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Serum protein profile studies of cervical cancers in monitoring of tumor response to radiotherapy using HPLC-LIF: A pilot study
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Medical Laser Application 24 (2009) 165–174 www.elsevier.de/mla
Serum protein profile studies of cervical cancers in monitoring of tumor response to radiotherapy using HPLC-LIF: A pilot study Mamidipudi Srinivasa Vidyasagara,, Maheedhar Kodalia,b, Prathima N. Balub, Gunjan Baijala, Bejadi Manjunath Vadhirajaa, Rani A. Bhatc, Donald Jerard Fernandesa, Chilakapati Murali Krishnad a
Department of Radiotherapy and Oncology, Shirdi Sai Baba Cancer Hospital and Research Center, Kasturba Medical College, Manipal University, Manipal 576104, Karnataka, India b Center for Atomic and Molecular Physics, Manipal University, Manipal 576104, Karnataka, India c Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576104, Karnataka, India d Chilakapati Laboratory, Cancer Research Institute, Advanced Center for Treatment Research and Education in Cancer (ACTREC), Kharghar, Sector ‘22’, Navi Mumbai 410 208, Maharastra, India Received 6 February 2009; accepted 6 May 2009
Abstract Objective: Exploration of the feasibility of serum protein profiles for monitoring tumor radioresponse in cervical cancers using HPLC-LIF system. Materials and methods: Twenty-one subjects were recruited in the study. Out of them 7 were healthy, 14 were cervical cancer patients who undertook fractionated radiotherapy (RT) with 2 Gy per fraction over 25 fractions, for 5 weeks followed by 2 applications of intracavitary brachytherapy once a week. Blood collected from above subjects was processed to obtain serum. Serum chromatograms of ‘normal’ (n ¼ 7) and conspicuous probes before RT (n ¼ 14, ‘malignant’) and 24 h after second fraction of RT (n ¼ 13, ‘2-RT’), were recorded using an In-house-built HPLC-LIF set-up. Data were analyzed in two approaches: (1) classical method using relative intensities of selected peaks, (2) principal component analysis (PCA). Clinical assessment of tumor radioresponse was carried out 4 months after first fraction of RT and the degree of the tumor shrinkage was determined as an index of radioresponsiveness (complete response (CR): 100% shrinkage, partial response (PR): Z50% shrinkage, and no response (NR): r50% shrinkage) which was further correlated with the analysis of 2-RT serum chromatograms. Results: Normal vs. malignant chromatograms demonstrated pronounced differences in the 800–1800 s region. Malignant vs. 2-RT chromatograms showed minute variations in the 1300–1800 s region. Our analysis, in both of the approaches, produced clear differentiation between ‘normal’ and ‘malignant’, whereas differentiation between ‘malignant’ and ‘2-RT’ was minimal. Clinical evaluation of the tumor radioresponse yielded that out of 13 patients (one patient discontinued the radiotherapy) ten showed CR, two showed PR and one NR. In case of prediction of tumor radioresponse, analysis of the 2-RT chromatograms produced only minor differentiation among CR, PR and NR groups.
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E-mail addresses: [email protected], [email protected] (M.S. Vidyasagar). 1615-1615/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.mla.2009.05.005
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Conclusion: Protein profiling of serum samples differentiated ‘normal’ from ‘malignant’, but could not differentiate ‘malignant’ from ‘2-RT’. Also this technique has limited application in prediction of tumor radioresponse. r 2009 Elsevier GmbH. All rights reserved. Keywords: Radiation therapy; Tumor markers; Cervical cancer; Principal component analysis (PCA)
Introduction Radiotherapy is the treatment of choice for locally advanced stages of cervical cancer. Tumors of same clinical stage and histological type may respond differently to the same treatment. This could be due to intrinsic factors such as DNA aneuploidy, S-phase fraction, proliferation kinetics, tumor vascularity and hypoxia and glutathione content [1–3]. Advances in molecular biology and high-throughput technologies have provided the hope of discovering novel biological markers in screening, early diagnosis, and prediction of tumor response to therapy [4]. Glutathione (GSH) levels of serum and tissue are probable biomarkers for prediction of tumor radioresponse in cervical cancers [2]. Several proteomics-based studies suggest that serum levels of squamous cell carcinoma antigen (SCC), cancer antigen CA125 [5,6] and other proteins like gasderminlike protein (GSDML) [7], which belongs to gasdermindomain containing protein family, could be used as biomarkers in screening as well as evaluation of therapeutic response in cervical cancers. Studies also suggest that HPV E6 and E7 oncoproteins might help in development of cervical cancer-specific markers [4,8]. However, immunoassays are generally applicable only for detection of a single marker at a time. Detection of several individual markers, in a single run could enhance the chances of early detection as well as monitoring response to the treatment and hence tailoring the bestpossible treatment with respect to tumor kinetics. Optical spectroscopic methods such as Raman spectroscopy [9,10], Fourier transform infrared (FTIR) spectroscopy [11–14] and laser-induced fluorescence (LIF) spectroscopy [15–18] have been shown to be promising alternatives in less invasive diagnosis of cervical cancers. The advantages of optical-based methods are: non/minimally invasive, rapid, no/minimal sample pretreatment, highly objective, opted for in vivo/ in situ conditions and provide information on biochemical changes. In our earlier study we have demonstrated the feasibility of discrimination among formalin-fixed normal and malignant tissues (before radiotherapy and after a second fraction of radiotherapy) using Raman microspectroscopy [19]. Very recently we have reported the feasibility of prediction of tumor radioresponse by a conventional Raman spectroscopic method [20,21]. However, these studies could only provide information on possible spectral markers of heterogeneous tissue
samples and the methods have only limited application to more homogenous serum samples. In view of above considerations, a combined highperformance liquid chromatography (HPLC) and LIF system has been developed in our laboratory [22,23]. Combination of ultra-sensitive LIF with highly efficient separation techniques such as HPLC could be useful to detect ultra-trace amounts (femtomoles) of individual biomarkers in complex biological systems. This makes it an attractive modality for application in this field and also to be applied to both serum and tissue homogenates [24,25]. Using this technique, a few proteomics-based studies have been reported in the diagnosis of oral [23], breast [24] and cervical cancers [25]. In the present study, we have explored the feasibility of differentiation between serum samples from normal (healthy) and cervical cancer patients, as well as the predictive utility of serum samples collected after a second fraction of radiotherapy (2-RT) using principal components analysis (PCA) as a discriminating tool. The results obtained in this study are presented and discussed in this paper. This study was approved by MAHE ethical clearance committee.
Materials and methods Patients and radiotherapy regimen Twenty-one study participants (age range: 35–67 years) were recruited in the study after informed consent. Of these, 14 were cancer patients with biopsyproven squamous cell carcinoma of uterine cervix (FIGO stage IIIB) and 7 were age-matched healthy volunteers who served as the control group. The cervical cancer patients underwent a radiotherapy (RT) regimen at the Department of Radiotherapy and Oncology, Kasturba Hospital, Manipal, which spanned over 4 months from the first fraction of RT till clinical evaluation of tumor radioresponse. The patients were subjected to fractionated external beam radiotherapy (EBRT) of 50 Gy, in total in 25 fractions, at the rate of 2 Gy per fraction, 5 days a week for a total of 5 weeks using a linear accelerator. The patients were then allowed a 2-week rest period for the acute reactions to subside. Afterwards 2 applications of remote after-loaded high-dose rate (HDR) intracavitary brachytherapy was
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administered for two weeks to point-A region, at dose rate of 8.5 Gy once a week. After 1 month rest, the patients were subjected to per vaginal, per rectal and per speculum clinical examinations manually in order to assess tumor response to radiotherapy. As an index of radio responsiveness, the degree of the tumor shrinkage was determined. A 100% shrinkage of the tumor volume was graded as ‘complete response’ (CR), shrinkage volume of Z50% was graded as ‘partial response’ (PR) and anything below 50% was considered as ‘no response’ (NR) according to the WHO’s recommendation [26]. After clinical evaluation of 13 patients (one patient abandoned the radiotherapy), 10 patients showed complete response (CR), 2 showed partial response (PR) and 1 no response (NR).
