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Chemoradiation in locally advanced Ca Cx: Effect on NGAL levels


1 Department of Radiotherapy, Pt. BD Sharma PGIMS, Rohtak, Haryana, India
2 Department of Biochemistry, Pt. BD Sharma PGIMS, Rohtak, Haryana, India

Date of Submission03-Sep-2018
Date of Decision17-Apr-2019
Date of Acceptance11-May-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Kiran Dahiya,
778/28, Bharat Colony, Rohtak - 124 001, Haryana
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_569_18

 > Abstract 


Introduction: Neutrophil gelatinase-associated lipocalin (NGAL) has been reported to be unregulated in many cancers and to suppress tumor suppressor genes like p53 leading to cell proliferation. Studies to report its relationship with carcinoma cervix (Ca Cx) are still scant.
Materials and Methods: Serum NGAL levels were analyzed in 30 patients of histopathologically proven locally advanced Ca Cx at the time of diagnosis and 3 weeks after standard chemoradiation by enzyme-linked immunosorbent assay. These patients underwent either brachytherapy or supplementary external beam radiotherapy (EBRT) depending on the response of treatment. The results were analyzed statistically by applying Student's paired t-test.
Results: No statistically significant difference (P > 0.05) was observed in patients of Ca Cx before and after treatment or when compared stage wise, histopathological grade wise, or response wise. But the levels were found to increase when duration of treatment was ≥8 weeks (P = 0.040) and to decrease significantly when duration of treatment was <8 weeks (P = 0.0052). The NGAL levels also increased significantly after treatment in patients who received EBRT and supplementary radiotherapy (P = 0.019) while the pre- and post-treatment difference in NGAL levels was not statistically significant in patients who received EBRT + intracavitary brachytherapy (P> 0.05).
Conclusion: As the duration as well as modality of treatment is quite important in Ca Cx, shorter duration associated with better results and lower NGAL levels, NGAL might prove to be a useful biomarker although further studies are needed to support the claim.

Keywords: Biomarker, carcinoma cervix, chemoradiation, duration of treatment, neutrophil gelatinase associated lipocalin



How to cite this URL:
Dhankhar R, Chhabra S, Dahiya K, Ghalaut VS, Singh S, Gupta K. Chemoradiation in locally advanced Ca Cx: Effect on NGAL levels. J Can Res Ther [Epub ahead of print] [cited 2020 Feb 29]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=277244




 > Introduction Top


Neutrophil gelatinase-associated lipocalin (NGAL) is also known as lipocalin-2 (LCN-2), oncogene 24p3, human neutrophil lipocalin, uterocalin, siderocalin, and neu-related lipocalin. It is a protein encoded by LCN-2 gene on chromosome 9 and belongs to the lipocalin protein family. This family is a large group of small extracellular proteins with a variety of physiological functions. NGAL exists as a 25 kDa monomer, 46 kDa disulfide-linked homodimer, and 135 kDa disulfide-linked heterodimer with neutrophil gelatinase.[1] Recently, it has emerged as a biomarker for several benign and malignant diseases. Upregulation of NGAL has been found to increase the invasiveness of breast, bladder, gastric, gynecological, thyroid, lung, esophageal, colon cancer, and chronic myelogenous leukemia while decreasing invasiveness in pancreatic and oral cancer.[2],[3] It has also been reported that upregulation of NGAL increases cell proliferation of cervical and lung cancer cells, while its downregulation reduces cell proliferation of these tissues.[2],[4]

Cervical cancer is a major gynecological cancer which involves uncontrolled cell division and tissue invasiveness of the female uterine cervix. It is the third leading cause of cancer-related mortality among women worldwide. Cervical cancer is induced by persistent infections with oncogenic human papilloma viruses (HPVs). While HPV infection is an indispensable factor, it is not sufficient to cause cancer. The majority of acute HPV infections induce low-grade precursor lesions that are cleared spontaneously after several months in more than 90% of cases, and <10% eventually progress to high-grade lesions or invasive cancer.[5] Upregulation of NGAL in higher grade cervical lesions is likely to lead to suppression of wild-type p53 by the HPV E6 oncoprotein. Suppression of p53 results in elimination of p53 block of NGAL transcription.[6] In a study, NGAL mRNA and proteins levels were found to be significantly higher in nonlymph node metastasis group and in lymph node metastasis group than in normal control group. Furthermore, the levels of matrix-metalloproteinase-9, a protease involved in tumor invasion and metastasis, and NGAL mRNA as well as protein were significantly elevated in lymph node metastasis group as compared with nonlymph node metastasis group.[7]

