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Lung ISSN 0341-2040Volume 192Number 6 Lung (2014) 192:967-974DOI 10.1007/s00408-014-9641-2

Evaluation of the Extent of PulmonaryCysts and Their Association withFunctional Variables and Serum Markersin Lymphangioleiomyomatosis (LAM)

Bruno Guedes Baldi, Mariana SponholzAraujo, Carolina Salim GonçalvesFreitas, Gustavo Borges da Silva Teles,Ronaldo Adib Kairalla, et al.

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INTERSTITIAL LUNG DISEASE

Evaluation of the Extent of Pulmonary Cysts and TheirAssociation with Functional Variables and Serum Markersin Lymphangioleiomyomatosis (LAM)

Bruno Guedes Baldi • Mariana Sponholz Araujo • Carolina Salim Goncalves Freitas •

Gustavo Borges da Silva Teles • Ronaldo Adib Kairalla • Olıvia Meira Dias •

Daniel Antunes Silva Pereira • Suzana Pinheiro Pimenta • Carlos Roberto Ribeiro Carvalho

Received: 2 June 2014 / Accepted: 1 September 2014 / Published online: 9 September 2014

� Springer Science+Business Media New York 2014

Abstract

Purpose Although computed tomography (CT) has been

used previously to assess disease severity in lymphangio-

leiomyomatosis (LAM), the associations between the extent

of pulmonary cysts on CT and six-minute walk test (6MWT),

matrix metalloproteinases (MMPs) and vascular endothelial

growth factor (VEGF-D) are not well established. We per-

formed a cross-sectional study to quantify the extent of

pulmonary cysts in CT and to establish their correlations

with pulmonary function tests (PFTs), 6MWT results,

including a composite index (desaturation–distance ratio,

DDR), and levels of VEGF-D and MMPs in LAM.

Methods Twenty-three LAM patients underwent CT

scanning to automatically quantify the extent of pulmonary

cysts and performed PFTs and 6MWT. Serum levels of

MMP-2, MMP-9, and VEGF-D were also measured.

Results The severity of pulmonary cystic involvement was

mild (the extent of cysts was 6.8 %) and correlated best with

FEV1/FVC (r = -0.84), residual volume (r = 0.66), DLCO

(r = -0.82), the DDR index (r = 0.77), and desaturation

during the 6MWT (r = -0.81). There was a weak

correlation with VEGF-D (r = 0.45), but no association was

found with MMP-2 and MMP-9.

Conclusions The severity of pulmonary cystic involve-

ment was mild in this sample of LAM patients and corre-

lated best with airway obstruction, air trapping, reduced

DLCO, the DDR index, and desaturation during the 6MWT.

Serum VEGF-D cannot be completely defined as a valu-

able marker of disease severity and there may be a mech-

anism independent of MMPs to explain the formation of

pulmonary cysts.

Keywords Computed tomography � Exercise �Lymphangioleiomyomatosis � Matrix metalloproteinase �Pulmonary function tests � Vascular endothelial growth

factor

Introduction

Lymphangioleiomyomatosis (LAM) is characterized by

atypical muscle cell proliferation, which can lead to vas-

cular and airway obstruction and formation of cysts. The

extent of pulmonary involvement in LAM varies from a

few scattered cysts to diffuse destruction of the lungs [1, 2].

An obstructive pattern, air trapping, and a reduced dif-

fusion capacity of the lungs for carbon monoxide (DLCO)

are the main findings in pulmonary function tests (PFTs) in

LAM [1–4]. Chest computed tomography (CT) shows

diffuse, thin-walled pulmonary cysts, together with pneu-

mothorax and pleural effusion [5–10].

PFTs are used most frequently to assess lung disease

severity, progression and response to treatment modalities;

forced expiratory volume in the first second (FEV1), and

DLCO are the main measurements recorded [1, 11–13].

However, there are some limitations to the use of PFTs,

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00408-014-9641-2) contains supplementarymaterial, which is available to authorized users.

