Relationship between lymphedema after breast cancer treatment and biophysical characteristics of the affected tissue.

The treatment of breast cancer is often complicated by lymphedema of the upper limbs. Standard lymphedema evaluation methodologies are not able to measure tissue fibrosis. The ultrasound aspects related to tissue microstructures of lymphedema are neglected in clinical evaluations. The objective of this study was to identify and measure the degree of impairment, topography, and biophysical alterations of subcutaneous lymphedema tissue secondary to the treatment of breast cancer by ultrasonography. Forty-two women at a mean age of 58 (±9.7) years, with unilateral lymphedema due to breast cancer treatment, were evaluated. The upper limbs were divided into affected (affected by lymphedema) and control (contralateral limb). Each limb was subdivided into seven areas, defined by perimetry, evaluated in pairs. The biophysical characteristics thickness, entropy, and echogenicity were evaluated by ultrasonography. The results showed a significant difference in the echogenicity and thickness variables between the affected and unaffected upper limb, in all the extent of the upper limb, while entropy showed no significant difference. The findings indicate that the data presented were consistent both in identifying and measuring the degree of impairment and biophysical changes in the subcutaneous tissue of lymphedema secondary to the treatment of breast cancer.

Introduction

Lymphedema secondary to the treatment of breast cancer is a chronic and recurrent condition involving the lymphatic and blood systems [1,2]. The dysfunctional lymphatic system becomes less capable of performing the complete resorption of large protein molecules, and these remain in the interstitial space. The consequent tissue fibrosis and the increasing accumulation of fluid and proteins in this space can trigger neurological alterations such as pain or paresthesia, distortion in the shape of the limb, and increased risk of related complications [3,4]. Chronic lymphedema causes physical deficiencies and psychological stress, which worsens with the progression of the dysfunction, to reduce the discomfort of the patient and improve the quality of life, an accurate diagnosis of lymphedema is essential for prognosis and treatment planning [5].

The evaluation of limb volume is considered the gold standard for measuring irregular edema and is considered the main routine measure for the diagnosis and evaluation of the evolution of lymphedema treatments [6,7]. However, these measures only characterize the external shape of the related upper limb, not having the sensitivity necessary to identify the intrinsic alterations of the affected tissue. The exclusively clinical diagnosis of lymphedema does not indicate consistent thresholds for the difference between the upper limbs, or between different evaluators. Subjective measures such as palpation and patient perception are not consistent in the evaluation of lymphedema [8].

Other medical imaging modalities are applied to diagnose lymphedema [9,10]. Among these, lymphoscintigraphy is the main imaging modality to evaluate circulatory dysfunctions of the lymphatic system, considered the standard for decades [11]. Nonetheless, it does not identify biophysical alterations such as stiffness, topography, and size of the limb with lymphedema, one of the main causes of discomfort and impairment of limb function in patients [12].

Chronic lymphedema induces complications such as inflammation, fat tissue hypertrophy, fibrosis, and recurrent infections [13]. These variables can be taken into account in the analysis of the ultrasound image of the affected tissue [8,14]. The topography, texture, and stiffness of the tissue that may directly influence the responses resulting from different therapeutic interventions have not been established yet. The control of lymphedema is fundamental, as it is considered a definitive condition [4], to prevent related comorbidities, minimizing complications with adequate treatment, as well as helping to monitor the progression of the dysfunction, assisting in the prognosis, and in the evaluation of effects resulting from different therapeutic interventions.

The objective of this study was to identify and measure the degree of impairment, topography, and biophysical alterations of subcutaneous lymphedema tissue secondary to the treatment of breast cancer by ultrasonography. For that, thickness, entropy, and echogenicity in seven points of the upper limb were examined and measured, with a subsequent comparison between the affected and unaffected upper limbs.

Materials and methods

Participants

The study was designed a type of cohort design of women enrolled between December/2017 and December/2018. The patients were recruited at REMA (Center for Teaching, Research, and Assistance in the Rehabilitation of Mastectomized Patients) at the University of São Paulo at Ribeirão Preto, College of Nursing (EERP-USP). Interested individuals completed a personal screening of the disease and treatment. 125 women with lymphedema from breast cancer treatment were invited and elected to participate in this study, 83 of them were excluded by the established criteria and 42 patients were allocated in this study. All participants provided institutionally approved, written, informed consent under a study protocol approved by the Ethics and Research Committee of the Medical School of Ribeirão Preto of the University of São Paulo (FMRP/USP), CAAE Process 65981216.4.0000.5440.