The main modalities of treatment for carcinoma cervix (Ca Cx) include surgery, radiotherapy (RT), or chemotherapy either alone or in combination. Surgery has long been a standard therapy for early-stage (I–IIa) disease, but limitations in the diagnosis of early lymph node metastasis of invasive cervical cancer still remain.[8] This disease is still in the search of good biomarkers for early metastasis or indicators of successful treatment. NGAL has shown some potential to fit into this role as suggested by reports in literature, though still very few.


 > Materials and Methods Top


The present study was conducted on 30 previously untreated, histopathologically proven patients of Ca Cx, attending the outpatient department of RT of the institute for definitive treatment by chemoradiation therapy after obtaining informed consent and approval from the institutional board of studies. All the patients were evaluated by complete history, general physical examination, and complete systemic examination including gynecological examination (per-speculum, per-vaginal, and per-rectal examination). The assessment of patient's general condition was done using Karnofsky Performance Status (KPS). Hematological assessment was done by analyzing complete hemogram on automated instrument and biochemical assessment by renal and liver functions on autoanalyzer (Randox Suzuka) using standard kit methods. Radiological assessment including chest X-ray, ultrasonography of abdomen and pelvis, and computed tomography was done in all patients. Whenever clinically indicated, intravenous pyelogram, cystoscopy, rectosigmoidoscopy, magnetic resonance imaging scan of abdomen, and pelvis were carried out. The patients were staged according to International Federation of Gynecologists and Obstetricians staging system.[9]

Based on the above assessment, the patients for the study were selected depending on the following criteria: KPS ≥70; complete hemogram with hemoglobin >8 g/dL, total leukocyte count >4000/mm3, platelet count >100,000/mm3; normal renal function tests with blood urea <40 mg/dL, and serum creatinine <1.5 mg/dL; normal liver function tests with aspartate transaminase <35 IU/L, alanine transaminase <40 IU/L), and locally advanced Ca Cx. The patients having any of the following conditions were excluded from the study: distant metastases; pregnant or lactating patient; and associated comorbid conditions such as renal disease, liver disease, gastrointestinal disease, heart disease, or endocrinal disorders.

The patients before treatment were designated as Group I and after treatment were designated as Group II. These patients received concomitant chemoradiation in the form of external radiation to whole pelvis 50 Gy/25#/5 weeks either by parallel opposed anteroposterior fields or by “Box-technique” along with injection cisplatin 40 mg/m2 IV every week for a total of 6 courses starting from day 0 of RT. These patients underwent either brachytherapy or supplementary external RT depending on the response of treatment. The blood sample was taken 3 weeks after the completion of whole treatment. Serum NGAL levels were estimated in both the groups by a commercial enzyme-linked immunosorbent assay kit for human NGAL.[10]

From the commencement of treatment, all the patients included in the study were carefully and regularly assessed on weekly basis. Detailed clinical evaluation of each patient for the tolerance to the treatment delivered was done by thorough clinical examination. Tumor response was assessed by World Health Organization response criteria. The major study endpoints were local pelvic disease control and treatment related morbidity. After completion of concomitant chemoradiation, every patient was assessed for clinical response and residual disease, depending on which the further treatment was given. In patients suitable for applicator placement, high-dose rate (HDR) brachytherapy with 3 fractions of 6 Gy each to point A was given at weekly intervals by Ir-192 microselectron HDR remote-controlled afterloading brachytherapy machine. In patients not suitable for brachytherapy, supplementary external beam RT (EBRT) with 20 Gy/10 fractions/2 weeks was given.

