E-ISSN 2228-8082

Siriraj Medical Journal

The world-leading biomedical science of Thailand

Volume 74, Number 1, January 2022

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Thai Association for Gastrointestinal Endoscopy

International Association of Surgeons Gastroenterologists & Oncologists Thailand Chapter

https://he02.tci-thaijo.org/index.php/sirirajmedj/index

E-mail: sijournal92@gmail.com

MONTHLY

E-ISSN 2228-8082

SMJ

Volume 74, Number 1, January 2022

Siriraj Medical Journal

The world-leading biomedical science of Thailand

ORIGINAL ARTICLE REVIEW ARTICLE

By Doonyaporn Wongsawaeng, et al.

Indexed by

Thai Association for Gastrointestinal Endoscopy

International Association of Surgeons Gastroenterologists & Oncologists Thailand Chapter

https://he02.tci-thaijo.org/index.php/sirirajmedj/index

E-mail: sijournal92@gmail.com

Siriraj Medical Journal SMJ

Volume 74, Number 1, January 2022

ORIGINAL ARTICLE

1 Goal-Directed Therapy to Improve Gross Motor Function and the Quality of Life of Children with Cerebral Palsy: A Randomized Controlled Trial

Suwannika Palee, et al.

11 Effectiveness of Ayurved Siriraj Prasa-Nam-Nom Recipe on Breast Milk Volume in Early Postpartum Women: A Randomized, Double-Blind, Placebo-Controlled Trial Pharuhas Chanprapaph, et al.

19 Clinical Characteristics of Female Patterned Hair Loss in Patients Attending Hair Clinic in Thailand

Warayuwadee Amornpinyo, et al.

27 Aflibercept as Adjunctive Treatment for Filtration Surgery in Neovascular Glaucoma

Naris Kitnarong, et al.

34 A Study of Early Parenteral Nutritional Support and Factors Associated with Clinical Outcomes in Major Pediatric Burn Patients

Weerawat Seubmora, et al.

40 Factors Predicting Survival in Ruptured Hepatocellular Carcinoma Treated with Surgical Resection

Charnwit Assawasirisin, et al.

48 Prevalence and Factors Associated with the Loss of PTEN Expression in Patients with Lung Cancer

Thiva Kiatpanabhikul, et al.

REVIEW ARTICLE

64 A Scenario of COVID-19 Disease on Mental Health Among the General Age-groups

Somadatta Das, et al.

68 Cardiovascular Presentation in Pheochromocytoma: What We Should be Aware

Rizki Amalia, et al.

SMJ

SIRIRAJ MEDICAL JOURNAL

www.smj.si.mahidol.ac.th

First Editor: Ouay Ketusinh Emeritus Editors: Somchai Bovornkitti, Adulya Viriyavejakul, Sommai Toongsuwan, Nanta Maranetra, Niphon Poungvarin, Prasit Watanapa, Vithya Vathanophas, Pipop Jirapinyo, Sanya Sukpanichnant, Somboon Kunathikom

Executive Editor: Prasit Watanapa Editorial Director: Manee Rattanachaiyanont

Managing Editor: Gulapar Srisawasdi, Chenchit Chayachinda

Editor-in-Chief: Thawatchai Akaraviputh

Associate Editor: Varut Lohsiriwat, Prapat Wanitpongpan Online Editor: Puttinun Patpituck

International Editorial Board

Philip Board (Australian National University, Australia) Richard J. Deckelbaum (Columbia University, USA) Yozo Miyake (Aichi Medical University, Japan)

Yik Ying Teo (National University of Singapore, Singapore) Harland Winter (Massachusetts General Hospital, USA)

Philip A. Brunell (State University of New York At Buffalo, USA) Noritaka Isogai (Kinki University, Japan)

Yuji Murata (Aizenbashi Hospital, Japan)

Keiichi Akita (Tokyo Medical and Dental University Hospital, Japan) Shuji Shimizu (Kyushu University Hospital, Japan)

David S. Sheps (University of Florida, USA)

Robin CN Williamson (Royal Postgraduate Medical School, UK) Tai-Soon Yong (Yonsei University, Korea)

Anusak Yiengpruksawan (The Valley Robotic Institute, USA) Stanlay James Rogers (University of California, San Francisco, USA) Kyoichi Takaori (Kyoto University Hospital, Japan)

Tomohisa Uchida (Oita University, Japan)

Yoshiki Hirooka (Nagoya University Hospital, Japan)

Hidemi Goto (Nagoya University Graduate School of Medicine, Japan) Kazuo Hara (Aichi Cancer Center Hospital, Japan)

Shomei Ryozawa (Saitama Medical University, Japan) Christopher Khor (Singapore General Hospital, Singapore) Yasushi Sano (Director of Gastrointestinal Center, Japan) Mitsuhiro Kida (Kitasato University & Hospital, Japan) Seigo Kitano (Oita University, Japan)

Ichizo Nishino (National Institute of Neuroscience NCNP, Japan)

Masakazu Yamamoto (Tokyo Women’s Medical University, Japan) Dong-Wan Seo (University of Ulsan College of Medicine, Korea) George S. Baillie (University of Glasgow, UK)

G. Allen Finley (Delhousie University, Canada)

Sara Schwanke Khilji (Oregon Health & Science University, USA) Matthew S. Dunne (Institute of Food, Nutrition, and Health, Switzerland) Marianne Hokland (University of Aarhus, Denmark)

Marcela Hermoso Ramello (University of Chile, Chile) Ciro Isidoro (University of Novara, Italy)

Moses Rodriguez (Mayo Clinic, USA)

Robert W. Mann (University of Hawaii, USA)

Wikrom Karnsakul (Johns Hopkins Children’s Center, USA)

Frans Laurens Moll (University Medical Center Ultrecht, Netherlands) James P. Dolan (Oregon Health & Science University, USA)

John Hunter (Oregon Health & Science University, USA) Nima Rezaei (Tehran University of Medical Sciences, Iran) Dennis J. Janisse (Subsidiary of DJO Global, USA)

Folker Meyer (Argonne National Laboratory, USA)

David Wayne Ussery (University of Arkansas for Medical Sciences, USA) Intawat Nookaew (University of Arkansas for Medical Sciences, USA)

Victor Manuel Charoenrook de la Fuente (Centro de Oftalmologia Barraquer, Spain)

Karl Thomas Moritz

(Swedish University of Agricultural Sciences, Sweden)

Nam H. CHO (University School of Medicine and Hospital, Korea)

Watchara Kasinrerk (Chiang Mai University, Thailand)

Editorial Board

Pornchai O-Charoenrat (Siriraj Hospital, Mahidol University, Thailand)

Rungroj Krittayaphong (Siriraj Hospital, Mahidol University, Thailand) Wiroon Laupattrakasem (Khon Kaen University, Thailand)

Anuwat Pongkunakorn (Lampang Hospital, Thailand) Nopporn Sittisombut (Chiang Mai University, Thailand)

Vasant Sumethkul (Ramathibodi Hospital, Mahidol University, Thailand) Yuen Tanniradorm (Chulalongkorn University, Thailand)

Saranatra Waikakul (Siriraj Hospital, Mahidol University, Thailand) Pa-thai Yenchitsomanus (Siriraj Hospital, Mahidol University, Thailand) Surapol Issaragrisil (Siriraj Hospital, Mahidol University,Thailand) Jaturat Kanpittaya (Khon Kaen University, Thailand)

Suneerat Kongsayreepong (Siriraj Hospital, Mahidol University, Thailand)

Statistician: Saowalak Hunnangkul (Mahidol University, Thailand)

Nopphol Pausawasdi (Siriraj Hospital, Mahidol University, Thailand) Supakorn Rojananin (Siriraj Hospital, Mahidol University, Thailand) Jarupim Soongswang (Siriraj Hospital, Mahidol University, Thailand) Suttipong Wacharasindhu (Chulalongkorn University, Thailand) Prapon Wilairat (Mahidol University, Thailand)

Pornprom Muangman (Siriraj Hospital, Mahidol University, Thailand)

Ampaiwan Chuansumrit

(Ramathibodi Hospital, Mahidol University, Thailand)

Sayomporn Sirinavin

(Ramathibodi Hospital, Mahidol University, Thailand)

Vitoon Chinswangwatanakul

(Siriraj Hospital, Mahidol University, Thailand)

Medical Illustrator: Chananya Hokierti (Nopparat Rajathanee Hospital, Thailand)

Online Assistant: Surang Promsorn, Wilailuck Amornmontien, Hatairat Ruangsuwan Editorial Office Secretary: Amornrat Sangkaew

SIRIRAJ MEDICAL JOURNAL is published bimonthly, 6 issues a year (Jan-Feb, Mar-Apr, May-Jun, Jul-Aug, Sep-Oct and Nov-Dec) and distributed by the end of the last month of that issue.

SIRIRAJ MEDICAL JOURNAL is listed as a journal following the Uniform Requirements for Manuscripts Submitted to Biomedical Journals (URM) by the International Committee of Medical Journal Editors (ICMJE) since 9 July 2010 [http://www.icmje.org/journals.html].

Goal-Directed Therapy to Improve Gross Motor Function and the Quality of Life of Children with Cerebral Palsy: A Randomized Controlled Trial

Suwannika Palee, M.D.**, Teerada Ploypetch, M.D.*, Kingkaew Pajareya, M.D.*, Suttirat Timdang, BSc*.

*Department of Rehabilitation Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand, **Department of Rehabilitation Medicine, Faculty of Medicine, Naresuan University, Phitsanulok 65000, Thailand.

ABSTRACT

Background: The multiplicity of interventions for the treatment of cerebral palsy (CP) can cause confusion about which are most suited to certain individuals. Hypothesis is that goal-directed therapy (GDT) can guide integrating therapies to improve clinical outcomes compared with conventional therapy (CT).

Materials and Methods: A prospective, assessor-blinded, randomized controlled trial was done with 23 children with CP (mean age, 4 years 4 months old; SD 1y4mo), who were divided into groups according to their level of gross motor function: GDT and CT. Both groups received 12 physiotherapy (PT) sessions and advice on daily home programs. The GDT group additionally had a team meeting to set a specific goal, and PT programs were shaped toward that goal. Assessments were done at baseline and after treatments, using the Thai-version Gross Motor Function Measure (GMFM-66), CP-Quality of Life (CP-QOL), caregiver burden, and home program compliance. Results: After the treatments, the GDT group showed significant improvements in GMFM-66, CP-QOL, and caregiver burden, while the CT group revealed improvements in caregiver burden and some domains of the GMFM, including sitting and crawling & kneeling. Comparisons between groups found GDT was more effective than CT in improving GMFM-66 and CP-QOL. Home program compliance was higher in the GDT (69%) than the CT group (42%).

Conclusion: GDT demonstrated clear gains for children with CP regarding gross motor function and QOL improvements. Team communication toward a customized goal was crucial, empowering the children and their caregivers to comply with home programs to achieve the set goal.

Keywords: Cerebral palsy; goal-directed therapy; gross motor function; quality of life; caregiver burden (Siriraj Med J 2022; 74: 1-10)

INTRODUCTION

Cerebral palsy (CP) is one of the most common causes of childhood physical disability1 and is caused by a non-progressive lesion to the immature brain.2 This syndrome causes the dysfunctional control of movement and posture, while perception, vision, learning, and language are also frequently affected.3 These characteristics

can influence the child’s ability to learn and perform everyday life activities4, and also the quality of life (QOL) of patients and their caregivers are affected by these disabilities.5 Treatment program should promote the improvement of the patient’s motor function, and facilitate their participation in activities and adaptation to daily living, with the ultimate goal of improving their QOL.6

Corresponding author: Teerada Ploypetch E-mail: teerada.plo@mahidol.ac.th

Received 23 August 2021 Revised 29 September 2021 Accepted 9 October 2021 ORCID ID: https://orcid.org/0000-0002-5834-6800 http://dx.doi.org/10.33192/Smj.2022.1

Numerous treatments for CP are available, which can be overwhelming for patients and families, and even make it challenging for healthcare professionals to make a clinical decision on what is the most suitable treatment for each child. Recent systematic reviews provide evidence-based guidance for assessing interventions for children with CP, such as the Evidence Alert Traffic Light system.7 Here, green-light-go interventions are inferred to be effective with a higher level of evidence, while yellow-light interventions show uncertain effects and require outcome measures to monitor progress, and red-light interventions are considered ineffective and therefore to be avoided.7 However, this does not suggest that every child with CP should undergo all green-light interventions for best practice since CP has a heterogeneity of symptoms and clinical status. The key to success is doing the right things with the right child at the right time.8

Goal-directed therapy (GDT) approach could be a solution to this overwhelming situation. GDT focuses on comprehensive treatment strategies that serve as stepping stones to achieving individual goals, which along the way promote functional performance and a gradual independence in everyday life activities.9,10,11 The approach consists of the identification of individual needs of both the child and the family, assessments of the child’s performance and capacity, setting goals that are meaningful tasks and relevant despite the child’s level of gross motor function12, and the development of individual-tailored treatment programs.13 Setting specific goals is a means to enhance awareness of the objectives of the therapy and can affect the treatment performance by focusing attention, directing effort, increasing motivation, and enabling the development of strategies to achieve the set goals.13 There is evidence showing an improvement in basic motor abilities and self-care in young children with CP after undergoing GDT, and a decrease in their need for caregiver assistance for self-care and mobility.13,14 However, in previous research, the research methodology either did not involve a randomized controlled trial (RCT) and the duration and frequency of treatment in the GDT group setting were three hours per session, five days a week, for a period of three weeks14 or did and treatment was three days a week for three consecutive months.13 Additionally, the treatment used in previous studies may be too onerous for patients in many developing countries because of certain socioeconomic problems and human resource issues15, such as parents finding it inconvenient to bring their children to the hospital because of the cost of travel, lack of time, and limited number of therapists available.16,17

Consequently, an RCT was performed to compare GDT and conventional therapy (CT), with reducing the amount of interventions performed at the hospital and encouraging the use of home programs, compatible with the available resources. Currently, conventional therapy is mostly practiced through a physiatrist, who authorizes prescribed treatment programs via the medical records after assessment of a patient and conveyed to the respective rehabilitation team. In contrast, GDT includes a family-team meeting organized to set specific goal(s), with the treatment programs then shaped to meet the set goal. We hypothesized that even with both groups receiving the same amount of hospital programs, GDT would be more effective than CT for improving gross motor function, QOL, caregiver burden, and compliance with home programs.

MATERIALS AND METHODS

Study design

This prospective, assessor-blinded RCT was conducted between April 2018 and April 2019. The study protocol was approved by the Institutional Review Board (COA no. Si 675/2017), and registered with the Thai Clinical Trials Registry (registration no. TCTR20180419002). Forty-five children with CP from a pediatric rehabilitation unit of the university hospital were evaluated for eligibility (Fig 1), with written informed consent obtained from their parents of all participants. The participants were stratified by their severity of CP using Gross Motor Function Classification System (GMFCS)18, as assessed by a study doctor, and then randomly allocated into either the intervention group (GDT) or control group (CT) by drawing random numbers from a sealed envelope. Computer-generated randomization with block sizes of 4 was used to create the random numbers. The participants were requested to maintain their other treatments, such as oral antispastic medications and orthoses that could be considered co-interventions; however, these could be changed as a medical necessity. The researcher regularly monitored and recorded whether additional therapies were used by the participants.

Participants

Eligible participants were children with CP, GMFCS levels I–IV, aged 1–6 years old, able to understand basic instructions and communicate pain or discomfort, and having caregivers with Thai language literacy who were able to complete the questionnaires. The exclusion criteria were specifically patients with uncontrolled epilepsy, history of fractures, serial casting, orthopedic surgery or chemoneurolysis intervention within 6 months

Intention to treat analysis

Analyzed (n=11)

Analyzed (n=12)

Failed to follow-up (n=2) (due to personal reasons)

Failed to follow-up (n=1) (due to pneumonia)

Stratified randomized (n=23)

Excluded (n=22)

• Did not meet the inclusion criteria (n=20)

• Declined to participate (n=2)

Assessed for eligibility (n=45)

Allocation

Control (n=12)

Conventional therapy group

Intervention (n=11)

Goal directed therapy group

Follow-up

Enrollment

Fig 1. Consolidated Standards of Reporting Trails (CONSORT) diagram showing the flow of participants in the study

prior to the study, known unstable cardiovascular or pulmonary diseases, and any contraindication for receiving physiotherapy (PT), or declined to participate.

The sample size calculation was based on a previous study.13 The nQuery, with a type-I error at 0.05 and 80% power of test, indicated that at least 10 participants per group would be required to detect a statistically significant difference in gross motor function improvement between the two groups. The recruitment process was on a first- come-first-served basis until the target number of 23 children had been reached, to compensate for potential loss to follow-up about 10%.

Interventions

Attending physiatrists evaluated all participants and prescribed individualized PT programs, such as hydrotherapy, bicycling, strengthening and stretching exercises, balance and mobility training, modalities, and tools applications. Both groups received 50 minutes of

PT for 12 sessions. The therapists assigned daily home exercise programs suitable for each patient. Caregivers were responsible for the home programs and for recording their compliance in a logbook (Fig 2).

For GDT group, an additional process was included, which was a team conference involving the physiatrist, physical therapist, child, and caregivers, where an individualized goal was agreed for each patient. The goal-development process included an assessment of the child’s performance and motor capacity, identification of a specific, measurable, achievable, relevant, and timed (SMART) goal, and the conception of the goal-attainment scale (GAS).14,19 Only one goal was set as the most crucial and relevant to the child and family’s needs, and that was possible to achieve based on each child’s assessed performance. PT programs were adjusted specifically for goal achievement. The GAS was used to objectively identify and follow progress. For example, if the goal was to sit independently for 30 seconds, the programs emphasized

Fig 2. A logbook for recording home program compliance.

exercises to promote core/trunk muscle strengthening and sitting balance. Others programs, such as stretching heel cords or mobilization, were still considered crucial, but were not intensively assigned because these were not related to the set goal. The individualized specific goal was presented in the cover of the logbook for the GDT group to remind all the team and family members of that goal.

Outcome measurements

A study doctor retrieved demographic data, GMFCS level, type of CP, and co-morbidities. The primary outcome of this study was the improvement of gross motor function after the therapy. A blinded physical therapist, who had experience but was not involved in any of the therapy sessions, assessed the participants using the Gross Motor Function Measure (GMFM-66) Thai version20, a standardized tool for measuring gross motor function in children with CP that contains 66 items for assessing gross motor ability, including: (1) lying and rolling, (2) sitting, (3) crawling and kneeling,

(4) standing, and (5) walking. The final GMFM-66 score was calculated with a computer-scoring program: the Gross Motor Ability Estimator. The maximum score is 100, and a higher score means better gross motor function. Here, a change in GMFM-66 score of 1.58 was reported as a clinically meaningful improvement, and a score change of 3.71 could discriminate between great and not great improvement.21

Secondary outcomes were the children’s QOL, caregiver burden, and home program compliance, assessed by the CP-QOL Questionnaire Parent Proxy Thai version22, the CP Caregiver Burden Thai version23, and logbook scoring (Fig 2), respectively. Those questionnaires and a survey for

the caregivers’ demographic data were self-administered by the caregivers, with the primary caregiver, as the main person the child spends most time with, the preferred candidate to answer the questionnaires, or the other caregivers if the primary caregiver was not available. Outcome measurements were recorded at baseline and immediately after the 12th session of therapy.

In the GDT group, goal achievement was evaluated after completion of all the therapy sessions using the GAS, a tool for identifying specific goals and measuring progress. This assessment was done by the physical therapist who provided the GDT. The GAS consists of 6 grades:

-3, for worse than at the start (i.e., a deterioration); -2, for equal to at the start; -1, for less than expected; 0, for expected goal; +1, for somewhat more than expected; and +2, for much more than expected.24

Statistical analysis

The analyses were performed using SPSS Statistics version 18.0 (SPSS, Inc., Chicago, IL, USA). A p-value

<0.05 was regarded as statistically significant. Data are presented as the number and percentage for categorical variables, and the mean ± standard deviation (SD) for continuous variables. The baseline patients’ characteristics and the results of both groups were assessed for normality using the Kolmogorov–Smirnov test. Chi-square or Fisher’s exact test were used to compare categorical variables. For continuous variables, the Student’s t-test was used to compare parametric data, and the Mann– Whitney U test was used to compare nonparametric data. Statistical analysis was finally performed by intention to treat analysis. Missing data were replaced by the last observation value.

RESULTS

The study participants comprised 23 children with CP (mean age, 4 years 4 months old; SD 1y4mo). The mean time interval between baseline and final assessments for all was 93.1 (SD 36.3) days. The participants’ baseline characteristics are presented in Table 1. Participants in both groups received co-interventions, such as oral antispastic medications and orthotic use, with no statistically significant difference between the groups.

All participants in the GDT group had a team meeting to set a SMART goal related to gross motor function. All showed an improvement from baseline according to their individualized GAS, except one child that was lost to follow-up due to pneumonia. Overall, 6 from 11 children achieved their goals as expected or more than expected (GAS>0).

Comparison of the outcomes before and after the therapies is demonstrated in Table 2. The GDT group showed substantial improvements in GMFM- 66 total score and all aspects of gross motor function except for walking & running. The CT group showed no significant improvement in GMFM-66 total score, but improvements in sitting and crawling & kneeling. CP-QOL was significantly increased only in the GDT group, while caregiver burden was decreased after the treatments in both groups.

