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## **Meet the editor**

Dr Alberto Vannelli, MD, is a lecturer in Oncological Lymphology at Università degli Studi of Milan, Italy. He received his university degree with a dissertation on "Assisted circulation by extracorporeal bypass techniques in liver transplantation", and his specialization in General Surgery with a dissertation on "Conservative surgery in rectal cancer". He received both degrees from the Univer-

sity of Milan, Italy. From 2001 he was an attending physician in Colorectal Division at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan, Italy. Vannelli first became interested in the lymphatic circulation and began seminal studies on the pathophysiology of lymphadenectomy, pelvic lymphedema and pelvic surgery. Since 2011 he has been a consultant surgeon in the General Surgery Division at Valduce Hospital in Como, Italy.

Contents

Introductory **A Brief Overview of Lymphology:**  Chapter **Past, Present and Future 1**  Alberto Vannelli

Fredrik Berglund

Chapter 4 **Preparing for and Coping with** 

M. Elise Radina and Mei R. Fu

Chapter 5 **Pelvic Lymphedema in Rectal Cancer 89**  Alberto Vannelli and Luigi Battaglia

Chapter 3 **Arm Lymphedema as a** 

Chapter 1 **Strategies in Modulating Lymphedema 13** 

Chapter 2 **Titanium and Yellow Nail Syndrome 23** 

**Consequence of Breast Cancer Therapy 31**  A. Gabriella Wernicke, Yevgeniya Goltser, Michael Shamis and Alexander J. Swistel

**Breast Cancer-Related Lymphedema 53** 

Jin-Hong Chang, Joshua H. Hou, Sandeep Jain and Dimitri T. Azar

## Contents


**Introductory Chapter** 

**Past, Present and Future** 

Alberto Vannelli

*Italy* 

**A Brief Overview of Lymphology:** 

*Foundation IRCCS "National Institute of Cancer", Milan* 

*Faculty Lecturer in lymphology, University of the Study of Milan,* 

The human body has two circulatory systems. These are the cardiovascular system and the lymphatic system. They are part of the immune system comprising a network of conduits called lymphatic vessels that carry a clear fluid called lymph (from Latin lympha "water") towards the heart. There are many milestones in the history of the lymphatic system. The lymphoid system can be broadly divided into the conducting system and the lymphoid tissue. The conducting system carries the lymph and consists of tubular vessels that include the lymph capillaries, the lymph vessels, and the right and left thoracic ducts. The lymphoid tissue is primarily involved in immune responses and consists of lymphocytes and other white blood cells enmeshed in connective tissue through which the lymph passes. Regions of the lymphoid tissue that are densely packed with lymphocytes are known as lymphoid follicles. Lymphoid tissue can either be structurally well organized as lymph nodes or may consist of loosely organized lymphoid follicles known as the mucosa-associated lymphoid tissue (MALT). Lymph vessels called lacteals are present in the lining of the gastrointestinal tract, predominantly in the small intestine. While most other nutrients absorbed by the small intestine are passed on to the portal venous system to drain via the portal vein into the liver for processing, fats (lipids) are passed on to the lymphatic system to be transported to the blood circulation via the thoracic duct. (There are exceptions, for example medium-chain triglycerides are fatty acid esters of glycerol that passively diffuse from the gastrointestinal tract to the portal system.) The enriched lymph originating in the lymphatics of the small intestine is called chyle. As the blood circulates, fluid leaks out into the body tissues. This fluid is important because it carries food to the cells and waste back to the bloodstream. The nutrients that are released to the circulatory system are processed by the liver, having passed through the systemic circulation. The lymph system is a one-way system, transporting interstitial fluid back to blood. Lymphatics were discovered by chance, but were misunderstood for a very long time. Up to the early twentieth century the accurate description of lymphatics was deemed necessary to promote advances in oncology (Rouviere, 1932). The study of lymphatic drainage of various organs is important in diagnosis, prognosis, and treatment of cancer. The lymphatic system, because of its physical proximity to many tissues of the body, is responsible for carrying cancerous cells between the various parts of the body in a process called metastasis. The intervening lymph nodes can trap the cancer cells. If they are not successful in destroying the cancer cells the nodes

**1. Introduction** 

## **Introductory Chapter**

## **A Brief Overview of Lymphology: Past, Present and Future**

#### Alberto Vannelli

*Foundation IRCCS "National Institute of Cancer", Milan Faculty Lecturer in lymphology, University of the Study of Milan, Italy* 

#### **1. Introduction**

The human body has two circulatory systems. These are the cardiovascular system and the lymphatic system. They are part of the immune system comprising a network of conduits called lymphatic vessels that carry a clear fluid called lymph (from Latin lympha "water") towards the heart. There are many milestones in the history of the lymphatic system. The lymphoid system can be broadly divided into the conducting system and the lymphoid tissue. The conducting system carries the lymph and consists of tubular vessels that include the lymph capillaries, the lymph vessels, and the right and left thoracic ducts. The lymphoid tissue is primarily involved in immune responses and consists of lymphocytes and other white blood cells enmeshed in connective tissue through which the lymph passes. Regions of the lymphoid tissue that are densely packed with lymphocytes are known as lymphoid follicles. Lymphoid tissue can either be structurally well organized as lymph nodes or may consist of loosely organized lymphoid follicles known as the mucosa-associated lymphoid tissue (MALT). Lymph vessels called lacteals are present in the lining of the gastrointestinal tract, predominantly in the small intestine. While most other nutrients absorbed by the small intestine are passed on to the portal venous system to drain via the portal vein into the liver for processing, fats (lipids) are passed on to the lymphatic system to be transported to the blood circulation via the thoracic duct. (There are exceptions, for example medium-chain triglycerides are fatty acid esters of glycerol that passively diffuse from the gastrointestinal tract to the portal system.) The enriched lymph originating in the lymphatics of the small intestine is called chyle. As the blood circulates, fluid leaks out into the body tissues. This fluid is important because it carries food to the cells and waste back to the bloodstream. The nutrients that are released to the circulatory system are processed by the liver, having passed through the systemic circulation. The lymph system is a one-way system, transporting interstitial fluid back to blood. Lymphatics were discovered by chance, but were misunderstood for a very long time. Up to the early twentieth century the accurate description of lymphatics was deemed necessary to promote advances in oncology (Rouviere, 1932). The study of lymphatic drainage of various organs is important in diagnosis, prognosis, and treatment of cancer. The lymphatic system, because of its physical proximity to many tissues of the body, is responsible for carrying cancerous cells between the various parts of the body in a process called metastasis. The intervening lymph nodes can trap the cancer cells. If they are not successful in destroying the cancer cells the nodes

A Brief Overview of Lymphology: Past, Present and Future 3

Bartholin (1616-1680) and Olof Rudbeck (1630-1702) published numerous studies on lymphatics and disagreed over their distribution (Hagelin, 1989). Jean Pecquet (1622-1681) described the confluence of abdominal lymph trunks and the cysterna chyli at the origin of the thoracic duct in a dog (Pecquet, 1651). Eleven years later, Frederik Ruysch (1638-1731) established the existence of valves in the lymphatic vessels. He presented this in a small book dedicated to his three much-admired teachers, Franciscus De Le Boe, Johann van

The Italian anatomist Paolo Mascagni (1755-1815) studied at Siena under Pietro Tabarrini (1702-1780). His most successful research was on lymphatics, and led first to the submission of preliminary results in 1784, in respect of which the French Academy of Sciences awarded a prize, and second, to the publication of one of the most striking atlases in 1787. By using a very simple method (a tubular needle bent at right angle), he was able to discover about fifty per cent of all the lymphatic vessels now known (Norman, 1978). Moreover, he established that every lymph vessel must in its course enter at least one lymph node; disproved the existence of arterial and venous lymph vessels, and concluded that the

Mascagni's procedure for the injection of superficial lymph vessels was widely used in the following century. In order to avoid filling the deep lymph vessels during the injection of the superficial ones, Mascagni recommended injecting glue in the arteries first, then cooling the specimen. In this way, all lymph vessels (superficial and deep) became collapsed. For the selective injection of the superficial vessels, he warmed up the surface of the specimen so that the glue could soften, therefore making the superficial lymph vessels permeable again. Ernest Alexandre Lauth (1803-1837) belonged to a famous Strasbourg family of anatomists. He studied under Vincent Fohmann (1794-1837), and carried out researches on the lymph system in birds (1825) and people (1824, 1829). In his 1829 handbook, he gives a detailed account of the injection and preparation of lymph vessels, which may be summarized as follows: the choice of the body to be prepared is very important. It should be young, robust,

If the body is fat, lymph vessels will be difficult to identify and Lauth recommends arterial injection with lukewarm water, so that the tissues become infiltrated. Another procedure is to inject arteries and veins with wax and to let the specimen soak for some days. The development of gas in the lymph vessels will then make them more visible, and the previous injection of wax allows an easy differentiation from arterial and venous vessels. This is the way the English anatomist William Cruikshank discovered the lymph vessels of

Finally, Lauth reminds us that hypertrophied organs make the dissection of lymph vessels easier. According to Lauth, the lymph vessels of a gravid uterus are thicker in diameter than the feather of a crow. In the nineteenth century, the lymph vessels were usually injected with mercury. Lauth stresses the fact that mercury must be as pure as possible, and must in any case be filtered through a piece of chamois leather. To check the purity of the mercury, he put a drop of mercury on an inclined plate. If the drop slid without dirtying the plate, the mercury was pure enough for the injection. If not, it meant it contained traces of lead or tin and could not be used for anatomical preparations. However, other products could also be used. For the injection of thick lymph vessels (thoracic duct or right lymphatic duct), Lauth

Home and Florentius Schuyl (Ruysch, 1665; Luyendijk-Elshout, 1964).

lymphatic system originates from all the cavities and surfaces of the body.

and should preferably have died of an acute episode.

the heart and the uterus in 1786.

may become sites of secondary tumours. As editor of this book it is my intention in this brief introductory chapter to provide a sampling of some of the varied topics related to the discipline of lymphology. Whetting the readers' appetite for this subject will enable them to better enjoy the many superb multi-authored chapters written with an international perspective that follow.

