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Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 521-526
Circulating free DNA as a marker of response to chemoradiation in locally advanced head and neck squamous cell carcinoma

1 Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Vibhuti Khand, Gomti Nagar, Lucknow, Uttar Pradesh, India
2 Department of Radiotherapy, Dr. Ram Manohar Lohia Institute of Medical Sciences, Vibhuti Khand, Gomti Nagar, Lucknow, Uttar Pradesh, India
3 Department of Radiology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Vibhuti Khand, Gomti Nagar, Lucknow, Uttar Pradesh, India

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Date of Submission06-Jan-2020
Date of Decision07-Apr-2020
Date of Acceptance18-Apr-2020
Date of Web Publication28-Oct-2020


Context: Liquid biopsy has moved from bench to bedside as a non-invasive biomarker for early diagnosis and monitoring treatment response. Objective: This study investigated the role of circulating free DNA (cfDNA) as a diagnostic marker in locally advanced head and neck squamous cell carcinoma (HNSCC) and in monitoring response to chemoradiation therapy. Materials and Methods: Serum was collected from treatment naïve, histopathologically diagnosed tumors in 24 HNSCC cases and 16 normal controls. CfDNA levels were quantified using β globin gene amplification. Results: The cfDNA level was significantly elevated in HNSCC (992.67 ± 657.43 ng/mL) as compared to healthy controls (60.65 ± 30.42 ng/mL, P = <0.001). The levels of cfDNA did not significantly correlate with TNM stage, lymph node involvement and grade. In responders, percentage decrease in cfDNA levels was 9.57% and 29.66%, whereas in nonresponders percentage increase was 13.28% and 24.52% at the end of three months of follow-up. Conclusion: Our study adds to the evidence that cfDNA levels are significantly higher in HNSCC cases and provides some evidence that levels increase with tumor progression. CfDNA may be a promising prospective non-invasive marker to predict response in patients undergoing chemo-radiotherapy.

Keywords: Chemotherapy, circulating free DNA, head and neck squamous cell carcinoma, liquid biopsy, real-time PCR

How to cite this article:
Verma T, Kumari S, Mishra S, Rastogi M, Tiwari V, Agarwal GR, Anand N, Husain N. Circulating free DNA as a marker of response to chemoradiation in locally advanced head and neck squamous cell carcinoma. Indian J Pathol Microbiol 2020;63:521-6

How to cite this URL:
Verma T, Kumari S, Mishra S, Rastogi M, Tiwari V, Agarwal GR, Anand N, Husain N. Circulating free DNA as a marker of response to chemoradiation in locally advanced head and neck squamous cell carcinoma. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 Dec 9];63:521-6. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/521/299303

   Introduction Top

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related deaths and account for 90% of head and neck cancers.[1] It is significant public health disorder in developed and in developing countries including India and is sixth leading cause of cancer by incidence and eighth by death.[2] In 2012, approximately 700,000 new HNSCC cases were reported worldwide.[3] Even with current therapeutic interventions, the prognosis for HNSCC is quite poor and 5-year survival ranges from 25%-60%.[4] Delayed diagnosis is a well-documented challenge and diagnosis at an advanced stage, eventually impacts its prognosis and survival.[5] In last three decades, incidence of human papilloma virus (HPV) associated oropharyngeal cancer has increased[6] and is reported to have a higher survival rate compared to HPV-negative counterparts.[7],[8] However, patient's outcome are heterogeneous in cases of HPV associated HNSCC.

For early tumor detection many biochemical, molecular, and pathological biomarkers have been tested with promising potential. Chemokine's and their receptors, melanoma-associated gene, p53, and eukaryotic translation factor 4E, have been assessed for prognoses. However, uses of these biomarkers are limited by lack of sensitivity and specificity suggesting the need for further research and clinical validation.[9]

CfDNA serves as a non-invasive biomarker for the diagnosis of cancers and quantification may help in optimizing medical practice.[10],[11] CfDNA has been reported in serum, plasma, induced sputum, bronchial lavage, milk, urine, and stool.[12] Release of cfDNA occurs via active release from diseased cells, apoptosis, necrosis and from the interface of the tumor and adjacent non-tumor cells; however, its release in circulation is not very clear.[10],[13],[14] In stage IV breast cancer CfDNA has been reported as an independent prognostic factor and can be used to monitor therapeutic response.[15] This study was carried out to evaluate pretreatment cfDNA levels and its kinetics during and after completion of chemoradiation in patients with HNSCC with the aim to discriminate responders and nonresponders.

