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.     

Rapid analysis of disease state in liquid human serum combining infrared spectroscopy and “digital drying”

In recent years, the diagnosis of brain tumors has been investigated with attenuated total reflection‐Fourier transform infrared (ATR‐FTIR) spectroscopy on dried human serum samples to eliminate spectral interferences of the water component, with promising results. This research evaluates ATR‐FTIR on both liquid and air‐dried samples to investigate “digital drying” as an alternative approach for the analysis of spectra obtained from liquid samples. Digital drying approaches, consisting of water subtraction and least‐squares method, have demonstrated a greater random forest (RF) classification performance than the air‐dried spectra approach when discriminating cancer vs control samples, reaching sensitivity values higher than 93.0% and specificity values higher than 83.0%. Moreover, quantum cascade laser infrared (QCL‐IR) based spectroscopic imaging is utilized on liquid samples to assess the implications of a deep‐penetration light source on disease classification. The RF classification of QCL‐IR data has provided sensitivity and specificity amounting to 85.1% and 75.3% respectively.     

En‐face optical coherence tomography for the detection of cancer in prostatectomy specimens: Quantitative analysis in 20 patients

The increase histopathological evaluation of prostatectomy specimens rises the workload on pathologists. Automated histopathology systems, preferably directly on unstained specimens, would accelerate the pathology workflow. In this study, we investigate the potential of quantitative analysis of optical coherence tomography (OCT) to separate benign from malignant prostate tissue automatically. Twenty fixated prostates were cut, from which 54 slices were scanned by OCT. Quantitative OCT metrics (attenuation coefficient, residue, goodness‐of‐fit) were compared for different tissue types, annotated on the histology slides. To avoid misclassification, the poor‐quality slides, and edges of annotations were excluded. Accurate registration of OCT data with histology was achieved in 31 slices. After removing outliers, 56% of the OCT data was compared with histopathology. The quantitative data could not separate malignant from benign tissue. Logistic regression resulted in malignant detection with a sensitivity of 0.80 and a specificity of 0.34. Quantitative OCT analysis should be improved before clinical use.     

A critical assessment of SELDI-TOF-MS for biomarker discovery in serum and tissue of patients with an ovarian mass

ABSTRACT: BACKGROUND: Less than 25% of patients with a pelvic mass who are presented to a gynecologist will eventually be diagnosed with epithelial ovarian cancer. Since there is no reliable test to differentiate between different ovarian tumors, accurate classification could facilitate adequate referral to a gynecological oncologist, improving survival. The goal of our study was to assess the potential value of a SELDI-TOF-MS based classifier for discriminating between patients with a pelvic mass. METHODS: Our study design included a well-defined patient population, stringent protocols and an independent validation cohort. We compared serum samples of 53 ovarian cancer patients, 18 patients with tumors of low malignant potential, and 57 patients with a benign ovarian tumor on different ProteinChip arrays. In addition, from a subset of 84 patients, tumor tissues were collected and microdissection was used to isolate a pure and homogenous cell population. RESULTS: Diagonal Linear Discriminant Analysis (DLDA) and Support Vector Machine (SVM) classification on serum samples comparing cancer versus benign tumors, yielded models with a classification accuracy of 71-81% (cross-validation), and 73-81% on the independent validation set. Cancer and benign tissues could be classified with 95-99% accuracy using cross-validation. Tumors of low malignant potential showed protein expression patterns different from both benign and cancer tissues. Remarkably, none of the peaks differentially expressed in serum samples were found to be differentially expressed in the tissue lysates of those same groups. CONCLUSION: Although SELDI-TOF-MS can produce reliable classification results in serum samples of ovarian cancer patients, it will not be applicable in routine patient care. On the other hand, protein profiling of microdissected tumor tissue may lead to a better understanding of oncogenesis and could still be a source of new serum biomarkers leading to novel methods for differentiating between different histological subtypes.     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.      

