Review Article
Raman spectroscopy in cervical cancers: An update ABSTRACT Cervical cancer is the third most common cancer among women worldwide. Developing countries contribute more than 80% towards global burden. Over the last 2 decades, Raman spectroscopy (RS) has been actively pursued for cervical cancer detection. In view of latest development in Raman spectroscopic applications in cervical cancers, especially in vivo studies, an update of the same is presented in this article. This articles opens with a brief note on Anatomy of cervix followed by Etiology, and conventional Screening and Diagnosis of Cervical cancers. In subsequent sections, brief description of Theory and Instrumentation of RS is followed by a review of recent developments in cervical cancer detection; with emphasis on cell lines, exfoliated cells, ex vivo and in vivo, and therapeutic response monitoring applications in cervical cancer. KEY WORDS: Biomedical applications, cervical cancers, raman spectroscopy
INTRODUCTION Cervix is the lower third of the uterus that extends into vagina. The part of the cervix that projects into the vagina is called as ectocervix, which is covered by nonkeratinized, stratified, squamous epithelium. The uterine part of cervix is called as endocervix, which is covered with mucus‑secreting columnar epithelium. Pictorial depiction of cervical anatomy is shown in Figure 1. The cervical transformation zone is an area of metaplastic tissue between the squamous epithelium of the vagina and the glandular tissue of the endocervical canal. Squamocolumnar junction has unique susceptibility to Human Papillomavir us (HPV)‑induced neoplastic transformation leading to cancer.[1] Cervical cancer is the third most frequent cancer among women worldwide and commonest female cancer in developing countries, including India. Developing countries contribute more than 80% to global cervical cancer burden. [2] While, in developed countries, cervical cancer incidence and mortality rates have declined due to introduction of cervical cancer screening campaigns, highlighting the significance of screening.[3] Papanicolaou test (Pap test), exfoliated cervical‑vaginal cytology, is the most practiced screening method. However, Pap test being laborious and is also known to suffer from some known limitations like low sensitivity and demands stringent quality assurance. Other known screening technologies are HPV testing, liquid‑based cytology, automated 10
cervical screening tool, and visual inspection of cervix after applying Lugol’s iodine (VILI) or acetic acid (VIA). [4] In routine clinical practice, an abnormal Pap smear is followed by colposcopic‑guided biopsies for confirmatory diagnosis. Though histopathological examination of excised biopsies remains as the gold standard of cervical cancer diagnosis, current conventional screening/diagnosis tools are also known to suffer from several disadvantages like tedious sample processing, long output duration, and the interobserver variability.[5] Therefore, conscious efforts are made for the development of minimally invasive and effective screening/diagnostic methodology that can probe the endogenous biomolecular properties of cells/tissue for early cancer detection. These studies represent vital advancements towards more efficient detection of cervical cancers. Among various methods, spectroscopy‑based approaches like fluorescence, diffuse reflectance, Fourier transformed infrared spectroscopy (FTIR), and Raman spectroscopy (RS) are being actively explored as potential stand alone or adjuvant diagnostic tools. [6‑12] RS represents a unique technique capable of label‑free and nondestructively probing endogenous biomolecules, for example, proteins, lipids, carbohydrates, and nucleic acids; to determine highly specific diagnostic information.[9‑12] The current review presents an update of latest developments of application of RS in cervical cancer detection based on cells, ex vivo, in vivo, and therapeutic response monitoring.
S. Rubina, C. Murali Krishna1 Chilakapati Laboratory, Advanced Centre for Treatment Research and Education in Cancer, Kharghar, Navi‑Mumbai, 1 Scientific Officer ‘F’ and Principal Investigator, Chilakapati Laboratory, Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi‑Mumbai, Maharashtra, India For correspondence: Dr. Murali C. Krishna, Scientific Officer ‘E’ and Principal Investigator, Chilakapati Laboratory, Advanced Center for Treatment Research and Education in Cancer, Tata Memorial Center, Kharghar, Sector 22, Navi Mumbai ‑ 410 210, Maharashtra, India. E‑mail: mchilakapati@ actrec.gov.in, [email protected]
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.154065 PMID: *** Quick Response Code:
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Figure 1: Pictorial depiction of cervix anatomy
BASIC PRINCIPLE AND INSTRUMENTATION RS is based on light scattering phenomenon. Light scattering can be explained as two‑photon process, the incident and scattered photon. Most often, scatter photons are of same energy to that of incident photon (elastic/Rayleigh scattering), but photons occasionally gain or lose energy resulting in frequency shift (inelastic/Raman scattering). Raman scattering is a weak phenomenon involving approximately one in every 106–108 scattered photons.[10] A typical Raman spectrometer consists of highly monochromatic excitation source, optical path, Rayleigh rejection filters, monochromator/dispersing elements, and detector.[10] Typically, excitation (laser beam) photons are coupled to the sample by appropriate optical path. Scattered photons from the sample are gathered, dispersed, and detected to transform the photons into spectrum. Advent of modern and high‑end technologies, the modern biomedical Raman spectrometers employ lasers, high throughput spectrographs, and multichannel charged coupled devices (CCDs). CELL‑BASED DIAGNOSIS BY RS IN CERVICAL CANCERS Classification of HPV +ve and HPV −ve cell lines High risk human papillomavirus (HR‑HPV) is a well‑known etiological factor of cervical cancers. HPV‑induced cervical carcinogenesis includes HPV infection, persistence of virus infection, precancers, and eventual invasion. Persistence of virus is linked to the development of a high‑grade precursor lesion or “precancer”. Although identification of HPV presence has clinical significance, it is pertinent to note that all HPV infections may not lead to cervical cancers after clearance of HPV infection.[13] Recently, high‑risk HPV strains testing have been incorporated into routine cervical cancer screening for menopausal females in developed countries. Hence, a brief note on studies related to Raman spectroscopic detection of HR‑HPV is presented. The first study on Raman spectroscopic identification of HPV‑16 virus was reported by Jess et al. They
observed variations in Raman bands of deoxyribonucleic acid (DNA) and proteins, which were consistent with HPV gene expression and cellular changes associated with neoplasia.[14] This study could discriminate between normal keratinocytes and keratinocytes expressing HPV‑16 E7 with 93% sensitivity and specificity. Subsequent study in the same line investigated biochemical changes in cells caused by high‑risk HPV strands. This study explored differences between the cells with high, medium, and low HPV copy number.[15] It was observed by authors that RS can discriminate between the cell lines with HPV presence and their copy number as well as elucidate cellular differences originating from proteins, nucleic acids, and lipids. Another study evaluated the ability of RS to detect the presence of HPV and differences between specific HPV strains using cell lines and patient specimens.[16] This study indicated that spectra of cell culture and patient samples exhibit statistically significant differences. This could be attributed to the fact that real‑life specimen and experimental cell lines are maintained in different environmental niche, hence can have differences in their biochemical constituents. Nevertheless, this study indicated that RS can detect HPV and differentiate among specific HPV strains. We have also investigated differences in HPV‑positive and ‑negative cell lines using RS. We have recorded Raman spectra of HPV‑18 positive HeLa, HPV‑16‑positive SiHa, and HPV negative C33A cell lines. Spectra were acquired using 40× objective coupled with HE‑785 Raman instrument (Jobin‑Yvon‑Horiba, France). Mean Raman spectra of C33A, HeLa, and SiHa cells are shown in Figure 2a. HPV negative (C33A) cells and HPV‑positive (HeLa and SiHa) cells showed distinct differences at amide I and δ CH2 region. Minor variations in amide III region were also observed, whereas no significant differences between HPV‑positive cells were discernible. A possible explanation for this observation could be that HPV infection eventually leads to oncoprotein expression resulting in differences in protein compositions in the host cells.[15] Principal component- linear discriminant analysis (PC‑LDA) gave well‑separated clusters with classification efficiency of ~95% [Figure 2b]. Findings of the study corroborate with earlier report and demonstrate subtle but significant differences that exist between HPV‑positive and ‑negative cell lines. Although HPV detection has clinical significance, as mentioned earlier, it is important to note that very few HPV‑infected subjects eventually develop cancer.[13] Hence, it is important to understand HPV‑induced cell changes leading to neoplasia. Further, the delineation observed for HPV‑positive and negative cell lines may not be entirely due to HPV presence. This could be because of the fact that the observed spectral variation in HPV‑positive and ‑negative cell lines can be due to differences in cell lines itself. This is quite clear from one of our RS studies on randomly mixed cell populations.[17] Raman spectral profile very much varies with cell lines and same can be explored for cell typing. Therefore, further studies on