Comparison of the improvements between the groups is shown in Table 3. The GDT group had significantly higher improvement in GMFM-66 total score and in walking & running subscale compared with the CT group. The increments of CP-QOL in the GDT group were significantly higher than in the CT group, while the decrements of caregiver burden were not different between groups.

Home program compliance was higher in GDT (69%) than CT (42%) group (p=0.010). All participants in the GDT group complied with performing more than half of the assigned tasks; while in the CT group, only 16% complied. Examples of logbook are shown. A participant in GDT group aimed to walk with a walker and well complied with walking exercise at home (Fig 3), while one in CT group revealed moderate compliance (Fig 4).

DISCUSSION

GDT showed superior outcomes compared with CT in improving gross motor function and the QOL of children with CP, consistent with previous studies13,14,19, even if the dose of treatment at the hospital in this study was limited to just once or twice a week for 12 sessions. GDT facilitated good compliance with home exercise

programs, whereby the children still received a high intensity of therapies from their caregivers. Healthcare professionals acted as a coach and trained the caregivers as therapists, which could be practically applied in patients with socioeconomic issues limiting them visiting hospital frequently.16 This model may also applied in low- to middle-income countries with limited human resources. GDT resulted in a higher improvement in gross motor function, when compared with CT. Although the amount of therapies performed in the hospital was similar in both groups, the GDT group managed to achieve a greater dosage of therapeutic exercise at home. It is known that strength and muscle endurance affect gross motor outcomes and depend on the amount of intervention the patient receives, so consistently performing exercises can increase strength and endurance.25,26 In addition, the PT program can be set to integrate specific activities to promote goal achievement. Therefore, those could be reasons for the greater improvement of gross motor outcomes in this group. Gross motor function improvement after GDT was clinically meaningful: the average change of GMFM-66 of 11.7±6.8 showed great improvement21, most likely due to the very young age of the participants in this study, which is well-known to offer a higher chance of gross motor improvement.21 It is known that the standard practice of rehabilitation

is goal oriented. Treatment programs in CT should be also for serving goals, considering that physiatrists prescribed those for individuals. The lower outcomes might be from ineffective communication about the goals. As a result, team members could not realize what programs to focus on and a lack of motivation to comply. The predominant home program compliance in GDT group was possibly due to the team meeting including a number of key elements for enhancing adherence to the prescribed home programs, especially effective goal setting. Here, a SMART goal was set by consensus between the multidisciplinary team and the patients’ caregivers, which helped tailoring the goals to the patients’ needs. One goal was chosen for each patient that was possible for them to achieve easily to encourage and motivate their adherence to physiotherapy rather than setting more difficult or multiple goals.27 The use of GAS encouraged communication and collaboration between the team members, and facilitated patient and family involvement.28 Moreover, the team meeting expedited effective communication, which enhanced reciprocal relationships, ameliorated problems, and facilitated caregiver/parental empowerment by their engagement with the professionals, who were perceived as collaborators instead of authoritative experts. Empowerment, motivation,

TABLE 1. Baseline characteristics of the participants and their caregivers.

1Mean ± SD, 2GMFCS = Gross Motor Function Classification System.

TABLE 2. Outcomes comparison within the goal-directed therapy (GDT) and conventional therapy (CT) groups.

1 Mean ± SD, *p-value ≤ 0.05.

TABLE 3. Comparison of the improvements after the completion of goal-directed therapy (GDT) and conventional therapy (CT).

1 Mean ± SD, *p-value ≤ 0.05.

Fig 3. An example of logbook of a participant in the goal-directed therapy (GDT) group

Fig 4. An example of logbook of a participant in the conventional therapy (CT) group

and reciprocal relationships are known determinants of effective parent-delivered therapy in children with CP.29 Caregivers’ perception of their children’s QOL increased after GDT. Previous studies reported gross motor function, cognitive level, complications, pain, and parental stress as factors associated with the QOL of children with CP.5,30,31 Therefore, the improved QOL in the present study was possibly related to the improvement of gross motor function after treatment. GDT was more effective toward improving gross motor function; consequently, the QOL increment in the GDT group was higher than in the CT. Additionally, effective communication between

the multidisciplinary professionals and caregivers via the team meeting in GDT possibly reduced caregivers’ stress level, which might have influenced how they reported their child’s QOL.32,33

Interestingly, caregiver burden was improved in both groups. Considering that the caregivers had to bring their child to the hospital for the PT programs and they were also instructed to spend time doing home exercise programs, in addition to their routine time spent helping the child with daily activities, like eating, personal care, etc., this seemed to be their socio-structural constraints or the objective burdens.34 Another aspect of caregiver

burden is the subjective burden or emotional distress, which may diminish over time by being able to identify positive aspects of their special parenthood.34 Family empowerment through home program education was applied to all the caregivers in this study. The results support the findings of a previous study in Thailand, whereby interventions that empowered caregivers’ ability to care for their child could reduce caregiver burden.23 There are limitations in this study. The physical therapists could not blinded due to the nature of the intervention. Consequently, the therapists who believed in GDT might have put in more effort during the therapy sessions. However, this might be another key to the success of GDT, in that the therapists, as key team members, would do their best for each child to achieve their set goal. Such a potential bias could be reduced by using a blinded assessor and an objective primary assessment tool. Considering baseline parameters, topography, duration of treatment and initial GMFM seemed to favor GDT although those were not statistically significant difference between groups. The nature of children continues to develop over time. Pattern of paralysis and gross motor function at baseline were predictors for good outcome after some interventions.35,36 Children with a lower initial GMFM achieved higher improvement after selective dorsal rhizotomy.36 Consequently, assessing a longer period of follow-up and the factors associated with the success of

GDT could be very interesting for future studies.

CONCLUSION

GDT demonstrated clear gains for children with CP for improving their gross motor function and QOL. The keys to success are: 1) customized SMART goal setting, 2) focus on integrative therapies to serve meeting that goal, 3) effective communication among multidisciplinary professionals, caregivers, and the child, and 4) family empowerment. In limited resource settings where hospital PT programs could be utilized just once or twice a week, GDT still showed impressive outcomes. Home programs could be a solution to maximize the intensity of interventions. Patients and caregivers are an important part of success, especially once they are motivated and voluntarily make a commitment to comply with home programs to achieve their goals.

ACKNOWLEDGMENT

This study is supported by Siriraj Research Development Fund (Managed by Routine to Research: R2R), IO = R016135014. The treatments and clinical data are parts of the Cerebral Palsy Clinical Tracer, responsible by Siriraj Pediatric Rehabilitation Unit.

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19. Türker D, Korkem D, Ozal C, Kerem Günel M, Karahan S. The effects of neurodevelopmental (Bobath) therapy based goal directed therapy on gross motor function and functional status of children with cerebral palsy. International Journal of Therapies and Rehabilitation Research. 2015;4(4):9-20.

20. Hensangvilai K, Yankai A, Angsupaisal M, Intachom R. Reliability in using the Modified Gross Motor Function Measurement-66 Thai version by Physical Therapy students. Journal of medical technology and physical therapy. 2009;21(2):170-85.

21. Wang HY, Yang YH. Evaluating the responsiveness of 2 versions of the gross motor function measure for children with cerebral palsy. Arch Phys Med Rehabil. 2006;87(1):51-6.

22. Suwanna K, Prasertsukdee S, Khajornchaikul P. Test-Retest Reliability and Internal Consistency of Cerebral Palsy Quality of Life Questionnaire (Thai version). Thai Journal of Physical Therapy. 2014;36:60-9.

23. Chumsri S, Chaimongkol N, Sanunruangsak S. Effects of the child caregiver's empowerment promoting program on ability and burden to care for childern with cerebral palsy. The Journal of Faculty of Nursing Burapha University. 2014;22(4):35-46.

24. Steenbeek D, Ketelaar M, Galama K, Gorter JW. Goal Attainment Scaling in paediatric rehabilitation: a report on the clinical training of an interdisciplinary team. Child Care Health Dev. 2008;34(4): 521-9.

25. Giessing J, Eichmann B, Steele J, Fisher J. A comparison of low volume ‘high-intensity-training’ and high volume traditional resistance training methods on muscular performance, body composition, and subjective assessments of training. Biol Sport. 2016;33:241-9.

26. Radaelli R, Fleck SJ, Leite T, Leite RD, Pinto RS, Fernandes L, et al. Dose-response of 1, 3, and 5 sets of resistance exercise on strength, local muscular endurance, and hypertrophy. J Strength Cond Res. 2015;29(5):1349-58.

27. Argent R, Daly A, Caulfield B. Patient Involvement With

    Home-Based Exercise Programs: Can Connected Health Interventions Influence Adherence? JMIR Mhealth Uhealth. 2018;6(3):e47.

28. Turner-Stokes L. Goal attainment scaling (GAS) in rehabilitation: a practical guide. Clin Rehabil. 2009;23(4):362-70.

29. Lord C, Rapley T, Marcroft C, Pearse J, Basu A. Determinants of parent-delivered therapy interventions in children with cerebral palsy: A qualitative synthesis and checklist. 2018;44(5):659- 69.

30. Sritipsukho P, Mahasup N. Correlations between gross motor functions and health-related quality of life in Thai children with spastic diplegia. J Med Assoc Thai. 2014;97 Suppl 8:S199- 204.

31. Arnaud C, White-Koning M, Michelsen SI, Parkes J, Parkinson K, Thyen U, et al. Parent-reported quality of life of children with cerebral palsy in Europe. Pediatrics. 2008;121(1):54-64.

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33. Rapp M, Eisemann N, Arnaud C, Ehlinger V, Fauconnier J, Marcelli M, et al. Predictors of parent-reported quality of life of adolescents with cerebral palsy: A longitudinal study. Res Dev Disabil. 2017;62:259-70.

34. Uldall P. Chapter 20 - Everyday life and social consequences of cerebral palsy. In: Olivier Dulac ML, Harvey BS, editors. Handbook of Clinical Neurology. Volume 111: Elsevier; 2013. p. 203-7.

35. Fazzi E, Maraucci I, Torrielli S, Motta F, Lanzi G. Factors predicting the efficacy of botulinum toxin-A treatment of the lower limb in children with cerebral palsy. J Child Neurol. 2005;20(8):661-6.

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Effectiveness of Ayurved Siriraj Prasa-Nam-Nom Recipe on Breast Milk Volume in Early Postpartum Women: A Randomized, Double-Blind, Placebo- Controlled Trial

Pharuhas Chanprapaph, M.D.*, Chantanat Thippayacharoentam, B.ATM.**, Apirada Iam-am, B.ATM.**, Natchagorn Lumlerdkij, Ph.D.**, Pravit Akarasereenont, Ph.D. **,******, Tawee Laohapand, M.D.**, Sudarat Bangkha, B.N.S.***, Nhupat Kumleemak, B.N.S.***, Sudarat Piyophirapong, M.D.****, Akarin Nimmannit, M.D.*****, Thapthep Thippayacharoentam, M.ATM.**

*Breastfeeding Division, Department of Obstetrics and Gynaecology, Faculty of Medicine Siriraj Hospital, Mahidol University, **Center of Applied

Thai Traditional Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, ***Obstetric and Gynaecological Nursing Unit, Siriraj Hospital, Mahidol University, ****Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, *****Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, ******Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

ABSTRACT

Objective: To explore the effectiveness of Ayurved Siriraj Prasa-Nam-Nom (ASPNN) recipe on breast milk production in early postpartum women.

Materials and Methods: Fifty-four normal vaginal term delivery mothers who had inadequate milk volume were enrolled into this randomized, double-blind, placebo-controlled trial. All participants received ASPNN or placebo 1,500 mg three times/day for 3 days in the hospital and 7 days at home. Primary outcomes, including breast milk volume, %creamatocrit, and level of prolactin, were evaluated on day 1 and day 3. Satisfaction scores, adverse effects, and types of breastfeeding were also determined.

Results: On day 3, milk volume was increased in both groups. The median volume of ASPNN group was 19 ml, while that of the placebo group was 30 ml. The median %creamatocrit of ASPNN and placebo group were 7.17% and 6.98%, respectively. Mean serum prolactin levels of ASPNN and placebo group were 321.76 ± 114.23 ng/ml and 323.78 ± 116.68 ng/ml, respectively. Although the effects were not different from the placebo, the reduction of prolactin in ASPNN was lower. Minor adverse effects included skin rash and mild diarrhea. Exclusive breastfeeding rate on day 11 in ASPNN and placebo group were 92.6 % and 88.5%, respectively.

Conclusion: Short term ASPNN supplementation produced no direct effect on breast milk volume, creamatocrit, and serum prolactin. It was safe and might help maintaining serum prolactin. A future trial with more participants and longer period should be conducted to confirm the effect of ASPNN on breast milk quantity and quality.

Keywords: Creamatocrit; galactagogue; prolactin; inadequate breast milk volume; Prasa-nam-nom (Siriraj Med J 2022; 74: 11-18)

Corresponding author: Thapthep Thippayacharoentam E-mail address: thapthep.thi@mahidol.edu

Received 18 August 2021 Revised 18 October 2021 Accepted 27 October 2021 ORCID ID: https://orcid.org/0000-0002-6396-3950 http://dx.doi.org/10.33192/Smj.2022.2

INTRODUCTION

Breastfeeding is standard method for infants feeding. Exclusive breastfeeding for at least 6 months has been globally promoted as a standard infant feeding by the United Nations International Children’s Emergency Fund (UNICEF) and the World Health Organization (WHO) through a huge campaign “Ten Steps to Successful Breastfeeding”. Multiple lines of evidence support that breastfeeding improves health of infants and mothers. It decreases not only the incidence but also the severity of neonatal infections and reduces postnatal mortality rate.1 However, WHO reported in 2018 that only about 44% of infants worldwide are exclusively breastfed.2 Breastfeeding failure is a result of multifactorial determinants, including negative reaction toward breastfeeding at work, negative attitude toward colostrum in some societies, fatigue and intensity from work, short maternity leave (<6 weeks), poor advice on breastfeeding position and latching, as well as insufficient supports.3 Other factors also include short nipple length4 and perception of babies’ dissatisfaction of breastmilk and inadequate milk volume.5,6

Human milk production can be successfully increased by means of psychological support, stress-relief-techniques and medications.6 Plasma concentration of prolactin (PRL) plays an important role in human milk production. Dopamine is a physiologic inhibitor of PRL release. Therefore, domperidone which acts as a hypothalamic dopaminergic receptor blocker has been widely used as a galactagogue to promote lactogenesis. The drug was reported to enhance PRL.7

Thai traditional medicine (TTM) is a holistic approach to take care of human health. There are several TTM methods used for postpartum women, such as hot salt pot compression, massages, hot herbal charcoal seats and herbal medicines.8 Prasa-Nam-Nom is a Thai herbal galactagogue mixture which has been traditionally used to improve milk production. This herbal formula helps increasing blood circulation and results in enhancing milk secretion and purifying its content. Prasa-Nam- Nom recipe has been listed in TTM text books as a safe herbal medicine and has been used for more than three decades. Various preparations of Prasa-Nam-Nom are widely described and accepted as a galactagogue, including our Ayurved Siriraj Prasa-Nam-Nom (ASPNN) recipe. The ingredients of ASPNN include garden spurge (Euphobia hirta Linn.), weeping fig (Ficus benjamina Linn.), rauwolfia (Rauvolfia serpentina Linn. Benth. ex Kurz.), and wild ginger (Zingiber zerumbet (L.) Smith.). Among these, E.hirta exhibited galactagogue effects in vivo by enhancing milk production, prolactin secretion, and the development of lobuloalveolar system of mammary glands.9,10

Despite its increasing popularity, there is limited evidence to support its uses. Tuntratuang (2017) reported that a Prasa-Nam-Nom used in Nong Bua Lamphu Hospital (no detail on its composition) increased breast milk volume of the mothers after caesarean section better than domperidone at 24, 48, and 72 hours.11 Jankaew and Narumitmontri (2020) reported that a herbal recipe containing Anthocephalus chinensis (Lam.) A. Rich ex Walp. and Diospyros variegata Kurz administered five grams thrice a day for one week enhanced breast milk flow at 32 and 40 hours after the delivery better than domperidone.12

The objectives of this study were to explore the effectiveness of ASPNN recipe on breast milk production in women with inadequate milk volume during early postpartum period. Furthermore, its safety and impact on the level of serum prolactin hormone and the milk quality were also investigated.

MATERIALS AND METHODS

Study design

This randomized, double-blind, placebo-controlled study was conducted at the postpartum ward, Department of Obstetrics and Gynaecology, Siriraj Hospital, Mahidol University, Bangkok, Thailand between November 2010 and December 2012. The research protocol was reviewed and approved to be ethical by the Siriraj Institutional Review Board No. 743/2554 (EC3). An inform consent was obtained from each participant before the trial began. The study was registered with Thai Clinical Trials Registry with the project number of TCTR20190218004.

Participants

Subjects were enrolled if they were eligible to the inclusion criteria: age of ≥18 years old; first vaginal delivery at term; milk volume at 24-hour after the delivery

<49 ml/breast pump.13 The exclusion criteria included having medical disorders (hypertension, type 2 diabetes, thyrotoxicosis and hypothyroidism); previous allergy to ASPNN or its ingredients; inability to eat vegetables or spicy food; severe breastfeeding problems (abnormal breast anatomy, short nipples, mastitis, breast abscess and severe fetal tongue-tie); taking other medications (domperidone, metoclopramide, antidepressive drugs, caffeine and alcohol). Subjects were withdrawn if they were unable to take 90% of total drugs or had gastrointestinal unwanted effects.

Sample size calculation

According to our previous study, normal milk volume on day three after the delivery was 49 ± 18 ml/ breast pump.13 In this study, an increase in milk production

up to 15 ml was considered to be clinically significant. By setting the probability of 2-sided type I error and the power of test at 0.05 and 80%, respectively, a minimum of 24 subjects in each group was required. To prevent loss to follow-up cases, 10% more of participants was added which resulted in the total number of 54 participants.

Randomization

For patient allocation, the treatment codes were randomly generated prior to the trial by a computer software nQuery Advisor® version 7 (Statistical Solutions Ltd., Cork, Ireland, United Kingdom). The codes were block randomized into two groups of the ASPNN group and the placebo group and kept strictly confidential in sequence in sealed opaque envelopes by a research nurse who had no responsibility for patient care in this study. The nurse would open each envelope for the treatment assignment without knowing the identity of the drugs administered by either the physicians or the patients.

Intervention

This study was conducted in a double-blind fashion. Each participant received three 500-mg capsules of either ASPNN or placebo thrice a day (4,500 mg/day)

30 minutes before meal for three days. All subjects equally obtained three meals and two snacks with total calories of 2,100 kcal daily. On day three, 2 milliliters of breast milk were collected for fat content evaluation and 6 milliliters of blood were taken for PRL level assessment. The volunteers were requested to answer a questionnaire regarding their satisfaction to the taken medication. Prior to the hospital discharge, participants in each group received more medications to continue their treatment for another seven days based on the traditional prescription of Prasa-nam-nom decoction, which is 7 - 10 days. On day eleven, they were interviewed by phone regarding any unwanted effects and type of infant feeding (Fig 1).

Participants’compliance was assessed by interviewing and counting the remaining capsules. Subjects in both groups received the standard postpartum care in the similar environment and were well-supported for breastfeeding by the same lactation consultant nurse. During the study period, they were encouraged to breastfeed every 2-3 hours in proper latch-on position and correct techniques. They also obtained the same amount of food and water in one day while a food and water chart were daily recorded for monitoring.

Fig 1. Study flow diagram based on the CONSORT 2010 flow diagram

Drug preparation

The 500-mg of ASPNN and identical-looking placebo capsules were prepared under the good manufacturing practice by Ayurved Siriraj Manufacturing Unit of Herbal Medicine and Products, Center of Applied Thai Traditional Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University (Bangkok, Thailand). All herbal ingredients were selected, washed and oven-dried at 45-55 °C for 6-8 hours. Then, they were chopped, grounded and filtered into brown fine powder. The mixtures were weighed and filled into 500-mg green opaque capsules using a capsule filling machine. Placebo capsules were made with green opaque capsule shells with the same appearance with those used to make ASPNN, but filled with fine granular flour, magnesium stearate and talcum. Both ASPNN and placebo capsules were similarly packed in sealed opaque envelopes. Then the capsules were tested for weight variation and disintegration to ensure that they met with the specification. To ensure the similarity of the given medications, all herbal and placebo capsules were sufficiently produced for the whole study within the same batch. All capsules were stored in a cool dry place at room temperature during the study period.

Breast milk volume collection and measurement

Breast milk was collected on day 1 before the drug administration and on day 3 before discharge using an electrical breast pump (Lactina Electric Plus breastpump by Medela CO. Switzerland) with moderate pumping power for 15 minutes on each side. The milk volume was presented as mL of breastmilk from both sides per pump (Fig 2). The collected breast milk was later brought to the infants.

Creamatocit measurement

Creamatocrit is a method widely used in lactation research. It is used to determine lipid content and energy content in breast milk.14 The assessment technique requires a hematocrit centrifuge machine, a standard glass capillary tube and a hematocrit reader. 75 µl of collected milk was transferred into a glass capillary tube which was sealed one end by clay and centrifuged for 30 minutes at a speed of 3,000 rpm. The creamatocrit value was evaluated on day 1 before the drug administration and on day 3 before discharge and demonstrated by a percentage of the length of the cream layer to the length of the milk column in the tube (Fig 3).

