| In 1998, Kenonen J et al introduced on Nature Medicine a novel technology named tissue chip, also called tissue microarray, which is now considered to be another important biochip technology followed the advent of gene chip and protein chip. Tissue microarray makes it possible that dozens or hundreds of samples be detected simultaneously by either immunohistochemistry or in situ hybridization experiments on a single tissue slice. Molecular pathology represented by the two key experiments would therefore be further standardized, become more objective and can be subjected to high through-put screening. As a vital branch of biochips, tissue microarray is becoming an important tool for studying gene phenotype and function, which would help to accelerate the translation of molecular genetics, genomics and proteomics research findings to clinical applications.Tissue microarray technique provides an unprecedented high through-put method in the human post-genomics era. On the research of gene function, it is essential to reveal the tissue location of each oncology related gene and theirprotein expression. Also is it essential to evaluate large numbers of molecular targets for their diagnostic, predictive or prognostic value in clinical oncology'7"9^. Tissue microarray technology meets the need above exactly. Tissue microarray, combined with gene chip and traditional tissue technology, provides a strong strategy for the identification and characterization of differently expressed genes and its protein expression in cancer tissues, as well as a effective method for therapy fl0]. Also it can be used in research for the correlation between molecular changes and clinical pathological characters in cancers t3OlPreparations of paraffin blocks for tissue microarray are similar to traditional pathological methods that are still mainly accomplished by handworks. Normally, Beecher Instrument (MTA-I / MTA-II) is required in assistant with these manipulations. Although Beecher Instrument helps researchers a lot, about 90% of the work is still made by hand, as in the research of routine pathological slices, but much harder, because of the high capacity of tissue microarray [11]. Repetitive work, which seriously compromises the efficiency of tissue microarray paraffin blocks making, renders the preparation process a bottleneck for the development and application of this new technique.As a high throughput technique, tissue microarray can not only be used to determine the in situ expression profiles of a certain biomarker in multiple tissues, but also be adopted to examine a variety of genes or proteins in a large number of samples, producing multiple tissue in-situ data conveniently and rapidly, which is more effective, abstemious, and comparative^12' 13] in comparison to traditional method. However, analysis of these experimental results is to date still limited to manmade or semi-automatism analysis function^141, which evidently restricts the efficiency of tissue microarray because of slower analysis speed and possible human interference. Results thus obtained often require critical reexaminations by researchers. Therefore, a highly efficient and objective data analysis method, which facilitates the screening of clinicallyuseful biomarkers, is in urgent needs for tissue microarray technique, and it is becoming another bottleneck for the extension and application of tissue microarray.Artificial neural network (ANN) , as an information analysis technique, arose in 1950s to 1960s and made great progress in 1980s. It deals with complex nonlinear problems possessing the ability of self-learning, which means the capability to summarize rule from experimental data in hands by itself. Nowadays, it has been widely used in pattern recognition and some other fields. As reported[15'16>31], ANN has its original value and advantage in distinguishing tumor tissue type, estimating curative effect and predicting recovery. High speed in analyzing such a large data, if combined with tissue microarray, can greatly advance examinations at the level of tissue in situ.To overcome the above mentioned disadvantages, present study attempts to establish a new method to make tissue array preparations and validate its feasibility experimentally. In addition to simplify working craft, reducing cost and promoting extension of this technique, we would use this newly-built platform to construct tissue microarray paraffin blocks from 9 kinds of tumors and colorectal cancer progression series respectively, and to investigate the expressions of a single bio-mark in multiple tumors and multiple bio-marks in single tumor series. By means of the tissue microarray paraffin block of colorectal carcinoma progression series, the stress signal pathway in this process is to be validated on tissue in situ level, which is discovered by Cancer Institute Zhejiang University fl8l Furthermore, we would combine tissue chip technology with ANN to analysis the experimental data we obtained, in an effort to explore the value the combination of these two high through put techniques and to provide new insights into developing and applying tissue microarray.Part I Development and Feasibility Validation of ZM-1 Tissue Microarray Paraffin Block