Potential of mid-infrared spectroscopy as a non-invasive diagnostic test in urine for endometrial or ovarian cancer.

The current lack of an accurate, cost-effective and non-invasive test that would allow for screening and diagnosis of gynaecological carcinomas, such as endometrial and ovarian cancer, signals the necessity for alternative approaches. The potential of spectroscopic techniques in disease investigation and diagnosis has been previously demonstrated. Here, we used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to analyse urine samples from women with endometrial (n = 10) and ovarian cancer (n = 10), as well as from healthy individuals (n = 10). After applying multivariate analysis and classification algorithms, biomarkers of disease were pointed out and high levels of accuracy were achieved for both endometrial (95% sensitivity, 100% specificity; accuracy: 95%) and ovarian cancer (100% sensitivity, 96.3% specificity; accuracy 100%). The efficacy of this approach, in combination with the non-invasive method for urine collection, suggest a potential diagnostic tool for endometrial and ovarian cancers.

Synchrotron- and focal plane array-based Fourier-transform infrared spectroscopy differentiates the basalis and functionalis epithelial endometrial regions and identifies putative stem cell regions of human endometrial glands.

The cyclical process of regeneration of the endometrium suggests that it may contain a cell population that can provide daughter cells with high proliferative potential. These cell lineages are clinically significant as they may represent clonogenic cells that may also be involved in tumourigenesis as well as endometriotic lesion development. To determine whether the putative stem cell location within human uterine tissue can be derived using vibrational spectroscopy techniques, normal endometrial tissue was interrogated by two spectroscopic techniques. Paraffin-embedded uterine tissues containing endometrial glands were sectioned to 10-µm-thick parallel tissue sections and were floated onto BaF2 slides for synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy and globar focal plane array-based FTIR spectroscopy. Different spectral characteristics were identified depending on the location of the glands examined. The resulting infrared spectra were subjected to multivariate analysis to determine associated biophysical differences along the length of longitudinal and crosscut gland sections. Comparison of the epithelial cellular layer of transverse gland sections revealed alterations indicating the presence of putative transient-amplifying-like cells in the basalis and mitotic cells in the functionalis. SR-FTIR microspectroscopy of the base of the endometrial glands identified the location where putative stem cells may reside at the same time pointing towards νsPO2- in DNA and RNA, nucleic acids and amide I and II vibrations as major discriminating factors. This study supports the view that vibration spectroscopy technologies are a powerful adjunct to our understanding of the stem cell biology of endometrial tissue. Graphical abstract ᅟ.

ATR-FTIR spectroscopy coupled with chemometric analysis discriminates normal, borderline and malignant ovarian tissue: classifying subtypes of human cancer.

Surgical management of ovarian tumours largely depends on their histo-pathological diagnosis. Currently, screening for ovarian malignancy with tumour markers in conjunction with radiological investigations has a low specificity for discriminating benign from malignant tumours. Also, pre-operative biopsy of ovarian masses increases the risk of intra-peritoneal dissemination of malignancy. Intra-operative frozen section, although sufficiently accurate in differentiating tumours according to their histological type, increases operation times. This results in increased surgery-related risks to the patient and additional burden to resource allocation. We set out to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, combined with chemometric analysis can be applied to discriminate between normal, borderline and malignant ovarian tumours and classify ovarian carcinoma subtypes according to the unique spectral signatures of their molecular composition. Formalin-fixed, paraffin-embedded ovarian tissue blocks were de-waxed, mounted on Low-E slides and desiccated before being analysed using ATR-FTIR spectroscopy. Chemometric analysis in the form of principal component analysis (PCA), successive projection algorithm (SPA) and genetic algorithm (GA), followed by linear discriminant analysis (LDA) of the obtained spectra revealed clear segregation between benign versus borderline versus malignant tumours as well as segregation between different histological tumour subtypes, when these approaches are used in combination. ATR-FTIR spectroscopy coupled with chemometric analysis has the potential to provide a novel diagnostic approach in the accurate diagnosis of ovarian tumours assisting surgical decision making to avoid under-treatment or over-treatment, with minimal impact to the patient.

Fourier-transform infrared spectroscopy coupled with a classification machine for the analysis of blood plasma or serum: a novel diagnostic approach for ovarian cancer.