Fig 2. (a) breast milk volume was measured before the drug administration and on day 3 and (b) an electrical breast pump

Fig 3. Creamatocit measurement (a) a hematocrit centrifuge machine and (b) a hematocrit reader.

Prolactin level assessment

Serum prolactin levels (PRL) were determined on day 1 before the drug administration and on day 3 before discharge using Prolactin II assay (Roche Diagnostic) and analyzed by Modular Analytics E170 module (Roche Diagnostic, Mannheim, Germany). This technique was an electrochemiluminescence immunoassay based on the sandwich principle.

Satisfaction scores, adverse effects, and types of breastfeeding

Satisfaction scores and adverse effects were determined on day 3. After a discharge from the hospital, the participants continued the intervention for another 7 days at home. Then satisfaction scores, adverse events, and types of breastfeeding were assessed on day 11 by a telephone interview. The participants rated their satisfaction with odor and flavor of the intervention on a scale of 1-5, where 1 = very unsatisfied, 2 = unsatisfied, 3 = neutral, 4 = satisfied, and 5 = very satisfied. The participants were asked with an open-ended question for self-report adverse effects. Lastly, they were asked for types of breastfeeding whether they fed the baby with only breast milk (exclusive breastfeeding) or combine with formula supplementation (partial breastfeeding).

Statistical analysis

All statistical analyses were performed using SPSS version 18 for Windows software (SPSS, Inc., Chicago, IL). All data were presented as mean ± SD or median (min, max). Independent t- test or Mann-Whitney U test were used to compare these values between two groups. Paired t-test or Wilcoxon Signed Rank test were used to compared results before - after of each group. Satisfaction scores and adverse effects were presented as

case number and percentage. The comparison between two groups was performed using Pearson Chi- square. Breastfeeding types on day 11 were presented as case number and percentage. The comparison was performed with Fisher’s exact test. The statistical significance was set at p <0.05.

RESULTS

From the total enrolled 61 women, there were 54 eligible women who were randomly allocated into the placebo group or the ASPNN group. On day three, one case in the placebo group dropped out. Therefore, the data from only 53 cases were used for statistical analysis (Fig 1).

At the beginning, maternal age, body mass index, total gestational weight gain, baby birth weight, pumped milk volume, creamatocrit and serum PRL level in both groups were similar (Table 1).

After three days of the interventions, the median milk volume in both groups remarkably improved from

0.15 to 19 ml in the ASPNN group and 0.3 to 30 ml in the placebo group (Table 2). Maximum milk volume of the ASPNN group was higher than that of the placebo group. Breast milk volume, increased milk volume, creamatocrit and PRL of both groups were not difference. Serum PRL of the ASPNN group on day 3 was not different from day 1 (p = 0.31), while that of the placebo group significantly reduced (p =0.03).

ASPNN drug was highly accepted by the women in terms of its odor and flavor (Table 3). Minor adverse effects were reported, including skin rash and mild diarrhea, with spontaneous recovery (Table 4). The percentage of exclusive breastfeeding on day 11 of the ASPNN group was 92.6%, whereas that of the placebo group was 88.5%.

TABLE 1. Demographic data (before the treatment)

Characteristic

ASPNN

(n = 27)

Placebo

(n = 26)

P-value

Body mass index (kg/m2), mean ± SD 20.57 ± 2.91 20.53 ± 2.52 0.48

Age (years), mean ± SD 23.74 ± 4.95 24.27 ± 5.38 0.36

Baby birth weight (g), mean ± SD 3,013.33 ± 288. 42 2,981.15 ± 311.58 0.35

Total gestational weight gain (kg), mean ± SD 12.54 ± 3.96 12.89 ± 3.43 0.37

Day-1 creamatocrit (%), median (min, max) 7.25 (2.58, 14.23) 6.03 (2.13, 12.34) 0.47

Day-1 breast milk volume (ml), median (min, max) 0.15 (0, 5) 0.3 (0, 4.1) 0.94

Day-1 prolactin (ng/mL), mean ± SD 339.18 ± 164. 21 386.54 ± 192.61 0.17

TABLE 2. Breast milk volume, creamatocrit and serum prolactin level on day 3 in each group and the changes

TABLE 3. Satisfaction with odor and flavor of the interventions (day 3)

Value is presented by number of cases (%)

TABLE 4. Self - reported adverse effects

Value is presented as number of cases (%)

(The is no p-value on day 11 because the adverse effect is a constant)

DISCUSSION

Compromised lactation is originated from several factors, including preglandular, glandular or postglandular problems.15 Phase II lactogenesis occurs 3 - 7 days after the delivery. In this phase, the quantity of breast milk becomes higher at the end of the first week after the delivery. However, many early-postpartum women perceive that they might have insufficient breastmilk and this leads them to stop breastfeeding. Therefore, it is necessary to initiate a successful breastfeeding as soon as possible to encourage exclusive breastfeeding later.16 In this study, we focused on the implementation of ASPNN, which has been used to stimulate breast milk production in Thai traditional medicine, in order to help early-postpartum women initiating breastfeeding.

ASPNN administration for 3 days showed no statistically significant effect to breast milk volume compared to the placebo. This might due to the short duration of the intervention, which was limited by the usual hospital stay. Galactagogues, such as domperidone or metoclopramide, are usually prescribed for 5 - 28 days.17 A study from Nong Bua Lamphu Hospital showed that Prasa-Nam-Nom could increase milk volume in 72 hours.11 However, this study measured the 24 - hour milk volume, while our study measured milk volume from only one pump in a day.

Lipid is a crucial component of breast milk which provides energy to infants. Creamatocrit measurement is simple, inexpensive, and effective to determine lipid content and calories in breast milk. There was also a strong relationship between the creamatocrit value and the infants’ body weight increment.14 The percentage of fat component in breast milk was improved in both groups (25 - 38%). Interestingly, there was a trend of increased creamatocrit in the ASPNN group compared to the placebo group. According to the TTM knowledge, ASPNN has been prescribed in order to promote milk production and to purify the milk. Therefore, further investigation should be conducted to explore the effect of ASPNN on quality of breast milk.

PRL is among reproductive hormones that are responsible for milk production and secretion and becomes a pharmacological target for galactagogues.17 Although ASPNN showed no statistically significant effect on PRL concentrations, there was a trend that the herbal medicine could maintain PRL level which is important for a successful breastfeeding. The level of PRL is high in the first two hours after delivery and declines after that. Infant suckling stimulates PRL.17 In our study, the participants were encouraged to breastfeed their babies every 2-3 hours and this should help maintaining PRL

level. However, the placebo group showed a significant reduction in PRL. This suggests that ASPNN might be beneficial for lactogenesis by its impact on PRL.

After discharged, all participants continued their treatment for another week and were interviewed by phone regarding any adverse effects and types of infant feeding. This study shows that short term use of ASPNN is safe for lactating women with only minor and recoverable adverse effects. Its safety for longer period of use is still needed to monitor. The rates of exclusive breastfeeding in both groups were high, which might due to the effectiveness of overall lactation program that could initiate breastfeeding in early postpartum phase.

Traditionally, Prasa-Nam-Nom decoction in conjunction with other treatments, including body massages, hot salt pot, hat charcoal seat, herbal hot spa (Yu-fai) are given to nursing mother for 4 - 6 weeks. This holistic care approach aims to relieve muscle pain, restore the body balance, improve blood circulation, promote perineum wound healing and uterine involution.8 ASPNN decoction has been used in Ayurved Siriraj Clinic of Applied Thai Traditional Medicine. However, the decoction is inconvenient and difficult to drink. The preparation process is also difficult to control when the participants were at home. Therefore, ASPNN capsule was used in this study instead to ensure that every participant in ASPNN group receive similar intervention. According to the satisfaction survey, the capsule was well-accepted similarly to the placebo.

The advantage of this study was that we designed the experiment to minimize interference to the participants. The breastfeeding was encouraged but voluntarily performed. The milk collection was done only once a day to avoid disturbing baby suckling. Maternal food and water intake were monitored as these affects milk production. Not only the quantity of breast milk, the quality was also assessed. Although the results were not significant, the information can be used in future research.

The limitation of this study was that the main outcomes were determined on day 3 but ASPNN may produce significant benefits later. Although many factors affecting breast milk production and other outcomes were controlled or monitored in the current study (eg. frequency of infant suckling, proper latch-on techniques, monitoring of food and water intake), some uncontrollable factors still exist, such as maternal stress, exact food and water intake, and exact times of milk and blood collection. Future clinical trials should be performed with larger sample size and evaluate outcomes on day 7 afterward. The milk and blood collection times should be similar in all participants. Even though management of maternal

stress was routinely performed in the postpartum ward, it should also be assessed.

CONCLUSION

Short term supplementation of ASPNN produce no direct effect on breast milk volume, creamatocrit, and serum prolactin. It was safe and might help maintaining serum prolactin. A future trial with more participants and longer period should be conducted to confirm the effect of ASPNN on breast milk quantity and quality.

ACKNOWLEDGEMENT

The authors would like to thank Mr.Suthipol Udompunthurak (Division of Clinical Epidemiology, Office of Research and Development, Faculty of Medicine Siriraj Hospital) for advice in the statistical analysis and Dr. Manmas Vannabhum (Center of Applied Thai Traditional Medicine, Faculty of Medicine Siriraj Hospital) for suggestion in writing the manuscript.

Abbreviations

ASPNN: Ayurved Siriraj Prasa-Nam-Nom; PRL: Prolactin level; TTM: Thai traditional medicine

Funding

This study is supported by Siriraj Research Development Fund (Managed by Routine to Research: R2R), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Authors’contributions

PC : analyzed and interpreted the data and contributed in writing the manuscript.

CT : collected the data and performed creamatocit measurement.

AI : collected the data and performed creamatocit measurement.

NL : conducted the production of medicines, analyzed the data and contributed in writing the manuscript.

PA : contributed in planning the study protocol and provided information for data analysis.

TL : contributed in planning and development of this research.

SB : recruited the participants, collected blood and breast milk, and gave the treatment.

NK : recruited the participants, collected blood and breast milk, and gave the treatment.

SP : performed Prolactin level assessment.

AN : contributed in planning and development of this research.

TT : analyzed the data and contributed in planning the study protocol and in writing the manuscript.

All : authors read and approved the final manuscript.

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4. Chanprapaph P, Luttarapakul J, Siribariruck S, Boonyawanichkul

   S. Outcome of non-protractile nipple correction with breast cups in pregnant women: a randomized controlled trial. Breastfeed Med. 2013;8(4):408-12.

5. Li R, Fein SB, Chen J, Grummer-Strawn LM. Why mothers stop breastfeeding: mothers’ self-reported reasons for stopping during the first year. Pediatrics. 2008;122 Suppl 2:S69-76.

6. Brown CR, Dodds L, Legge A, Bryanton J, Semenic S. Factors influencing the reasons why mothers stop breastfeeding. Can J Public Health. 2014;105(3):e179-85.

7. Domperidone. Drugs and Lactation Database (LactMed). Bethesda (MD): National Library of Medicine (US); 2006.

8. Suwannatrai U, Tungsukrutai P, Visesmanee S, Stieanrut P, Boriboonhirunsarn D, Akarasereenont P, et al. The efficacy of hot salt pot compression in postpartum women: a randomized controlled trial. J Thai Trad Altern Med. 2014;12(2):144-58.

9. Maya D, Bale B, Samson G, Basile T, Raymond B. Phytochemical composition, toxicity, antioxidant and lactogenic activities of Euphorbia hirta (L.). Int J of Adv Res. 2018;6(8):322-35.

10. Koko B, Konan A, Kouacou F, Djétouan J, Amonkan A. Galactagogue effect of Euphorbia hirta (Euphorbiaceae) aqueous leaf extract on milk production in female Wistar rats. JBM. 2019;7(9).

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12. Jankaew K, Narumitmontri T. The effects of herbal milk nourishing medication on the levels of milk flow in postpartum mothers. TJPHS. 2020;3(2):41-51.

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14. Lin HY, Hsieh HY, Chen HH, Chiu HY, Lin HC, Su BH. Efficacy of creamatocrit technique in evaluation of premature infants fed with breast milk. Pediatr Neonatol. 2011;52(3):130-4.

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17. Grzeskowiak LE, Wlodek ME, Geddes DT. What evidence do we have for pharmaceutical galactagogues in the treatment of lactation insufficiency?-a narrative review. Nutrients. 2019;11(5):974.

Clinical Characteristics of Female Patterned Hair Loss in Patients Attending Hair Clinic in Thailand

Warayuwadee Amornpinyo, M.D., Rattapon Thuangtong, M.D., Supisara Wongdama, M.D., Daranporn Triwongwaranat, M.D.

Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

ABSTRACT

Objective: To study the clinical features and associated factors of female pattern hair loss (FPHL) in premenopausal and menopausal women patients.

Materials and Methods: This is a retrospective chart review of FPHL patients visited hair clinic, Siriraj Hospital from June 2012 to May 2015. Demographic data, family history and history of hair loss were evaluated. Factors associated with FPHL were analysed.

Results: There were 267 patients (180 premenopausal women and 87 menopausal women) in this study. The mean age of onset of patients was 35.5±12 years (premenopausal FPHL) and 60.5±7 years (menopausal FPHL). Positive family history of androgenetic alopecia (AGA) was 48.3%, mainly in first-degree relatives. The data showed an increased incidence of FPHL with advancing age. The most common presentation is Ludwig grade I. The study showed that patients also have dyslipidemia (16.9%), hypertension (16.5%), diabetes mellitus (10.9%), hypothyroidism (4.9%), anemia (3.7%), and hyperthyroidism (2.9%). In multivariate analysis, significant associations were found between low ferritin level <70 µg/L and premenopausal FPHL (OR 5.51, 95% CI 2.26-15.14, P = 0.01).

Conclusion: Maternal family history of AGA seems to have a greater influence on premenopausal FPHL. Low serum ferritin levels <70 µg/L were significantly associated with FPHL in premenopausal women.

Keywords: Female pattern hair loss; androgenetic alopecia; family history; ferritin; premenopause; menopause (Siriraj Med J 2022; 74: 19-26)

Abbreviations

OR : Odds ratio

CI : confidence Interval

INTRODUCTION

Female pattern hair loss (FPHL) is a common cause of non-scarring alopecia, resulted from the miniaturization of hair follicles. This condition presents as a diffuse hair loss and prominent in the frontal, crown and parietal areas. In contrast to other scalp diseases such as tinea capitis and scalp psoriasis, the patients typically present with patchy hair loss and erythematous plagues with silvery scales, respectively.1,2 Patients with FPHL have a lower

self-image and a lower quality of life. The prevalence of FPHL is varied among ethnicities and increases with age.3 The overall prevalence is lower in the Asian population (5.6% in the Korean study of Paik et al.)3 compare to Caucasians (19% in the US study of Mansouri et al.).4 In addition, one study recently conducted in Thailand reported the prevalence of disorders of skin appendages was 1.0% during the five-year period.5

Corresponding author: Daranporn Triwongwaranat Email: d.triwongwaranat@gmail.com

Received 29 September 2021 Revised 22 October 2021 Accepted 1 November 2021 ORCID ID: https://orcid.org/0000-0001-7388-1249 http://dx.doi.org/10.33192/Smj.2022.3

The pathophysiology of this condition is not well established. The role of androgens in FPHL is different from male pattern hair loss. Individuals with 5-α reductase deficiency or androgen insensitivity syndrome still have FPHL.6 Estrogen may also play a protective role through inhibition of 5-α reductase which explain the higher prevalence in menopausal group.7 Other findings that associated to this condition such as insulin resistance, metabolic syndrome, coronary artery disease, hypothyroidism, hyperprolactinemia, breast feeding, ultraviolet light exposure were reported.8-12 The diagnosis can be made mainly by history taking and physical examination. Skin biopsy is not necessary except for differentiating it from other causes of hair loss. The laboratory evaluations for thyroid function, ferritin level, and androgen state are essential in some cases with suspicious history.

There were mostly publications about the prevalence and risk factors related to FPHL in Western and few Asian studies from South Korea, Taiwan, China and India.3,10,13,14 As prominent differences in the prevalence and factors of female pattern hair loss in difference countries and race are known. This study investigates about these aspects in different backgrounds which might have various impacts on the effects of these factors. For providing more information of FPHL management in the future.

MATERIALS AND METHODS

Study design

A retrospective chart review of female pattern hair loss patients visited hair clinic, Department of Dermatology, Siriraj Hospital, Mahidol University, a tertiary center in Bangkok, Thailand from June 2012 to May 2015 was performed. The study protocol was approved by the Siriraj Institutional Review Boards. The diagnosis of female pattern hair loss was made by a dermatologist based on history taking, physical examination of pattern of hair loss based on Ludwig type and excluded other causes of hair loss. The data obtained from medical records including demographic data, family history of pattern hair loss, medical history, underlying disease, menstruation history, pregnancy and lactation history in past 12 months, significant weight loss in 6 months, features of hyperandrogenism (e.g. seborrhea, severe acne, infertility, hirsutism, overweight), detailed history of hair loss, physical examination by using Ludwig’s classification and laboratory investigation (hemoglobin, hematocrit, creatinine, HbA1C, thyroid stimulating hormone, serum ferritin, antinuclear antibody, antithyroid antibody) at diagnosis were noted.

Sample size calculation and statistical analysis

Since no previous study compared clinical characteristics between premenopausal and menopausal FPHL patients, the sample size was calculated based on the family history of AGA in FPHL patients. Previous studies in Korean and China reported 45.2% and 32.4% of FPHL patients had family history of AGA, respectively.3,15 The estimated family history of AGA in FPHL patients was 40% with 6% error, therefore a total sample size of 257 patients was needed.

Data was analysed using statistical package for social science (SPSS) software version 18 statistical programs. The chi-square test was performed to compare categorical data. P values using the traditional cutoff of P less than

0.05 were calculated to determine statistical significance. A logistic regression and multivariable regression model were used to evaluate the relationship of each possible risk factor to FPHL.

RESULTS

There were 316 patients with a medical record diagnosis of ICD10 L64.9 Androgenetic alopecia, unspecified in Hair clinic, Department of Dermatology, Siriraj Hospital, Mahidol University from June 2012 to May 2015. The subjects were divided into 2 groups by the first presentation of FPHL, premenopause and menopause. Premenopause is defined as the whole of the reproductive period before the menopause, while menopause is defined as the permanent cessation of menstruation.16 Of the 316 patients, 49 patients diagnosed with alopecia areata were excluded. 267 eligible patients (180 premenopausal women and 87 menopausal women) met the criteria for this study.

Characteristics of FPHL

Of the 267 patients, the mean age of hair loss onset among all patients was 43.5±15 years. The mean age of onset of premenopausal FPHL patients was 35.5±12 years, while 60.5±7 years was noted in menopausal FPHL patients (Table 1).

Family history

Both male and female relatives with androgenetic alopecia (AGA) counted as family history. A positive family history of AGA was found in 129 of 267 (48.3%) patients and mainly 121 of 267 (45.3%) patients noted

in history of first-degree relatives. 66 of 267 (24.7%) patients reported a family history of AGA in paternal relatives, 27 of 267 (10.1%) patients in maternal relatives, 36 of 267 (13.5%) patients in both paternal and maternal relatives and 138 of 267 (51.7%) patients had no family history of AGA (Table 1).

TABLE 1. Characteristics of female pattern hair loss patients: Medical profiles

*Significant at P = 0.05, Data expressed as mean±SD or n (%); AGA, androgenetic alopecia; OCP, oral contraceptive pill.

Association between FPHL and possible risk factors

In the univariate analysis, many factors were associated with having early onset FPHL. Statistically significant associations were noted between premenopausal FPHL and these factors: dyslipidemia (OR 0.15, 95% CI 0.07-

0.30, P = 0.01), diabetes mellitus (OR 0.14, 95% CI 0.06-

0.35, P = 0.01), hypertension (OR 0.01, 95% CI 0.03-0.15,

P = 0.01), family history of AGA (OR 1.72, 95% CI 1.02-

2.90, P = 0.04), maternal family history (OR 3.06, 95% CI 1.02-9.14, P = 0.03), low level of thyroid stimulating hormone <0.27 µIU/ml (OR 0.14, 95% CI 0.03-0.77,

P = 0.01), low ferritin level <70 µg/L (OR 2.81, 95% CI

1.15-6.86, P = 0.02) and positive antinuclear antibody (OR 0.16, 95% CI 0.03-1.06, P = 0.04) (Table 2).

Independent factors were analysed by multivariable regression model. Significant associations were found between low ferritin level <70 µg/L and premenopausal FPHL (OR 5.51, 95% CI 2.26-15.14, P = 0.01) (Table 3).

Past medical history

There were 2 patients (0.8%) with each history of postpartum and lactation within 1 year and only one patient (0.4%) with significant weight loss in 6 months. 14 of 267 (5.2%) patients reported history of taking oral contraceptive pill within 6 months. Regular menstruation was found in 223 of 267 (83.2%) of patients. Only 6 patients (2.3%) had got the features of hyperandrogenism. There were common medical problems among patients included dyslipidemia (16.9%), hypertension (16.4%),

diabetes mellitus (10.9%), hypothyroidism (4.9%), anemia

(3.7%) and hyperthyroidism (2.9%) in respectively

(Table 1).