The collected data were entered in the MS Excel spreadsheet, coded appropriately. Paired Student's t-test was applied for analyzing the data.


 > Results Top


All the patients were in the age range of 35–70 years with the median age being 54.9 years. Overall, 25 (83.4%) patients were from rural areas while 5 (16.6%) of the patients belonged to urban background. As per personal habits, 28 (93.3%) patients were nonsmokers while only 2 (6.6%) of the patients were smokers. None of the patient was alcoholic.

Based on KPS, 7 (23.4%) of patients presented with KPS of 70, 13 (43.3%) of patients presented with KPS of 80, and 10 (33.3%) of patients presented with KPS of 90.

Common presenting symptoms were bleeding per vaginum (P/V) in 12 (40%) patients and discharge P/V in 17 (56.7%) patients while 1 (3.3%) patient presented with other symptoms.

Overall, 2 (6.7%) of patients had well-differentiated squamous cell carcinoma (WDSCC), majority, i.e., 23 (76.6%) had moderately differentiated squamous cell carcinoma (MDSCC), 3 (10%) had poorly differentiated carcinoma (PDSCC), and 2 (6.7%) had adenocarcinoma (AC). Total squamous cell carcinoma patients reported were 28 (93.3%). As per staging, 14 (46%) patients presented with Stage II B, 15 (50%) patients with Stage III, and only one patient (3.3%) presented in Stage IV A.

The levels of blood urea were found to be 30.63 ± 7.65 mg% with a nonsignificant negative coefficient of correlation (r = −0.178, P = 0.078) with NGAL levels. Similarly, serum creatinine levels were found to be 0.96 ± 0.03 mg%, and coefficient of correlation was again found to be nonsignificant (r = 0.0914, P = 0.102) with serum NGAL levels.

The comparison of levels of NGAL in different groups has been presented in the form of [Table 1]. Histograms have been used to present stagewise [Figure 1], histopathological gradewise [Figure 2], and durationwise [Figure 3] and [Figure 4] distribution of NGAL levels and according to the type of radiation used [Figure 5] and [Figure 6] in patients of Ca Cx.
Figure 1: Graphic representation of the stage wise distribution of neutrophil gelatinase-associated lipocalin levels in patients of carcinoma cervix

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Figure 2: Graphic representation of the histological distribution of neutrophil gelatinase-associated lipocalin levels in patients of carcinoma cervix

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Figure 3: Graphic representation of the groupwise distribution of neutrophil gelatinase-associated lipocalin levels in patients of carcinoma cervix with <8 weeks of treatment

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Figure 4: Graphic representation of the groupwise distribution of neutrophil gelatinase-associated lipocalin levels in patients of carcinoma cervix with more than or equal to 8 weeks of treatment

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Figure 5: Graphic representation of the group wise distribution of neutrophil gelatinase-associated lipocalin levels in patients of carcinoma cervix who received radiation in the form of external beam radiotherapy + intracavitary brachytherapy

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Figure 6: Graphic representation of the group wise distribution of neutrophil gelatinase-associated lipocalin levels in patients of carcinoma cervix who received radiation in the form of external beam radiotherapy + supplementary radiotherapy

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Table 1: Neutrophil gelatinase-associated lipocalin levels (ng/mL) in patients of carcinoma cervix before (Group I) and after treatment (Group II) expressed as mean±standard deviation

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 > Discussion Top


Before commenting on the findings regarding NGAL levels in different groups, let us have a look on the other aspects of the study. The present study has shown that most of the Ca Cx patients were in the age group of 41–60 years (63.3%). As per Indian reports, this cancer strikes early, the incidence rises in 30–34 years of age and peaks at 55–65 years, with a median age of 38 years (age 21–67 years).[11] The small variation in age-wise distribution might be due to enrollment of only locally advanced Ca Cx patients in study group. Haryana is an agricultural state, and majority of population live in rural areas, and this is reflected in the participants of the study. Majority of the participants (93.4%) were nonsmokers, and all of them were nonalcoholic. Smoking is a risk factor for Ca Cx,[12] but this could not be established by this study due to small sample size and inclusion of only locally advanced cases. As per literature, squamous cell carcinomas accounts for over 90% of all cervical malignancies. Of all, 7%–10% of cervical carcinomas are AC, and 1%–2% are the clear-cell, mesonephric type.[12] Our study also reflected the same results showing 93.3% patients of squamous cell carcinoma and 6.7% patients of AC.