Sample collection and preparation Blood samples were collected from the 14 cervix cancer patients before treatment with radiotherapy (referred to as ‘malignant’). Another blood sample from the same cancer patients (n ¼ 13) was collected 24 h after the second day of treatment, i.e. 24 h after exposure of two fractions of external radiation therapy (cumulative dose: 4 Gy) (referred to as ‘2-RT’). One sample was unavailable due to discontinuation of the treatment. Blood samples from the 7 healthy volunteers were employed as controls (referred to as ‘normal’). Blood samples collected from study participants referred to above were allowed to clot in standing tubes at room temperature and the supernatant (serum) was transferred to centrifuge tubes. The serum was further
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centrifuged at 5000 rpm for 10 min to remove traces of blood. Where necessary, these samples were stored at 80 1C and passively thawed to room temperature just before the experiment. The serum thus obtained was immediately subjected to HPLC-LIF studies. The serum samples were diluted 200 times using HPLC-grade ultrapure water and 20 ml of the diluted sample were injected into the HPLC system to record the chromatogram. The chromatograms of serum samples (normal, malignant and 2-RT) thus recorded were analyzed.
HPLC-LIF set-up Fig. 1 shows the HPLC-LIF set-up, which was used for the measurement of the chromatograms. The HPLC system (Hewlett Packard Model series 1100, USA) is comprised of a degasser (G13224), pump (G1311A), and manual injector (Model no. 7725, Rheodyne). A reversed-phase diphenyl narrow-bore column (Vydac 219TP52), which is used for separation of proteins was connected to this system using PEEK tubing (Upchurch Scientific). Once the serum proteins are separated in the HPLC column, the effluent from the column is eluted out through a quartz capillary (G1600-64311, Hewlett Packard) of 75 mm diameter (flow rate 0.2 ml/min) using finger-tight fittings (F-130X, Upchurch Scientific) and 1/16-in unions (Agilent) by increasing the gradient concentration of organic modifier (acetonitrile) from 30–60% with a run time of 60 min. An intracavity frequency-doubled Ar+ laser (Innova 90C FreD, Coherent) of 257 nm and 15 mW obtained by doubling 514.5 nm with a beta-barium borate (BBO) crystal was used to induce fluorescence excitation at 340 nm.
Fig. 1. Schematic layout of HPLC-LIF set-up.
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The laser beam is tightly focused onto the capillary so that proteins present in the effluent are excited due to interaction with laser beam and fluoresce as a result. The emitted fluorescence on one side of the capillary is collimated and collected by the collection optics, then focused onto the monochromator (DH10 SPEX, JobinYvon) set at 340 nm. The signal was chopped using a chopper (Model 651, EG&G Instruments) at 20 Hz for lock-in detection. The fluorescence was detected by a photomultiplier tube (Hamamatsu R 453) of 850 V coupled via preamplifier (Model 5113, EG&G Instruments) to lock-in amplifier (Model 7265, EG&G Instruments). The chromatogram was recorded via a lock-in chromatographic peak-detection arrangement, which was interfaced to the computer. On the other side of the capillary, fluorescence spectra of individual fluorophores were recorded by a fluorescence-spectroscopy set-up consisting of a SpectraPro-150 imaging spectrograph fitted with a 600 g/mm grating blazed for 300 nm, a CCD (Model RTE/CCD-128-HB, Roper Scientific), and collection and focusing lenses. The fluorescence from the proteins in the capillary was collected and collimated using a 2 cm lens and a further 5-cm lens was used for focusing the fluorescence on to the spectrograph. The spectra were then recorded on the CCD (Fig. 1.) [23,24].
Data analysis In order to explore the feasibility of classification for normal, malignant and 2-RT conditions, chromatograms were analyzed in two different ways: (1) the classical method using relative intensities of selected peaks and (2) multivariate statistical tool using principal components analysis. Chromatograms were subjected to several preprocessing steps before analysis. Spectra were backgroundcorrected by fitting a third-order polynomial to remove the interference of background fluorescence. In order to minimize/avoid run-to-run variations of peak positions, chromatograms were calibrated by shifting to injection peak along time scale (x-axis). In our study, the injection peak is considered as the internal standard as it appears in all chromatograms, including the blank. Therefore all the chromatograms were calibrated with respect to injection peak. After smoothing with 21 points by Savitsky–Golay method (to minimize the noise) the chromatograms were normalized with respect to human serum albumin (HSA)-peak (shown in asterisk, Fig. 2). These chromatograms were subjected to derivatization (first derivative) in order to minimize the background effect, which is widely used in FTIR spectroscopy. These first derivative spectra were interpolated to the region of interest before subjecting to PCA.
Fig. 2. Typical serum chromatogram of normal (a), malignant (b) and 2-RT samples (c). The chromatogram shows the plot of time (x-axis) vs. the intensity of signal (y-axis).
In the classical method, in order to understand relative intensity changes of fluorophores intensity plots were computed and displayed as scattered plots for the chromatograms of all different conditions. In the next approach, pretreated chromatograms were subjected to multivariate statistical tool PCA. In PCA, a mean of the spectra is formed and the differences of each spectrum from the mean are computed, which gives the variation of each spectrum from the mean. With n spectra, each having p data points, a [n p] matrix of these variations are computed. PCA is one of the dimensionality reduction techniques where a large amount of data is broken down into smaller sets of independent variables, and their contributions to the chromatogram vary from sample to sample depending on the concentration of the component peaks. ‘Eigenvalues’ are actually a measure of the importance of each factor for reconstructing the real chromatogram. Only few initial eigenvectors (principal components) represent the true variations in the given set of spectra, whereas contribution of the remaining principal components may be close to zero with no practical contribution. By solving the eigenvalue–eigenvector problem, the so-called ‘principal components (PC)’ or ‘factors’, the total % variance (contribution of the factors to the variations in the data set) and scores of factors were obtained for each spectrum. The scores for a given sample correspond to the contribution of each PC to the variation of that of each spectrum from the mean. Trial runs were carried out with several factors. The number of significant factors can be decided by several techniques such as eigenvalues, total % variance and spectral loadings. PCA was carried out under different conditions: entire range and selected regions (100–1400, 1250–2000, 1580–2700 s) and derivatives of the same regions using different number of factors. In our analysis, PCA of first derivative spectra in the region
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of 1250–2000 s region with 5 factors gave the best results when compared with entire region, 100–1400 and 1580–2700 s region. Thus, further analysis was carried out under 1250–2000 s region. The total % variance, eigenvalues and factor profiles were employed for standardizing the number of factors. Pretreatment steps and data analysis were carried out using algorithms implemented in Grams PLS plus/IQ, Galactic Industries Corporation, USA.
Results Serum chromatograms Typical serum chromatograms of normal, malignant and 2-RT conditions are shown in Fig. 2. The run time of 45 min proved to be ideal to evaluate the maximum number of peaks. An additional run time of 15 min (total 60 min) did not provide any extra information. Normal and malignant chromatograms show significant variations in the 800–1800 s region. Malignant and 2-RT chromatograms show minute variations in the 1300–1800 s region. It can be appreciated from Fig. 2 that peak 2, which is absent in normal chromatograms appeared in the malignant and 2-RT chromatograms. A broad shoulder in peak 5 is observed in the malignant chromatograms compared with the normal ones. Peak 10 that is present in normal chromatogram is missing in malignant ones and again reappeared in 2-RT condition. However, peak 9, which appeared in 2-RT chromatograms is missing in the normal and malignant ones. It should be noted that peak 6 was saturated in all three conditions.
Fig. 3. Analysis of the serum chromatograms based on the ratio of relative intensities of peaks 5 and 10. (a) Ratio of relative intensities of peak number 5 of B normal, ’ malignant and m 2-RT produced a ‘normal’ cluster. (b) Ratio of relative intensities of peak number 10 of ’ malignant and m 2-RT did not produce clear differentiation between both.
Classical data analysis In the classical method, relative intensities of several selected peaks were used for analysis of normal, malignant and 2-RT chromatograms. Of these, peaks 5 and 10 yielded similar and best results where normal serum chromatograms were clearly differentiated compared with malignant and 2-RT. As an example, the scattered plot of peak 5 for normal, malignant and 2-RT conditions produced 2 clusters. One cluster corresponds to the normal condition whereas the other one corresponds to malignant and 2-RT (Fig. 3a). Mean and standard deviation values of the normal, malignant and 2-RT intensities were 1.5170.23, 1.0670.35 and 1.0870.37, respectively. In order to verify the feasibility of differentiating malignant and 2-RT conditions, intensities of peak 10 for malignant and 2-RT samples were plotted next (Fig. 3b). Mean and standard deviation values of malignant and 2-RT intensities are 0.1270.16 and 0.5471.08.
