Search

Regeneration of CNS myelin involves differentiation of oligodendrocytes from oligodendrocyte progenitor cells. In multiple sclerosis, remyelination can fail despite abundant oligodendrocyte progenitor cells, suggesting impairment of oligodendrocyte differentiation. T cells infiltrate the CNS in multiple sclerosis, yet little is known about T cell functions in remyelination. We report that regulatory T cells (T$_{reg}$) promote oligodendrocyte differentiation and (re)myelination. T$_{reg}$-deficient mice exhibited substantially impaired remyelination and oligodendrocyte differentiation, which was rescued by adoptive transfer of T$_{reg}$. In brain slice cultures, T$_{reg}$ accelerated developmental myelination and remyelination, even in the absence of overt inflammation. T$_{reg}$ directly promoted oligodendrocyte progenitor cell differentiation and myelination in vitro. We identified CCN3 as a T$_{reg}$-derived mediator of oligodendrocyte differentiation and myelination in vitro. These findings reveal a new regenerative function of T$_{reg}$ in the CNS, distinct from immunomodulation. Although the cells were originally named 'T$_{reg}$' to reflect immunoregulatory roles, this also captures emerging, regenerative T$_{reg}$ functions.

SummaryBackground Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR. Objectives The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects. Methods Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function. Results Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.

OBJECTIVE: To examine, for the first time Bcl-2 expression in sequential (autogenous) oral mucosal biopsies taken from the same sites in a gender, risk-factor matched, Caucasoid sample, over a 21-year period. DESIGN: Retrospective immunocytochemical longitudinal study of archival serial biopsies. MATERIALS AND METHODS: Computer records were used to identify biopsy specimens derived from 12 patients. These were divided into four groups: (1) Histologically innocuous lesions which remained histologically innocuous. (2) Dysplastic lesions which remained dysplastic. (3) Histologically innocuous lesions which later progressed to squamous cell carcinoma (SCC). (4) Dysplastic lesions which later progressed to SCC. This represented 65 biopsies in total. Bcl-2 expression was studied using mouse antihuman BCL-2 oncoprotein clone 124 (Dako, Denmark). RESULTS: Generally, there was a lack of Bcl-2 immuno-reactivity in the epithelium, with one exception in dysplastic epithelium from a group (3) patient. CONCLUSION: These findings suggest that in our series, Bcl-2 is not expressed early in oral premalignant lesions and appears to contradict previous reports. Possible explanations for this disparity are considered.

Barrett’s esophagus (BE) is a precursor of esophageal adenocarcinoma (EAC). Both low-grade dysplasia (LGD) and high-grade dysplasia (HGD) are associated with an increased risk of progression to EAC. However, histological interpretation and grading of dysplasia (particularly LGD) is subjective and poorly reproducible. This study has combined whole slide imaging with DNA image cytometry to provide a novel method for the detection of abnormal DNA content through image analysis of tissue sections. A total of 20 cases were evaluated, including 8 negative for dysplasia (NFD), 6 LGD, and 6 HGD. Feulgen-stained esophageal sections were scanned in their entirety. Barrett’s mucosa was interactively chosen for automatic nuclei segmentation where irrelevant cell types were ignored. The combined DNA content histogram for all nuclei within selected image regions was then obtained. In addition, three histogram measurements were computed, including xER-5C, 2cDI, and DNA-MG. Visual evaluation suggested the shape of DNA content histograms from NFD, LGD, and HGD cases exhibiting identifiable differences. The histogram measurements, xER-5C, 2cDI, and DNA-MG, were shown to be effective in differentiating metaplastic from dysplastic cases with statistical significance. Moreover, they also successfully separated NFD, LGD, and HGD patients with statistical significance. Whole slide image cytometry is a novel and effective method for the detection of abnormal DNA content in BE. Compared with histological review, it is more objective. Compared with flow cytometry and cytology-preparation image cytometry, it is low cost, simple to use, only requires a single 1 μm section, and facilitates selection of tissue and topographical correlation. Whole slide image cytometry can detect differences in DNA content between NFD, LGD, and HGD patients in this cross-sectional study. Abnormal DNA content detection by whole slide image cytometry is a promising biomarker of progression that could affect future diagnostics in BE.

Raman spectroscopy is recognized as a tool for chemometric analysis of biological materials due to the high information content relating to specific physical and chemical qualities of the sample. Thirty cells belonging to two different prostatic cell lines, PNT1A (immortalized normal prostate cell line) and LNCaP (malignant cell line derived from prostate metastases), were mapped using Raman microscopy. A range of spectral preprocessing methods (partial least-squares discriminant analyses (PLSDAs), principal component analyses (PCAs), and adjacent band ratios (ABRs)) were compared for input into linear discriminant analysis to model and classify the two cell lines. PLSDA and ABR were able to correctly classify 100% of cells into benign and malignant groups, while PLSDA correctly classified a greater proportion of individual spectra. PCA was used to image the distribution of various biochemicals inside each cell and confirm differences in composition/distribution between benign and malignant cell lines. This study has demonstrated that PLSDAs and ABRs of Raman data can identify subtle differences between benign and malignant prostatic cells in vitro.

