Evaluation of the residual lung function after thoracoscopic segmentectomy compared with lobectomy

Accepted Manuscript Evaluation of the residual lung function after thoracoscopic segmentectomy compared with lobectomy Shinya Tane, MD, PhD, Wataru Nishio, MD, PhD, Yuki Nishioka, MD, Hiroki Tanaka, MD, Hiroyuki Ogawa, MD, PhD, Yoshitaka Kitamura, MD, Daisuke Takenaka, MD, PhD, Masahiro Yoshimura, MD, PhD PII:

S0003-4975(19)31005-7

DOI:

https://doi.org/10.1016/j.athoracsur.2019.05.052

Reference:

ATS 32789

To appear in:

The Annals of Thoracic Surgery

Received Date: 23 October 2018 Revised Date:

1 May 2019

Accepted Date: 20 May 2019

Please cite this article as: Tane S, Nishio W, Nishioka Y, Tanaka H, Ogawa H, Kitamura Y, Takenaka D, Yoshimura M, Evaluation of the residual lung function after thoracoscopic segmentectomy compared with lobectomy, The Annals of Thoracic Surgery (2019), doi: https://doi.org/10.1016/ j.athoracsur.2019.05.052. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Evaluation of the residual lung function after thoracoscopic segmentectomy

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compared with lobectomy Running head: Pulmonary function after segmentectomy

Shinya Tane1), MD, PhD, Wataru Nishio1),MD, PhD, Yuki Nishioka1),MD, Hiroki Tanaka1),MD,

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Hiroyuki Ogawa1), MD, PhD, Yoshitaka Kitamura1) MD,

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Daisuke Takenaka2), MD, PhD, Masahiro Yoshimura1) MD, PhD

1) Division of Chest Surgery, Hyogo Cancer Center

2) Division of Diagnostic Radiology, Hyogo Cancer Center 1), 2) 13-70, Kitaoji-cho, Akashi City, Japan

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*Address for Correspondence: Wataru Nishio, MD, PhD. Division of Chest Surgery, Hyogo Cancer Center

13-70, kitaoji-cho, Akashi city, Japan, Zip code 673-0021

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E-mail: [email protected]

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Classifications: Computed tomography/CAT scan/CT scan; Pulmonary function; Thoracoscopy/VATS

Word count: 4456 words.

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Abstract Background: Segmentectomy has shown a beneficial effect on preserving the lung function after resection. However, the preservable lung volume and changes after thoracoscopic segmentectomy remain unknown.

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We compared the residual lung function after thoracoscopic segmentectomy and lobectomy, using a novel three-dimensional computed tomography (3DCT)-based volumetric method.

Methods: Seventy-four patients who received thoracoscopic segmentectomy were matched to the 74

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patients who received thoracoscopic lobectomy. Spirometry and computed tomography were performed before and six months after resection, and the ipsilateral residual preserved and non-operated lobe volume,

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and the contralateral lung volume were calculated using 3D-CT. The percentage of actual/predicted regional forced expiratory volume in 1 second (the preservation rate) in each lobe (measured by volumetry and spirometry), was compared with the extent of resection and procedural difficulty (typical or atypical segmentectomy).

Results: The postoperative lung function was significantly more well-preserved in segmentectomy than in

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lobectomy. After segmentectomy and lobectomy, the regional forced expiratory volume in 1 second of the ipsilateral unaffected lobe were increased in comparison to the preoperative value, while that of the residual lobe rescued by segmentectomy was decreased. The preservation rates of the residual and

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unaffected lobes were inversely and positively corelated, respectively, with the extent of the resected segment. The preservation rates of the residual lobe after typical or atypical segmentectomy were not

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significantly different.

Conclusions: Although the decrease in the actual lung function of the residual lobe was greater than predicted and increased with increasing extent of resection, segmentectomy preserved the whole lung function better than lobectomy.

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During the past two decades, segmentectomy has evolved as an established treatment for peripheral early non-small-cell lung cancer. Segmentectomy has been thought to be advantageous because it preserves the lung function, and allows patients to benefit from future resection of metachronous lung

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cancer (1). Although the indications should be carefully taken into account for this type of resection for preventing locoregional recurrence in patients with non-small-cell lung cancer (2), segmentectomy performed by either open or thoracoscopic approaches has gained popularity among surgeons over the

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years. Thus, aside from oncologic compromise, it is important to precisely define the functional status, as this lung sparing benefit is the biggest strength of segmentectomy. However, how the lung function of

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each lobe, including the residual lobe rescued by segmentectomy, can be preserved and are changed after segmentectomy compared to lobectomy has not been investigated.