In our study, NGAL levels were observed to have a weak correlation with both serum creatinine and serum urea levels (P > 0.05). Literature suggests that serum NGAL levels are elevated in acute kidney injury resulting from a wide variety of insults to the kidney ranging from ischemia to toxins. This rise occurs early and depends on both the cause and the extent of renal damage. For instance, following ischemic injury, NGAL levels in the kidney tissue rises by nearly 10 fold within 3 h.[13],[14] Our observations show that serum blood urea and serum creatinine are related weakly with serum NGAL values.

Literature suggests that 47% patients with cervical cancer present at Stage I, 28% at Stage II, 21% at Stage III, and 4% at Stage IV.[12] Since the present study has included only patients with locally advanced carcinoma (Stage II-B, III, and IV-A), stage at presentation cannot be commented on. It has been shown in literature that normal NGAL serum concentration is about 20 ng/mL, and it is probably related to physiological neutrophil production and limited liver, spleen, and kidney expression.[14] The expression of NGAL is significantly increased in several solid and hematological tumors and has been shown to correlate with both tumor characteristics and disease outcome.[15] In our study also, the serum NGAL levels have increased with progressive stages of Ca Cx, but the difference in serum NGAL levels in different stages has not been found to be statistically significant (P > 0.05).

The mean levels of serum NGAL in posttreatment group (Group II) in different stages has been found to be increased as compared to the levels in pretreatment group (Group I), but the difference is not found to be statistically significant (P > 0.05). No study is available in literature to show any relation between serum NGAL values and stage of Ca Cx. Literature suggests that radiation and chemotherapy may induce reactive oxygen species and subsequent downstream NGAL transcription. RT could result in the synthesis of NGAL in cancer cells which may lead to the development of therapy-resistant cells. NGAL leads to a significant upregulation in the expression of the antioxidant enzymes superoxide dismutase and heme oxygenase.[16],[17] This antioxidant role of NGAL might be the possible reason for increase in serum NGAL values in all stages of Ca Cx after completion of treatment. Literature also suggests that the role of HPV as a causative agent of cervical cancer is well established. As per a report, upregulation of NGAL was also related to HPV detection and showed a linear relationship to high-risk HPV load.[18] The possible role of HPV infection and hence increased serum NGAL levels in these patients after treatment in all stages cannot be ruled out. These facts need to be evaluated in further studies. Possible reason for increased NGAL levels after treatment in WDSCC and MDSCC, though statistically not significant (P > 0.05), may be associated HPV infection in Ca Cx patients, but reason for decrease in serum NGAL values in PDSCC and AC remains unknown and needs to be analyzed in future studies.

The mean value of serum NGAL in pretreatment group in 4 patients who received EBRT + supplementary EBRT was 396 ± 93.70 ng/mL, and posttreatment serum NGAL value was 686 ± 59.71 ng/mL. This difference was statistically significant (P = 0.019) while the difference was not statistically significant in 26 patients who received EBRT + intracavitary brachytherapy (P = 0.968). Possible reason for this may be better tumor control with brachytherapy as compared to supplementary RT after EBRT. Poor tumor control after supplementary RT might have resulted in statistically significant increased serum NGAL levels. No study in literature has shown any relation between serum NGAL and treatment modality used. Further studies are needed for confirmation.