Currently available screening tests do not deliver the required sensitivity and specificity for accurate diagnosis of ovarian or endometrial cancer. Infrared (IR) spectroscopy of blood plasma or serum is a rapid, versatile, and relatively non-invasive approach which could characterize biomolecular alterations due to cancer and has potential to be utilized as a screening or diagnostic tool. In the past, no such approach has been investigated for its applicability in screening and/or diagnosis of gynaecological cancers. We set out to determine whether attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy coupled with a proposed classification machine could be applied to IR spectra obtained from plasma and serum for accurate class prediction (cancer vs. normal). Plasma and serum samples were obtained from ovarian cancer cases (n = 30), endometrial cancer cases (n = 30) and non-cancer controls (n = 30), and subjected to ATR-FTIR spectroscopy. Four derived datasets were processed to estimate the real-world diagnosis of ovarian and endometrial cancer. Classification results for ovarian cancer were remarkable (up to 96.7%), whereas endometrial cancer was classified with a relatively high accuracy (up to 81.7%). The results from different combinations of feature extraction and classification methods, and also classifier ensembles, were compared. No single classification system performed best for all different datasets. This demonstrates the need for a framework that can accommodate a diverse set of analytical methods in order to be adaptable to different datasets. This pilot study suggests that ATR-FTIR spectroscopy of blood is a robust tool for accurate diagnosis, and carries the potential to be utilized as a screening test for ovarian cancer in primary care settings. The proposed classification machine is a powerful tool which could be applied to classify the vibrational spectroscopy data of different biological systems (e.g., tissue, urine, saliva), with their potential application in clinical practice.

High contrast images of uterine tissue derived using Raman microspectroscopy with the empty modelling approach of multivariate curve resolution-alternating least squares.

Approaches that allow one to rapidly understand tissue structure and functionality in situ remain to be developed. Such techniques are required in many instances, including where there is a need to remove with a high degree of confidence positive tumour margins during surgical excision. As biological tissue has little contrast, gold standard confirmation of surgical margins is conventionally undertaken by histopathological diagnosis of tissue architecture via optical microscopy. Vibrational spectroscopy techniques, when coupled to sophisticated computational analyses, are capable of constructing bio-molecular contrast images of unstained tissue. To assess the relative applicability of a range of candidate algorithms to distinguish the in situ bio-molecular structures of a complex tissue, the empty modelling approach of multivariate curve resolution-alternating least squares (MCR-ALS) was compared to hierarchical cluster analysis (HCA) or principal component analysis (PCA). Such chemometric analyses were applied to Raman images of benign (tumour-adjacent) endometrium, stage I and stage II endometrioid cancer. Re-constructed images from the in situ bio-molecular tissue architectures highlighted features associated with glandular epithelium, stroma, glandular lumen and myometrium. Of the tested chemometric analyses, MCR-ALS provided the best bio-molecular contrast images, superior to those derived following HCA or PCA, with clear and defined margins of histological features. Iteratively-resolved spectra identified wavenumbers responsible for the contrast image. Wavenumbers 1234 cm(-1) (Amide III), 1390 cm(-1) (CH(3) bend), 1675 cm(-1) (Amide I/lipid), 1275 cm(-1) (Amide III), 918 cm(-1) (proline) and 936 cm(-1) (proline, valine and proteins) were responsible for generating the majority of the contrast within MCR-ALS-generated images. Applications of sophisticated computational analyses coupled with vibrational spectroscopy techniques have the potential to lend novel functionality insights into bio-molecular structures in vivo.

Fourier-transform infrared spectroscopy discriminates a spectral signature of endometriosis independent of inter-individual variation.