This study analyzed the chromatin organization state in histologically normal urothelium in patients with synchronous papillary carcinoma using digital texture analysis. The quantitative evaluation was carried out on hematoxylin and eosin-stained sections from 17 cases of urothelial papillary carcinoma in which a simultaneous biopsy specimen featuring histologically normal urothelium was available. Five bladder biopsy specimens of histologically normal urothelium from patients with prostate pathology in whom cystoscopy revealed a normal bladder mucosa were also analyzed. Karyometry showed that the 17 cases of papillary carcinoma, morphologically classified according to the 1973 World Health Organization scheme, belonged to a continuous spectrum or trend curve spanning grade 1 to grade 3. An abnormal pattern and distribution of the nuclear chromatin was seen in the normal-looking urothelium from the 17 bladders with papillary lesions. When this population was plotted along the trend curve, it occupied an intermediate position between the normal samples and samples from grade 1 carcinoma. When the nuclei were considered individually, the changes were detected only in a subpopulation of nuclei with chromatin alteration pointing toward that seen in grade 1 cases, even though distinct from them. In conclusion, karyometry can detect an abnormal chromatin pattern and distribution in the normal-looking urothelium adjacent to papillary carcinoma. Such alterations correspond to the so-called “malignancy-associated change.”

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

The evaluation of prostate cancer histology following hormonal therapy often represents a diagnostic problem for the pathologist. Previous studies have shown that an inference or Bayesian belief network (BBN) offers a descriptive classifier useful for the accurate analysis of morphological changes in individual cases of prostate neoplasia. Three different BBNs were evaluated in 94 cancer foci present in 20 radical prostatectomy (RP) specimens and in the matching biopsies in which the initial diagnosis of prostatic adenocarcinoma was made. Ten RP specimens were from patients treated with total androgen ablation or combination endocrine therapy (CET) before surgery. The first and second BBN allowed the identification with high certainty of the cancer foci present in the biopsies and RP specimens, as well as their Gleason grade, the belief value often being close to 1.0. The results of the second BBN showed a good correspondence between the Gleason grade given in the biopsies and that in the RP specimens, except in the surgical material of the treated patients, in which upgrading was always present. The third BBN showed the existence of three subgroups in treated RP specimens, one with morphological effect, another with poor effect, and the third with the histology of untreated (i.e. unaffected) cancer. In conclusion, an inference network-based analysis allows the characterization of treated prostate cancers according to the degree of histopathological change.

Aims Metallothioneins (MTs) are a group of ubiquitous low molecular weight proteins with a high affinity for heavy metal ions. The aim of the present study was to investigate MT expression in a series of endometrial carcinomas. We wished to determine whether MT expression in endometrial carcinoma was related to established prognostic factors such as tumour grade, stage and histological type. We also wanted to establish if high MT expression in curettings of endometrial carcinoma was predictive of high expression in the subsequent hysterectomy specimen. Methods and results Sixty-three cases of endometrial carcinoma were included in the study. These comprised 57 endometrioid adenocarcinomas (15 grade 1, 25 grade 2, 17 grade 3), three papillary serous adenocarcinomas, two mucinous adenocarcinomas and one clear cell adenocarcinoma. Forty-five tumours were stage I, 10 were stage II and eight were stage III. In 28 cases, diagnostic endometrial curettings, performed prior to hysterectomy, were available for study. Immunohistochemical staining was performed using the anti-MT monoclonal antibody E9. The intensity and distribution of MT staining were assessed using a semiquantitative method. This resulted in an intensity distribution (ID) score out of a maximum of 300. The mean ID score of grade 1 and 2 endometrioid adenocarcinomas was 67 and 63, respectively, while for grade 3 tumours the mean ID score was 114. This was statistically significant (P = 0.05). The three papillary serous adenocarcinomas had high ID scores with a mean of 208. The mean ID score of stage I tumours was 69. This was lower than those of stage II and III tumours which had mean ID scores of 116 and 128, respectively. However, these differences were not statistically significant (P = 0.288). A significant correlation was observed between MT ID scores in endometrial curettings and in the subsequent hysterectomy (P = 0.013). Conclusions MT isoforms can be demonstrated in most endometrial adenocarcinomas. High MT ID scores are associated with high grade and high stage endometrial adenocarcinomas and with the aggressive papillary serous adenocarcinoma. Whether this is of value as an independent prognostic factor has yet to be established.

Primary synovial chondromatosis (PSC) is thought to be a cartilaginous metaplasia, but it may recur locally and malignant change has been reported. Histologically, the cartilage is usually cellular, with binucleate forms. These findings suggest that the disease is not simply a metaplasia but imply a proliferative component. In this study, immunohistochemical detection of Ki-67 protein using an antigen retrieval microwave heating technique and DNA image cytometry (VIDAS image analysis system) has been used to assess the proliferative activity in 20 cases of PSC and the results have been compared with those obtained in other cartilage tissues: ten enchondromas, ten chondrosarcomas, and ten samples of normal articular cartilage. There was no detectable staining for Ki-67 protein in cases of PSC or in benign tissues, but there was a significant association between Ki-67 labelling index and grade in the chondrosarcomas (P