Thoracoscopic segmentectomy was introduced in our institution in 2012 and is even applied in cases of complex segmentectomy, such as posterior basal segmentectomy which requires high levels of training and expertise (3). In such cases, three-dimensional computed tomography (3D-CT) images are

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needed to identify the intersegmental plane during the operation. Besides the assessment of the preoperative simulation of the vasculature, 3D-CT reconstruction is a reliable tool for predicting the postoperative lung function (4). A volumetric analysis via quantitative 3D-CT imaging is fast, accurate,

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and technically simple and yields a more accurate prediction in comparison to the conventional segment counting method (5). This method enables estimating the functional contribution of a specific lung lobe. In

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the present study, this method was applied to calculate the volume of lobe, and we compared the residual lung function after thoracoscopic segmentectomy and lobectomy and analyzed the changes in the residual lobe function after thoracoscopic segmentectomy.

Material and Methods Patients

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We examined 74 consecutive patients who underwent thoracoscopic segmentectomy at Hyogo Cancer Center between January 2012 and December 2017. These patients were paired with another 74 patients who underwent thoracoscopic lobectomy during the same period, based on their sex, age, smoking history,

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tumor location and pulmonary function, using propensity score matching (Table 1). Our basic selection criteria for thoracoscopic segmentectomy were as follows: (1) patients with peripheral small-sized nonsmall-cell lung cancer with ground glass opacity indicating clinical T1a/bN0M0 based on the eighth

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edition Lung Cancer Stage Classification, (2) patients with compromised resection who were considered poor candidates for lobectomy because of their limited cardiopulmonary reserve and (3) patients with

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pulmonary metastases or benign lesions.

All of the patients who received thoracoscopic segmentectomy and lobectomy underwent computed tomography and a lung function test before and six months after the operation. Patients treated with induction chemoradiotherapy or who had a history of lung surgery were excluded from this study. The Hyogo Cancer Center institutional review board approved the study and each participant provided their

Operative procedure

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informed consent.

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All lobectomy and segmentectomy procedures were performed by the thoracoscopic approach. In our institution, thoracoscopic segmentectomy is performed with the aid of simulated images, as we described

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previously (4). A representative image is shown in Figure 1B. Briefly, the operation was generally performed through four port sites (2–4 cm) without rib spreading. We first ligated and resected the segmental artery and detached the bronchus to allow it to be dissected with a stapler after inflation of the lung on the operating side. The inflation–deflation line gradually became clear when the lung was recollapsed. The intersegmental veins were identified with reference to the preoperative simulation and the inflation–deflation line, and the pulmonary parenchyma was dissected from the hilum towards the periphery with electrocautery and a stapling device—so that the surgical margin from the tumor was sufficiently secured, while the inter-segmental veins were preserved. 4

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CT and three-dimensional lung image construction All plain chest CT examinations were performed with 16- or 80-multidetector row CT (MDCT) scanners

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(Aquilion 16 or Prime, Toshiba Medical Systems, Otawara Japan). The entire lung was scanned from the lung apex to the diaphragm during a single breath hold at end-inspiration. The scan parameters of the MDCT examination were as follows: 130kVp, 150mAs, collimation 1mm×16, rotation 0.5s, or 120kVp,

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390-500mAs, collimation 0.5mm×80, rotation 0.35s, 512×512 matrix, FOV 320mm, reconstruction 1mm/1mm. After the initial non-contrast-enhanced-MDCT examination, 100 ml of iodine contrast

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material (Iopamiron 300; Bayer AG, Leverkusen, Germany) was administered to each patient. Three-dimensional imaging was reconstructed from the CT data using the SYNAPSE VINCENT software program (Fujifilm Corporation, Tokyo, Japan). Besides the capability of reconstructing 3D images of the pulmonary vessels and bronchial trees, this software program enables the calculation of the bronchial ventilation area using an algorithm based on the direction and diameter of the bronchi (6). This function

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allowed for the measurement of the volume of the resected lobe, the ipsilateral unaffected lobe and the contralateral lung using the preoperative CT data (Figure 1C, 1D). These volumes after thoracoscopic

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segmentectomy were also calculated from the postoperative CT data.