B. G. Baldi (&) � M. S. Araujo � C. S. G. Freitas �R. A. Kairalla � O. M. Dias � D. A. S. Pereira �S. P. Pimenta � C. R. R. Carvalho

Pulmonary Division, Heart Institute (InCor), University of Sao

Paulo Medical School, Dr. Eneas de Carvalho Aguiar Avenue,

44, Fifth floor, Sao Paulo 05403-900, Brazil

e-mail: bruno.guedes2@terra.com.br

G. B. da Silva Teles

Department of Radiology, University of Sao Paulo Medical

School, Sao Paulo, Brazil

123

Lung (2014) 192:967–974

DOI 10.1007/s00408-014-9641-2

Author's personal copy

such as the need for patient cooperation and inter-test

variability. Other methods that may be used to assess the

severity of lung involvement in LAM include histological

scoring, exercise testing, and CT [3, 5–11, 14, 15]. The

walking distance and oxygen desaturation during the six-

minute walk test (6MWT) may also be used to evaluate the

severity of pulmonary involvement in LAM [3]. However,

a composite index that uses the continuous peripheral

oxygen saturation (SpO2) combined with the walking dis-

tance may be a more reliable tool than the isolated evalu-

ation of these variables for functional assessment of LAM

patients [16].

Previous semiquantitative or quantitative studies have

used CT to evaluate lung disease severity in LAM and have

demonstrated correlations between the extent of pulmonary

cysts with PFTs [5–10]. CT is a non-invasive testing

method and requires minimal patient cooperation.

Serum levels of vascular endothelial growth factor

(VEGF-D), a lymphangiogenic growth factor, are increased

in most LAM patients. This marker seems to be associated

with disease severity and thus may potentially be useful for

assessing the course of the disease over the long term [17,

18]. Xu and co-authors have shown that VEGF-D levels are

correlated with a semiquantitative grade of cystic

involvement on CT [19]. Matrix metalloproteinases

(MMPs) are components of the extracellular matrix that

degrades some matrix substrates, including collagen and

elastin, in the pulmonary parenchyma. Previous studies

have shown that pulmonary lesions and serum from LAM

patients contain increased levels of MMPs, primarily

MMP-2 and MMP-9, which may play a role in the patho-

genesis of cystic lung destruction [13, 20–22]. Therefore, it

would be reasonable to correlate the extent of cysts on CT

with levels of MMPs in LAM patients.

The correlations between the extent of pulmonary cysts

and 6MWT parameters and levels of MMPs and VEGF-D are

not well characterized. Thus, the aims of this study were to

quantify the extent of pulmonary cysts using high-resolution

computed tomography (HRCT) images and to determine the

degree of correlations between the extent of cysts and

parameters of PFTs, the distance walked and oxygen desat-

uration during 6MWT, a composite index that uses the

desaturation during 6MWT combined with the walking

distance (desaturation–distance ratio, DDR), and serum

levels of VEGF-D, MMP-2, and MMP-9 in LAM patients.

Methods

Study Population

This cross-sectional study included 23 women with LAM

who were patients in our center between 2009 and 2011.

The diagnosis of LAM was confirmed by lung biopsy

(74 %), kidney biopsy (9 %), or by the combination of

typical HRCT findings with clinical features (17 %) [1].

No patients were evaluated during an exacerbation of dis-

ease or during an episode of pneumothorax or chylothorax.

This study was performed as part of an institutional

review board-approved protocol (0345/07), and all patients

gave written, informed consent.

Measurements

All measurements in each patient were performed within a

maximum period of 15 days.

Computed Tomography Scanning

All patients underwent CT scanning without intravenous

contrast injection. Ten-detector and 16-detector MDCT

scanners were used (MX8000 IDT10 or IDT16, Philips

Medical Systems, Cleveland, OH, USA). Scan data were

obtained in spiral mode with 10 9 0.75 mm or

16 9 0.75 mm collimation, with patients in prone position

and holding their breath at the end of inspirations. Expo-

sure settings were 150–280 mAs at 120 or 140 kVp,

depending on the participants weight. Axial images were

reconstructed with 1.0 mm thickness at 1.0 mm incre-

ments. All scans were reconstructed with a soft recon-

struction filter using a 512 9 512 matrix.