   Materials and Methods Top

Patient selection

The study group included of 27 histopathologically proven cases of HNSCC and 16 normal controls matching inclusion criteria and giving informed consent [Figure 1]. Treatment naïve cases planned for chemoradiation were recruited for the study from the department of radiotherapy, Dr. Ram Manohar Lohia Institute of Medical Sciences (RMLIMS) Lucknow and tested in molecular lab of the department of Pathology. Institutional Ethics Committee clearance was obtained. Hematoxylin and eosin stained biopsy tissues were analyzed to identify the type of tumor, grade (well, moderately and poorly differentiated). Computed tomography (CT) or Magnetic Resonance Imaging (MRI) was done to assess the location and size of the lesion and lymph node involvement. The clinical investigations, tumor size, node involvement and any evidence of metastasis and clinical TNM staging was done as per the AJCC cancer staging manual for head and neck cancers 8th edition.
Figure 1: Flow chart for case recruitment testing and response assessment

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Sample collection

3.5 mL of peripheral blood was collected from the cases and controls in silica gel vials (B.D Vacutainer, UK) before treatment (S1), 3 weeks after the commencement of chemotherapy (1 week after completion of three doses) (S2) and three months after commencement of Chemotherapy (S3). Serum was separated by centrifugation and stored at –80°C until further processing. Peripheral blood was collected in all 30 pretreatment (S1) and immediate postchemotherapy (S2) samples. Diagnostic evaluation was done in 27 cases and response assessment at 3 months postchemoradiation was done in 24 cases, whereas 3 cases were lost to follow-up.

Response assessment

Evaluation was done according to the WHO response assessment criteria. A case was categorized as a complete responder (CR) on complete disappearance of the lesion, partial responder (PR) with at least 50% decrease in lesion, stable disease when neither PR no PD criteria where met and as a progressive disease (PD) when there was a 25% increase in the lesion with no CR, PR or SD documented before increased disease, or appearance of new lesion(s). For the purpose of statistical analysis CR and PR were clubbed as responders and SD and PD as nonresponders.

CfDNA extraction and quantification

CfDNA DNA isolation was done with 800 μl of serum sample as described by Kumari et al.[16] Quantification of cfDNA level was done by SYBR Green real-time PCR by amplification of β-globin gene compared to standard curve plotted by TaqMan Control Human Genomic DNA (Invitrogen, USA) as described by Kumari et al.[16] A negative control (without DNA) was set up in each run.

Statistical analysis

Statistical analysis was performed using the SPSS (Statistical Package for the Social Sciences) software package, version 21.0. Categorical variables were presented as number and percentage (%) and continuous variables as mean ± SD and median. Quantitative variables were compared using unpaired t test between two groups and one-way analysis of variance (ANOVA) test between three groups. A value of P < 0.05 was considered to be statistically significant.

   Results Top

Demographic and clinicopathological characteristics

The demographic and clinicopathological characteristics of HNSCC cases are summarized in [Table 1]. Smokeless tobacco, gutka, and paan masala addiction was evident in approximately 77.7% cases, whereas smokers (bidi/cigarette) formed 63% of the total study group. Oral cavity sites including tongue, buccal mucosa and lower alveolus was the most common site involved by tumor (59.25%) followed by larynx (22.24%) and oropharynx (18.51%). Histological grade was moderately differentiated in 51.85% and fair number of cases presented with T4 disease stage (55.56%) and clinical lymph node status of N2 (51.85%).
Table 1: Association of cfDNA Level with demographic and clinico.pathological characteristics in HNSCC patients

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CfDNA level in cases and controls

The cfDNA level of cases and controls is summarized in [Table 2]. The mean (±SD) cfDNA levels in normal control and carcinoma patients was significantly different (P < 0.001) [Figure 2]a.
Table 2: cfDNA levels (ng/mL) of cases and control groups

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Figure 2: (a) Box plot showing cfDNA levels of responders vs. nonresponders. Groups were compared by unpaired t test. (b) Distribution of percentage change in the cfDNA levels with treatment

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Association of cfDNA level with demographic and clinico-pathological characteristics in cases with HNSCC

