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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 8-12

Spectral technique for monitoring cervical cancer treatment following radiotherapy

1 Department of Radiation Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India
2 Department of Physics, College of Education, Imam Abdul Rahman Bin Faisal University, Dammam, Saudi Arabia
3 Department of Physics and Astronomy, College of Science, King Saud University, Riyadh, Saudi Arabia

Date of Submission27-Sep-2019
Date of Decision14-Dec-2019
Date of Acceptance11-Feb-2020
Date of Web Publication20-Apr-2020

Correspondence Address:
Dr. Siddanna R Palled
Department of Radiation Oncology, Kidwai Memorial Institute of Oncology, Bengaluru - 560 056, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/oji.oji_43_19

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Background: The pre- and post-treatment evaluation of cervical cancer is usually done by clinical examination and radiological imaging depending on the facility available. The biochemistry of tumor tissues gets dramatically altered after chemoradiation, and such changes could be monitored by the spectral analysis of blood and urine for biochemical component. Aim: This study aims to evaluate the pre- and post-treatment biochemical changes through spectral analysis of blood and urine samples. Materials and Methods: Sixty-nine diagnosed cases of cervical carcinoma were taken for the study. The pre- and post-treatment evaluation of disease was done by clinical examination and radiological imaging. The biochemical component of blood and urine samples of all patients was analyzed spectroscopically before and after radiotherapy by exciting at 400 nm and capturing the emission spectrum over the range of 425 nm–675 nm. Results: The majority of cervical carcinoma patients were clinical International Federation of Gynecology and Obstetrics Stage IIIB followed by Stage IIB and Stage IB consisting of 49.28%, 33.33%, and 8.70% of cases, respectively. Rest of 8.70% of patients were postoperative. The initial results were found to be encouraging with good correlation (up to 66.67%) between spectral biomarker measurement, and the clinical and abdominal ultrasound scan monitoring. Conclusion: This proof of concept study with a limited number of patients, there was good clinical correlation and supplementary information for monitoring the patients. Spectral biomarker analysis could become a reliable, inexpensive tool complementing or supplementing expensive techniques like computed tomography scan.

Keywords: Biochemical components, cervical cancer, fluorescence spectra, posttreatment monitoring

How to cite this article:
Palled SR, Aldaleeli NY, Ganesh K M, Vadivel M, AlSalhi S M. Spectral technique for monitoring cervical cancer treatment following radiotherapy. Oncol J India 2020;4:8-12

How to cite this URL:
Palled SR, Aldaleeli NY, Ganesh K M, Vadivel M, AlSalhi S M. Spectral technique for monitoring cervical cancer treatment following radiotherapy. Oncol J India [serial online] 2020 [cited 2020 Aug 13];4:8-12. Available from: http://www.ojionline.org/text.asp?2020/4/1/8/282836

  Introduction Top

Cervical cancer is the second-most common cancer in India, accounting for 22.8% of all cancer cases in women.[1],[2] The 5-year survival rate for early-stage cervical cancer is 92% and it drops to 56% or less in locoregionally advanced cases and barely 10%–15% in metastatic disease. The high mortality rate from cervical cancer globally can be reduced through a comprehensive approach that includes prevention, early diagnosis, and effective screening and treatment programs. Currently, vaccines that protect against common cancer-causing types of human papillomavirus can significantly reduce the risk of cervical cancer.  Pap smear More Detailss and visual inspection with acetic acid at regular intervals are advocated as screening tests to diagnose cervical cancer before the onset of symptoms. The usual procedure for detection, diagnosis, and posttreatment monitoring are as follows clinical examination, ultrasound, computed tomography (CT) scans, magnetic resonance imaging (MRI), and sometimes positron emission tomography scans depending on availability of resources.[3]

In malignancy, blood and biopsy of tumor are tested, which help to determine the characteristics of the tumor, i.e., aggressiveness, rate of growth, and degree of abnormality. Tumor markers can be elevated and detected in the blood, urine, or body tissues of some patients with certain types of cancer. It may be used as complementary tests along with other essential staging investigations to follow the course of the disease, to measure the effect of treatment, and to check for recurrence.

A few biomolecules in the human body have endogenous fluorescence, with a unique combination of excitation and emission maxima, which taken together can serve as a fingerprint for the molecule. The relative concentration of these biomolecules serves as a marker to assess the presence and stage of cancer in some organ or tissue of the body, and also, the particular types of cancer in the body. A study by Sordillo et al. found there was marked increases level of tryptophan in the breast carcinoma samples when compared to the normal breast tissues.[4] Spectral profiles from the cancerous and normal tissues were substantially different in a rat tissue model study, showing characteristic principal and secondary maxima. These peaks were assigned to fluorophores (flavins and porphyrins).[5] Drezek et al. tried to understand the contributions of nicotinamide adenine dinucleotide (NADH) and collagen to cervical tissue fluorescence spectra using modeling study, in which NADH fluorescence increases with dysplasia, and collagen decreases with dysplasia.[6]

