classification ( p < 0.001). Survival time in patients with a low neovascularization coefficient was significantly better than the survival time in patients with a high neovascularization coefficient ( p = 0.021). VEGF expression did not correlate with clinicopathologic data ( p > 0.05) or with patient survival ( p > 0.05). The tumors with a high neovascularization coefficient did not have significantly elevated VEGF expression. Based on a strong quantitative computer evaluation program, the present study indicates that neovascularization has an important impact on the survival of patients with Barrett's carcinoma. However, VEGF does not appear to be the vascular growth factor stimulating neovascularization in Barrett's carcinoma patients.

Effects of 17alpha-ethinylestradiol on the reproduction of the cladoceran species Ceriodaphnia reticulata and Sida crystallina.

Single-species tests allow the assessment of chronical effects of endocrine disruptors on organisms under laboratory conditions. In the current study, three-generation tests with Ceriodaphnia reticulata and Sida crystallina were carried out to examine the influence of the synthetic hormone 17alpha-ethinylestradiol (EE) on the reproduction of these cladoceran species. For each species, six different concentrations (10-500 microg/l EE) and two controls were tested with eight replicates for a duration of 4 weeks. The test was initiated by transferring one neonate individual into a test vessel which was incubated under standardized conditions. Every 2 days, the medium was renewed and life history parameters such as survivorship of the adults and juveniles, clutch size, first appearance and number of produced offspring were investigated. Acute toxicity tests showed that C. reticulata (EC50 (24 h) 1814 microg/l) was more sensitive towards the substance compared to S. crystallina (EC50 (24 h) >4100 microg/l). The juvenile phase of S. crystallina was significantly shorter at concentrations above 100 microg/l EE. For C. reticulata, 17alpha-ethinylestradiol caused a higher mortality of the newly hatched juveniles at EE concentrations above 200 microg/l. No effects were found for mortality of adult animals, birth rate, number of juveniles per female and net reproduction rate of S. crystallina and C. reticulata. Thus, sublethal effects on parental generation exposed to EE lead to disturbances in reproduction and to affection of their offspring. Negative consequences for the population dynamic cannot be excluded, e.g. the decrease of a population.