Three-dimensional (3D) reconstruction can be used to study the structural relationships between various tissue components and can help in the understanding of disease processes. Examples of perineural invasion have been reconstructed in two cases of adenocarcinoma of the extrahepatic bile duct. Ninety-six serial 5 microns sections were taken from both cases stored on file and 3D images were constructed using a Kontron VIDAS image analysis system. Both cases showed continuity of tumour cells within the perineural space. The isolated islands of malignant glands seen by conventional microscopy were shown to be in continuity with larger tumour cell masses via a complex branching network. In addition, direct continuity was demonstrated between malignant glands within the perineural space and those within the surrounding stromal tissue. During growth, the tumour appeared to have followed the plane of least resistance, although the availability of the perineural space may itself have been shaped by pressure effects and/or proteolytic enzyme secretion. Three-dimensional reconstruction of perineural invasion in adenocarcinoma of the extrahepatic bile ducts shows the value of this technique in demonstrating the structural relationships between the tumour and the host nerve bundle.

The assessment of cell proliferation in colorectal tissue may provide information with both prognostic and therapeutic implications. A variety of methods are available, including flow cytometric estimations of S phase fraction, immunohistochemical and autoradiographic visualization of exogenous and endogenous proliferation proteins, and morphological and stathmokinetic techniques. There is some correlation between Dukes stage and proliferation state features, and there is increased proliferative activity throughout the adenoma–carcinoma sequence. Data on cell proliferation rates are difficult to obtain. When correctly applied, the metaphase arrest technique remains the ‘gold standard’ of measuring proliferation, but its usefulness in clinical practice is limited. Recent studies have employed dual measurement flow cytometry and double labelling techniques to produce rate data.

Prostatic intraepithelial neoplasia (PIN) diagnosis and grading are affected by uncertainties which arise from the fact that almost all knowledge of PIN histopathology is expressed in concepts, descriptive linguistic terms, and words. A Bayesian belief network (BBN) was therefore used to reduce the problem of uncertainty in diagnostic clue assessment, while still considering the dependences between elements in the reasoning sequence. A shallow network was used with an open-tree topology. with eight first-level descendant nodes for the diagnostic clues (evidence nodes), each independently linked by a conditional probability matrix to a root node containing the diagnostic alternatives (decision node). One of the evidence nodes was based on the tissue architecture and the others were based on cell features. The system was designed to be interactive, in that the histopathologist entered evidence into the network in the form of likelihood ratios for outcomes at each evidence node. The efficiency of the network was tested on a series of 110 prostate specimens. subdivided as follows : 22 cases of non-neoplastic prostate or benign prostatic tissue (NP), 22 PINs of low grade (PINlow), 22 PINs of high grade (PINhigh), 22 prostatic adenocarcinomas with cribriform pattern (PACcri), and 22 prostatic adenocarcinomas with large acinar pattern (PAClgac). The results obtained in the benign and malignant categories showed that the belief for the diagnostic alternatives is very high, the values being in general more than 0.8 and often dose to 1.0. When considering the PIN lesions, the network classified and graded most of the cases with high certainty. However, there were some cases which showed values less than 0.8 (13 cases out of 44), thus indicating that there are situations in which the feature changes are intermediate between contiguous categories or grades. Discrepancy between morphological grading and the BBN results was observed in four out of 44 PIN cases : one PINlow was classified as PINhigh and three PlNhigh were classified as PINlow. In conclusion, the network can grade PIN lesions and differentiate them from other prostate lesions with certainty. In particular, it offers a descriptive classifier which is readily implemented and which allows the use of linguistic, fuzzy variables.

Basal cell adenoma is a well recognized histopathologic variant of monomorphic adenoma of salivary gland, but in recent years there have been occasional reports of malignant basal cell tumors of major salivary glands. We present a case report of one such basal cell adenocarcinoma arising in the submandibular gland and discuss the differential diagnosis and distinction from a basal cell adenoma. We also review the published literature on basal cell adenocarcinoma. This is the first case of basal cell adenocarcinoma to be studied in detail by both immunohistochemistry and electron microscopy. There has been debate in the past as to the cell of origin of salivary gland basal cell tumors and specifically as to whether myoepithelial differentiation occurs in these tumors. The immunohistochemical and ultrastructural features of this case provide evidence for a dual population of ductal and myoepithelial cells. A flow cytometric analysis of nuclear deoxyribonucleic acid content showed the presence of DNA aneuploidy within one tissue block.

Thirteen nuclear and cellular morphometric variables were measured in 312 cases of colorectal adenocarcinoma. All variables, except nuclear shape factors, differed significantly (P

The main curative therapy for patients with nonsmall cell lung cancer is surgery. Despite this, the survival rate is only 50%, therefore it is important to more efficiently diagnose and predict prognosis for lung cancer patients. Raman spectroscopy is useful in the diagnosis of malignant and premalignant lesions. The aim of this study is to investigate the ability of Raman microscopy to diagnose lung cancer from surgically resected tissue sections, and predict the prognosis of these patients. Tumor tissue sections from curative resections are mapped by Raman microscopy and the spectra analzsed using multivariate techniques. Spectra from the tumor samples are also compared with their outcome data to define their prognostic significance. Using principal component analysis and random forest classification, Raman microscopy differentiates malignant from normal lung tissue. Principal component analysis of 34 tumor spectra predicts early postoperative cancer recurrence with a sensitivity of 73% and specificity of 74%. Spectral analysis reveals elevated porphyrin levels in the normal samples and more DNA in the tumor samples. Raman microscopy can be a useful technique for the diagnosis and prognosis of lung cancer patients receiving surgery, and for elucidating the biochemical properties of lung tumors.