The image interpretation and data analysis

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The regional FEV1.0 was measured from the volumetric and spirometric parameters using the following formula:

FEV1.0 × (the regional subjected lung volume / the whole lung volume). The predicted FEV1.0 of the resected segments, the ipsilateral preserved segments in the residual lobe, the ipsilateral unaffected lobe, and the contralateral lung were calculated using the following formula: Preoperative FEV1.0 × (the subjected lung volume / whole lung volume measured from preoperative 3D5

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CT). Similarly, the actual FEV1.0 of the residual preserved lobe, the ipsilateral unaffected lobe, and the

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contralateral lung were calculated using the following formula: Actual FEV1.0 × (the subjected lung volume / whole lung volume measured from postoperative 3D-CT).

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The percentage of actual / predicted FEV1.0 of each lobe was defined as the preservation rate.

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Statistical analysis

All statistical analyses were performed using the JMP software program (Version 8, SPSS Inc., Chicago, IL, USA). All values in the text and tables were expressed as the mean ± standard error of the mean (SEM).

For the clinical variables of the operation time, blood loss, the extent of resection (1-segment or >1-

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segment) and procedural difficulty (typical or atypical segmentectomy), the difference between 2 groups with or without propensity score matching was analyzed by the 2-tailed Student’s t-test or the MannWhitney U test.

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A linear regression analysis was performed to test the relationship between the continuous variables. P

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values of < 0.05 were considered to indicate statistical significance.

Results

Operative results

The patient characteristics, and the results of the overall operation and resected segment are summarized in Table 1 and 2. The average operation time and the amount of bleeding were 184 ± 21 minutes and 45.3 ± 5.2 ml in segmentectomy, and 191 ± 6 minutes and 63.2 ± 8.0 ml in lobectomy, respectively.

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Regarding postoperative complications, three patients presented with prolonged air leak, one patient presented with empyema, one presented with arterial fibrillation, and one presented with pneumonia in segmentectomy, while six patients presented with prolonged air leak, one presented with both hemothorax

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and arterial fibrillation, and one with arterial fibrillation in lobectomy. The complication rate was not markedly different between the 2 groups (8.1% versus 10.8%, p = 0.41). No postoperative deaths occurred

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in these populations.

The postoperative lung function with segmentectomy and lobectomy

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In the segmentectomy group, the average of preoperative and postoperative FEV1.0 was 2.24 ± 0.07 L and 2.04 ± 0.06 L, respectively. In the lobectomy group, the average of preoperative and postoperative FEV1.0 values were 2.25 ± 0.07 L and 1.83 ± 0.06 L, respectively. The percentage of post-/preoperative FEV1.0 was 81.7% in the lobectomy group and 91.9% in the segmentectomy group, indicating that segmentectomy significantly preserved the postoperative lung function over lobectomy (p < 0.01).

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Figure 2 shows the change in the pulmonary function with segmentectomy and lobectomy. After thoracoscopic segmentectomy, the regional FEV1.0 of the ipsilateral unaffected lobe was significantly increased compared to the preoperative value (p=0.02). In contrast, the regional FEV1.0 of the residual

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lobe rescued by segmentectomy was decreased significantly (p<0.01). After thoracoscopic lobectomy, the tendency of the postoperative functional change was similar to that in segmentectomy. The regional

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FEV1.0 of the ipsilateral unaffected lobe was increased compared to the preoperative value (p = 0.01). However, one lobe was completely resected after lobectomy (while some segments were spared after segmentectomy), and this could explain the difference observed in the postoperative lung function between segmentectomy and lobectomy. Next, we focused on the left upper lobe, as the left upper lobe is one of the biggest lobes, and segmentectomies such as upper division and lingular segmentectomy have been well established by thoracic surgeons worldwide. The percentage of post-/preoperative FEV1.0 was 97% in left S1+2, 82% in upper division, 86% in lingular segmentectomy, and 73% in left upper lobectomy. In all cases, the 7

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percentage of post-/preoperative FEV1.0 in segmentectomy were significantly higher than in left upper lobectomy (p<0.01, respectively), indicating that segmentectomy could be a meaningful approach in terms

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of preserving the postoperative lung function (Figure 3).