The sample was calculated based on the study by Suehiro et al. (2014) [15], with a statistical power of 80% and an alpha error of 0.05. The program used was StateMate 2 (GraphPad Software® v 2.0), resulting in n = 35, and to compensate for possible losses, a sample of 42 was considered.

The outcome of the measurement of lymphedema was obtained through indirect measurement of volume, determined by the upper limb’s circumference. The limb volume was calculated from the circumference measurements, treating each segment of the limb as a pair of circumferences, formed by the measurement points of the circumference of the seven points of the arm and forearm, called truncated cones. Lymphedema was considered when there was a difference greater than 2 cm in the perimetry of two or more predetermined points on the affected limb compared to the contralateral limb [16].

Inclusion criteria were women over 21 years old who underwent treatment for unilateral breast cancer, had unilateral lymphedema from treatment for breast cancer, according to criteria of Sander et al., (2001). Women with skin conditions, adjuvant treatment in progress, diagnosis of metastasis or unexplained edema in the arm not involved were excluded. None of the participants was under intensive treatment for lymphedema.

Lymphedema measurements

Assessment of member volume

Limb volume was evaluated by indirect measurement, resulting from the evaluation of the perimetry of both upper limbs and the sum of the approximate volume of six truncated cones, formed of the measurement of the circumferences of the seven points on the arm and forearm. The sum of these six parts provided the total volume of the limb. The method of measuring the indirect volume has good levels of intra- and inter-examiner reliability, with values of intraclass correlation coefficient (ICC) of 0.99 [16].

Evaluation of biophysical characteristics by ultrasound

Ultrasound images (US) were obtained using Ultrasonics ultrasound equipment (Sonix RP, Richmond, BC, Canada), with a linear transducer of 9–15 MHz, gain 70%, depth 5.0 cm. The B-mode image and the RF (radio frequency) signal were collected simultaneously. A phantom (biological tissue-mimicking material), produced from a 2.5 cm thick styrene—ethylene—butylene—styrene copolymer (SEBS), was used between the transducer and the skin, both surfaces coupled with ultrasound gel, to standardize the collections.

The signal was collected in the anterior region of the affected upper limb and of the unaffected limb by lymphedema in the seven points already pre-defined for perimetry, we considered a line that was made to standardize these points starting from the acromion and passing through the midpoint between the epicondyles and the styloid process of the radius and ulna. Other studies that quantify the characteristics of the subcutaneous tissue use the anterior region of the forearm [17,18]. This is a relatively level surface where there is the convenience of positioning the US transducer (Fig 1).

Fig 1

a) Illustrative image of the points used for elastography; b) Placement of the US pad and transducer.

Source: Personal records.

In each US image, the region of interest (ROI) was defined as the distance between two echogenic lines, as described in previous studies defining the subcutaneous tissue, the thickness of this region, and the measurements of echogenicity and entropy [19-21].

The ultrasound signal from the affected and unaffected upper limb, 30 frames were obtained for each point, for each of the seven predefined points. These images were saved in both B and RF (Radiofrequency) modes. For this analysis, the B mode (.b8) file was used. For processing the ultrasound images, computational routines in the Matlab environment (TheMathWorks, Inc., Natick, MA) were used to obtain three images for each point and transform them into another extension for analysis (.png). The images in (.png) were analyzed using the Image J software. The Straight function present in the software to measure distance was used to quantify the thickness of the tissue affected by lymphedema in pixels. For entropy and echogenicity analysis, ROI was initially defined using the Polygon selection tool. In this ROI, standard measurements of entropy and echogenicity were implemented with a new plugin that was added and used in the software (Fig 2).

Fig 2

Image of thickness, echogenicity and entropy evaluation in Image J.

Source: Personal records.

Statistical analysis

The data were submitted to exploratory analysis, which had the primary objective of synthesizing a series of values of the same nature, allowing a global view of the variation of these values, organizing and describing the data in tables with descriptive measures.