Characterisation of DNA methylation status using spectroscopy (mid‐IR versus Raman) with multivariate analysis

Methylation status plays important roles in the regulation of gene expression and significantly influences the dynamics, bending and flexibility of DNA. The aim of this study was to determine whether attenuated total reflection Fourier‐transform infrared (ATR‐FTIR) or Raman spectroscopy with subsequent multivariate analysis could determine methylation patterning in oligonucleotides variously containing 5‐methylcytosine, cytosine and guanine bases. Applied to Low‐E reflective glass slides, 10 independent spectral acquisitions were acquired per oligonucleotide sample. Resultant spectra were baseline‐corrected and vector normalised over the 1750 cm^–1–760 cm^–1 (for ATR‐FTIR spectroscopy) or the 1750 cm^–1–600 cm^–1 (for Raman spectroscopy) regions. Data were then analysed using principal component analysis (PCA) coupled with linear discriminant analysis (LDA). Exploiting this approach, biomolecular signatures enabling sensitive and specific discrimination of methylation patterning were derived. For DNA sequence and methylation analysis, this approach has the potential to be an important tool, especially when material is scarce. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Investigation of human pancreatic cancer tissues by Fourier Transform Infrared Hyperspectral Imaging

Fourier‐transform infrared hyperspectral imaging (FTIR‐HSI) provides hyperspectral images containing both morphological and chemical information. It is widely applied in the biomedical field to detect tumor lesions, even at the early stage, by identifying specific spectral biomarkers. Pancreatic neoplasms present different prognoses and are not always easily classified by conventional analyses. In this study, tissue samples with diagnosis of pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumor were analyzed by FTIR‐HSI and the spectral data compared with those from healthy and dysplastic samples. Multivariate/univariate approaches were complemented to hyperspectral images, and definite spectral markers of the different lesions identified. The malignant lesions were recognizable both from healthy/dysplastic pancreatic tissues (high values of phospholipids and triglycerides with shorter, more branched and less unsaturated alkyl chains) and between each other (different amounts of total lipids, phosphates and carbohydrates). These findings highlight different metabolic pathways characterizing the different samples, well detectable by FTIR‐HSI.     

Fiber attenuated total reflection infrared spectroscopy of kidney tissue during live surgery

More than 90% of solid kidney tumors are cancerous and have to be treated by surgical resection where surgical outcomes and patient prognosis are dependent on the tumor discrimination. The development of alternative approaches based on a new generation of fiber attenuated total reflection (ATR) probes could aid tumor identification even under intrasurgical conditions. Herein, fiber ATR IR spectroscopy is employed to distinguish normal and cancerous kidney tissues. Freshly resected tissue samples from 34 patients are investigated under nearly native conditions. Spectral marker bands that allow a reliable discrimination between tumor and normal tissue are identified by a supervised classification algorithm. The absorbance values of the bands at 1025, 1155 and 1240 cm^?1 assigned to glycogen and fructose 1,6‐bisphosphatase are used as the clearest markers for the tissue discrimination. Absorbance threshold values for tumor and normal tissue are determined by discriminant analysis. This new approach allows the surgeon to make a clinical diagnosis.     

Segregation of ovarian cancer stage exploiting spectral biomarkers derived from blood plasma or serum analysis: ATR‐FTIR spectroscopy coupled with variable selection methods