makerI. The devising of ZM-1 Tissue Microarray Paraffin Block makerNowadays, people in and out of China usually use Tissue Microarray Paraffin Block maker made by Beecher Company (MTA-I / MTA-II) to prepare the tissue microarray paraffin block. But this method has some shortcomings as follows:? To sampling tissue specimens, need repetitive operation one by one, which has low efficiency;(2) Need prepare blank recipient paraffin block and paraffin in a high quality;? Low conformity between tissue samples and recipient block paraffin, which results in tissue points lost;? Need rigid locating equipment, which is very expensive;(5) To master the instrument facture, one needs good training, which is bad for extension and prevalence.To overcome such disadvantages, we devised a new tissue microarrayparaffin block making platform------ZM-1 Tissue Microarray Paraffin Blockmaker (Pat.App.No.: 200410025645.7;Pat.App.No.: 200420037020.8). The ZM-1 tissue microarray paraffin block maker has many advantages as below:? Simplify the donor block sampling craft, need not repetitive operation one by one, which enhances the efficiency;? Need not blank recipient paraffin block, and no extra requirement for paraffin quality, the convention paraffin of pathology can be suitable used. The instrument can be performed easily without any related training;? All the specimen cylinders arraying and the TMA block shaping are finished in only one step so that the specimen cylinders and the paraffin of the TMA block are much easier to be incorporated and the tissue points lost reduced;@ Need no rigid locating equipment, which makes it cheap and easier toextend and prevalent;(D ZM-1 tissue microarray paraffin block maker possesses independent knowledge of property. It has been protected by Nation Novel Application Patents, and we are currently applying Nation Inventing Patents (Pat.App.No.: 200410025645.7;Pat.App.No.: 200420037020.8).II. Feasibility Validation of ZM-1 Tissue Microarray Paraffin Block makerThe convention method need prepare blank recipient paraffin block in advance, which requires the extra quality of paraffin. Only high density paraffin (Leica Co.) with adequate hard and toughness is suitable. To validate the feasibility of ZM-1 tissue microarray paraffin block maker, the convention paraffin and high density paraffin were all selected. A tissue microarray paraffin block with 106 tissue points was made by ZM-1 and convention paraffin, meanwhile a tissue microarray paraffin block with 116 tissue points was made by convention method—MTA-I and high quality paraffin. Comparing the quality of paraffin blocks by two methods, we found:(D ZM-1 tissue microarray paraffin block maker needs not prepare blank recipient paraffin block in advance, so that the efficiency is increased. It took only 30 minutes for one person to make a tissue microarray paraffin block with 106 tissue points by ZM-1, meanwhile no less than 2 hours are essential to make a tissue microarray paraffin block with 116 tissue points by MTA-I;(2) ZM-1 tissue microarray paraffin block maker has no extra requirement of paraffin quality, and the convention paraffin of pathology can be suitable used.(3) By means of ZM-1, all the specimen cylinders arraying and the TMA block shaping are finished simultaneously so that the specimen cylinders and the paraffin of the TMA block are much easier to be incorporated and the tissue points lost reduced. Random selecting 3 HE stained slides respectively, the tissue points lost of slides by ZM-1 is only 0.94%(l+0+2/106x3), and that ofslides by MTA-I is 10.06%(ll+8+16/116x3);(|) The arraying-board of ZM-1 was devised instead of rigid locating equipment in MTA-I, which makes it cheap and easier to extend and prevalent.Part II The research on tissue microarray technology and related tumor gene expressionI. Expression spectrum of COX-2 among multiple tumorsMany research showed that COX-2 was closely related with oncogenesis and progression of tumor. Presently, tumor therapy focus on COX-2 was being paid much more attention. Anyway there were still some inconsistencies about the expression level of COX-2. as reported by Soslow et al[19], the positive rate of COX-2 in breast carcinoma was 56%, meanwhile the result reported by Ristjmaki A et al*20^ was only 37.4%. These inconsistencies were closely correlated with different experiment condition, different reagents and so on. Therefore it was necessary to investigate the expression of COX-2 thoroughly. By means of tissue microarray technology, multiple different tumors could be assembled in only one slice, in which investigation in tissue in situ such as immunohistochemistry, hybridization and so on in situ could be implemented simultaneously. Since the difference of experiment condition was eliminated, the result was much more reliable112' 13l By ZM-1 tissue microarray paraffin block maker, a tissue paraffin block with multiple tumors were prepared, which included esophagus, gastric carcinoma, colorectal carcinoma, liver carcinoma, breast carcinoma, kidney carcinoma, lung carcinoma thyroid carcinoma and bladder carcinoma. Using it, the expression of COX-2 was detected under the same space and experiment condition. The result showed: the positives