The residual lung function after segmentectomy

In the segmentectomy group, the percentage of post-/preoperative FEV1.0 in the 1-segment group was

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significantly higher than that in the >1-segment group (95% versus 87%; p<0.01) (Figure 4A). In addition, the regional FEV1.0 of the residual lobe in the 1-segment group was significantly higher than that in the

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>1-segment group (0.36L versus 0.28L, p<0.01) (Figure 4B).

The preservation rate of the residual lobe rescued by segmentectomy in the >1-segment group was lower than that of the 1-segment group (66.1% versus 84.6%; p<0.01) (Figure 5A). The preservation rate of the residual lobe was inversely correlated with the extent of the resected segment (r = 0.55, p < 0.01) (Figure 5B). These results indicated that segmentectomy decreases the actual lung function of the residual lobe

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rather than the preoperatively predicted value, as the extent of the resected segment increases. In contrast, the preservation rate of the unaffected lobe in the >1-segment group was higher than that in the 1-segment group (130.2% versus 110.4%; p<0.01) (Figure 5C). The preoperative FEV1.0/FVC

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showed no significant difference between the 2 groups (73.2% versus 73.6%; p=0.91), suggesting that postoperative compensatory lung growth might not be related to the preoperative pulmonary function. The

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preservation rate of the residual lobe was inversely correlated with the extent of the resected segment (r = 0.47, p < 0.01) (Figure 5D). According to the extent of the resected segment, the unaffected ipsilateral lobe was inflated, which limited the expansion of the residual lobe.

The residual lung function according to the procedure The preservation rate of the residual lobe was 90.7% ± 3.8% in the bilateral S6, 82.8% ± 5.4% in the right S2, 82.9% ± 4.8% in the left S1+2, 63.4% ± 3.4% in the left upper division, 78.4% ± 4,4% in the lingular segment, and 70.4% ± 3.4% in posterior basal segmentectomy such as S9+10 and S10. When assessment 8

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was limited to the left lower lobe, the preservation rate of left S1+2 and upper division segmentectomy was significantly lower in comparison to lingular segmentectomy (p = 0.03).Next, all cases were divided into typical (n=58) and atypical segmentectomy (n=17) groups. Typical segmentectomy included bilateral

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S6, left upper division, and left lingular segmentectomies, and all other types of segmentectomies were defined as atypical segmentectomy. The preservation rate of the residual lobe in the typical and atypical segmentectomy groups did not differ to a statistically significant extent (75.5% versus 75.9%; p=0.930)

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(Figure 6), indicating that, even in atypical segmentectomy requiring a difficult procedure, our method of

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thoracoscopic segmentectomy is feasible for preserving the postoperative lung function.

Comment

One of the suggested benefits of segmentectomy is the relative preservation of the vital pulmonary function. However, previous studies comparing segmentectomy and lobectomy have reported some conflicting results regarding the preservation of the postoperative lung function (7, 8). These conflicting

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results among these reports may depend on the operative procedure (thoracotomy versus thoracoscopic surgery), the extent of the resected segment, the method of examining the postoperative lung function and the timing of the examination. In the present study, even after adjustment, patients who underwent

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thoracoscopic segmentectomy demonstrated a greater pulmonary function in comparison to those who underwent thoracoscopic lobectomy, which is the similar to the result of a recent report (9). Total

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thoracoscopic surgery has an advantage over thoracotomy for preserving the postoperative lung function because this approach facilitates the preservation of respiratory muscle such as the intercostal muscle. Moreover, recent advances in operative techniques, instruments, and perioperative management might contribute to improving the postoperative lung function. However, the method of preserving the lung function of each lobe and the changes that take place after thoracoscopic segmentectomy have not been investigated. To the best of our knowledge, our study is the first case to examine the postoperative respiratory function after total thoracoscopic segmentectomy using CT volumetry.

