Comprehensive mutation analysis in colorectal flat adenomas

Background

Flat adenomas are a subgroup of colorectal adenomas that have been associated with a distinct biology and a more aggressive clinical behavior compared to their polypoid counterparts. In the present study, we aimed to compare the mutation spectrum of 14 cancer genes, between these two phenotypes.

Methods

A consecutive series of 106 flat and 93 polypoid adenomas was analyzed retrospectively for frequently occurring mutations in “hot spot” regions of KRAS, BRAF, PIK3CA and NRAS, as well as selected mutations in CTNNB1 (β-catenin), EGFR, FBXW7 (CDC4), PTEN, STK11, MAP2K4, SMAD4, PIK3R1 and PDGFRA using a high-throughput genotyping technique. Additionally, APC was analyzed using direct sequencing.

Results

APC mutations were more frequent in polypoid adenomas compared to flat adenomas (48.5% versus 30.3%, respectively, p = 0.02). Mutations in KRAS, BRAF, NRAS, FBXW7 and CTNNB1 showed similar frequencies in both phenotypes. Between the different subtypes of flat adenomas (0-IIa, LST-F and LST-G) no differences were observed for any of the investigated genes.

Conclusion

The lower APC mutation rate in flat adenomas compared to polypoid adenomas suggests that disruption of the Wnt-pathway may occur via different mechanisms in these two phenotypes. Furthermore, in contrast to previous observations our results in this large well-defined sample set indicate that there is no significant association between the different morphological phenotypes and mutations in key genes of the RAS-RAF-MAPK pathway.     

Cytology of colorectal adenomas

A study of the cytological appearances of benign and malignant colorectal adenomatous polyps is reported. The aim of the study was to characterize the cytological features of adenomatous polyps and predict the likelihood of malignancy using cytology. A five grade classification of colorectal cytology has been developed and the characteristic appearances of cells from adenomatous polyps are described. The reproducibility of cytological diagnosis based on this classification has been tested in 120 smears from normal mucosa and adenomatous polyps (including polyp cancers). Correlation with histology was achieved in 88% and correlation of the cytological diagnosis between two observers was achieved in 84%. We conclude that cytology can be used reliably as an adjunct to histology in the assessment of malignancy of adenomatous polyps.     

Digital immunohistochemistry wizard: image analysis-assisted stereology tool to produce reference data set for calibration and quality control

Background

Digital image analysis (DIA) enables better reproducibility of immunohistochemistry (IHC) studies. Nevertheless, accuracy of the DIA methods needs to be ensured, demanding production of reference data sets. We have reported on methodology to calibrate DIA for Ki67 IHC in breast cancer tissue based on reference data obtained by stereology grid count. To produce the reference data more efficiently, we propose digital IHC wizard generating initial cell marks to be verified by experts.

Methods

Digital images of proliferation marker Ki67 IHC from 158 patients (one tissue microarray spot per patient) with an invasive ductal carcinoma of the breast were used. Manual data (mD) were obtained by marking Ki67-positive and negative tumour cells, using a stereological method for 2D object enumeration. DIA was used as an initial step in stereology grid count to generate the digital data (dD) marks by Aperio Genie and Nuclear algorithms. The dD were collected into XML files from the DIA markup images and overlaid on the original spots along with the stereology grid. The expert correction of the dD marks resulted in corrected data (cD). The percentages of Ki67 positive tumour cells per spot in the mD, dD, and cD sets were compared by single linear regression analysis. Efficiency of cD production was estimated based on manual editing effort.

Results

The percentage of Ki67-positive tumor cells was in very good agreement in the mD, dD, and cD sets: regression of cD from dD (R²=0.92) reflects the impact of the expert editing the dD as well as accuracy of the DIA used; regression of the cD from the mD (R²=0.94) represents the consistency of the DIA-assisted ground truth (cD) with the manual procedure. Nevertheless, the accuracy of detection of individual tumour cells was much lower: in average, 18 and 219 marks per spot were edited due to the Genie and Nuclear algorithm errors, respectively. The DIA-assisted cD production in our experiment saved approximately 2/3 of manual marking.