Ovarian cancer is a solid tumor and a leading cause of mortality. Diagnostic tools for the detection of early stage (stage I) ovarian cancer are urgently needed. For this purpose, attenuated total reflection Fourier‐transform infrared spectroscopy (ATR‐FTIR) coupled with variable selection methods, successive projection algorithm or genetic algorithm (GA) combined with linear discriminant analysis (LDA), were employed to identify spectral biomarkers in blood plasma or serum samples for accurate diagnosis of different stages of ovarian cancer, histological type and segregation based on age. Three spectral datasets (stage I vs. stage II–IV; serous vs. non‐serous carcinoma; and, ≤60 years vs. >60 years) were processed: sensitivity and specificity required for real‐world diagnosis of ovarian cancer was achieved. Toward segregating stage I vs. stage II–IV, sensitivity and specificity (plasma blood) of 100% was achieved using a GA‐LDA model with 33 wavenumbers. For serous vs. non‐serous category (plasma blood), the sensitivity and specificity levels, using 29 wavenumbers by GA‐LDA, were remarkable (up to 94%). For ≤60 years and >60 years categories (plasma blood), the sensitivity and specificity, using 42 wavenumbers by GA‐LDA, gave complete accuracy (100%). For serum samples, sensitivity and specificity results gave relatively high accuracy (up to 91.6% stage I vs. stage II–IV; up to 93.0% serous vs. non‐serous; and, up to 96.0% ≤60 years vs. >60 years) using several wavenumbers. These findings justify a prospective population‐based assessment of biomarkers signatures using ATR‐FTIR spectroscopy as a screening tool for stage of ovarian cancer. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:832–839, 2015     

Simplifying the assessment of human breast cancer by mapping a micro‐scale heterogeneity index in optical coherence elastography

Surgical treatment of breast cancer aims to identify and remove all malignant tissue. Intraoperative assessment of tumor margins is, however, not exact; thus, re‐excision is frequently needed, or excess normal tissue is removed. Imaging methods applicable intraoperatively could help to reduce re‐excision rates whilst minimizing removal of excess healthy tissue. Optical coherence elastography (OCE) has been proposed for use in breast‐conserving surgery; however, intraoperative interpretation of complex OCE images may prove challenging. Observations of breast cancer on multiple length scales, by OCE, ultrasound elastography, and atomic force microscopy, have shown an increase in the mechanical heterogeneity of malignant breast tumors compared to normal breast tissue. In this study, a micro‐scale mechanical heterogeneity index is introduced and used to form heterogeneity maps from OCE scans of 10 ex vivo human breast tissue samples. Through comparison of OCE, optical coherence tomography images, and corresponding histology, malignant tissue is shown to possess a higher heterogeneity index than benign tissue. The heterogeneity map simplifies the contrast between tumor and normal stroma in breast tissue, facilitating the rapid identification of possible areas of malignancy, which is an important step towards intraoperative margin assessment using OCE.

     

Infrared attenuated total reflection spectroscopic surface analysis of bovine‐tail intervertebral discs after UV‐light‐activated riboflavin‐induced collagen cross‐linking

The tensile strength of the intervertebral disc (IVD) is mainly maintained by collagen cross‐links. Loss of collagen cross‐linking combined with other age‐related degenerative processes contributes to tissue weakening, biomechanical failure, disc herniation and pain. Exogenous collagen cross‐linking has been identified as an effective therapeutic approach for restoring IVD tensile strength. The current state‐of‐the‐art method to assess the extent of collagen cross‐linking in tissues requires destructive procedures and high‐performance liquid chromatography. In this study, we investigated the utility of infrared attenuated total reflection (IR‐ATR) spectroscopy as a nondestructive analytical strategy to rapidly evaluate the extent of UV‐light‐activated riboflavin (B2)‐induced collagen cross‐linking in bovine IVD samples. Thirty‐five fresh bovine‐tail IVD samples were equally divided into five treatment groups: (a) untreated, (b) cell culture medium Dulbecco's Modified Eagle's Medium only, (c) B2 only, (d) UV‐light only and (e) UV‐light‐B2. A total of 674 measurements have been acquired, and were analyzed via partial least squares discriminant analysis. This classification scheme unambiguously identified individual classes with a sensitivity >91% and specificity >92%. The obtained results demonstrate that IR‐ATR spectroscopy reliably differentiates between different treatment categories, and promises an excellent tool for potential in vivo, nondestructive and real‐time assessment of exogenous IVD cross‐linking.     

