Interaction of Chemotherapy and Radiotherapy in Altering the Morphology of Subcortical Structures in Patients With Nasopharyngeal Carcinoma

Neuroimaging studies have found signicant structural alterations of the cerebral cortex in patients with nasopharyngeal carcinoma (NPC) following radiotherapy (RT) or concomitant chemoradiotherapy (CCRT), while their effects on the morphology of subcortical structures remain largely unknown. In this study, we investigated the subcortical morphological alterations between three groups: 56 untreated NPC patients (pre-RT group), 37 RT-treated NPC patients (post-RT group), and 108 CCRT-treated NPC patients (post-CCRT group). Using FSL-FIRST, we found that, compared with the pre-RT group, the post-CCRT group exhibited morphological atrophy in the bilateral thalamus, bilateral putamen, left pallidum, and left caudate and morphological ination in the left caudate, while the post-RT group only exhibited morphological atrophy in the bilateral thalamus. We also found a signicant negative correlation between the maximum dosage of RT for temporal lobes and the morphological changes of the bilateral thalamus in treated NPC patients. These results indicated that there may be an interaction between RT and CT that can cause subcortical damage.


Introduction
Nasopharyngeal carcinoma (NPC) is an uncommon cancer arising from the nasopharynx epithelium. It is an endemic disease with a high incidence rate in east and southeast Asia (especially in southern China) (L. L. Tang et al., 2016). To date, radiotherapy (RT) has been considered the primary treatment modality for NPC ( Shi, & Peng, 2012). Moreover, it has been shown that concomitant chemo-radiotherapy (CCRT) is associated with more severe side effects on the brain than RT alone (Wang et al., 2019) or CT alone (Simo et al., 2016).
In recent years, advances in neuroimaging techniques have provided new and exciting avenues for us to noninvasively characterize the macroscopic changes of brain parenchyma in vivo. In NPC, most of previous neuroimaging studies were conducted attempting to examine the effects of RT on brain structure and function. For example, studies using structural MRI data have revealed that compared with patients before RT, patients with NPC have signi cantly decreased gray matter after RT, mainly involving the temporal lobe, hippocampus and the cerebellum, and that the gray matter decreases were dose-and A DTI-based network study found that brain global network properties (e.g. clustering coe cient, normalized characteristic path length, normalized clustering coe cient) decreased in patients with NPC within 6 months after RT, compared with those without RT (Q. Chen et al., 2020). Using resting-state fMRI, some studies have found that the cerebellum and some regions of default mode network, including the hippocampus, temporal lobe, and posterior cingulate cortex (PCC), were associated with altered functional connectivity to other regions in (S. C. Chen  Compared with patients with NPC who did not receive RT, RT-treated NPC patients had aberrant regional homogeneity (ReHo) values in cerebellum, temporal lobe and insula and aberrant ReHo values had different dynamic changes patterns over time (Yang et al., 2019). In contrast, less attention has been paid to the effect of CT on the brain. Using multimodal MRI data, our recent study investigated the effects of RT and CT on the cortical morphology and functional connectivity in patients with NPC, and found that chemotherapy potentially facilitated the occurrence of radiation encephalopathy in treated NPC patients (Y. Zhang et al., 2019). One question remains, however, as to whether RT and CT could affect the morphology of subcortical structures in patients with NPC.
In the present study, we aimed to investigate the effects of CT and RT on the morphology of subcortical structures in patients with NPC. Speci cally, we used structural MRI data to characterize the shape abnormalities of subcortical nuclei in a cohort of patients with NPC who had undergone RT (Post-RT) or CCRT (Post-CCRT), as compared to untreated NPC patients (Pre-RT). We hypothesized that, compared with Pre-RT group, Post-RT group would show signi cant abnormalities in the shape of the subcortical nuclei including the thalamus, while Post-CCRT group would show more extensive changes in the shape of the subcortical nuclei including the thalamus.