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a
b
Figure 2: Raman studies of HPV +ve and –ve cell lines. (a) Average Raman spectra of HPV +ve (solid line) and −ve cell line (Dotted lines) and (b) 3D scatter plot for Hela, SiHa, and C33A cells. HPV = Human papillomavirus, 3D = Three‑dimensional
HPV‑induced‑neoplastic changes in the same type of cells are warranted. RS of exfoliated cells Wong et al., were first to report the potential of infrared (IR) spectroscopy to classify normal and abnormal cervical exfoliated cells.[18] Several subsequent IR studies of exfoliated cervical cells have explored cell pellet and single cell approach. [19‑24] However, presence of water in biological specimens is a serious hurdle in FTIR spectroscopy; hence, dried samples were used for study. Due to which same cells could not be used for Pap staining and parallel sample for Pap staining may not be ideal for cytological correlation, as abnormal cell content in an ‘abnormal’ smear can vary. To circumvent these limitations, we have explored feasibility of classification of cervical cell specimens using RS.[25] This study could distinguish normal and abnormal exfoliated specimens with 86 and 84% classification efficiencies, respectively. However, major concern is spectra being influenced by the presence of blood in abnormal specimen, which may lead to false interpretations, as bleeding also occur in several noncancerous conditions. Further studies on red blood corpuscles (RBC) lysis buffer‑treated specimens also gave classification efficiency of about 79 and 78% for normal and abnormal smears, respectively. Thus, indicating efficacy of RS in classifying normal and abnormal exfoliated specimens in both treated and untreated conditions. Even though findings of this study are quite comparable to conventional Pap test, efficacy of this approach can be further improved by developing robust model by selectively accruing specimens with very higher number of abnormal cells. Spectra acquired from cell pellet then can be compared against developed model and sample wherein even a spectrum matches with cancer model can be assigned as cancer, similar to the standard practice in histopathology and cytology. In conventional cytology, cell smears containing few tumor cells are treated as cancer. As mentioned earlier, Raman’s studies on cell pellets have also 12
shown the feasibility of classifying a single cell type in a mixed cancer cell population, HPV detection, and discrimination of wild and multidrug resistant cell type. Thus, prospective studies on pure cancerous and precancerous specimens to build true standard models and validation through blinded specimens on clinically significant sample size can further pave RS as a tool in cervical cancer. EX VIVO STUDIES Raman spectroscopic studies on ex vivo tissue are carried out, either by conventional RS or Raman microspectroscopy. Conventional Raman spectroscopic studies on tissues are important as they mimic the in vivo conditions. However, to improve the understanding of Raman spectral signatures, Raman microspectroscopic studies are ideal. This is because Raman microscope can facilitate site‑specific and in‑depth evaluations. This section will elaborate on Raman spectroscopic ex vivo studies of cervical cancer tissues. Conventional Raman spectroscopic studies In vitro Raman spectra of fresh, frozen, and preserved cervix tissue biopsies have been reported in the literature.[26‑29] Ex vivo RS study on cervix tissue carried out as early as 1998, indicated potential advantage of RS to detect cervical precancers.[26] They concluded that empirically selected normalized intensities can differentiate precancers from other tissues with sensitivity and specificity of 88 and 92%, respectively. However, unbiased multivariate methods gave the sensitivity of 82% and specificity of 92%. Raman algorithms can potentially separate benign abnormalities such as inflammation and metaplasia from precancers. They reported that the tissue spectra showed the molecular features of collagen, nucleic acids, phospholipids, and glucose l‑phosphate. In 2006, our group reported a study on ex vivo cervix tissues by RS.[27] We have observed that Raman spectra of normal cervix tissue are characterized by strong, broad amide I, broader amide III,
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and strong peaks at 853 and 938 cm−1. This was attributed to structural proteins such as collagen. While prominent features of malignant tissue spectra with respect to normal tissue were relatively weaker and sharper amide I, minor red shift in δCH2 and sharper amide III which indicated the presence of DNA, lipids, and noncollagenous proteins. PCA combined with multiparametric limit test was used for discriminating different pathological conditions in cervical cancer which produced 99.5% sensitivity and specificity. Raman microspectroscopic study on cervix tissue The architecture of tissue consists of 8 to 10 layers of cells called as epithelium resting on connective tissue which is rich in collagen. While epithelium is very thin; it consists of basal, intermediate, and superficial layer. Probing volume and the kind of cells and tissue molecular composition plays significant role in determining Raman spectral signature. Previous Raman spectroscopic studies on cervix tissue biopsies reported presence of collagenous features in normal tissue spectra and contribution of Raman signal arising from the collagenous rich connective tissue cannot be ruled out. Whereas the Raman signal of tumor tissue biopsies contributed more of DNA, lipid, and noncollagenous protein features which can be of tumor cells. Raman imaging on tissue sections is very demanding due to the extremely long acquisition time for pixel‑by‑pixel acquisition. However, recent technological advancement in Raman imaging has improved the acquisition time.[30] Previous study on tissue sections included spectral acquisition from different layers of tissue. This is a comparative Raman and synchrotron IR (SR‑IR) spectroscopy study on parallel cervical cancer specimens.[29] In their study, frozen and dewaxed formalin paraffin preserved tissues were used and discrimination between different cell types in normal cervical tissue was reported. The spectra of invasive carcinoma showed a marked difference from normal cervical epithelial cells. It is observed that spectral differences identified with the onset of carcinogenesis include increased nucleic acid contributions and decreased glycogen levels. Subsequent study investigated the potential of RS as a diagnostic tool to detect biochemical changes associated to cervical cancer progression.[30] In their study, Raman spectra were acquired from different point of dewaxed 10 µm section and Raman spectra of pure compound of proteins, nucleic acids, lipids, and carbohydrates were employed in order to gain an insight into the biochemical composition of cells and tissues. It is reported that spectra of basal cells show strong bands at 724, 779, and 1,578 cm−1, which are characteristics of nucleic acids. Spectra of epithelial cells showed characteristic glycogen bands at 482, 849, 938, 1,082, and 1,336 cm−1; whereas, spectra of connective tissue showed characteristic collagen bands at 850, 940, and 1,245 cm −1. Absence of glycogen bands, presence of characteristic nucleic acid band, and increase in the intensity of the amide I band in the spectra of invasive carcinoma has been demonstrated. Spectral features of premalignant specimens also showed the nucleic acid bands