Fig. 4. PCA of first derivative serum chromatograms in the 1250–2000 s region. (a) Unsupervised analysis of B normal, ’ malignant and m 2-RT produced clear classification of ‘normal’ cluster compared with the other two conditions. (b) Unsupervised analysis of ’ Malignant and m 2-RT could not produce clear classification.
Multivariate statistics (PCA) In our second approach, PCA of first derivative spectra in the 1250–2000 s region with 5 factors yielded the best results. PCA was carried out in a three-step process. In the first step, spectra of all three conditions (normal, malignant and 2-RT) were pooled and analyzed. The analysis once again produced two clusters. One cluster corresponds to normal while the other one corresponds to malignant and 2-RT (Fig. 4a). The differentiation is based on score of factor 1. Mean
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Fig. 5. Unsupervised analysis of 2-RT first derivative serum chromatograms in the 1250–2000 s region in the lines of prediction of tumor response to radiotherapy could not yield clear differentiation between m complete response (CR), B partial response (PR) and no response (NR).
and standard deviation values of scores of factor 1 for normal, malignant and 2-RT were 0.1770.17, 0.0370.16 and 0.0970.15, respectively. Mean and standard deviation values of score of factor 2 for normal, malignant and 2-RT are 0.0570.17, 0.0170.16 and 0.0570.15, respectively. In the second step, malignant and 2-RT chromatograms were pooled and analyzed in order to verify the feasibility of classification between malignant and 2-RT (Fig. 4b). Mean and standard deviation values for the score of factor 1 for malignant and 2-RT serum chromatograms are 0.0170.19 and 0.0170.16, respectively and mean and standard deviation values for the score of factor 2 are 0.0270.02 and 0.0270.15, respectively. As a third step, in order to verify the feasibility of prediction of tumor response to radiotherapy i.e. to differentiate CR, PR and NR-groups, unsupervised analysis of only 2-RT chromatograms was carried out (Fig. 5).
Discussion Radiotherapy is a major treatment modality for locally advanced stages of carcinoma of uterine cervix. The altered response of tumors to standard radiotherapy regimen is believed to be due to several intrinsic factors such as DNA aneuploidy, S-phase fraction and proliferation kinetics, tumor vascularity and hypoxia and glutathione content. Tumor response to radiotherapy may vary in patients with the same clinical stage and histological type [20]. If one can predict the tumor radioresponse prior to, or at an early stage of the treatment, there is still a possibility for the clinician to plan for alternative therapeutic modalities. In recent years, there has been an increased interest in developing techniques that allow the rapid and accurate monitoring of tumor response to radiotherapy. Several of such studies using biochemical parameters, such as the total GSH content [1], the surviving fraction (SF2),
the single cell gel electrophoresis (SCGE) assay [27], the potential doubling time (Tpot) and the micronucleus assay [28], have been useful for prediction of the treatment response to radiotherapy. However, none of these methods have been established in routine clinical practice due to findings contrary to the positive studies [29]. Due to this we used the HPLC-coupled LIF method to verify the feasibility of monitoring of tumor response to radiotherapy. The main advantage of this method over Raman and LIF is that it can detect subfemtomole levels of protein that can be used for prediction of tumor response early in the course of treatment. The sensitivity can be enhanced by several orders of magnitude if required. However, very few studies have been reported in the literature using this system [22,24,25]. To best of our knowledge, no attempts have been made in the monitoring of tumor response to radiotherapy using this set-up. Hence in the present study we have attempted to monitor tumor response to radiotherapy by less invasive serum samples using this technique. In our earlier study we have demonstrated the efficacy of Raman spectroscopy in prediction of tumor response to radiotherapy using 2-RT tissues [20,21]. In this study we have demonstrated the prediction of a tumor response to radiotherapy in terms of responding (CR, PR) and non-responding (NR) groups. However, Raman spectra of any given class, such as proteins, will be similar; hence it is difficult to identify the changes that take place in protein decomposition. Also as mentioned in the introduction, Raman studies could only provide information on possible spectral markers of heterogeneous tissue samples and these methods have limited application for more homogenous serum samples. Even though spectroscopy in its various forms has been shown to be an important component of noninvasive diagnosis of various cancers, the coupling of a LIF with HPLC system appears to be challenging. It brings to the forefront the prospect of not only diagnosing cases early but also being able to tailor treatments very early in the original treatment plan based on the response to radiotherapy. As mentioned before, this could eventually help us in intensifying treatment schedules for patients showing inadequate response or in choosing different treatment protocols for patients with no response thus sparing them additional morbidity and cost. As has already been said, the laser beam is tightly focused on the capillary tube through which individual proteins are being eluted and this leads to excitation of the fluorophores present in the proteins. Proteins which incorporate the aromatic amino acids tryptophan, tyrosine and phenylalanine in their superstructure are known to exhibit fluorescence. In an earlier study it has been demonstrated that the excitation wavelength of 257 nm was absorbed by all three amino acids and
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resulted in emission wavelengths of 303, 282 and 350 nm for tyrosine, phenylalanine, and tryptophan, respectively [22]. In this study, fluorescence spectra of individual fluorophores were recorded by the fluorescence-spectroscopy system on one side of the capillary and chromatograms using a monochromator-coupled PMT and lock-in amplifier system on the other side [22]. However, in the present study our main focus is on identification of differences among chromatographic patterns of different conditions (normal, malignant and 2-RT) for monitoring of tumor response to radiotherapy. The study was conducted and results were obtained with these factors in mind. Several different peaks indicate the presence of various proteins in the serum samples. In our earlier studies by co-injection method, using commercially available pure proteins, three proteins (transferrin, HSA, and creatine kinase) were ascertained [22–25]. These peaks are known to be common for all samples. The HSA peak, approximately at 1666-s region, is used for normalization of chromatograms as the relative intensity of this peak is constant in all samples. However, little attempt has been made to identify various other peaks of the chromatogram. Thus, the peak numbers ascertained in the present study are merely there to compare the typical chromatographic patterns of normal, malignant and 2-RT conditions. The chromatograms of the specimens in our pilot study showed certain important differences (Fig. 2). Peak 2 appeared in malignant specimens while it was absent in the normal ones. Peak 10 was present in all normal specimens whereas it is absent in malignant specimens. These presence of peak 2 and absence of peak 10 may be correlated to being a spectroscopic tumor marker. These results were in agreement with the earlier reports [25]. The reappearance of peak 10 in 2-RT samples is interesting because it may be an indication of the return to normalcy in patients with cervical cancer. Peak 9 was unique in the sense that it was present only in the treated (2-RT) samples. Its correlation with response to radiation is uncertain. It might be postulated that this peak may be a marker of radiationinduced effects to malignant tissues, as radiotherapy was the only different factor between the treated and the non-treated groups (‘malignant’ and ‘normal’). However, the difference in the protein profile patterns between malignant and 2-RT samples was minute, which was also reflected in our analysis (discussed in later part of the section). This might be due to the fact that samples were taken very early in the treatment. Samples collected later during the course of radiotherapy may magnify these changes allowing for definitive conclusions. It is a well-known fact that radiation-induced effects are mediated via free radical generation. The body