Endometriosis is the growth of endometrial tissue outside of the uterine cavity. Its aetiology remains obscure, and it is difficult to diagnose ranging from asymptomatic to debilitating disease. Mid-infrared (IR) spectroscopy has become recognised as a potential clinical diagnostic tool. Biomolecules absorb mid-IR (4000 cm(-1) to 400 cm(-1)) and from this, a biochemical-cell fingerprint in the form of an absorbance spectrum can be derived. We set out to determine if IR spectroscopy could be used to identify underlying biochemical differences between endometrial tissues growing outside of the uterus (ectopic) from endometrial tissue of the uterus (eutopic). For comparative purposes, endometrial tissues from endometriosis-free women were also obtained (benign eutopic). Attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy or transmission FTIR microspectroscopy was employed for spectral acquisition. Principal component analysis (PCA)-linear discriminant analysis (LDA) was used for chemometric analysis. A clear segregation was exhibited between the three categories independent of inter-individual confounding differences. Importantly, there was a marked difference between eutopic endometrial tissue from patients with or without endometriosis. This indicates that IR spectroscopy coupled with multivariate analysis (e.g., PCA-LDA) may provide a non-invasive diagnostic tool for endometriosis. By analysing the underlying biochemistry of these endometrial tissues, this approach may facilitate a better understanding of this pathology.

Detecting Endometrial Cancer by Blood Spectroscopy: A Diagnostic Cross-Sectional Study.

Endometrial cancer is the sixth most common cancer in women, with a rising incidence worldwide. Current approaches for the diagnosis and screening of endometrial cancer are invasive, expensive or of moderate diagnostic accuracy, limiting their clinical utility. There is a need for cost-effective and minimally invasive approaches to facilitate the early detection and timely management of endometrial cancer. We analysed blood plasma samples in a cross-sectional diagnostic accuracy study of women with endometrial cancer (n = 342), its precursor lesion atypical hyperplasia (n = 68) and healthy controls (n = 242, total n = 652) using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and machine learning algorithms. We show that blood-based infrared spectroscopy has the potential to detect endometrial cancer with 87% sensitivity and 78% specificity. Its accuracy is highest for Type I endometrial cancer, the most common subtype, and for atypical hyperplasia, with sensitivities of 91% and 100%, and specificities of 81% and 88%, respectively. Our large-cohort study shows that a simple blood test could enable the early detection of endometrial cancer of all stages in symptomatic women and provide the basis of a screening tool in high-risk groups. Such a test has the potential not only to differentially diagnose endometrial cancer but also to detect its precursor lesion atypical hyperplasia-the early recognition of which may allow fertility sparing management and cancer prevention.

Elevated expression of CYP1A1 and gamma-SYNUCLEIN in human ectopic (ovarian) endometriosis compared with eutopic endometrium.

Endometriosis is a debilitating disease in which apoptotic, genetic, immunological, angiogenic and environmental factors have been implicated. Endocrine-disrupting agents (e.g. dioxins) might be involved. Dioxins, via the arylhydrocarbon receptor (AhR), induce estrogen-metabolizing enzymes CYP1A1 and CYP1B1. Elevated expression of gamma-SYNUCLEIN (gamma-SYN) has been associated with hormone-related conditions. Tissue sets consisting of eutopic and ectopic (ovarian) endometrium from patients with stage 3 or 4 endometriosis were obtained. Following RNA extraction and reverse transcription, quantitative real-time reverse transcriptase-polymerase chain reaction was performed for anti-apoptotic B-cell leukaemia/lymphoma 2 (BCL-2), CYP1A1, CYP1B1, estrogen receptor (ER)alpha, ER beta and gamma-SYN. Immunohistochemical analyses for gamma-syn, ER alpha, ER beta and CYP1A1 were also conducted. A 3-9-fold increase in intra-individual expression of CYP1A1 in ectopic (ovarian) endometrium compared with eutopic tissue was observed; immunohistochemical analyses pointed to CYP1A1 being localized to the glandular epithelium. This intra-individual expression profile was not observed for CYP1B1 or BCL-2. However, a 5-53-fold intra-individual increase in gamma-SYN expression was also demonstrated in six of nine tissue sets (a further two showed an increase that was not considered significant) when comparing ectopic to eutopic endometrium; gamma-syn positivity was associated with endothelial cells. An elevation in ER beta was also noted when comparing ectopic to eutopic endometrium; with regard to ER alpha, this was inconsistent. These results suggest an up-regulation of dioxin-inducible CYP1A1 and gamma-SYN occurs in endometriosis. Whether gamma-syn may be a novel diagnostic marker for endometriosis remains to be ascertained.

Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium.