at 724, 779, 852, 1366 and 1578 cm−1. Spectral differences of basal cells to that of tumor were investigated. It is vital to note that abnormality in the cells of basal layer develop the neoplastic tumor cells.[31] Hence, it is important to probe the differences between basal and tumor cells, as these cells are in dividing phase though proliferative index of tumor cells are high as compared to the basal cells. Another study on the Raman‑based optical diagnosis of normal cervix, inflammatory cervix (cervicitis), and cervical intraepithelial neoplasia (CINI) found major alterations in the 857 cm−1 (CCH deformation aromatic); 925 cm−1 (C‑C stretching); 1,247 cm−1 (CN stretch, NH bending of Amide III); 1,370 cm−1 (CH2 bending); and 1,525 cm−1 (C = C = C = N stretching) vibrational bands.[32] This study corroborated earlier reported spectral profiles. Another in vitro Raman microspectroscopy study on normal and cancerous cervical human tissue section observed the spectral features associated with collagen (775–975 cm−1) in normal squamous cells, which were below the detection limit in cancer.[33] In this study, Raman chemical maps, using 775–975 cm−1 and 2,800–3,100 cm−1 spectral regions, of cancer and normal cells in the cervical epithelium have also been found to show good correlation with each other. Thus, as described above, several studies not only demonstrated efficacy of Raman microspectroscopic approach in classifying normal and pathological cervical condition but also its utility in understanding differences at biomolecular level. IN VIVO STUDIES Several fiber‑probe in vivo Raman studies for diagnosis of different cancers, including cervical cancers, have appeared in the literature.[32,34‑49] In 1998, a fiberoptic in vivo Raman study of cervix to delineate precancer lesions from the normal cervix was reported.[39] In this study, in vivo Raman spectra of cervix could be acquired in 90 s. Authors concluded that the increase in the laser power could reduce the integration time to less than 20 s for measuring in vivo Raman spectra of cervix. Moreover, another pilot clinical trial by the same group suggested that in vivo Raman spectra resemble to that of in vitro cervix tissue.[40] The study has also concluded that cervical epithelial cells may contribute to tissue spectra at 1,330 cm−1, a region associated with DNA and epithelial cells probably do not contribute to tissue spectra at 1,454 cm−1, a region associated with collagen and phospholipids. In this study Raman spectral acquisition time was 60–180 s, which was relatively longer than the clinically acceptable time. Another study, using clinical feasible time (5 s), could distinguish between high‑grade dysplasia and benign with sensitivity and specificity of 89 and 81%, respectively.[41] A subsequent in vivo study could discriminate between normal ectocervix, normal endocervix, low‑grade dysplasia, and high‑grade dysplasia with 95% classification efficiency.[42] Moreover, this study showed that RS can differentiate among the different precancers with improved sensitivity of 98% and specificity of 96% in conjunction with multiclass discrimination algorithms like maximum representation and discrimination feature (MRDF)
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and sparse multinomial logistic regression (SMLR). Subsequent study looked at variations in normal cervical spectra due to menopausal status and time point in the menstrual cycle.[43] The study wherein the data stratified by menopausal state resulted in improvement of accurate classification of low grade squamous intraepithelial lesion (LGSIL) to 97% from 74%.[44] This makes RS much closer to clinical use. Another study has explored utility of high wave number (HW) Raman spectra in detection of precancer cervix.[45] This study demonstrated the diagnostic algorithms based on principal components analysis and LDA together with the leave‑one‑patient‑out cross‑validation method yielded a diagnostic sensitivity of 93.5% and specificity of 97.8% for dysplasia identification. Another study suggested that RS in conjunction with genetic algorithm‑partial least squares‑discriminant analysis (GA‑PLS‑DA) with double cross‑validation (dCV) methods has potential to provide clinically significant discrimination between normal and precancer cervix.[46] Variability such as race/ethnicity, body mass index (BMI), parity, and socioeconomic status was also explored.[47] Findings of the study suggest that BMI and parity have greatest impact; whereas, race/ethnicity and socioeconomic status have a limited effect. Another study reported that simultaneous fingerprint (FP)/HW confocal RS has potential to early diagnosis and detection of cervical precancer in vivo.[48] This study demonstrated integrated FP and HW in vivo Raman signals of a cervix can be classified with 85% sensitivity and 81% specificity. Also spectral differences between normal and dysplastic cervix can be ascribed to protein, lipids, glycogen, nucleic acids, and water content. More recent confocal studies have demonstrated great potential of in vivo RS to improve early diagnosis of cervical precancers.[49] Confocal RS coupled with PCA‑LDA modeling yielded a sensitivity and specificity of 81 and 87%, respectively, for in vivo discrimination of dysplastic cervix. It is also concluded that the best classification can be achieved using confocal RS compared to the composite near infrared autofluorescence (NIR AF)/RS or NIR AF spectroscopy alone. This study illustrated that confocal RS has great potential to improve early in vivo diagnosis of cervical precancers during clinical colposcopy.
In vivo Raman applications in cervical cancers have been enlisted in Table 1. As table suggests, general trend of lesser spectral acquisition time with increased laser power is indicative of technological advancement as well as closer to clinical applicability. As mentioned earlier, Raman spectral signature depends on the probing volume and spectral acquisition site of tissue. Most of the in vivo studies on cervical cancers have focused on detection of premalignant patches. Considering thin epithelium of cervix and the probing volume/ depth could be the important criterion; thus as reported by recent studies, using confocal Raman probes may have great potential to improve early diagnosis. THERAPEUTIC APPLICATION OF RS IN CERVICAL CANCERS Most work on RS to date has focused on diagnostic applications, particularly the early detection of cancer and precancerous changes in cervix. RS also has enormous potential in monitoring the result of treatment, that is, tumor therapeutic response. This section highlights the efforts towards therapeutic application of RS in cervical cancers using cells and tissue specimens. Studies on ex vivo cervical cell lines and treatment response We have also explored potentials of RS in classifying HeLa cells irradiated at different doses and its correlation with biological assays (clonogenic and γ‑H2AX assay).[50] PCA gave exclusive clusters for HeLa cells irradiated at low doses, that is, 2Gy, 4Gy, and control. But overlapping clusters have been observed for HeLa cells irradiated at higher doses, that is, 6, 8, and 10Gy. Preliminary work supports the feasibility of RS to classify HeLa cells that were subjected to different doses of radiation and results showed good correlation with biochemical assays. Studies on ex vivo tissues and treatment response Approximately 80% of cervical cancer patients present at advanced stages (stage IIA and above) in developing countries like India. The 5‑year disease free survival rate of less than 60% are reported for these cancers. Concurrent
Table 1: In vivo Raman spectroscopic studies of cervical cancer diagnosis Acquisition time (s) 90 60-180 5 5
Laser power (mW) 15 15 80 80
Number of cases 13 79 90
3 3 1 2-3 1
80 80 100 80 100
120 133 46 75 29
1
100
44
1
100
84
Major findings In vivo Raman spectra can be measured from cervix Ex vivo and in vivo RS exhibits similarities Classification of high‑grade squamous dysplasia and normal Classification of ectocervix, endocervix, and, low‑grade and high‑grade dysplasia shows multiclass algorithm is better for classifications Incorporation of hormonal status improves classification Incorporation of menopausalstatus improves classification Highwavenumber can detect cervical dysplasia Body mass index and parity have the greatest impact on classification genetic algorithm‑partial least squares‑discriminant analysis with double cross‑validation identify cervical dysplasia Simultaneous fingerprint and high wavenumber has potential to detect cervical dysplasia Confocal Raman spectroscopy has potential to improve early diagnosis
Reference number [39] [40] [41] [42] [43] [44] [45] [47] [46] [48] [49]
RS=Raman spectra, GA‑PLS‑DA=Genetic algorithm‑Partial least squares‑Discriminant analysis