Quantitative Analysis

Quantification of cystic lesions was obtained by CT den-

sitovolumetry using a computer program (Advantage

Workstation Thoracic VCAR software; GE Medical Sys-

tems, Milwaukee, WS, USA) that allows for the automatic

segmentation of the lungs and airways. To obtain the

volume of pulmonary cysts, a threshold value of -950 HU

was applied to the segmented lungs to select pixels

between -1000 and -950 HU. The total lung volume, the

volume occupied by cysts and the ratio of the abnormal

cyst volume to the total lung volume were calculated

automatically. The image analysis and a manual correction

of the results to exclude bronchiectasis or residual pneu-

mothorax from cystic voxels were performed by a thoracic

radiologist (G.B.S.T) who was blind to the results from the

other exams. The method and the computer program used

to quantify the extent of cysts were very similar to those

performed by Avila and co-authors [7].

Pulmonary Function Tests

All measurements were obtained using standard methods

[23–25]. Spirometry was performed using a calibrated

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pneumotachograph (Medical Graphics Corporation, MGC,

St. Paul, MN, USA, 2005), while lung volumes and DLCO

values were obtained with a body plethysmograph (Elite

Dx, Elite SeriesTM; MGC). The following variables were

obtained: forced vital capacity (FVC), FEV1, total lung

capacity (TLC), residual volume (RV), and DLCO. Pre-

dicted values were derived from the Brazilian population

[26–28].

Six-Minute Walk Test

Patients performed the 6MWT in accordance with standard

guidelines [29]. Oxygen saturation, as measured by pulse

oxymeter holter (Nonin WristOx� 3100Plymouth, MN,

USA), was obtained at rest and at the end of exercise.

During the 6MWT (360 s), SpO2 was recorded every 2 s

using the pulse oxymeter holter. All SpO2 obtained during

the 6MWT were entered into a computer program, nVI-

SION� software (Plymouth, MN, USA), and exported to an

EXCEL worksheet. Each recorded SpO2 was subtracted

from 100 % (maximal SpO2) and plotted on a graph that

showed the desaturation area (DAO2). The DDR index was

obtained using the ratio between the desaturation area and

the walking distance (Fig. 1). Patients who presented a

high DDR index had the worst performance during exercise

[16].

The walking distance was expressed as the percentage of

that predicted for the Brazilian population [30].

Serum MMP-2, MMP-9, and VEGF-D

Serum MMP-2, MMP-9, and VEGF-D levels were mea-

sured by enzyme-linked immunosorbent assay (ELISA;

R&D Systems, Minneapolis, MN, USA). The detection

limits of the assays were 156.2 pg/mL for MMP-2 and

15.6 pg/mL for VEGF-D and for MMP-9.

Statistical Analysis

Data are reported as the mean ± SD for variables with

normal distribution, as the median (25th–75th percentiles)

for variables with non-normal distribution or as numbers

(percentiles). Spearman correlation coefficients were cal-

culated to measure the degrees of association between the

extent of pulmonary cysts in HRCT and PFT and 6MWT

results, DDR index, and levels of VEGF-D, MMP-2, and

MMP-9. Data were analyzed with SigmaStat version 3.5

(Systat Software, Inc., San Jose, CA, USA).

Results

Twenty-three LAM patients were included in the study.

Clinical features are described in Table 1. The mean age of

the patients enrolled was 46 ± 8 years. Three (13 %)

patients had tuberous sclerosis, 11 (48 %) had renal angi-

omyolipoma, and 16 (70 %) had a history of pneumotho-

rax. Six (26 %) patients were ex-smokers (all of whom had

smoked less than 10 pack-years). Eight (35 %) patients

Fig. 1 Desaturation–distance ratio (DDR): calculated using the ratio

between DAO2 [dotted area—obtained by subtraction between each

recorded peripheral oxygen saturation (SpO2) at every 2 s from

100 % (maximal SpO2)] and the walking distance

Table 1 Clinical features and pulmonary function tests (n = 23)