Objective: The study summarizes results of karyometric measurements in epithelial cells of the colorectal mucosa to document evidence of a field effect of preneoplastic development among patients with colorectal adenocarcinoma or adenoma. Methods: Karyometric analyses were done on high-resolution images of histologic sections from 48 patients with colorectal adenocarcinomas and 44 patients with adenomas and on images from matching normal-appearing mucosa directly adjacent to such lesions, at a 1-cm and 10-cm distance from the lesions or from the rectal mucosa of adenoma patients, as well as from 24 healthy normal controls with no family history of colonic disease. Results: The nuclei recorded in the histologically normal-appearing mucosa of patients with either colorectal adenoma or adenocarcinoma exhibited differences in karyometric features in comparison with nuclei recorded in rectal mucosa from patients who were free of a colonic lesion. These differences were expressed to the same extent in tissue adjacent to the lesions and in normal-appearing tissue as distant as the rectum. Conclusions: The nuclear chromatin pattern may serve as an integrating biomarker for a preneoplastic development. The field effect might provide an end point in chemopreventive intervention trials. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2704–16)

OBJECTIVE To explore the nuclear chromatin phenotype, overall epigenetic mechanisms, chromatin remodelling enzymes and their role as diagnostic biomarkers in prostate lesions, using high-resolution computerized quantitative digital image analysis (DIA). MATERIALS AND METHODS A tissue microarray (TMA) was constructed using paraffin wax-embedded prostatic tissues from 78 patients, containing 30 cores of benign prostatic hyperplasia (BPH), 10 of low-grade prostatic intraepithelial neoplasia (LGPIN), 38 of prostate adenocarcinoma, 20 of BPH tissue excised at 0.6–1 mm from LGPIN lesions, and 10 of BPH prostatic tissues obtained 0.6–1 mm from high-grade PIN (HGPIN) lesions. Chromatin phenotype was assessed on haematoxylin-stained sections using high-resolution texture analysis. For quantitative immunohistochemistry, antibodies raised against acetylated histone H3 lysine 9 (AcH3K9), 5′methylcytidine (5MeC) and the chromatin remodelling ATPase ISWI (SNF2H and SNF2L) were used. The immunodensity was measured using DIA to determine the epigenetic profile of the cases. At least 60 nuclei were measured from each case. RESULTS There were many statistically significant differences in staining intensity and nuclear distribution patterns in chromatin phenotype and immunostaining (p ≤ 0.001). These changes allowed the differentiation between the various pathological subgroups with a classification accuracy of 76–100% using chromatin phenotype or immunostaining measuring epigenetic and chromatin remodelling changes (5MeC, AcH3K9 and ISWI). In PIN lesions, there was a high chromatin content with DNA-hypermethylation, while in prostatic adenocarcinoma there was a lower chromatin content with DNA-hypomethylation and H3K9-hypoacetylation. There was significantly more ISWI protein in neoplastic tissues. There were malignancy-associated changes (MAC) in chromatin phenotype and overall epigenetic events in BPH tissues adjacent to PIN lesions. CONCLUSIONS The present study confirms the ability of high-resolution computerized digital imaging of nuclear texture features to detect changes in chromatin phenotype, epigenetic events and the presence of chromatin remodelling, factors that can be used to distinguish between different prostatic pathologies, i.e. BPH, LGPIN, HGPIN and prostate adenocarcinoma, and further allow the detection of MAC near PIN lesions. These results provide a base for future diagnostic applications of DIA combined with immunohistochemistry. Our experiments underscore the importance of epigenetic mechanisms during carcinogenesis. Further studies are needed to elucidate the complex interplay between chromatin structure, its modifications and the progression of prostate cancer.

Background: Nuclear texture analysis measures phenotypic changes in chromatin distribution within a cell nucleus, while the alkaline Comet assay is a sensitive method for measuring the extent of DNA breakage in individual cells. The authors aim to use both methods to provide information about the sensitivity of cells to ionizing radiation. Methods: The alkaline Comet assay was performed on six human bladder carcinoma cell lines and one human urothelial cell line exposed to γ-radiation doses from 0 to 10 Gy. Nuclear chromatin texture analysis of 40 features was then performed in the same cell lines exposed to 0, 2, and 6 Gy to explore if nuclear phenotype was related to radiation sensitivity. Results: Comet assay results demonstrated that the cell lines exhibited different levels of radiosensitivity and could be divided into a radiosensitive and a radioresistant group at >6 Gy. Using stepwise discriminant analysis, a subset of important nuclear texture features that best discriminated between sensitive and resistant cell lines were identified A classification function, defined using these features, correctly classified 81.75% of all cells into their radiosensitive or radioresistant groups based on their pretreatment chromatin phenotype. Posttreatment chromatin changes also varied between cell lines, with sensitive cell lines showing a relaxed chromatin conformation following radiation, whereas resistant cell lines exhibited chromatin condensation. Conclusions: The authors conclude that the alkaline Comet assay and nuclear texture methodologies may prove to be valuable aids in predicting the response of tumor cells to radiotherapy. © 2006 International Society for Analytical Cytology

