Diagnosis Of The Chronic Lymphocytic Leukemia (CLL) Using A Raman-Based Scanner Optimized For Blood Smear Analysis (M3s Project)

Introduction/ Background In hematology, actual diagnosis of B chronic lymphocyte-leukemia (CLL) is based on the microscopic analysis of cell morphology from patient blood smear. However, new photonic technologies appear promising to facilitate and improve the early diagnosis, prognostic and monitoring of personalized therapy. The development of automated diagnostic approaches could assist clinicians in improving the efficiency and quality of health services, but also reduce medical costs. Aims The M3S project aims at improving the diagnosis and prognosis of the CLL pathology by developing a multimodal microscopy platform, including Raman spectrometry, dedicated to the automatic analysis of lymphocytes. Methods Blood smears were prepared on glass slides commonly used in pathology laboratories for microscopy. Two types of sample per patient were prepared: a conventional blood smear and a deposit of “pure” lymphocyte subtypes (i.e. normal B, CLL B, T and NK), sorted out in flow cytometry by using the negative double labeling technique. The second sample is used for the construction of a database of spectral markers specific of these different cell types. The preparations were analyzed with the multimodal machine which combines i) a Raman micro-spectrometer, equipped with a 532nm diode laser excitation source; ii) a microscope equipped with 40x and 150x lenses and a high precision xyz motorized stage for scanning the blood smear, and localizing x-y coordinates of representative series (~100 for each patient) of lymphocyte cells before registering three Raman spectra; these cells of interest being previously localized by an original method based on the morphology analysis. After the Raman acquisitions, the conventional blood smears were submitted to immunolabelling using specific antibodies. For the establishment of the Raman classifiers, this post-acquisition treatment was used as reference to distinguish the different lymphocyte sub-populations. Raman data were then analyzed using chemometric processing and supervised statistical classifiers in order to construct a spectral library of markers highly specific of the lymphocyte type and status (normal or pathological). Results Currently, a total of 60 patients (CLL and healthy) were included in the study. Various classification methods such as LDA (Linear Discriminant Analysis), PLS-DA (Partial Least Square Discriminant Analysis), RF (Random Forest) and SVM (Support Vector Machine), were tested in the purpose to distinguish tumoral B lymphocytes from other cell types. These classification algorithms were combined with feature selection approaches. The best performances were around 70% of correct identification when a three-class model (B-CLL vs B-normal vs T and NK lymphocytes) was considered, and 80% in case of a two-class model (B-CLL vs B-normal lymphocytes). These encouraging results demonstrate the potential of Raman micro-spectroscopy coupled to supervised classification algorithms for leukemic cell classification. The approach can find interest more generally in the field of cyto-hematology. Further developments will concern the integration of additional modality such as Quantitative Phase Imaging on one hand to speed the exploration process of cells of interest to be probed, and on the other hand to extract additional characteristics likely to be informative for CLL diagnosis. In addition, the identification of prognostic markers will be investigated by confronting the photonic data to clinical patient information.

Proteomic Interrogation of Androgen Action in Prostate Cancer Cells Reveals Roles of Aminoacyl tRNA Synthetases

Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen-treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer

Suppression of Estrogen Receptor Transcriptional Activity by Connective Tissue Growth Factor

Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER) that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF) physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs

Characterization of Type I and IV Collagens by Raman Microspectroscopy: Identification of Spectral Markers of the Dermo-Epidermal Junction

Abstract. Type I and IV collagens are important constituents of the skin. Type I collagen is found in all dermal layers in high proportion, while type IV collagen is localized in the basement membrane of the dermo-epidermal junction (DEJ). These proteins are strongly altered during aging or cancer progression. Although they possess amino acid compositions which, are close, they present also important structural differences inducing specific physicochemical properties. Raman spectroscopy is based on a nondestructive interaction of the light with the matter. This technique permits to probe the intrinsic molecular composition of the samples without staining or particular preparation. The aim of our research is to study the correlation between the molecular conformations of type I and IV collagens and their Raman features. We showed that signals specific of each protein can be revealed and that they translate structural differences between the two collagens. From this collagens spectral characterization, the analysis of skin sections also permitted to identify spectral markers of dermis, epidermis, and epidermis/dermis interface. These preliminary results represent basic data for further studies, particularly to probe skin molecular alterations induced by chronologic aging

