Detection of pathological aortic tissues by infrared multispectral imaging and chemometrics.

Processing of multispectral images is becoming an important issue, especially in terms of data mining for disease diagnosis. We report here an original image analysis procedure developed in order to compare 42 infrared multispectral images acquired on human ascending aortic healthy and pathological tissues. Each image contained about 2500 infrared absorption spectra, each composed of 1641 variables (wavenumbers). To process this large data set, we have restricted the spectral window used to the 1800-950 cm(-1) spectral range and selected 100 spectra from the aortic media, which is the most altered part of the aortic tissue in aneurysms. Prior to this selection, a spectral quality test was performed to eliminate 'bad' spectra. Our data set was first subjected to a discriminant analysis, which allowed separation of aortic tissues in two groups corresponding respectively to normal and aneurysmal states. Then a K-means analysis, based on 20 groups, allowed reconstruction of infrared images using false-colours and discriminated between pathological and healthy tissues. These results demonstrate the usefulness of such data processing methods for the analysis and comparison of a set of spectral images.

Analysis of structural changes in normal and aneurismal human aortic tissues using FTIR microscopy.

Aortic aneurisms are frequently asymptomatic but can induce dramatic complications. The diagnosis is only based on the aortic diameter and not on a structural and compositional basis. In this preliminary study, we propose infrared microspectroscopy to nondestructively probe normal and aneurismal human aortas. Spectra from 19 human ascending aortic biopsies (10 normal and 9 aneurismal) were acquired using infrared microspectroscopy. A 1500 x 150 microm(2) area of each 7-microm thick cryosection was investigated using a 30-microm spatial resolution with a total of about 200 spectra per sample. Spectral differences between normal and aneurismal tissues were mainly located in spectral regions related to proteins, such as elastin and collagen, and proteoglycans (1750-1000 cm(-1)). Tissue heterogeneity and sample classification have been evaluated using hierarchical cluster analysis of individual or mean spectra and their second derivative. Using spectral range related to proteins, 100% of good classification was obtained whereas the proteoglycan spectral range was less discriminant. This in vitro study demonstrates the potential of such technique to differentiate between normal and aneurismal aortas using selected spectral ranges. Future investigations will be focused on these specific spectral regions to determine the role of elastin and collagen in the discrimination of normal and pathological aortas.

Expression of lumican, a small leucine-rich proteoglycan with antitumour activity, in human malignant melanoma.

BACKGROUND: The family of small leucine-rich proteoglycans (SLRPs), which includes decorin, lumican, biglycan and fibromodulin, constitutes an abundant component of the skin extracellular matrix. We previously demonstrated that human lumican inhibits melanoma growth and progression in a mouse experimental model, by regulating cell migration, proliferation and apoptosis. AIM: The aim of this study was to investigate the expression of lumican and decorin in human malignant melanoma and adjacent peritumoral tissue, to understand better their role in the control of growth and invasion of human melanoma. METHODS: Expression of both proteoglycans was studied by immunohistochemistry using specific antibodies in 34 malignant melanomas, 12 Hutchinson's melanotic freckles and 4 cutaneous metastatic melanomas. RESULTS: We showed that lumican and decorin are located in the dermis and in the peritumoral stroma of malignant melanoma, but are not found in melanoma cells or dense tumour tissue. In the healthy dermis, distant from the tumour, the increasing ratio of lumican to decorin was inversely correlated with the proliferation of the tumour cells (P = 0.035). The comparison of the level of expression of lumican protein in superficial vs. nodular subtypes of malignant melanomas showed a decrease of lumican but not decorin in the peritumoral stroma of nodular subtypes. In the peritumoral stroma, the level of expression of lumican but not decorin decreased significantly (P = 0.016) with increasing Clark levels. In addition, immunocytochemical and reverse transcription PCR analyses of malignant melanoma cell lines (A-375, HT-144) and of MRC-5 and dermal fibroblasts from healthy donors in vitro confirmed that dermal fibroblasts are responsible for lumican and decorin synthesis in skin. CONCLUSIONS. Lumican may regulate vertical progression of human malignant melanoma, but further study is necessary to clarify the antitumour mechanism and the downstream signal transduction pathways involved.

Vibrational spectroscopy studies of formalin-fixed cervix tissues.