Previous treatment history

50 of 267 (18.7%) patients reported history of previous treatment for FPHL. Topical minoxidil was used in majority of patients (18.4%) and one patient (0.4%) had been treated with oral antiandrogen (Table 1).

History of hair loss

For most patients, the time from hair loss onset to seeing a hair specialist was longer than one year. There was previous history of hair manipulation in 32 of 267 (11.9%) patients including hair coloring (8.6%),

curling (7.5%), straightening (3.8%) and hair dryer (0.4%)

(Table 1).

Overall, the age-specific prevalence of female pattern hair loss (FPHL) increased with advancing age and peaked in age group 50-59 years (24.7%). Most patients (61.0%) were Ludwig grade I, followed by grade II (29.2%) and

grade III (9.7%). The age-specific prevalence of severe hair loss (Ludwig grade ≥II) was relatively increasing; however, it was slightly lower in those aged 40-49 years. There were mostly recorded Ludwig grade I in patients with age group 0-19 years and Ludwig grade ≥ II in age group 50-59 years (Fig 1).

DISCUSSION

As prominent differences in the prevalence and factors of female pattern hair loss in difference countries and race are known. No study regarding the clinical characteristics and factors associated with FPHL in Thailand has been reported. This study investigated in difference background which might have various impacts on the effects of these factors. In this study demonstrated the trend of increased incidence of FPHL with advancing age and Ludwig grade I was the most common type, similar to previous studies in other population.3,15,17-22 Ludwig grade ≥ II were more common in patients aged ≥50 years. This may be due to the fact that less cosmetically concern about hair loss in older adults and higher Ludwig grade with advancing age may be influenced by the duration of disease progression. Although most patients in this study were premenopausal cases, the trend of increased incidence of FPHL with advancing age was observed.

FPHL can occur in any time of life. The mode of inheritance in AGA has not been characterised. There were previous reports of higher prevalence of AGA in patients with paternal rather than maternal inheritance, similarly to this study.20,23 Moreover, female patients were more likely to have positive maternal family history.23,24 Specifically, this study demonstrated that positive maternal family history was statistically significantly associated with premenopausal FPHL. This may be due to some inherited genes from maternal side. Further studies are needed to determine the association of early onset FPHL and family history of AGA in FPHL patients. The youngest patient with a positive maternal family history of AGA was 16 years old. These results suggested that paternal family history of AGA has more effect on FPHL than maternal family history of AGA. However, maternal family history of AGA was seem to be greater influence in premenopausal FPHL.

Hypertension, insulin resistance and increased cardiovascular risk have been described associated with early onset FPHL compared with healthy subjects.9 Possible explanations were higher aldosterone, C-protein, D-dimer and insulin levels in women with FPHL.9 This study demonstrated that menopausal women with FPHL had higher rate of hypertension, dyslipidemia, diabetes mellitus. There may be explained by more commonly

TABLE 2. Univariate analyses of risk factors associated with early onset female pattern hair loss

*Significant at P = 0.05, Data expressed as mean±SD or n (%); OR, odds ratio; CI, confidence interval

**Positive ANA pattern in premenopausal FPHL including fine speckle with titer of 1:640, fine speckle with titer of 1:160, fine speckle with titer of 1:100, fine speckle with borderline titer, borderline ANA, titer of 1:2560 with no data of ANA pattern and no data of ANA pattern and titer

***Positive ANA pattern in menopausal FPHL including fine speckle with titer of 1:640, fine speckle with titer of 1:320, nucleolar with titer of 1:320, fine speckle with borderline titer and borderline ANA

AGA, androgenetic alopecia; OCP, oral contraceptive pill; TSH, thyroid stimulating hormone; ANA, antinuclear antibody

TABLE 3. Multivariate analyses of risk factors associated with early onset female pattern hair loss

*Significant at P = 0.05, Data expressed as mean±SD or n (%); OR, odds ratio; CI, confidence interval AGA, androgenetic alopecia; OCP, oral contraceptive pill

Fig 1. Age-specific prevalence of female pattern hair loss (FPHL)

increasing frequency of those in elderly patients. However, further comparative study will be needed to evaluate the association between hypertension, insulin resistance and increased cardiovascular risk to FPHL.

Previous studies have noted the relationship of iron deficiency and FPHL. Whether defined iron deficiency by using serum ferritin level, the definition has ranged from serum ferritin level less than or equal to 15 μg/L, less than or equal to 40 μg/L. The sensitivity of using

those cut-off level of serum ferritin level was 59%, 98% in respectively, with about 99%, 98% specificity based on comparison with complete absence of iron staining on bone marrow aspiration.25-27 Coenen et al.25 noted that all patients with serum ferritin less than 70 μg/L had a lack of macrophage and/or sideroblast iron stains on bone-marrow aspiration and would be considered to have iron deficiency anemia. Rushton et al.28 reported that there was increased scalp hair shedding and decreased

hair volume in women with serum ferritin less than of equal to 70 μg/L. In 2003, Kantor et al.29 found a lower mean serum ferritin level in FPHL patients who was under 40 years old (mean = 37.3 μg/L; n = 52) than control subjects (mean = 59.5 μg/L; n = 11). In this study, mean level of ferritin in FPHL patients was higher (mean = 212.15±206 μg/L) compared to previous studies.29,30 There did seem to be lower mean of ferritin level in menopausal (mean = 84.94±100.9 μg/L; n = 87) than premenopausal (mean = 201.67±248.5 μg/L; n = 180) FPHL patients. The results could be related to differences in the diet, genetic, ethnic variations of the study populations and lack of data pertaining to FPHL in our country. Furthermore, this study demonstrated that serum ferritin levels <70 µg/L were significantly associated with FPHL in premenopausal women which was similar to previous studies.28,31 However, some studies did not support the association between FPHL and iron deficiency.32-34 No established guideline for routine investigations and role of iron supplement therapy in women with FPHL. Due to various cut-off levels of ferritin, we commonly prescribed iron supplement to FPHL patients with serum ferritin levels <70 µg/L based on previous studies25,28,34 and several reasons. First, cut-off level of ferritin >70 could cover more patients with iron deficiency anemia. Moreover, we also found that giving iron supplement in FPHL patients with serum ferritin level <70 µg/L helped their hair regrowth and decreased hair shedding. However, additional comparative study with larger sample sizes of the correlation between FPHL and iron deficiency status or responsiveness of oral iron supplement are necessary to answer the efficacy of iron supplement therapy in FPHL patient.

The limitation of the present study are some missing data due to retrospective approach. Furthermore, the study is from a single center and may not accurately represent the general population. Further case control design or population based descriptive study helps to identify the exact prevalence and risk factors of FPHL patients.

CONCLUSION

In conclusion, this study described clinical characteristics of FPHL and the factors associated with FPHL in premenopausal and menopausal female. Maternal family history of AGA was seem to be greater influence in premenopausal FPHL. Moreover, low serum ferritin levels <70 µg/L were significantly associated with FPHL in premenopausal women.

Conflicts of Interest: None declared

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4. Mansouri P, Mortazavi M, Eslami M, Mazinani M. Androgenetic alopecia and coronary artery disease in women. Dermatol Online J 2005;11:2.

5. Chaiyamahapurk S, Warnnissorn P. Prevalence and Pattern of Diseases of The Skin and Subcutaneous Tissue in A Primary Care Area in Thailand. Siriraj Med J. 2021;73:357-62.

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7. Brough KR, Torgerson RR. Hormonal therapy in female pattern hair loss. Int J Womens Dermatol. 2017;3:53-7.

8. Arias-Santiago S, Gutierrez-Salmeron MT, Buendia-Eisman A, Girón-Prieto MS, Naranjo-Sintes R. Hypertension and aldosterone levels in women with early-onset androgenetic alopecia. Br J Dermatol 2010;162:786-9.

9. Arias-Santiago S, Gutierrez-Salmeron MT, Castellote-Caballero L, Buendía-Eisman A, Naranjo-Sintes R. Androgenetic alopecia and cardiovascular risk factors in men and women: a comparative study. J Am Acad Dermatol 2010;63:420-9.

10. Su LH, Chen LS, Chen HH. Factors associated with female pattern hair loss and its prevalence in Taiwanese women: a community-based survey. J Am Acad Dermatol 2013;69:e69- 77.

11. Vincent M, Yogiraj K. A Descriptive Study of Alopecia Patterns and their Relation to Thyroid Dysfunction. Int J Trichology 2013; 5:57-60.

12. Zaki MS, Ahmed IZ. Female pattern hair loss: the relation to metabolic syndrome in premenopausal women. J Egypt Woman Dermatol Soc 9:18-21.

13. Shilpashree P, Clarify S, Shashidhar T. An epidemiological study of female pattern hair loss at a referral centre in South India. Indian J Clin Exp Dermatol 2016;2:106-10.

14. Wang TL, Zhou C, Shen YW, Wang XY, Ding XL, Tian S, et al. Prevalence of androgenetic alopecia in China: a community- based study in six cities. Br J Dermatol 2010;162:843-7.

15. Xu F, Sheng YY, Mu ZL, Lou W, Zhou J, Ren YT, et al. Prevalence and types of androgenetic alopecia in Shanghai, China: a community-based study. Br J Dermatol 2009;160:629-32.

16. Sherman S. Defining the menopausal transition. Am J Med. 2005;118 Suppl 12B:3-7.

17. Singal A, Sonthalia S, Verma P. Female pattern hair loss. Indian J Dermatol Venereol Leprol. 2013;79:626-40.

18. Gan DC, Sinclair RD. Prevalence of male and female pattern hair loss in Maryborough. J Investig Dermatol Symp Proc 2005;10: 184-9.

19. Ramos PM, Miot HA. Female Pattern Hair Loss: a clinical and pathophysiological review. An Bras Dermatol. 2015;90:529-43.

    
    

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22. Ravikiran SP, Syrti C, T S, An epidemiological study of female pattern hair loss at a referral centre in South India. Indian J Clin Exp Dermatol. 2016;2:106-10.

23. Lee WS, Lee HJ. Characteristics of androgenetic alopecia in asian. Ann Dermatol. 2012;24:243-52.

24. Salman KE, Altunay IK, Kucukunal NA, Cerman AA. Frequency, severity and related factors of androgenetic alopecia in dermatology outpatient clinic: hospital-based cross-sectional study in Turkey. An Bras Dermatol 2017;92:35-40.

25. Coenen JL, van Dieijen-Visser MP, van Pelt J, van Deursen CT, Fickers MM, van Wersch JW, et al. Measurements of serum ferritin used to predict concentrations of iron in bone marrow in anemia of chronic disease. Clin Chem 1991;37:560-3.

26. Hallberg L, Bengtsson C, Lapidus L, Lindstedt G, Lundberg PA, Hultén L. Screening for iron deficiency: an analysis based on bone-marrow examinations and serum ferritin determinations in a population sample of women. Br J Haematol 1993;85:787- 98.

27. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood 1997;89:1052-7.

28. Rushton DH, Ramsay ID. The importance of adequate serum ferritin levels during oral cyproterone acetate and ethinyl oestradiol treatment of diffuse androgen-dependent alopecia in women. Clin Endocrinol (Oxf) 1992;36:421-7.

29. Kantor J, Kessler LJ, Brooks DG, Cotsarelis G. Decreased serum ferritin is associated with alopecia in women. J Invest Dermatol 2003;121:985-8.

30. Raichur S, Pandit A, Malleshappa A. Correlation of serum ferritin levels, in female patients with chronic diffuse hair loss: A cross sectional study. Indian J Health Sci Biomed Res. 2017; 10:190-5.

31. Park SY, Na SY, Kim JH, Cho S, Lee JH. Iron plays a certain role in patterned hair loss. J Korean Med Sci. 2013;28:934-8.

32. Bregy A, Trueb RM. No association between serum ferritin levels >10 microg/l and hair loss activity in women. Dermatology 2008;217:1-6.

33. Sinclair R. There is no clear association between low serum ferritin and chronic diffuse telogen hair loss. Br J Dermatol 2002;147: 982-4.

34. Trost LB, Bergfeld WF, Calogeras E. The diagnosis and treatment of iron deficiency and its potential relationship to hair loss. J Am Acad Dermatol 2006;54:824-44.

    
    

    Aflibercept as Adjunctive Treatment for Filtration Surgery in Neovascular Glaucoma

    
    

    

    

    

    Naris Kitnarong, M.D., MBA., Janyawassamon Kittipiriyakul, M.D., Anuwat Jiravarnsirikul, M.D.

    Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

    
    

    
    

    ABSTRACT

    Objective: To investigate intravitreal aflibercept (IVA) injection as an adjunctive treatment to trabeculectomy with mitomycin C (TMC) and panretinal photocoagulation (PRP) for neovascular glaucoma (NVG).

    Materials and Methods: PRP and IVA (2 mg/0.05 ml) injection were given, and TMC was performed within 2 weeks after IVA. Additional PRP, laser suture lysis, subconjunctival 5-fluorouracil injection, and bleb needling were performed after TMC if indicated. Best corrected visual acuity (BCVA), intraocular pressure (IOP), surgical complications, and number of anti-glaucoma medications were collected.

    Results: Five eyes from 5 consecutive patients were included. Two eyes had proliferative diabetic retinopathy (PDR), 2 central retinal vein occlusion, and 1 ocular ischemic syndrome (OIS) (mean initial IOP: 46.8±6.8 mmHg). NVI regression occurred in one eye after PRP alone, and in one eye after PRP and IVA resulting in a good IOP control with topical medical therapy. The other 3 underwent TMC. The preoperative IOP was 34 (OIS), 54 (PDR), and 50 (PDR) mmHg. The 3-month postoperative IOP decreased to 8, 8, and 4 mmHg, respectively, and to 21, 10, and 6 mmHg, respectively, at the last visit. Only the one OIS eye required postoperative topical IOP-lowering medications. Final BCVA was improved, unchanged, and decreased in 2, 2, and 1 eye, respectively. No intraoperative/postoperative complications or NVI recurrence were observed (mean follow-up: 10.7 months).

    Conclusion: Intravitreal aflibercept was shown to be a potentially effective additional treatment to PRP and TMC in patients with NVG.

    
    

    Keywords: Aflibercept; adjunctive treatment; filtration surgery; neovascular glaucoma (Siriraj Med J 2022; 74: 27-33)

    
    

    INTRODUCTION

    Neovascular glaucoma (NVG) is a devastating secondary glaucoma characterized by the development of neovascularization of the iris (NVI) and anterior chamber angle (NVA), which causes obstruction of aqueous outflow and increased intraocular pressure. It is estimated that 97% of NVG patients develop neovascularization as a result of posterior segment ischemia.1,2 The common causes of this condition include proliferative diabetic retinopathy (PDR), central retinal vein occlusion (CRVO), and ocular ischemic syndrome (OIS).1,3 Ocular ischemia

    leads to the production of pro-angiogenic factors that diffuse into the anterior segment and cause NVI, NVA, and fibrovascular membranes.4 Vascular endothelial growth factor (VEGF) is one of the pro-angiogenic factors found in the ocular fluids of both PDR patients and NVG patients.4 Panretinal photocoagulation (PRP) is a standard treatment for retinal ischemic conditions that is combined with treatment of the underlying disease.5 However, PRP causes the death of healthy retinal cells, permanently diminishes visual fields, and gradually regresses the neovascularization. The use of

    
    

    
    

    

    Corresponding author: Naris Kitnarong E-mail: naris.kit@mahidol.ac.th

    Received 26 June 2021 Revised 21 September 2021 Accepted 25 September 2021 ORCID ID: https://orcid.org/ 0000-0002-6041-4868 http://dx.doi.org/10.33192/Smj.2022.4

    
    

    anti-VEGF, such as bevazicumab and ranibizumab, has been reported to rapidly reduce the ischemic process, reverse neovascularization, and limit ocular tissue damage.6,7 Treatment of NVG with combination PRP, intravitreal anti-VEGF, and trabeculectomy with mitomycin C (TMC) was reported to reduce intraoperative bleeding and improve surgical outcomes.8-11 In 2011, the US Food and Drug Administration approved aflibercept (Eylea®; Regeneron, New York / Bayer, Berlin, Germany) for the treatment of neovascular age-related macular degeneration (AMD).12 Aflibercept is a novel recombinant fusion protein that is made up of portions of vascular endothelial growth factor receptor 1 (VEGFR 1) and VEGFR 2 fused to the Fc portion of human immunoglobulin G1 and VEGF- receptor ligand binding elements. Aflibercept exhibits higher affinity for VEGF-A/-B compared to previously- known anti-VEGF molecules, and binds to all of the VEGF isoforms (VEGF-B and -C, placental growth factor-1/-2).13 The aim of this study was to investigate the effects of intravitreal aflibercept (IVA) injection as an adjunctive treatment to TMC and PRP in patients with NVG.

    
    

    MATERIALS AND METHODS

    This prospective study included NVG patients who consecutively presented at the Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand during November 2018 to October 2019. The study protocol was reviewed and approved by the Siriraj Institutional Review Board (SIRB) (certificate of approval number Si726/2018). Written informed consent was obtained from all study patients prior to participation. All patients received PRP and maximum tolerated IOP reduction, including systemic acetazolamide. Aflibercept was injected intravitreally (2 mg in 0.05 ml) through the pars plana in the operating theater within 1 week after the first session of PRP if NVI still persisted. TMC was carried out within 2 weeks after IVA in eyes with intraocular pressure (IOP) greater than 21 mmHg with maximal anti-glaucoma medications.

    
    

    Surgical technique

    All surgeries were performed by NK. The operated eye was draped and then a lid speculum was placed. Xylocaine hydrochloride 2% was injected subconjunctivally at the superonasal quadrant. A fornix-based conjunctival flap was created by disinsertion of the conjunctiva and Tenon’s capsule at the 2-3 o’clock position. Oblique relaxing incisions at one side facilitated adequate scleral exposure. Diathermy was applied to control the hemostasis of the sclera. A no.15 surgical blade was used to create a partial

    thickness triangular scleral flap 3.5 (base) x 3.5 (height) mm. The cellulose sponge soaked with mitomycin C (MMC) 0.4 mg/ml was placed over the sclera under the Tenon’s capsule and the conjunctiva for 2-3 minutes. The duration of MMC application was based upon the preoperative evaluation of potential risks for surgical failure, including the status of the conjunctiva over the operating area and the patient’s age. Generally, a thicker Tenon’s capsule and/or a younger age underwent a longer application of MMC. The MMC at the surgical area was then extensively washed out with balance saline solution (BSS).

    A 20-gauge needle was used to performed paracentesis at the nasal or temporal clear cornea. The sclerotomy was done using a 15-degree blade. A Kelly Descemet’s punch may be needed to widen the sclerotomy for an adequate size. Peripheral iridectomy was performed then the scleral flap was sutured with 10-0 nylon sutures at its apex and sides. The number and the location of sutures were customized for each patient to facilitate optimal aqueous outflow. The fornix-based conjunctival flap was repositioned to the limbus, tightly anchored with round needle 10-0 nylon sutures, and the relaxing incisions were closed with a continuous suture. The surgical area was tested for leaks before the injection of subconjunctival dexamethasone.

    
    

    Postoperative procedure

    A combination of topical 1% prednisolone and antibiotics were administered 4-6 times a day for 7 days. A topical eye drop containing a combination of antibiotics and dexamethasone was applied 4 times a day for the following 1 month, and then reduced to twice a day thereafter. Best corrected visual acuity (BCVA), IOP, number of anti-glaucoma medications, and the present of NVI were compared between pre- and post- IVA, and before and after TMC. The intraoperative and postoperative complications were also noted. The patients were scheduled for follow-up at 1 day, 1 week, and every 4-8 weeks. Each postoperative visit involved a full eye examination and Seidel test for the presence of bleb leak. Postoperative interventions such as laser suture lysis, 5-fluorouracil (5-FU) injection, bleb needling, or any other procedures were performed under surgeon (NK) consideration.

    
    

    RESULTS

    Five eyes from 5 patients with a mean age of 70.8 years were enrolled in this study during the November 2018 to August, 2019 study period. One patient was male and 4 patients were female. The underlying causes of NVG

    
    

    included 2 eyes with PDR, 2 eyes with CRVO, and 1 eye with OIS. The eye with OIS underwent several sessions of PRP before participation. The partial regression of NVI had been observed. The mean IOP at presentation was 46.8±6.8 mmHg. After the first session of PRP in eyes without prior PRP, regression of NVI observed in one eye with CRVO, and the IOP was controlled with topical anti-glaucoma medications. The remaining 4 eyes received IVA due to the minimal regression and the persistence of NVI. The mean IOP among those 4 eyes was 37.9 mmHg before IVA, and 36.1 at 1 week after IVA. There was no observed complication related to IVA. Iris neovascularization was absolutely regressed within 1 week in all 4 eyes after IVA. Intraocular pressure was controlled in 1 eye with CRVO after PRP and IVA with maximal topical therapy. The remaining 3 eyes (2 PDR, 1 OIS) underwent TMC. The preoperative IOP of each patient was 34 (OIS), 54 (PDR), and 50 (PDR) mmHg (mean: 46 mmHg). Postoperatively, the IOPs decreased to 8, 8, and 4 mmHg (mean: 6.7 mmHg), respectively,

    at 3 months, and to 21, 10, and 6 mmHg (mean: 12.3 mmHg), respectively, at the last visit. Only the eye with OIS required postoperative medications. The mean follow-up among all five operated eyes was 10.7 months. No intraoperative or postoperative complication was noted. The BCVA was improved in 2 eyes, unchanged in 2 eyes, and decreased in 1 eye. There was no recurrence of NVI or significant symptoms in any of the 5 study eyes. Pre- and post-procedural characteristics compared among the 5 study eyes/patients are shown in (Table 1). Change in IOP over time from baseline compared among the 3 patients who underwent TMC is shown in (Fig 1).