#### **2. Past**

Hippocrates was one of the first people to mention the lymphatic system in the 5th century BC. In his work "On Joints," he briefly mentioned the lymph nodes in one sentence. Rufus of Ephesus, a Roman physician, identified the axillary, inguinal and mesenteric lymph nodes as well as the thymus during the 1st to 2nd century AD (Ambrose, 2006).

Erasistrate, the most famous Alexandrian anatomist, observed lymph vessels in animals, but he mistook them for arteries. Another anatomist, Herophile mistook them for veins. However, the latter also referred to "glandular bodies", which must be regarded as the current lymph nodes. The first mention of lymphatic vessels was in 3rd century BC by Herophilos, a Greek anatomist living in Alexandria, who incorrectly concluded that the "absorptive veins of the lymphatics", by which he meant the lacteals (lymph vessels of the intestines), drained into the hepatic portal veins, and thus into the liver (Ambrose, 2006). Findings of Ruphus and Herophilos were further propagated by the Greek physician Galen, who described the lacteals and mesenteric lymph nodes which he observed in his dissection of apes and pigs in the 2nd century AD. Until the 17th century, ideas of Galen were most prevalent. Accordingly, it was believed that the blood was produced by the liver from chyle contaminated with ailments by the intestine and stomach, to which various spirits were added by other organs, and that this blood was consumed by all the organs of the body.

This theory required that the blood be consumed and produced many times over. His ideas remained unchallenged until the 17th century, and even then were defended by some physicians (Fanous et al, 2007). In the 16th century, Nicolas Massa briefly described some lymph vessels of the human kidneys (1536), and Bartolomeo Eustachi (1564) made the first accurate description of the thoracic duct in a horse, which he called "Vena alba thoracis". Eustachi only observed its thoracic course and its connection with the subclavian or internal jugular vein. In the 17th century, many famous European anatomists such as Gaspare Aselli, Thomas Bartholin, Olof Rudbeck, Jean Pecquet and Frederik Ruysch made important contributions to the knowledge of the lymphatic system (Arvy & Rivet, 1976).

The Italian surgeon Gaspare Aselli was a pupil of his famous compatriot Gabriele Falloppio. In 1622, he discovered the lacteal vessels while displaying the abdominal viscera of a dog at an anatomical demonstration. As it happened, the animal had been fed shortly before the dissection, and Aselli could therefore observe milky "fibers" running on the mesentery. He first mistook them for nerves and decided to cut them in the course of his demonstration. He then observed a whitish liquid flowing out of these "fibers" and came to the conclusion that they were vessels. Some days later, he began to verify his observations in other animal species: cats, lambs, cows, pigs, and even a horse he bought only for this purpose (Regis & Kaoru, 1971). Unfortunately, before his death in 1626, Aselli could not confirm the existence of lacteal vessels in humans. Aselli's book was published posthumously under the editorship of Alessandro Tadini and Luigi Settala (1627). The Danish anatomists Thomas

may become sites of secondary tumours. As editor of this book it is my intention in this brief introductory chapter to provide a sampling of some of the varied topics related to the discipline of lymphology. Whetting the readers' appetite for this subject will enable them to better enjoy the many superb multi-authored chapters written with an international

Hippocrates was one of the first people to mention the lymphatic system in the 5th century BC. In his work "On Joints," he briefly mentioned the lymph nodes in one sentence. Rufus of Ephesus, a Roman physician, identified the axillary, inguinal and mesenteric lymph nodes

Erasistrate, the most famous Alexandrian anatomist, observed lymph vessels in animals, but he mistook them for arteries. Another anatomist, Herophile mistook them for veins. However, the latter also referred to "glandular bodies", which must be regarded as the current lymph nodes. The first mention of lymphatic vessels was in 3rd century BC by Herophilos, a Greek anatomist living in Alexandria, who incorrectly concluded that the "absorptive veins of the lymphatics", by which he meant the lacteals (lymph vessels of the intestines), drained into the hepatic portal veins, and thus into the liver (Ambrose, 2006). Findings of Ruphus and Herophilos were further propagated by the Greek physician Galen, who described the lacteals and mesenteric lymph nodes which he observed in his dissection of apes and pigs in the 2nd century AD. Until the 17th century, ideas of Galen were most prevalent. Accordingly, it was believed that the blood was produced by the liver from chyle contaminated with ailments by the intestine and stomach, to which various spirits were added by other organs, and that this blood was consumed by all the organs of the body.

This theory required that the blood be consumed and produced many times over. His ideas remained unchallenged until the 17th century, and even then were defended by some physicians (Fanous et al, 2007). In the 16th century, Nicolas Massa briefly described some lymph vessels of the human kidneys (1536), and Bartolomeo Eustachi (1564) made the first accurate description of the thoracic duct in a horse, which he called "Vena alba thoracis". Eustachi only observed its thoracic course and its connection with the subclavian or internal jugular vein. In the 17th century, many famous European anatomists such as Gaspare Aselli, Thomas Bartholin, Olof Rudbeck, Jean Pecquet and Frederik Ruysch made important

The Italian surgeon Gaspare Aselli was a pupil of his famous compatriot Gabriele Falloppio. In 1622, he discovered the lacteal vessels while displaying the abdominal viscera of a dog at an anatomical demonstration. As it happened, the animal had been fed shortly before the dissection, and Aselli could therefore observe milky "fibers" running on the mesentery. He first mistook them for nerves and decided to cut them in the course of his demonstration. He then observed a whitish liquid flowing out of these "fibers" and came to the conclusion that they were vessels. Some days later, he began to verify his observations in other animal species: cats, lambs, cows, pigs, and even a horse he bought only for this purpose (Regis & Kaoru, 1971). Unfortunately, before his death in 1626, Aselli could not confirm the existence of lacteal vessels in humans. Aselli's book was published posthumously under the editorship of Alessandro Tadini and Luigi Settala (1627). The Danish anatomists Thomas

contributions to the knowledge of the lymphatic system (Arvy & Rivet, 1976).

as well as the thymus during the 1st to 2nd century AD (Ambrose, 2006).

perspective that follow.

**2. Past** 

Bartholin (1616-1680) and Olof Rudbeck (1630-1702) published numerous studies on lymphatics and disagreed over their distribution (Hagelin, 1989). Jean Pecquet (1622-1681) described the confluence of abdominal lymph trunks and the cysterna chyli at the origin of the thoracic duct in a dog (Pecquet, 1651). Eleven years later, Frederik Ruysch (1638-1731) established the existence of valves in the lymphatic vessels. He presented this in a small book dedicated to his three much-admired teachers, Franciscus De Le Boe, Johann van Home and Florentius Schuyl (Ruysch, 1665; Luyendijk-Elshout, 1964).

The Italian anatomist Paolo Mascagni (1755-1815) studied at Siena under Pietro Tabarrini (1702-1780). His most successful research was on lymphatics, and led first to the submission of preliminary results in 1784, in respect of which the French Academy of Sciences awarded a prize, and second, to the publication of one of the most striking atlases in 1787. By using a very simple method (a tubular needle bent at right angle), he was able to discover about fifty per cent of all the lymphatic vessels now known (Norman, 1978). Moreover, he established that every lymph vessel must in its course enter at least one lymph node; disproved the existence of arterial and venous lymph vessels, and concluded that the lymphatic system originates from all the cavities and surfaces of the body.

Mascagni's procedure for the injection of superficial lymph vessels was widely used in the following century. In order to avoid filling the deep lymph vessels during the injection of the superficial ones, Mascagni recommended injecting glue in the arteries first, then cooling the specimen. In this way, all lymph vessels (superficial and deep) became collapsed. For the selective injection of the superficial vessels, he warmed up the surface of the specimen so that the glue could soften, therefore making the superficial lymph vessels permeable again.