Optical histopathology is fast emerging as a potential tool in cancer diagnosis. Fresh tissues in saline are ideal samples for optical histopathology. However, evaluation of suitability of ex vivo handled tissues is necessitated because of severe constraints in sample procurement, handling, and other associated problems with fresh tissues. Among these methods, formalin-fixed samples are shown to be suitable for optical histopathology. However, it is necessary to further evaluate this method from the point of view discriminating tissues with minute biochemical variations. A pilot Raman and Fourier transform infrared (FTIR) microspectroscopic studies of formalin-fixed tissues normal, malignant, and after-2-fractions of radiotherapy from the same malignant cervix subjects were carried out, with an aim to explore the feasibility of discriminating these tissues, especially the tissues after-2-fractions of radiotherapy from other two groups. Raman and FTIR spectra exhibit large differences for normal and malignant tissues and subtle differences are seen between malignant and after-2-fractions of radiotherapy tissues. Spectral data were analyzed by principal component analysis (PCA) and it provided good discrimination of normal and malignant tissues. PCA of data of three tissues, normal, malignant, and 2-fractions after radiotherapy, gave two clusters corresponding to normal and malignant + after-2-fractions of radiotherapy tissues. A second step of PCA was required to achieve discrimination between malignant and after-2-fractions of radiotherapy tissues. Hence, this study not only further supports the use of formalin-fixed tissues in optical histopathology, especially from Raman spectroscopy point of view, it also indicates feasibility of discriminating tissues with minute biochemical differences such as malignant and after-2-fractions of radiotherapy.

FTIR and Raman microspectroscopy of normal, benign, and malignant formalin-fixed ovarian tissues.

Ovarian cancer is the sixth most common cancer among women worldwide, and mortality rates from this cancer are higher than for other gynecological cancers. This is attributed to a lack of reliable screening methods and the inadequacy of treatment modalities for the advanced stages of the disease. FTIR and Raman spectroscopic studies of formalin-fixed normal, benign, and malignant ovarian tissues have been undertaken in order to investigate and attempt to understand the underlying biochemical changes associated with the disease, and to explore the feasibility of discriminating between these different tissue types. Raman spectra of normal tissues indicate the dominance of proteins and lower contents of DNA and lipids compared to malignant tissues. Among the pathological tissues studied, spectra from benign tissues seem to contain more proteins and less DNA and lipids compared to malignant tissue spectra. FTIR studies corroborate these findings. FTIR and Raman spectra of both normal and benign tissues showed more similarities than those of malignant tissues. Cluster analysis of first-derivative Raman spectra in the 700-1700 cm(-1) range gave two clear groups, one corresponding to malignant and the other to normal+benign tissues. At a lower heterogeneity level, the normal+benign cluster gave three nonoverlapping subclusters, one corresponding to normal and two for benign tissues. Cluster analysis of second-derivative FTIR spectra in the combined spectral regions of 1540-1680 and 1720-1780 cm(-1) resulted into two clear clusters corresponding to malignant and normal+benign tissues. The cluster corresponding to normal+benign tissues produced nonoverlapping subclusters for normal and benign tissues at a lower heterogeneity level. The findings of this study demonstrate the feasibility of Raman and FTIR microspectroscopic discrimination of formalin-fixed normal, benign, and malignant ovarian tissues.

[Ocular melanoma: immunohistochemical evaluation of the potentially predictive markers of prognosis. Preliminary results]. / Mélanome oculaire: évaluation immunohistochimique de marqueurs potentiellement prédictifs du pronostic. Résultats préliminaires.