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chemoradiotherapy (CCRT) is the choice of treatment for stage IIA and other locally advanced tumors. Details of typical treatment regimen and its response evaluation are elaborated elsewhere.[51] Radiation resistance is a serious hurdle in CCRT and there are no methods to predict tumor response at early stages of treatment regimen. Hence, efforts are also made towards development of new method which is objective, rapid, and preferably noninvasive. Vibrational microspectroscopic studies on formalin‑fixed malignant cervix tissue sections has demonstrated feasibility of classifying formalin‑fixed tumor tissues which was collected before and 24 h after patient was exposed to 2nd fraction (2RT) of CCRT.[52] It is observed that classification between normal, pre‑, and post‑RT biopsies could be achieved, since all subjects accrued in the study were complete responders, no conclusion on prediction of treatment response could be drawn. Subsequent investigations on cervix biopsies, before and after 2nd radiation fraction of CCRT, it was observed that PCA of post-CCRT spectra could be classified into responding (complete and partial response) and nonresponding conditions. But untreated tumor spectra failed to provide any classification‑based tumor response to CCRT.[53] 5RT and 10RT, that is, tissue collected after 5th fraction of treatment and 10th fraction of radiation treatment were explore and pattern of classification has been same as 2RT, described above. A pilot study based on 42 tissues using fiberoptic‑based Raman setup, with an outlook for an in vivo approach demonstrated that normal tissue can be efficiently classified from pre‑ and post‑treated tumor biopsies and PCA of complete responder, partial responder, and nonresponder showed the tendency of classification.[51] The results of this study further support the feasibility of prospective noninvasive in vivo RS of tumor radioresponse. However, this study needs to be further validated on larger sample size as well as on diverse population. CONCLUSIONS Cervical cancers are one of the leading cancers and major cause of death among women in developing world, including India. In view of known limitations, development of new approaches for rapid, objective, and noninvasive/minimally invasive methods are actively being pursued. Several Raman spectroscopic studies demonstrated applicability of these methods in screening and therapeutic monitoring of cervical cancers. Raman studies of HPV detection have been reported, but further studies are warranted. As is known, HPV detection is one of the latest conventional methods of screening. For prospective evaluation, Raman‑based label free approaches can be adjuvant to HPV cytological‑based screening. Pap test is the workhorse for cervical screening. Proof of concept for Raman‑based screening of cervical exfoliated cells has been demonstrated. In this case, large‑scale studies are desired for further validations. Robust and highly‑specific Raman methods for identification of even normal cervical
smears can reduce the workload on conventional pathology/ cytology; fatigue is often attributed to be major reason for false reporting. Several groups have demonstrated in vivo Raman identification of cancers and precancers in clinically acceptable time. Our own recent study suggests that vagina can be utilized as control for in vivo Raman studies. [54] This could be very useful in screening at remote and rural health camps where colposcope may not be available to identify healthy control areas in cervix. This makes one step forward towards noninvasive, rapid, and objective Raman‑based screening. Very limited studies, mostly by us, so far have demonstrated utility of RS in identifying responders/nonresponders to CCRT of cervix tumors. Hence, further studies are needed to validate this approach. REFERENCES 1. Schiffman M, Wentzensen N. Human papillomavirus infection and the multistage carcinogenesis of cervical cancer. Cancer Epidemiol Biomarkers Prev 2013;22:553‑60. 2. Sankaranarayanan R, Ferlay J. Worldwide burden of gynaecological cancer: The size of the problem. Best Pract Res Clin Obstet Gynaecol 2006;20:207‑25. 3. López‑Gómez M, Malmierca E, de Górgolas M, Casado E. Cancer in developing countries: The next most preventable pandemic. The global problem of cancer. Crit Rev Oncol Hematol 2013;88:117‑22. 4. Darwish AM, Abdulla SA, Zahran KM, Abdel‑Fattah NA. Reliability of unaided naked‑eye examination as a screening test for cervical lesions in a developing country setup. J Low Genit Tract Dis 2013;17:182‑6. 5. de Boer P, Adam JA, Buist MR, vab de Vijver MJ, Rasch CR, Stoker J, et al. Role of MRI in detecting involvement of the uterine internal os in uterine cervical cancer: Systematic review of diagnostic test accuracy. Eur J Radiol 2013;82:e422‑8. 6. Francisco AL, Correr WR, Pinto CA, Kurachi C, Kowalski LP. PP021: Fluorescence spectroscopy for evaluation of safety margins in individuals with squamous cell carcinoma of the oral cavity surgically treated. Oral Oncol 2013;49:S100. 7. Spliethoff JW, Evers DJ, Klomp HM, van Sandick JW, Wouters MW, Nachabe R, et al. Improved identification of peripheral lung tumors by using diffuse reflectance and fluorescence spectroscopy. Lung Cancer 2013;80:165‑71. 8. Lee S, Kim K, Lee H, Jun CH, Chung H, Park JJ. Improving the classification accuracy for IR spectroscopic diagnosis of stomach and colon malignancy using non‑linear spectral feature extraction methods. Analyst 2013;138:4076‑82. 9. Zeng H. Real‑time in vivo Raman spectroscopy‑technology development and clinical applications in early cancer detection. Bio‑Optics: Design and application. Opt Soc Am 2013. Available from: http://dx.doi.org/10.1364/BODA.2013.BM2A.1 [Last accessed on 2013 Apr 18]. 10. Tu Q, Chang C. Diagnostic applications of Raman spectroscopy. Nanomedicine 2012;8:545‑58. 11. Brauchle E, Schenke‑Layland K. Raman spectroscopy in biomedicine – non‑invasive in vitro analysis of cells and extracellular matrix components in tissues. Biotechnol J 2013;8:288‑97. 12. Krafft C, Sergo V. Biomedical applications of Raman and infrared spectroscopy to diagnose tissues. Spectroscopy 2006;20:195‑218. 13. Chen HC, Schiffman M, Lin CY, Pan MH, You SL, Chuang LC, et al. CBCSP‑HPV Study Group. Persistence of type‑specific human papillomavirus infection and increased long‑term risk of cervical cancer. J Natl Cancer Inst 2011;103:1387‑96. 14. Jess PR, Smith DD, Mazilu M, Dholakia K, Riches AC, Herrington CS.
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Early detection of cervical neoplasia by Raman spectroscopy. Int J Cancer 2007;121:2723‑8. 15. Ostrowska KM, Malkin A, Meade A, O’Leary J, Martin C, Spillane C, et al. Investigation of the influence of high‑risk human papillomavirus on the biochemical composition of cervical cancer cells using vibrational spectroscopy. Analyst 2010;135:3087‑93. 16. Vargis E, Tang YW, Khabele D, Mahadevan‑Jansen A. Near‑infrared Raman microspectroscopy detects high‑risk human papilloma viruses. Transl Oncol 2012;5:172‑9. 17. Krishna CM, Sockalingum GD, Kegelaer G, Rubin S, Kartha VB, Manfait M. Micro‑Raman spectroscopy of mixed cancer cell populations. Vib Spectrosc 2005;38:95‑100. 18. Wong PT, Wong RK, Caputo TA, Godwin TA, Rigas B. Infrared spectroscopy of exfoliated human cervical cells: Evidence of extensive structural changes during carcinogenesis. Proc Natl Acad Sci USA 1991;88:10988‑92. 19. Fung Kee Fung M, Senterman M, Eid P, Faught W, Mikhael NZ, Wong PT. Comparison of Fourier‑transform infrared spectroscopic screening of exfoliated cervical cells with standard papanicolaou screening. Gynecol Oncol 1997;66:10‑5. 20. Cohenford MA, Rigas B. Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: Implications for tumor biology. Proc Natl Acad Sci U S A 1998;95:15327‑32. 21. Wood BR, Quinn MA, Tait B, Ashdown M, Hislop T, Romeo M, et al. FTIR microspectroscopic study of cell types and potential confounding variables in screening for cervical malignancies. Biospectroscopy 1998;4:75‑91. 22. Sindhuphak R, Issaravanich S, Udomprasertqul V, Srisookho P, Warakamin S, Sindhuphak S, et al. A new approach for the detection of cervical cancer in Thai women. Gynecol Oncol 2003;90:10‑4. 23. Romeo MJ, Quinn MA, Burden FR, Mcnaughton D. Influence of benign cellular changes in diagnosis of cervical cancer using IR microspectroscopy. Biopolymers 2002;67:362‑6. 24. Martin FL, Fullwood NJ. Raman vs. Fourier transform spectroscopy in diagnostic medicine. Proc Natl Acad Sci U S A 2007;104:E1. 25. Rubina S, Maheswari A, Deodhar KK, Bharat R, Krishna CM. Raman spectroscopic study on classification of cervical cell specimens. Vib Spectrosc 2013;68:115‑21. 26. Mahadevan‑Jansen A, Mitchell MF, Ramanujam N, Malpica A, Thomsen S, Utzinger U, et al. Near‑Infrared Raman spectroscopy for in vitro detection of cervical precancers. Photochem Photobiol 1998;68:123‑32. 27. Krishna CM, Prathima NB, Malini R, Vadhiraja BM, Rani AB, Fernandes DJ, et al. Raman spectroscopy studies for diagnosis of cancers in human uterine cervix. Vib Spectrosc 2006;41:136‑41. 28. Faolain EO, Hunter MB, Byrne JM, Kelehan P, Byrne HJ, Lyng FM. The potential of vibrational spectroscopy in the early detection of cervical cancer: An exciting emerging field. Proceedings of Opto Ireland 2005. SPIE 2005;5826:25‑36. 29. Lyng FM, Faolain EO, Conroy J, Meade AD, Knief P, Duffy B, et al. Vibrational spectroscopy for cervical cancer pathology, from biochemical analysis to diagnostic tool. Exp Mol Pathol 2007;82:121‑9. 30. Downes A, Elfick A. Raman spectroscopy and related techniques in biomedicine. Sensors (Basel) 2010;10:1871‑89. 31. Boon ME, Suurmeijer AJ. The precursor lesions. The Pap Smear. 3rd ed. Amsterdam: Harwood Academic Publishers GmbH; 1996. p. 99‑137. 32. Martinho Had S, Monteiro da Silva CM, Yassoyama MC, Andrade Pde O, Bitar RA, Santo AM, et al. Role of cervicitis in the Raman‑based optical diagnosis of cervical intraepithelial neoplasia. J Biomed Opt 2008;13:054029‑6. 33. Kamemoto LE, Misra AK, Sharma SK, Goodman MT, Luk H, Dykes AC, et al. Near‑infrared micro‑Raman spectroscopy for in vitro detection of cervical cancer. Appl Spectrosc 2010;64:255‑61.