Tamoxifen has been used in the management of receptor-positive breast cancer for >20 years. Usage confers an elevated risk of developing endometrial carcinoma. Its mechanism of carcinogenicity remains unresolved with controversy as to whether or not this is mediated through a genotoxic mechanism. Usage is not only associated with an elevated occurrence of endometrioid endometrial carcinoma, but also type 2 and mixed epithelial-stromal tumours (MESTs) that have a poorer prognosis. Following hysterectomy, endometrial tissues (n=18) classified as benign (n=6), non-tamoxifen-associated carcinoma (n=6) and tamoxifen-associated carcinoma (n=6) were obtained; quantitative gene expression was performed. Employing real-time RT-PCR, the relative gene expressions of phase I/II metabolic enzymes CYP1A2, CYP1B1 and CYP3A4, cathechol-O-methyltransferase (COMT) and SULT2A1 were ascertained. Measurable mRNA transcripts, especially for those genes associated with tamoxifen bioactivation, were quantifiable in all the tissues examined. Whether this is evidence that generation of genotoxic tamoxifen metabolites may occur in human endometrial tissue remains to be ascertained.

Raman spectroscopic techniques to detect ovarian cancer biomarkers in blood plasma.

Robust diagnosis of ovarian cancer is crucial to improve patient outcomes. The lack of a single and accurate diagnostic approach necessitates the advent of novel methods in the field. In the present study, two spectroscopic techniques, Raman and surface-enhanced Raman spectroscopy (SERS) using silver nanoparticles, have been employed to identify signatures linked to cancer in blood. Blood plasma samples were collected from 27 patients with ovarian cancer and 28 with benign gynecological conditions, the majority of which had a prolapse. Early ovarian cancer cases were also included in the cohort (n = 17). The derived information was processed to account for differences between cancerous and healthy individuals and a support vector machine (SVM) algorithm was applied for classification. A subgroup analysis using CA-125 levels was also conducted to rule out that the observed segregation was due to CA-125 differences between patients and controls. Both techniques provided satisfactory diagnostic accuracy for the detection of ovarian cancer, with spontaneous Raman achieving 94% sensitivity and 96% specificity and SERS 87% sensitivity and 89% specificity. For early ovarian cancer, Raman achieved sensitivity and specificity of 93% and 97%, respectively, while SERS had 80% sensitivity and 94% specificity. Five spectral biomarkers were detected by both techniques and could be utilised as a panel of markers indicating carcinogenesis. CA-125 levels did not seem to undermine the high classification accuracies. This minimally invasive test may provide an alternative diagnostic and screening tool for ovarian cancer that is superior to other established blood-based biomarkers.

Tamoxifen: important considerations of a multi-functional compound with organ-specific properties.

Tamoxifen remains a frontline treatment for hormone-responsive breast cancer despite its use being associated with a 2-7-fold elevated risk of developing endometrial carcinoma. Several groups have investigated whether tamoxifen induces DNA-damaging (genotoxic) versus non-genotoxic mechanisms. Some studies point to the presence of tamoxifen-DNA adducts while others suggest otherwise. In many of these studies, the histological sub-type has not been considered; as type 1 carcinomas are associated with PTEN and KRAS2 mutations whereas type 2 carcinomas exhibit TP53 and ERBB-2 mutations, the absence of this information makes comparisons between such independent investigations difficult. An examination of the sub-types of endometrial carcinoma points to histological and mechanistic distinctions between sporadic and tamoxifen-associated disease; this could suggest differing aetiologies. On this basis, we propose a dual mechanism of action highlighted by the different patterns of endometrial carcinoma sub-types. Tamoxifen may initially be pro-oestrogenic in the endometrium giving rise to elevated type 1 endometrioid carcinoma occurrence whereas after long-term use, there is an increase of type 2 disease or malignant mixed mullerian tumours associated with a hormone-independent mechanism of action. Despite these associated risk factors, and the introduction of new selective oestrogen receptor modulators (SERMs), we suggest that the organ-specific pleiotrophic effects of tamoxifen mean that this effective therapeutic agent for breast cancer will continue to have significant usage. The focus of future research should concentrate on the different aetiologies of tamoxifen-associated endometrial carcinomas while efforts continue to develop future SERMs.

IR microspectroscopy: potential applications in cervical cancer screening.