When photons of a particular wavelength interact with biomolecules, most photons are scattered as radiation with or without shifts in their wavelengths, i.e., Raman or Raleigh scattering, respectively. A few photons are absorbed by the biomolecules which results in the excited states. The molecules stay at the excited states for a few nanoseconds and subsequently release energy as fluorescence radiation. Raman shifts provide unique measures of vibrational energy levels but fluorescence the electronic energy levels of molecules. Based on the presence and intensities of these signals, the relative proportions of these biomolecules could be determined. Such assays provide significant information about disorders or diseases associated with the tissues or fluids of a particular subject. A number of related studies have been published for the diagnosis of cancers and blood disorders, based on the spectral features of biomolecular components of blood and urine.[7],[8],[9],[10],[11],[12],[13],[14],[15],[16]

With this background, we had conducted a study to assess treatment response in cervical cancer by monitoring biomarkers with optical biopsy, i.e., fluorescence spectral technique on blood and urine biochemical components which is an emerging novel technique based on the interaction of light with biomolecules. The spectral features have been compared before and after a course of treatment, specifically radiotherapy.

  Materials and Methods Top

This was a prospective observational study conducted at the Department of Radiation Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, from July 2012 to August 2014. A total of 69 patients were included under the study with the age ranges from 25 to 65 years and the median age of presentation being 50 years. All the patients were informed about the investigation, and proper consents were obtained. The Ethical Clearance for this investigation was obtained from the Institutional Review Board of Kidwai Memorial Institute of Oncology, Bengaluru, India. Among the individuals, the normal controls used in this study were friends and relatives of the patients who accompanied them.

The fluorescent biomolecules considered here include tryptophan, reduced nicotinamide adenine dinucleotide, and flavins. The crux of this technique is that malignancy upsets biochemistry of cells and also the intercellular matrix, and these changes can be quantized by native fluorescence analysis of these bio fluorophores involved, either in tissue or in body fluids.[17],[18],[19],[20],[21] A modification of fluorescence spectroscopy is synchronous fluorescence excitation spectroscopy, where the excitation and emission gratings are synchronously rotated with an offset of fixed difference in wavelength.[19] Since the excitation changes continuously over a range of wavelengths, several closely spaced fluorophores, commonly found in a multi-component matrix such as blood plasma, are resolved. This may be more appropriately called as Stokes shift spectroscopy when the offset is equal to the Stokes shift (the difference between the emission and excitation peaks); then, the individual molecules in a composite system could be unmistakably identified.

A conventional spectrofluorometer, such as Perkin Elmer (LS 50 or 55) is capable of collecting excitation, emission, and synchronous spectra in the range of 200–800 nm. The current spectroscopic experiment was used to acquire fluorescence emission spectra (FES) of acetone extracts of red blood cells (RBCs) from 425 to 700 nm upon excitation at 400 nm with a spectral width of 10 nm (Xe lamp source). The same instrument was used to obtain synchronous fluorescence excitation spectra (SFXS) of different biomolecules of blood plasma (e.g., tryptophan, NADH, and flavins) from 200 to 700 nm. This was performed using an offset of 70 nm between excitation and emission gratings.[13],[14],[15],[16],[17],[18],[19]

Briefly, 5 ml blood was collected from each individual in an ethylene diamine tetraacetic acid vial containing standard anticoagulant. The collected blood vials were gently rocked five times and centrifuged for 15 min at 3000 rpm to separate plasma from cellular components. The top supernatant yielded greenish-yellow liquid, plasma, which was collected and subjected to SFXS and FES. The bottom thick, semisolid paste of blood containing mostly RBC was lysed with acetone (1:2 v: V) and centrifuged to obtain a clear, transparent liquid. This contained the biomolecules for FES fluorescence study of RBC.[16],[17],[18],[19],[20],[21],[22] In contrast, first voided urine was collected in sterile vial from each individual and used without any treatment.

  Results Top

It is important to emphasize that of 69 patients taken in the study, 49.28% of patients were with Stage IIIB, 33.33% patients with Stage IIB, 8.70% patients with Stage IB, and 8.70% patients were postoperative. There was an initial good correlation between treatment responses and spectral outcome observed only in 46 patients (66.67%) with spectral data in the form of R1 and R3 and clinical and sonological correlation. In other 18 patients, the biomarker levels stayed at abnormal level indicative of substantial residual disease, mostly in Stage IIIB patients with large tumor size, and in 5 of the postoperative patients, the level was not correlating clinically and also with ultrasound findings. This indicates that the disease at the time of presentation is advanced and possibly systemic spread might have occurred. Correlation of results could not be done with CT during this study period due to the waiting period for CT. In future, we will study the outcome in correlation with CT/MRI correlation.