Récepteur des androgènes : Pathologie moléculaire

Les androgènes sont responsables de la différenciation, du développement et du maintien du phénotype masculin en participant à la régulation de nombreux organes androgéno-dépendants, à travers un récepteur nucléaire, le récepteur des androgènes (RcA). Après liaison de la testostérone ou de la dihydrotestosterone, le RcA se fixe sur des séquences d’ADN spécifiques et active la transcription de gènes-cibles. Des progrès considérables ont été accomplis dans les domaines moléculaires et structuraux du RcA comme dans celui de la régulation transcriptionnelle des gènes androgéno-régulés. Ils permettent d’envisager une meilleure approche de la relation structure/fonction du RcA à partir de l’analyse moléculaire des syndromes d’insensibilité aux androgènes (l’étiologie la plus fréquente des pseudo-hermaphrodismes masculins), des cancers de la prostate hormono-résistants, de l’amyotrophie spinobulbaire (Maladie de Kennedy), de stérilité isolée.La génétique moléculaire permet aujourd’hui d’affirmer le diagnostic de l’une de ces affections par l’identification d’une mutation du gène du RcA, par la confirmation de ses conséquences in vitro après reconstruction de la mutation, par l’analyse tri-dimensionnelle du RcA et des conséquences de la mutation sur la structure du RcA.Dans le domaine des insensibilités aux androgènes, l’absence d’identification de mutation du RcA a fait envisager la responsabilité d’anomalie de cofacteurs transcriptionnels. Dans ce sens, l’analyse du profil d’expression de ces cofacteurs couplée à l’expertise de la fonctionnalité du RcA endogène sur fibroblastes du patient s’avère prometteuse. En réalité, la mise en évidence de gènes-cibles (au niveau des organes génitaux externes) constituera l’étape fondamentale pour évaluer le degré de l’action cellulaire et moléculaire des androgènes

Application Of Raman Microscopy For The Diagnosis Of The Chronic Lymphocytic Leukemia (Cll) : PS01.01 | ePoster Session I

INTRODUCTION / BACKGROUND: In hematology, actual diagnosis of B chronic lymphocyte-leukemia (CLL) is based on the microscopic analysis of cell morphology from patient blood smear. However, new photonic technologies appear promising to facilitate and improve the early diagnosis, prognostic and monitoring of personalized therapy. The development of automated diagnostic approaches could assist clinicians in improving the efficiency and quality of health services, but also reduce medical costs. AIMS: The M3S project aims at improving the diagnosis and prognosis of the CLL pathology by developing a multimodal microscopy platform, including Raman spectrometry, dedicated to the automatic analysis of lymphocytes. METHODS: Blood smears were prepared on glass slides commonly used in pathology laboratories for microscopy. Two types of sample per patient were prepared: a conventional blood smear and a deposit of “pure” lymphocyte subtypes (i.e. normal B, CLL B, T and NK), sorted out in flow cytometry by using the negative double labelling technique. The second sample is used for the construction of a database of spectral markers specific of these different cell types. The preparations were analyzed with the multimodal machine which combines i) a Raman micro-spectrometer, equipped with a 532nm diode laser excitation source; ii) a micro- scope equipped with 40x and 150x lenses and a high precision xyz motorized stage for scanning the blood smear, and localizing x-y coordinates of representative series (~100 for each patient) of lymphocyte cells before registering three Raman spectra; these cells of interest being previously localized by an original method based on the morphology analysis. After the Raman acquisitions, the conventional blood smears were submitted immunolabelling using specific antibodies. For the establishment of the Raman classifiers, this post-acquisition treatment was used as reference to distinguish the different lymphocyte sub-populations. Raman data were then analyzed using chemometric processing and supervised statistical classifiers in order to construct a spectral library of markers highly specific of the lymphocyte type and status (normal or pathological). RESULTS: Currently, a total of 60 patients (CLL and healthy) were in- cluded in the study. Various classification methods such as LDA (Linear Discriminant Analysis), PLS-DA (Partial Least Square Discriminant Analysis), RF (RandomForest) and SVM (Support Vector Machine), were tested in the purpose to distinguish tumoral B lymphocytes from other cell types. These classification algorithms were combined with feature selection approaches. The best performances were around 70% of correct identification when a three-class model (B-CLL vs B-normal vs T and NK lymphocytes) was considered, and 80% in case of a two-class model (B-CLL vs B-normal lymphocytes). These encouraging results demonstrate the potential of Raman micro-spectroscopy coupled to supervised classification algorithms for leukemic cell classification. The approach can find interest more generally in the field of cyto-hematology. Further developments will concern the integration of additional modality such as Quantitative Phase Imaging on one hand to speed the exploration process of cells of interest to be probed, and on the other hand to extract additional characteristics likely to be informative for CLL diagnosis. In addition, the identification of prognostic markers will be investigated by confronting the photonic data to clinical patient information

Proteomic profiling of epididymis and vas deferens: identification of proteins regulated during rat genital tract development.