Screening exfoliative cytology for early dysplastic cells reduces incidence and mortality from squamous carcinoma of the cervix. In the developed world, screening programmes have adopted a 3-5 years recall system. In its absence, cervical cancer would be the second most common female cancer in these regions; instead, it is currently eleventh. However, there exist a number of limitations to the smear test even given the removal of contaminants using liquid-based cytology. It is prohibitively expensive, labour-intensive and subject to inaccuracies that give rise to significant numbers of false negatives. There remains a need for novel approaches to allow efficient and objective interrogation of exfoliative cytology. Methods that variously exploit infrared (IR) microspectroscopy are one possibility. Using IR microspectroscopy, an integrated 'biochemical-cell fingerprint' of the lipid, protein and carbohydrate composition of a biomolecular entity may be derived in the form of a spectrum via vibrational transitions of individual chemical bonds. Powerful statistical approaches (e.g. principal component analysis) now facilitate the interrogation of large amounts of spectroscopic data to allow the extraction of what may be small but extremely significant biomarker differences between disease-free and pre-malignant or malignant samples. An increasing wealth of literature points to the ability of IR microspectroscopy to allow the segregation of cells based on their disease status. We review the current evidence supporting its diagnostic potential in cancer biology.

A biospectroscopic analysis of human prostate tissue obtained from different time periods points to a trans-generational alteration in spectral phenotype.

Prostate cancer is the most commonly-diagnosed malignancy in males worldwide; however, there is marked geographic variation in incidence that may be associated with a Westernised lifestyle. We set out to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) or Raman spectroscopy combined with principal component analysis-linear discriminant analysis or variable selection techniques employing genetic algorithm or successive projection algorithm could be utilised to explore differences between prostate tissues from differing years. In total, 156 prostate tissues from transurethral resection of the prostate procedures for benign prostatic hyperplasia from 1983 to 2013 were collected. These were distributed to form seven categories: 1983-1984 (n = 20), 1988-1989 (n = 25), 1993-1994 (n = 21), 1998-1999 (n = 21), 2003-2004 (n = 21), 2008-2009 (n = 20) and 2012-2013 (n = 21). Ten-µm-thick tissue sections were floated onto Low-E (IR-reflective) slides for ATR-FTIR or Raman spectroscopy. The prostate tissue spectral phenotype altered in a temporal fashion. Examination of the two categories that are at least one generation (30 years) apart indicated highly-significant segregation, especially in spectral regions containing DNA and RNA bands (≈1,000-1,490 cm(-1)). This may point towards alterations that have occurred through genotoxicity or through epigenetic modifications. Immunohistochemical studies for global DNA methylation supported this. This study points to a trans-generational phenotypic change in human prostate.

Determination using synchrotron radiation-based Fourier transform infrared microspectroscopy of putative stem cells in human adenocarcinoma of the intestine: corresponding benign tissue as a template.

The epithelial-cell layer lining the two morphologically and functionally distinct segments of the mammalian intestinal tract, small intestine, and colon is constantly being renewed. This renewal is necessitated by a harsh lumen environment and is hypothesized to be driven by a small population of stem cells (SCs) that are believed to reside at the base of intestinal crypts. A lack of specific markers has hampered previous attempts to identify their exact location. We obtained tissue sections containing small intestine and colon crypts derived from normal (benign) or adenocarcinoma (AC) human intestine. The samples were floated onto BaF2 windows and analyzed using synchrotron radiation-based Fourier transform infrared microspectroscopy via an aperture size of 10 × 10 µm. Derived infrared (IR) spectral data was then analyzed using principal component analysis and/or linear discriminant analysis. Hypothesized cell types (as a function of aperture location along the length of individual crypts) within benign crypts were classed based on exploratory unsupervised IR spectral point clustering. Scores plots derived from individual small intestine crypts consistently generated one or two distinct spectra that clustered away from the remaining cell categories; these were retrospectively classed as "distinct base region" spectra. In these plots, a clear progression of locations along crypt lengths designated as from putative stem cells (SCs) to transit-amplifying (TA) cells to terminally differentiated (TD) cells was observed in benign small intestine and colon crypts. This progression of spectral points was crypt specific, pointing away from a unifying cell lineage model in human intestinal crypts. On comparison of AC-derived spectra versus corresponding benign, a subpopulation of AC-derived spectra suggested a putative SC-like spectral fingerprint; remaining IR spectra were classed as exhibiting TA cell-like or TD cell-like spectral characteristics. These observations could point to a cancer SC phenotype; an approach capable of identifying their in situ location has enormous therapeutic applications.