This digital texture analysis-based study evaluates the chromatin organization state in flat and cribriform high-grade prostatic intraepithelial neoplasia (PIN), in the adjacent normal looking secretory epithelium and in the co-occurring adenocarcinoma. Digital texture analysis (karyometry) was carried out on hematoxylin and eosin-stained sections from 24 radical prostatectomy specimens with high-grade PIN (12 with flat and 12 with cribriform architectural pattern, respectively) and cancer. Quantification was also conducted on the normal looking secretory epithelium. Discriminant analysis and the nonsupervised learning algorithm P-index were used to identify suitable subsets of features useful for the discrimination and classification of pathological groups and to explore multivariate data structure in the pathological subgroups. The average nuclear abnormality increases monotonically from the histologically normal appearing secretory epithelium to high-grade PIN and to adenocarcinoma. The nuclei from the so-called perimeter compartment of the flat high-grade PIN lesions show a higher nuclear abnormality compared to the nuclei of the cribriform high-grade PINs. Discriminant analysis shows that flat and cribriform high-grade PINs fall into two populations. Processing by the nonsupervised learning algorithm P-index revealed the existence of three well-defined, distinct subpopulations of nuclei of different chromatin phenotype. In the flat high-grade PIN lesions the proportions of nuclei in the three subpopulations are 16.5% (low abnormality), 25.0% (mid abnormality) and 58.5% (high abnormality), respectively. In the cribriform high-grade PIN lesions, 100% of the nuclei are in the mid-abnormality subpopulation. These differences are also discernible in the co-occurring adenocarcinoma and the histologically normal appearing secretory epithelium. To conclude, karyometry and statistical analysis detect the existence of distinct cell subpopulations of different chromatin packaging and phenotype, with the nuclei from the flat high-grade PIN lesions, adjacent normal looking epithelium and co-occurring adenocarcinoma expressing a greater nuclear abnormality than in the specimens with cribriform high-grade PIN.

Previous studies have revealed considerable interobserver and intraobserver variation in the histological classification of preinvasive cervical squamous lesions. The aim of the present study was to develop a decision support system (DSS) for the histological interpretation of these lesions. Knowledge and uncertainty were represented in the form of a Bayesian belief network that permitted the storage of diagnostic knowledge and, for a given case, the collection of evidence in a cumulative manner that provided a final probability for the possible diagnostic outcomes. The network comprised 8 diagnostic histological features (evidence nodes) that were each independently linked to the diagnosis (decision node) by a conditional probability matrix. Diagnostic outcomes comprised normal; koilocytosis; and cervical intraepithelial neoplasia (CIN) I, CIN II, and CIN III. For each evidence feature, a set of images was recorded that represented the full spectrum of change for that feature. The system was designed to be interactive in that the histopathologist was prompted to enter evidence into the network via a specifically designed graphical user interface (i-Path Diagnostics, Belfast, Northern Ireland). Membership functions were used to derive the relative likelihoods for the alternative feature outcomes, the likelihood vector was entered into the network, and the updated diagnostic belief was computed for the diagnostic outcomes and displayed. A cumulative probability graph was generated throughout the diagnostic process and presented on screen. The network was tested on 50 cervical colposcopic biopsy specimens, comprising 10 cases each of normal, koilocytosis, CIN I, CIN II, and CIN III. These had been preselected by a consultant gynecological pathologist. Using conventional morphological assessment, the cases were classified on 2 separate occasions by 2 consultant and 2 junior pathologists. The cases were also then classified using the DSS on 2 occasions by the 4 pathologists and by 2 medical students with no experience in cervical histology. Interobserver and intraobserver agreement using morphology and using the DSS was calculated with kappa statistics. Intraobserver reproducibility using conventional unaided diagnosis was reasonably good (kappa range, 0.688 to 0.861), but interobserver agreement was poor (kappa range, 0.347 to 0.747). Using the DSS improved overall reproducibility between individuals. Using the DSS, however, did not enhance the diagnostic performance of junior pathologists when comparing their DSS-based diagnosis against an experienced consultant. However, the generation of a cumulative probability graph also allowed a comparison of individual performance, how individual features were assessed in the same case, and how this contributed to diagnostic disagreement between individuals. Diagnostic features such as nuclear pleomorphism were shown to be particularly problematic and poorly reproducible. DSSs such as this therefore not only have a role to play in enhancing decision making but also in the study of diagnostic protocol, education, self-assessment, and quality control.