of COX-2 in these tumors were 66.67%, 66.67%, 73.33%, 72.73%, 70.00%, 80.00%, 64.29%, 60.00% and 41.67% respectively;In esophagus, gastric carcinoma, colorectal carcinoma, lung carcinoma thyroid carcinoma and bladdercarcinoma, the expression of COX-2 was not related with lymphonode metastasis(P>0.05);The expression of COX-2 is correlated with the metastasis of liver carcinoma (P=0.024, <0.05);and is not correlated with the ER expression of breast carcinoma (P=0.500> >0.05) .II. Multiple molecular marks among colorectal carcinoma progressionBy means of tissue microarray technology, different stages' specimens among the progression of one kind tumor could be assembled into one same tissue microarray paraffin block, which could be acquired hundreds of same glides. Using these glides, multiple molecular marks could be detected in tissue in situ under the same experiment condition. Since the specimens in these glides were not any difference, the experiment result was much more comparable^12*13' so that the molecular mark with more clinical value could be easier to screening. Using ZM-1 tissue microarray parafiSn block maker, a tissue microarray paraffin block with colorectal mucosa, adenoma and colorectal carcinoma series was prepared for multiple molecular marks investigation under the same experiment condition. The test results showed as below:(D The protein expressions of ST13 and PTEN among colorectal mucosa, adenoma and colorectal carcinoma reduce gradually. The protein expression of PTEN has no significant difference among colorectal mucosa, adenoma and colorectal carcinoma(P>0.05).(2) The protein expression of P53, Cathepin D, Bcl-2 and Survivin among colorectal mucosa, adenoma and colorectal carcinoma is raise up gradually.(3) The protein expression of ST13, P53, Cathepin D, Bcl-2 and Survivin is closed related with colorectal carcinoma progression due to their significant difference among colorectal mucosa, adenoma and colorectal carcinoma(P<0.05).? The protein expression of ST13, P53, Cathepin D, Bcl-2 and Survivin is not related with FAP adenoma family history because they have nosignificant difference between FAP adenoma and sporadic adenoma (P>0.05), and so does between FAP adenoma canceration and sporadic colorectal carcinoma (P>0.05).III. The stress signal pathway and the colorectal carcinoma progressionAs "molecular chaperones" of function proteins, heat shock protein (HSP) could be able to protect the vital movement^1'72i. Recent research showed that HSP could participate in oncology immune reaction, and mediate tumor proliferation. Colorectal cancer progression is complex process which involves multiple-gene and multiple-stage. The in-depth study of colorectal cancer pathogenesis is of significant importance to improve its clinical efficacy and to achieve complete success in treatment. We have found that heat shock proteins expressed highly in colorectal cancer by gene chip technique^. In order to find out the exact role of heat shock proteins played in colorectal carcinoma progression, we prepared the tissue microarray paraffin block of colorectal cancer progression series by ZM-1 tissue microarray paraffin block maker. Using the tissue microarray block, expressions of HSFlmRNAs HSP27n HSP70 and HSP90ct in different stages of colorectal carcinoma progression were detected in tissue in situ under the same space and experiment condition. The test result showed as below:? The positive expression of HSP27 in colorectal adenoma is significanthigh than that in colorectal mucosa and colorectal carcinoma (P<0.05), andthat is significant high in FAP adenoma specially(P<0.01);? The positive expression of HSP70 in colorectal carcinoma is significanthigh than that in colorectal mucosa and colorectal adenoma (PO.05), andthat is no significant difference in FAP adenoma canceration and sporadiccolorectal cancer (P>0.05);(D The positive expressions of HSP90a in colorectal adenoma andcolorectal carcinoma are significant high than those in colorectal mucosa(P<0.05), and those are significant high in FAP adenoma and FAP adenoma canceration specially(P<0.01);? The positive expressions of HSFlmRNA in colorectal adenoma and colorectal carcinoma are significant high than those in colorectal nucosa (P<0.05);but those are on significant difference between FAP adenoma and sporadic adenoma, and so does between FAP adenoma canceration and sporadic colorectal cancer.Above experiment data validated that the activation of stress signal pathway was enhanced during colorectal cancer progression. The expressions of HSP27 and HSP90a were related with FAP adenoma family history, meanwhile the expressions of HSFlmRNA and HSP70 were not related with FAP adenoma family history.Part III The application of bioinformatics in experimentdata analysis of tissue microarrayColorectal cancer poses a severe threat to human health, and as suggested by the epidemiology research data, its incidence is increasing with each yearp4> 25\ However, significant breakthrough has yet been made in the treating strategies. Early diagnosis and treatment remains the critical factor in improving the clinical efficacy in treating colorectal cancer. At present, the pathological diagnosis for colorectal cancer still