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Various methods such as conventional segment-counting, CT, and lung perfusion scintigraphy with a fusion of single-photon emission computed tomography (SPECT) have been used to investigate the regional pulmonary function. Among these, a volumetric analysis via quantitative CT imaging is fast,

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technically simple and is performed by analyzing available data from chest CT scans, which is available in any case, since it is routinely performed in all cases of lung cancer. CT volumetry was able to predict the residual pulmonary function more accurately than the segment-counting method. The predictive capability

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of quantitative CT imaging has also been compared to perfusion scanning, and both methods yielded similar results, with small differences either in favor of perfusion scanning or quantitative CT imaging (10,

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11).

Since we introduced total thoracoscopic segmentectomy in 2012, we have used a 3D-CT software program for virtual simulation in all thoracoscopic segmentectomy cases. It has been demonstrated that 3D-CT simulation can visualize the intra-segmental and inter-segmental veins, the target segmental bronchi and the pulmonary arteries, which makes it easy to detect the intersegmental plane (12). An

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accurate understanding of the anatomy of the intersegmental plane is also useful for confirming the tumor location within an anatomic segment and predicting the surgical margins. Thus, besides calculating the lung volume, the use of 3D-CT is beneficial for thoracic surgeons to perform appropriate anatomical

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segmentectomy and curative resection (12, 13).

In this study, the actual lung function of the residual lobe was lower than the predicted value. Three

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main reasons have been considered for this result. First, when the pulmonary parenchyma was dissected from the hilum towards the periphery preserving the intersegmental vein, the cutting of the intersegmental plane was performed using both an electrocautery device and a stapler, which might have restricted the reexpansion of the preserved segments. Moreover, when cutting the intersegmental plane, we dissected the peripheral parenchyma into the rescued segment to secure a tumor-free margin. These surgical procedures would decrease the actual lung function of the residual lobe. Second, anatomical displacement of the remaining lobe after segmentectomy could influence the preservation rate of the residual lobe. In this study, upper division segmentectomy spared the lower 10

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residual lobe function to a greater extent than was predicted preoperatively, probably because the upward displacement of the lingula could cause an angulation of the lingular bronchus, leading to disturb the recovery of FEV1.0. This is consistent with the change in the bronchial angle through the upward

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displacement of the remaining lung and the diaphragm following right upper lobectomy, which can lead to deformity of the residual bronchus, resulting in a decreased postoperative lung function (14). In contrast, the residual lobe function was the most preserved after S6 segmentectomy, suggesting that the shape of

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the preserved segments (Basal segment) may be amenable to inflation without anatomical displacement after resection.

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Third, the space left by resected segment was mainly filled with the ipsilateral non-operated lobes, meaning that the lung function of the non-operated lobe will increase after segmentectomy. The phenomenon is called compensatory lung growth; however, the extent of compensatory lung growth after lung resection is not fully understood. Several reports using an experimental pneumonectomy model have suggested that increased mechanical stress and strain on the remaining lung induces adaptive responses to

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augment oxygen transport, including regenerative growth of the alveolar tissue when the strain exceeds a critical threshold after lung resection (15). In this study, the larger extent of resection of the segment, the greater the increase in the lung function of the ipsilateral non-affected lobe. Thus, besides preserving the

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segment in the operated lobe, segmentectomy has the potential to increase the non-affected lobe function through compensatory lung growth. Further studies should be performed to clarify this physiological

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mechanism after thoracoscopic segmentectomy. Regarding the influence of technical difficulty on the postoperative lung function, the preservation rate of the residual lobe in the typical and atypical segmentectomy groups did not differ to a statistically significant extent. The technical difficulty depends on the resected segment. For instance, S6 segmentectomy, which has single intersegmental dissection surface, would be considered easy segmentectomy because the dissection is simply performed along the single intersegmental surface. On the other hand, S10 segmentectomy, which involves dissection at multiple surfaces at an acute angle, and in which the point of divergence of the bronchus and vessels is located deeply, is considered to be difficult 11

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segmentectomy. In this study, all the segmentectomy procedures were divided into typical and atypical segmentectomy according to the procedural difficulty, and the preservation rate of the operated lobe was compared. As a result, the postoperative preservation rate of atypical segmentectomy did not differ from

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that of typical segmentectomy, demonstrating that our technical procedure seemed to be feasible in terms of preserving the postoperative lung function.