    
    

    DISCUSSION

    The primary goal of management of NVG associated with ischemic retinopathy is to control IOP and the ischemic conditions of the retina. The treatments for NVG consist mainly of PRP, endolaser, or retinal cryotherapy to decrease oxygen consumption by ischemic retina.4 PRP is a major procedure that involves ablating the retina to control retinal ischemia, which results in regression of neovascularization of both the anterior and posterior segments.5 However, the regression of ocular neovascularization may not be rapid enough to limit further ocular tissue damage, such as the peripheral anterior synechiae, and NVG. Among the 5 cases in our series, regression of NVI occurred after PRP alone in one eye, and after combination PRP and IVA in one eye. IOP could be controlled in these 2 eyes with topical IOP lowering medications alone. This may be explained by the fact that prompt treatment in the early stage

    of the disease to eliminate the progression of ocular neovascularization can prevent further ocular complications. The effects of intraocular administration of anti-VEGFs in NVG included rapid regression of neovascularization, IOP reduction, and improved surgical outcome.6,8-11 In several previous reports, the regression of NVI usually observed within 1 week after administration of anti- VEGF.14,15 This effect may persist for several weeks (4 to 28 weeks). According to the half-life of anti-VEGFs and their transitory effects, multiple injections may be needed. The transient effect of anti-VEGF underscores the demand for a simultaneous treatment to eradicate the source of angiogenic factors, such as retinal photocoagulation. The combined treatment of intravitreal anti-VEGF and retinal ablation to control the retinal ischemic condition is theoretically more effective.16 Anti-VEGF rapidly, but transiently reduces neovascularization in the early treatment stage, whereas PRP prolongedly controls ischemia of the retina over the long-term. In addition, intravitreal anti-VEGFs have demonstrated their benefit as an alternative treatment for NVG patients in whom retinal ablation could not be applied due to inadequate visualization. We considered administering anti-VEGF as soon as possible, especially in eyes in which the status of the ocular media contraindicates retinal treatment. Our group previously reported that application of PRP and anti-VEGF may be considered simultaneously during the same visit to expedite the regression of neovascularization.11 Bevacizumab (Avastin®; Genentech, Inc., South San Francisco, CA, USA) is a recombinant humanized monoclonal antibody to VEGF. It is approved as an anti-angiogenic agent for the treatment of metastatic colorectal cancer in combination with chemotherapy.17,18 Off-label use of intraocular injection of bevacizumab has been reported as a treatment for macular degeneration, PDR, retinal vascular occlusive disorder, and NVG.6,7,19 Ranibizumab (Lucentis®; Genentech) is a recombinant humanized antibody antigen-binding fragment (Fab) that was approved for the treatment of neovascular AMD by the US Food and Drug Administration due to its ability to neutralize all active forms of VEGF-A.20,21 Intravitreal bevacizumab (IVB) and intravitreal ranibizumab (IVR) have been reported as adjunctive treatment in NVG not only to reduce rubeosis iridis, but also to improve the surgical outcome of glaucoma surgeries.8-11 Aflibercept is the most recently introduced anti-VEGF for the treatment of neovascular AMD.22,23 According to our review of the literature, aflibercept has not yet been widely studied as a treatment for NVG. The additional effect of this anti- VEGF compared to the previous anti-VEGFs is that it has a longer half-life, and a higher binding affinity for

    
    

    
    

    

    
    

    TABLE 1. Pre- and post-procedural characteristics compared among the 5 study eyes/patients.

    
    

    
    

    Case number	1	2	3	4	5
    Age (years)	80	73	78	54	69
    Gender	Female	Female	Female	Male	Female
    Lateralization	RE	LE	RE	LE	LE
    Underlying condition	CRVO	CRVO	OIS	PDR	PDR
    Initial BCVA (logMar)	1.0	1.6	1.0	2.0	2.0
    Initial IOP (mmHg)	42	52	36	54	50
    No. of medications	4	3	4	4	4
    Lens	IOL	NS2+	IOL	IOL	IOL
    Interventions	PRP	PRP/IVA	PRP/IVA/TMC	PRP/IVA/TMC	PRP/IVA/TMC
    Days between PRP and IVA	-	1	272	4	Same day
    BCVA at last visit (logMar)	1.0	1.2	1.0	0.3	3.0
    IOP at last visit (mmHg)	13	12	10	8	1
    Numbers of medications at last visit	4	3	3	0	0
    Follow-up (months)	6.0	1.1	31.6	20.2	17.0

    
    

    Abbreviations : RE, right eye; LE, left eye; CRVO, central retinal vein occlusion; OIS, ocular ischemic syndrome; PDR, proliferative diabetic retinopathy; BCVA, best-collected visual acuity; logMar, logarithm of the minimum angle of resolution; IOP, intraocular pressure; IOL, intraocular lens; NS2+, mild nuclear sclerosis cataract; PRP, panretinal photocoagulation; IVA, intravitreal aflibercept; TMC, trabeculectomy with mitomycin C

    
    

    

    
    

    Fig 1. Change in intraocular pressure (IOP) over time from baseline compared among patient 3 (ocular ischemic syndrome [OIS], patient 4 (proliferative diabetic retinopathy [PDR], and patient 5 (PDR) (Abbreviations: mmHg, millimeters of mercury; TMC, trabeculectomy with mitomycin C).

    
    

    VEGF-A. The use of aflibercept can, therefore, decrease the frequency of injections.24 Often times, IOP may not be adequately controlled with anti-glaucoma medications alone in recalcitrant NVG despite the regression of ocular neovascularization. This may due to permanent damage to the aqueous drainage channel. When this happens, surgical intervention is required. Three eyes in our series subsequently underwent TMC due to uncontrolled IOP after PRP and IVA. Our study demonstrated that aflibercept injected intravitreally before TMC resulted in prompt regression of NVI, which resulted in reduced intraoperative bleeding and good postoperative IOP control. Moreover, there was no recurrence of NVI during the mean follow-up of 10.7 months, and no complication related to IVA and TMC was observed. The eye with OIS had localized filtration bleb resulted in occasional IOP spike compared to the eyes with PDR. The OIS eye underwent bleb needling with 5-FU injection 3 times. This difference in IOP control and the filtration bleb morphology may due to the nature of underlying disease. Trabeculectomy has long been the gold standard surgical treatment for glaucoma.25,26 In NVG, the success of filtration surgery needs both the neovascularization control and wound healing modulation at the filtering site. Several studies have reported frequent intraoperative complications, subsequently poor surgical success of TMC in NVG, especially in eyes with persistent ocular neovascularization.2,27 The management of NVG with a combination treatment of PRP, intravitreal anti-VEGF injection, and TMC has been reported as an alternative option.8-11 In addition to its anti-VEGF property, anti- VEGF may provide an additional effect on the wound- healing modulation at the filtering area. Anti-VEGF influenced an inhibitory effect on fibroblast activity and wound healing response that may improve the surgical success of trabeculectomy in NVG.28,29 Our findings have shown the potential of IVA as an adjunctive treatment in filtration surgery for NVG. In 3 patients who underwent TMC, IVA induced preoperative regression of NVI, which minimized intraoperative bleeding and postoperative inflammation, and this resulted in the enhanced success of TMC. There were no significant local or systemic side effects associated with IVA in our series. That said, the side effects and complications from the use of anti-VEGF include central retinal arterial occlusion, endophthalmitis, retinal detachment, increased intraocular pressure, conjunctival hemorrhage, and cataract.30-33 Several studies reported short-term or long-term IOP elevation after intravitreal anti-VEGF injection.34,35 There was no clinically significant change in IOP observed in our study. The surgical result was encouraging in terms

    of IOP control and visual outcome. At last visit, all eyes had IOP under 21 mmHg with or without topical IOP lowering medications, and had improved or preserved visual acuity. Moreover, recurrence of neovascularization was not detected during the follow-up period (range: 16- 62 weeks). We hypothesized that in addition to reduced VEGF production from PRP and anti-VEGF injection, VEGF had a new drainage channel via trabeculectomy. The new VEGF drainage channel may explain the non- recurrence of neovascularization, the favorable surgical outcome, and the need for repeat anti-VEGF injection. However, owing to the short-term follow-up period in this study, the possibility of long-term recurrent neovascularization still exists. All eyes in our study demonstrated benefits from IVA and filtration at the last follow-up, including being symptom-free. Most of the eyes had retained visual acuity and had good IOP control.

    Preoperative IVA combined with TMC appears to be a safe and effective management for IOP controlling in NVG. Although its effect is temporary, aflibercept may provide an adjunctive effect to PRP because of its swift and dramatic biologic effect. The application of IVA may also be considered before performing the glaucoma drainage device surgery. The long-term outcome of this technique is still undetermined. The repeated application of IVA may be needed to achieve a long-term effect.

    
    

    Limitations and strengths

    Despite the small sample size, the short-term study period, and the lack of a control group, this is the first study to report a series of NVG eyes who underwent TMC after IVA. Further study with a larger sample size, a longer follow-up period with a control group should be conducted to evaluate the safety profile and the long- term efficacy of this technique.

    
    

    CONCLUSIONS

    Preoperative intravitreal administration of aflibercept was shown to be a potentially effective additional treatment to PRP and TMC in patients with NVG. Moreover, IVA was found to be safe and effective for controlling IOP and preserving visual acuity. The rapid regression of NVI minimized intraoperative complications during trabeculectomy, and may have improved the short- term surgical result after TMC. All eyes in this series remained symptom-free, preserved their visual acuity, and had good IOP control. A further study with a long- term follow-up in a larger study population needs to be conducted to evaluate the long-term outcomes.

    
    

    ACKNOWLEDGEMENTS

    The authors gratefully acknowledge the patients who participated in this study.

    Conflict of interest declaration

    All authors declare no personal or professional conflicts of interest, and no financial support from the companies that produce and/or distribute the drugs, devices, or materials described in this report.

    
    

    Funding disclosure

    This was an unfunded study.

    
    

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    12. Heier JS, Brown DM, Chong V, Korobelnik JF, Kaiser PK, Nguyen QD, et al. Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration. Ophthalmology. 2012;119(12): 2537-48.

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    14. Iliev ME, Domig D, Wolf-Schnurrbursch U, Wolf S, Sarra GM. Intravitreal bevacizumab (Avastin) in the treatment of neovascular glaucoma. Am J Ophthalmol. 2006;142(6):1054-6.

    15. Vatavuk Z, Bencic G, Mandic Z. Intravitreal bevacizumab for neovascular glaucoma following central retinal artery occlusion. Eur J Ophthalmol. 2007;17(2):269-71.

    16. Olmos LC, Sayed MS, Moraczewski AL, Gedde SJ, Rosenfeld PJ, Shi W, et al. Long-term outcomes of neovascular glaucoma treated with and without intravitreal bevacizumab. Eye (Lond). 2016;30(3):463-72.

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    21. Gillies MC, Campain A, Walton R, Simpson JM, Arnold JJ, Guymer RH, et al. Time to Initial Clinician-Reported Inactivation of Neovascular Age-Related Macular Degeneration Treated Primarily with Ranibizumab. Ophthalmology. 2015;122(3):589- 94 e1.

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    23. Bakall B, Folk JC, Boldt HC, Sohn EH, Stone EM, Russell SR, et al. Aflibercept therapy for exudative age-related macular degeneration resistant to bevacizumab and ranibizumab. Am J Ophthalmol. 2013;156(1):15-22 e1.

    24. Papadopoulos Z. Aflibercept: A review of its effect on the treatment of exudative age-related macular degeneration. Eur J Ophthalmol. 2019;29(4):368-78.

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    27. Kiuchi Y, Sugimoto R, Nakae K, Saito Y, Ito S. Trabeculectomy with mitomycin C for treatment of neovascular glaucoma in diabetic patients. Ophthalmologica. 2006;220(6):383-8.

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    34. Hoguet A, Chen PP, Junk AK, Mruthyunjaya P, Nouri-Mahdavi K, Radhakrishnan S, et al. The Effect of Anti-Vascular Endothelial Growth Factor Agents on Intraocular Pressure and Glaucoma: A Report by the American Academy of Ophthalmology. Ophthalmology. 2019;126(4):611-22.

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        A Study of Early Parenteral Nutritional Support and Factors Associated with Clinical Outcomes in Major Pediatric Burn Patients

        
        

        

        

        

        Weerawat Seubmora, M.D.*, Harikrishna K.R. Nair, M.D., Ph.D.**, Kusuma Chinaroonchai, M.D.***

        *Department of Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand, **Wound Care Unit, Department of Internal Medicine, Kuala Lumpur Hospital, Kuala Lumpur, Malaysia, ***Division of Trauma Surgery, Department of Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

        
        

        
        

        ABSTRACT

        Objectives: to study parenteral nutrition within 7 days post admission (early PN) and factors affecting clinical outcomes in major pediatric burn patients.

        Materials and Methods: A retrospective study was conducted regarding pediatric burn patients who had over 15% of their total body surface area (TBSA) with second- or third-degree burns. All the patients were classified as requiring early PN support or non-early PN support.

        Results: 124 major pediatric burns were reviewed. Eighty-six patients (65.2%) were male, and their median age was three years (0.3-15 years). Early PN showed no association with LOS (p=0.480) or a 30-day mortality (p=0.529). The children’s age, wound infections, and abdominal distension were the independent associated factors of LOS (p=0.025, 0.001, and 0.003 respectively). Pneumonia and urinary tract infection were independent factors associated with 30-day mortality (p=0.025 and N/A, respectively).

        Conclusions: Early PN in acute pediatric burns was not associated with LOS or 30-day mortality. It can be considered as options of nutritional support in acute, major pediatric burns. Effective management of wound infections and abdominal distension may reduce LOS.

        
        

        Keywords: Early PN; EN; major pediatric burn; nutritional support; treatment outcomes (Siriraj Med J 2022; 74: 34-39)

        
        

        INTRODUCTION

        Optimal nutritional support is considered one of the key treatments of major pediatric burns, since malnutrition and inadequate nutrient delivery have been associated with bad clinical outcomes.1,2 Moreover, children have limited macronutrient stores and relatively higher energy requirements than adults who are admitted into the intensive care unit; these factors can lead to substantial caloric and macronutrient deficits.3,4 Feeding is thought to

        attenuate the metabolic stress response, prevent oxidative cellular injury, and modulate immune responses. This has led to a shift from nutritional support as adjunctive care to an actual therapy in cases of major pediatric burns. The enteral route is preferred for providing nutrition.5 However, major pediatric burn patients are often too ill to be fed normally by mouth, and nasogastric or nasoduodenal tube-feeding is often not well- tolerated due to gastric dysmotility or ileus from acute, severe

        
        

        

        Corresponding author: Kusuma Chinaroonchai E-mail: doctorchinaroonchaik@hotmail.com

        Received 11 October 2021 Revised 14 October 2021 Accepted 25 October 2021 ORCID ID: https://orcid.org/0000-0002-7764-3368 http://dx.doi.org/10.33192/Smj.2022.5

        
        

        stress. Interruption of enteral feeding also frequently occurs for various reasons, such as medical or surgical contraindications, or radiologic, bedside, or surgical procedures.6 Severe burn injury causes a persistent and prolonged hypermetabolic state and increased catabolism, which result in increased muscle-wasting and cachexia. The metabolic rates of burn patients can surpass twice the norm, and failure to supply these energy requirements causes impaired wound healing, organ dysfunction, and susceptibility to infection. Accurate assessment and provision of nutritional needs is essential for these patients.7 Therefore, parenteral nutrition (PN) is often initiated to supplement the insufficient enteral intake. Published guidelines on the timing and thresholds of initiation, as well as the composition and doses of supplemental PN, vary widely.8-10 Moreover, concerns about overfeeding have led to even more uncertainty. A recent survey showed significant differences in nutritional practices in PICUs worldwide, in terms of macronutrient goals, estimation of energy requirements, timing of nutrient delivery, and thresholds for starting supplemental PN. The latest guideline suggests late initiation of PN support (after 7 days) in PICU due to the result of better treatment outcome and lower PN related complication which studied mainly in non-burn population.11,12

        
        

        Objective

        To study parenteral nutrition within 7 days post admission (early PN) and any factors with clinical outcomes in major pediatric burn patients

        
        

        MATERIALS AND METHODS

        Study design and population

        This was a retrospective study involving the review of pediatric burns charts for patients admitted to the Burn Unit at Siriraj Hospital in Bangkok from January, 1999 to September, 2019. This study was conducted under the Siriraj Institutional Review Board’s (SIRB) approval (COA no. Si 834/2019). Patients age ≤ 15 years who were injured ≥ 15% of their total body surface area (TBSA) with second- or third-degree burns were enrolled. All patients were given enteral nutrition (EN) as soon as they had a stable hemodynamic within 24 hours after admission with calculated goal of daily caloric requirement by using Curreri junior formula. The patients received all types of parenteral nutrition within seven days after admission to achieve calculated goal of daily caloric requirement; those children were classified as being in the early PN group. The others were in the non-early PN group. Patients with an incomplete medical record were excluded from analysis.

        Statistic analysis

        The data was analyzed by SPSS, version 18.0. The demographic data was presented in frequency, percentage, and median. The length of hospital stay (LOS) and 30- day mortality between the early PN and non-early PN groups were determined using the odds ratio (OR). The independent factors associated with the LOS were analyzed by using the linear regression coefficient, and adjusted factors were analyzed using the backward multiple linear regression model. Fisher’s exact test and the Mann- Whitney U test statistical analysis were performed with a univariable analysis of factors associated with 30-day mortality. The independent factors associated with 30- day mortality were reported in OR using binary logistic regression analysis, and the adjusted factors by using the backward multiple linear regression model. A P value of 0.05 was considered statistically significant.

        
        

        RESULTS

        124 patients’ data were analyzed. Twenty-two patients (17.7%) were identified in the early PN group, and 102 patients (82.3%) were in the non-early PN group. The medium age was three years old. The degree of burns were as follows: second- degree burns: 120 (96.8%) patients; third-degree burns: 1 (0.8%) patient; and mixed second- and third-degree burns: 3 (2.4%) patients. The median percentage of those with whose total body surface area was burned (%TBSA) was 30 (15, 90). The type of burns consisted of scald burns (87, or 70.2%); flame burns (36, or 29.0%); and other (1, or 0.8%). (Table 1)

        The comparisons of 30-day mortality and the LOS of patients who were early PN (n=22) vs. non-early PN (n=102) are reported in Table 2. There was no statistically significant difference in the 30-day mortality [1 (4.5%) vs 4 (3.9%), P=1.000] and median LOS [42 (12, 70) vs. 49 (2, 143), P=0.086] between the groups. The early PN group had a slightly shorter LOS.

        A multivariate logistic regression with forward selection revealed that early PN [-3.564 (-13.516, 6.388) (p=0.480)] had no statistically significant effect on the LOS, while wound infection [13.567 (5.734, 21.399)

        (p=0.001)], abdominal distension [13.460 (4.528, - 22.391)

        (p=0.003)], and age <1 year [0.817 (0.104, 1.530) (p=0.025)]

        significantly affected the LOS. (Table 3)

        The overall 30-day mortality of pediatric burns in our center was 17.7 %. A univariate analysis with Fisher’s exact test or the Mann-Whitney U test forward selection showed that the early PN group had no statistically significant effect on 30-day hospital mortality, in contrast to the non-early PN group [1 (20.0%) vs. 21 (17.6%) (p=1.000)]. However, a few factors did have a statistically

        
        

        
        

        

        
        

        TABLE 1. Characteristic of major pediatric burn patients.

        
        

        Characteristics	N = 124
        Sex, male, n (%)	82 (66.1%)
        Median age (min, max) (years)	3 (0.3, 15)
        Groups, n (%) Early PN	22 (17.7%)
        Non-early PN	102 (82.3%)
        Degree of burn, n (%) Second-degree	120 (96.8%)
        Third-degree	1 (0.8%)
        Second- & third-degree	3 (2.4%)
        Median % TBSA (min, max)	30 (15, 90)
        Burn type, n (%) Scald burn	87 (70.2%)
        Flame burn	36 (29.0%)
        Other	1 (0.8%)
        Abbreviations: PN = parenteral nutrition, % TBSA = % Total body surface area

        
        

        
        

        

        TABLE 2. Length of hospital stay (LOS) and 30-day mortality in the early PN vs. non-early PN groups of major pediatric burn patients.

        
        

        Outcome		Groups	p-value
        	Early PN (n=22)	Non-early PN (n=102)
        LOS (days) Median (min, max)	42 (12, 70)	49 (2, 143)	0.086
        30-day mortality, n (%)	1 (4.5%)	4 (3.9%)	1.000

        Abbreviations: PN = parenteral nutrition

        
        

        

        
        

        TABLE 3. Independent factors associated with Length of hospital stay (LOS).