Epididymis and vas deferens form part of the male internal genital tract and are dependent on androgens for their growth and development. To better understand the molecular action of androgens during male genital tract development, protein expression profiles were generated using two-dimensional gels, for rat epididymides and vasa deferentia isolated on embryonic days (E) 17-21. Proteins that were differentially expressed between E17 and E21 were cut from the gels, digested into tryptic peptides and analyzed on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Using this approach, 20 proteins could be identified that were regulated in time and were categorized into cytoskeletal proteins, nuclear proteins, transport proteins, chaperones, and enzymes (mainly glycolytic). Furthermore, epididymides and vasa deferentia isolated on E19 were cultured in vitro in the absence or presence of 10 nm of the synthetic androgen R1881, for 9, 24, and 48 h. Under these conditions, regulation and posttranslational modification were observed for glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, heterogeneous nuclear ribonucleoprotein A2/B1 and heterogeneous nuclear ribonucleoprotein A3, similar to the observed changes in vivo. In addition, posttranslational modification of RhoGDI1 (also named RhoGDIalpha) was found in response to androgen. Androgen-induced posttranslational modification of RhoGDI1 and glycolytic enzymes may be an important functional link between signaling pathways and cytoskeletal rearrangements in control of growth and development of the male internal genital tract

Proteomic profiling of epididymis and vas deferens: identification of proteins regulated during rat genital tract development.

Epididymis and vas deferens form part of the male internal genital tract and are dependent on androgens for their growth and development. To better understand the molecular action of androgens during male genital tract development, protein expression profiles were generated using two-dimensional gels, for rat epididymides and vasa deferentia isolated on embryonic days (E) 17-21. Proteins that were differentially expressed between E17 and E21 were cut from the gels, digested into tryptic peptides and analyzed on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Using this approach, 20 proteins could be identified that were regulated in time and were categorized into cytoskeletal proteins, nuclear proteins, transport proteins, chaperones, and enzymes (mainly glycolytic). Furthermore, epididymides and vasa deferentia isolated on E19 were cultured in vitro in the absence or presence of 10 nm of the synthetic androgen R1881, for 9, 24, and 48 h. Under these conditions, regulation and posttranslational modification were observed for glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, heterogeneous nuclear ribonucleoprotein A2/B1 and heterogeneous nuclear ribonucleoprotein A3, similar to the observed changes in vivo. In addition, posttranslational modification of RhoGDI1 (also named RhoGDIalpha) was found in response to androgen. Androgen-induced posttranslational modification of RhoGDI1 and glycolytic enzymes may be an important functional link between signaling pathways and cytoskeletal rearrangements in control of growth and development of the male internal genital tract

Effect of substrate choice and tissue type on tissue preparation for spectral histopathology by Raman microspectroscopy

Raman spectroscopy is a non-destructive, non-invasive, rapid and economical technique which has the potential to be an excellent method for the diagnosis of cancer and understanding disease progression through retrospective studies of archived tissue samples. Historically, biobanks are generally comprised of formalin fixed paraffin preserved tissue and as a result these specimens are often used in spectroscopic research. Tissue in this state has to be dewaxed prior to Raman analysis to reduce paraffin contributions in the spectra. However, although the procedures are derived from histopathological clinical practice, the efficacy of the dewaxing procedures that are currently employed is questionable. Ineffective removal of paraffin results in corruption of the spectra and previous experiments have shown that the efficacy can depend on the dewaxing medium and processing time. The aim of this study was to investigate the influence of commonly used spectroscopic substrates (CaF2, Spectrosil quartz and low-E slides) and the influence of different histological tissue types (normal, cancerous and metastatic) on tissue preparation and to assess their use for spectral histopathology. Results show that CaF2 followed by Spectrosil contribute the least to the spectral background. However, both substrates retain paraffin after dewaxing. Low-E substrates, which exhibit the most intense spectral background, do not retain wax and resulting spectra are not affected by paraffin peaks. We also show a disparity in paraffin retention depending upon the histological identity of the tissue with abnormal tissue retaining more paraffin than normal