Diagnostic segregation of human brain tumours using Fourier-transform infrared and/or Raman spectroscopy coupled with discriminant analysis.

The most common initial treatment received by patients with a brain tumour is surgical removal of the growth. Precise histopathological diagnosis of brain tumours is to some extent subjective. Furthermore, currently available diagnostic imaging techniques to delineate the excision border during cytoreductive surgery lack the required spatial precision to aid surgeons. We set out to determine whether infrared (IR) and/or Raman spectroscopy combined with multivariate analysis could be applied to discriminate between normal brain tissue and different tumour types (meningioma, glioma and brain metastasis) based on the unique spectral "fingerprints" of their biochemical composition. Formalin-fixed paraffin-embedded tissue blocks of normal brain and different brain tumours were de-waxed, mounted on low-E slides and desiccated before being analyzed using attenuated total reflection Fourier-transform IR (ATR-FTIR) and Raman spectroscopy. ATR-FTIR spectroscopy showed a clear segregation between normal and different tumour subtypes. Discrimination of tumour classes was also apparent with Raman spectroscopy. Further analysis of spectral data revealed changes in brain biochemical structure associated with different tumours. Decreased tentatively-assigned lipid-to-protein ratio was associated with increased tumour progression. Alteration in cholesterol esters-to-phenylalanine ratio was evident in grade IV glioma and metastatic tumours. The current study indicates that IR and/or Raman spectroscopy have the potential to provide a novel diagnostic approach in the accurate diagnosis of brain tumours and have potential for application in intra-operative diagnosis.

Expression of ERα, its ERαΔ3 Splice Variant and γ-SYNUCLEIN in Ovarian Cancer: A Pilot Study.

AIMS: Ovarian cancer has the highest mortality of any gynaecological malignancy; this is due to rapid peritoneal spread of tumour cells and neovascularization. Understanding the mechanisms underlying this is critical to developing early diagnostic or treatment strategies. We devised a pilot study to examine the role of γ-SYNUCLEIN (γ-SYN), oestrogen receptor (ER)α, and the splice variant ERαΔ3. METHODOLOGY: With ethical approval, ovarian tissue was collected from patients (n=24) undergoing oopherectomy for non-ovarian pathology or primary surgery for suspected ovarian cancer. Quantitative gene expression analysis was employed for γ-SYN, ERα, and ERαΔ3. To identify the in situ localization, immunofluorescence for γ-syn was carried out. RESULTS: Ovarian tumour tissue exhibited an elevated expression of γ-SYN and high-grade tumours had an elevated ERαΔ3:ERα ratio compared with benign tissue. The majority of previous studies point to the γ-syn protein being present in epithelial cells of high-grade disease. Our study supports this, but additionally we conclusively identify its presence in the endothelial cells of vasculature surrounding low-grade disease; immunofluorescence was strongest in the apical cells surrounding the lumen. CONCLUSION: Our results demonstrate for the first time that there are readily-expressed levels of γ-SYN and ERαΔ3 in normal ovarian tissue and ovarian tumours. In high-grade disease, γ-syn and an elevated ERαΔ3:ERα ratio might confer metastatic potential to the tumourigenic cells and promote neoangiogenesis. Future in vitro studies might be necessary to delineate such a mechanism, which could potentially be the basis of early intervention.

Elevated oestrogen receptor splice variant ERαΔ5 expression in tumour-adjacent hormone-responsive tissue.

Susceptibility to prostate or endometrial cancer is linked with obesity, a state of oestrogen excess. Oestrogen receptor (ER) splice variants may be responsible for the tissue-level of ER activity. Such micro-environmental regulation may modulate cancer initiation and/or progression mechanisms. Real-time reverse transcriptase (RT) polymerase chain reaction (PCR) was used to quantitatively assess the levels of four ER splice variants (ERαΔ3, ERαΔ5, ERß2 and ERß5), plus the full-length parent isoforms ERα and ERß1, in high-risk [tumour-adjacent prostate (n = 10) or endometrial cancer (n = 9)] vs. low-risk [benign prostate (n = 12) or endometrium (n = 9)], as well as a comparison of UK (n = 12) vs. Indian (n = 15) benign prostate. All three tissue groups expressed the ER splice variants at similar levels, apart from ERαΔ5. This splice variant was markedly raised in all of the tumour-adjacent prostate samples compared to benign tissues. Immunofluorescence analysis for ERß2 in prostate tissue demonstrated that such splice variants are present in comparable, if not greater, amounts as the parent full-length isoform. This small pilot study demonstrates the ubiquitous nature of ER splice variants in these tissue sites and suggests that ERαΔ5 may be involved in progression of prostate adenocarcinoma.

