Diagnostic potential of near-infrared Raman spectroscopy in the colon: differentiating adenomatous from hyperplastic polyps

BACKGROUND: Near-infrared Raman spectroscopy is a promising optical technique for GI tissue diagnosis. This study assessed the diagnostic potential of near-infrared Raman spectroscopy in the colon by evaluating its ability to distinguish between adenomatous and hyperplastic polyps. METHODS: Ex vivo and in vivo Raman spectra of colon polyps were collected by using a custom-built, fiber-optic, near-infrared Raman spectroscopic system. Multivariate statistical techniques, including principal component analysis and linear discriminant analysis, were used to develop diagnostic algorithms for classifying colon polyps based on their spectral characteristics. With the number of samples available, spectral classification of polyps was tested by using a leave-one-out, cross-validation method. RESULTS: Fifty-four ex vivo Raman spectra were analyzed (20 hyperplastic, 34 adenomatous). The spectral-based diagnostic algorithms identified adenomatous polyps with 91% sensitivity, 95% specificity, and 93% accuracy. In vivo, adenomas (n = 10) were distinguished from hyperplastic polyps (n = 9) with 100% sensitivity, 89% specificity, and 95% accuracy. CONCLUSIONS: Near-infrared Raman spectroscopy differentiated adenomatous from hyperplastic polyps with high diagnostic accuracy. To our knowledge, this is the first demonstration of the potential of near-infrared Raman spectroscopy for differentiation of colonic polyps during GI endoscopy.     

Expression of c-kit protein in proliferative lesions of human breast: sexual difference and close association with phosphotyrosine status

PURPOSE: The c-kit gene which codes transmembrane tyrosine kinase receptor protein plays an important role in several types of normal and/or neoplastic human tissues. We examined the expression patterns of c-kit protein in proliferative lesions of human breast tissues in both sexes. METHODS: The localization of c-kit protein was examined immunohistochemically in human breast, consisting of 366 normal tissue, 156 benign lesions (fibroadenoma, fibrocystic change, intraductal papilloma, benign phyllodes tumor, and gynecomastia), 13 borderline diseases (atypical ductal hyperplasia, atypical lobular hyperplasia, and borderline malignant phyllodes tumor), and 197 malignant lesions (non-invasive and/or invasive ductal carcinoma and malignant phyllodes tumor). RESULTS: In normal tissues and benign proliferative lesions, c-kit product was consistently detected on epithelial cell membranes and/or cytoplasms regardless of gender difference. In contrast, we failed to find c-kit product in female borderline epithelial lesions, including atypical lobular hyperplasia, or in female malignant lesions, except for two carcinomas. In situ hybridization analysis of c-kit mRNA in female tissues gave results comparable to those obtained by immunohistochemistry. On the other hand, c-kit product was consistently detected in male benign and malignant proliferative lesions. Apart from the female breast carcinomas which lacked c-kit, c-kit expression was almost always accompanied by positivity for phosphotyrosine in the breast tissues examined, suggesting possible phosphorylation of tyrosine residues of the c-kit receptor protein. CONCLUSIONS: Loss of c-kit product was related to malignant transformation in female breast, but not in the case of male breast. We suggest that the oncogenesis pathway of breast epithelium is different between males and females in terms of c-kit expression.     

Intravascular ultrasound combined with Raman spectroscopy to localize and quantify cholesterol and calcium salts in atherosclerotic coronary arteries