Aims: Intracellular folate deficiency has been implicated in colonic carcinogenesis in epidemiological studies and animal and human cancer models. Our aim was to determine the effect of folate supplementation on patients with recurrent adenomatous polyps using rectal mucosal cell proliferation as a biomarker. Patients and methods: Eleven patients with recurrent adenomatous polyps of the colon were randomised into a treatment group (n=6) receiving a dietary supplement of 2 mg folic acid per day for three months and a control group (n=5) receiving a placebo. Rectal biopsies where taken at 10 cm from the anal verge prior to supplementation and repeated at four, 12, and 18 weeks from the start of the supplementation. Each biopsy was immediately incubated in culture medium enriched with bromodeoxyuridine (BrdU). The S phase cells which incorporated BrdU into their DNA were identified following immunohistochemical staining. Twenty five orientated crypts were identified for each time point and the number and position of BrdU positive and BrdU negative cells were counted. BrdU labelling indices (LIs) were calculated for the entire crypt and for each of five equal compartments running consequently from the base to the luminal surface. Results: The LI of the treatment group (9.1 (6.7, 12.3)) and the control group (9.3 (7.8, 10.3)) were comparable at the start. Over the duration of the supplementation period, LI in the control group did not alter significantly (9.3 (7.8, 10.3) v 9.6 (8.9, 10.4)). However, LI of the folate treated group was lowered after 12 weeks of supplementation (9.1 (6.7, 12.3) v 7.4 (5.3, 9.6)). Analysis of the LI for compartments within the crypt showed that the most significant drop in number of proliferating cells was in the upper most regions of the crypt. Conclusion: These data indicate that (a) folate supplementation decreases colonic mucosal cell proliferation in a high risk group for colon cancer and (b) the most significant reduction takes place at the luminal aspect of the crypt.

Antisense oligonucleotides (AO) downregulate Bcl-2 protein expression in various tumours if good target cell uptake is achieved. In this study, uptake of FITC labelled AO (FITC-AO) directed at Bcl-2 was examined in: (1) the RT4 bladder tumour cell line; (2) normal pig urothelium, and (3) human superficial bladder tumours.In the RT4 cell line, uptake of FITC-AO, FITC-scrambled and FITC-sense oligonucleotides were quantified by flow cytometry at 4-hour intervals over 24 h. Uptake of FITC-AO was assessed in normal pig urothelium by flow cytometry after FITC-AO was infused for 1 h. Uptake of FITC AO was assessed in samples from 14 human superficial bladder tumours which were maintained in an ex vivo model. In samples from 6 tumours, uptake at 4 h was assessed using fluorescence microscopy. In samples from 8 separate tumours uptake every 4 h within the first 24-hour incubation period was assessed by flow cytometry.In the RT4 cell line the FITC-AO, FITC-scrambled and FITC-sense oligonucleotide uptake was similar. Disaggregated cells from the normal urothelium of the 3 pigs exhibited 33, 46 and 51% of cells staining positively for FITC-AO as determined by flow cytometry. All 6 tumour samples had detectable intracellular FITC-AO by fluorescence microscopy at 4 h. In the 8 tumours examined over the 24-hour incubation period, there was a range of percentages of positively staining cells. However, most tumours had a monotonic increase in intracellular fluorescence intensity that plateaued 16 h post-infusion.Antisense Bcl-2 oligonucleotides were readily taken up by superficial bladder cancer cells but the heterogeneous uptake in tumour samples needs to be considered when assessing the bioavailability of these drugs.

The quantitative assessment of apoptotic index (AI) and mitotic index (MI) and the immunoreactivity of p53, bcl-2, p21, and mdm2 were examined in tumour and adjacent normal tissue samples from 30 patients with colonic and 22 with rectal adenocarcinoma. Individual features and combined profiles were correlated with clinicopathological parameters and patient survival data to assess their prognostic value. Increased AI was significantly associated with increased bcl-2 expression (p

The epithelial tumours of the adult kidney, in particular renal cell carcinoma (RCC), are a variety of neoplasms that can be classified by morphology and genotype. Although most are well characterised, typical and less typical tumour variants are recognised. There is evidence to indicate that stage is one of the most important prognostic factors, irrespective of tumour subtype. However, the appropriate handling of nephrectomy specimens is essential for accurate evaluation of diagnostic and prognostic factors in RCC. The problem of how to achieve more objective nuclear grading is still unresolved. The use of diagnostic decision support systems offers the possibility of a flexible approach to this problem, while still utilising morphological criteria. The histopathological analysis remains important, but new techniques of molecular and cell biology will be providing new tools of extraordinary power to sharpen the diagnosis and give it a biological interpretation.

Background: Prostate cancer develops over an extended period of time. Until recently, the events initiating the process and the developments concomitant with the evolution towards invasive disease were largely unknown. Methods: Analytical and quantitative methods are applied to provide insights into certain individual molecular events and their effects on the complex multiple feedback system of cellular metabolism and regulation in prostate neoplasia. Results: Prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) are associated with or possibly preceded by changes in the chromatin of secretory cell nuclei. The changes are detectable with a Bayesian belief network and quantifiable by computer image analysis in prostatic tissue that still appears histologically normal. In addition, normal-looking prostate epithelium shows some molecular changes similar to those present in the associated preneoplastic and neoplastic lesions. Such changes are also occasionally present in normal prostate glands without PIN and cancer. Conclusions: The subtle morphological and molecular changes of normal-looking epithelium might be seen as the onset of the development of prostatic neoplasia.