After tabulating the variables, the Shapiro-Wilk normality test was applied to analyze the distribution. The echogenicity and entropy variables presented non-parametric distribution; when Wilcoxon was applied. The thickness variable presented normal distribution, so the related T-test was applied. In all calculations, a critical level of 5% (p<0.05) was fixed, and data processing was performed by SPSS software, version 17.0.

Results

The general characteristics and the characteristics relative to the surgical treatment of patients submitted to the evaluation of lymphedema by ultrasound are present in Table 1.

Table 1

General characteristics of patients with lymphedema.

Characteristics
Age (years)	58 (9.7)
BMI (Kg/m 2 )	31.86 (6.05)
Type of surgery	Mastectomy	24
	Quadrantectomy	10
	Nodulectomy	8
Side Surgery	Right	23
	Left	19
Axillary Lymphadenectomy	Yes	42
	No	0

*Values presented as mean (standard deviation). BMI = Body mass index.

The results regarding echogenicity (capacity of the tissue to reflect an echo of the ultrasound wave, indicative of the density of the subcutaneous tissue) of the upper limb affected and unaffected by lymphedema are present in Table 2. The affected upper limb showed greater echogenicity at all points, except for point 7 (wrist), probably due to the presence of tendons in the region.

Table 2

Values referring to echogenicity in the affected upper limb (MSA) and unaffected upper limb (MSNA) at the seven predetermined points.

Points	MSA	MSNA	p
1	51.94 (41.51, 50.13)	44.91 (37.88, 63.87)	<0.001
2	56.76 (47.72, 78.74)	54.76 (45.68, 70.11)	0.001
3	61.55 (51.25, 81.28.)	57.07 (48.60, 73.35)	<0.001
4	55.34 (46.94, 93.82)	55.54 (42.08, 75.42)	0.004
5	52.28 (42.66, 83.55)	51.89 (42.08, 75.06)	0.040
6	53.49 (42.91, 89.31)	47.28 (39.05, 68.01)	0.001
7	62.32 (53.09, 88.69)	64.00 (53.70, 92.70)	0.161

*Values presented in median (first quartile, third quartile). MSA: Affected upper limb by lymphedema. MSNA: Unaffected upper limb by lymphedema.

The results regarding entropy (a measurement of subcutaneous tissue disorder) of the upper limb affected and unaffected by lymphedema are present in Table 3. This variable did not vary significantly among limbs affected or not.

Table 3

Values referring to entropy in the affected upper limb (MSA) and unaffected upper limb (MSNA) at the seven predetermined points.

Points	MSA	MSNA	p
1	1.87 (1.84, 1.99)	1.89 (1.83, 2.07)	0.061
2	1.89 (1.85, 2.08)	1.89 (1.85, 2.04)	0.472
3	1.89 (1.86, 2.07)	1.88 (1.85, 2.08)	0.091
4	1.89 (1.82, 1.99)	1.87 (1.85, 2.08)	0.738
5	1.88 (1.85, 2.03)	1.89 (1.85, 2.07)	0.965
6	1.88 (1.85, 2.03)	1.88 (1.83, 2.02)	0.574
7	1.87 (1.83, 2.01)	1.84 (1.82, 2.05)	0.213

*Values presented in median (first quartile, third quartile). MSA: Affected upper limb by lymphedema. MSNA: Unaffected upper limb by lymphedema.

The data regarding the thickness of the subcutaneous tissue of the upper limb affected by lymphedema was significantly higher at all points except at point 4 (elbow), compared to the unaffected upper limb (Table 4).

Table 4

Pixel thickness values of the subcutaneous tissue in the affected upper limb (MSA) and unaffected upper limb (MSNA) at the seven predetermined points.

Points	MSA	MSNA	p
1	99.36 (31.62)	84.59 (36.32)	<0.001
2	94.34 (32.94)	78.81 (38.37)	0.009
3	83.16 (34.39)	62.99 (34.98)	<0.001
4	102.50 (212.37)	50.60 (26.32)	0.110
5	87.73 (32.46)	56.61 (25.47)	<0.001
6	102.87 (35.80)	74.90 (40.44)	<0.001
7	69.24 (34.22)	48.56 (34.36)	<0.001

*Values presented as mean (standard deviation). MSA: Affected upper limb by lymphedema. MSNA: Unaffected upper limb by lymphedema.