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instantly tries to counteract this effect by enhancing the production of free radical scavengers in order to protect from the deleterious effects of the free radicals. Nevertheless, the aim of the radiotherapy is to overcome the system’s counteraction in order to get rid of the cancerous cells. From radiobiological models it will be appreciated that repair of radiation-induced damage commences only after 4–6 h of treatment. Since we have collected samples 24 h after the second fraction of RT, the body had a considerable amount of time to repair these radiation-induced effects. Thus, the radiationinduced effects that have persisted after the repair process i.e. 24 h after treatment might have contributed to the minute variations in the protein profile patterns among treated and untreated malignant samples. It was seen in the classical method of analysis that the mean values relative intensities of the peak 5 for normal, malignant and 2-RT specimens were 1.51 (SD 0.23), 1.06 (SD 0.35), and 1.08 (SD 0.37), respectively (Fig. 3a). This indicates that the variation in relative intensity of the peak for normal and malignant is quite large and diminution of peak 5 (mean of malignantonormal) maybe a surrogate marker of malignancy. It is evident from the Fig. 3b, even though the mean values of peak 5 for malignant and 2-RT specimens are not significantly different, that there is slight upward trend (mean of 2-RT 4mean of malignant). This may again be a harbinger of the normal state and observation of this peak is warranted at a later stage in the course of treatment. Similar observations were made for peak 10. It is well-known that optical methods are amenable to several multivariate statistical tools such as hierarchical cluster analysis (HCA), artificial neural networks (ANN), k-nearest neighbor (KNN), maximum representation and discrimination feature (MRDF), principal components analysis available for data mining. However, as mentioned earlier, we have opted for PCA as a discriminating tool in our second approach of analysis. PCA is a well-known dimensionality reduction technique where large spectral data are reduced into small number of independent variations known as factors or principal components and contributions of these factors are called scores. Scores of factor were employed as discriminating parameters [30]. As stated in the data analysis section, analysis was carried out in full region, selected regions and derivatives of the same. As mentioned earlier, derivatization was more advantageous because the background was automatically corrected. Background in the chromatographic runs could arise from laser light scattered from capillary walls, fluorescence of the organic modifier (acetonitrile) in the gradient, PMT dark count, etc. Due to background fluorescence, a slight inclined slope was observed in the chromatograms. In this case a baseline correction by a third-order polynomial might induce artifacts. Hence we have opted for derivatization (widely used in
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FTIR spectroscopy) of chromatograms to minimize baseline/background-induced artifacts. As mentioned in the data analysis section, in our analysis PCA of first derivative spectra in the 1250–2000 s region, with 5 factors yielded best results and further analysis was carried out under these conditions. PCA was carried out in three-step process. In the first step, chromatograms of all three conditions, normal, malignant and 2-RT, were pooled and analyzed (Fig. 4a). These results show that both scores of factors 1 and 2 contribute to classification of normal and cancerous conditions (before and after 2-RT). It is evident from the figure that mean scores of factors 1 and 2 were positive for normal subjects and negative for the malignant and 2-RT, thus facilitating differentiation between the normal and cancerous conditions. However, the treated and untreated cervical neoplasia could not show clear differentiation. As can be seen from Fig. 4b, in the second step of analysis there was a slight decrease in the mean values of factors 1 and 2 when compared to malignant and 2-RT samples. This again suggests that samples taken later in the course of treatment may yield definitive results. This pilot study is important because it suggests that certain peaks, like peaks 2 and 10, could be probable markers for malignancy while peaks 5 and 10 may suggest response to radiotherapy. Peak 9 may suggest radiation-induced damage and may predict the severity of radiation reactions. As a third step we have carried out PCA of 2-RT serum chromatograms in order to verify the feasibility of prediction of tumor response to radiotherapy (CR, PR and NR) at an early stage of the treatment (Fig. 5). It is evident from the figure that PR and NR groups together tended to form a separate cluster from CR group. However, the CR groups outweigh this separation. It has to be noted the number of serum samples for PR (n ¼ 2) and NR (n ¼ 1) groups was very less. However, the above findings require confirmation in a larger trial. Correlation of peaks with clinical response to treatment may definitely predict the magnitude or range of values of relative intensities at which response to treatment can be ascertained. The use of serum samples collected in a later course of therapy may be useful in establishing the role of HPLC-LIF technique as a predictive marker of radioresponse.
Conclusions The findings of the study presented here suggest that protein profile studies of less invasive serum samples can clearly differentiate normal and malignant conditions. However, the protein profiling of malignant conditions failed to differentiate from 2-RT. This could be
explained as samples collected after 2-RT might be early enough to observe radiation-induced therapeutic effects. It has also been demonstrated that this technique has a limited application in prediction of tumor response to radiotherapy using 2-RT samples. Prospective studies with blood samples collected later in the course of the treatment could differentiate from malignant condition using this technique. Further, by obtaining larger data sets of different response conditions in the later course of treatment, the potential of this technique in prediction of tumor response to radiotherapy needs to be verified.
Acknowledgements The work was carried out under the Department of Atomic Energy; Board of Research in Nuclear Sciences (DAE-BRNS), Govt. of India project entitled ‘Laser spectroscopy as predictor of tumor response to radiotherapy in cervical cancer’ (Grant number 2003/34/17/ BRNS/1903). Authors acknowledge technical support of Ms. Keerti and Mr. Chetan Anand. Dr. C. Santosh, Incharge, Center for Atomic and Molecular Physics (CAMP) is also acknowledged. One of the authors (MK) is grateful to DAE-BRNS for a Research Fellowship.
Zusammenfassung Bestimmung von Serumproteinprofilen mittels HPLC¨ berwachung der Tumoransprechrate bei LIF zur U Radiotherapie des Zervixkarzinoms: Eine Pilotstudie Zielsetzung: Durchfu¨hrung einer Machbarkeitsstudie mit dem Ziel zu pru¨fen, inwieweit die Bestimmung von Serumproteinprofilen mittels eines kombinierten HPLCLIF-Systems zur U¨berwachung der Tumoransprechrate bei Radiotherapie des Zervixkarzinoms geeignet ist. Material und Methoden: 21 Studienteilnehmerinnen wurden in die Untersuchungen involviert: 7 davon waren gesund, 14 mussten sich aufgrund eines diagnostizierten Zervixkarzinoms fu¨r eine Dauer von 5 Wochen einer fraktionierten Radiotherapie (RT) mit 25 Einzeldosen a´ 2 Gy unterziehen gefolgt von 2 intrakavita¨ren Bestrahlungen im Abstand von einer Woche. Den Probandinnen wurden Blutproben entnommen, um das Blutserum chromatografisch zu untersuchen. Insgesamt wurden von den gesunden Patientinnen 7 ‘‘normale’’ Blutproben und von den Krebspatientinnen 14 sogenannte maligne’’ Proben vor Beginn der ’’ Radiotherapie sowie 13 sogenannte ‘‘2-RT’’-Proben 24 h nach der zweiten Bestrahlung (1 Patientin unterbrach die Behandlung) entnommen, aufbereitet und
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mittels eines selbstentwickelten HPLC-LIF-Systems vermessen und analysiert. Die Serumchromatogramme wurden auf zwei Arten ausgewertet: (1) klassisch durch den Vergleich der relativen Intensita¨ten auffa¨lliger Peaks und (2) mittels der Hauptkomponentenanalyse (Principal component analysis, PCA). Zusa¨tzlich wurde nach Abschluss der Radiotherapie, 4 Monate nach der ersten Bestrahlung, die Tumoransprechrate bestimmt (Tumorschrumpfung ¼ 100%: complete response (CR), Tumorschrumpfung Z50%: partial response (PR), Tumorschrumpfung r50%: no response (NR)) und mit den 2-RT-Chromatogrammen korreliert. Ergebnisse: Ein Vergleich der normalen und malignen Serumchromatogramme zeigte im Bereich 800–1800 s ausgepra¨gte Unterschiede. Zwischen den malignen und 2-RT-Chromatogrammen wurden nur minimale Unterschiede im Bereich 1300–1800 s festgestellt. Beide Auswerteverfahren (klassisch, PCA) konnten klar zwischen normal’’ und maligne’’ unterscheiden, ’’ ’’ jedoch nicht zwischen ‘‘maligne’’ und ‘‘2-RT’’. Von den 13 komplett behandelten Patientinnen zeigten 10 einen vollsta¨ndigen Tumorru¨ckgang (CR), 2 einen teilweisen Ru¨ckgang (PR) und eine Patientin kein Ansprechen auf die Therapie (NR). Durch Analyse der 2-RT-Chromatogramme konnte keine eindeutige Differenzierung zwischen diesen drei Patientengruppen erzielt werden. Zusammenfassung: Durch die Proteinanalyse von Serumproben konnte zwischen ‘‘normal’’ und ‘‘maligne’’ unterschieden werden, nicht jedoch zwischen maligne’’ ’’ und ‘‘2-RT’’. Eine Vorhersage der Ansprechrate auf die Bestrahlung anhand der 2-RT-Chromatogramme war nicht eindeutig mo¨glich. Schlu¨sselwo¨rter: Bestrahlungstherapie; Tumormarker; Zervixkarzinom; Hauptkomponentenanalyse (PCA)