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34. Bergholt MS, Zheng W, Huang Z. Development of a multiplexing fingerprint and high wave number Raman spectroscopy technique for real time in vivo tissue Raman measurements at endoscopy. J Biomed Opt 2013;18:030502. 35. Bergholt MS, Zheng W, Ho KY, Teh M, Yeoh KG, So JB, et al. Fiber‑optic Raman spectroscopy probes gastric carcinogenesis in vivo at endoscopy. J Biophotonics 2013;6:49‑59. 36. Lui H, Zhao J, McLean D, Zeng H. Real‑time Raman spectroscopy for in vivo skin cancer diagnosis. Cancer Res 2012;72:2491‑500. 37. Crow P, Molckovsky A, Stone N, Uff J, Wilson B, Wongkeesong LM. Assessment of fiberoptic near‑infrared Raman spectroscopy for diagnosis of bladder and prostate cancer. Urology 2005;65:1126‑30. 38. Fenn MB, Xanthopoulos P, Pyrgiotakis G, Grobmyer SR, Pardalos PM, Hench LL. Raman spectroscopy for clinical oncology. Adv Opt Technol 2011;2011:1‑20. 39. Mahadevan‑Jansen A, Mitchell MF, Ramanujam N, Utzinger U, Richads‑kortum R. Development of a fiber optic probe to measure NIR Raman spectra of cervical tisue in vivo. Photochem Photobiol 1998;68:427‑31. 40. Utzinger U, Heintzelman DL, Mahadevan‑Jansen A, Malpica A, Follen M, Richards‑Kortum R. Near‑infrared Raman spectroscopy for in vivo detection of cervical precancers. Appl Spectrosc 2001;55:955‑9. 41. Robichaux‑Viehoever A, Kanter E, Shappell H, Billheimer D, Jones H 3rd, Mahadevan‑Jansen A. Characterization of Raman spectra measured in vivo for the detection of cervical dysplasia. Appl Spectrosc 2007;61:986‑93. 42. Kanter EM, Majumder S, Vargis E, Robichaux‑Viehoever A, Kanter GJ, Shappell H, et al. Multiclass discrimination of cervical precancers using Raman spectroscopy. J Raman Spectrosc 2009;40:205‑11. 43. Kanter EM, Majumder S, Kanter GJ, Woeste EM, Mahadevan‑Jansen A. Effect of hormonal variation on Raman spectra for cervical disease detection. Am J Obstet Gynecol 2009;200:512.e1‑5. 44. Kanter EM, Vargis E, Majumder S, Keller MD, Woeste E, Rao GG, et al. Application of Raman spectroscopy for cervical dysplasia diagnosis. J Biophotonics 2009;2:81‑90. 45. Mo J, Zheng W, Low JJ, Ng J, Ilancheran A, Huang Z. High wave number Raman spectroscopy for in vivo detection of cervical dysplasia. Anal Chem 2009;81:8908‑15. 46. Duraipandian S, Zheng W, Ng J, Low JJ, Ilancheran A, Huang Z. In vivo diagnosis of cervical precancer using Raman spectroscopy and genetic algorithm techniques. Analyst 2011;136:4328‑36. 47. Vargis E, Byrd T, Logan Q, Khabele D, Mahadevan‑Jansen A. Sensitivity of Raman spectroscopy to normal patient variability. J Biomed Opt 2011;16:117004. 48. Duraipandian S, Zheng W, Ng J, Low JJ, Ilancheran A, Huang Z. Simultaneous fingerprint and high‑wave number confocal Raman spectroscopy enhances early detection of cervical precancer in vivo. Anal Chem 2012;84:5913‑9. 49. Duraipandian S, Zheng W, Ng J, Low JJ, Ilancheran A, Huang Z. Near‑infrared‑excited confocal Raman spectroscopy advances in vivo diagnosis of cervical precancer. J Biomed Opt 2013;18:067007. 50. Sathe P, Domnic D, Sastri Goda J, Chopra S, Krishna CM. International onference on Radiation Biology (ICRB‑2012) on cosmic radiation to cancer therapeutics and 11th Biennial Meeting of the Indian Society of Radiation Biology. J Can Res Ther 2012;8:468‑506. 51. Rubina S, Vidyasagar M, Krishna CM. Raman spectroscopic study on prediction of treatment response in cervical cancers. J Innov Opt Health Sci 2013;6:1350014‑22. 52. Krishna CM, Sockalingum GD, Vadhiraja BM, Maheedhar K, Rao AC, Rao L, et al. Vibration spectroscopy studies of formalin‑fixed cervix tissues. Biopolymers 2007;85:214‑21. 53. Vidyasagar M, Maheedhar K, Vadhiraja BM, Fernendes DJ, Kartha VB, Krishna CM. Prediction of radiotherapy response in cervix cancer by Raman spectroscopy: A pilot study. Biopolymers 2008;89:530‑7.
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54. Rubina S, Dora TK, Chopra S, Maheshwari A, Kedar KD, Bharat R, et al. In vivo Raman spectroscopy of human uterine cervix: Exploring the utility of vagina as an internal control. J Biomed Opt 2014;19:087001.
Cite this article as: Rubina S, Krishna CM. Raman spectroscopy in cervical cancers: An update. J Can Res Ther 2015;11:10-7. Source of Support: Nil, Conflict of Interest: None declared.
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Raman spectroscopy in cervical cancers: An update ABSTRACT Cervical cancer is the third most common cancer among women worldwide. Developing countries contribute more than 80% towards global burden. Over the last 2 decades, Raman spectroscopy (RS) has been actively pursued for cervical cancer detection. In view of latest development in Raman spectroscopic applications in cervical cancers, especially in vivo studies, an update of the same is presented in this article. This articles opens with a brief note on Anatomy of cervix followed by Etiology, and conventional Screening and Diagnosis of Cervical cancers. In subsequent sections, brief description of Theory and Instrumentation of RS is followed by a review of recent developments in cervical cancer detection; with emphasis on cell lines, exfoliated cells, ex vivo and in vivo, and therapeutic response monitoring applications in cervical cancer. KEY WORDS: Biomedical applications, cervical cancers, raman spectroscopy
INTRODUCTION Cervix is the lower third of the uterus that extends into vagina. The part of the cervix that projects into the vagina is called as ectocervix, which is covered by nonkeratinized, stratified, squamous epithelium. The uterine part of cervix is called as endocervix, which is covered with mucus‑secreting columnar epithelium. Pictorial depiction of cervical anatomy is shown in Figure 1. The cervical transformation zone is an area of metaplastic tissue between the squamous epithelium of the vagina and the glandular tissue of the endocervical canal. Squamocolumnar junction has unique susceptibility to Human Papillomavir us (HPV)‑induced neoplastic transformation leading to cancer.[1] Cervical cancer is the third most frequent cancer among women worldwide and commonest female cancer in developing countries, including India. Developing countries contribute more than 80% to global cervical cancer burden. [2] While, in developed countries, cervical cancer incidence and mortality rates have declined due to introduction of cervical cancer screening campaigns, highlighting the significance of screening.[3] Papanicolaou test (Pap test), exfoliated cervical‑vaginal cytology, is the most practiced screening method. However, Pap test being laborious and is also known to suffer from some known limitations like low sensitivity and demands stringent quality assurance. Other known screening technologies are HPV testing, liquid‑based cytology, automated 10
cervical screening tool, and visual inspection of cervix after applying Lugol’s iodine (VILI) or acetic acid (VIA). [4] In routine clinical practice, an abnormal Pap smear is followed by colposcopic‑guided biopsies for confirmatory diagnosis. Though histopathological examination of excised biopsies remains as the gold standard of cervical cancer diagnosis, current conventional screening/diagnosis tools are also known to suffer from several disadvantages like tedious sample processing, long output duration, and the interobserver variability.[5] Therefore, conscious efforts are made for the development of minimally invasive and effective screening/diagnostic methodology that can probe the endogenous biomolecular properties of cells/tissue for early cancer detection. These studies represent vital advancements towards more efficient detection of cervical cancers. Among various methods, spectroscopy‑based approaches like fluorescence, diffuse reflectance, Fourier transformed infrared spectroscopy (FTIR), and Raman spectroscopy (RS) are being actively explored as potential stand alone or adjuvant diagnostic tools. [6‑12] RS represents a unique technique capable of label‑free and nondestructively probing endogenous biomolecules, for example, proteins, lipids, carbohydrates, and nucleic acids; to determine highly specific diagnostic information.[9‑12] The current review presents an update of latest developments of application of RS in cervical cancer detection based on cells, ex vivo, in vivo, and therapeutic response monitoring.