Ernest Alexandre Lauth (1803-1837) belonged to a famous Strasbourg family of anatomists. He studied under Vincent Fohmann (1794-1837), and carried out researches on the lymph system in birds (1825) and people (1824, 1829). In his 1829 handbook, he gives a detailed account of the injection and preparation of lymph vessels, which may be summarized as follows: the choice of the body to be prepared is very important. It should be young, robust, and should preferably have died of an acute episode.

If the body is fat, lymph vessels will be difficult to identify and Lauth recommends arterial injection with lukewarm water, so that the tissues become infiltrated. Another procedure is to inject arteries and veins with wax and to let the specimen soak for some days. The development of gas in the lymph vessels will then make them more visible, and the previous injection of wax allows an easy differentiation from arterial and venous vessels. This is the way the English anatomist William Cruikshank discovered the lymph vessels of the heart and the uterus in 1786.

Finally, Lauth reminds us that hypertrophied organs make the dissection of lymph vessels easier. According to Lauth, the lymph vessels of a gravid uterus are thicker in diameter than the feather of a crow. In the nineteenth century, the lymph vessels were usually injected with mercury. Lauth stresses the fact that mercury must be as pure as possible, and must in any case be filtered through a piece of chamois leather. To check the purity of the mercury, he put a drop of mercury on an inclined plate. If the drop slid without dirtying the plate, the mercury was pure enough for the injection. If not, it meant it contained traces of lead or tin and could not be used for anatomical preparations. However, other products could also be used. For the injection of thick lymph vessels (thoracic duct or right lymphatic duct), Lauth

A Brief Overview of Lymphology: Past, Present and Future 5

this condition does not develop until months or even years after the therapy has concluded. Lymphedema may also be associated with accidents or certain diseases or problems that may inhibit the lymphatic system from functioning properly. In tropical areas of the world, a common cause of secondary lymphedema is filariasis, a parasitic infection. It can also be caused by the compromising of the lymphatic system, as a result of cellulitis. While the exact cause of primary lymphedema is still unknown, it generally occurs due to the poorly developed or missing lymph nodes or channels in the body. Lymphedema may be present at birth, develop at the onset of puberty (praecox), or not become apparent for many years into adulthood (tarda). In men, lower-limb primary lymphedema is most common, occurring in one or both legs. Some cases of lymphedema may be associated with other vascular abnormalities. Secondary lymphedema affects both men and women. In women, it is most prevalent in the upper limbs after breast cancer surgery and lymph node dissection, occurring in the arm on the side of the body in which the surgery is performed. Head and neck lymphedema can be caused by surgery or radiation therapy for tongue or throat cancer. It may also occur in the lower limbs or groin after surgery for colon, ovarian or uterine cancer, in which removal of lymph nodes or radiation therapy is required. Surgery or treatment of prostate, colon and testicular cancers may result in secondary lymphedema, particularly when lymph nodes have been removed or damaged. The onset of secondary lymphedema in patients who have had cancer surgery has also been linked to aircraft flight (likely due to decreased cabin pressure). For cancer survivors, therefore, wearing a prescribed and properly fitted compression garment may help decrease swelling during air travel. Some cases of lower-limb lymphedema have been associated with the use of tamoxifen, due to the blood clots and deep vein thrombosis (DVT) that can be caused by this medication. Resolution of the blood clots or DVT is needed before lymphedema treatment

Today the treatment for lymphedema varies depending on the severity of the edema and the degree of fibrosis of the affected limb. Most people with lymphedema follow a daily regimen of treatment as suggested by their physician or certified lymphedema therapist. The most common treatments for lymphedema are a combination of manual compression lymphatic massage, compression garments or bandaging. Complex decongestive physiotherapy is an empiric system of lymphatic massage, skin care, and compressive garments. Although a combination treatment program may be ideal, any of the treatments

Elastic compression garments are worn by people with lymphedema on the affected limb following complete decongestive therapy to maintain edema reduction. Depending on the therapist's discretion, a compression garment may be custom-fitted or purchased in over-

Compression garments are meant to be worn every day to maintain edema reduction and must be replaced on a regular basis. Support garments may be the only garment of choice

Compression bandaging, also called wrapping, is the application of several layers of padding and short-stretch bandages to the involved areas. Short-stretch bandages are preferred over long-stretch bandages (such as those normally used to treat sprains) as the long-stretch bandages cannot produce the proper therapeutic tension necessary to safely

can be initiated.

can be done individually.

the-counter, standard sizes.

for patients with Scrotal edema.

writes that plaster yields good results provided one takes care of the orientation of the valves. In addition, he reminds us that his colleague Andre Marie Constant Dumeril made successful injections of lymph vessels with milk, provided the specimen was not intended to be preserved by drying. Other anatomists injected black ink by blowing into a tube connected to a thin glass needle. This procedure was used for the first study of lymphatics in Japan (Husiya & Waran, 1805). To inject mercury into the superficial lymph vessels, Lauth carefully removed a small square of skin, located a vessel, made a small incision in its wall with a lancet, and introduced a thin glass or steel tube into the vessel. Owing to the high number of anastomoses, three injections are sufficient to inject all superficial lymph vessels of the lower limb: one injection on the hallux, a second on the fifth toe, and a third one behind the medial malleolus. For the same reason, three or four injections in various parts of the hand will fill all superficial lymph vessels of the upper limb (here he is at variance with Mascagni, who wrote that at least twenty injections are necessary for a whole limb).

Marie Philibert Constant Sappey was a French anatomist born in Cernon, near the city of Bourg-en-Bresse. He conducted numerous studies on the lymphatic system up to the middle of the 19th century. In the second edition of his treatise on anatomy in 1869, he gives a summary of the important points that have to be taken into consideration for a successful injection of the lymphatics. Sappey differs with Lauth regarding the choice of body. Sappey recommends the body of man who died of a chronic disease because it will be sufficiently emaciated. The body of a child should only be used to study the lymphatics of the head, the tongue, the soft palate, the gums or the scrotum. Regardless of the specimen, however, Sappey thought that the injection of lymphatics should always be carried out in summer because a high temperature makes the progression of mercury easier.

#### **3. Present**

In today's world the interest in lymphology can be considered extraordinary, even in view of the increasing incidence of lymphedema, and covering all ages and lymphatic malformations, troncular and extra-troncular. Several national scientific societies, associations and study groups, which fall within the guidelines of the International Society of Lymphology and the International Union of Phlebology, are contributing to the awareness of these problems and to their solutions, which the international scientific community has already been studying for a long time. The study and the management of the lymphatic diseases requires the co-operation of many specialists such as lymphologists, internists, oncologists, dermatologists, vascular internists, phlebologists, physicians specializing in infective diseases, radiologists, gynecologists, surgeons, vascular surgeons, plastic surgeons, nurses, physiotherapists, gymnastic instructors and diet instructors.

The lymphatic system clears away infection and keeps your body fluids in balance. If it's not working properly, fluid builds in your tissues and causes swelling called lymphedema. Other lymphatic system problems can include infections, blockage, and cancer. Lymphedema results from an alteration of lymphatic vessels as a consequence of malformation (primary) or mechanical damage (secondary) (Warren et al, 2007), consistent with an equal distribution in the upper and lower limbs, neck, scrotum and pubis (Purushotham et al, 2007; Fang et al 2008). It is most frequently seen after lymph node dissection, surgery or radiation therapy, in which damage to the lymphatic system is caused during the treatment of cancer, most notably breast cancer. In many patients with cancer,

writes that plaster yields good results provided one takes care of the orientation of the valves. In addition, he reminds us that his colleague Andre Marie Constant Dumeril made successful injections of lymph vessels with milk, provided the specimen was not intended to be preserved by drying. Other anatomists injected black ink by blowing into a tube connected to a thin glass needle. This procedure was used for the first study of lymphatics in Japan (Husiya & Waran, 1805). To inject mercury into the superficial lymph vessels, Lauth carefully removed a small square of skin, located a vessel, made a small incision in its wall with a lancet, and introduced a thin glass or steel tube into the vessel. Owing to the high number of anastomoses, three injections are sufficient to inject all superficial lymph vessels of the lower limb: one injection on the hallux, a second on the fifth toe, and a third one behind the medial malleolus. For the same reason, three or four injections in various parts of the hand will fill all superficial lymph vessels of the upper limb (here he is at variance with

Mascagni, who wrote that at least twenty injections are necessary for a whole limb).

because a high temperature makes the progression of mercury easier.

**3. Present** 

Marie Philibert Constant Sappey was a French anatomist born in Cernon, near the city of Bourg-en-Bresse. He conducted numerous studies on the lymphatic system up to the middle of the 19th century. In the second edition of his treatise on anatomy in 1869, he gives a summary of the important points that have to be taken into consideration for a successful injection of the lymphatics. Sappey differs with Lauth regarding the choice of body. Sappey recommends the body of man who died of a chronic disease because it will be sufficiently emaciated. The body of a child should only be used to study the lymphatics of the head, the tongue, the soft palate, the gums or the scrotum. Regardless of the specimen, however, Sappey thought that the injection of lymphatics should always be carried out in summer

In today's world the interest in lymphology can be considered extraordinary, even in view of the increasing incidence of lymphedema, and covering all ages and lymphatic malformations, troncular and extra-troncular. Several national scientific societies, associations and study groups, which fall within the guidelines of the International Society of Lymphology and the International Union of Phlebology, are contributing to the awareness of these problems and to their solutions, which the international scientific community has already been studying for a long time. The study and the management of the lymphatic diseases requires the co-operation of many specialists such as lymphologists, internists, oncologists, dermatologists, vascular internists, phlebologists, physicians specializing in infective diseases, radiologists, gynecologists, surgeons, vascular surgeons, plastic surgeons, nurses, physiotherapists, gymnastic instructors and diet instructors.