relies on the analysis of the morphous changes in the related tissues and cells;therefore subjective factor such as specialty knowledge and clinical experience of a certain pathological doctor might compromise the accuracy of the results. In addition, this method is far from enough for the unambiguous diagnosis of the colorectal tumor in early stage, for which different researcher might come up to different diagnosis. Quite often, other methods such as the immunohistochemistry experiments are necessary to assist the diagnosis. In the post-genomics era, an increasing number of tumor associated genes have been revealed. Many colorectal canerrelated genes have also been discovered and some are even beginning to be applied into the diagnosis in clinical pathology126"291. Nevertheless, large variations of each given marker do exist;rendering every single marker insufficient for an accurate diagnosis. For many markers, inconsistent results are found among different laboratories, which might derive from variations in reagents sources or experimental conditions. To circumvent these problems, we made the tissue microarray of colorectal caner progress series by ZM-1 tissue microarray instrument to examine the in situ expression of 10 markers including P53 in tissue. Our approach benefits from the controlled experimental conditions and therefore the expression profiles under the same spatial and temporal conditions are obtained. Also we adopted the bioinformatics method to process the tissue microarray data to establish the Artificial Neural Network, thus produced a novel diagnosis model. Our results showed:? Single molecular mark's ANN-BP diagnosis model could not accurately distinguish colorectal mucosa, colorectaJ adenoma and colorectal carcinoma;colorectal mucosa, FAP adenoma and FAP adenoma canceration;colorectal mucosa, sporadic adenoma and sporadic colorectal carcinoma simultaneously (Fig. 3-2—Fig.3-6);(D The accuracy of selected multiple marks' ANN-BP diagnosis models are better than that of single molecular mark's ANN-BP diagnosis model (Tab.3-2).Although these diagnosis models need further to be regulated and optimized, the test data above has already thrown out that dealing the microarray data by the bioinformatics method is more effective and of higher efficiency, which could significantly improve the accuracy of diagnosis by data from in situ tissue marker detection.Conclusion:1. ZM-l tissue microarray paraffin block maker can make the preparation of the tissue microarray paraffin block simple, without extra requirement for the paraffin quality and precise orientation devices. Compared to traditional techniques, this one can reduce the lost of tissue points sharply to keep the tissue microarray paraffin block quality. With the low cost and easy manipulation, this technique may be applied broadly.2. Detected in tissue in situ under the same space and experiment condition, COX-2 has high expression in many tumor tissues including esophagus, gastric carcinoma, colorectal carcinoma, liver carcinoma, breast carcinoma, kidney carcinoma, lung carcinoma thyroid carcinoma and bladder carcinoma. In esophagus, gastric carcinoma, colorectal carcinoma, lung carcinoma thyroid carcinoma and bladder carcinoma, the expression of COX-2 was not related with lymphonode metastasis. The expression of COX-2 is correlated with the metastasis of liver carcinoma, and is not correlated with the ER expression of breast carcinoma.3. By means of tissue microarray technology, expressions of multiple molecular marks in different stages of colorectal carcinoma progression were investigated under the same same space and experiment condition. The results showed as below:? The protein expression of ST13 and PTEN among colorectal mucosa, adenoma and colorectal carcinoma reduce gradually. The protein expression of PTEN has no significant difference among colorectal mucosa, adenoma and colorectal carcinoma(P>0.05).? The protein expression of P53, Cathepin D, Bcl-2 and Survivin among colorectal mucosa, adenoma and colorectal carcinoma is raise up gradually. (3) The protein expression of ST13, P53, Cathepin D, Bcl-2 and Survivin is closed related with colorectal carcinoma progression due to their significant difference among colorectal mucosa, adenoma and colorectalcarcinoma(P<0.05).? The protein expression of ST13, P53, Cathepin D, Bcl-2 and Survivinis not related with FAP adenoma family history because they have nosignificant difference between FAP adenoma and sporadic adenoma(P>0.05),and so does between FAP adenoma canceration and colorectalcarcinoma(P>0.05).4. The experiments on the level of tissue in situ validated that the activation of stress signal pathway was enhanced during colorectal cancer progression. The expressions of HSP27 and HSP90a were related with FAP adenoma family history, meanwhile the expressions of HSFlmRNA and HSP70 were not related with FAP adenoma family history.5. By means of bioinformatics, the analysis of experiment data from tissue microarray be enhanced much more scientific and efficiency. The combination of tissue microarray and artificial neural network technique improve the diagnosis of the markers for tissue in situ.
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