Discussion

Fibrosis is a histological characteristic of lymphedema; however, its measurement in the patient sometimes is still performed through clinical measures, such as palpation [22]. This study aimed to measure fibrosis of the subcutaneous tissue in lymphedema secondary to the treatment of breast cancer by ultrasonography.

The results showed that both thickness and echogenicity are measured through the differentiation between the upper limb affected by lymphedema and the unaffected upper limb, showing all the degree of impairment of the affected tissue either by the increase in size, in case of thickness, or fibrosis developed, quantified by employing the echogenicity variable. These findings demonstrate how the use of ultrasound images can be a differential in the diagnosis and treatment of lymphedema. Once this is a definitive condition with negative repercussions on physical, psychological, and emotional well-being, lymphedema must be controlled to prevent comorbidities [4]. Ultrasonography is considered a promising method [8] and our results confirm that this tool is effective in the evaluation and characterization of biophysical alterations inherent to lymphedema.

Understanding the degree of structural involvement of the affected tissue can help to monitor the progression of the dysfunction, assisting in the prognosis, as well as assessing the effects of different therapeutic interventions. In the present study, there was a concern about analyzing the lymphedema distribution topography; the findings showed that the regions closest to the elbow presented greater differences, unlike from the wrist in terms of the amount of fibrosis.

Due to fibrosis and the blocking of lymphatic channels in the regions near the elbow of an upper limb affected by lymphedema, there is an important impairment of lymphatic circulation. The lymphatic pathway of this limb presents significant differences such as obliteration of superficial lymphatic vessels, dermal reflux, uncommon communication between the superficial and deep lymphatics, and interval lymph nodes. These alterations seem to facilitate lymphatic drainage after sporadic blockage of the lymph, especially proximal in the limb [23].

The circulatory function is influenced by the drainage of superficial and deep lymphatic vessels and is often interrupted in some regions of lymphedema, triggering superficial collateralization with retrograde flow to the lymphatic vessels of the skin. Lymphatic fluid stasis is associated with the accumulation of interstitial fluid in the subcutaneous tissue and skin and also with an accumulation of proteins and glycosaminoglycans, the retained interstitial fluid subsequently stimulates the production of collagen, which leads to skin thickness and subcutaneous fibrosis of soft tissues [24].

Subjective measures such as palpation and patient perception are not consistent for assessing lymphedema fibrosis. Furthermore, the diagnosis exclusively based on volume does not point to consistent thresholds for the difference between the upper limbs, or for the evaluation of professionals [8]. In phantom testing in a reduced number of patients using 2D ultrasound, there were encouraging initial results; deformation values of the arms affected by lymphedema were much higher than those of normal arms [25]. In the present study, it is possible to observe a difference in the thickness of the subcutaneous tissue by ultrasound. Although volume and palpation are routinely measured for diagnosis and evaluation of treatment evolution, they are very inconsistent, supporting the argument that they should not be the only objective measurement of lymphedema.

Both entropy, a measure of randomness within the ultrasound image reflecting the degree of organization or disorganization of tissue, and echogenicity, a measure of the capacity of the tissue reflecting an echo of the ultrasound wave, serve as an indicator of the density of the tissue and may be able to discriminate the affected and unaffected side of lymphedema [8,14]. Ashikaga et al. (2005) showed a preference for the measurement of entropy to characterize image texture, which was seen by increasing the values of entropy on the affected side. However, Johson et al. (2016) observed a lower entropy. The difference between these studies is the different types of surgery evaluated.

Entropy measurements were less variable in this study, so it was not significant between members. The previously mentioned studies only evaluated a predetermined ROI (region of interest) of a few millimeters. This study is a pioneer in evaluating the entire subcutaneous tissue area of the assessed points. This choice may have been a determinant of why the difference between limbs was not significant in the entropy variable, as this is a randomization measure of the ultrasound image, which reflects how much the tissue is organized or not [26]. Therefore, larger regions could have more variations, once in the subcutaneous tissue there are also other structures such as fat, and not only fibrosis. It is important to note that most of the population studied are overweight or obese, which is common in surviving breast cancer patients [12,27].