Conclusions

Digital IHC wizard enabled DIA-assisted stereology to produce reference data in a consistent and efficient way. It can provide quality control measure for appraising accuracy of the DIA steps.

     

Color image analysis for quantifying renal tumor angiogenesis

OBJECTIVE: To segment and quantify microvessels in renal tumor angiogenesis based on a color image analysis method and to improve the accuracy and reproducibility of quantifying microvessel density. STUDY DESIGN: The segmentation task was based on a supervised learning scheme. First, 12 color features (RGB, HSI, I1I2I3 and L*a*b*) were extracted from a training set. The feature selection procedure selected I2L*S features as the best color feature vector. Then we segmented microvessels using the discriminant function made using the minimum error rate classification rule of Bayesian decision theory. In the quantification step, after applying a connected component-labeling algorithm, microvessels with discontinuities were connected and touching microvessels separated. We tested the proposed method on 23 images. RESULTS: The results were evaluated by comparing them with manual quantification of the same images. The comparison revealed that our computerized microvessel counting correlated highly with manual counting by an expert (r = 0.95754). The association between the number of microvessels after the initial segmentation and manual quantification was also assessed using Pearson's correlation coefficient (r = 0.71187). The results indicate that our method is better than conventional computerized image analysis methods. CONCLUSION: Our method correlated highly with quantification by an expert and could become a way to improve the accuracy, feasibility and reproducibility of quantifying microvessel density. We anticipate that it will become a useful diagnostic tool for angiogenesis studies.     

Nucleolar PTEN in human colorectal adenomas

Molecular Biology Reports -     

Digital image analysis in pathology: benefits and obligation

Pathology has recently entered the era of personalized medicine. This brings new expectations for the accuracy and precision of tissue-based diagnosis, in particular, when quantification of histologic features and biomarker expression is required. While for many years traditional pathologic diagnosis has been regarded as ground truth, this concept is no longer sufficient in contemporary tissue-based biomarker research and clinical use. Another major change in pathology is brought by the advancement of virtual microscopy technology enabling digitization of microscopy slides and presenting new opportunities for digital image analysis. Computerized vision provides an immediate benefit of increased capacity (automation) and precision (reproducibility), but not necessarily the accuracy of the analysis. To achieve the benefit of accuracy, pathologists will have to assume an obligation of validation and quality assurance of the image analysis algorithms. Reference values are needed to measure and control the accuracy. Although pathologists' consensus values are commonly used to validate these tools, we argue that the ground truth can be best achieved by stereology methods, estimating the same variable as an algorithm is intended to do. Proper adoption of the new technology will require a new quantitative mentality in pathology. In order to see a complete and sharp picture of a disease, pathologists will need to learn to use both their analogue and digital eyes.     

Cathepsin D expression in colorectal adenocarcinomas and adenomas

The aim of this study was to investigate the role of cathepsin D in colorectal cancer. For this purpose cathepsin D expression was evaluated by means of immunohistochemistry in stromal and tumor cells of 31 colorectal carcinomas and 29 adenomas. Cytoplasmic cathepsin D expression of tumor cells was present in 90.3% of the carcinoma cases and various degrees of stromal cell cathepsin D expression were present in all cases. In the adenomas, the epithelial cells and stromal cells expressed cathepsin D in 68.96% and 96.55% of cases, respectively. The staining intensity was always weaker in the adenomas. When the stromal and tumor cell cathepsin D expression in the adenocarcinoma and adenoma cases were compared, a statistically significant difference was observed in the staining of stromal cells. Furthermore, stromal cathepsin D expression in the adenocarcinomas was related to tumor stage when the carcinomas were divided into low and high stage. Cathepsin D expression in stromal cells may be an important indicator of poor prognosis in colorectal adenocarcinomas.     