Another interesting finding of the study is that serum NGAL value has significantly decreased when total duration of treatment was ≤8 weeks (P = 0.040). Similarly, when total duration of treatment was >8 weeks, serum NGAL values increased significantly (P = 0.0052). Various studies in literature have shown that overall treatment time for treatment of Ca Cx should be as short as possible. It has also been mentioned in these studies that lower pelvic control and survival rates are observed when treatment duration is prolonged.[12] When the treatment duration is <8 weeks, the tumor cells do not get time to repopulate or to repair sublethal damage. In such cases, better tumor control is achieved which has been shown by statistically significant decrease in serum NGAL in our study. As the treatment duration is increased, the tumor cells get time to repopulate and hence lower pelvic control. Hence, serum NGAL can be used as prognostic marker in Ca Cx patients. Further studies are needed for confirmation of the role of serum NGAL values in pelvic control of disease.

This study observed that serum NGAL levels, after complete treatment, were decreased in patients with complete clinical response (n = 18), but the difference in serum NGAL values was not statistically significant (P = 0.801) while in patients with partial response (n = 11) or nonresponding patients (n = 1), serum NGAL levels were found to be increased, but the difference was again not statistically significant (P = 0.0749). Possible reason for not getting statistically significant decrease in serum NGAL levels in complete clinical response group might be subclinical microscopic disease, still present, after treatment. No previous study in literature has shown any relation between serum NGAL values and clinical response to treatment. Since our study group is small (n = 30), this parameter needs further evaluation.

As total treatment duration and modality of treatment used is of major concern in the management of Ca Cx, which can be assessed by serum NGAL levels, NGAL may act as a prognostic marker though the claim needs to be reinforced by further supporting studies in this regard.


 > Conclusion Top


On the basis of the present observations, it is concluded that NGAL is affected by duration as well as modality of treatment for carcinoma cervix. It might be considered a potential biomarker for this disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Strong RK, et al. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 2002;10:1033-43.  Back to cited text no. 1
    
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Candido S, Maestro R, Polesel J, Catania A, Maira F, Signorelli SS, et al. Roles of neutrophil gelatinase-associated lipocalin (NGAL) in human cancer. Oncotarget 2014;5:1576-94.  Back to cited text no. 3
    
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Chung IH, Wu TI, Liao CJ, Hu JY, Lin YH, Tai PJ, et al. Overexpression of lipocalin 2 in human cervical cancer enhances tumor invasion. Oncotarget 2016;7:11113-26.  Back to cited text no. 4
    
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Syrjänen S, Naud P, Sarian L, Derchain S, Roteli-Martins C, Tatti S, et al. Up-regulation of lipocalin 2 is associated with high-risk human papillomavirus and grade of cervical lesion at baseline but does not predict outcomes of infections or incident cervical intraepithelial neoplasia. Am J Clin Pathol 2010;134:50-9.  Back to cited text no. 6
    
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Chung IH, Chen CY, Lin YH, Chi HC, Huang YH, Tai PJ, et al. Thyroid hormone-mediated regulation of lipocalin 2 through the met/FAK pathway in liver cancer. Oncotarget 2015;6:15050-64.  Back to cited text no. 7
    
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Hsu WL, Shueng PW, Jen YM, Wu CJ, Hwang JM, Chang LP, et al. Long-term treatment results of invasive cervical cancer patients undergoing inadvertent hysterectomy followed by salvage radiotherapy. Int J Radiat Oncol Biol Phys 2004;59:521-7.  Back to cited text no. 8
    
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Sreedevi A, Javed R, Dinesh A. Epidemiology of cervical cancer with special focus on India. Int J Womens Health 2015;7:405-14.  Back to cited text no. 12
    
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Viau A, El Karoui K, Laouari D, Burtin M, Nguyen C, Mori K, et al. Lipocalin 2 is essential for chronic kidney disease progression in mice and humans. J Clin Invest 2010;120:4065-76.  Back to cited text no. 13
    
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Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 2003;14:2534-43.  Back to cited text no. 14
    
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Monisha J, Padmavathi G, Bordoloi D, Roy NK, Kunnumakkara AB. Neutrophil gelatinase-associated lipocalin (NGAL): A promising biomarker for cancer diagnosis and a potential target for cancer therapeutics. J Cell Sci Mol Biol 2014;1:106.  Back to cited text no. 15
    
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