The lymphatic system clears away infection and keeps your body fluids in balance. If it's not working properly, fluid builds in your tissues and causes swelling called lymphedema. Other lymphatic system problems can include infections, blockage, and cancer. Lymphedema results from an alteration of lymphatic vessels as a consequence of malformation (primary) or mechanical damage (secondary) (Warren et al, 2007), consistent with an equal distribution in the upper and lower limbs, neck, scrotum and pubis (Purushotham et al, 2007; Fang et al 2008). It is most frequently seen after lymph node dissection, surgery or radiation therapy, in which damage to the lymphatic system is caused during the treatment of cancer, most notably breast cancer. In many patients with cancer, this condition does not develop until months or even years after the therapy has concluded. Lymphedema may also be associated with accidents or certain diseases or problems that may inhibit the lymphatic system from functioning properly. In tropical areas of the world, a common cause of secondary lymphedema is filariasis, a parasitic infection. It can also be caused by the compromising of the lymphatic system, as a result of cellulitis. While the exact cause of primary lymphedema is still unknown, it generally occurs due to the poorly developed or missing lymph nodes or channels in the body. Lymphedema may be present at birth, develop at the onset of puberty (praecox), or not become apparent for many years into adulthood (tarda). In men, lower-limb primary lymphedema is most common, occurring in one or both legs. Some cases of lymphedema may be associated with other vascular abnormalities. Secondary lymphedema affects both men and women. In women, it is most prevalent in the upper limbs after breast cancer surgery and lymph node dissection, occurring in the arm on the side of the body in which the surgery is performed. Head and neck lymphedema can be caused by surgery or radiation therapy for tongue or throat cancer. It may also occur in the lower limbs or groin after surgery for colon, ovarian or uterine cancer, in which removal of lymph nodes or radiation therapy is required. Surgery or treatment of prostate, colon and testicular cancers may result in secondary lymphedema, particularly when lymph nodes have been removed or damaged. The onset of secondary lymphedema in patients who have had cancer surgery has also been linked to aircraft flight (likely due to decreased cabin pressure). For cancer survivors, therefore, wearing a prescribed and properly fitted compression garment may help decrease swelling during air travel. Some cases of lower-limb lymphedema have been associated with the use of tamoxifen, due to the blood clots and deep vein thrombosis (DVT) that can be caused by this medication. Resolution of the blood clots or DVT is needed before lymphedema treatment can be initiated.

Today the treatment for lymphedema varies depending on the severity of the edema and the degree of fibrosis of the affected limb. Most people with lymphedema follow a daily regimen of treatment as suggested by their physician or certified lymphedema therapist. The most common treatments for lymphedema are a combination of manual compression lymphatic massage, compression garments or bandaging. Complex decongestive physiotherapy is an empiric system of lymphatic massage, skin care, and compressive garments. Although a combination treatment program may be ideal, any of the treatments can be done individually.

Elastic compression garments are worn by people with lymphedema on the affected limb following complete decongestive therapy to maintain edema reduction. Depending on the therapist's discretion, a compression garment may be custom-fitted or purchased in overthe-counter, standard sizes.

Compression garments are meant to be worn every day to maintain edema reduction and must be replaced on a regular basis. Support garments may be the only garment of choice for patients with Scrotal edema.

Compression bandaging, also called wrapping, is the application of several layers of padding and short-stretch bandages to the involved areas. Short-stretch bandages are preferred over long-stretch bandages (such as those normally used to treat sprains) as the long-stretch bandages cannot produce the proper therapeutic tension necessary to safely

A Brief Overview of Lymphology: Past, Present and Future 7

lymphangioplasty enteromesenteric flap omental transfer (pedicled portion of omentum transposed to the affected limb); third - modified liposuction that has been developed in Sweden in recent years to remove adipose tissue associated with longstanding lymphedema, primarily in the upper region. This last technique is meant to reduce the volume of a limb and does not cure lymphedema. Compression garments and compression bandages must

Low-level laser therapy (LLLT) was cleared by the US Food and Drug Administration (FDA) for the treatment of lymphedema in November 2006 (El Segundo, 2006). According to the US National Cancer Institute, studies suggest that low-level laser therapy may be effective in reducing lymphedema in a clinically meaningful way for some women. Two cycles of laser treatment were found to be effective in reducing the volume of the affected arm, extracellular fluid, and tissue hardness in approximately one-third of patients with post-mastectomy lymphedema at three months post-treatment. Suggested rationales for laser therapy include a potential decrease in fibrosis, stimulation of macrophages and the immune system, and a possible role in encouraging lymphangiogenesis (Carati et al,

Lymphedema is a chronic and irreversible disease with significant negative consequences for the patient, as the resulting aesthetic deformities may make it impossible to continue their career and may lead to social isolation. In this book the authors deal with novel strategies in lymphedema. Still today, physicians and surgeons diagnose lymphedema relatively infrequently and the literature relating to the prevalence of lymphedema is limited. However it is correct to assume that in the future there will be an increase in

Jin-Hong Chang in her chapter, presents lymphangiogenesis as a complex process that involves the interplay of many molecules with redundant mechanisms. The author deals with specific molecules that have shown the most promise as therapeutic targets including the vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), COX-2 selective inhibitors, tumor necrosis factor (TNF)-α, and transforming growth factor

Primary congenital lymphedema (Milroy disease), representing 10 per cent of primary, is present at birth and associated with an autosomal dominant familial history (Dahlberg et al, 1983). Mutations in the FLT4 gene cause some cases of Milroy disease. The FLT4 gene provides instructions for producing a protein called vascular endothelial growth factor receptor 3 (VEGFR-3), which regulates the development and maintenance of the lymphatic system. About 10 to 15 per cent of people with a mutation in the FLT4 gene do not develop the features of Milroy disease. Today, some of the possible treatments for Milroy's disease from various sources may include: exercise and elevation of extremity, elastic stockings, proper skin hygiene, antibiotics, benzathine penicillin, split-thickness skin grafts, and pedicular transfer of skin. Genetic testing is becoming almost practical, defining a limited number of specific hereditary syndromes with discrete gene mutations such as lymphedemadistichiasis (FOXC2), some forms of Milroy disease, and

incidence in primary as well as in secondary lymphedema.

hypotrichosislymphedema-telangiectasis (SOX18).

still be worn after the operation.

2003).

**4. Future** 

(TGF)-β.

reduce lymphedema and may in fact end up producing a tourniquet effect. During activity, whether exercise or daily activities, the short-stretch bandages enhance the pumping action of the lymph vessels by providing increased resistance for them to push against. This encourages lymphatic flow and helps to soften fluid-swollen areas.

Compression pump technology utilizes a multi-chambered pneumatic sleeve with overlapping cells to promote movement of lymph fluid. Pump therapy may be used in addition to other treatments such as compression bandaging and manual lymph drainage. In many cases, pump therapy may help soften fibrotic tissue and therefore potentially enable more efficient lymphatic drainage. Sequential pump therapy may also be used as a home treatment method, usually as part of a regimen also involving compression garments or wrapping. A Stanford University medical study showed that patients receiving the combined modalities of complete decongestive therapy or manual lymph drainage (MLD/CDT) and pneumatic pumping had a greater overall reduction in limb volume than patients receiving only MLD/CDT (Szuba et al, 2002). However, some therapists have begun to raise concern that compression pumps can cause genital swelling when used on persons with leg lymphedema.

Complete decongestive therapy (CDT) is a primary tool in lymphedema management consisting of manual manipulation of the lymphatic ducts, short-stretch compression bandaging, therapeutic exercise, and skin care (National Lymphedema Network Medical Advisor Committee, 2011). The technique was pioneered by Emil Vodder in the 1930s for the treatment of chronic sinusitis and other immune disorders. Initially, CDT involves frequent visits to a certified therapist with a doctor's prescription. Once the lymphedema is reduced, increased patient participation is required for ongoing care, along with the use of elastic compression garments and non-elastic directional flow foam garments. Manual manipulation of the lymphatic ducts consists of gentle, rhythmic massaging of the skin to stimulate the flow of lymph and its return to the blood circulation system. In the blood's passage through the kidneys, the excess fluid is filtered out and eliminated from the body through urination. The treatment is very gentle and a typical session will involve drainage of the neck, trunk, and involved extremity (in that order), lasting approximately 40 to 60 minutes. CDT is generally effective on non-fibrotic lymphedema and less effective on more fibrotic legs, although it has been shown to help break up fibrotic tissue.