Mean age, years 46 ± 8

Age at diagnosis, years 39 ± 7

Presence of TSC 3 (13 %)

Renal angiomyolipoma 11 (48 %)

History of pneumothorax 16 (70 %)

History of chylothorax 2 (9 %)

Ex-smokers 6 (26 %)

FEV1, L 2.09 ± 0.66

% predicted 76 ± 25

FVC, L 3.03 ± 0.45

% predicted 91 ± 14

FEV1/FVC 0.68 ± 0.19

RV, L 1.88 ± 0.61

% predicted 138 ± 44

TLC, L 4.99 ± 0.74

% predicted 105 ± 14

RV/TLC 0.37 ± 0.08

DLCO, mL/min/mmHg 16.7 ± 6.6

% predicted 64 ± 25

Values are the mean ± SD or percentage (%)

DLCO lung diffusing capacity for carbon monoxide, FEV1 forced

expiratory volume in the first second, FVC forced vital capacity, RV

residual volume, TLC total lung capacity, TSC tuberous sclerosis

complex

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Fig. 2 a, b Axial CT

image and coronal minIP

reconstruction show diffuse

pulmonary cysts in a 63-year-

old woman with LAM. c,

d Axial and coronal CT images

with data segmentation. The

percentage of the total lung area

occupied with voxels with an

attenuation of -950 HU or

lower (depicted in blue) is 19 %

Fig. 3 a, b Axial CT image and

coronal minIP reconstruction

show few scattered pulmonary

cysts in a 55-year-old woman

with LAM. c, d Axial and

coronal CT images with data

segmentation. The percentage

of the total lung area occupied

with voxels with an attenuation

of -950 HU or lower (depicted

in blue) is 0.08 %

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were receiving a gonadotropin-releasing hormone agonist,

but no patient was using doxycycline or mTOR inhibitors.

High-Resolution Computed Tomography

The median extent of cysts on HRCT was 6.8 %

(1.8–16.1 %). Images from a LAM patient with moderate

impairment and 19 % of the total lung area occupied with

cysts are shown in Fig. 2, and images from a LAM patient

with normal PFTs and 0.08 % of the total lung area

occupied with cysts are shown in Fig. 3.

Pulmonary Function Tests

Baseline pulmonary function data are described in Table 1.

Nine (39 %) patients had normal PFTs. The most common

abnormalities identified in PFTs were reduced DLCO

(52 %), an obstructive pattern (48 %), and air trapping

(35 %).

Mean FEV1, FEV1/FVC, and DLCO were 76 ± 25 %,

0.68 ± 0.19, and 64 ± 25 % predicted, respectively.

Six-Minute Walk Test

The distance walked was 508 ± 112 m (94 ± 21 % pre-

dicted). The minimum SpO2 at the end of the 6MWT was

89 ± 8 %, whereas the oxygen desaturation from rest to

the end of the test was 6 ± 5 % The median DDR index

was 4 (Table 2). Ten patients (43 %) had significant oxy-

gen desaturation (a decline of at least 4 % in SpO2) during

exercise.

Serum MMP-2, MMP-9, and VEGF-D

The median VEGF-D serum level was 464 pg/mL

(317–1782 pg/mL), and 11 (48 %) patients had elevated

levels of this marker (cut-off = 574 pg/mL). The serum

levels of MMP-2 and MMP-9 were 0 pg/mL (0–778 pg/

mL) and 1017 ± 408 ng/mL, respectively.

Associations Between the Extent of Pulmonary Cysts

and Other Variables

The extent of cysts on HRCT correlated best with FEV1/

FVC (r = -0.84, p \ 0.001), DLCO (r = -0.82,

p \ 0.001), RV (r = 0.66, p \ 0.001), DDR index

(r = 0.77, p \ 0.001), and desaturation during the 6MWT

(r = -0.81, p \ 0.001). A weak correlation was found

with VEGF-D serum dosage (r = 0.45, p = 0.03). There

was no correlation with the distance walked and with

MMP-2 and MMP-9 serum dosage (Table 3; Fig. 1OR,

2OR and 3OR in the online resource).