        
        

        Factors	Unadjusted coefficient (95%CI)	p-value	Adjusted coefficient (95%CI)	p-value
        Early PN	-8.778(-19.077, 1.521)	0.094	-3.564 (-13.516, 6.388)	0.480
        Age < 1 year	1.248(0.525, 1.971)	0.001	0.817 (0.104, 1.530)	0.025
        Wound infection	16.652(8.887, 24.417)	<0.001	13.567 (5.734, 21.399)	0.001
        Abdominal distension	16.208(6.571, 25.846)	0.001	13.460(4.528, - 22.391)	0.003

        Abbreviations: PN = parenteral nutrition

        
        

        significant effect on 30-day mortality: the duration of ventilator use [8 (1, 34) vs. 0 (0, 55) (p<0.001)]; pneumonia

        [5 (100%) vs. 12 (10.1%) (p<0.001)]; sepsis [5 (100%) vs.

        16 (13.4%) (p<0.001)]; urinary tract infection [4 (80.0%) vs. 8 (6.7%) (p<0.001)]; and percentage of burn area [70 (43,90) vs. 28 (15,80) (p=0.002)]. (Table 4)

        A multivariate logistic regression with forward selection revealed that the early PN group [0.356 (0.014, 8.901) (p=0.529)] had no statistically significant effect on 30-day mortality, but urinary tract infection (UTI) [34.750 (1.641-735.686) (p=0.023)] and pneumonia

        did. (Table 5) Pneumonia had a statistically significant effect on 30-day mortality, but the number calculator reported this as non-shown (N/A) because the program could not identify this data.

        DISCUSSION

        Major pediatric burns in our center found more common in preschool age. They got injury from hot water and liquid more than flame. These findings are reported the same around the world except in our center male victims are more common.13,14 They may cause from normal pattern of preschool children development to explore new thing with touching and gripping anything without fear of danger due to lack of experience. Most of small children love to play with water, so kettle, pot and pan are thought to be one of the favorite toys for them. This awareness should be raised in parents for injury prevention purpose.

        Feeding intolerance is one of the common problems in critically ill trauma patients. Instability of hemodynamic,

        
        

        

        
        

        TABLE 4. Univariable analysis of factors associated with 30-day mortality in major pediatric burn patients.

        
        

        
        

        Factor 30-day mortality p-valuea
        	Yes (n=5)	No (n=119)
        Early PN, n (%)	1 (20.0%)	21 (17.6%)	1.000
        Duration of ventilator use Median (min, max)	8 (1, 34)	0 (0, 55)	<0.001
        Pneumonia	5 (100%)	12 (10.1%)	<0.001
        Sepsis	5 (100%)	16 (13.4%)	<0.001
        UTI	4 (80.0%)	8 (6.7%)	<0.001
        % TBSA Burn Median (min, max)	70 (43,90)	28 (15,80)	0.002

        aFisher’s exact test or the Mann-Whitney U test

        Abbreviations: PN = parenteral nutrition, %TBSA = % Total body surface area

        
        

        

        TABLE 5. Independent factors associated with 30-day mortality.

        
        

        
        

        Factors	Unadjusted OR (95% CI)	p-value	Adjusted OR (95% CI)	p- value
        Early PN	1.167(0.124, 10.974)	0.893	0.356(0.014, 8.901)	0.529
        UTI	55.500(5.532, 556.775)	0.001	34.750(1.641-735.686)	0.023
        Pneumonia	N/A	N/A	N/A	N/A

        
        

        

        Abbreviations: PN = parenteral nutrition, UTI = urinary tract infection

        
        

        ileus from massive fluid and electrolytes disturbance from post resuscitation aftermath of exsanguinating bleeding combining with post injury response by inflammatory cytokines flush worsen intestinal continuity and mobility. Major burn is considered as severe injury that create systematic change in all system of body. Abrupt change of extravascular electrostatic pressure and intravascular oncotic pressure result in rapid decrease of intravascular volume which partly pathophysiology of burn shock. Adequate fluid resuscitation lessen outcome of end organ failure like kidney injury and dead.15 Intestinal dysfunction of acute major burns can cause from non-burn tissue edema effect from post burn systemic response, electrolytes imbalance and hypoalbuminemia. Major burns standard nutrition recommendation of very early enteral nutrition initiation and caloric goal achievement in a week16-18 might not success in every case.19

        PN is one of the options to supply calories to reach daily goal in enteral intolerance and offer some specific nutrients support. For energy providing purpose, it can be used as partial caloric supplement or main supply source of daily energy requirement. In adult critically ill patient including adult major burns, late initiation of PN after first 7 days is recommended in low nutritional risk patient that did not achieve caloric goal via enteral route or contraindicated of enteral nutrition (EN). Early PN is encouraged only when EN is not feasible in high nutritional risk or severely malnourished patients.12,16,17 Standard nutritional guideline for critically ill children patient also recommend the same, but specialized population like major pediatric burns cannot apply into this guideline.5 This exception cause from the lack of evidence in the treatment outcome of supplement PN support in this group of patient. The threshold of PN initiation in pediatric is lower (1-3 days in infants and 4-5 days in older children) than adult due to low energy reserve and growth and development effect.20 The current recommendation based on the evidence that most studies reported no significant improving outcome, but trend to increase complication from PN such as infection like catheter related blood steam infection (CRBSI).21-23

        PN role in major adult burn is limited that was reported increased mortality without modulate inflammatory response.24,25 The outcomes of comparing the early and late initiation of PN in major pediatric burn patients are lack of evidence biggest trial like a multicenter PEPaNIC RCT (Early versus Late Parenteral Nutrition in the Pediatric ICU) conducted in 2016 enrolled only 10 cases of burn. The study conclusion from PEPaNIC RCT is not good to apply in major pediatric burn population. No previous studies in Thailand had reported the compared outcomes

        of early PN support in major pediatric burn patients. Our study found no statistically significant difference in LOS and 30-day mortality between the groups of major pediatric burn patients with and without early PN support. Our findings support the less restricted use of early PN in major pediatric burn patients who, according to the recent guidelines, qualify as critically ill pediatric patients were preserved and recommended PN only for high nutrition risk patients and their trend in late initiation more than early.11,26

        We found that the main factors associated with LOS are age less than one year, wound infection, and abdominal distension. Burn injury in infants is more complicated to treat than the injury in the same severity in older child patient. Abdominal distension can lead to feeding problem that can prolong disease natural history and LOS. The duration of ventilator use, and/or the presence of pneumonia, sepsis, and/or urinary tract infection, and the percentage of burned skin affects 30- day mortality. Overall, the 30-day mortality rate in our center was 17.7%, compared with 21.3%27 and 31.3%28 from other studies.

        There are a few limitations in this study. First, as this is a retrospective, observational, single-center study, our findings may not be generalizable to other institutions or ethnic populations. The investigators relied on available documentation to determine the number of days of poor nutrient intake prior to patients’ PN initiation. Also, a prior power-analysis calculation was not conducted, and the small number of in-hospital deaths may have affected the outcomes analysis.

        
        

        CONCLUSION

        Early parenteral nutritional support for major pediatric burn patients had no effect on their length of hospital stay or 30-day mortality rate. The patients’ age, wound infection, and abdominal distension were independent factors associated with their length of hospital stay. The duration of ventilator use, along with pneumonia, sepsis, urinary tract infection, and the percentage of burned skin were independent factors associated with 30-day mortality.

        
        

        ACKNOWLEDGEMENTS

        Thank you, Ms. Sasima Tongsai, Ph.D., for your great work and guidance in the statistical analysis, and to Mr. Tanut Sornmanapong for facilitating the research- coordinating process. Additionally, this research would not have been possible without the significant help from the medical records staff, our surgery team, and various medical students.

        
        

        Conflicts of interest: It is hereby declared that there are no conflicts of interest.

        
        

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        9. Fivez T, Kerklaan D, Mesotten D, Verbruggen S, Joosten K, Van den Berghe G. Evidence for the use of parenteral nutrition in the pediatric intensive care unit. Clin Nutr. 2017;36(1):218- 23.

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        12. Joosten K, Embleton N, Yan W, Senterre T; ESPGHAN/ESPEN/ ESPR/CSPEN working group on pediatric parenteral nutrition. ESPGHAN/ESPEN/ESPR/CSPEN guidelines on pediatric parenteral nutrition: Energy. Clin Nutr. 2018 Dec;37(6 Pt B): 2309-14.

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Factors Predicting Survival in Ruptured Hepatocellular Carcinoma Treated with Surgical Resection

Charnwit Assawasirisin, M.D., Pholasith Sangserestid, M.D., Yongyut Sirivatanauksorn, M.D., Somchai Limsrichamrern, M.D., Prawat Kositamongkol, M.D., Prawej Mahawithitwong, M.D., Chutwichai Tovikkai, M.D., Wethit Dumronggittigule, M.D.

Hepato-Pancreato-Biliary and Transplant Surgery unit, Division of General Surgery, Department of Surgery, Faculty of Medicine Siriraj Hospital,

Mahidol University, Bangkok 10700, Thailand.

ABSTRACT

Objective: Today, ruptured hepatocellular carcinoma (HCC) is a less frequently encountered problem globally due to availability of cancer surveillance protocols for the high-risk population. However, in Thailand, a number of patients do not enroll in screening programs, leading to high rates of ruptured complications. In fit-for-surgery and clinically stable patients, hepatectomy means long-term survival. This study aimed to identify predictive factors of survival in resected patients.

Materials and Methods: A retrospective review of patients with ruptured HCC who underwent liver resection between January 2013 and December 2019 at Siriraj Hospital was performed. The clinical data and outcomes of patients was analyzed.

Results: A total of forty-two patients with ruptured HCC underwent resection or 9.8% of all operable HCC cases. There were 6 patients (14.3%) who suffered from postoperative liver failure and one patient (2.4%) died within 30 days. Overall survival (OS) and recurrence-free survival (RFS) were 90%, 64%, 52% and 42.5%, 24%, 16% at 1, 3, and 5 years, respectively. The factors affecting OS were tumor size ≥10 cm, vascular invasion, and positive resection margin.

Conclusion: Surgical resection in ruptured HCC provides long-term survival. Predicting factors affecting overall survival were large tumor size, vascular invasion, and positive resection margin. Patient selection is a key for better patient’s outcomes.

Keywords: Ruptured hepatocellular carcinoma; surgical resection; risk factors; survival (Siriraj Med J 2022; 74: 40-47)

INTRODUCTION

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver and the sixth most common worldwide.1 In Thailand, HCC is the most common malignancy in males and second-most malignancy in females.1 Although most HCC patients are asymptomatic,

some patients suffer with intra-abdominal bleeding due to a ruptured tumor. The incidence rate of ruptured HCC varies among countries, however, it is more common in Asia than in the West where the rate fluctuates between 3% to 26%.2 In Thailand, the incidence rate is approximately 12.4%.3 The outcomes of patients with ruptured HCC

Corresponding author: Pholasith Sangserestid E-mail: pholasith.san@mahidol.ac.th

Received 7 September 2021 Revised 15 October 2021 Accepted 27 October 2021 ORCID ID: https://orcid.org/0000-0002-5975-1840 http://dx.doi.org/10.33192/Smj.2022.6

varies widely depending on the treatment and other factors such as performance status, vital signs on arrival, amount of intraperitoneal hemorrhage, comorbidities and background liver parenchyma disease. Moreover, outcomes of liver resection are significantly better than other treatments with a mortality rate of 4.4% compared to 85-100%, respectively.4,5 In some reports, patients with ruptured HCC who had surgical resection had similar overall survival (OS) compared to non-ruptured HCC patients.6-9 In this study, our aim was to identify predictive factors that affect survival rate in ruptured HCC patients

undergoing surgical resection.

MATERIALS AND METHODS

This retrospective study was performed in patients diagnosed with ruptured HCC who had liver resection at Siriraj Hospital between January 2013 and December 2019. The patients were diagnosed with ruptured HCC through preoperative imaging such as ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI), or by the intraoperative finding of hemoperitoneum. The decision to perform a surgery depended on the patient’s condition and the attending surgeon. While major hepatectomy was defined as resection of 3 Couinaud’s segments or more, minor hepatectomy was defined as resection of less than 3 Couinaud’s segments. After a successful liver resection, patients had to follow-up with postoperative imaging using either CT or MRI every 3-6 months to evaluate the surgical outcome and to detect the recurrence of HCC. The definition of HCC recurrence is the appearance of hypervascular patterns in a CT or MRI scan with early enhancement in the arterial phase and rapid washout during the porto-venous phase. The patients who were diagnosed with recurrence were evaluated for the possibility of curative treatments such as resection or ablation. Trans-arterial chemoembolization (TACE), trans-arterial radioembolization (TARE) or other systemic therapies were offered if definitive treatments were not possible. The outcomes we focused on in this study included overall survival (OS) and disease-free survival (DFS). While OS was defined as the length of time from date of operation to the date of death or last follow-up DFS was defined as the time from the date of operation to the date of confirmed HCC recurrence. An extrahepatic recurrence or peritoneal seeding tumor was confirmed by imaging or pathological reports. The characteristics of patients with ruptured HCC were analyzed to determine related factors influencing outcomes. This study was approved by the human research committee of Faculty of Medicine Siriraj Hospital, Mahidol University. Variable data was expressed as mean ± standard

deviation (SD) and number (percentage). The OS and DFS values were calculated using the Kaplan-Meier survival analysis. Meanwhile, the univariable and forward stepwise multivariable Cox regression analysis were performed to investigate predictors and all the data was analyzed using SPSS software, version 17.0 [IBM, Illinois].

RESULTS

Between January 2013 and December 2019, a total of 460 patients with HCC underwent surgical resection at Siriraj Hospital. Of the total, 45 patients (9.8%) were categorized as those presenting clinical signs of HCC. However, three patients were excluded from the study due to the incomplete recording of data. Therefore, a total of 42 patients were included in this analysis. Most of patients were males (78.6%) and the average age of patients was 59.5. Fourteen patients (33.3%) showed signs of hypovolemic shock and of this total, 10 (71.4%) required emergency trans-arterial embolization (TAE). Regardless, 6 patients from the non-hypovolemic shock group also underwent TAE due to ongoing bleeding. The average tumor size was 7.9 cm and remarkably, almost all the patients had liver function status defined as Child-Pugh class A (97.6%). The patient demographic data is described in Table 1. Major liver resections were performed in 11 patients (26.2%) and median intraoperative blood loss was 600 mL (range 80 - 5,000 mL). Twenty-six patients (61.9%) had vascular invasion; 7 patients (16.7%) had a positive resection margin. The intraoperative and pathologic outcomes are described in Table 2.

Regarding the postoperative period, one patient (2.4%) died within 30 days due to post-hepatectomy liver failure. The postoperative morbidity rate was 28.6% (12/42) and the median length of stay 6 days (range 4 - 58 days). The median overall survival (OS) was 61.1 months and median follow-up time 22 months. The overall survival was 90%, 64%, 52% at 1, 3, and 5 years respectively (Fig 1A). Meanwhile, recurrence-free survival (RFS) was 42.5%, 24%, 16% at 1, 3, and 5 years respectively (Fig 1B). Twenty-nine patients (69%) had a recurrence of HCC at the median time of 7.5 months. The type of recurrence was intrahepatic recurrence (17/29, 58.6%), intrahepatic and extrahepatic recurrence (10/29, 34.5%), and extrahepatic recurrence (2/42, 6.9%). Seven patients (16.7%) suffered from peritoneal recurrence. Risk factors that affected OS and RFS were analyzed with a univariable and multivariable Cox regression analysis. At least eight factors (univariable Cox regression analysis) affected OS: the preoperative factors were - tumor size ≥10 cm, macrovascular invasion, hematocrit <30%, time-to-surgery ≤7 days, intraoperative factors - blood

TABLE 1. Demographic data.

Characteristics n = 42

Patient’s characteristics 59.5 ± 13.1

Age (years) Gender

Male 33 (78.6%)

Female 9 (21.4%)

Tumor size (cm) 7.9 ± 3.2

Macrovascular invasion 4 (9.5%)

No. of tumor

1 37 (88.1%)

2 3 (7.1%)

3 2 (4.8%)

Hypovolemic shock 14 (33.3%)

Preoperative embolization 16 (38.1%)

MELD score 8 (6-14)

Child-Pugh

grade A 41 (97.6%)

grade B 1 (2.4%)

Time to surgery

≤7 days 12 (28.6%)

>7 days 30 (71.4%)

TABLE 2. Intraoperative and pathologic result.

Major hepatectomy = liver resection ≥3 Couinaud’s segments

Data was presented as mean ± SD, median (range) or number (percentage)

A B

Fig 1. A- Kaplan-Meier estimation of overall survival of all patients. 5-year OS was 52%.;

B- Kaplan-Meier estimation of recurrence-free survival of all patients. 5-year DFS was 16%.

loss > 1,000 mL, blood transfusion, and pathological factors - vascular invasion, positive resection margin (Table 3). Using a multivariable analysis, only three factors i.e. tumor size ≥10 cm, vascular invasion, and positive resection margin were documented for OS. There were 3 pathological factors affecting RFS; i.e. vascular invasion, positive resection margin, and satellite lesion (Table 4). Among these factors, tumor size ≥10 cm, time- to-surgery ≤7 days, vascular invasion, positive resection margin were found to be major factors leading to worst OS outcome (Fig 2).

DISCUSSION

Spontaneous rupturing of HCC is a catastrophic complication with a high mortality rate. Today, patients with HCC receive treatment early due to worldwide implementation of surveillance programs in high-risk patients, resulting in less people presenting ruptured HCC. However, the incidence rate of ruptured HCC in Asian countries is higher than in Western countries. In our institution, Siriraj Hospital, the ruptured HCC rate was 9.8% of all resectable HCC cases, which is comparable to reports from other Asian countries such as Hong Kong (9%)10, Taiwan (26%)11, and Japan (2.3%).12 Ruptured HCC usually shows aggressive tumor biology. According to our data, there was high rate of vascular invasion (61.9%) and 9 patients (21.4%) had a large tumor of over 10 cm. In this study, patients with tumor size ≥10 cm, vascular invasion, and positive tumor

margin had significant shorter overall survival rate.

This study provided results of long-term survival with low morbidity in select surgical candidates. However,

almost all operable cases had a well-preserved liver function (CTP-A, 97.6%) and most operations were partial hepatectomies (73.8%). The strategy of using non-urgent surgical resections despite tumor bleeding was stopped, either spontaneously or by embolization. The surgery was performed after patient condition was stabilized. Surgery performed within 7 days significantly affected the overall survival. Moreover, post-hepatectomy liver failure was encountered only in 6 patients (14.3%) with only one case of mortality (2.4%). The 5-year overall survival was 52%, which is better than results in several other studies4,6,7 and comparable with some large series.8,13 This study strongly demonstrates that surgical resection plays a role in the treatment of ruptured HCC patients including those at the advanced tumor stage. Peritoneal recurrence is uncommon in non-ruptured HCC when compared to the high rates of ruptured HCC. Peritoneal recurrence was found only in 16.7% of patients in this study and this was comparable to previous studies which reported a rate between 11 - 40% with median time to recurrence being 6 - 11 months.14-18 Ruptured HCC and time-to-surgery were the two most common deciding factors for peritoneal recurrence, however, there is some supportive and contradictive evidence regarding this.14,15,19,20 Most ruptured HCC patients usually presented poor liver function and were excluded from liver resection. Therefore, the number of ruptured HCC patients who could be candidates for liver resection was limited. Nevertheless, a further prospective study with a large number of ruptured HCC patients, including a multicenter study should be carried out to clarify additional predictive factors of ruptured HCC patients treated with surgical resection.

Factors

Univariable analysis

Multivariable analysis

TABLE 3. factors affecting overall survival of ruptured HCC undergoing resection by univariable and multivariable Cox regression analysis.

Factors

Univariable analysis

95% CI

Multivariable analysis

HR

p-value

HR

95% CI

p-value

TABLE 4. factors affecting recurrence of ruptured HCC undergoing resection by univariable and multivariable Cox regression analysis.

A B

C D

Fig 2. Kaplan-Meier estimation of overall survival of patients. A: Patients with Tumor size ≥ 10 cm had significantly poor OS (median OS

61.1 months [size <10 cm] vs 15.8 months [size ≥10 cm], p = 0.01); B: Time-to-Surgery ≤7 days had significantly poor OS (median OS 34.8 months [≤7 days] vs Not reach [>7 days], p <0.01); C: HCC with microvascular invasion had significantly poor OS (median OS 34.8 months [with vascular invasion] vs not reach [without vascular invasion], p <0.01); D: Patients with positive resection margin had significantly poor OS (median OS 61.1 months [free resection margin] vs 4.2 months [positive resection margin], p = 0.01)

CONCLUSIONS

Surgical resection in ruptured HCC provides long- term survival. Predicting factors affecting overall survival were large tumor size, vascular invasion, and positive resection margin. Patient selection is a key for better patient’s outcomes.

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17. Uchiyama H, Minagawa R, Itoh S, Kajiyama K, Harimoto N, Ikegami T, et al. Favorable Outcomes of Hepatectomy for Ruptured Hepatocellular Carcinoma: Retrospective Analysis of Primary R0-Hepatectomized Patients. Anticancer Res. 2016; 36(1):379-85.

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19. Kwak MS, Lee JH, Yoon JH, Yu SJ, Cho EJ, Jang ES, et al. Risk factors, clinical features, and prognosis of the hepatocellular carcinoma with peritoneal metastasis. Dig Dis Sci. 2012;57(3): 813-9.