DNA quantification in colorectal carcinoma using flow and image analysis cytometry

Numerous studies using flow cytometry (FCM) have shown that DNA quantification and ploidy classification can provide information of prognostic significance for patients with colorectal carcinoma; recent advances in image analysis cytometry (image cytometry, ICM) provide a new, alternative technique for DNA quantification. This study investigated whether (1) patients with colorectal carcinomas that exhibit a diploid pattern of DNA distribution have improved five-year survival statistics as compared to their non-diploid counterparts and (2) ICM provides quantitative data comparable to that obtained by FCM. DNA quantification and ploidy classification of 27 cases of primary colorectal carcinoma was performed on archival paraffin-embedded tissue by both FCM and ICM; 70% (19) of the tumors were classified as nondiploid by ICM while 56% (15) were similarly classified by FCM. Diploid tumors were associated with Dukes' stage A while nondiploid tumors were associated with Dukes' stage D. The overall five-year survival rate was 75% for patients with ICM diploid tumors and 67% for patients with FCM diploid tumors. The five-year survival was only 53% for patients with nondiploid tumors identified by both techniques. This study confirmed that DNA quantification is an important prognostic indicator for patients with colorectal carcinoma. It also showed that ICM provides data comparable to that of FCM and may be more sensitive.     

Digital cell image analysis of Ewing's sarcoma.

We analyzed the lesional tissue in Ewing's sarcoma by means of a cell image processor computing 15 nuclear parameters in order to quantify the morphologic variability of the tumor cell nuclei. To this end we combined 32 cases (350-400 Feulgen-stained nuclei analyzed per case) in a data file, which was then subjected to principal component and canonical analyses. We found considerable heterogeneity within the cell nucleus population of Ewing's sarcomas. Indeed, after the arbitrary subdivision of the file into two cell classifiers (CC1 and CC2 cell types) on the basis of the first canonical function, the 32 Ewing's sarcomas showed a great deal of variability in the proportion of CC1 and CC2 cell nucleus types. The nuclei of the CC1 type had a more finely textured chromatin when compared to the CC2 type, the nuclei of which exhibited a more granular chromatin pattern. Additionally, these 32 Ewing's sarcomas were characterized by three distinct DNA histogram types. Eight tumors displayed a diploid nonproliferating DNA histogram pattern (type A), 11 a diploid proliferating (type B) and 13 an aneuploid (type C) DNA histogram profile. We found a highly significant relationship between these DNA histogram types and the CC1:CC2 cell type percentage ratio. The eight Ewing's sarcomas with a type A DNA histogram contained a significantly higher proportion of CC1 cell type nuclei as compared to the 13 tumors with a type C DNA histogram, which contained a great proportion of CC2 cell type nuclei.     

Confocal stereology and image analysis: methods for estimating geometrical characteristics of cells and tissues from three-dimensional confocal images

A short review of confocal stereology and three-dimensional image analysis is presented, pointing out the achievements accomplished in this field by the Department of Biomathematics (Institute of Physiology, Prague). One of the methods of confocal stereology, the fakir method for surface area estimation, developed by this laboratory, is described. Methods for automatic measurement of geometrical characteristics of microscopical structures, based on 3-D image processing or surface triangulation, are discussed and compared with interactive stereological methods. Three-dimensional reconstruction programs and software implementation of stereological and digital methods as well as their practical applications are presented. The future trends are discussed.     