Echogenicity is a measure of the ability of the tissue to reflect an echo of the ultrasound wave, higher echogenicity indicates that the tissue has a higher density suggestive of higher fibrosis. Studies [28,29] have shown that upper limbs with lymphedema resulting from the treatment of breast cancer increased or altered the echogenicity in subcutaneous tissue. However, the authors did not quantify the variable. The measurement of echogenicity through subcutaneous ultrasonography can improve the accuracy of diagnosis and can be used to monitor the progress and severity of lymphedema in the forearm [30]. The present study quantified and demonstrated that the analysis of echogenicity can be an important tool in the evaluation of the degree of fibrosis of the affected upper limb.

Previous studies [14,26] have only evaluated two measurement points, one in the arm and other in the forearm, or even an additional measurement close to the elbow [8]. To achieve the proposed objectives, this study included seven measurement points, necessary to evaluate irregular edemas such as lymphedema. Differences were observed in our results such as a significant increase in the thickness of the subcutaneous tissue measured by ultrasonography in the upper limb, different in distinct areas affected by lymphedema.

Previous studies [8,14] have used 4 mm of depth for the analysis of their images, but because this ROI is too small, it only registers skin and the most superficial region of the subcutaneous tissue. This limitation was observed by Johson et al. (2016), who considers that important information regarding the subcutaneous tissue may have been lost. In the present study, the 5.0 cm depth was used, thus, excluding the possibility of a loss of information regarding the subcutaneous tissue. The phantom thickness of 2.5 cm is another reason for the choice of using a depth of 5.0 cm.

One limitation of the tissue was the use of a transducer smaller than 20 MHz, which presents a lower spatial resolution and consequent visualization of structures close to it. The resolution for this limitation was to make a phantom used as a spacer between the transducer and the skin to minimize potential resolution problems that would affect the accuracy of the images since it is a transducer smaller than 20 MHz.

The understanding of the involvement of the tissue affected by lymphedema, in a thorough way, can help to monitor the progression of the dysfunction, assisting in the prognosis, as well as in the evaluation of effects resulting from different therapeutic interventions. The diagnostic ultrasound technique can help understand how the treatments act on the intrinsic tissue affected by lymphedema, which is fundamental for both clinical performance and research. Therefore, the analysis of the ultrasound image seems to be a promising tool for both the evaluation and monitoring of different interventions.

Conclusion

The findings indicated that the data presented were consistent in identifying the biophysical alterations of subcutaneous lymphedema tissue secondary to the treatment of breast cancer. The thickness of the subcutaneous tissue was increased in all measurements except for the elbow, which is not such a complacent region. However, entropy in these parameters still showed no difference between the affected and unaffected upper limbs. Echogenicity, quantified to measure fibrosis was increased in all upper limbs except for the wrist.

27 Oct 2020

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18 Jan 2021

PONE-D-20-33153

RELATIONSHIP BETWEEN LYMPHEDEMA AFTER BREAST CANCER TREATMENT AND BIOPHYSICAL CHARACTERISTICS OF THE AFFECTED TISSUE

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Dear Dr. Perez,

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Reviewer #1: Perez et al. presented the evaluation of lymphedema after breast cancer by using ultrasonography. They focused on tissue thickness, echogenicity, and entropy. These components seem to be a kind of worn-out technique. Still, it is of note that they mention fibrosis caused by lymphedema. Once fibrosis has developed, it is quite difficult to treat it. To choose treatment options, their technique may be useful in determining whether fibrosis is already well established or not. However, I have several major concerns about this report the authors should meet.

Major concern 1

The authors evaluated values of ultrasonography on lymphedema, just compared with unaffected side of upper limbs. It is not so difficult to decide whether there is lymphedema or not via subjective measures. It is much more important to evaluate how much lymphedema progresses. Therefore, they need to illustrate the association between values of ultrasonography and the progression of lymphedema. The author should analyze the differences between patients. They need to illustrate whether the index proposed in the manuscript can reflect the severity of the lymphedema.

Major concern 2

They stated that clinical problem in lymphedema after breast cancer lies in lack of diagnostic tools, but there are actually much more serious problems, including the difficulty in controlling lymphedema and stopping the fibrosis, and lack of treatment options. They need to point out how their technique would contribute to the future treatment of lymphedema.

Minor point

Tissue Elastgraphy is also attracting more and more attention as a non-invasive measurement for fibrosis. Does the author’s method correlate to these other methods?

Reviewer #2: This study showed the echogenicity and the thickness of subcutaneous tissue of the upper limb on ultrasonography (US) related to lymphedema after breast cancer treatment.