PURPOSE: To study the characteristics of certain biological parameters, many of which have been recently discovered, to assess their possible contribution to the prognosis of ocular melanoma. MATERIAL: and methods: 25 patients with ocular and orbital melanoma treated at the Reims Regional Hospital since 1993 were included in the retrospective study. The surgical specimens were put in formalin 10%, then in paraffin. Five proteins were analyzed retrospectively: topoisomerase I, topoisomerase IIalpha, ICBP90, Ki67, and P53 on 200 cells per case. RESULTS: Of the 25 tumors from 14 men and 11 women, 15 were iris and ciliary body melanomas and ten choroidal melanomas. Histopathologic analysis showed 12 spindle-cell-type melanomas, seven epithelioid-cell-type melanomas and six mixed-cell-type melanomas. The immunohistochemical study found a significant difference in distribution between the cellular types for the topoisomerase IIalpha and Ki67 antibodies. Eight patients had metastasis, six of whom died. The metastasis came from three mixed-cell-type melanomas, three epithelioid-cell-type melanomas, and two spindle-cell-type melanomas. On the other hand, six cases of sclera infiltration were discovered among these eight patients. CONCLUSION: Several prognostic factors seem to be involved in the progression of melanoma: sclera infiltration seems to be essential in our study. Its rate seems to increase with the tumor size and in epithelioid-cell-type melanomas. The correlation between the different immunomarkers and the appearance of metastasis has not yet been verified.

In-vitro analysis of normal and aneurismal human ascending aortic tissues using FT-IR microspectroscopy.

FTIR microspectroscopy has shown to be a proven tool in the investigation of many tissue types. We have used this spectroscopic approach to analyse structural differences between normal and aneurismal aortic tissues and also aortas from patients with congenital anomalies like aortic bicuspid valves. Spectral analysis showed important variations in amide I and II regions, related to changes in alpha-helix and beta-sheet secondary structure of proteins that seem to be correlated to structural modifications of collagen and elastin. These proteins are the major constituents of the aortic wall associated to smooth muscular cells. The amide regions have thus been identified as a marker of structural modifications related to these proteins whose modifications can be associated to a given aortic pathological situation. Both univariate (total absorbance image and band ratio) and multivariate (principal components analysis) analyses of the spectral information contained in the infrared images have been performed. Differences between tissues have been identified by these two approaches and allowed to separate each group of aortic tissues. However, with univariate band ratio analysis, the pathological group was found to be composed of samples from aneurismal aortas associated or not with an aortic bicuspid valve. In contrast, PCA was able to separate these two types of aortic pathologies. For other groups, PCA and band ratio analysis can differentiate between normal, aneurismal, and none dilated aortas from patients with a bicuspid aortic valve.

[Nano-biocaptures for research and diagnostics in inflammation diseases and cancer]. / Nano-biocapteurs pour la recherche et les diagnostics des maladies inflammatoires et du cancer.

As part of the ongoing search for ways to decrease the mortality of different pathological conditions related to cancer and inflammatory diseases, nanotechnologies currently under evaluation offer potentially attractive tools for innovative methodologies for early diagnosis, new bioimaging techniques and therapeutic strategies. Nano-tools can be employed for various functions, such as the detection of lesions at very early stages of disease development, extremely precise anatomical localization, or evaluation of the efficacy of medications specifically targeted against cells and pathological tissues. We have synthesized homogeneous CdSe/ZnS (core/shell) highly fluorescent nanocrystals (NC) detectable as individual nanoparticules with a routine fluorescent microscope. These NC are at least 10-fold brighter than the best organic fluorophores and at least 1000-fold more stable against photobleaching than AlexaFluor, for example. When conjugated with proteins, DNA or with drugs, NCs may be excited with the light of any wavelength from UV through visible spectral region providing a range of fluorescence colors depending on their diameter. These properties provide excellent perspectives for high through-put multiplexing and long-term tracking of labeled precursors for days or even weeks. We present here NC applications for ultrasensitive detection of p-glycoprotein, cytokeratins, LCA, Ki67, etc. both on the cellular level and in pathological human surgical specimens.

Evaluation of the suitability of ex vivo handled ovarian tissues for optical diagnosis by Raman microspectroscopy.