结直肠腺瘤及结直肠癌患者肠道中梭杆菌属与产丁酸菌的定量研究

【目的】通过观察梭杆菌属(Fusobacterium spp.)和两株产丁酸菌(Eubacterium rectale、Faecalibacterium prausnitzii)在结直肠癌患者及结直肠腺瘤患者粪便样品中的丰度差异,研究梭杆菌属和产丁酸菌数量变化在结直肠腺瘤和结直肠癌发生发展中的作用和意义。【方法】收集结直肠癌患者(n=19)、结直肠腺瘤患者(n=12)及健康人(n=19)3组粪便样品,提取细菌基因组DNA,利用实时荧光定量PCR技术定量检测3组样品中梭杆菌属(Fusobacterium spp.)、直肠真杆菌(Eubacterium rectale)、普拉梭菌(Faecalibacterium prausnitzii)以及总菌的16S rRNA基因的拷贝数,然后利用秩和检验两两比较3组样品中目标菌群的数量和丰度差异。【结果】结直肠癌组的梭杆菌属丰度显著高于结直肠腺瘤组(P=0.013)和健康组(P=0.000),结直肠腺瘤组的梭杆菌属丰度显著高于健康组(P=0.002);结直肠腺瘤组普拉梭菌的丰度显著低于健康组(P=0.033);结直肠腺瘤组的总菌16S rRNA基因拷贝数也显著低于健康组(P=0.002);直肠真杆菌的水平在3组样品间没有显著差异。【结论】与健康人的粪便样品相比,结直肠腺瘤病人的粪便中产丁酸菌普拉梭菌数量下降,而结直肠腺瘤和结直肠癌病人的粪便样品中梭杆菌属数量增加;梭杆菌属和产丁酸菌数量上的变化提示它们可能与结直肠腺瘤和结直肠癌的发生密切相关。     

Quantification of angiogenesis by a computerized image analysis system in renal cell carcinoma

OBJECTIVE: To ascertain whether tumor angiogenesis quantitated by a computerized image analysis system correlates with clinical outcome in renal cell carcinoma. STUDY DESIGN: Microvessels were immunohistochemically labeled with antibodies to CD34 in sections from 62 cases of renal cell carcinoma. Computerized image analysis was used to evaluate the mean microvessel count (MMC) and mean percentage microvessel area (MPMA). RESULTS: MMC ranged from 19.3 to 315.0, while MPMA was 0.6-17.9%. There was a highly significant correlation between MMC and MPMA (r = .867, P < .01). Although MMC and MPMA decreased with increasing nuclear grade and TNM stage, this difference failed to achieve statistical significance. No statistically significant differences in survival were found for MMC or MPMA. CONCLUSION: Our results indicate that computerized image analysis can evaluate accurately tumor angiogenesis, but tumor angiogenesis in renal cell carcinoma does not provide significant prognostic information in renal cell carcinoma.     

Karyometry in rectal mucosa of patients with previous colorectal adenomas

OBJECTIVE: To determine whether karyometric measurements taken in biopsies from histologically normal-appearing rectal mucosa could serve as a biomarker for the risk of recurrence of polyps. MATERIALS AND METHODS: Biopsies were taken from the rectal mucosa of cases with a prior history of colonic polyps at the baseline of the study. In 57 cases recurrent polyps occurred (R cases); in 72 cases no recurrent disease was found at the end of the study (NR cases). From each biopsy 100 nuclei were recorded at high resolution. After segmentation, feature extraction and selection of a discriminating subset of features, a number of discriminant functions were derived. Also, measures of nuclear abnormality were computed. RESULTS: The differences in karyometricfeature values for nuclei from biopsies of cases with recurrent or nonrecurrent disease were very small and not notably expressed in the majority of nuclei. It was possible by focusing on nuclei showing clear deviations from normal to derive a discriminant function that exhibited a shift for the NR and R data sets. The distributions of discriminant function scores were then subjected to a second-order discriminant analysis to separate cases according to recurrence status. This function showed a statistically highly significant correlation with recurrence. At one extreme of its score distribution were 11 of 57 cases that had a recurrence, and at the other extreme were 8-10 of 72 cases that had no recurrence. The distributions of nuclear abnormality values for these subsets of cases were drastically different, with an average value of 1.72 for the group that may be at high risk for another recurrence and 1.02 for the group possibly at low risk. All cases with a prior history of colonic polyps showed a nuclear abnormality deviating from normal. CONCLUSION: Measurement of a sample of 100 nuclei from the rectal mucosa will suggest, for approximately 10% of cases, that a high risk for recurrence of adenomatous polyps exists and, for a slightly lower proportion, confirm that the nuclei deviate only slightly from those from individuals with no history of colonic polyps. For the majority of cases with a prior history of adenoma, the nuclei in the biopsy show a notable deviation from normal, but the deviation is practically the same for cases that had a recurrence and those that did not. However, a tentative discriminant function (DF I,3) derived from the characteristics of the extreme cases correctly classified approximately 64% of nonrecurrent and 83% of recurrent cases using a Bayesian decision boundary.     

DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P less than 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80-1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 +/- 0.28. The mean DNA index for the three cases of "carcinoma in adenoma" with invasion of the stalk of the adenoma was 1.52 +/- 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.     

Digital image analysis of different crown shape of Platycladus orientalis

Platycladus orientalis (L.) Franco is a beautifully crown-shaped evergreen tree species used for ornamental cultivation. These trees are also important afforestation plants at hill sites containing calcareous parent rocks and exhibit high tolerance to drought and barren sites. However, Platycladus orientalis trees with abnormal crowns, such as fusiform and torch-form, have been identified at sites with extreme drought and barren hills in Shandong, China, although the abnormal crowns does not reduce the ornamental value of these trees. In the present study, we used the RGB imaging and geographical statistical analyses through the construction of meteorological indices. The results indicate that variations of abnormal Platycladus orientalis crowns are associated with both the internal metabolism of these trees and the external environment. Crown shapes are strongly affected by the local dry, hot and windy meteorological environment, particularly individuals planted at poor hill site conditions. In response to extreme events of drought, high temperature and strong winds, the twigs and scale leaves of Platycladus orientalis typically wilt from the lower part to the upper part of the crown. The fusiform and torch-form crowns are formed through the wilting of partial twigs and scale leaves to avoid the entire wilt of the trees, thereby saving the life of the tree at the expense of partial twigs and scale leaves.     

Digital image analysis of Zostera marina leaf injury

Current methods for assessing leaf injury in Zostera marina (eelgrass) utilize subjective indexes for desiccation injury and wasting disease. Because of the subjective nature of these measures, they are inherently imprecise making them difficult to use in quantifying complex leaf injuries from multiple sources. We have developed a method using color digital photography of eelgrass leaves which are then manipulated using image processing programs and analyzed using geographic digital image analysis. The resulting false color images are then assigned by the user into uninjured and injured groupings which may then be reported as a percentage of leaf area affected. If images are rectified, leaf area (cm²) of injured and uninjured leaf segments may be determined. Although this method is time consuming and still requires some subjective judgments, it does allow for precise analysis of highly complex leaf injuries and has the potential to be a substantial improvement over existing leaf injury indexes.     

Particle detection,number estimation,and feature measurement in gene transfer studies: optical fractionator stereology integrated with digital image processing and analysis

Assessing the efficacy of in vivo gene transfer often requires a quantitative determination of the number, size, shape, or histological visualization characteristics of biological objects. The optical fractionator has become a choice stereological method for estimating the number of objects, such as neurons, in a structure, such as a brain subregion. Digital image processing and analytic methods can increase detection sensitivity and quantify structural and/or spectral features located in histological specimens. We describe a hardware and software system that we have developed for conducting the optical fractionator process. A microscope equipped with a video camera and motorized stage and focus controls is interfaced with a desktop computer. The computer contains a combination live video/computer graphics adapter with a video frame grabber and controls the stage, focus, and video via a commercial imaging software package. Specialized macro programs have been constructed with this software to execute command sequences requisite to the optical fractionator method: defining regions of interest, positioning specimens in a systematic uniform random manner, and stepping through known volumes of tissue for interactive object identification (optical dissectors). The system affords the flexibility to work with count regions that exceed the microscope image field size at low magnifications and to adjust the parameters of the fractionator sampling to best match the demands of particular specimens and object types. Digital image processing can be used to facilitate object detection and identification, and objects that meet criteria for counting can be analyzed for a variety of morphometric and optical properties.