Coronary intravascular ultrasound (IVUS) can assess arterial wall architecture and localize large intravascular deposits, but it does not provide quantitative chemical information, which is essential in the evaluation of atherosclerotic lesions. Previously, it has been shown that Raman spectroscopy can be used to accurately quantify the relative weights of cholesterol, calcium salts, triglycerides, and phospholipids in homogenized arterial tissue. In the present study, we explore some benefits of combining IVUS and Raman spectroscopy to evaluate the intact arterial wall. IVUS images were collected in vitro from human coronary arterial segments in various stages of disease (n=7). The images were divided into radial segments (11 to 28 per image, 332 in total), each of which was classified visually as calcified or noncalcified tissue. The arteries were opened longitudinally, and Raman spectra were collected from locations at 0. 5-mm intervals across the arterial luminal circumference. The spectra were used to calculate the chemical composition of the arterial wall at the examined locations. Generally, locations containing large amounts of calcium salts, as determined with Raman spectroscopy, were classified as calcified with IVUS. However, small calcific deposits (<6% of weight) were not readily detected with IVUS. The amounts and location of cholesterol determined with Raman spectroscopy were correlated closely with the presence of cholesterol observed by histochemistry, but these deposits could not be located accurately by IVUS. The combination of Raman spectroscopy and IVUS applied in vitro provides detailed information about the amount and location of calcific deposits and lipid pools in atherosclerotic plaques. Future advances in optical fiber technology may allow simultaneous collection of Raman spectra and IVUS images through the same catheter in vivo.     

Identification of Raman spectroscopic markers for the characterization of normal and adenocarcinomatous colonic tissues

Raman spectroscopy has been recognised as a valuable analytical tool in biological and medical research. This technique allows probing molecular vibrations of samples without external labels or extensive preparation. This non-destructive optical technique can provide rapid and objective and reproducible measurements of sample biochemistry and identify variations that occur between healthy and diseased tissues. In fact, biochemical changes within tissue may either initiate disease or occur as a result of the disease process. The qualitative analysis of such changes provides important clues in the search for a specific diagnosis and the quantitative analysis of biochemical abnormalities is important in measuring the extent of the disease process, designing therapy and evaluating the efficacy of treatment. In this paper, we discuss one medical application of near-infrared Raman microspectroscopic imaging as a diagnostic tool to investigate, ex vivo, the changes between normal and adenocarcinomatous human colonic tissues. Multivariate statistical analysis was applied on these measured data to identify the molecular composition and distribution of lipids, proteins, mucus and collagens in normal and malignant tissue. Unsupervised hierarchical cluster analysis shows two unsupervised distinct clusters that were assigned to normal and adenocarcinomatous in accordance with conventional histopathological examination. The spectral images allowed good correlation between pseudo-color Raman and histopathological features.     

A Raman-based endoscopic strategy for multiplexed molecular imaging

Endoscopic imaging is an invaluable diagnostic tool allowing minimally invasive access to tissues deep within the body. It has played a key role in screening colon cancer and is credited with preventing deaths through the detection and removal of precancerous polyps. However, conventional white-light endoscopy offers physicians structural information without the biochemical information that would be advantageous for early detection and is essential for molecular typing. To address this unmet need, we have developed a unique accessory, noncontact, fiber optic-based Raman spectroscopy device that has the potential to provide real-time, multiplexed functional information during routine endoscopy. This device is ideally suited for detection of functionalized surface-enhanced Raman scattering (SERS) nanoparticles as molecular imaging contrast agents. This device was designed for insertion through a clinical endoscope and has the potential to detect and quantify the presence of a multiplexed panel of tumor-targeting SERS nanoparticles. Characterization of the Raman instrument was performed with SERS particles on excised human tissue samples, and it has shown unsurpassed sensitivity and multiplexing capabilities, detecting 326-fM concentrations of SERS nanoparticles and unmixing 10 variations of colocalized SERS nanoparticles. Another unique feature of our noncontact Raman endoscope is that it has been designed for efficient use over a wide range of working distances from 1 to 10 mm. This is necessary to accommodate for imperfect centering during endoscopy and the nonuniform surface topology of human tissue. Using this endoscope as a key part of a multiplexed detection approach could allow endoscopists to distinguish between normal and precancerous tissues rapidly and to identify flat lesions that are otherwise missed.     