Surgical techniques for correcting lymphedema may be excisional or physiological. However, surgery for lymphedema does not cure the disease or eliminate the need for decongestive treatment. Surgical treatment is used only in extreme cases in order to reduce the weight of the affected limb, to help minimize the frequency of inflammatory attacks, to improve cosmesis, and to potentially reduce the risk of secondary angiosarcoma (National Lymphedema Network Medical Advisor Committee, 2011). Although surgery has been shown to reduce edema in the short-term, there is a lack of evidence to suggest that it is beneficial in the long-term. Excisional techniques include three steps: first - circumferential excision of the lymphedematous tissue followed by skin grafting (Charles technique), longitudinal removal of the affected segment of skin and subcutaneous tissue and primary closure (Homans technique), excision of subcutaneous tissue and tunneling of a dermal flap through the fascia into a muscular compartment of the leg (Thompson technique); second lympholymphatic anastomosis (autologous lymphatic grafts to bridge obstructed lymphatic segments), lymphovenous shunt (anastomosis of lymphatic channels to veins), lymphangioplasty enteromesenteric flap omental transfer (pedicled portion of omentum transposed to the affected limb); third - modified liposuction that has been developed in Sweden in recent years to remove adipose tissue associated with longstanding lymphedema, primarily in the upper region. This last technique is meant to reduce the volume of a limb and does not cure lymphedema. Compression garments and compression bandages must still be worn after the operation.

Low-level laser therapy (LLLT) was cleared by the US Food and Drug Administration (FDA) for the treatment of lymphedema in November 2006 (El Segundo, 2006). According to the US National Cancer Institute, studies suggest that low-level laser therapy may be effective in reducing lymphedema in a clinically meaningful way for some women. Two cycles of laser treatment were found to be effective in reducing the volume of the affected arm, extracellular fluid, and tissue hardness in approximately one-third of patients with post-mastectomy lymphedema at three months post-treatment. Suggested rationales for laser therapy include a potential decrease in fibrosis, stimulation of macrophages and the immune system, and a possible role in encouraging lymphangiogenesis (Carati et al, 2003).

#### **4. Future**

6 Novel Strategies in Lymphedema

reduce lymphedema and may in fact end up producing a tourniquet effect. During activity, whether exercise or daily activities, the short-stretch bandages enhance the pumping action of the lymph vessels by providing increased resistance for them to push against. This

Compression pump technology utilizes a multi-chambered pneumatic sleeve with overlapping cells to promote movement of lymph fluid. Pump therapy may be used in addition to other treatments such as compression bandaging and manual lymph drainage. In many cases, pump therapy may help soften fibrotic tissue and therefore potentially enable more efficient lymphatic drainage. Sequential pump therapy may also be used as a home treatment method, usually as part of a regimen also involving compression garments or wrapping. A Stanford University medical study showed that patients receiving the combined modalities of complete decongestive therapy or manual lymph drainage (MLD/CDT) and pneumatic pumping had a greater overall reduction in limb volume than patients receiving only MLD/CDT (Szuba et al, 2002). However, some therapists have begun to raise concern that compression pumps can cause genital swelling when used on

Complete decongestive therapy (CDT) is a primary tool in lymphedema management consisting of manual manipulation of the lymphatic ducts, short-stretch compression bandaging, therapeutic exercise, and skin care (National Lymphedema Network Medical Advisor Committee, 2011). The technique was pioneered by Emil Vodder in the 1930s for the treatment of chronic sinusitis and other immune disorders. Initially, CDT involves frequent visits to a certified therapist with a doctor's prescription. Once the lymphedema is reduced, increased patient participation is required for ongoing care, along with the use of elastic compression garments and non-elastic directional flow foam garments. Manual manipulation of the lymphatic ducts consists of gentle, rhythmic massaging of the skin to stimulate the flow of lymph and its return to the blood circulation system. In the blood's passage through the kidneys, the excess fluid is filtered out and eliminated from the body through urination. The treatment is very gentle and a typical session will involve drainage of the neck, trunk, and involved extremity (in that order), lasting approximately 40 to 60 minutes. CDT is generally effective on non-fibrotic lymphedema and less effective on

more fibrotic legs, although it has been shown to help break up fibrotic tissue.

Surgical techniques for correcting lymphedema may be excisional or physiological. However, surgery for lymphedema does not cure the disease or eliminate the need for decongestive treatment. Surgical treatment is used only in extreme cases in order to reduce the weight of the affected limb, to help minimize the frequency of inflammatory attacks, to improve cosmesis, and to potentially reduce the risk of secondary angiosarcoma (National Lymphedema Network Medical Advisor Committee, 2011). Although surgery has been shown to reduce edema in the short-term, there is a lack of evidence to suggest that it is beneficial in the long-term. Excisional techniques include three steps: first - circumferential excision of the lymphedematous tissue followed by skin grafting (Charles technique), longitudinal removal of the affected segment of skin and subcutaneous tissue and primary closure (Homans technique), excision of subcutaneous tissue and tunneling of a dermal flap through the fascia into a muscular compartment of the leg (Thompson technique); second lympholymphatic anastomosis (autologous lymphatic grafts to bridge obstructed lymphatic segments), lymphovenous shunt (anastomosis of lymphatic channels to veins),

encourages lymphatic flow and helps to soften fluid-swollen areas.

persons with leg lymphedema.

Lymphedema is a chronic and irreversible disease with significant negative consequences for the patient, as the resulting aesthetic deformities may make it impossible to continue their career and may lead to social isolation. In this book the authors deal with novel strategies in lymphedema. Still today, physicians and surgeons diagnose lymphedema relatively infrequently and the literature relating to the prevalence of lymphedema is limited. However it is correct to assume that in the future there will be an increase in incidence in primary as well as in secondary lymphedema.

Jin-Hong Chang in her chapter, presents lymphangiogenesis as a complex process that involves the interplay of many molecules with redundant mechanisms. The author deals with specific molecules that have shown the most promise as therapeutic targets including the vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), COX-2 selective inhibitors, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β.

Primary congenital lymphedema (Milroy disease), representing 10 per cent of primary, is present at birth and associated with an autosomal dominant familial history (Dahlberg et al, 1983). Mutations in the FLT4 gene cause some cases of Milroy disease. The FLT4 gene provides instructions for producing a protein called vascular endothelial growth factor receptor 3 (VEGFR-3), which regulates the development and maintenance of the lymphatic system. About 10 to 15 per cent of people with a mutation in the FLT4 gene do not develop the features of Milroy disease. Today, some of the possible treatments for Milroy's disease from various sources may include: exercise and elevation of extremity, elastic stockings, proper skin hygiene, antibiotics, benzathine penicillin, split-thickness skin grafts, and pedicular transfer of skin. Genetic testing is becoming almost practical, defining a limited number of specific hereditary syndromes with discrete gene mutations such as lymphedemadistichiasis (FOXC2), some forms of Milroy disease, and hypotrichosislymphedema-telangiectasis (SOX18).

A Brief Overview of Lymphology: Past, Present and Future 9

The third group of primary lymphedema is lymphedema tarda. It occurs in individuals older than 35 years (Kinmonth et al, 1957). Out of all patients with primary lymphedema, 10 per cent have lymphedema tarda. The cause of lymphedema tarda is a break in the FOXC2 gene. Decongestive therapy is the most widely accepted form of treatment. Today there is no cure for lymphedema tarda, but the condition can be managed by early diagnosis and

Even in this group, the emerging era of molecular lymphology will result in improved

Secondary lymphedema develops as a consequence of disruption or obstruction of the lymphatic pathways by surgery or other disease processes. Secondary lymphedema is much more common than the primary form. Its global incidence can be ascribed, predominantly, to filariasis, which accounts for over 90 million afflicted individuals (Laharya et al, 2011). Nevertheless, there is a growing number of lymphedema cases that are arising as a consequence of neoplastic disease, both through direct lymphatic invasion and, iatrogenically, through treatment of the neoplasm. It has been reported to occur within days and up to 30 years after treatment for breast cancer (Shaw et al, 2007). Cancer rates could further increase by 50 per cent to 15 million new cases in the year 2020 and we can assume that incidences of lymphedema will increase in the future. The most frequent causes are breast cancer in lymphedema of the arm, and prostate cancer in disease of the leg (Smith et al, 1963). In rare cases, lymphedema can lead to a form of cancer called lymphangiosarcoma, although the mechanism of carcinogenesis is not understood. Lymphedema-associated lymphangiosarcoma is called Stewart-Treves syndrome. Lymphangiosarcoma most frequently occurs in cases of long-standing lymphedema. The incidence of angiosarcoma is estimated to be 0.45 per cent in patients living five years after radical mastectomy (Chopra 2007). Lymphedema is also associated with a low-grade form of cancer called retiform

Moreover, another rare case of lymphedema is the Yellow nail syndrome, defined (Samman and White 1964) as a combination of slow growing, discoloured nails. In this book Fredrik Berglund deal with this rare syndrome referring to several papers which mention the exposure to titanium implants and titanium dioxe preceding the development of yellow nails and also return to normal conditions after withdrawal of the drugs. The author suggests that nail changes and defective lymph drainage are related, and considers that "nail changes are believed to be the result of defective lymph drainage". Since titanium is always present in the nails of these patients, it is more tempting to consider the nail changes as a toxic reaction to titanium. Since lymphedema is disfiguring, causes difficulties in daily living and can lead to lifestyle becoming severely limited, it may also result in psychological

Gabriella Wernicke, as a board certified radiation oncologist, presents a careful review about the lymphedema in post-operative breast cancer. In 2008, the American Cancer Society published the results of this study entitled "Preoperative Assessment Enables the Early Diagnosis and Successful Treatment of Lymphedema". The study revealed that early diagnosis of lymphedema in breast cancer patients (called stage 0 in the article) associated with an early intervention, a compression sleeve and gauntlet for one month, led to a return to pre-operative baseline status. In a five year follow-up, patients remained at their

understanding, evaluation and treatment in clinical lymphology.

hemangioendothelioma (a low-grade angiosarcoma).

treatment.

distress.