Serum copper,zinc levels,and copper

Serum copper and zinc levels were determined in 20 healthy women and in 100 women with gynecological tumors. Malignant and benign tumor cases were separated according to their postoperative, histopathological examinations. The stages of malignant and benign tumors were also established histologically. Seventy benign and 30 malignant genital tumors (carcinoma of cervix in situ, cervix, ovary endometrium, and vulva) of the patients were differentiated histopathologically. The serum Cu/Zn ratios of patients were increased significantly from the control group (0.32±0.35) to the benign group (1.22±0.63) and from the benign group to the malignant group (2.24±1.03). Nine of 30 malignant cases were determined as false negative (30%) and 15 of 70 benign cases were determined as false positive (14.2%) according to the serum Cu/Zn ratios of patients. Serum copper levels of 30 malignant and 10 benign tumor cases showed linear correlation with serum ceruloplasmin values.     

Comparative studies of nuclear DNA content in benign and malignant thyroid lesions

Currently available data suggest that DNA aneuploidy is associated with aggressive behavior of and unfavorable prognosis in several malignant human tumors as compared with diploid malignancies. However, the diagnostic and prognostic importance of flow cytometric DNA measurements in the case of thyroid neoplasms remains controversial. Therefore, the aim of our study was to evaluate utility of DNA index (DI) and proliferative index (PI) in distinguishing benign from malignant thyroid lesions taking into account the possible influence of intra-tumor heterogeneity and tissue preparation mode on DNA flow-cytometry measurements. A retrospective study was performed on 71 paraffin-embedded specimens from 57 patients with benign and malignant thyroid pathologies: 13 colloid goitres, 12 parenchymatous goitres, 19 adenomas and 13 carcinomas. In 14 of 57 cases two separate specimens taken from different areas of the same lesion were analysed and DNA parameters were compared. Additionally, flow cytometry DNA analysis was parallelly performed on 3 adjacent but differently processed tissue sections (fresh, formalin-fixed and paraffin-embedded) taken from each of 26 surgically excised thyroid lesions. DNA content was also analysed in both fresh and formalin-fixed twin specimens of normal pig thyroid glands (N = 6). We demonstrated that all tumors diagnosed as thyroid carcinomas were associated with abnormal nuclear DNA content although aneuploidy was not found specific to malignant thyroid tumors. Aneuploid samples of benign thyroid lesions exhibited higher proliferative activity, expressed as mean PI values, than diploid ones. In carcinomas the mean PI values were significantly higher than in benign lesions, independently whether they concerned aneuploid or diploid tissues. Considering intra-tumor heterogeneity, the flow cytometric DNA parameters can be assumed as reproducible despite differences in the mode of tissue fixation and preparation for analysis.     

Rapid diagnosis of malignant pleural mesothelioma and its discrimination from lung cancer and benign exudative effusions using blood serum

Malignant pleural mesothelioma (MPM), an aggressive cancer associated with exposure to fibrous minerals, can only be diagnosed in the advanced stage because its early symptoms are also connected with other respiratory diseases. Hence, understanding the molecular mechanism and the discrimination of MPM from other lung diseases at an early stage is important to apply effective treatment strategies and for the increase in survival rate. This study aims to develop a new approach for characterization and diagnosis of MPM among lung diseases from serum by Fourier transform infrared spectroscopy (FTIR) coupled with multivariate analysis. The detailed spectral characterization studies indicated the changes in lipid biosynthesis and nucleic acids levels in the malignant serum samples. Furthermore, the results showed that healthy, benign exudative effusion, lung cancer, and MPM groups were successfully separated from each other by applying principal component analysis (PCA), support vector machine (SVM), and especially linear discriminant analysis (LDA) to infrared spectra.     

Three‐dimensional mapping of the attenuation coefficient in optical coherence tomography to enhance breast tissue microarchitecture contrast

Effective intraoperative tumor margin assessment is needed to reduce re‐excision rates in breast‐conserving surgery (BCS). Mapping the attenuation coefficient in optical coherence tomography (OCT) throughout a sample to create an image (attenuation imaging) is one promising approach. For the first time, three‐dimensional OCT attenuation imaging of human breast tissue microarchitecture using a wide‐field (up to ~45 × 45 × 3.5 mm) imaging system is demonstrated. Representative results from three mastectomy and one BCS specimen (from 31 specimens) are presented with co‐registered postoperative histology. Attenuation imaging is shown to provide substantially improved contrast over OCT, delineating nuanced features within tumors (including necrosis and variations in tumor cell density and growth patterns) and benign features (such as sclerosing adenosis). Additionally, quantitative micro‐elastography (QME) images presented alongside OCT and attenuation images show that these techniques provide complementary contrast, suggesting that multimodal imaging could increase tissue identification accuracy and potentially improve tumor margin assessment.     