This study describes a new technique for the visualisation and quantitation of glandular epithelial cell proliferation in gastrointestinal mucosa using computerised three-dimensional reconstruction. The tissue used in this study was colorectal biopsy tissue infiltrated in vitro with bromodeoxyuridine (BrdU), although the method could be applied to any gastrointestinal site labelled with any specific marker for cell proliferation. The method is as follows. Five-micron-thick serial sections (> 100) were cut from colorectal biopsies infiltrated in vitro with BrdU. After labelling all the sections for BrdU-positive cells using standard immunohistochemistry, colorectal glands were identified which were completely sectioned within the series. Each microscopic image of the sectioned gland was orientated, digitised and stored using a Kontron image analyser. On each of the stored images, the crypt profile, the positive cells and the negative cells were interactively marked and digitally stored. Using three-dimensional (3-D) reconstruction software, the outer surface of the crypt, the total positive and the total negative fractions could be viewed in three dimensions. The total BrdU-positive cell number could be automatically calculated for the complete crypt or, alternatively, compartmental analysis of the labelling pattern within the crypt could be obtained. This represents a powerful technique: it does not require orientation, it can be carried out on complex glandular structures and is not affected by the biases involved in measuring labelling indices from single tissue sections. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6

DNA analysis was assessed by densitometry for 281 cases of colorectal adenocarcinoma. Detection of aneuploidy in a single case rose from 65% if one, to 92.5% when three or more sections, were analysed. Although aneuploid tumours had significantly larger nuclear areas than near diploid tumours (p = 0.009), densitometric measurements showed no association with clinicopathological variables. DNA content determined by densitometry was compared with that from flow cytometry on 465 tissue sections from 241 cases. Aneuploidy assessed by flow cytometry was significantly associated with that determined by densitometry (p < 0.01 for all comparisons), ploidy state being similar in 381 sections (82%, kappa = 0.63, p < 0.001), and 187 cases (77.6%, kappa = 0.57, p < 0.001). Univariate survival analysis showed that DNA densitometric variables had no significant association with survival in (a) all cases, (b) cases without lymph node metastases, or (c) cases without distant metastases. Multivariate regression analysis of densitometric and clinicopathological variables identified Dukes's stage, patient age, and tumour differentiation as the combination of variables most closely related to survival. Densitometric measurement of DNA content could not significantly improve on the prognostic model containing these three variables. It is concluded that, although the assessment of DNA content by densitometry is comparable with that of flow cytometry, conventional histological variables remain the best predictors of prognosis in colorectal cancer.

Morphometric image analysis of nuclear features was performed on tissue from 46 patients who had had curative resections for gastric cancer. Clinical, pathological, flow cytometric, and follow-up data were available for these patients, which were drawn from a larger, previously reported series. The morphometric data were compared with patient survival, clinico-pathological status, and DNA ploidy. Univariate survival analysis revealed that morphometric parameters were not significantly related to survival, but examination of clinico-pathological data showed lymph node involvement, involvement of the resection margin, and lymphatic invasion to be significantly associated (P < 0.01) with patient prognosis. Multivariate survival analysis using the Cox model found only lymph node and resection margin involvement to be independently related to survival. Comparison of morphometric results with the clinico-pathological parameters showed various features, relating to nuclear size, and its variation to be significantly associated (P < 0.01) with the presence of lymphatic invasion, resection margin involvement, and tumour pattern (intestinal/diffuse). A comparison of morphometry with flow cytometric analysis in these cases showed that nuclear size was not significantly related to either DNA aneuploidy or the DNA proliferative index.

A cell-counting technique and a photographic-based scoring system for assessing inflammatory cell infiltration were compared for agreement in 275 endoscopic gastric antral biopsies from 55 dyspeptic patients. Each biopsy was independently scored 0-5 for polymorphs and mononuclear cells using both methods, and a mean score was derived for each patient. There was a highly significant correlation between the mean patient scores obtained using each method (P less than 0.001). Agreement statistics showed that the mean score difference (cell-counting minus photographic score) was negative for polymorphonuclear and mononuclear cells, indicating that the photographic method yielded higher mean patient scores. Scatterplots of difference versus mean by the two methods showed that only 3.6 per cent of scores fell outside +/- 2 SD of the mean difference. The use of standard photomicrographs to grade superficial antral gastritis therefore provides accurate results, comparable to those obtained by cell counting, yet is a faster and much cheaper technique.

Inflammatory cell counting in endoscopic biopsy sections was carried out on duodenal mucosal samples from defined sites in patients with duodenal ulcer, duodenitis but no ulcer, non-ulcer dyspepsia, and asymptomatic controls using computer linked image analysis. The variables measured included polymorphonuclear and mononuclear cells per mm of superficial epithelium and per mm2 lamina propria. Duodenal ulcer crater margin and mucosal biopsy specimens from endoscopically inflamed mucosa in the group with duodenitis but no ulcer showed significantly higher inflammatory cell counts than endoscopically normal non-ulcer dyspepsia and control mucosa. Biopsy specimens from non-ulcer dyspepsia patients showed significantly higher lamina propria polymorphs than control group mucosa. Endoscopically normal duodenal ulcer and duodenitis but no ulcer mucosa also showed significantly higher acute and chronic inflammatory cell counts than controls. The prevalence of Helicobacter pylori in duodenal biopsy specimens was low (0-22%) and unrelated to local inflammatory response. Despite histological appearances, duodenal biopsy specimens from non-ulcer dyspepsia patients showed significantly higher inflammatory cell infiltration than control specimens, suggesting that at least some represent part of a spectrum of subclinical peptic disease.