Derivation of a subtype-specific biochemical signature of endometrial carcinoma using synchrotron-based Fourier-transform infrared microspectroscopy.

Endometrial carcinoma consists of endometrioid (type I) and serous papillary (SP; type II) subtypes; a rarer form is malignant mixed müllerian tumours (MMMT; type II/mixed). We set out to determine whether one might be able to biochemically signature these subtypes using Fourier-transform infrared (FTIR) microspectroscopy and distinguish non-tamoxifen associated from tamoxifen-associated cases. Paraffin-embedded blocks were obtained from non-tamoxifen associated cases reported as endometrioid (n=7), SP (n=4) or MMMT (n=4). From tamoxifen-associated cases, endometrioid (n=1), SP (n=3) and MMMT (n=4) blocks were retrieved; benign tissues (n=3) were also analysed. Exploiting synchrotron-based radiation, sections (10-microm thick) on BaF(2) windows were interrogated through a 10 microm x 10 microm aperture. Point spectra were derived from >or=10 locations in each of six glandular elements per tissue; a further 20 stromal spectra were obtained. Following normalisation to Amide I, average spectra (1800-900 cm(-1)) per gland or stroma were analysed for variance using principal component analysis (PCA) and linear discriminant analysis (LDA). In scores plots, segregation of spectra from different subtypes or benign tissues was noted and it proved possible to distinguish tamoxifen-associated cases. In the PCA-LDA loadings plots, the wavenumbers that highlighted variance for benign or endometrioid carcinoma tissues were in the protein region (1800-1480 cm(-1)) whereas those contributing most to SP or MMMT segregation were primarily in the DNA/RNA region (1425-900 cm(-1)) of the vibrational spectrum. Our results suggest that the application of FTIR microspectroscopy is a powerful new approach in disease diagnosis and characterisation.

FTIR Microspectroscopy Coupled with Two-Class Discrimination Segregates Markers Responsible for Inter- and Intra-Category Variance in Exfoliative Cervical Cytology.

Infrared (IR) absorbance of cellular biomolecules generates a vibrational spectrum, which can be exploited as a "biochemical fingerprint" of a particular cell type. Biomolecules absorb in the mid-IR (2-20 mum) and Fourier-transform infrared (FTIR) microspectroscopy applied to discriminate different cell types (exfoliative cervical cytology collected into buffered fixative solution) was evaluated. This consisted of cervical cytology free of atypia (i.e. normal; n = 60), specimens categorised as containing low-grade changes (i.e. CIN1 or LSIL; n = 60) and a further cohort designated as high-grade (CIN2/3 or HSIL; n = 60). IR spectral analysis was coupled with principal component analysis (PCA), with or without subsequent linear discriminant analysis (LDA), to determine if normal versus low-grade versus high-grade exfoliative cytology could be segregated. With increasing severity of atypia, decreases in absorbance intensity were observable throughout the 1,500 cm(-1) to 1,100 cm(-1) spectral region; this included proteins (1,460 cm(-1)), glycoproteins (1,380 cm(-1)), amide III (1,260 cm(-1)), asymmetric (nu(as)) PO(2) (-) (1,225 cm(-1)) and carbohydrates (1,155 cm(-1)). In contrast, symmetric (nu(s)) PO(2) (-) (1,080 cm(-1)) appeared to have an elevated intensity in high-grade cytology. Inter-category variance was associated with protein and DNA conformational changes whereas glycogen status strongly influenced intra-category. Multivariate data reduction of IR spectra using PCA with LDA maximises inter-category variance whilst reducing the influence of intra-class variation towards an objective approach to class cervical cytology based on a biochemical profile.