Protein profile study of breast‐tissue homogenates by HPLC‐LIF

Proteomics is a promising approach for molecular understanding of neoplastic processes including response to treatment. Widely used 2D‐gel electrophoresis/Liquid chromatography coupled with mass spectrometry (LC‐MS) are time consuming and not cost effective. We have developed a high‐sensitivity (femto/subfemtomoles of protein/20 μl) High Performance Liquid Chromatography‐Laser Induced Fluorescence HPLC‐LIF instrument for studying protein profiles of biological samples. In this study, we have explored the feasibility of classifying breast tissues by multivariate analysis of chromatographic data. We have analyzed 13 normal, 17 malignant, 5 benign and 4 post‐treatment breast‐tissue homogenates. Data was analyzed by Principal Component Analysis PCA in both unsupervised and supervised modes on derivative and baseline‐corrected chromatograms. Our findings suggest that PCA of derivative chromatograms gives better classification. Thus, the HPLC‐LIF instrument is not only suitable for generation of chromatographic data using femto/subfemto moles of proteins but the data can also be used for objective diagnosis via multivariate analysis. Prospectively, identified fractions can be collected and analyzed by biochemical and/or MS methods. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detection of oral squamous cell carcinoma with ex vivo fluorescence confocal microscopy: Sensitivity and specificity compared to histopathology

Real‐time microscopic imaging of freshly excised tissue enables a rapid bedside‐pathology. A possible application of interest is the detection of oral squamous cell carcinomas (OSCCs). The aim of this study was to analyze the sensitivity and specificity of ex vivo fluorescence confocal microscopy (FCM) for OSCCs and to compare confocal images visually and qualitatively with gold standard histopathology. Two hundred eighty ex vivo FCM images were prospectively collected and evaluated immediately after excision. Every confocal image was blindly assessed for the presence or absence of malignancy by two clinicians and one pathologist. The results were compared with conventional histopathology with hematoxylin and eosin staining. OSCCs were detected with a very high sensitivity of 0.991, specificity of 0.9527, positive predictive value of 0.9322 and negative predictive value of 0.9938. The results demonstrate the potential of ex vivo FCM in fresh tissue for rapid real‐time surgical pathology.     

Protein expression changes in ovarian cancer during the transition from benign to malignant

Epithelial ovarian carcinoma has in general a poor prognosis since the vast majority of tumors are genomically unstable and clinically highly aggressive. This results in rapid progression of malignancy potential while still asymptomatic and thus in late diagnosis. It is therefore of critical importance to develop methods to diagnose epithelial ovarian carcinoma at its earliest developmental stage, that is, to differentiate between benign tissue and its early malignant transformed counterparts. Here we present a shotgun quantitative proteomic screen of benign and malignant epithelial ovarian tumors using iTRAQ technology with LC-MALDI-TOF/TOF and LC-ESI-QTOF MS/MS. Pathway analysis of the shotgun data pointed to the PI3K/Akt signaling pathway as a significant discriminatory pathway. Selected candidate proteins from the shotgun screen were further confirmed in 51 individual tissue samples of normal, benign, borderline or malignant origin using LC-MRM analysis. The MRM profile demonstrated significant differences between the four groups separating the normal tissue samples from all tumor groups as well as perfectly separating the benign and malignant tumors with a ROC-area of 1. This work demonstrates the utility of using a shotgun approach to filter out a signature of a few proteins only that discriminates between the different sample groups.     

Micropatterning and characterization of electrospun poly(ε‐caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126. © 2010 Wiley Periodicals, Inc.