The present study is associated with several limitations. First, we used FEV1.0 as the pulmonary

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functional parameter, since a few guidelines proposed by the American College of Chest Physicians recommended the use of the FEV1.0 to estimate the amount of functional lung tissue that would be lost

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with surgical resection in NSCLC patients with a preoperative FEV1.0 value in order to predict the postoperative lung function (16). However, other parameters such as forced volume capacity and oxygen diffusing capacity were not compared.Second, we evaluated the postoperative lung function only at six months after lung resection. A chronological evaluation at later points such as one or five years will be needed to confirm the long-term functional benefit of segmentectomy over lobectomy. Third, although we

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evaluated the postoperative lung function separately in each type of segmentectomy, the number and size of each type of segmentectomy were too small. A larger case series and comparative study should be performed to determine the real significance of thoracoscopic segmentectomy for preserving the

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postoperative pulmonary function.

In summary, despite the fact that the actual lung function of the residual lobe was less well-

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preserved than had been predicted before surgery and that the reduction increased with the extent of resection, thoracoscopic segmentectomy helped preserve the pulmonary function by sparing some segments of lung tissue that would have otherwise been removed by lobectomy. This phenomenon was seen irrespective of the technical difficulty, indicating that our thoracoscopic segmentectomy procedure is feasible for preserving the postoperative lung function.

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Table 1 Characteristics

Segmentectomy

Lobectomy

Number

74

74

Age in year

70.3

69.7

Male / Female

45 / 29

45 / 29

Smoking history (Yes/No)

40 / 33

40 / 33

Lung cancer

72

74

Benign diseases

2

0

FVC

3.17 ± 0.10

3.08 ± 0.10

0.85

FEV1.0

2.24 ± 0.07

2.25 ± 0.07

0.82

Operation time (min)

184 ± 21

191 ± 6

0.39

Amount of bleeding (ml)

45.3 ± 5.2

63.2 ± 8.0

0.04

Left upper lobe Left lower lobe

(%)

7

15

15

38

38

14

0.15

14

complications 6 (8.1)

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Postoperative

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Right lower lobe

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Resected Location Right upper lobe

0.65

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Diagnosis

p-value

8 (10.8)

Prolonged air leak *

3

6

Pneumonia

1

0

Empyema

1

0

Hemothorax

0

1

Arterial fibrillation

1

2

15

0.41

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FVC, Forced Expiratory capacity FEV1.0, forced expiratory volume in 1.0 second

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*Prolonged air leak was defined as air leak lasting longer than 7 days or the use of pleurodesis.

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Table 2. Resected Segments Resected segment

Data (n = 74)

S3

1

S6

8

S8

2

S8+9

2

S9+10

2

S10

1

Left S1+2

7

S1+2+3

22

S6

8 10 1 1 2

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S9+10

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S8

S10

1

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S3+4+5 S4+5

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6

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S2

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Right

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Figure legends

Figure 1. A representative case of right S2 segmentectomy.

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A. The computed tomography findings showing the tumor (adenocarcinoma) located in the right S2 B. An interoperative view in which the intersegmental plane is dissected while preserving the intersegmental vein between S2 and S3.

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C,D. Three dimensional volumetric images of the right lung before and after S2 segmentectomy.

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Figure 2.

A change in the regional forced expiratory volume in 1.0 second in each lobe before and after thoracoscopic segmentectomy and lobectomy.

Figure 3.

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A comparison of the percentage of postoperative forced expiratory volume in 1.0 second divided by preoperative forced expiratory volume in 1.0 second among left S1+2, upper division, lingular

Figure 4.

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segmentectomy and left upper lobectomy.

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A. A comparison of the percentage of postoperative forced expiratory volume in 1.0 second divided by preoperative forced expiratory volume in 1.0 second between in the 1-segment and >1-segment groups. B. A comparison of the regional forced expiratory volume in 1.0 second of the residual lobe between in the 1-segment and >1-segment groups.

Figure 5. A. A comparison of the preservation rate of the residual lobe in the 1-segment and >1-segment groups. B. The relationship between the extent of the resected area and the preservation rate of the residual lobe. 18

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C. A comparison of the preservation rate of the ipsilateral unaffected lobe in the 1-segment and >1segment groups. D. The relationship between the extent of the resected area and the preservation rate of the ipsilateral

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unaffected lobe.

Figure 6. A comparison of the preservation rate of the residual lobe in the typical and atypical

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segmentectomy groups.

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SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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