[5]
[6]
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Medical Laser Application 24 (2009) 165–174 www.elsevier.de/mla
Serum protein profile studies of cervical cancers in monitoring of tumor response to radiotherapy using HPLC-LIF: A pilot study Mamidipudi Srinivasa Vidyasagara,, Maheedhar Kodalia,b, Prathima N. Balub, Gunjan Baijala, Bejadi Manjunath Vadhirajaa, Rani A. Bhatc, Donald Jerard Fernandesa, Chilakapati Murali Krishnad a
Department of Radiotherapy and Oncology, Shirdi Sai Baba Cancer Hospital and Research Center, Kasturba Medical College, Manipal University, Manipal 576104, Karnataka, India b Center for Atomic and Molecular Physics, Manipal University, Manipal 576104, Karnataka, India c Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576104, Karnataka, India d Chilakapati Laboratory, Cancer Research Institute, Advanced Center for Treatment Research and Education in Cancer (ACTREC), Kharghar, Sector ‘22’, Navi Mumbai 410 208, Maharastra, India Received 6 February 2009; accepted 6 May 2009
Abstract Objective: Exploration of the feasibility of serum protein profiles for monitoring tumor radioresponse in cervical cancers using HPLC-LIF system. Materials and methods: Twenty-one subjects were recruited in the study. Out of them 7 were healthy, 14 were cervical cancer patients who undertook fractionated radiotherapy (RT) with 2 Gy per fraction over 25 fractions, for 5 weeks followed by 2 applications of intracavitary brachytherapy once a week. Blood collected from above subjects was processed to obtain serum. Serum chromatograms of ‘normal’ (n ¼ 7) and conspicuous probes before RT (n ¼ 14, ‘malignant’) and 24 h after second fraction of RT (n ¼ 13, ‘2-RT’), were recorded using an In-house-built HPLC-LIF set-up. Data were analyzed in two approaches: (1) classical method using relative intensities of selected peaks, (2) principal component analysis (PCA). Clinical assessment of tumor radioresponse was carried out 4 months after first fraction of RT and the degree of the tumor shrinkage was determined as an index of radioresponsiveness (complete response (CR): 100% shrinkage, partial response (PR): Z50% shrinkage, and no response (NR): r50% shrinkage) which was further correlated with the analysis of 2-RT serum chromatograms. Results: Normal vs. malignant chromatograms demonstrated pronounced differences in the 800–1800 s region. Malignant vs. 2-RT chromatograms showed minute variations in the 1300–1800 s region. Our analysis, in both of the approaches, produced clear differentiation between ‘normal’ and ‘malignant’, whereas differentiation between ‘malignant’ and ‘2-RT’ was minimal. Clinical evaluation of the tumor radioresponse yielded that out of 13 patients (one patient discontinued the radiotherapy) ten showed CR, two showed PR and one NR. In case of prediction of tumor radioresponse, analysis of the 2-RT chromatograms produced only minor differentiation among CR, PR and NR groups.
Corresponding author. Tel.: +91 820 2922579; fax: +91 820 2571651.
E-mail addresses: [email protected], [email protected] (M.S. Vidyasagar). 1615-1615/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.mla.2009.05.005
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Conclusion: Protein profiling of serum samples differentiated ‘normal’ from ‘malignant’, but could not differentiate ‘malignant’ from ‘2-RT’. Also this technique has limited application in prediction of tumor radioresponse. r 2009 Elsevier GmbH. All rights reserved. Keywords: Radiation therapy; Tumor markers; Cervical cancer; Principal component analysis (PCA)
Introduction Radiotherapy is the treatment of choice for locally advanced stages of cervical cancer. Tumors of same clinical stage and histological type may respond differently to the same treatment. This could be due to intrinsic factors such as DNA aneuploidy, S-phase fraction, proliferation kinetics, tumor vascularity and hypoxia and glutathione content [1–3]. Advances in molecular biology and high-throughput technologies have provided the hope of discovering novel biological markers in screening, early diagnosis, and prediction of tumor response to therapy [4]. Glutathione (GSH) levels of serum and tissue are probable biomarkers for prediction of tumor radioresponse in cervical cancers [2]. Several proteomics-based studies suggest that serum levels of squamous cell carcinoma antigen (SCC), cancer antigen CA125 [5,6] and other proteins like gasderminlike protein (GSDML) [7], which belongs to gasdermindomain containing protein family, could be used as biomarkers in screening as well as evaluation of therapeutic response in cervical cancers. Studies also suggest that HPV E6 and E7 oncoproteins might help in development of cervical cancer-specific markers [4,8]. However, immunoassays are generally applicable only for detection of a single marker at a time. Detection of several individual markers, in a single run could enhance the chances of early detection as well as monitoring response to the treatment and hence tailoring the bestpossible treatment with respect to tumor kinetics. Optical spectroscopic methods such as Raman spectroscopy [9,10], Fourier transform infrared (FTIR) spectroscopy [11–14] and laser-induced fluorescence (LIF) spectroscopy [15–18] have been shown to be promising alternatives in less invasive diagnosis of cervical cancers. The advantages of optical-based methods are: non/minimally invasive, rapid, no/minimal sample pretreatment, highly objective, opted for in vivo/ in situ conditions and provide information on biochemical changes. In our earlier study we have demonstrated the feasibility of discrimination among formalin-fixed normal and malignant tissues (before radiotherapy and after a second fraction of radiotherapy) using Raman microspectroscopy [19]. Very recently we have reported the feasibility of prediction of tumor radioresponse by a conventional Raman spectroscopic method [20,21]. However, these studies could only provide information on possible spectral markers of heterogeneous tissue
samples and the methods have only limited application to more homogenous serum samples. In view of above considerations, a combined highperformance liquid chromatography (HPLC) and LIF system has been developed in our laboratory [22,23]. Combination of ultra-sensitive LIF with highly efficient separation techniques such as HPLC could be useful to detect ultra-trace amounts (femtomoles) of individual biomarkers in complex biological systems. This makes it an attractive modality for application in this field and also to be applied to both serum and tissue homogenates [24,25]. Using this technique, a few proteomics-based studies have been reported in the diagnosis of oral [23], breast [24] and cervical cancers [25]. In the present study, we have explored the feasibility of differentiation between serum samples from normal (healthy) and cervical cancer patients, as well as the predictive utility of serum samples collected after a second fraction of radiotherapy (2-RT) using principal components analysis (PCA) as a discriminating tool. The results obtained in this study are presented and discussed in this paper. This study was approved by MAHE ethical clearance committee.
Materials and methods Patients and radiotherapy regimen Twenty-one study participants (age range: 35–67 years) were recruited in the study after informed consent. Of these, 14 were cancer patients with biopsyproven squamous cell carcinoma of uterine cervix (FIGO stage IIIB) and 7 were age-matched healthy volunteers who served as the control group. The cervical cancer patients underwent a radiotherapy (RT) regimen at the Department of Radiotherapy and Oncology, Kasturba Hospital, Manipal, which spanned over 4 months from the first fraction of RT till clinical evaluation of tumor radioresponse. The patients were subjected to fractionated external beam radiotherapy (EBRT) of 50 Gy, in total in 25 fractions, at the rate of 2 Gy per fraction, 5 days a week for a total of 5 weeks using a linear accelerator. The patients were then allowed a 2-week rest period for the acute reactions to subside. Afterwards 2 applications of remote after-loaded high-dose rate (HDR) intracavitary brachytherapy was
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administered for two weeks to point-A region, at dose rate of 8.5 Gy once a week. After 1 month rest, the patients were subjected to per vaginal, per rectal and per speculum clinical examinations manually in order to assess tumor response to radiotherapy. As an index of radio responsiveness, the degree of the tumor shrinkage was determined. A 100% shrinkage of the tumor volume was graded as ‘complete response’ (CR), shrinkage volume of Z50% was graded as ‘partial response’ (PR) and anything below 50% was considered as ‘no response’ (NR) according to the WHO’s recommendation [26]. After clinical evaluation of 13 patients (one patient abandoned the radiotherapy), 10 patients showed complete response (CR), 2 showed partial response (PR) and 1 no response (NR).