The future holds promise that such testing, combined with careful phenotypic descriptions, will become routine to classify familial lymphangiodysplastic syndromes and other congenital/genetic-dysmorphogenic disorders characterized by lymphedema, lymphangiectasia, and lymphangiomatosis. In addition, there are many other clinical syndromes with lymphedema as a component, and these may have genes identified in the future. The emerging era of molecular lymphology will result in improved understanding, evaluation and treatment in clinical lymphology; dramatic progress has been made towards effective targeted molecular therapies for lymphedema. Vascular endothelial growth factors C (VEGF-C) and their receptors (VEGFR-3) are known to be the primary players, but these molecules work in sync with other factors. Currently, no specific molecular treatment options are available for clinical use; however, promising results from animal trials suggest a role for VEGF-C gene therapy in treatment of lymphedema. Other results indicate that other factors such as COX-2, MMP-9, and interstitial flow dynamics may also be important in future management of lymphedema. Combination therapies such as stem cell implantation, skin grafting, and lymph node transfer in conjunction with VEGF-C therapy may further expand the effectiveness of future therapies as well. Design of a drug to treat lymphedema will require an effective animal model that accurately mimics lymphedema in humans, and further evaluation of the metastatic risk of inducing lymphangiogenesis in cancer patients is also needed. Due to the proliferative efforts of researchers over the last decade, effective treatments for lymphedema in humans may soon be a reality. However, while the addition of growth (or inhibitory) factors is attractive, the availability of these treatments in the future is uncertain at this time and should be conducted in the context of co-morbid conditions (presence of cancer, cancer treatments, drug regimens).

The second group of primary lymphedema is lymphedema praecox (Meige disease) that represents 80 per cent of primary lymphedema and occurs (estimated incidence 1.15 in 100,000 people younger than 20) after birth but before the age of 35. The age of onset is generally in adolescence (Wheeler, et al 1981). There is, unfortunately, no cure but, occasionally, patients will improve with time. BOTOX injections may help with the blepharospasm and can be used to suppress mouth movements but it is no cure. Some patients are benefited by anticholinergics such as Artane (trihexphenidyl) or Cogentin (benztropine) and few are benefited by muscle relaxants such as Lioresal (baclofen). Anticonvulsants such as Tegretol (carbamazepine) have also been employed with sporadic benefit. The only causative genes so far identified for the non-congenital primary lymphoedemas are the transcription factor FOXC2, where mutations are known to produce lymphoedema with distichiasis, and SOX18 in the very rare condition hypotrichosislymphoedema-telangiectasia. A recent study has examined the FOXC2 gene by sequence analysis in 23 affected individuals with Meige disease (Rezaie et al, 2008). A novel truncating mutation (c.563-584del) was identified in one family and found to segregate with the disease in eight affected relatives over three generations. Although the affected patient, initially selected for mutation screening from this family, had lymphoedema without distichiasis, all but one of his affected relatives who carried the FOXC2 mutation did have accessory eyelashes originating from their meibomian glands. This is further confirmation that of the primary lymphoedemas, only lymphoedema with distichiasis is caused by FOXC2 mutations.

The future holds promise that such testing, combined with careful phenotypic descriptions, will become routine to classify familial lymphangiodysplastic syndromes and other congenital/genetic-dysmorphogenic disorders characterized by lymphedema, lymphangiectasia, and lymphangiomatosis. In addition, there are many other clinical syndromes with lymphedema as a component, and these may have genes identified in the future. The emerging era of molecular lymphology will result in improved understanding, evaluation and treatment in clinical lymphology; dramatic progress has been made towards effective targeted molecular therapies for lymphedema. Vascular endothelial growth factors C (VEGF-C) and their receptors (VEGFR-3) are known to be the primary players, but these molecules work in sync with other factors. Currently, no specific molecular treatment options are available for clinical use; however, promising results from animal trials suggest a role for VEGF-C gene therapy in treatment of lymphedema. Other results indicate that other factors such as COX-2, MMP-9, and interstitial flow dynamics may also be important in future management of lymphedema. Combination therapies such as stem cell implantation, skin grafting, and lymph node transfer in conjunction with VEGF-C therapy may further expand the effectiveness of future therapies as well. Design of a drug to treat lymphedema will require an effective animal model that accurately mimics lymphedema in humans, and further evaluation of the metastatic risk of inducing lymphangiogenesis in cancer patients is also needed. Due to the proliferative efforts of researchers over the last decade, effective treatments for lymphedema in humans may soon be a reality. However, while the addition of growth (or inhibitory) factors is attractive, the availability of these treatments in the future is uncertain at this time and should be conducted in the context of co-morbid conditions

The second group of primary lymphedema is lymphedema praecox (Meige disease) that represents 80 per cent of primary lymphedema and occurs (estimated incidence 1.15 in 100,000 people younger than 20) after birth but before the age of 35. The age of onset is generally in adolescence (Wheeler, et al 1981). There is, unfortunately, no cure but, occasionally, patients will improve with time. BOTOX injections may help with the blepharospasm and can be used to suppress mouth movements but it is no cure. Some patients are benefited by anticholinergics such as Artane (trihexphenidyl) or Cogentin (benztropine) and few are benefited by muscle relaxants such as Lioresal (baclofen). Anticonvulsants such as Tegretol (carbamazepine) have also been employed with sporadic benefit. The only causative genes so far identified for the non-congenital primary lymphoedemas are the transcription factor FOXC2, where mutations are known to produce lymphoedema with distichiasis, and SOX18 in the very rare condition hypotrichosislymphoedema-telangiectasia. A recent study has examined the FOXC2 gene by sequence analysis in 23 affected individuals with Meige disease (Rezaie et al, 2008). A novel truncating mutation (c.563-584del) was identified in one family and found to segregate with the disease in eight affected relatives over three generations. Although the affected patient, initially selected for mutation screening from this family, had lymphoedema without distichiasis, all but one of his affected relatives who carried the FOXC2 mutation did have accessory eyelashes originating from their meibomian glands. This is further confirmation that of the primary lymphoedemas, only lymphoedema with distichiasis is caused by

(presence of cancer, cancer treatments, drug regimens).

FOXC2 mutations.

The third group of primary lymphedema is lymphedema tarda. It occurs in individuals older than 35 years (Kinmonth et al, 1957). Out of all patients with primary lymphedema, 10 per cent have lymphedema tarda. The cause of lymphedema tarda is a break in the FOXC2 gene. Decongestive therapy is the most widely accepted form of treatment. Today there is no cure for lymphedema tarda, but the condition can be managed by early diagnosis and treatment.

Even in this group, the emerging era of molecular lymphology will result in improved understanding, evaluation and treatment in clinical lymphology.

Secondary lymphedema develops as a consequence of disruption or obstruction of the lymphatic pathways by surgery or other disease processes. Secondary lymphedema is much more common than the primary form. Its global incidence can be ascribed, predominantly, to filariasis, which accounts for over 90 million afflicted individuals (Laharya et al, 2011). Nevertheless, there is a growing number of lymphedema cases that are arising as a consequence of neoplastic disease, both through direct lymphatic invasion and, iatrogenically, through treatment of the neoplasm. It has been reported to occur within days and up to 30 years after treatment for breast cancer (Shaw et al, 2007). Cancer rates could further increase by 50 per cent to 15 million new cases in the year 2020 and we can assume that incidences of lymphedema will increase in the future. The most frequent causes are breast cancer in lymphedema of the arm, and prostate cancer in disease of the leg (Smith et al, 1963). In rare cases, lymphedema can lead to a form of cancer called lymphangiosarcoma, although the mechanism of carcinogenesis is not understood. Lymphedema-associated lymphangiosarcoma is called Stewart-Treves syndrome. Lymphangiosarcoma most frequently occurs in cases of long-standing lymphedema. The incidence of angiosarcoma is estimated to be 0.45 per cent in patients living five years after radical mastectomy (Chopra 2007). Lymphedema is also associated with a low-grade form of cancer called retiform hemangioendothelioma (a low-grade angiosarcoma).