Colored Tattoo Ink Screening Method with Optical Tissue Phantoms and Raman Spectroscopy

Due to the increasing popularity of tattoos among the general population, to ensure their safety and quality, there is a need to develop reliable and rapid methods for the analysis of the composition of tattoo inks, both in the ink itself and in already existing tattoos. This paper presents the possibility of using Raman spectroscopy to examine tattoo inks in biological materials. We have developed optical tissue phantoms mimicking the optical scattering coefficient typical for human dermis as a substitute for an in vivo study. The material employed herein allows for mimicking the tattoo-making procedure. We investigated the effect of the scattering coefficient of the matrix in which the ink is located, as well as its chemical compositions on the spectra. Raman surface line scanning has been carried out for each ink in the skin phantom to establish the spatial gradient of ink concentration distribution. This ensures the ability to detect miniature concentrations for a tattoo margin assessment. An analysis and comparison of the spectra of the inks and the tattooed inks in the phantoms are presented. We recommend the utilization of Raman spectroscopy as a screening method to enforce the tattoo ink safety legislations as well as an early medical diagnostic screening tool.     

Immunohistochemical profile of the sodium/iodide symporter in thyroid,breast, and other carcinomas using high density tissue microarrays and conventional sections

Extrathyroidal cancers could potentially be targeted with (131)I, if the Na(+)/I(-) symporter (NIS) were functional. Using immunohistochemical methods we probed 1278 human samples with anti-NIS antibody, including 253 thyroid and 169 breast conventional whole tissue sections (CWTS). Four high density tissue microarrays containing a wide variety of breast lesions, normal tissues, and carcinoma cores were tested. The results of the normal microarray were corroborated in 50 CWTS. Nineteen of 34 normal tissues, including bladder, colon, endometrium, kidney, prostate, and pancreas, expressed NIS. Nineteen of 25 carcinomas demonstrated NIS immunopositivity; 55.7% of 479 carcinoma microarray cores expressed NIS, including prostate (74%), ovary (73%), lung (65%), colon (62.6%), and endometrium (56%). NIS protein was present in 75% benign thyroid lesions, 73% thyroid cancers, 30% normal-appearing, peritumoral breasts, 88% ductal carcinomas in situ, and 76% invasive breast carcinoma CWTS. Comparatively, breast microarray cores had lower immunoreactivity. Plasma membrane immunopositivity was confirmed in thyrocytes, salivary ductal, gastric mucosa, and lactating mammary cells. In other tissues, immunoreactivity was predominantly intracellular, particularly in malignant lesions. Thus, NIS is present in many normal epithelial tissues and is predominantly expressed intracellularly in many carcinomas. Elucidating the regulatory mechanisms that render NIS functional in extrathyroidal carcinomas may make (131)I therapy feasible.     

Identification of Raman spectroscopic markers for the characterization of normal and adenocarcinomatous colonic tissues

Raman spectroscopy has been recognised as a valuable analytical tool in biological and medical research. This technique allows probing molecular vibrations of samples without external labels or extensive preparation. This non-destructive optical technique can provide rapid and objective and reproducible measurements of sample biochemistry and identify variations that occur between healthy and diseased tissues. In fact, biochemical changes within tissue may either initiate disease or occur as a result of the disease process. The qualitative analysis of such changes provides important clues in the search for a specific diagnosis and the quantitative analysis of biochemical abnormalities is important in measuring the extent of the disease process, designing therapy and evaluating the efficacy of treatment. In this paper, we discuss one medical application of near-infrared Raman microspectroscopic imaging as a diagnostic tool to investigate, ex vivo, the changes between normal and adenocarcinomatous human colonic tissues. Multivariate statistical analysis was applied on these measured data to identify the molecular composition and distribution of lipids, proteins, mucus and collagens in normal and malignant tissue. Unsupervised hierarchical cluster analysis shows two unsupervised distinct clusters that were assigned to normal and adenocarcinomatous in accordance with conventional histopathological examination. The spectral images allowed good correlation between pseudo-color Raman and histopathological features.     

Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens.

We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.     