Sample collection and preparation Blood samples were collected from the 14 cervix cancer patients before treatment with radiotherapy (referred to as ‘malignant’). Another blood sample from the same cancer patients (n ¼ 13) was collected 24 h after the second day of treatment, i.e. 24 h after exposure of two fractions of external radiation therapy (cumulative dose: 4 Gy) (referred to as ‘2-RT’). One sample was unavailable due to discontinuation of the treatment. Blood samples from the 7 healthy volunteers were employed as controls (referred to as ‘normal’). Blood samples collected from study participants referred to above were allowed to clot in standing tubes at room temperature and the supernatant (serum) was transferred to centrifuge tubes. The serum was further
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centrifuged at 5000 rpm for 10 min to remove traces of blood. Where necessary, these samples were stored at 80 1C and passively thawed to room temperature just before the experiment. The serum thus obtained was immediately subjected to HPLC-LIF studies. The serum samples were diluted 200 times using HPLC-grade ultrapure water and 20 ml of the diluted sample were injected into the HPLC system to record the chromatogram. The chromatograms of serum samples (normal, malignant and 2-RT) thus recorded were analyzed.
HPLC-LIF set-up Fig. 1 shows the HPLC-LIF set-up, which was used for the measurement of the chromatograms. The HPLC system (Hewlett Packard Model series 1100, USA) is comprised of a degasser (G13224), pump (G1311A), and manual injector (Model no. 7725, Rheodyne). A reversed-phase diphenyl narrow-bore column (Vydac 219TP52), which is used for separation of proteins was connected to this system using PEEK tubing (Upchurch Scientific). Once the serum proteins are separated in the HPLC column, the effluent from the column is eluted out through a quartz capillary (G1600-64311, Hewlett Packard) of 75 mm diameter (flow rate 0.2 ml/min) using finger-tight fittings (F-130X, Upchurch Scientific) and 1/16-in unions (Agilent) by increasing the gradient concentration of organic modifier (acetonitrile) from 30–60% with a run time of 60 min. An intracavity frequency-doubled Ar+ laser (Innova 90C FreD, Coherent) of 257 nm and 15 mW obtained by doubling 514.5 nm with a beta-barium borate (BBO) crystal was used to induce fluorescence excitation at 340 nm.
Fig. 1. Schematic layout of HPLC-LIF set-up.
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The laser beam is tightly focused onto the capillary so that proteins present in the effluent are excited due to interaction with laser beam and fluoresce as a result. The emitted fluorescence on one side of the capillary is collimated and collected by the collection optics, then focused onto the monochromator (DH10 SPEX, JobinYvon) set at 340 nm. The signal was chopped using a chopper (Model 651, EG&G Instruments) at 20 Hz for lock-in detection. The fluorescence was detected by a photomultiplier tube (Hamamatsu R 453) of 850 V coupled via preamplifier (Model 5113, EG&G Instruments) to lock-in amplifier (Model 7265, EG&G Instruments). The chromatogram was recorded via a lock-in chromatographic peak-detection arrangement, which was interfaced to the computer. On the other side of the capillary, fluorescence spectra of individual fluorophores were recorded by a fluorescence-spectroscopy set-up consisting of a SpectraPro-150 imaging spectrograph fitted with a 600 g/mm grating blazed for 300 nm, a CCD (Model RTE/CCD-128-HB, Roper Scientific), and collection and focusing lenses. The fluorescence from the proteins in the capillary was collected and collimated using a 2 cm lens and a further 5-cm lens was used for focusing the fluorescence on to the spectrograph. The spectra were then recorded on the CCD (Fig. 1.) [23,24].
Data analysis In order to explore the feasibility of classification for normal, malignant and 2-RT conditions, chromatograms were analyzed in two different ways: (1) the classical method using relative intensities of selected peaks and (2) multivariate statistical tool using principal components analysis. Chromatograms were subjected to several preprocessing steps before analysis. Spectra were backgroundcorrected by fitting a third-order polynomial to remove the interference of background fluorescence. In order to minimize/avoid run-to-run variations of peak positions, chromatograms were calibrated by shifting to injection peak along time scale (x-axis). In our study, the injection peak is considered as the internal standard as it appears in all chromatograms, including the blank. Therefore all the chromatograms were calibrated with respect to injection peak. After smoothing with 21 points by Savitsky–Golay method (to minimize the noise) the chromatograms were normalized with respect to human serum albumin (HSA)-peak (shown in asterisk, Fig. 2). These chromatograms were subjected to derivatization (first derivative) in order to minimize the background effect, which is widely used in FTIR spectroscopy. These first derivative spectra were interpolated to the region of interest before subjecting to PCA.
Fig. 2. Typical serum chromatogram of normal (a), malignant (b) and 2-RT samples (c). The chromatogram shows the plot of time (x-axis) vs. the intensity of signal (y-axis).
In the classical method, in order to understand relative intensity changes of fluorophores intensity plots were computed and displayed as scattered plots for the chromatograms of all different conditions. In the next approach, pretreated chromatograms were subjected to multivariate statistical tool PCA. In PCA, a mean of the spectra is formed and the differences of each spectrum from the mean are computed, which gives the variation of each spectrum from the mean. With n spectra, each having p data points, a [n p] matrix of these variations are computed. PCA is one of the dimensionality reduction techniques where a large amount of data is broken down into smaller sets of independent variables, and their contributions to the chromatogram vary from sample to sample depending on the concentration of the component peaks. ‘Eigenvalues’ are actually a measure of the importance of each factor for reconstructing the real chromatogram. Only few initial eigenvectors (principal components) represent the true variations in the given set of spectra, whereas contribution of the remaining principal components may be close to zero with no practical contribution. By solving the eigenvalue–eigenvector problem, the so-called ‘principal components (PC)’ or ‘factors’, the total % variance (contribution of the factors to the variations in the data set) and scores of factors were obtained for each spectrum. The scores for a given sample correspond to the contribution of each PC to the variation of that of each spectrum from the mean. Trial runs were carried out with several factors. The number of significant factors can be decided by several techniques such as eigenvalues, total % variance and spectral loadings. PCA was carried out under different conditions: entire range and selected regions (100–1400, 1250–2000, 1580–2700 s) and derivatives of the same regions using different number of factors. In our analysis, PCA of first derivative spectra in the region
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of 1250–2000 s region with 5 factors gave the best results when compared with entire region, 100–1400 and 1580–2700 s region. Thus, further analysis was carried out under 1250–2000 s region. The total % variance, eigenvalues and factor profiles were employed for standardizing the number of factors. Pretreatment steps and data analysis were carried out using algorithms implemented in Grams PLS plus/IQ, Galactic Industries Corporation, USA.
Results Serum chromatograms Typical serum chromatograms of normal, malignant and 2-RT conditions are shown in Fig. 2. The run time of 45 min proved to be ideal to evaluate the maximum number of peaks. An additional run time of 15 min (total 60 min) did not provide any extra information. Normal and malignant chromatograms show significant variations in the 800–1800 s region. Malignant and 2-RT chromatograms show minute variations in the 1300–1800 s region. It can be appreciated from Fig. 2 that peak 2, which is absent in normal chromatograms appeared in the malignant and 2-RT chromatograms. A broad shoulder in peak 5 is observed in the malignant chromatograms compared with the normal ones. Peak 10 that is present in normal chromatogram is missing in malignant ones and again reappeared in 2-RT condition. However, peak 9, which appeared in 2-RT chromatograms is missing in the normal and malignant ones. It should be noted that peak 6 was saturated in all three conditions.
Fig. 3. Analysis of the serum chromatograms based on the ratio of relative intensities of peaks 5 and 10. (a) Ratio of relative intensities of peak number 5 of B normal, ’ malignant and m 2-RT produced a ‘normal’ cluster. (b) Ratio of relative intensities of peak number 10 of ’ malignant and m 2-RT did not produce clear differentiation between both.
Classical data analysis In the classical method, relative intensities of several selected peaks were used for analysis of normal, malignant and 2-RT chromatograms. Of these, peaks 5 and 10 yielded similar and best results where normal serum chromatograms were clearly differentiated compared with malignant and 2-RT. As an example, the scattered plot of peak 5 for normal, malignant and 2-RT conditions produced 2 clusters. One cluster corresponds to the normal condition whereas the other one corresponds to malignant and 2-RT (Fig. 3a). Mean and standard deviation values of the normal, malignant and 2-RT intensities were 1.5170.23, 1.0670.35 and 1.0870.37, respectively. In order to verify the feasibility of differentiating malignant and 2-RT conditions, intensities of peak 10 for malignant and 2-RT samples were plotted next (Fig. 3b). Mean and standard deviation values of malignant and 2-RT intensities are 0.1270.16 and 0.5471.08.