S. Rubina, C. Murali Krishna1 Chilakapati Laboratory, Advanced Centre for Treatment Research and Education in Cancer, Kharghar, Navi‑Mumbai, 1 Scientific Officer ‘F’ and Principal Investigator, Chilakapati Laboratory, Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi‑Mumbai, Maharashtra, India For correspondence: Dr. Murali C. Krishna, Scientific Officer ‘E’ and Principal Investigator, Chilakapati Laboratory, Advanced Center for Treatment Research and Education in Cancer, Tata Memorial Center, Kharghar, Sector 22, Navi Mumbai ‑ 410 210, Maharashtra, India. E‑mail: mchilakapati@ actrec.gov.in, [email protected]
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.154065 PMID: *** Quick Response Code:
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Figure 1: Pictorial depiction of cervix anatomy
BASIC PRINCIPLE AND INSTRUMENTATION RS is based on light scattering phenomenon. Light scattering can be explained as two‑photon process, the incident and scattered photon. Most often, scatter photons are of same energy to that of incident photon (elastic/Rayleigh scattering), but photons occasionally gain or lose energy resulting in frequency shift (inelastic/Raman scattering). Raman scattering is a weak phenomenon involving approximately one in every 106–108 scattered photons.[10] A typical Raman spectrometer consists of highly monochromatic excitation source, optical path, Rayleigh rejection filters, monochromator/dispersing elements, and detector.[10] Typically, excitation (laser beam) photons are coupled to the sample by appropriate optical path. Scattered photons from the sample are gathered, dispersed, and detected to transform the photons into spectrum. Advent of modern and high‑end technologies, the modern biomedical Raman spectrometers employ lasers, high throughput spectrographs, and multichannel charged coupled devices (CCDs). CELL‑BASED DIAGNOSIS BY RS IN CERVICAL CANCERS Classification of HPV +ve and HPV −ve cell lines High risk human papillomavirus (HR‑HPV) is a well‑known etiological factor of cervical cancers. HPV‑induced cervical carcinogenesis includes HPV infection, persistence of virus infection, precancers, and eventual invasion. Persistence of virus is linked to the development of a high‑grade precursor lesion or “precancer”. Although identification of HPV presence has clinical significance, it is pertinent to note that all HPV infections may not lead to cervical cancers after clearance of HPV infection.[13] Recently, high‑risk HPV strains testing have been incorporated into routine cervical cancer screening for menopausal females in developed countries. Hence, a brief note on studies related to Raman spectroscopic detection of HR‑HPV is presented. The first study on Raman spectroscopic identification of HPV‑16 virus was reported by Jess et al. They
observed variations in Raman bands of deoxyribonucleic acid (DNA) and proteins, which were consistent with HPV gene expression and cellular changes associated with neoplasia.[14] This study could discriminate between normal keratinocytes and keratinocytes expressing HPV‑16 E7 with 93% sensitivity and specificity. Subsequent study in the same line investigated biochemical changes in cells caused by high‑risk HPV strands. This study explored differences between the cells with high, medium, and low HPV copy number.[15] It was observed by authors that RS can discriminate between the cell lines with HPV presence and their copy number as well as elucidate cellular differences originating from proteins, nucleic acids, and lipids. Another study evaluated the ability of RS to detect the presence of HPV and differences between specific HPV strains using cell lines and patient specimens.[16] This study indicated that spectra of cell culture and patient samples exhibit statistically significant differences. This could be attributed to the fact that real‑life specimen and experimental cell lines are maintained in different environmental niche, hence can have differences in their biochemical constituents. Nevertheless, this study indicated that RS can detect HPV and differentiate among specific HPV strains. We have also investigated differences in HPV‑positive and ‑negative cell lines using RS. We have recorded Raman spectra of HPV‑18 positive HeLa, HPV‑16‑positive SiHa, and HPV negative C33A cell lines. Spectra were acquired using 40× objective coupled with HE‑785 Raman instrument (Jobin‑Yvon‑Horiba, France). Mean Raman spectra of C33A, HeLa, and SiHa cells are shown in Figure 2a. HPV negative (C33A) cells and HPV‑positive (HeLa and SiHa) cells showed distinct differences at amide I and δ CH2 region. Minor variations in amide III region were also observed, whereas no significant differences between HPV‑positive cells were discernible. A possible explanation for this observation could be that HPV infection eventually leads to oncoprotein expression resulting in differences in protein compositions in the host cells.[15] Principal component- linear discriminant analysis (PC‑LDA) gave well‑separated clusters with classification efficiency of ~95% [Figure 2b]. Findings of the study corroborate with earlier report and demonstrate subtle but significant differences that exist between HPV‑positive and ‑negative cell lines. Although HPV detection has clinical significance, as mentioned earlier, it is important to note that very few HPV‑infected subjects eventually develop cancer.[13] Hence, it is important to understand HPV‑induced cell changes leading to neoplasia. Further, the delineation observed for HPV‑positive and negative cell lines may not be entirely due to HPV presence. This could be because of the fact that the observed spectral variation in HPV‑positive and ‑negative cell lines can be due to differences in cell lines itself. This is quite clear from one of our RS studies on randomly mixed cell populations.[17] Raman spectral profile very much varies with cell lines and same can be explored for cell typing. Therefore, further studies on
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a
b
Figure 2: Raman studies of HPV +ve and –ve cell lines. (a) Average Raman spectra of HPV +ve (solid line) and −ve cell line (Dotted lines) and (b) 3D scatter plot for Hela, SiHa, and C33A cells. HPV = Human papillomavirus, 3D = Three‑dimensional
HPV‑induced‑neoplastic changes in the same type of cells are warranted. RS of exfoliated cells Wong et al., were first to report the potential of infrared (IR) spectroscopy to classify normal and abnormal cervical exfoliated cells.[18] Several subsequent IR studies of exfoliated cervical cells have explored cell pellet and single cell approach. [19‑24] However, presence of water in biological specimens is a serious hurdle in FTIR spectroscopy; hence, dried samples were used for study. Due to which same cells could not be used for Pap staining and parallel sample for Pap staining may not be ideal for cytological correlation, as abnormal cell content in an ‘abnormal’ smear can vary. To circumvent these limitations, we have explored feasibility of classification of cervical cell specimens using RS.[25] This study could distinguish normal and abnormal exfoliated specimens with 86 and 84% classification efficiencies, respectively. However, major concern is spectra being influenced by the presence of blood in abnormal specimen, which may lead to false interpretations, as bleeding also occur in several noncancerous conditions. Further studies on red blood corpuscles (RBC) lysis buffer‑treated specimens also gave classification efficiency of about 79 and 78% for normal and abnormal smears, respectively. Thus, indicating efficacy of RS in classifying normal and abnormal exfoliated specimens in both treated and untreated conditions. Even though findings of this study are quite comparable to conventional Pap test, efficacy of this approach can be further improved by developing robust model by selectively accruing specimens with very higher number of abnormal cells. Spectra acquired from cell pellet then can be compared against developed model and sample wherein even a spectrum matches with cancer model can be assigned as cancer, similar to the standard practice in histopathology and cytology. In conventional cytology, cell smears containing few tumor cells are treated as cancer. As mentioned earlier, Raman’s studies on cell pellets have also 12
shown the feasibility of classifying a single cell type in a mixed cancer cell population, HPV detection, and discrimination of wild and multidrug resistant cell type. Thus, prospective studies on pure cancerous and precancerous specimens to build true standard models and validation through blinded specimens on clinically significant sample size can further pave RS as a tool in cervical cancer. EX VIVO STUDIES Raman spectroscopic studies on ex vivo tissue are carried out, either by conventional RS or Raman microspectroscopy. Conventional Raman spectroscopic studies on tissues are important as they mimic the in vivo conditions. However, to improve the understanding of Raman spectral signatures, Raman microspectroscopic studies are ideal. This is because Raman microscope can facilitate site‑specific and in‑depth evaluations. This section will elaborate on Raman spectroscopic ex vivo studies of cervical cancer tissues. Conventional Raman spectroscopic studies In vitro Raman spectra of fresh, frozen, and preserved cervix tissue biopsies have been reported in the literature.[26‑29] Ex vivo RS study on cervix tissue carried out as early as 1998, indicated potential advantage of RS to detect cervical precancers.[26] They concluded that empirically selected normalized intensities can differentiate precancers from other tissues with sensitivity and specificity of 88 and 92%, respectively. However, unbiased multivariate methods gave the sensitivity of 82% and specificity of 92%. Raman algorithms can potentially separate benign abnormalities such as inflammation and metaplasia from precancers. They reported that the tissue spectra showed the molecular features of collagen, nucleic acids, phospholipids, and glucose l‑phosphate. In 2006, our group reported a study on ex vivo cervix tissues by RS.[27] We have observed that Raman spectra of normal cervix tissue are characterized by strong, broad amide I, broader amide III,