The association of cfDNA levels with demographic and clinico-pathological characteristics of HNSCC patients is summarized in [Table 1]. In HNSCC patients, the mean cfDNA level was lower among the age group of ≤50 years than higher aged patients (>50 years) although the difference was not significant (0.807). The mean cfDNA level was higher in oral cavity cancers (including buccal mucosa, tongue and alveolus) followed by laryngeal tumors and oropharyngeal tumors. The mean cfDNA level was higher in T4 versus T3; however, this difference was not statistically significant (P = 0.250). In addition, mean cfDNA levels were higher in stage IV as compared to stage III; however, the difference was also not statistically significant (P = 0.681).

Diagnostics of cfDNA level in cases with HNSCC

ROC curve analysis was performed to discriminate the HNSCC cases from normal controls. At a cutoff point of ≥150 ng/mL, cfDNA value significantly discriminated HNSCC cases from controls with a 100.00% sensitivity (95%CI = 87.23–100.00) and 100% specificity (95% CI = 79.41–100.00). Calculating cut offs using the criteria of mean ± 2SD of controls the cut off was also calculated at ≥121 ng/mL. At this cut off the sensitivity and specificity was 100%, respectively (data not shown).

Response assessment

Response evaluation was done in 24 patients at 3 months from chemoradiation. Complete response (CR) was observed in four patients (16.6%) with no detectable lesions. In PR 4 patients showed 50% decrease in tumor size on laryngoscopy, whereas two had decreased oral lesions documented in CT scan and on clinical evaluation. Among the 11 cases with PD, 5 presented with a clinically overt lesion extending up to the skin, four had nodal metastasis which was confirmed on biopsy and two showed new metastasis in the lung and bone. Ten cases (42%) were grouped as responders and 14 (58%) as nonresponders. There was no significant difference in demographic and clinicopathological characteristics of responders and nonresponders [Table 2].

CfDNA and response to therapy

The mean (±SD) cfDNA levels of pretreatment (S1), 3 weeks after commencement of chemotherapy (1 week after completion of three doses) (S2) and three months after commencement of chemotherapy (S3) were 1008 ± 694, 1096 ± 972, and 1305 ± 1051 ng/mL, respectively. The mean cfDNA levels in S1, S2 and S3 were higher in the nonresponders as compared to responders though the difference did not reach statistical significance [Table 3]. In responders, percentage decrease in cfDNA levels after chemotherapy was 9.57% at 6 weeks and 29.66% at the end of three months of follow-up, whereas in nonresponders a percentage increase in the cfDNA levels was observed after chemotherapy and was 13.28% at 6 weeks and 24.52% at the end of three months of follow-up [Figure 2]b. Individual trends in 10 responders showed a decreasing trend in, 7 cases, whereas in 3 cases there was an increasing trend. Nonresponders showed an overall increase in the levels of cfDNA [Figure 3].
Table 3: Pretreatment (S1), postchemotherapy (S2), and follow-up cfDNA (S3) values

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Figure 3: Scatter gram showing cfDNA levels of responders in pretreatment, postchemoradiotherapy, and follow-up patients in (a) responders and (b) nonresponders. Box plot showing cfDNA level of responders vs. nonresponders: (c) pretreatment (S1), (d) postchemotherapy (S2), and (e) follow-up (S3)

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   Discussion Top

To the best of our knowledge, this is the first study that has analyzed change in the levels of cfDNA in HNSCC with treatment with the objective to use CfDNA for evaluation of response in cases undergoing chemoradiation. Circulating tumor DNA may serve as an emerging biomarker in HNSCC. Minimally invasive liquid biopsy allows monitoring of the tumor burden during treatment. Often it is difficult to predict response to chemoradiation in the initial stages of treatment where necrosis and edema prevent an accurate assessment of reduction in tumor burden. This study was therefore undertaken to the diagnostic utility of cfDNA in monitoring response to chemoradiation in HNSCC.