Moreover, another rare case of lymphedema is the Yellow nail syndrome, defined (Samman and White 1964) as a combination of slow growing, discoloured nails. In this book Fredrik Berglund deal with this rare syndrome referring to several papers which mention the exposure to titanium implants and titanium dioxe preceding the development of yellow nails and also return to normal conditions after withdrawal of the drugs. The author suggests that nail changes and defective lymph drainage are related, and considers that "nail changes are believed to be the result of defective lymph drainage". Since titanium is always present in the nails of these patients, it is more tempting to consider the nail changes as a toxic reaction to titanium. Since lymphedema is disfiguring, causes difficulties in daily living and can lead to lifestyle becoming severely limited, it may also result in psychological distress.

Gabriella Wernicke, as a board certified radiation oncologist, presents a careful review about the lymphedema in post-operative breast cancer. In 2008, the American Cancer Society published the results of this study entitled "Preoperative Assessment Enables the Early Diagnosis and Successful Treatment of Lymphedema". The study revealed that early diagnosis of lymphedema in breast cancer patients (called stage 0 in the article) associated with an early intervention, a compression sleeve and gauntlet for one month, led to a return to pre-operative baseline status. In a five year follow-up, patients remained at their

A Brief Overview of Lymphology: Past, Present and Future 11

Clarke, AC.; (1962) Hazards of prophecy :The failure of imagination in Profiles of the Future,

Dahlberg, P.J.; Borer, W.Z.; Newcomer, K.L. & Yutuc, W.R. (1983). Autosomal or X-linked

El Segundo, C.A. (2006). Low Level Laser FDA Cleared for the Treatment of Lymphedema,

Fang, Y.; He, Y. & Liu, Z. (2008). Negative pressure in pharyngo-oral cavity can treat

Fanous, M.Y.; Phillips, A.J. & Windsor, J.A. (2006). Mesenteric lymph: the bridge to future

Gummere, RM. (1917-25). 3 vols. Volume I. Epistle LXIV, In: *Lucius Annaeus Seneca. Moral* 

Hagelin, O. (1989). *Rare and important medical books in the library of the Swedish Society of Medicine*, Svenska Lakaresallskapet, ISBN 9781174702020, Stockholm, Swedish Husiya, K. & Waran, Iwa. (1805). Reprinted by Teizo O (*Japan Society for History of Medicine*), Ishi Yaku Shuppan Kabushiki Gaisha ISBN: 426370441X , Tokyo, Japan Kinmonth, J.B.; Taylor, G.W.; Tracy, G.D. & Marsh, J.D. Primary Lymphoedema. Clinical

Lahariya, C.; Tomar, S.S. (2011). How endemic countries can accelerate lymphatic filariasis

Luyendijk-Elshout, A.M. (1964). *Introduction in: Dilucidatio valvularam in vasis lymphaticis et* 

National Lymphedema Network Medical Advisor Committee. (2011). In: *Position statement of the national lymphedema network*, 20 November 2010, Available from:

Norman, J. (1978). *Medicine and the life sciences*. In Catalogue No.4, Jeremy Norman & Co, Inc, ISBN : 0825617103 9780825617102, San Francisco, United State of America Pecquet, J. (1651). *Experimenta nova anatomica, quibus incognitum chyli receptaculum, et ab eo per* 

Purushotham, AD.; Bennett Britton, TM.; Klevesath, MB.; Chou, P.; Agbaje, OF. & Duffy,

http://www.lymphnet.org/pdfDocs/nlntreatment.pdf

*Surgery*, Vol.246, N.1, (July 2007), pp. 42-5, ISSN 1528-1140

*Genetics*. N.16(1), (September 1983), pp. 99-104, ISSN: 0148-7299

http://www.prweb.com/releases/2006/12/prweb487900.htm

recessive syndrome of congenital lymphedema, hypoparathyroidism, nephropathy, prolapsing mitral valve, and brachytelephalangy. *American Journal of Medical* 

lymphedema and related disorders. *Medical Hypotheses*, Vol.70, N.4, (October 2008),

management of critical illness. *Journal Of Pancreas*, Vol.9, N.8(4), (July 2007), pp.

*Epistles. Translated by Richard M. Gummere*. The Loeb Classical Library, pp. 117,

and lymphangiographic studies of a series of 107 patients in which the lower limbs were affected. British Journal of Surgery. N.45(189), (July 1957), pp.1–10, ISSN:

elimination? An analytical review to identify strategic and programmatic interventions. *Journal of Vector Borne Diseases.* N.48(1), (March 2011), pp. 1-6,

*lacteis*. In Dutch Classics on History of Science, XI, Ed. B. De Graaf, ISBN:

*thoracem in ramos usque subclavis vasa lactea deteguntur*, Apud Sebastianum Cramoisy

SW. (2007). Lymph node status and breast cancer-related lymphedema. *Annal of* 

Harper & Row, ISBN 0-445-04061-0, New York

In: *Press Release Distribution*, Available from:

Cambridge, Mass.: Harvard UP, Retrived from

<http://www.stoics.com/seneca\_epistles\_book\_1.html>

pp.886-7, ISSN 0306-9877

374-99, ISSN 1590-8577

1365-2168

ISSN:0972-9062

9060044290, Nieuwkoop, Netherland

et Gabrielem Cramoisy, Paris, France

preoperative baseline, suggesting that preclinical detection of lymphedema can halt if not reverse its progression (Stout Gergich et al, 2008).

The chapter: "Preparing for and Coping with Breast Cancer-related Lymphedema", is aimed at addressing real and present concerns for both patients and their family members regarding breast cancer-related lymphedema. Research interests include roles of patients in familial and social relationships within the specific context of their own family members' or friends' illness experiences with emphasis on lymphedema and lymphedema-related symptoms.

The group of Fondazione IRCCS "Istituto dei tumori" of Milan presents an innovative theory on a new model of lymphedema: pelvic lymphedema

This lymphedema might be a consequence of mechanical pelvic injury or of the altered lymphatic system caused by such injury. The extra-peritoneal pelvic area is sited between the peritoneum, covering the pelvic organs, and the pelvic diaphragm. In the pelvic cavity the peritoneum is separated from the walls which delimit the cavity by the surrounding and supporting fatty extra-peritoneal tissue. Pelvic lymphadenectomy may lead to damaged lymphatic vessels with subsequent pelvic malfunction within a few weeks post-surgery, if undiagnosed and untreated, and can progress to a chronic pelvic dysfunction (Vannelli et al, 2009).

In conclusion I recommend remembering that "but even if the old masters have discovered everything, one thing will be always new - the application and the scientific study and classification of the discoveries made by others." (Gummere, 1917-28). Lymphangiogenesis is a complex process that involves the interplay of many molecules with redundant mechanisms and, though a solid body of evidence exists, significant research is ultimately still needed. Moreover, to date, lymphedema is frequently undiagnosed even in teaching centres (Schuchhardt C, 1997), and it seems likely that many surgical interventions have not been adequately studied with respect to lymphatic damage and their consequences. Is the future of lymphedema near? Clarke's Second Law is: "The only way of discovering the limits of the possible is to venture a little way past them into the impossible" (Clarke, 1962). Lymphedema issues this challenge.

#### **5. References**


preoperative baseline, suggesting that preclinical detection of lymphedema can halt if not

The chapter: "Preparing for and Coping with Breast Cancer-related Lymphedema", is aimed at addressing real and present concerns for both patients and their family members regarding breast cancer-related lymphedema. Research interests include roles of patients in familial and social relationships within the specific context of their own family members' or friends' illness experiences with emphasis on lymphedema and lymphedema-related

The group of Fondazione IRCCS "Istituto dei tumori" of Milan presents an innovative

This lymphedema might be a consequence of mechanical pelvic injury or of the altered lymphatic system caused by such injury. The extra-peritoneal pelvic area is sited between the peritoneum, covering the pelvic organs, and the pelvic diaphragm. In the pelvic cavity the peritoneum is separated from the walls which delimit the cavity by the surrounding and supporting fatty extra-peritoneal tissue. Pelvic lymphadenectomy may lead to damaged lymphatic vessels with subsequent pelvic malfunction within a few weeks post-surgery, if undiagnosed and untreated, and can progress to a chronic pelvic dysfunction (Vannelli et al,

In conclusion I recommend remembering that "but even if the old masters have discovered everything, one thing will be always new - the application and the scientific study and classification of the discoveries made by others." (Gummere, 1917-28). Lymphangiogenesis is a complex process that involves the interplay of many molecules with redundant mechanisms and, though a solid body of evidence exists, significant research is ultimately still needed. Moreover, to date, lymphedema is frequently undiagnosed even in teaching centres (Schuchhardt C, 1997), and it seems likely that many surgical interventions have not been adequately studied with respect to lymphatic damage and their consequences. Is the future of lymphedema near? Clarke's Second Law is: "The only way of discovering the limits of the possible is to venture a little way past them into the impossible" (Clarke, 1962).