A pilot Raman microspectroscopy study of formalin-fixed, paraffin-embedded, and deparaffinized sections from the same ovarian normal and malignant tissues was carried out. This approach was considered in order to evaluate the suitability of these ex vivo tissue handling procedures in discrimination as well as biochemical characterization. The spectra of formalin-fixed normal and malignant tissues exhibited no contamination due to formalin, which is indicated by the absence of strong formalin peaks; spectral features also show significant differences for normal and malignant tissues. The differences between spectral profiles of deparaffinized normal and malignant tissues are subtle and spectra show few residual sharp peaks of paraffin. Complete dominance of paraffin swamping signals from tissues was observed in the spectra of paraffin-embedded tissues. Principal components analysis (PCA), which was employed for discrimination of tissue type, provided good discrimination for formalin-fixed and paraffin-embedded tissue spectra. PCA of deparaffinized tissues resulted in a poor classification with significant overlap among the clusters. Thus, this study indicates that formalin fixation is the most suitable among the three procedures employed in the study. Significant differences between spectral profiles of normal and malignant formalin-fixed tissues can not only be exploited for discrimination but can also provide information on biochemical characteristics of the tissues. Deparaffinized tissues provide poor discrimination and information on tissue biochemistry is lost. Paraffin-embedded tissues may provide good discrimination, but predominance of paraffin in the spectra could jeopardize biochemical characterization. Prospectively, as a result of the better availability of paraffin-embedded tissues and problems associated with frozen sectioning of formalin-fixed tissues, the results of this study using paraffin-embedded tissues are very encouraging.

Micro-Raman spectroscopy for optical pathology of oral squamous cell carcinoma.

Micro-Raman spectra of formalin-fixed oral squamous normal and carcinoma tissues, stored at room temperature for 2 months, have been recorded. Spectra were recorded both in the epithelial and subepithelial regions of the tissues. No noticeable spectral contamination due to formalin was observed. Very significant differences between spectra of normal epithelial and malignant epithelial samples were found. No such differences in spectra of subepithelial malignant and subepithelial normal samples could be observed. This study shows that spectra from the epithelial region changes drastically because of malignancy-induced biochemical changes in this region. Major differences between normal and malignant spectra seem to arise from the protein composition, conformational/structural changes, and possible increase in protein content in malignant epithelia. The differences between normal epithelial and subepithelial spectra, as expected, arise mainly from the collagen in subepithelial tissue. Principal component analysis of the combined sets of spectra-epithelial and subepithelial, normal and malignant- showed that very good discrimination can be achieved by Raman microspectroscopy. This study thus validates the suitability of formalin-fixed tissues for optical pathology in oral malignancy.

Inhibition of multidrug resistance by immunisation with synthetic P-glycoprotein-derived peptides.

Overexpression of the membrane glycoprotein (P170) represents the most common multidrug resistance (MDR) mechanism in cancer therapy. Specific auto-antibodies to extracellular loops 1, 2 and 4 of murine P170 were elicited in mice using palmitoylated synthetic peptides reconstituted in liposomes, with or without Lipid A, and resuspended in alum. IgM antibodies were detected 14 days following the first injection and IgG1 became predominant after the third challenge. Animals did not show any auto-immune symptoms or induced toxicity up to 18 months after the immunisation. Previous immunisations of mice using liposomes with MDR1 peptides increases the efficacy of chemotherapy treatments with doxorubicin and vinblastine against P388 R cells with increase of 77% in the survival half time in the immunised group. Sera from the immunised mice were also effective in reducing cellular resistance to vinblastine and doxorubicin in vitro. Taken together, these data suggest that this immunisation approach might have potential clinical applications.

A method for simultaneous quantification of monoclonal antibody Ki-67 and DNA content by flow cytometry. Application to breast carcinomas.

OBJECTIVE: To seek a method assessing Ki-67 immunostaining and DNA content by flow cytometry simultaneously. STUDY DESIGN: The murine monoclonal antibody Ki-67 (Ki) identifies a nuclear protein complex expressed by all nonquiescent tumor cells. Since the antigen detected by Ki is quite labile in most fixation and embedding protocols, a new method for simultaneous quantification of nuclear Ki immunofluorescence and DNA content by flow cytometry was developed. Unfixed, solid tumor cells are permeabilized only with saponin to preserve Ki antigen. The percentage of Ki-positive cell subpopulations calculated by subtraction of the related aspecific fixation histogram gives optimal results more rapidly than by cytogram analysis. Application to breast carcinoma shows the feasibility of the method. RESULTS: Significant correlations between Ki staining and the S-phase fraction were observed. Mean Ki labelling rates of aneuploid tumors were significantly higher than those of the diploid tumors, and significant differences between histologic types were found. CONCLUSION: This technique can be considered a fast, sensitive and optimal method to evaluate the proliferative activity of breast carcinomas and possibly of other solid tumors in a department of pathology.