Fig. 4. PCA of first derivative serum chromatograms in the 1250–2000 s region. (a) Unsupervised analysis of B normal, ’ malignant and m 2-RT produced clear classification of ‘normal’ cluster compared with the other two conditions. (b) Unsupervised analysis of ’ Malignant and m 2-RT could not produce clear classification.
Multivariate statistics (PCA) In our second approach, PCA of first derivative spectra in the 1250–2000 s region with 5 factors yielded the best results. PCA was carried out in a three-step process. In the first step, spectra of all three conditions (normal, malignant and 2-RT) were pooled and analyzed. The analysis once again produced two clusters. One cluster corresponds to normal while the other one corresponds to malignant and 2-RT (Fig. 4a). The differentiation is based on score of factor 1. Mean
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Fig. 5. Unsupervised analysis of 2-RT first derivative serum chromatograms in the 1250–2000 s region in the lines of prediction of tumor response to radiotherapy could not yield clear differentiation between m complete response (CR), B partial response (PR) and no response (NR).
and standard deviation values of scores of factor 1 for normal, malignant and 2-RT were 0.1770.17, 0.0370.16 and 0.0970.15, respectively. Mean and standard deviation values of score of factor 2 for normal, malignant and 2-RT are 0.0570.17, 0.0170.16 and 0.0570.15, respectively. In the second step, malignant and 2-RT chromatograms were pooled and analyzed in order to verify the feasibility of classification between malignant and 2-RT (Fig. 4b). Mean and standard deviation values for the score of factor 1 for malignant and 2-RT serum chromatograms are 0.0170.19 and 0.0170.16, respectively and mean and standard deviation values for the score of factor 2 are 0.0270.02 and 0.0270.15, respectively. As a third step, in order to verify the feasibility of prediction of tumor response to radiotherapy i.e. to differentiate CR, PR and NR-groups, unsupervised analysis of only 2-RT chromatograms was carried out (Fig. 5).
Discussion Radiotherapy is a major treatment modality for locally advanced stages of carcinoma of uterine cervix. The altered response of tumors to standard radiotherapy regimen is believed to be due to several intrinsic factors such as DNA aneuploidy, S-phase fraction and proliferation kinetics, tumor vascularity and hypoxia and glutathione content. Tumor response to radiotherapy may vary in patients with the same clinical stage and histological type [20]. If one can predict the tumor radioresponse prior to, or at an early stage of the treatment, there is still a possibility for the clinician to plan for alternative therapeutic modalities. In recent years, there has been an increased interest in developing techniques that allow the rapid and accurate monitoring of tumor response to radiotherapy. Several of such studies using biochemical parameters, such as the total GSH content [1], the surviving fraction (SF2),
the single cell gel electrophoresis (SCGE) assay [27], the potential doubling time (Tpot) and the micronucleus assay [28], have been useful for prediction of the treatment response to radiotherapy. However, none of these methods have been established in routine clinical practice due to findings contrary to the positive studies [29]. Due to this we used the HPLC-coupled LIF method to verify the feasibility of monitoring of tumor response to radiotherapy. The main advantage of this method over Raman and LIF is that it can detect subfemtomole levels of protein that can be used for prediction of tumor response early in the course of treatment. The sensitivity can be enhanced by several orders of magnitude if required. However, very few studies have been reported in the literature using this system [22,24,25]. To best of our knowledge, no attempts have been made in the monitoring of tumor response to radiotherapy using this set-up. Hence in the present study we have attempted to monitor tumor response to radiotherapy by less invasive serum samples using this technique. In our earlier study we have demonstrated the efficacy of Raman spectroscopy in prediction of tumor response to radiotherapy using 2-RT tissues [20,21]. In this study we have demonstrated the prediction of a tumor response to radiotherapy in terms of responding (CR, PR) and non-responding (NR) groups. However, Raman spectra of any given class, such as proteins, will be similar; hence it is difficult to identify the changes that take place in protein decomposition. Also as mentioned in the introduction, Raman studies could only provide information on possible spectral markers of heterogeneous tissue samples and these methods have limited application for more homogenous serum samples. Even though spectroscopy in its various forms has been shown to be an important component of noninvasive diagnosis of various cancers, the coupling of a LIF with HPLC system appears to be challenging. It brings to the forefront the prospect of not only diagnosing cases early but also being able to tailor treatments very early in the original treatment plan based on the response to radiotherapy. As mentioned before, this could eventually help us in intensifying treatment schedules for patients showing inadequate response or in choosing different treatment protocols for patients with no response thus sparing them additional morbidity and cost. As has already been said, the laser beam is tightly focused on the capillary tube through which individual proteins are being eluted and this leads to excitation of the fluorophores present in the proteins. Proteins which incorporate the aromatic amino acids tryptophan, tyrosine and phenylalanine in their superstructure are known to exhibit fluorescence. In an earlier study it has been demonstrated that the excitation wavelength of 257 nm was absorbed by all three amino acids and
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resulted in emission wavelengths of 303, 282 and 350 nm for tyrosine, phenylalanine, and tryptophan, respectively [22]. In this study, fluorescence spectra of individual fluorophores were recorded by the fluorescence-spectroscopy system on one side of the capillary and chromatograms using a monochromator-coupled PMT and lock-in amplifier system on the other side [22]. However, in the present study our main focus is on identification of differences among chromatographic patterns of different conditions (normal, malignant and 2-RT) for monitoring of tumor response to radiotherapy. The study was conducted and results were obtained with these factors in mind. Several different peaks indicate the presence of various proteins in the serum samples. In our earlier studies by co-injection method, using commercially available pure proteins, three proteins (transferrin, HSA, and creatine kinase) were ascertained [22–25]. These peaks are known to be common for all samples. The HSA peak, approximately at 1666-s region, is used for normalization of chromatograms as the relative intensity of this peak is constant in all samples. However, little attempt has been made to identify various other peaks of the chromatogram. Thus, the peak numbers ascertained in the present study are merely there to compare the typical chromatographic patterns of normal, malignant and 2-RT conditions. The chromatograms of the specimens in our pilot study showed certain important differences (Fig. 2). Peak 2 appeared in malignant specimens while it was absent in the normal ones. Peak 10 was present in all normal specimens whereas it is absent in malignant specimens. These presence of peak 2 and absence of peak 10 may be correlated to being a spectroscopic tumor marker. These results were in agreement with the earlier reports [25]. The reappearance of peak 10 in 2-RT samples is interesting because it may be an indication of the return to normalcy in patients with cervical cancer. Peak 9 was unique in the sense that it was present only in the treated (2-RT) samples. Its correlation with response to radiation is uncertain. It might be postulated that this peak may be a marker of radiationinduced effects to malignant tissues, as radiotherapy was the only different factor between the treated and the non-treated groups (‘malignant’ and ‘normal’). However, the difference in the protein profile patterns between malignant and 2-RT samples was minute, which was also reflected in our analysis (discussed in later part of the section). This might be due to the fact that samples were taken very early in the treatment. Samples collected later during the course of radiotherapy may magnify these changes allowing for definitive conclusions. It is a well-known fact that radiation-induced effects are mediated via free radical generation. The body