Increased levels of cfDNA in other malignancies as compared to healthy controls have been reported, with 18 times higher values in lung,[17],[18],[19] 3.5 times higher in breast,[20] 5 times higher in colon,[21] 3 times higher in prostate[22] and 14 times higher in gall bladder[16] cancer. In our study using a β-globin assay, we have observed that mean level of cfDNA in HNSCC was about 16-fold higher than the healthy control group. Our study group included cases that had a large tumor burden presenting in stages III and IV as per the AJCC criteria. This bias was inevitable as chemoradiation is the treatment of choice in advanced stage HNSCC, whereas cases with resectable disease in stage I and II undergo surgical wide/radical excision. Mazurek et al.[23] have reported higher cfDNA levels in HNSCC patients as compared to age-matched controls. CfDNA levels showed significant association with stage IV and with N2-3 nodal disease (P = 0.015). In our study group mean cfDNA levels was higher in T4 lesions as compared to T3 and stage IV as compared to stage III; however, the difference was statistically not significant (P = 0.259, 0.681). We also attempted to characterize the utility of baseline cfDNA levels for detecting malignancy. ROC analysis showed a sensitivity and specificity of 100% in differentiation of cases from healthy controls.

We have used clinical TNM to determine the prognosis of the patients as cases did not undergo resection. Depth of invasion, pattern of invasion, perineural invasion, lymphovascular-invasion, lymphoid response, and tumor budding that predict lymph node metastasis in OSCC patients used by us in The Aditi-Nuzhat Lymph node Prediction score (ANLPS)[24] could not be evaluated due to histological constraints.

Assessment of tumor burden is a key feature of the clinical evaluation of response taking into accounts both tumor shrinkage (objective response) and disease progression. WHO response evaluation criteria were used in HNSCC cases (ref). As per the WHO criteria total 24 patients were categorized as CR (n = 4), PR (n = 06), PD (n = 11) and Static disease (n = 03). For the purpose of analysis CR and PR were clubbed as responders (n = 10) and PD and SD as nonresponders (n = 14). We have observed a general trend of cfDNA rise with progressive and static disease and decrease with complete and partial response; however, this was not observed in all cases.

The percentage decrease in cfDNA levels after chemo-therapy was 9.57% and 29.66% at three months of follow-up in the responders. Similarly in nonresponders the percentage increase in the cfDNA levels after chemotherapy was 13.28% and 24.52% at three months of follow-up. These findings support the hypothesis of cfDNA may help to predict response; an increasing value following treatment indicating a nonresponder. Changes in CfDNA levels have not been reported in HNSCC cases in terms of response. In a study of Gautschi et al.[25] observed that increasing cfDNA concentrations were significantly associated with different stages, tumor progression after chemotherapy in Non-Small Cell Lung Cancer patients. However, there were no significant decreases in total cfDNA levels in patients who had radiologic response to chemotherapy. Studies by Kumar et al.[26] and Pan et al.[27] have shown significant decreases in total plasma cfDNA in lung cancer cases that had radiologic response to chemotherapy. In both these studies changes in cfDNA levels were not able to differentiate patients with stable disease from those with PD which is in concordance with our study. Localized tumor growth and invasion often damages adjacent normal tissue, resulting in the release of cfDNA. This may explain early increase following RT; however, with a long term follow-up the cf DNA levels would decrease further.

In 6 of our cases there was a rapid increase in cfDNA values after chemo-radiotherapy which eventually decreased in follow-up treatments. This may have occurred due to rapid cell death (apoptosis and necrosis with release of a large amount of tumor cell DNA into the circulation, which later decreased with tumor regression. In three of our cases posttreatment imaging revealed an apparent complete response, but cfDNA remained stable. This may have occurred due to progression of micro metastatic disease that compensated for the volume of local tumor eliminated by radiation therapy. These patients developed metastases in multiple organs several months later, clinically confirming the cfDNA result. Future may look at longer duration of follow-up to establish changing trends with evolution of disease in progression free survival or recurrence and metastasis. Further mutations such as EGFR, KRAS, and TP53 genomic instability and methylation status may be assessed, which were beyond the scope of this study, to specifically define tumor origin of the cfDNA.

This study is limited by the number of cases included. Postsurgical treatment evolution of cfDNA levels may also be interesting to study.

   Conclusion Top

Our study adds to the evidence that cfDNA levels are significantly elevated in patients with HNSCC may reflect tumor progression in HNSCC. CfDNA appears to be promising prospective noninvasive marker to predict response in patients undergoing chemo-radiotherapy.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Nuzhat Husain
Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow - 226 010, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_28_20

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  [Table 1], [Table 2], [Table 3]


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