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and strong peaks at 853 and 938 cm−1. This was attributed to structural proteins such as collagen. While prominent features of malignant tissue spectra with respect to normal tissue were relatively weaker and sharper amide I, minor red shift in δCH2 and sharper amide III which indicated the presence of DNA, lipids, and noncollagenous proteins. PCA combined with multiparametric limit test was used for discriminating different pathological conditions in cervical cancer which produced 99.5% sensitivity and specificity. Raman microspectroscopic study on cervix tissue The architecture of tissue consists of 8 to 10 layers of cells called as epithelium resting on connective tissue which is rich in collagen. While epithelium is very thin; it consists of basal, intermediate, and superficial layer. Probing volume and the kind of cells and tissue molecular composition plays significant role in determining Raman spectral signature. Previous Raman spectroscopic studies on cervix tissue biopsies reported presence of collagenous features in normal tissue spectra and contribution of Raman signal arising from the collagenous rich connective tissue cannot be ruled out. Whereas the Raman signal of tumor tissue biopsies contributed more of DNA, lipid, and noncollagenous protein features which can be of tumor cells. Raman imaging on tissue sections is very demanding due to the extremely long acquisition time for pixel‑by‑pixel acquisition. However, recent technological advancement in Raman imaging has improved the acquisition time.[30] Previous study on tissue sections included spectral acquisition from different layers of tissue. This is a comparative Raman and synchrotron IR (SR‑IR) spectroscopy study on parallel cervical cancer specimens.[29] In their study, frozen and dewaxed formalin paraffin preserved tissues were used and discrimination between different cell types in normal cervical tissue was reported. The spectra of invasive carcinoma showed a marked difference from normal cervical epithelial cells. It is observed that spectral differences identified with the onset of carcinogenesis include increased nucleic acid contributions and decreased glycogen levels. Subsequent study investigated the potential of RS as a diagnostic tool to detect biochemical changes associated to cervical cancer progression.[30] In their study, Raman spectra were acquired from different point of dewaxed 10 µm section and Raman spectra of pure compound of proteins, nucleic acids, lipids, and carbohydrates were employed in order to gain an insight into the biochemical composition of cells and tissues. It is reported that spectra of basal cells show strong bands at 724, 779, and 1,578 cm−1, which are characteristics of nucleic acids. Spectra of epithelial cells showed characteristic glycogen bands at 482, 849, 938, 1,082, and 1,336 cm−1; whereas, spectra of connective tissue showed characteristic collagen bands at 850, 940, and 1,245 cm −1. Absence of glycogen bands, presence of characteristic nucleic acid band, and increase in the intensity of the amide I band in the spectra of invasive carcinoma has been demonstrated. Spectral features of premalignant specimens also showed the nucleic acid bands
at 724, 779, 852, 1366 and 1578 cm−1. Spectral differences of basal cells to that of tumor were investigated. It is vital to note that abnormality in the cells of basal layer develop the neoplastic tumor cells.[31] Hence, it is important to probe the differences between basal and tumor cells, as these cells are in dividing phase though proliferative index of tumor cells are high as compared to the basal cells. Another study on the Raman‑based optical diagnosis of normal cervix, inflammatory cervix (cervicitis), and cervical intraepithelial neoplasia (CINI) found major alterations in the 857 cm−1 (CCH deformation aromatic); 925 cm−1 (C‑C stretching); 1,247 cm−1 (CN stretch, NH bending of Amide III); 1,370 cm−1 (CH2 bending); and 1,525 cm−1 (C = C = C = N stretching) vibrational bands.[32] This study corroborated earlier reported spectral profiles. Another in vitro Raman microspectroscopy study on normal and cancerous cervical human tissue section observed the spectral features associated with collagen (775–975 cm−1) in normal squamous cells, which were below the detection limit in cancer.[33] In this study, Raman chemical maps, using 775–975 cm−1 and 2,800–3,100 cm−1 spectral regions, of cancer and normal cells in the cervical epithelium have also been found to show good correlation with each other. Thus, as described above, several studies not only demonstrated efficacy of Raman microspectroscopic approach in classifying normal and pathological cervical condition but also its utility in understanding differences at biomolecular level. IN VIVO STUDIES Several fiber‑probe in vivo Raman studies for diagnosis of different cancers, including cervical cancers, have appeared in the literature.[32,34‑49] In 1998, a fiberoptic in vivo Raman study of cervix to delineate precancer lesions from the normal cervix was reported.[39] In this study, in vivo Raman spectra of cervix could be acquired in 90 s. Authors concluded that the increase in the laser power could reduce the integration time to less than 20 s for measuring in vivo Raman spectra of cervix. Moreover, another pilot clinical trial by the same group suggested that in vivo Raman spectra resemble to that of in vitro cervix tissue.[40] The study has also concluded that cervical epithelial cells may contribute to tissue spectra at 1,330 cm−1, a region associated with DNA and epithelial cells probably do not contribute to tissue spectra at 1,454 cm−1, a region associated with collagen and phospholipids. In this study Raman spectral acquisition time was 60–180 s, which was relatively longer than the clinically acceptable time. Another study, using clinical feasible time (5 s), could distinguish between high‑grade dysplasia and benign with sensitivity and specificity of 89 and 81%, respectively.[41] A subsequent in vivo study could discriminate between normal ectocervix, normal endocervix, low‑grade dysplasia, and high‑grade dysplasia with 95% classification efficiency.[42] Moreover, this study showed that RS can differentiate among the different precancers with improved sensitivity of 98% and specificity of 96% in conjunction with multiclass discrimination algorithms like maximum representation and discrimination feature (MRDF)
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and sparse multinomial logistic regression (SMLR). Subsequent study looked at variations in normal cervical spectra due to menopausal status and time point in the menstrual cycle.[43] The study wherein the data stratified by menopausal state resulted in improvement of accurate classification of low grade squamous intraepithelial lesion (LGSIL) to 97% from 74%.[44] This makes RS much closer to clinical use. Another study has explored utility of high wave number (HW) Raman spectra in detection of precancer cervix.[45] This study demonstrated the diagnostic algorithms based on principal components analysis and LDA together with the leave‑one‑patient‑out cross‑validation method yielded a diagnostic sensitivity of 93.5% and specificity of 97.8% for dysplasia identification. Another study suggested that RS in conjunction with genetic algorithm‑partial least squares‑discriminant analysis (GA‑PLS‑DA) with double cross‑validation (dCV) methods has potential to provide clinically significant discrimination between normal and precancer cervix.[46] Variability such as race/ethnicity, body mass index (BMI), parity, and socioeconomic status was also explored.[47] Findings of the study suggest that BMI and parity have greatest impact; whereas, race/ethnicity and socioeconomic status have a limited effect. Another study reported that simultaneous fingerprint (FP)/HW confocal RS has potential to early diagnosis and detection of cervical precancer in vivo.[48] This study demonstrated integrated FP and HW in vivo Raman signals of a cervix can be classified with 85% sensitivity and 81% specificity. Also spectral differences between normal and dysplastic cervix can be ascribed to protein, lipids, glycogen, nucleic acids, and water content. More recent confocal studies have demonstrated great potential of in vivo RS to improve early diagnosis of cervical precancers.[49] Confocal RS coupled with PCA‑LDA modeling yielded a sensitivity and specificity of 81 and 87%, respectively, for in vivo discrimination of dysplastic cervix. It is also concluded that the best classification can be achieved using confocal RS compared to the composite near infrared autofluorescence (NIR AF)/RS or NIR AF spectroscopy alone. This study illustrated that confocal RS has great potential to improve early in vivo diagnosis of cervical precancers during clinical colposcopy.