20. Kow AW, Kwon CH, Song S, Shin M, Kim JM, Joh JW. Risk factors of peritoneal recurrence and outcome of resected peritoneal recurrence after liver resection in hepatocellular carcinoma: review of 1222 cases of hepatectomy in a tertiary institution. Ann Surg Oncol. 2012;19(7):2246-55.

Prevalence and Factors Associated with the Loss of PTEN Expression in Patients with Lung Cancer

Thiva Kiatpanabhikul, M.D.*, Wasakorn Bunyayothin, M.D.**

*Department of Medicine, **Department of Pathology, Charoenkrung Pracharak Hospital, Bangkok 10120, Thailand.

ABSTRACT

Objectives: Phosphatase and tensin homolog (PTEN) is a major tumor suppressor gene and is involved in cell survival control. PTEN loss of expression (PTEN-) is associated with a poor outcome. Our study investigated the prevalence of PTEN- in terms of its characteristics and disease prognosis for lung cancer patients.

Materials and Methods: In total, 167 tissue blocks from lung cancer patients at Chareonkrung Pracharak Hospital between January 2010 and December 2020 were studied through immunohistochemistry staining (IHC) for PTEN expression. The clinicopathological factors, IHC features, and epidermal growth factor receptor (EGFR) status were analyzed in association with PTEN- in term of prognosis and the overall survival (OS).

Result: Adenocarcinoma was the major subtype (85.6%) and most patients (90.6%) were diagnosed at stage IV of lung cancer. The prevalence of PTEN- was 66.5%. A location at the left lower lobe (LLL) location and the absence of tumor-infiltrating lymphocytes (TILs) were significantly associated with PTEN- (p=0.039, p=0.046), while the smoking was likely correlated but not statistically significant (p=0.09). The median OS for PTEN- was not significantly different from PTEN+ (8.88 vs 7.20 months, p=0.38). However, smoking, Eastern cooperative oncology group (ECOG) status and primary symptoms were significantly associated with poorer OS.

Conclusion: The prevalence of PTEN- was higher in our studies. Absent TILs and a LLL location were independent factors associated with PTEN-. However, a right upper lobe (RUL) location with PTEN- tended to have a poor prognosis. Interestingly, better survival was found in active smokers with PTEN-. Further survival studies in cases with no TILs lesions and active smokers in associations PTEN expression and other immune-related biomarkers, such as programmed death–ligand 1 (PD-L1), are warranted.

Keywords: PTEN; immunohistochemistry; tumor infiltrating lymphocytes; lung cancer (Siriraj Med J 2022; 74: 48-63)

INTRODUCTION

Lung cancer is the leading cause of cancer death worldwide, and the second leading cancer in males and females. In 2019, lung cancer was responsible for the death of 1.37 million people in the United States.1 In Thailand, lung cancer is the third most common cancer in males, accounting for 24.74% of all cancers, and the fourth most common cancer in females, accounting for 7.26% of all cancers, while the most common cancers

overall are hepatobiliary cancer in males and breast cancer in females.2

Lung cancer can be classified as either small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC, which is more common (accounting for 85% of lung cancers), can be sub-classified as adenocarcinoma (AC, 40%), squamous cell carcinoma (SC, 25%-30%), and large cell carcinoma (10%-15%).3,4 Regarding its genetic and molecular basis, the dysregulation of cell growth

Corresponding author: Thiva Kiatpanabhikul E-mail: mdping143@gmail.com

Received 9 October 2021 Revised 3 November 2021 Accepted 17 November 2021 ORCID ID: https://orcid.org/0000-0002-5139-5781 http://dx.doi.org/10.33192/Smj.2022.7

and development may predispose some patients to lung cancer as well as determine its severity.5 Tumor suppressor genes (TSGs), in particular PTEN (phosphatase and tensin homolog), are considered significant controllers of cell survival and the cell cycle process; thus their dysregulation may lead to the development of several cancers, including lung cancer.6-10 PTEN, located on chromosome 10q23, regulates the cell cycle via the PI3K/AKT pathway. In the development of lung cancer, PTEN dysfunction, including its loss of function, protein instability, and somatic mutation, has been noted to be associated with the malignant transformation of lung cells.

Several studies reported that approximately 41.2%- 43.7% of lung cancer patients have a loss of PTEN expression.11-13 Moreover, PTEN dysregulation was found to be associated with an advanced tumor stage, partly due to the anchorage-independent growth mechanism.14 In lung cancer patients, the loss of PTEN expression (PTEN-) made negatively regulates phosphatidylinositol 3 phosphate level in the P13K/AKT pathway15, and this alteration may be associated with the development of lung cancer.16 PTEN loss of expression (PTEN-) was related to characteristics of NSCLC patients, including male, a previous history of smoking, and lung cancer with a poorly differentiated type, increased lymph node involvement, high distant metastasis, and late-stage, and thus it is a factor for a poor prognosis.17 Further, patients with a combination of the PTEN- and p-AKT+ have a lower 5-year survival rate and median survival time. Though the associations between the loss of PTEN expression and SCLC is still unclear. Alterations in the PTEN pathway have been regularly reported in SCLC.18 In the genetic mice model, the PTEN inactivation could accelerate the SCLC with more aggressive behavior.19 PTEN- is also an independent poor prognostic factor for NSCLC.11-13 Furthermore, lung cancer patients with a loss of PTEN expression and the positive epidermal growth factor receptor (EGFR) mutation may be resistant to chemotherapy and targeted therapy.20,21

Consequently, our study aimed to explore the prevalence of PTEN loss of expression in Thai lung cancer patients, and also its associated clinical characteristics and possible effects on disease prognosis.

MATERIALS AND METHODS

This retrospective study was approved by the Human Research and Ethics Committees of Bangkok Metropolitan Administration, Bangkok, Thailand. (No. S013h/63_EXP). Informed consent was waived because the study involved anonymous data extraction with no direct patient or public involvement.

Study population

In total, 191 lung cancer patients underwent either transbronchial biopsy (53.9%), transcutaneous biopsy (24.6%), lobectomy (3.6%), or tissue biopsy (lymph node, skin, bone, mass) (18%) at the Charoenkrung Pracharak Hospital between 1 January 2010 and 31 December 2020 and were enrolled in the study. The inclusion criteria were patients aged >15 years old diagnosed with lung cancer by histopathological studies. If there was insufficient tissue for the further analysis of PTEN expreesion, the patients were excluded from the study. The patients’ demographics and clinical characteristics, including age, gender, smoking, associated symptoms (such as chronic cough, progressive dyspnea, hemoptysis, and chest pain), Eastern cooperative oncology group (ECOG) performance status, tumor size and its primary location, histopathological study, duration of follow-up, and living status, were collected. Ancillary immunohistochemical reports, including thyroid transcription factor 1 (TTF – 1) and epidermal growth factor receptor (EGFR) mutation, were also collected.

Histopathology and immunohistochemistry (IHC) for PTEN

Formalin-fixed paraffin-embedded (FFPE) blocks were cut 4 μm in thickness by microtomy (Thermo Fisher Scientific HM355S automated microtomy). Slides were stained by hematoxylin and eosin (H&E). All the previous H&E slides were reviewed for confirming the diagnosis and evaluating the tissue adequacy for further PTEN immunohistochemical study by two pathologists at pathologists at the Department of Pathology, Charoenkrung Pracharak Hospital, blinded to the patient information and clinical data. The laboratory was certified for Laboratory Academic Standards by The Royal College of Pathologist of Thailand. Histopathological parameters, including histologic subtype and tumor-infiltrating lymphocytes (TILs), were evaluated. Unstained whole-section FFPE slides from 167 FFPE blocks were heated at 70 0C for 30 min in a hot air oven (Memmert UF55) before running them in a Ventana BenchMark XT automatic sample preparation system (serial number KPXT715667). The IHC staining process included deparaffinization by EZ prep (LOT#G13961) and cell conditioning by CC1 (LOT#G06580) for 56 min and antigen retrieval by a primary peroxidase inhibitor. Rabbit monoclonal primary antibody VENTANA PTEN clone SP218 (LOT#G27111) was used as the primary antibody in this study. Automated incubation was performed for 16 min. OptiView HQ links and OptiView HQ Universal links were mixed to ensure amplified signals, followed

by use of the Ventana OptiView TM Universal DAB Detection Kit. Counterstaining was performed by dyeing with Ventana Hematoxylin II for 12 minutes, followed by bluing reagent for 6 minutes. The oil was removed from each slide by soap and the slide was subsequently dehydrated with 95% ethyl alcohol, absolute alcohol, and xylene, respectively. Finally, a coverslip was placed on the slide and it was then mounted with mounting media.

IHC scoring of PTEN

The IHC score for PTEN was modified from the previous literature.11 The interpretation and scoring of PTEN IHC was performed in terms of either the intensity of staining or the average number of positive tumor cells, as independently evaluated by two pathologists. PTEN IHC slides were visually scored using a bright field microscope (Olympus BX43) under an objective lens (40x magnification) and eye pieces lens (10x magnification and field number 22). The positive internal control staining included the bronchial epithelial cells and stromal cells. Five 40x high-power fields were selected to includes 200 cell counts. The interpretation of PTEN expression involved the cytoplasmic and/or nuclear staining pattern. The average percentage of positive tumor cells is reported as the following: 0 = no tumor cells stained, 1 = 10%-20% of cells stained, 2 = 20%-50% cells stained, and 3 = >50% of cells stained. The intensity of positive cell staining was categorized as follows: 0 = no appreciable staining in the cells, 1 = barely detectable staining as compared with normal stromal cells, 2 = readily appreciable brown staining distinctly marking the cell cytoplasm/or nucleus, and 3 = dark brown staining in the cytoplasm and/or nucleus. PTEN IHC scoring was rendered, with regard to the calculated results for the intensity and the average percentage of positive tumor cells, ranging from 0 to 9. A score of 2 or less is defined as negative PTEN IHC expression, whereas 3 or greater is defined as positive PTEN IHC expression.

Using 200-400x (a 10x eyepieces and a 20-40x objective lens) microscopic magnification, tumor infiltrating lymphocytes (TILs) are the percentages of TILs in the stromal compartment (% stromal TILs), defined as the area of mononuclear cells (including lymphocyte and plasma cell) infiltration, between the cancer cells with no direct contact, in the stromal tissue. The 10% of more stromal TILs is considered positive.22

Statistical analysis

Data were analyzed for the prevalence of PTEN loss of expression (PTEN-) in lung cancer, the associations

between PTEN loss of expression and the clinicopathological factors, IHC features, and the EGFR type status. The parametric statistical analysis was performed using SPSS version 26 software (IBM Corp., Armonk, NY). The patients’ demographic and clinical characteristics were expressed as a numbers, percentages, median, and mean and standard deviation (SD). Categorical variables were analyzed by Pearson chi-square test and Fisher’s exact test for the normally distributed data. A p-value less than

1. was considered statistically significant. Kaplan–Meier analysis and the log-rank test were used to analyze the results from the survival study. Cox proportional hazard regression was applied to determine the PTEN loss of expression and the variables affecting the survival status. According to Seol-Bong Yoo et al.13, the prevalence of the PTEN loss of expression in NSCLC was 42.4% and was used to calculated the sample size. In this study, there were only 8 patients with SCLC; therefore, only descriptive statistics were applied. Moreover, no prevalence of PTEN loss of expression was previously reported.

   
   

   RESULTS

   Among the 191 lung cancer patients enrolled on the study, 24 were excluded because 23 had no available FFPE blocks and one who had inadequate tumor tissue for the IHC study. Subsequently, a total of 167 patients were included in the study for the data analysis. Two pathologists independently interpreted the PTEN IHC staining on each slide. For the first 40 slides, the interpretation kappa values were 0.975 for the inter-observer reliability and 0.95 for the intra-observer reliability, accordingly. For the overall 167 slides, the inter-observer reliability was 0.98. Three discordantly interpreted slide results between the two pathologists were re-evaluated and further discussed until consensus was reached on each slide (positive or negative PTEN staining)

   The mean age of the patients was 64.8 ± 11.77 years old, with a median age of 65.0 years old (ranging from 32-93 years), with 51.5% having an age ≥65 years old. The majority of patients were male (57.5%). Out of the 167 lung cancers cases, NSCLC was accounted for 159 cases (95.2%) and SCLC 8 cases (4.8%). Lung cancer was the most commonly found in patients who had never smoked group (46.1%), while its incidence in those with a smoking history group, which was active (37.7%), secondhand (6.6%), or former smokers (9.6%), was 53.9%. Clinical symptoms included chronic cough (77.2%), progressive dyspnea (68.3%), hemoptysis (19.8%), and chest pain (24.6%), and the most common ECOG score at the time of diagnosis was 1 (54.5%), respectively. Among

   
   

   the NSCLC cases, 85.6% of patients had adenocarcinoma subtypes, and 90.6% were diagnosed at an advanced stage (stage IV). Moreover, all 8 SCLC cases were involved extensive disease (ED). Approximately half the patients (57.6%) had a tumor sized 3-7 cm at the greatest diameter. Regarding of the location of the lung lobes, primary lung cancer was mostly located at the right upper lung

   area (34.1%). Surprisingly, most lung cancer tissues (64.1%) displayed no tumor-infiltrating lymphocytes. The prevalence of PTEN loss of expression was 66.5%. Most lung cancer tissues were stained positive for TTF-1 (77.5%) and Napsin A staining (63.2%). Regarding the EGFR mutation, 46 cases (47.8%) were located on either EXON 19 (68.2%) or EXON 21 (18.2%).

   
   

   

   
   

   Fig 1. A: NSCLC with TILs (dark arrow) (H&E, x40); B: Normal bronchial epithelium (white arrow) and stromal cell (dark arrow) with nuclear and cytoplasmic staining of PTEN. PTEN Intensity was categorized as follows: C: 0 if no appreciable intensity stain. D: 1 if barely stain; E: 2 if appreciable brown; F: 3 if dark brown stain in the cytoplasm and/or nucleus. The different percentages of PTEN IHC were demonstrated as follows: G: no tumor cell staining or 0%; H: 10% - 20%, I: 20% - 50%; J: more than 50% in the NSCLC patients.

   
   

   
   

   

   
   

   TABLE 1. Demographics, clinical characteristics, and histopathological and immunohistochemical studies of the patients with lung cancer.

   
   

   Characteristic variables Number Percentage (%)

   

   Age (years) (n = 167)

   Gender (n = 167)

   
   

   Male	96	57.5
   Female	71	42.5

   
   

   ≥65	86	51.5
   <65	81	48.5
   Smoking (n = 167)
   Never smoked	77	46.1
   Active smoker	63	37.7
   Secondhand smoker	11	6.6
   Former smoker	16	9.6
   Chronic cough (n = 167)	129	77.2
   Hemoptysis (n = 167)	134	80.2
   Progressive dyspnea (n = 167)	114	68.3
   Weight loss (n = 167)	100	59.9
   Chest pain (n = 167)	41	24.6
   ECOG status (n = 167)
   0	8	4.8
   1	91	54.5
   2	36	21.6
   3	21	12.6
   4	11	6.6
   Histological type of lung cancer (n = 167)
   NSCLC	159	95.2
   SCLC	8	4.8
   NSCLC subtype (n = 159)
   Adenocarcinoma	143	89.9
   Squamous cell carcinoma	12	7.5
   Large cell neuroendocrine carcinoma	2	1.3
   Other (e.g. carcinosarcoma)	2	1.3
   Tumor size (n = 167) (cm.)
   <3	15	9.3
   3–7	95	58.6
   >7	52	32.1
   Location of the primary tumor (n = 167)
   RUL	57	34.1
   RML	20	12
   RLL	31	18.6
   LUL	32	19.1
   LLL	26	15.6
   Center	1	0.6

   
   

   

   
   

   TABLE 1. Demographics, clinical characteristics, and histopathological and immunohistochemical studies of the patients with lung cancer. (Continue)

   
   

   Characteristic variables Number Percentage (%)

   Tumor-infiltrating lymphocytes (TILs) (n = 167)

   

   Histological pattern (n = 167)

   Present 60 35.9

   
   

   Acinar	89	53.3
   Papillary	8	4.8
   Solid	61	36.5
   Lepidic	9	5.4
   Degree of differentiation (n = 167) Moderate	157	94
   Poor	10	6
   PTEN expression (n = 167)
   Loss of expression	111	66.5
   TTF-1 IHC (n = 142)
   Positive staining	110	77.5
   EGFR mutation (n = 46)	22	47.8
   Exon 19	15	68.2
   Exon 20	1	4.5
   Exon 21	4	18.2
   Double mutation	2	9.1
   Treatment (1st line)
   1st line chemotherapy	79	47.3
   1st line tyrosine kinase inhibitor (TKI)	12	7.2
   1st line surgery	3	1.8
   1st line radiotherapy	15	9
   no definite treatment/palliative care	58	34.7
   Living status
   Alive	23	13.77
   Dead	144	86.23

   
   

   Abbreviations: ECOG = Eastern Cooperative Oncology Group performance status, NSCLC = non-small cell lung cancer, SCLC = small cell lung cancer, PTEN = phosphatase and tensin homolog, TTF-1 = thyroid transcription factor 1, EGFR = epidermal growth factor receptor, RUL=Right upper lung, RML=Right middle lung, RLL=Right lower lung, LUL=Left upper lung, and LLL=Left lower lung

   
   

   Correlation between PTEN expression and survival time

   In terms of the correlation between the PTEN status and clinical outcome, PTEN- was less common in lung cancer that was primarily located at the left lower lobe (LLL), compared to at the right upper lobe (RUL) (p = 0.039, OR = 0.36), and in the absence of TILs (p = 0.045, OR = 1.96). Whereas an age <65 years old and smoking were likely correlated with the PTEN status (p = 0.056, OR = 0.22 and p = 0.089, OR = 1.75, respectively).

   According to the multivariate analysis, the absence of TILs (p = 0.017, adjusted OR = 2.5), location at the LLL (p = 0.026, adjusted OR = 0.297), and age <65 years old (p = 0.04, adjusted OR = 0.47) were independent factors correlated with the PTEN loss of expression.

   In addition, SCLC and smoking behavior were also marginally significantly associated with the PTEN loss of expression (p = 0.054 and 0.089, respectively).

   The median follow-up time was 8.04 months (range, 0.01–94.80). Most patients (65.3%) had received specific treatments (including 1st line chemotherapy (47.3%), 1st line radiotherapy (13.8%), 1st line tyrosine kinase inhibitor (7.2%), or surgery for primary cancer (1.8%)), while the remaining 34.7% had received no aggressive treatment due to their poor baseline status. The median overall survival (mOS) was 8.88 months, with 2- and 5-year overall survival rates of 19.7% and 7.4%, respectively. Of note, 27 patients (16.17%) had CNS/spine metastasis at the 1st diagnosis.

   
   

   

   
   

   TABLE 2. Association among clinical status, immunohistochemical study, EGFR mutation of primary lung cancer, and PTEN expression.