The results were explained in the following set of figures. As shown in [Figure 1], the FES of plasma of normal control, a typical cervical patient before and after treatment was presented. Note it is customary to represent the y axis as relative intensity of fluorescence.
Figure 1: (a) Fluorescence emission spectra of blood plasma of normal control. (b) Fluorescence emission spectra of blood plasma of a cervical cancer patient with advanced stage disease. (c) Fluorescence emission spectra of blood plasma of a cervical cancer patient before and after treatment (good regression)

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[Figure 1]a showed typical FES of blood plasma of a normal control, in which there are two peaks can be seen, one at 475 nm and other at 525 nm. The one at 475 nm is due to the coenzyme NADH and that at 525 nm is due to another coenzyme FAD. The intensity ratio (R1) between these two (I525/I475) was about 1.1 for normal. [Figure 1]b shows the FES of cervical cancer patients before treatment. It is markedly different with 525 nm peak becoming predominant. If we take the ratio R1 (I525/I475) here, it is 3 times higher for a cancer patient.

The initial results for 46 patients are shown in [Figure 1]c which was similar FES of plasma of another patient before and after treatment. In this case, R1 was 1.8 before treatment and 0.8 after treatment, almost similar to the normal range shown for comparison. This means, for this particular patient, the treatment had been effective as the biomarker levels are reaching the normal range. These patients' data were clinically and sonologically correlated as there was no detectable disease.

[Figure 2] showed FES of blood plasma of advanced with extensive tumor. It can be seen that this ratio R1 (I525/I45) is 10 for this patient before treatment, but had come down to 2 after treatment indicating partial recovery with significant residual disease. Furthermore, note the biomarker levels for normal control for comparison.
Figure 2: Fluorescence emission spectra of blood plasma of cancer cx patient before and after treatment (poor recovery)

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In the next set of figures [Figure 3], the cancer indicative biomarkers were explored in the acetone extract of RBC, three major spectral bands were observed with peaks at 470, 585, and 630 nm. The spectra were normalized to the intensity at 470 nm (i.e., the background Raman spectrum of acetone). The intensity ratio between the peaks at 630 nm and 585 nm (i.e., the neutral and basic forms of porphyrin) is a measure of oxygen-carrying capacity. This measurement was different for blood components of normal control and advanced stage of cervical cancer patient before and after treatment. To highlight the differences, a ratio parameter was defined as R2 = I630/I585. The presented ratio parameter was 1:1 for control group, whereas for advanced cancer patient, it was about 3:1 before treatment and 1:5 after treatment. Here again, there was a strong indication for the disease regression following the treatment (chemoradiotherapy).
Figure 3: Fluorescence emission spectra of acetone extract of red blood cell of a patient before and after treatment

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[Figure 4] showed the SFXS of urine. Here again, there were two peaks, one at 340 nm (due to NADH) and 450 nm (due to FAD). For normal, the ratio R3 between the two peaks is 4, but for the carcinoma cervix patient, it was 1:1 before treatment. This was because FAD had become stronger by three times and NADH weaker by two times. Note the remarkable change after treatment: For the same patient after treatment, the ratio had become again 3, indicating good recovery.
Figure 4: Synchronous fluorescence excitation spectrum of urine of a patient before and after treatment

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The range of ratio parameter for the normal and the cervical cancer patients before and after treatment can be better visualized for a typical ratio R1 in [Figure 5].
Figure 5: Distribution of R1 parameter for cancer cervix patients before and after treatment. Note the wide variation in R1 for untreated patients

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  Discussion Top

In the present study, pre- and post-treatment tumor monitoring was done by clinical examination, spectral analysis, and ultrasound abdomen and CT scan of the abdomen and pelvis was done only in few cases. The treatments received were concurrent chemoradiation or radiation therapy alone based on clinician decision which is decided by stage, performance status of the patient, logistics and other clinical parameters.

The crux of the investigation

In cancer, abnormal cell proliferation leads to angiogenesis, which could be sustained only by more oxygen content in the blood. This is reflected as the enhanced level of oxygen carrier molecule porphyrin in cancer patients.[5] This gets reduced when the treatment has been effective.

NADH is another important biomarker and has been shown many times that reduction in NADH is an indication of the onset of cancer because malignancy thrives by glycolysis which consumes a lot of NADH. This is called the Warburg effect. Such a reduction of NADH has been dramatically shown in blood plasma and even in urine when disease sets in. Whenever the treatment has been effective, NADH level went up and FAD level came down.[4],[6] We found an initial correlation in 46 patients (66.67%) in the study group with spectral data in the form of R1 and R3 and clinical and sonological correlation. One needs to do a large set of patient data to come up with conclusive evidence for disease monitoring.

  Conclusion Top

The spectral technique can be proposed to monitor the cervical cancer treatment monitoring based on the quantification of fluorescent biomolecules such as coenzyme NADH, FAD, and oxygen carrier protein porphyrin indicative of abnormal, growth of cells. The potential of using fluorescence spectral technique to measure the regression/progression of the cancer is the key to be a candidate for future clinical trials. This report is only preliminary and done as a proof of concept. More detailed study needs to be done in due course.


The authors would like to thank Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India, for all facilities offered and Imam Abdulrahman Bin Faisal University, for technical resources and support provided.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]


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