AIMS: Alterations in epithelial proliferation and apoptosis in colonic mucosa are associated with an increased risk of colon cancer. Deregulation of bcl-2 and bax are important factors in the control of cell turnover. The aim of this study was to analyze patterns of bcl-2 and bax expression and in mitosis and apoptosis in the progression to colorectal neoplasia, and to reconstruct colorectal crypts to visually demonstrate this deregulation. METHODS: 12 patients with a sigmoid adenomatous polyp and 12 low risk controls were studied. Mucosal biopsies were taken from the polyp, tissue adjacent to and distant from the polyp and from the sigmoid colon of controls. Apoptotic and mitotic figures, and cells expressing bcl-2 or bax were counted, and labeling indices calculated for each within each crypt. Results are expressed as total labeling index (TLI) and labeling index (L1) for each of the five compartments (cpts) in which colonic crypts were divided for mitotic index (MI), apoptotic index (AI), and bd-2 and bax expression. Sample individual crypts that were complete within serially sections were idemified. The appropriate cells were marked, the images captured and stored, and then whole crypts were reconstructed in 3D using Analyze3.1 to assess the intra-erypt relationships of each variable. RESULTS: There was no significant difference between TLI and LI for bcl-2 and bax for controls vs. distant mucosa. The TL1 and L1 were significantly higher for polyp vs adjacent vs. distant mucosa for both bcl-2 (cpts 1-3, p

Gastric antral endoscopic pinch biopsies from a group of dyspeptic patients were analysed for acute and chronic inflammatory cell numbers in the lamina propria and surface epithelial layer using computer-linked graphic tablet planimetry, and independently graded for Campylobacter pylori (CP) infection using a visual scoring system with grade 1 assessed as patchy epithelial infection and grade 2 as a continuous layer of organisms on the mucosal surface extending into gastric pits. The study group consisted of 36 patients (18 duodenitis; 18 non-ulcer dyspepsia). Within the 140 biopsies analysed, grade 1 and 2 biopsies had significantly higher acute and chronic inflammatory cell counts than CP-negative biopsies (grade 0) in lamina and surface epithelium (P less than 0.001). Acute inflammatory cell counts were significantly higher in the surface epithelium in grade 1 (P less than 0.05) and grade 2 biopsies (P less than 0.001) but chronic inflammatory cells were only higher in grade 1 (P less than 0.01). No significant differences were present between grade 1 and grade 2 biopsies for any parameter. This study confirms that highly significant quantitative differences in the inflammatory status are related to the presence of CP.

The purpose of this study was the quantitative assessment of mucosal inflammation and its relationship to Campylobacter pylori in gastric antral and body biopsies from patients with dyspepsia and controls. The study groups comprised patients with duodenal ulcer (DU; n = 20), duodenitis (DUN; n = 20), non-ulcer dyspepsia (NUD; n = 20). Using a semi-automatic, computer-linked image analyser (Kontron: MOP Videoplan), mucosal acute and chronic inflammatory cell densities were measured in defined gastric sites for each patient group and expressed as number per mm2 of lamina propria and number per mm length of epithelium. Measurements were also made on a group of asymptomatic controls (n = 9) who fulfilled strict exclusion criteria. All biopsies were analysed for the presence of Camplyobacter pylori (CP) with a Giemsa stain. Data between groups were compared using the Mann-Whitney U-test. In the antrum and body, the mononuclear cell count was significantly higher in lamina propria in DU patients than in DUN, NUD and controls. In the body, DU laminia propria mononuclear cell counts were higher than those of DUN and controls. Prevalence rates for CP for DU, DUN, and NUD were 94, 89, and 50 per cent for antral and 88, 83, and 56 per cent for body biopsies. Significant differences were present between CP-positive and negative subjects in the NUD group. Antral and body inflammation within these clinical groups shows a wide variation. Higher inflammatory cell counts in the DU group may reflect the prevalence of CP colonization.

Semi-automatic image analysis was used to assess the architectural features of normal colorectal mucosa and ulcerative colitis with and without dysplasia. Eight measured and derived morphometric variables were compared with the histological grading. The main data-set variation was due to: (1) the area of mucosa and epithelium per unit length of muscularis mucosae; (2) mean mucosal and epithelial height; and (3) the percentage epithelium and number of crypts per unit length of muscularis mucosae. Discriminant analysis using the variables mean epithelial height and mean lamina propria area per unit length of muscularis mucosae separated normals (n = 10) from high-grade dysplasia (8). The classification rule allocated low-grade dysplasia (8) to the high-grade category and 60% of regeneration cases (10) to the normal mucosa group. Scatter plots of the two discriminating variables separated normal and regenerative mucosa from dysplasia. Histological review of overlapping cases allowed redesignation of a high-grade dysplasia lesion as low grade. Architectural morphometry may be of use in assessing premalignant mucosal changes in ulcerative colitis as a guide to patient surveillance and therapy.