But there are some problems on this manuscript.

1) What is the definition of lymphedema? Please describe in the participant section.

2) In the last paragraph of the Discussion, the authors say that the US seems to be a promising tool for the monitoring of interventions, but this study did not assess the changes of US findings after treatment for lymphedema and the prognosis.

3) This study did not include the degree of impairment, therefore the first sentence of the conclusion seems inadequate.

Minor points

1) Table 1 law means right?

2) Table 2 -4, abbreviation of MSA and MSNA should be spelled out in the foot note.

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18 Feb 2021

Thank you for your valuable inputs towards our study. Therefore, the corrections respond to the recommendations of all reviewers. They emphasized the relevant data and enhanced comprehension of the manuscript.

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Answer: All participants provided institutionally approved, written, informed consent under a study protocol approved by the Ethics and Research Committee of the Medical School of Ribeirão Preto of the University of São Paulo (FMRP/USP), CAAE Process 65981216.4.0000.5440. This information was inserted in the manuscript and is highlighted in the document 'Revised Manuscript with Track Changes'.

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Answer: The patients were recruited at REMA (Center for Teaching, Research, and Assistance in the Rehabilitation of Mastectomized Patients) at the University of São Paulo at Ribeirão Preto, College of Nursing (EERP-USP). Interested individuals completed a personal screening of the disease and treatment. This information was inserted in the manuscript and is highlighted in the document 'Revised Manuscript with Track Changes'.

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

Review Comments to the Author

Reviewer #1: Perez et al. presented the evaluation of lymphedema after breast cancer by using ultrasonography. They focused on tissue thickness, echogenicity, and entropy. These components seem to be a kind of worn-out technique. Still, it is of note that they mention fibrosis caused by lymphedema. Once fibrosis has developed, it is quite difficult to treat it. To choose treatment options, their technique may be useful in determining whether fibrosis is already well established or not. However, I have several major concerns about this report the authors should meet.

Major concern

1. The authors evaluated values of ultrasonography on lymphedema, just compared with unaffected side of upper limbs. It is not so difficult to decide whether there is lymphedema or not via subjective measures. It is much more important to evaluate how much lymphedema progresses. Therefore, they need to illustrate the association between values of ultrasonography and the progression of lymphedema. The author should analyze the differences between patients. They need to illustrate whether the index proposed in the manuscript can reflect the severity of the lymphedema.

Answer: The data analyzed in diagnostic ultrasonography aimed to characterize thickness, echogenicity, and entropy measurements and their distribution in the upper limb affected and not affected by lymphedema. Subjective measures still seem insufficient to identify lymphedema since most measures are clinical and dependent evaluators (Johnson et al., 2016).

Regarding the measures presented and the disease progression, the greater the thickness, the greater the limb's volume, echogenicity may be related to the amount of collagen tissue, suggesting fibrosis, and the greater the entropy, the more significant tissue disorganization. To establish this relationship, the measurement of these measures is done first between the affected tissue and a phantom of known measures. Only after establishing this relationship is a comparison between the limbs.

The choice of a longitudinal study appears to be interesting, but it does not include this study's objectives. Lymphedema is a chronic disease, where the time of onset is still unknown, many patients take months to years to seek health care. Also, several factors can alter the volume and tissue fibrosis of lymphedema during the course of the disease, such as injuries, physical inactivity, and weight change, among others. Therefore, the data cannot infer the disease's progression since it is multifactorial.

Johnson KC, DeSarno M, Ashikaga T, Henry SM. Ultrasound and clinical measures for lymphedema. Lymphat Res Biol. 2016;14(1):8-17.

Major concern

2. They stated that clinical problem in lymphedema after breast cancer lies in lack of diagnostic tools, but there are actually much more serious problems, including the difficulty in controlling lymphedema and stopping the fibrosis, and lack of treatment options. They need to point out how their technique would contribute to the future treatment of lymphedema.

Answer: The treatments for lymphedema concentrate only on controlling the upper limb's volume, with little focus on tissue alteration. The diagnostic ultrasound technique can help understand how the treatments act on the intrinsic tissue affected by lymphedema, which is fundamental for both clinical performance and research. This information was inserted in the manuscript and is highlighted in the document 'Revised Manuscript with Track Changes'.