In vivo Raman applications in cervical cancers have been enlisted in Table 1. As table suggests, general trend of lesser spectral acquisition time with increased laser power is indicative of technological advancement as well as closer to clinical applicability. As mentioned earlier, Raman spectral signature depends on the probing volume and spectral acquisition site of tissue. Most of the in vivo studies on cervical cancers have focused on detection of premalignant patches. Considering thin epithelium of cervix and the probing volume/ depth could be the important criterion; thus as reported by recent studies, using confocal Raman probes may have great potential to improve early diagnosis. THERAPEUTIC APPLICATION OF RS IN CERVICAL CANCERS Most work on RS to date has focused on diagnostic applications, particularly the early detection of cancer and precancerous changes in cervix. RS also has enormous potential in monitoring the result of treatment, that is, tumor therapeutic response. This section highlights the efforts towards therapeutic application of RS in cervical cancers using cells and tissue specimens. Studies on ex vivo cervical cell lines and treatment response We have also explored potentials of RS in classifying HeLa cells irradiated at different doses and its correlation with biological assays (clonogenic and γ‑H2AX assay).[50] PCA gave exclusive clusters for HeLa cells irradiated at low doses, that is, 2Gy, 4Gy, and control. But overlapping clusters have been observed for HeLa cells irradiated at higher doses, that is, 6, 8, and 10Gy. Preliminary work supports the feasibility of RS to classify HeLa cells that were subjected to different doses of radiation and results showed good correlation with biochemical assays. Studies on ex vivo tissues and treatment response Approximately 80% of cervical cancer patients present at advanced stages (stage IIA and above) in developing countries like India. The 5‑year disease free survival rate of less than 60% are reported for these cancers. Concurrent
Table 1: In vivo Raman spectroscopic studies of cervical cancer diagnosis Acquisition time (s) 90 60-180 5 5
Laser power (mW) 15 15 80 80
Number of cases 13 79 90
3 3 1 2-3 1
80 80 100 80 100
120 133 46 75 29
1
100
44
1
100
84
Major findings In vivo Raman spectra can be measured from cervix Ex vivo and in vivo RS exhibits similarities Classification of high‑grade squamous dysplasia and normal Classification of ectocervix, endocervix, and, low‑grade and high‑grade dysplasia shows multiclass algorithm is better for classifications Incorporation of hormonal status improves classification Incorporation of menopausalstatus improves classification Highwavenumber can detect cervical dysplasia Body mass index and parity have the greatest impact on classification genetic algorithm‑partial least squares‑discriminant analysis with double cross‑validation identify cervical dysplasia Simultaneous fingerprint and high wavenumber has potential to detect cervical dysplasia Confocal Raman spectroscopy has potential to improve early diagnosis
Reference number [39] [40] [41] [42] [43] [44] [45] [47] [46] [48] [49]
RS=Raman spectra, GA‑PLS‑DA=Genetic algorithm‑Partial least squares‑Discriminant analysis
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Rubina and Krishna: Raman spectroscopy and cervical cancers
chemoradiotherapy (CCRT) is the choice of treatment for stage IIA and other locally advanced tumors. Details of typical treatment regimen and its response evaluation are elaborated elsewhere.[51] Radiation resistance is a serious hurdle in CCRT and there are no methods to predict tumor response at early stages of treatment regimen. Hence, efforts are also made towards development of new method which is objective, rapid, and preferably noninvasive. Vibrational microspectroscopic studies on formalin‑fixed malignant cervix tissue sections has demonstrated feasibility of classifying formalin‑fixed tumor tissues which was collected before and 24 h after patient was exposed to 2nd fraction (2RT) of CCRT.[52] It is observed that classification between normal, pre‑, and post‑RT biopsies could be achieved, since all subjects accrued in the study were complete responders, no conclusion on prediction of treatment response could be drawn. Subsequent investigations on cervix biopsies, before and after 2nd radiation fraction of CCRT, it was observed that PCA of post-CCRT spectra could be classified into responding (complete and partial response) and nonresponding conditions. But untreated tumor spectra failed to provide any classification‑based tumor response to CCRT.[53] 5RT and 10RT, that is, tissue collected after 5th fraction of treatment and 10th fraction of radiation treatment were explore and pattern of classification has been same as 2RT, described above. A pilot study based on 42 tissues using fiberoptic‑based Raman setup, with an outlook for an in vivo approach demonstrated that normal tissue can be efficiently classified from pre‑ and post‑treated tumor biopsies and PCA of complete responder, partial responder, and nonresponder showed the tendency of classification.[51] The results of this study further support the feasibility of prospective noninvasive in vivo RS of tumor radioresponse. However, this study needs to be further validated on larger sample size as well as on diverse population. CONCLUSIONS Cervical cancers are one of the leading cancers and major cause of death among women in developing world, including India. In view of known limitations, development of new approaches for rapid, objective, and noninvasive/minimally invasive methods are actively being pursued. Several Raman spectroscopic studies demonstrated applicability of these methods in screening and therapeutic monitoring of cervical cancers. Raman studies of HPV detection have been reported, but further studies are warranted. As is known, HPV detection is one of the latest conventional methods of screening. For prospective evaluation, Raman‑based label free approaches can be adjuvant to HPV cytological‑based screening. Pap test is the workhorse for cervical screening. Proof of concept for Raman‑based screening of cervical exfoliated cells has been demonstrated. In this case, large‑scale studies are desired for further validations. Robust and highly‑specific Raman methods for identification of even normal cervical
smears can reduce the workload on conventional pathology/ cytology; fatigue is often attributed to be major reason for false reporting. Several groups have demonstrated in vivo Raman identification of cancers and precancers in clinically acceptable time. Our own recent study suggests that vagina can be utilized as control for in vivo Raman studies. [54] This could be very useful in screening at remote and rural health camps where colposcope may not be available to identify healthy control areas in cervix. This makes one step forward towards noninvasive, rapid, and objective Raman‑based screening. Very limited studies, mostly by us, so far have demonstrated utility of RS in identifying responders/nonresponders to CCRT of cervix tumors. Hence, further studies are needed to validate this approach. REFERENCES 1. Schiffman M, Wentzensen N. Human papillomavirus infection and the multistage carcinogenesis of cervical cancer. Cancer Epidemiol Biomarkers Prev 2013;22:553‑60. 2. Sankaranarayanan R, Ferlay J. Worldwide burden of gynaecological cancer: The size of the problem. Best Pract Res Clin Obstet Gynaecol 2006;20:207‑25. 3. López‑Gómez M, Malmierca E, de Górgolas M, Casado E. Cancer in developing countries: The next most preventable pandemic. The global problem of cancer. Crit Rev Oncol Hematol 2013;88:117‑22. 4. Darwish AM, Abdulla SA, Zahran KM, Abdel‑Fattah NA. Reliability of unaided naked‑eye examination as a screening test for cervical lesions in a developing country setup. J Low Genit Tract Dis 2013;17:182‑6. 5. de Boer P, Adam JA, Buist MR, vab de Vijver MJ, Rasch CR, Stoker J, et al. Role of MRI in detecting involvement of the uterine internal os in uterine cervical cancer: Systematic review of diagnostic test accuracy. Eur J Radiol 2013;82:e422‑8. 6. Francisco AL, Correr WR, Pinto CA, Kurachi C, Kowalski LP. PP021: Fluorescence spectroscopy for evaluation of safety margins in individuals with squamous cell carcinoma of the oral cavity surgically treated. Oral Oncol 2013;49:S100. 7. Spliethoff JW, Evers DJ, Klomp HM, van Sandick JW, Wouters MW, Nachabe R, et al. Improved identification of peripheral lung tumors by using diffuse reflectance and fluorescence spectroscopy. Lung Cancer 2013;80:165‑71. 8. Lee S, Kim K, Lee H, Jun CH, Chung H, Park JJ. Improving the classification accuracy for IR spectroscopic diagnosis of stomach and colon malignancy using non‑linear spectral feature extraction methods. Analyst 2013;138:4076‑82. 9. Zeng H. Real‑time in vivo Raman spectroscopy‑technology development and clinical applications in early cancer detection. Bio‑Optics: Design and application. Opt Soc Am 2013. Available from: http://dx.doi.org/10.1364/BODA.2013.BM2A.1 [Last accessed on 2013 Apr 18]. 10. Tu Q, Chang C. Diagnostic applications of Raman spectroscopy. Nanomedicine 2012;8:545‑58. 11. Brauchle E, Schenke‑Layland K. Raman spectroscopy in biomedicine – non‑invasive in vitro analysis of cells and extracellular matrix components in tissues. Biotechnol J 2013;8:288‑97. 12. Krafft C, Sergo V. Biomedical applications of Raman and infrared spectroscopy to diagnose tissues. Spectroscopy 2006;20:195‑218. 13. Chen HC, Schiffman M, Lin CY, Pan MH, You SL, Chuang LC, et al. CBCSP‑HPV Study Group. Persistence of type‑specific human papillomavirus infection and increased long‑term risk of cervical cancer. J Natl Cancer Inst 2011;103:1387‑96. 14. Jess PR, Smith DD, Mazilu M, Dholakia K, Riches AC, Herrington CS.
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Rubina and Krishna: Raman spectroscopy and cervical cancers
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Journal of Cancer Research and Therapeutics - January-March 2015 - Volume 11 - Issue 1
Rubina and Krishna: Raman spectroscopy and cervical cancers
54. Rubina S, Dora TK, Chopra S, Maheshwari A, Kedar KD, Bharat R, et al. In vivo Raman spectroscopy of human uterine cervix: Exploring the utility of vagina as an internal control. J Biomed Opt 2014;19:087001.
Cite this article as: Rubina S, Krishna CM. Raman spectroscopy in cervical cancers: An update. J Can Res Ther 2015;11:10-7. Source of Support: Nil, Conflict of Interest: None declared.
Journal of Cancer Research and Therapeutics - January-March 2015 - Volume 11 - Issue 1
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