Subjects
This retrospective cross-sectional study included 201 patients with pathologically diagnosed NPC. They were divided into three groups according to the difference in treatment methods: the Pre-RT group (56 NPC patients who had not received treatment), the Post-RT group (37 NPC patients who had received RT) and the Post-CCRT group (108 NPC patients who had received CCRT)(Y. Zhang et al., 2019). To measure the effect of RT or CCRT on the structure of subcortical nuclei, other possible confounding factors (such as age, sex, time intervals between RT and sMRI examinations, RT technology, and maximum RT dosage to the temporal lobes) had to be evenly distributed among the groups. The inclusion criteria for all patients in this study were as follows: (1) Pathologically con rmed NPC patients; (2) normal-appearing brain parenchyma on MRI; (3) right-handedness; (4) more than 6 years of education; and (5) an age range from 20 to 60 years. The exclusion criteria for all patients in this study were as follows: (1) brain parenchymal invasion, (2) brain tumor, (3) prior substantial head trauma or surgery, (4) neurological or psychiatric illness, (5) alcoholism or drug abuse, (6) any other major intracranial disease(Zhang et al.,

2018).
For each NPC patient, the following clinical data were available: the Karnofsky Performance Status (KPS) score, the main side of NPC, clinical stage, RT techniques, maximum dosage of RT to the temporal lobes, Page 4/14 time intervals between RT and MRI examination, detailed information on the chemotherapy agents. All patients receiving RT were given Intensity-modulated radiation therapy (IMRT) or conventional twodimensional radiotherapy (2D-CRT), as shown in Table 1 Additional adjuvant chemotherapy regimen was 3 cycles of cisplatin (80 mg/m 2 intravenously on day 1) and uorouracil (4 g/m 2 in continuous intravenous infusion over 120 h). Additional neoadjuvant chemotherapy regimen was two cycles of cisplatin (80 mg/m 2 intravenously on day 1) and uorouracil (4 g/m 2 in continuous intravenous infusion over 120 h). This study was approved by the Medical Research Ethics Committee of Xiangya Hospital, Central South University and performed in line with the principles of the Declaration of Helsinki and its later amendments. Written informed consent was obtained from all subjects.  (2) The superscripts a and b denote data loss in 5 and 9 subjects respectively. *P < 0.05.

MRI Acquisitions
All MRI data, including axial T1-weighted images, T2-weighted images, and T2-weighted uid attenuated The speci c process of constructing the model is as follows. First, linear subcortical registration was applied in all images to achieve the correct alignment between the model and images. Second, while maintaining cross-subject vertex correspondence, a deformable 3D mesh was used to t the training images to obtain manually labelled information, and then iteratively update its vertex position. Third, appearance was modeled with normalized intensity, which was to sample each training intensity image along the surface normal of the mesh vertex. Finally, the variation in the mesh tting process for each image was modelled by vertex coordinates and intensity samples. Before the new image was tted with the model, the model was registered into the native space using inverse transformation, and then the optimal tting was obtained by maximizing the posterior probability. In this way, for each subject, we can obtain a surface mesh consisting of vertices and triangles. Because the subcortical surface meshes of all subjects were isomorphic, the comparisons between groups could be performed by examining group differences in the spatial location of each vertex. Although the vertices were in correspondence, the surface mesh was not in standard space, but in its native space. Before investigating group differences, the pose differences were removed by rigid alignment of the mean surface mesh in standard space, which meant that the sum-of-squares between a subject's surface mesh and the mean surface mesh were minimized. After that, multivariate F-tests were performed for each vertex separately to detect between-group localized shape difference. Vertex-wise correlation analysis was also performed between the shape of subcortical nuclei and the maximum dosage of RT for temporal lobes in treated NPC patients. False discovery rate (FDR) theory was used to correct for multiple comparisons.

Results
Comparisons of the vertex locations between the Pre-RT group and the Post-RT group showed that Post-RT group exhibited signi cant regional atrophy in the medial posterior segment of the bilateral thalamus (Fig. 1).
Similarly, compared with the Pre-RT group, the medial posterior segment of the bilateral putamen, bilateral thalamus, left pallidum, and the left caudate were substantially atrophied in the Post-CCRT group. Further, the Post-CCRT group showed a signi cant regional in ation in the lateral anterior segment of the left pallidum (Fig. 2).
In the post-treatment patient groups (including post-RT and post-CCRT groups), there was a signi cant negative correlation between the maximum dosage of RT for temporal lobes and the morphological changes of the superior segment of the bilateral thalamus (Fig. 3).