Light-induced autofluorescence spectroscopy for the endoscopic detection of esophageal cancer

BACKGROUND: Any innovative optical system that facilitates the early endoscopic detection of neoplastic change in the GI mucosa has the potential to greatly improve survival and quality of life for patients prone to have GI malignancies develop. The present article describes light-induced autofluorescence spectroscopy with violet-blue excitation light for in vivo diagnosis of cancerous tissue of the esophagus during routine endoscopy. METHODS: One hundred twenty-nine endogenous fluorescence spectra were obtained from normal mucosa and malignant lesions in 9 patients with squamous cell cancer and 4 with adenocarcinoma of the esophagus. Following spectrographic measurements, biopsy specimens were obtained for definitive classification of the spectra. A special light source capable of delivering either white or violet-blue light for excitation of tissue autofluorescence by means of an endoscope was used. Endogenous fluorescence spectra emitted by tissues were detected with a fiberoptic probe and analyzed with a spectrograph. RESULTS: Squamous cell cancer and adenocarcinoma of the esophagus exhibit specific changes in the emitted fluorescence spectra as compared with normal mucosa. Based on the results obtained in earlier studies, malignant and benign spectra were differentiated with the aid of a mathematical algorithm. By using this algorithm, a sensitivity of 97% and specificity of 95% were obtained for the diagnosis of esophageal carcinoma. CONCLUSIONS: Light-induced fluorescence spectroscopy is useful for the endoscopic detection of squamous cell cancer and adenocarcinoma of the esophagus. This spectroscopic study provides a basis for the design of a simplified autofluorescence imaging system for detection of esophageal neoplasms.     

Reconstruction of functionally normal and malignant human breast tissues in mice

The study of normal breast epithelial morphogenesis and carcinogenesis in vivo has largely used rodent models. Efforts at studying mammary morphogenesis and cancer with xenotransplanted human epithelial cells have failed to recapitulate the full extent of development seen in the human breast. We have developed an orthotopic xenograft model in which both the stromal and epithelial components of the reconstructed mammary gland are of human origin. Genetic modification of human stromal cells before the implantation of ostensibly normal human mammary epithelial cells resulted in the outgrowth of benign and malignant lesions. This experimental model allows for studies of human epithelial morphogenesis and differentiation in vivo and underscores the critical role of heterotypic interactions in human breast development and carcinogenesis.     

Clinical translational application of Raman spectroscopy to advance Benchside biochemical characterization to bedside diagnosis of esophageal diseases

Esophageal diseases result in significant mortality, morbidity, and health care costs worldwide. Current approaches to detect and monitor esophageal diseases have severe limitations. Advanced imaging technologies are being developed to complement current approaches to improve diagnostic, therapeutic and surveillance protocols in order to advance the field. Raman spectroscopy‐based technologies hold promise to increase the sensitivity for detection of diseased and high‐risk lesions in vitro and in vivo in real time. This technique allows for the investigation of microstructural changes and also facilitates the discovery of disease‐specific biochemical alterations with the potential to provide novel insights into the pathobiology of these conditions. Raman spectroscopy has been increasingly applied in precancerous and cancerous esophageal conditions. However, its application in benign esophageal diseases is still in the early stages. Continuing its application in cancerous and precancerous conditions and expanding its use to benign esophageal disorders could lay a foundation for integration of this technology in clinical practice and diagnostic paradigms and development of an accurate and cost‐effective tool for use in a clinical setting. Furthermore, Raman spectroscopy can also be used as an innovative technique to advance our understanding of the biochemical transformations associated with esophageal diseases and answer a myriad of fundamental questions in the field. In this review, we described the principles of Raman spectroscopy and instrumentation while providing an overview of current applications, challenges, and future directions in the context of esophageal diseases with an emphasis on its clinical translational application.     

PTEN mutation spectrum in breast cancers and breast hyperplasia

Purpose  

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently mutated human tumor suppressor genes. The present study aims to investigate the role of PTEN mutation in breast carcinogenesis by analyzing PTEN mutation spectrum and the protein expression in breast cancers, adjacent hyperplastic lesions, benign breast lesions and normal breast tissues.     