Minor point

Tissue Elastgraphy is also attracting more and more attention as a non-invasive measurement for fibrosis. Does the author's method correlate to these other methods?

Answer: The data may have a complementary relationship since elastography would measure the "hardness", replacing the palpation.

Reviewer #2: This study showed the echogenicity and the thickness of subcutaneous tissue of the upper limb on ultrasonography (US) related to lymphedema after breast cancer treatment.

But there are some problems on this manuscript

1) What is the definition of lymphedema? Please describe in the participant section.

Answer: The outcome of the measurement of lymphedema was obtained through indirect measurement of volume, determined by the upper limb's circumference. The limb volume was calculated from the circumference measurements, treating each segment of the limb as a pair of circumferences, formed by the measurement points of the circumference of the seven points of the arm and forearm, called truncated cones (Sander et al., 2002).

The method of measuring the indirect volume has acceptable levels of intra- and inter-examiner reliability, with intraclass correlation coefficient (ICC) values of 0.99, and is easily operational (Taylor et al., 2006). The final estimated excess volume corresponds to the sum of the differences between each point. Lymphedema was considered when there was a difference greater than 2 cm in the perimetry of two or more predetermined points on the affected limb compared to the contralateral limb (Sander et al., 2002). This information was inserted in the manuscript and is highlighted in the document 'Revised Manuscript with Track Changes'.

Sander AP, Hajer NM, Hemenway K, et al (2002) Upper- extremity volume measurements in women with lymphedema: a comparison of measurements obtained via water displacement with geometrically determined volume. Phys Ther. 82:1201–1212

Taylor R, Jayasinghe UW, Koelmeyer L, et al. (2006) Reliability and validity of arm volume measurements for assessment of lymphedema. Phys Ther 86:205–214

2) In the last paragraph of the Discussion, the authors say that the US seems to be a promising tool for the monitoring of interventions, but this study did not assess the changes of US findings after treatment for lymphedema and the prognosis.

Answer: Clinical measures such as volumetry and palpation are still the main measures for monitoring lymphedema treatments. Although volumetry has an excellent ability to measure the difference in volume between the limbs, it is only concerned with measuring an external characteristic. Simultaneously, palpation is not very reliable because it is a dependent evaluator. The subcutaneous tissue so affected by lymphedema, cannot be reliably represented by these clinical measures, so the treatments are focused on just decreasing volume. Although the recommendations of the guidelines for the diagnosis and prognosis of lymphedema may be typically delayed concerning new developments in the diagnostic field, the changes in the guidelines depend on robust evidence, necessary for the diagnostic ultrasound to be an integral part in monitoring the treatment of this dysfunction. Indeed, the immediate availability, the non-invasive nature, and the low cost can make this modality the first choice to establish lymphedema measures in the diagnosis and prognosis.

3) This study did not include the degree of impairment, therefore the first sentence of the conclusion seems inadequate.

Answer: As suggested in the first sentence of the conclusion, the measurement of the degree of impairment was removed, leaving the identification of biophysical changes. This information was inserted in the manuscript and is highlighted in the document 'Revised Manuscript with Track Changes'.

Minor points

1) Table 1 law means right?

Answer: In table 1 the word "law" has been replaced by "right". This information was inserted in the manuscript and is highlighted in the document 'Revised Manuscript with Track Changes'.

2) Table 2 -4, abbreviation of MSA and MSNA should be spelled out in the footnote.

Answer: The abbreviations MSA and MSNA were explained in the footnote and is highlighted in the document 'Revised Manuscript with Track Changes'.

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Reviewer #1: No

Reviewer #2: No

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

7 Feb 2022

RELATIONSHIP BETWEEN LYMPHEDEMA AFTER BREAST CANCER TREATMENT AND BIOPHYSICAL CHARACTERISTICS OF THE AFFECTED TISSUE

PONE-D-20-33153R1

Dear Dr. Perez,

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Additional Editor Comments (optional):

The authors responded and revised the article adequately.

Reviewers' comments:

9 Feb 2022

PONE-D-20-33153R1

RELATIONSHIP BETWEEN LYMPHEDEMA AFTER BREAST CANCER TREATMENT AND BIOPHYSICAL CHARACTERISTICS OF THE AFFECTED TISSUE

Dear Dr. Perez:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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