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instantly tries to counteract this effect by enhancing the production of free radical scavengers in order to protect from the deleterious effects of the free radicals. Nevertheless, the aim of the radiotherapy is to overcome the system’s counteraction in order to get rid of the cancerous cells. From radiobiological models it will be appreciated that repair of radiation-induced damage commences only after 4–6 h of treatment. Since we have collected samples 24 h after the second fraction of RT, the body had a considerable amount of time to repair these radiation-induced effects. Thus, the radiationinduced effects that have persisted after the repair process i.e. 24 h after treatment might have contributed to the minute variations in the protein profile patterns among treated and untreated malignant samples. It was seen in the classical method of analysis that the mean values relative intensities of the peak 5 for normal, malignant and 2-RT specimens were 1.51 (SD 0.23), 1.06 (SD 0.35), and 1.08 (SD 0.37), respectively (Fig. 3a). This indicates that the variation in relative intensity of the peak for normal and malignant is quite large and diminution of peak 5 (mean of malignantonormal) maybe a surrogate marker of malignancy. It is evident from the Fig. 3b, even though the mean values of peak 5 for malignant and 2-RT specimens are not significantly different, that there is slight upward trend (mean of 2-RT 4mean of malignant). This may again be a harbinger of the normal state and observation of this peak is warranted at a later stage in the course of treatment. Similar observations were made for peak 10. It is well-known that optical methods are amenable to several multivariate statistical tools such as hierarchical cluster analysis (HCA), artificial neural networks (ANN), k-nearest neighbor (KNN), maximum representation and discrimination feature (MRDF), principal components analysis available for data mining. However, as mentioned earlier, we have opted for PCA as a discriminating tool in our second approach of analysis. PCA is a well-known dimensionality reduction technique where large spectral data are reduced into small number of independent variations known as factors or principal components and contributions of these factors are called scores. Scores of factor were employed as discriminating parameters [30]. As stated in the data analysis section, analysis was carried out in full region, selected regions and derivatives of the same. As mentioned earlier, derivatization was more advantageous because the background was automatically corrected. Background in the chromatographic runs could arise from laser light scattered from capillary walls, fluorescence of the organic modifier (acetonitrile) in the gradient, PMT dark count, etc. Due to background fluorescence, a slight inclined slope was observed in the chromatograms. In this case a baseline correction by a third-order polynomial might induce artifacts. Hence we have opted for derivatization (widely used in
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FTIR spectroscopy) of chromatograms to minimize baseline/background-induced artifacts. As mentioned in the data analysis section, in our analysis PCA of first derivative spectra in the 1250–2000 s region, with 5 factors yielded best results and further analysis was carried out under these conditions. PCA was carried out in three-step process. In the first step, chromatograms of all three conditions, normal, malignant and 2-RT, were pooled and analyzed (Fig. 4a). These results show that both scores of factors 1 and 2 contribute to classification of normal and cancerous conditions (before and after 2-RT). It is evident from the figure that mean scores of factors 1 and 2 were positive for normal subjects and negative for the malignant and 2-RT, thus facilitating differentiation between the normal and cancerous conditions. However, the treated and untreated cervical neoplasia could not show clear differentiation. As can be seen from Fig. 4b, in the second step of analysis there was a slight decrease in the mean values of factors 1 and 2 when compared to malignant and 2-RT samples. This again suggests that samples taken later in the course of treatment may yield definitive results. This pilot study is important because it suggests that certain peaks, like peaks 2 and 10, could be probable markers for malignancy while peaks 5 and 10 may suggest response to radiotherapy. Peak 9 may suggest radiation-induced damage and may predict the severity of radiation reactions. As a third step we have carried out PCA of 2-RT serum chromatograms in order to verify the feasibility of prediction of tumor response to radiotherapy (CR, PR and NR) at an early stage of the treatment (Fig. 5). It is evident from the figure that PR and NR groups together tended to form a separate cluster from CR group. However, the CR groups outweigh this separation. It has to be noted the number of serum samples for PR (n ¼ 2) and NR (n ¼ 1) groups was very less. However, the above findings require confirmation in a larger trial. Correlation of peaks with clinical response to treatment may definitely predict the magnitude or range of values of relative intensities at which response to treatment can be ascertained. The use of serum samples collected in a later course of therapy may be useful in establishing the role of HPLC-LIF technique as a predictive marker of radioresponse.
Conclusions The findings of the study presented here suggest that protein profile studies of less invasive serum samples can clearly differentiate normal and malignant conditions. However, the protein profiling of malignant conditions failed to differentiate from 2-RT. This could be
explained as samples collected after 2-RT might be early enough to observe radiation-induced therapeutic effects. It has also been demonstrated that this technique has a limited application in prediction of tumor response to radiotherapy using 2-RT samples. Prospective studies with blood samples collected later in the course of the treatment could differentiate from malignant condition using this technique. Further, by obtaining larger data sets of different response conditions in the later course of treatment, the potential of this technique in prediction of tumor response to radiotherapy needs to be verified.
Acknowledgements The work was carried out under the Department of Atomic Energy; Board of Research in Nuclear Sciences (DAE-BRNS), Govt. of India project entitled ‘Laser spectroscopy as predictor of tumor response to radiotherapy in cervical cancer’ (Grant number 2003/34/17/ BRNS/1903). Authors acknowledge technical support of Ms. Keerti and Mr. Chetan Anand. Dr. C. Santosh, Incharge, Center for Atomic and Molecular Physics (CAMP) is also acknowledged. One of the authors (MK) is grateful to DAE-BRNS for a Research Fellowship.
Zusammenfassung Bestimmung von Serumproteinprofilen mittels HPLC¨ berwachung der Tumoransprechrate bei LIF zur U Radiotherapie des Zervixkarzinoms: Eine Pilotstudie Zielsetzung: Durchfu¨hrung einer Machbarkeitsstudie mit dem Ziel zu pru¨fen, inwieweit die Bestimmung von Serumproteinprofilen mittels eines kombinierten HPLCLIF-Systems zur U¨berwachung der Tumoransprechrate bei Radiotherapie des Zervixkarzinoms geeignet ist. Material und Methoden: 21 Studienteilnehmerinnen wurden in die Untersuchungen involviert: 7 davon waren gesund, 14 mussten sich aufgrund eines diagnostizierten Zervixkarzinoms fu¨r eine Dauer von 5 Wochen einer fraktionierten Radiotherapie (RT) mit 25 Einzeldosen a´ 2 Gy unterziehen gefolgt von 2 intrakavita¨ren Bestrahlungen im Abstand von einer Woche. Den Probandinnen wurden Blutproben entnommen, um das Blutserum chromatografisch zu untersuchen. Insgesamt wurden von den gesunden Patientinnen 7 ‘‘normale’’ Blutproben und von den Krebspatientinnen 14 sogenannte maligne’’ Proben vor Beginn der ’’ Radiotherapie sowie 13 sogenannte ‘‘2-RT’’-Proben 24 h nach der zweiten Bestrahlung (1 Patientin unterbrach die Behandlung) entnommen, aufbereitet und
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mittels eines selbstentwickelten HPLC-LIF-Systems vermessen und analysiert. Die Serumchromatogramme wurden auf zwei Arten ausgewertet: (1) klassisch durch den Vergleich der relativen Intensita¨ten auffa¨lliger Peaks und (2) mittels der Hauptkomponentenanalyse (Principal component analysis, PCA). Zusa¨tzlich wurde nach Abschluss der Radiotherapie, 4 Monate nach der ersten Bestrahlung, die Tumoransprechrate bestimmt (Tumorschrumpfung ¼ 100%: complete response (CR), Tumorschrumpfung Z50%: partial response (PR), Tumorschrumpfung r50%: no response (NR)) und mit den 2-RT-Chromatogrammen korreliert. Ergebnisse: Ein Vergleich der normalen und malignen Serumchromatogramme zeigte im Bereich 800–1800 s ausgepra¨gte Unterschiede. Zwischen den malignen und 2-RT-Chromatogrammen wurden nur minimale Unterschiede im Bereich 1300–1800 s festgestellt. Beide Auswerteverfahren (klassisch, PCA) konnten klar zwischen normal’’ und maligne’’ unterscheiden, ’’ ’’ jedoch nicht zwischen ‘‘maligne’’ und ‘‘2-RT’’. Von den 13 komplett behandelten Patientinnen zeigten 10 einen vollsta¨ndigen Tumorru¨ckgang (CR), 2 einen teilweisen Ru¨ckgang (PR) und eine Patientin kein Ansprechen auf die Therapie (NR). Durch Analyse der 2-RT-Chromatogramme konnte keine eindeutige Differenzierung zwischen diesen drei Patientengruppen erzielt werden. Zusammenfassung: Durch die Proteinanalyse von Serumproben konnte zwischen ‘‘normal’’ und ‘‘maligne’’ unterschieden werden, nicht jedoch zwischen maligne’’ ’’ und ‘‘2-RT’’. Eine Vorhersage der Ansprechrate auf die Bestrahlung anhand der 2-RT-Chromatogramme war nicht eindeutig mo¨glich. Schlu¨sselwo¨rter: Bestrahlungstherapie; Tumormarker; Zervixkarzinom; Hauptkomponentenanalyse (PCA)
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