Presence of luteinizing hormone/human chorionic gonadotropin receptors in male breast tissues

Receptors for LH/human chorionic gonadotropin (hCG) have been found in a variety of nongonadal tissues including the female breast. Using in situ hybridization and immunohistochemistry, we demonstrated the presence of LH/hCG receptor mRNA and protein in normal male breast tissue obtained at autopsy (n = 4) and archival samples of benign gynecomastia (n = 14) and male breast carcinoma (n = 5). Although the function of these receptors remains to be determined, the findings suggest the possibility that LH and hCG may play a role in the pathogenesis of male breast disorders.     

Raman spectroscopy for early real-time endoscopic optical diagnosis based on biochemical changes during the carcinogenesis of Barrett’s esophagus

Raman spectroscopy is a spectroscopic technique based on the inelastic scattering of monochromatic light that represents the molecular composition of the interrogated volume to provide a direct molecular fingerprint. Several investigations have revealed that confocal Raman spectroscopy can differentiate non-dysplastic Barrett’s esophagus from esophageal high-grade dysplasia and adenocarcinoma with high sensitivity and specificity. An automated on-line Raman spectral diagnostic system has made it possible to use Raman spectroscopy to guide accurate target biopsy instead of multiple random forceps-biopsies,this novel system is expected to improve in vivo precancerous diagnosis and tissue characterization of Barrett’s esophagus.     

Human mammary gland and breast carcinoma contain immunoreactive inhibin/activin subunits: evidence for a secretion into cystic fluid.

OBJECTIVE: Inhibins and activins are members of the transforming growth factor beta superfamily and are known to modulate the growth and differentiation of several cell types. The present study investigated the localization of inhibin and activin subunits in human normal and pathological breast tissues. DESIGN: A cross-sectional study comparing the expression of inhibin/activin subunits alpha, betaA and betaB in surgical specimens from women undergoing reductive mammoplasty (classified, according to the phase of the menstrual cycle, as follicular, luteal, or postmenopausal), and patients submitted to lumpectomy for fibrocystic disease, benign (intraductal papilloma, adenomyoepithelioma, and hamartoma) or malignant breast neoplams (intraductal, intralobular, and invasive carcinoma). METHODS: Immunohistochemistry was used to localize inhibin alpha and activin betaA and betaB subunits in the cytoplasm of epithelial cells of mammary glands. Dimeric activin A, inhibin A and inhibin B were measured by specific two-site enzyme immunoassay in the cystic fluid collected from patients with fibrocystic disease. RESULTS: An intense staining for the alpha inhibin subunit and a mild staining for betaA and betaB subunits were present in samples obtained from normal breast tissue regardless of menstrual cycle phase, and in fibrocystic disease and benign neoplasms. Carcinoma cells stained weakly to moderately for alpha subunit and were negative for betaA and betaB subunits. Fibrocystic disease was associated with absence of betaA subunit expression in normal epithelial cells and intense staining for all subunits in the apocrine cells. Immunoreactive inhibin A, inhibin B, and activin A were also present in cystic fluid, suggesting a local secretion of these proteins. CONCLUSION: These data suggest a local expression and secretion of inhibin and activin in human normal, fibrocystic disease and neoplastic breast tissues. The low expression of these proteins may facilitate abnormal cell proliferation in breast carcinoma.     

A comparison of vitamin D activity in paired non-malignant and malignant human breast tissues

Links between a low vitamin D status and an increased risk of breast cancer have been observed in epidemiological studies. These links have been investigated in human tissue homogenates and cultured cell lines. We have used non-malignant, malignant and normal reduction mammoplasty breast tissues to investigate the biological and metabolic consequences of the application of vitamin D to intact ex vivo human breast tissue. Tissues were exposed to 1α,25(OH)(2)D(3) (1,25D; active metabolite) and 25(OH)D (25D; pre-metabolite). Changes in mRNA expression and protein expression after vitamin D exposure were analysed. Results indicate that while responses in normal and non-malignant breast tissues are similar between individuals, different tumour tissues are highly variable with regards to their gene expression and biological response. Collectively, malignant breast tissue responds well to active 1,25D, but not to the inactive pre-metabolite 25D. This may have consequences for the recommendation of vitamin D supplementation in breast cancer patients.     