   
   

   Clinicopathological, immune-molecular features	Total n (%)	PTEN (+) n (%)	PTEN (-) n (%)	P-value
   Gender
   Male	96 (57.5)	33 (58.9)	63 (56.8)	0.79
   Female	71 (42.5)	23 (41.1)	48 (43.2)
   Age
   ≥65	86 (51.5)	23 (41.1)	63 (56.8)	0.056
   <65	81 (48.5)	33 (58.9)	48 (43.2)
   Smoking
   No smoking	77 (46.1)	31 (55.4)	46 (41.4)	0.089
   History of smoking	90 (53.9)	25 (44.6)	65 (58.6)
   Chronic cough
   Yes	129 (77.2)	41 (73.2)	88 (79.3)	0.37
   No	38 (22.8)	15 (26.8)	23 (20.7)
   Progressive dyspnea
   Yes	114 (68.3)	39 (69.6)	75 (67.6)	0.786
   No	53 (31.7)	17 (30.4)	36 (32.4)
   Hemoptysis
   Yes	33 (19.8)	12 (21.4)	21 (18.9)	0.70
   No	134 (80.2)	44 (78.6)	90 (81.1)
   Weight loss
   Yes	100 (59.9)	34 (60.7)	66 (59.5)	0.88
   No	67 (40.1)	22 (39.3)	45 (40.5)
   Chest pain
   Yes	41 (24.6)	15 (26.8)	26 (23.4)	0.63
   No	126 (75.4)	41 (73.2)	85 (76.6)
   Size
   <3 cm	15 (9.3)	2 (3.6)	13 (12.3)	0.12
   3–7 cm	95 (58.6)	39 (69.6)	56 (52.8)
   >7 cm	52 (32.1)	15 (26.8)	37 (34.9)

   
   

   

   
   

   TABLE 2. Association among clinical status, immunohistochemical study, EGFR mutation of primary lung cancer, and PTEN expression. (Continue)

   
   

   Clinicopathological, immune-molecular features	Total n (%)	PTEN (+) n (%)	PTEN (-) n (%)	P-value
   Primary site RUL (0)	57 (34.1)	17 (30.4)	40 (36)	0.31
   RML (1)	20 (12)	5 (8.9)	15 (13.5)
   RLL (2)	31 (18.6)	8 (14.3)	23 (20.7)
   LUL (3)	32 (19.2)	12 (21.4)	20 (18)
   LLL (4)	26 (15.6)	14 (25)	12 (10.8)
   Central (5)	1 (0.6)	-	1 (0.9)
   ECOG status 0	8 (4.8)	1 (1.8)	7 (6.3)	0.49
   1	91 (54.5)	30 (53.6)	61 (55)
   2	36 (21.6)	14 (25)	22 (19.8)
   3	21 (12.6)	7 (12.5)	14 (12.6)
   4	11 (6.6)	4 (7.1)	7 (6.3)
   Type NSCLC	159 (95.2)	56 (100)	103 (92.8)	0.05
   SCLC	8 (4.8)	-	8 (7.2)
   Stage (NSCLC) I	4 (2.5)	1 (1.8)	3 (2.9)	0.21
   II	4 (2.5)	1 (1.8)	3 (2.9)
   III	7 (4.4)	4 (7.3)	3 (2.9)
   IV	144 (90.6)	49 (89.1)	95 (91.3)
   Stage (small cell) Extensive disease	8 (100)	0 (0)	8 (100)	-
   NSCLC
   Adenocarcinoma	143 (89.9)	47 (83.9)	96 (93.2)	0.12
   Squamous cell CA	12 (7.5)	6 (10.7)	6 (5.8)
   Large cell / NE	2 (1.3)	1 (1.8)	1 (1)
   Other	2 (1.3)	2 (3.6)	-
   TILs Yes	60 (35.9)	26 (46.4)	34 (30.6)	0.045
   No	107 (64.1)	30 (53.6)	77 (69.4)
   TTF-1
   Yes	110 (77.5)	39 (79.6)	71 (76.3)	0.66
   No	32 (22.5)	10 (20.4)	22 (23.7)
   EGFR mutation Yes	22 (47.8)	11 (55)	11 (42.3)	0.39
   No	24 (52.2)	9 (45)	15 (57.7)
   EGFR mutation EXON 18	-	-	-	0.79
   EXON 19	15 (68.2)	8 (72.7)	7 (63.6)
   EXON 20	1 (4.5)	-	1 (9.1)
   EXON 21	4 (18.2)	2 (18.2)	2 (18.2)
   Double mutation	2 (9.1)	1 (9.1)	1 (9.1)
   Treatment 1st line chemotherapy	79 (86.8)	27 (87.1)	52 (86.7)	0.003
   1st line (TKI)	12 (13.2)	4 (12.9)	8 (13.3)

   Abbreviations: ECOG = Eastern Cooperative Oncology Group performance status, NSCLC = non-small cell lung cancer, SCLC = small cell lung cancer, PTEN = phosphatase and tensin homolog, TTF-1 = thyroid transcription factor 1, EGFR = epidermal growth factor receptor, RUL=Right upper lung

   
   

   Patients who had a history of smoking, chronic cough, progressive dyspnea, no hemoptysis, chest pain, weight loss, larger tumor size, and lower ECOG status had a lower mOS time than in the opposite group (p = 0.003, 0.005, 0.015, 0.008, 0.03, 0.001, 0.001, and 0.001,

   respectively). Nevertheless, gender, age, a subtype of lung cancer (adenocarcinoma vs squamous cell subtypes) and primary brain/spine metastasis, presence of TILs, TTF-1, and EGFR mutation showed no significant difference

   in the mOS time between the comparative populations (p >0.05).

   In all lung cancer patients, the mOS was 8.88 months (ranging from 0.01 to 94.8 months). There was no significant difference in mOS between the PTEN+ and PTEN– groups in NSCLC (7.20 vs. 8.88 months) (p = 0.38) and also no significant difference in mOS between the PTEN+ and PTEN– groups in adenocarcinoma (7.20 vs 9.96 months) (p = 0.23) (Fig 2.1 and 2.2).

   
   

   

   
   

   TABLE 3. Association among the clinicopathologic, immune-molecular features and median overall survival (mOS) and hazard ratio (HR) (n = 167).

   
   

   Clinicopathological, immune-molecular features	Total n (%)	mOS month	95% CI n (%)	P-value	Hazard ratio	95% CI n (%)	P-value
   Gender
   Male	96 (57.5)	7.68	5.54–9.82	0.368	reference	-	-
   Female	71 (42.5)	13.63	8.91–18.35		0.86	0.62–1.20	0.369
   Age
   ≥65	86 (51.5)	8.64	4.86–12.42	0.256	reference	-	-
   <65	81 (48.5)	9.96	7.25–12.67		0.823	0.60–1.15	0.258
   Smoking
   No smoking	77 (46.1)	14.57	8.83–20.31	0.003	reference	-	-
   History of smoking	90 (53.9)	6.36	4.62–8.10		1.64	1.18–2.29	0.004
   Chronic cough
   No	38 (22.8)	19.2	13.94–24.46	0.005	1.75	1.17–2.62	-
   Yes	129 (77.2)	7.2	5.20–9.2		reference	-	0.006
   Progressive dyspnea
   No	53 (31.7)	18.84	12.39–25.29	0.015	reference	-	-
   Yes	114 (68.3)	7.2	4.44–9.96		1.55	1.08–2.20	0.016
   Hemoptysis
   No	134 (80.2)	7.68	4.89–10.47	0.008	reference	-	-
   Yes	33 (19.8)	10.08	0–20.67		0.55	0.35–0.86	0.009
   Weight loss
   No	67 (40.1)	18.24	9.70–26.78	0.001	reference	-	-
   Yes	100 (59.9)	7.08	4.57–9.59		1.77	1.25–2.49	0.001
   Chest pain
   No	126 (75.4)	9.96	5.92–14	0.03	reference	-	-
   Yes	41 (24.6)	7.2	2.93–11.47		1.54	1.04–2.28	0.032

   
   

   

   
   

   TABLE 3. Association among the clinicopathologic, immune-molecular features and median overall survival (mOS) and hazard ratio (HR) (n = 167). (Continue)

   
   

   Clinicopathological, immune-molecular features	Total n (%)	mOS month	95% CI n (%)	P-value	Hazard ratio	95% CI n (%)	P-value
   ECOG status
   0	8 (4.8)	35.16	22.83–47.49	<0.01	reference	-	-
   1	91 (54.5)	14.52	10.19–18.85		2.28	0.92–5.66	0.076
   2	36 (21.6)	3.79	1.40–6.18		2.36	2.08–13.84	0.001
   3	21 (12.6)	5.88	2.63–9.13		5.99	2.21–16.23	<0.001
   4	11 (6.6)	4.6	0–9.52		3.92	1.30–11.84	0.016
   Primary site
   RUL (0)	57 (34.1)	16.03	9.81–22.26	0.024	reference	-	-
   RLL (2)	31 (18.6)	9.24	5.91–12.57		0.96	0.59–1.54	0.86
   LUL (3)	32 (19.2)	3.79	2.01–5.57		1.60	1.01–2.54	0.046
   LLL (4)	26 (15.6)	5.58	1.19–10.57		1.90	1.16–3.09	0.01
   Type
   Adenocarcinoma	143 (92.3)	9.24	6.49–11.99	0.26	reference	-	-
   Squamous cell CA	12 (7.7)	11.64	0.03–23.25		0.68	0.34–1.34	0.27
   Brain/spine metastasis
   No	140 (83.8)	9.24	6.39–12.09	0.31	reference	-	-
   Yes	27 (16.2)	6.72	3.60–9.84		1.254	0.81–1.95	0.32
   Size
   <3 cm	15 (9.3)	20.88	3.61–39.15	<0.01	0.45	0.23–0.88	0.02
   3–7 cm	95 (58.6)	11.28	7.44–15.12		reference	-	-
   >7 cm	52 (32.1)	5.28	3.23–7.33		2.13	1.46–3.10	<0.001
   TILs
   Yes	60 (35.9)	7.08	1.36–12.80	0.68	reference	-	-
   No	107 (64.1)	8.88	5.9–11.86		1.08	0.76–1.52	0.67
   TTF-1
   No	32 (22.5)	10.08	6.75–13.41	0.88	reference	-	-
   Yes	110 (77.5)	7.92	4.68–11.16		1.03	0.68–1.56	0.88
   PTEN
   No	56 (33.5)	7.2	1.97–12.43	0.38	reference	-	-
   Yes	111 (66.5)	8.8	6.62–11.14		0.85	0.60–1.22	0.38
   EGFR
   No	24 (52.2)	7.44	4.62–10.26	0.41	reference	-	-
   Yes	22 (47.8)	14.52	11.99–17.05		0.76	0.39–1.46	0.41

   
   

   

   Abbreviations: ECOG = Eastern Cooperative Oncology Group performance status, TILs = tumor-infiltrating lymphocytes, TTF-1 = thyroid transcription factor 1, PTEN = phosphatase and tensin homolog, EGFR = epidermal growth factor receptor.

   
   

   
   

   

   

   

   

   

   

   

   

   

   

   All lung cancer & PTEN mOS 7.20 vs 8.88 months (p=0.38) HR = 0.854 (95% CI 0.59 – 1.22, p = 0.38) Incidence rate PTEN + = 7.3/100/month PTEN - = 5.6/100/month
   	2.1
   PTEN - & smoking status mOS 22.2 vs 9.6 months (p<0.001) HR = 1.76 (95% CI 1.19-2.58, p = 0.004) Incidence rate smoking status yes = 6.9/100/month No = 4.4/100/month
   	2.3
   PTEN - & chronic cough mOS 22.9 vs 10.7 months (p<0.001) HR=1.84 (95%CI=1.1-3.33, P=0.045) Incidence rate chronic cough yes = 7.2/100/month no = 3.0/100/month
   	2.5

   Adenocarcinoma subtype & PTEN mOS 7.20 vs 9.96 months (p=0.23) HR = 0.79 (95% CI 0.54 – 1.16, p = 0.3) Incidence rate PTEN + = 7.4/100/month
   PTEN - = 5.8/100/month	2.2

   PTEN - & ECOG status mOS 32.3,18.4,8.9,7.2,5.5 mo. (p<0.05) HR 0.24 (95% CI 0.09-0.61, p=0.003) Incidence rate ECOG 0 = 2.3/100/month, 1= 4.8/100/month, 2=13.3/100/month, 3=12.0/100/month, 4=4.6/100/month
   	2.4
   PTEN - & chest pain mOS 19.2 vs 9.1 months (p<0.05) HR = 1.72 (95% CI 1.06 – 2.79, p = 0.03) Incidence rate chest pain yes = 10.9/100/month no = 4.9/100/month
   	2.6

   PTEN - & hemoptysis mOS 14.6 vs 31.0 months (p<0.001) HR = 0.54 (95% CI 0.3 – 0.97, p = 0.04) Incidence rate hemoptysis yes = 3.9/100/month hemoptysis no = 6.0/100/month
   	2.7

   PTEN - & Primary site mOS 18.2,10.2,20.2,8.0,14.6 months (p>0.05) Incidence rate RUL = 5.0/100/month RML=6.8/100/month RLL = 4.7/100/month LUL = 7.2/100/month LLL = 15.7/100/month
   	2.8
   Active smoker & PTEN status mOS 1.64 vs 6.12 months (p<0.012) HR = 0.48 (95% CI 0.27 – 0.86, p = 0.014) Incidence rate PTEN + = 12.9/100/month
   PTEN - = 7.6/100/month	2.10

   PTEN - & TILs mOS 17.6 vs 14.6 months (p>0.05) HR = 1.06 (95% C1 0.65-1.72, p = 0.81) Incidence rate TILs Yes = 5.8/100/month TILs No = 5.1/100/month
   	2.9

   Fig 2. The Kaplan–Meier graph demonstrating the mOS of the patients with lung cancer (2.1), adenocarcinoma subtypes (2.2), and active smoking (2.10) in association with the PTEN status. Fig 2.3–2.9 display the mOS of PTEN- correlated with the smoking status, EGOG status, present symptom (chronic cough, progressive dyspnea, hemoptysis), primary site, TILs.

   
   

   In the subgroup analysis of PTEN– patients, the mOS was significantly decreased with the presence of smoking (Fig 2.3). high ECOG status (Fig 2.4), chronic cough (Fig 2.5), chest pain (Fig 2.6) and hemoptysis (Fig 2.7). Nevertheless, there was no significant difference in the mOS depending on the primary tumor site (Fig 2.8). Fig 2.9 reveals that the mOS in the no TILs feature

   was longer than the mOS in the TILs group, but was no statistical difference. Among the active smokers, the mOS was longer in the PTEN- group than in the PTEN+ group (Fig 2.10). The mOS in either presence or absence of EGFR mutation was not significantly different (7.44 vs 14.52 months) (p = 0.41).

   
   

   DISCUSSION

   As the function of PTEN is to inhibit the Akt kinase activity in the kinase/Akt/mTOR pathway, PTEN deletion results in high levels of activated Akt, which brings out the G1 cell cycle and possible progress to pathogenesis of the NSCLC and subsequent metastasis.23 Furthermore, the absence of PTEN creates an immunosuppressive microenvironment, thus facilitating tumor growth and metastasis.24 PTEN- creates an immunosuppressive microenvironment.25 On the contrary, the presence of CD8+ T-lymphocyte infiltration is correlated with a better prognosis in several cancers.26 The expression of PTEN protein could also block the programmed cell death receptor-1 (PD-1). Consequently, several studies have reported the expression of PD-1 is related with the expression of PTEN.27

   Our study is the first to report the overall prevalence of PTEN- in Thai patients with lung cancer (66.5%), with a prevalence of 64.8% in NSCLC patients and 100% in

   SCLC patients. Previously, the prevalence of PTEN- in NSCLC patients was reported to be approximately 24%- 59.86%11,13,28-32, In the literature, adenocarcinoma (AC) is the most common subtype of NSCLC, representing approximately 40% of patients.33 However, in this study, the prevalence of AC subtypes was very high (84.4% in males and 87.3% in females), compared to the reported prevalence according to a SEER study in 201834 and in the 2015 report of the National Cancer Research Institute of Thailand2, which to be reported a rate of approximately 50%-60%.

   Of note, the percentages of AC and PTEN- in our study population were relatively high. Possible explanations for this include the higher cut-off score of 3 and higher we employed, similar to the study of Tang et al. in NSCLC patients11, whereas many previous studies utilized lower cut-off scores of 1-2.11,35-40 Table 4 described the PTEN expression in lung cancer patients from each study and their correlated parameters.

   
   

   

   
   

   TABLE 4. The studies of the PTEN loss of expression in lung cancer patients and their correlated clinical parameters.

   
   

   References	Histology	Finding	PTEN score	Number of patients	Related clinical parameters with PTEN-
   Soria et al.28	NSCLC	Protein loss	Intensity - absence	24% (30/125)	No related parameters
   Chang et al.41	NSCLC	Protein loss	< 3	59.86% (173/289)	LN metastasis, smoking status, low survival rate
   Yoo et al.42	NSCLC	Protein loss	No data	42.4% (122/288)	SC, smoking status, low progression free survival
   Scrima et al.43	NSCLC	Protein loss	0-25%	39% (41/104)	SC subtype
   Tang et al.11	NSCLC	Protein loss	0-2	46.1% (47/102)	Poor survival of p-AktS473 positive
   Goncharak et al.32	NSCLC	Protein loss	0-2	41.4% (43/104)	Advanced disease, LN metastasis, low survival rate
   Kim et al.37	AD, SC	Protein loss	0-2	37.4% (34/91)	High histological grade, pathological T stage, N stage, short survival in AD
   Thiva et al., These study	NSCLC	Protein loss	<3	64.8% (103/159)	Absence of TILs, poor location at LLL, age <65 years, smoking

   
   

   Moreover, we preferred the detection of PTEN expression by the IHC method over PTEN mRNA study as the method is more sensitive for studying overall survival. Further, in the meta-analysis, NSCLC patients with PTEN loss of expression had an unfavorable prognosis, whereas the results could not be demonstrated when the PTEN mRNA method was utilized.24

   In our study, patients with hemoptysis had a longer mOS than asymptomatic patients (p = 0.008). Thus, hemoptysis might prompt both patients and doctors’ concerns, thus allowing early investigation, followed by prompt treatment, eventually extending the overall mOS period. Furthermore, similar to the study of Port et al., our study indicated that a smaller tumor size and lower ECOG performance status were correlated with a prolonged mOS.44 Moreover, no significant difference in mOS was observed between the age groups and genders, corresponding with another study by Franceschini et al.45

   In NSCLC, no significant difference was seen in mOS between PTEN– and PTEN+ (p = 0.38). Though most studies expected a lower mOS in NSCLC patients with PTEN - 31,35-38,40, 42,46-50, some studies reported a longer mOS.51 The differences could be possibly due to different races and ethnicities, techniques to detect PTEN loss of expression (including polymerase chain reaction, fluorescence in situ hybridization, IHC), and cut-off score in IHC. Notably, in our study, a higher proportion of AC was observed, and this NSCLC subtype is more responsive to treatment.

   Interestingly, all the SCLC patients in our study were at an extensive stage with PTEN-, compared with those with the NSCLC subtype (p = 0.05). The mOS of SCLC was short, only 5.28 months (range. 0.96-9.61). Regarding previous reports on the prevalence of SCLC, possible SCLC-Y accounted for approximately 10% of all four subtypes. However, only in the SCLC-Y subtype is the oncogenesis related to the mTOR pathway and associated with PTEN loss of expression. Presumably, all the SCLC cases in our study were likely to be the SCLC-Y subtype. However, further studies to analyze the subtypes of the SCLC are required to demonstrate the actual prevalence of each subtype in Thai patients. In this study, there were no significant differences in mOS in patients with smoking history, a more advanced stage, and squamous cell type with PTEN- status (p = 0.089, 0.237, and 0.053); though previous studies demonstrated a correlation between smoking status41,52,53, stage13, and squamous cell subtype13,31, with PTEN-. In the PTEN- group in our study, patients with a smoking history had a longer mOS than those with a negative smoking history

   (p = 0.012; 6.12 vs 1.68 months, HR (PTEN +/-) = 0.48,

   95%CI 0.27-0.86, p = 0.014). Chang et al. also reported an unclear association between the PTEN status and smoking behavior.41 Of note, the frequency and intensity of smoking are known risk factors for lung cancer, but these were not extensively included in the analysis due to the limited data availability in the medical records.

   Interestingly, our study is the first to report that the location of the primary tumor at the RUL was significantly correlated with PTEN-, compared to at the LLL location (p = 0.03). Patients with the primary site at the RUL also had a significantly longer mOS than at the LUL and LLL locations (16.03 months vs. 3.79 months and 5.58

   months (p = 0.024) (LUL:HR 1.60, 95%CI 1.01–2.54, p

   = 0.046 and LLL:HR = 1.90, 95%CI 1.16–3.09, p = 0.01).

   For the primary location of NSCLC, Lee et al. reported that a lower lobe group had a higher mortality rate than non-lower lobe ones (48.6% and 40.3%, p <0.0001) with less frequent EGFR mutation.54 Lung cancer with the primary location at the upper lobe may contain different immunomolecular processes that might interfere with the survival outcomes. These hypotheses probably lead to the need for personalized medicine.

   In the PTEN+ group, the location of the primary site was significantly associated with the mOS, of which the RUL location had a longer mOS than the LLL location. However, this association was not found in the PTEN- group. Nevertheless, in the PTEN+ group, the primary location of the tumor at the RUL had a longer mOS than at the LLL location [32.4 months vs 14.8 months (p = 0.0001), HR 3.14, 95%CI 1.52–6.52, p = 0.002]. Therefore,

   further studies on the PTEN expression using the IHC of the patients with the primary location of lung cancer at the upper lobe may reveal its prognostic capability.

   For the histopathological studies, the PTEN- was found to be associated with the negative findings of TILs (p

   = 0.045). In our study, there was no significant difference in mOS using the TILs status (p = 0.67). On the contrary, Schalper et al. and Gao et al. reported better survival outcomes in NSCLC and triple-negative breast cancer with the presence of TILs, respectively.55,56 In our mOS study in NSCLC, no TILs feature was not a prognostic factor, probably not only the level of activated Akt in the mTOR pathway, influencing cellular survival, and other tumor suppressor functions, such as chromosome integrity and DNA repair57, take part in the survival process. Other confounding factors, such as a small sample size number, the method used to identify PTEN, and scoring of the PTEN expression by IHC, were influential in these studies.

   
   

   CONCLUSION

   The prevalence of the PTEN loss of expression in NSCLC in our study population was quite a bit higher than in previous studies. The pathological no TILs feature and RUL location were found to be independent factors associated with the PTEN loss of expression. The small cell subtype and the smoking group were nearly significantly related to negative PTEN staining. Smoking status, all symptoms, ECOG status, RUL location, and tumor size

   >7 cm were found to play unfavorable prognostic roles in the overall survival in NSCLC. In the subgroup study, PTEN loss of expression with RUL location tended to involve a poor prognosis. Interestingly, a better survival outcome was shown in the active smoker group with PTEN negative. Further survival study of the no TILs pathological status and active smoker subgroups in terms of PTEN expression and other immune-related biomarkers, such as IHC for programmed death–ligand 1 (PD-L1), with an adequate sample size and proper study design is warranted.

   
   

   ACKNOWLEDGMENTS

   We gratefully thank to Dr.Sirisanpang Yodavudh for academic advice, Mr. Supalarp Puangsa-art and Miss Waraporn Netprao for statistical analysis, Dr.Susama Chokesuwattanaskul for manuscript edition, Dr.Phatharaporn Kiatpanabhikul (my wife) for all kind support.

   
   

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