Discussion
In this study, we examined the subcortical shape abnormalities in patients with NPC who underwent RT or CCRT, compared with untreated NPC patients. Our main ndings can be summarized as follows. First, compared with the pre-RT group, patients in the post-RT group showed signi cant regional atrophy in the medial posterior segment of the bilateral thalamus. Second, compared with the pre-RT group, patients in the post-CCRT group showed signi cant regional atrophy in the medial posterior segment of the bilateral thalamus, bilateral putamen, left pallidum, and left caudate, and signi cant regional in ation in the lateral anterior segment of the left pallidum. Finally, in treated NPC patients, the maximum dosage of RT for temporal lobes was negatively correlated with the morphological changes of the superior segment of the bilateral thalamus. Taken together, these ndings suggest that RT and CT may interact in altering the morphology of subcortical structures in NPC. Of note, the thalamic area identi ed with signi cant regional atrophy in the post-CCRT group was much larger than that identi ed in the post-RT group, suggesting possible interactions between RT and CT. In addition, using the pooled data of the post-RT and post-CCRT groups, we also found a signi cant negative correlation between the morphological changes of the thalamus and the maximum dose of RT for temporal lobe, suggesting that the thalamic abnormalities could arise from the direct effects of RT or from abnormal afferent inputs from RT-lesioned temporal lobe via the reciprocal thalamo-cortical connections ( . Compared with the pre-RT group, the present study also found signi cant regional in ation in the lateral anterior segment of the left pallidum in the post-CCRT group. The nding of concurrent atrophy and in ation in the pallidum may indicate functional heterogeneity of different nuclei of the pallidum. However, the underlying mechanism of such alteration remains unclear and requires further investigation. Collectively, given that no signi cant shape abnormalities in putamen, pallidum and caudate were found in the post-RT group, we therefore speculate that the shape alterations of these subcortical structures may primarily occur as a result of CT. The exact pathophysiological mechanism that leads to these signi cantly morphological alterations of subcortical structures remains unclear. It is possible that multiple mechanisms, including DNA damage, apoptosis, in ammatory response and oxidative stress, vessel abnormity, and destruction of the bloodbrain barrier (BBB), are involved in the development of the brain injury after RT or CT(Lumniczky, Szatmari, & Safrany, 2017; X. Ren et al., 2019). In fact, in the studies of the mechanism of RT or CT, the in ammatory response mediated by reactive microglia has always been of particular interest. More speci cally, cellular debris from RT-or CT-damaged neuron could activate microglia, which in turn could release pro-in ammatory cytokines and other neurotoxic factors that damage neurons (Block, Zecca, & Hong, 2007). Similarly, there is evidence that reactive astrocytes induced by reactive microglia are also neurotoxic (Liddelow et al., 2017). Besides, increased BBB permeability caused by CCRT also seems to be able to indirectly exacerbate existing levels of in ammatory response (Zlokovic, 2008). However, we cannot exclude the involvement of other mechanisms in causing the morphologic alterations in treated NPC patients.
Functionally, the thalamus, as a hub, plays a particularly critical role in controlling the transmission and integration of information ow in the cortical and subcortical networks, which is the basis for its support of related cognitive functions (Hwang et

Conclusion
In this study, we investigated subcortical morphological alterations in the post-RT and post-CCRT groups compared with the pre-RT group. We found that, compared with the pre-RT group, the post-CCRT group exhibited morphological alterations in the bilateral thalamus, bilateral putamen, left pallidum, and left caudate while the post-RT group was only in the bilateral thalamus, suggesting that there may be an interaction between RT and CT for subcortical structural injury. Our ndings provide new insights into the optimization of treatment options for NPC.

Declarations
Con ict of interest statement The authors declare that they have no con ict of interest.

Ethics approval
This study was approved by the Medical Research Ethics Committee of Xiangya Hospital, Central South University and performed in line with the principles of the Declaration of Helsinki and its later amendments.

Consent to participate
Written informed consent was obtained from all subjects.

Consent for publication
Written informed consent for publication was obtained from all participants Availability of data and material The datasets generated during the current study are available from the corresponding author upon reasonable request from a quali ed investigator.

Code availability
Not applicable Authors' contributions Yuanchao Zhang and You-ming Zhang designed and organized the study. You-ming Zhang and Wei-hua Liao contributed to the data collection. Jian-ming Gao and Li Li analyzed the data together. Yuanchao Zhang prepared the gures. Yuanchao Zhang and Feibiao Nan drafted and revised the manuscript. All authors approved the submitted version.