CALCITONIN IN BREAST AND LUNG CANCER

Immunoreactive calcitonin was found in extracts of seven out of eight consecutive breast carcinomas and four selected lung carcinomas, but not in extracts of benign breast lesions or normal tissues. This suggests that the high plasma calcitonin levels observed in patients with a wide variety of cancers reflect ectopic production of calcitonin by cancer tissue, and that the radioimmunoassay for calcitonin could prove to be of value in the detection and management of malignant disease, particularly breast cancer.     

纤维支气管镜活检病变组织标本和痰标本Survivin基因的检测对肺癌的诊断价值

目的明确纤维支气管镜(以下简称纤支镜)活检病变组织和痰标本中SurvivinmRNA的检测在肺癌诊断中的意义。方法应用逆转录聚合酶链反应(RT PCR)法检测41例肺癌手术标本癌组织、癌旁组织和9例良性肺疾病病变组织手术标本,80例肺癌和30例良性肺疾病纤支镜活检病变组织标本及所有(160例)患者痰标本SurvivinmRNA表达情况,并与病理组织学、刷检细胞学和痰细胞学检查结果比较。结果肺癌手术切除标本癌组织SurvivinmRNA的阳性率为70.7%(29/41),高于癌旁组织[17.1%(7/41)]和良性肺疾病组织(1/9),差异有统计学意义(χ2值分别为23.97和10.93,P均<0.05),而癌旁组织与肺良性疾病组织相比,差异无统计学意义(χ2=0.20,P>0.05);纤支镜活检肺癌组织标本SurvivinmRNA的阳性率为63.8%(51/80),高于良性肺疾病的13.3%(4/30,χ2值为22.18,P<0.05);肺癌患者癌组织SurvivinmRNA表达与否及表达水平与患者年龄、性别及肿瘤的病理分型、分级、部位及转移情况均无明显相关性(P均>0.05)。肺癌患者痰标本SurvivinmRNA的阳性率是59.5%(72/121),癌细胞的检出率是47.1%(57/121);痰Survivin mRNA检测联合痰细胞学检查诊断肺癌的敏感性为80.2%(97/121),高于单独痰细胞学及单独痰SurvivinmRNA检测的敏感性(P均<0.05)。手术标本、纤支镜活检标     

Human colorectal cancers display abnormal Fourier-transform infrared spectra.

Fourier-transform infrared spectroscopy (FT-IR) was applied to the study of tissue sections of human colorectal cancer. Pairs of tissue samples from colorectal cancer and histologically normal mucosa 5-10 cm away from the tumor were obtained from 11 patients who underwent partial colectomy. All cancer specimens displayed abnormal spectra compared with the corresponding normal tissues. These changes involved the phosphate and C-O stretching bands, the CH stretch region, and the pressure dependence of the CH2 bending and C = O stretching modes. Our findings indicate that in colonic malignant tissue, there are changes in the degree of hydrogen-bonding of (i) oxygen atoms of the backbone of nucleic acids (increased); (ii) OH groups of serine, tyrosine, and threonine residues (any or all of them) of cell proteins (decreased); and (iii) the C = O groups of the acyl chains of membrane lipids (increased). In addition, they indicate changes in the structure of proteins and membrane lipids (as judged by the changes in their ratio of methyl to methylene groups) and in the packing and the conformational structure of the methylene chains of membrane lipids. The cell(s) of the malignant colon tissues responsible for these spectral abnormalities is unknown. Cultured colon adenocarcinoma cell lines displayed similarly abnormal FT-IR spectra. The diagnostic potential of the observed changes is discussed.