Discussion

This study investigated the extent of pulmonary cysts in

LAM and examined the degree to which this variable is

correlated with PFT parameters, 6MWT measurements,

and serum levels of VEGF-D, MMP-9, and MMP-2. The

main findings of this study are as follows: (1) the severity

of pulmonary cystic involvement was mild in the majority

of LAM patients; (2) the extent of pulmonary cysts cor-

related with airway obstruction, air trapping, reduced

DLCO, the DDR index, and desaturation during the 6MWT;

(3) VEGF-D cannot be completely defined as a marker of

Table 2 Variables obtained in the six-minute walk test

Distance, m 508 ± 112

Distance, % predicted 94 ± 21

Initial SpO2, % 95 ± 3

Minimum SpO2, % 89 ± 8

Reduction in SpO2, % 6 ± 5

DDR 4 (2–10)

Values are the mean ± SD or median (25th–75th percentiles)

DDR desaturation–distance ratio, SpO2 oxyhemoglobin saturation by

pulse oximetry

Table 3 Correlations between the extent of pulmonary cysts as

shown by computed tomography and parameters from pulmonary

function tests, six-minute walk test results, DDR and serum markers

Correlation

(r)

p CI 95 %

FEV1 (% predicted) -0.64 \0.001 -0.83 to -0.31

FEV1/FVC -0.84 \0.001 -0.93 to -0.65

RV (% predicted) 0.66 \0.001 0.34 to 0.84

RV/TLC 0.64 \0.001 0.31 to 0.83

DLCO (% predicted) -0.82 \0.001 -0.92 to -0.62

Distance walked (m) -0.28 0.19 -0.62 to 0.15

Distance walked (%

predicted)

-0.27 0.21 -0.61 to 0.16

Desaturation during

6MWT (%)

-0.81 \0.001 -0.92 to -0.60

DDR 0.77 \0.001 0.52 to 0.90

VEGF-D (pg/mL) 0.45 0.03 0.05 to 0.73

MMP-2 (pg/mL) 0.30 0.16 -0.13 to 0.63

MMP-9 (ng/mL) 0.01 0.98 -0.40 to 0.42

CI confidence interval, DDR desaturation–distance ratio, DLCO lung

diffusing capacity for carbon monoxide, FEV1 forced expiratory

volume in the first second, FVC forced vital capacity, MMP matrix

metalloproteinase, r Spearman correlation coefficient, RV residual

volume, TLC total lung capacity, VEGF-D vascular endothelial

growth factor D

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disease severity, because there was a weak association with

pulmonary cystic involvement; and (4) there was no cor-

relation between the extent of pulmonary cysts and MMP-2

and MMP-9 serum dosage.

Previous studies have shown that qualitative and quan-

titative HRCT may be used to evaluate disease severity in

LAM patients [7, 31]. Our study lends support to the idea

of HRCT as a valuable tool for assessing disease severity in

LAM; imaging showed that the disease was mild in our

sample of patients, with a cyst volume as a percentage of

the total lung volume of 6.8 % (range 0.08–32 %).

Several authors have demonstrated a correlation

between the extent of cysts on HRCT and airflow

obstruction and reduced DLCO [5–10]. Avila and co-

authors showed that the majority of patients had moderate

or severe involvement of pulmonary cysts on HRCT, which

correlated significantly with FEV1 and DLCO [8]. In the

present study, LAM patients had mild impairment in PFTs,

which showed a significant association with the extent of

pulmonary cysts on HRCT.

Our study is the first to show a correlation between the

extent of cysts and desaturation during the 6MWT and the

DDR index. However, we could not establish an associa-

tion with the distance walked in the 6MWT, in contrast

with a previous study [9]. We speculate that the reasons

responsible for desaturation during the 6MWT were ven-

tilatory limitation and gas exchange impairment, based on

a recent study, and that cysts may be related with desatu-

ration due to reduction in the area of gas exchange and air

trapping [3]. A possible explanation for the lack of corre-

lation found between the volume of pulmonary cysts and

desaturation during exercise in the study of Paciocco and

co-authors is that patients with Langerhans cell histiocy-

tosis were included in their analysis, and they showed less

desaturation during exercise. We speculate that the lack of

correlation found between the extent of cysts and the dis-

tance walked in 6MWT, and that a similar degree of cor-

relation with the extent of cysts between desaturation

during 6MWT and the DDR index in our study may be

explained by the fact that most patients performed regular

physical activity. In a recent study, it was speculated that

performance of regular physical activity was a possible

explanation for a similar maximal exercise capacity found

between patients with and without ventilatory limitation

and gas exchange impairment during exercise [3].

Our study has shown a weak correlation between VEGF-

D and the extent of cysts and, therefore, this marker cannot

be completely defined as a valuable tool to access disease

severity in LAM. However, a recent study reported that

levels of serum VEGF-D are higher in patients with greater

cystic involvement on semiquantitative CT [19]. Therefore,

future studies are still necessary to establish the role of

VEGF-D in evaluating disease severity. VEGF-D is

upregulated through mTOR signaling pathway, is expres-

sed by LAM cells, and is associated with LAM cell pro-

liferation and dissemination, and with lymphangiogenesis

[17–19].

For the first time, we have evaluated the association

between the extent of cysts and levels of serum MMPs. In

our study, MMP-2 and MMP-9 were not correlated with

the extent of pulmonary cysts on HRCT. It has been

speculated that the pathogenesis of cyst formation in LAM

involves an imbalance between MMPs and tissue inhibitor

of MMPs, with increased levels of MMPs. LAM cells

express MMPs, which promote the destruction of collagen

and elastin in the pulmonary parenchyma [13, 20–22].

However, our study suggests that there may be a mecha-

nism independent of MMPs that results in the development

of cysts and a possible explanation is a bronchiolar check-

valve mechanism caused by obstruction of airways by

LAM cells, resulting in overdistended alveoli.

Our study has limitations that need to be addressed.

Although we have demonstrated a correlation between the

percentage of cysts and PFT parameters, desaturation dur-

ing the 6MWT and the DDR index, it is not known whether

subsequent changes in CT will remain correlated with

changes in these functional parameters. Another limitation

is that we have not investigated the possibility that other

MMPs, such as MMP-1 or MMP-8, may be involved in

LAM. However, MMP-2 and MMP-9 were previously

shown to be the main MMPs associated with LAM [13, 20–

22]. An additional limitation of our study is that the method

used to assess the percentage of cysts may include

emphysema as areas between -1000 and -950 HU.

However, the presence of emphysema was most likely not

significant in our study, because all ex-smokers had smoked

less than 10 pack-years. Moreover, results were evaluated

excluding these six ex-smokers patients and were quite

similar to those described in this study (data not shown).

In summary, in this study, we found that the extent of

pulmonary cysts on HRCT is correlated with airway

obstruction, air trapping, reduced DLCO, desaturation dur-

ing the 6MWT and the DDR index. However, we have

found a weak association with serum levels of VEGF-D,

and no correlation with levels of serum MMP-2 and MMP-

9, which suggests that there may be a mechanism inde-

pendent of MMPs that determine the formation of pul-

monary cysts. Prospective studies are necessary to assess

the utility of serial HRCT to the measurement of the pro-

gression of LAM over time, to the impact on survival, and

to the assessment of patient responses to treatment

modalities in association with PFTs. Future studies are also

necessary to establish whether a new composite index

(DDR) obtained in the 6MWT is a more reliable tool than

other functional variables to assess lung disease severity

and the progression of LAM in the long term.

972 Lung (2014) 192:967–974

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Conflict of interest Bruno Guedes Baldi, Mariana Sponholz Ara-

ujo, Carolina Salim Goncalves Freitas, Gustavo Borges da Silva

Teles, Ronaldo Adib Kairalla, Olıvia Meira Dias, Daniel Antunes

Silva Pereira, Suzana Pinheiro Pimenta, and Carlos Roberto Ribeiro

Carvalho have no conflict of interest to declare.

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