Ambrose, C. (2006). Immunology's first priority dispute—An account of the 17th-century

Arvy, L. & Rivet, R. (1976). Marie Philibert Constant Sappey (1810-1896). The man and the

Carati, C.J.; Anderson, S.N.; Gannon, B.J. & Piller, N.B. (2003). Treatment of post-

Chopra, S.; Ors, F. & Bergin, D. (2007). MRI of angiosarcoma associated with chronic

Rudbeck–Bartholin feud. *Cellular Immunology*, Vol.242, N.1, (July 2006), pp. 1-8,

lymphologist . *Bulletin De l'Association Des Anatomistes (Nancy)*, Vol.60, N.168

mastectomy lymphedema with low-level laser therapy. *Cancer* Vol.15, N.98(6),

lymphoedema: Stewart Treves syndrome. *British Journal of Radiology* N.80(960),

reverse its progression (Stout Gergich et al, 2008).

theory on a new model of lymphedema: pelvic lymphedema

symptoms.

2009).

Lymphedema issues this challenge.

ISSN 1672-7681

(March 1976), pp. 63-79, ISSN 0376-6160

(September 2003), pp. 1114–22, ISSN: 1097-0142

(December 2007), pp. 310-3, ISSN: 1748-880X.

**5. References** 


<http://www.stoics.com/seneca\_epistles\_book\_1.html>


**1** 

*United States of America* 

**Strategies in Modulating Lymphedema** 

Jin-Hong Chang\*, Joshua H. Hou, Sandeep Jain and Dimitri T. Azar\* *Department of Ophthalmology and Visual Sciences, University of Illinois Chicago* 

Lymphedema is the accumulation of interstitial fluid within tissues due to the impairment of lymphatic function. Dysfunction can result from direct obstruction of lymphatic vessels, absence of lymphatic vessels, or inadequate lymphatic function. From congenital forms of lymphedema, such as Milroy disease, to acquired forms of lymphedema, such as filiarisis lymphedema or post-surgical lymphedema, lymphatic dysfunction contributes significantly

Due to the inherent difficulties in visualizing lymphatic channels, research in the pathogenesis and treatment of lymphedema has lagged behind similar investigations in vascular pathology. Over the past two decades, aggressive research efforts have vastly improved our understanding of the lymphatic system, but significant advances in medical therapies for lymphedema and lymphatic regeneration are still lacking. To date, the majority of treatment strategies for lymphedema (compression stockings, massage, and exercise) do not address the underlying molecular pathophysiology (Nakamura &

Several nonspecific pharmacological agents, such as selenium and benzo-pyrones, have been studied with limited success. A review of ten different trials of pharmacological therapies for lymphedema by Kligman *et al*. concluded that insufficient evidence exists to support the use of these nonspecific medical therapies at the moment (Kligman et al.,

With improvements in our understanding of the molecular mechanisms of lymphedema and lymphangiogenesis, however, an increasing number of potential pharmacological targets for specific therapies are being identified. Due to ongoing efforts by numerous researchers in the study of therapeutic lymphatic regeneration, significant progress is being made towards targeted pharmacological therapies for lymphedema. Specific molecules that have shown the most promise as therapeutic targets include the vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), cyclooxygenase 2 (COX-2) selective inhibitors, tumor necrosis factor (TNF)-α, and transforming growth

**1. Introduction** 

Rockson, 2008).

factor (TGF)-β.

Corresponding author

 \*

2004).

to the world's human disease burden.


## **Strategies in Modulating Lymphedema**

Jin-Hong Chang\*, Joshua H. Hou, Sandeep Jain and Dimitri T. Azar\* *Department of Ophthalmology and Visual Sciences, University of Illinois Chicago United States of America* 

#### **1. Introduction**

12 Novel Strategies in Lymphedema

Regis, O. & Kaoru M. (1997). Paolo Mascagni, Ernest Alexandra Lauth and Marie Philibert

Rezaie, T.; Ghoroghchian ,R.; Bell, R.; Brice, G.; Hasan, A.; Burnand, K.; Vernon, S.; Mansour,

Rouviere, P.H. (1932). *Anatomie des lymphatiques de l'homme*, Masson et Cie, ISBN 0-8016-

Ruysch, F. (1665). *Dilucidatio valvularum in vasis lymphaticis et lacteis,* ex officina Harmani

Schuchhardt, C. (1997). Lymphedema. An easy diagnosis - but frequently missed.

Shaw, C.; Mortimer, P.; Judd, P.A.; (2007). Randomized controlled trial comparing a low-fat

Smith, R.D.; Spittell, J.A. & Schirger, A. (1963). Secondary lymphedema of the leg: its

Stout Gergich, N.L.; Pfalzer, L.A.; McGarvey, C.; Springer, B.; Gerber, L.H. & Soballe, P.

Szuba, A.; Achalu, R. & Rockson, S.G. (2002). Decongestive lymphatic therapy for patients

Vannelli, A.; Battaglia, L.; Poiasina, E. & Leo E. Pelvic lymphedema: Truth or fiction? *Medical* 

Warren, AG.; Brorson, H.; Borud, LJ. & Slavin, SA. (2007). Lymphedema - a comprehensive

Wheeler, E.S.; Chan, V.; Wassman, R.; Rimoin, D.L. & Lesavoy, M.A. (1981). Familial

*Hypotheses*, Vol.72, N.3, (March 2009), pp. 267-70, ISSN 0306-9877

Vol.15, N.109 (10), (May 2007), pp. 1949-56, ISSN: 1097-0142

*Association.* N.185, (July 1963), pp. 116–18 ISSN: 1538-3598

N.95(11), (December 2002), pp.2260-7, ISSN: 1097-0142

(March 1981), pp. 362-4, ISSN: 1529-4242

*Fortschritte der Medizin*, Vol. 20, N.115, (August 1997), pp. 24, 27-31, ISSN 0946-5634

diet with a weight-reduction diet in breast cancer-related lymphedema. *Cancer*

characteristics and diagnostic implications. *Journal of the American Medical* 

(2008). Pre-operative assessment enables the early diagnosis and successful treatment of lymphedema. *Cancer*, Vol.15, N.112(12), (June 2008), pp. 2809-19 ISSN:

with breast carcinoma-associated lymphedema. A randomized, prospective study of a role for adjunctive intermittent pneumatic compression. *Cancer*, Vol.1,

review. *Annals of Plastic Surgery*, Vol.59, N.4, (October 2007), pp. 464-72, ISSN 1536-

lymphedema praecox: Meige's disease. *Plastic and Reconstructive Surgery*. N.67(3),

2171

4813

1097-0142

3708

3556-X, Paris, France

Gael, Hagae Comitis, Netherland

Constant Sappey on the Dissection and Injection of the Lymphatics. *Journal of the International Society for Plastination,* Vol.12, N.2 (February 1997), pp. 4-7, ISSN 1090-

S.; Mortimer, P.; Jeffery, S.; Child, A. & Sarfarazi, M. (2008), Primary nonsyndromic lymphoedema (Meige disease) is not caused by mutations in FOXC2. *European Journal of Human Genetics*. N.16(3), (March 2008), pp. 300-4, ISSN: 1018-

> Lymphedema is the accumulation of interstitial fluid within tissues due to the impairment of lymphatic function. Dysfunction can result from direct obstruction of lymphatic vessels, absence of lymphatic vessels, or inadequate lymphatic function. From congenital forms of lymphedema, such as Milroy disease, to acquired forms of lymphedema, such as filiarisis lymphedema or post-surgical lymphedema, lymphatic dysfunction contributes significantly to the world's human disease burden.

> Due to the inherent difficulties in visualizing lymphatic channels, research in the pathogenesis and treatment of lymphedema has lagged behind similar investigations in vascular pathology. Over the past two decades, aggressive research efforts have vastly improved our understanding of the lymphatic system, but significant advances in medical therapies for lymphedema and lymphatic regeneration are still lacking. To date, the majority of treatment strategies for lymphedema (compression stockings, massage, and exercise) do not address the underlying molecular pathophysiology (Nakamura & Rockson, 2008).

> Several nonspecific pharmacological agents, such as selenium and benzo-pyrones, have been studied with limited success. A review of ten different trials of pharmacological therapies for lymphedema by Kligman *et al*. concluded that insufficient evidence exists to support the use of these nonspecific medical therapies at the moment (Kligman et al., 2004).

> With improvements in our understanding of the molecular mechanisms of lymphedema and lymphangiogenesis, however, an increasing number of potential pharmacological targets for specific therapies are being identified. Due to ongoing efforts by numerous researchers in the study of therapeutic lymphatic regeneration, significant progress is being made towards targeted pharmacological therapies for lymphedema. Specific molecules that have shown the most promise as therapeutic targets include the vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), cyclooxygenase 2 (COX-2) selective inhibitors, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β.

<sup>\*</sup> Corresponding author

Strategies in Modulating Lymphedema 15

trafficking via *in vivo* bioluminescent imaging. Three days post-surgery, the animals were then treated with parenteral recombinant human VEGF-C generated from engineered DNA encoding the human VEGF homology domain (amino acid residues Thr103-Arg227) fused to a human CD33 signal peptide at the N-terminus and a 10x-histidine tag at the C-terminus. Upon examination, treated animals were found to have reversal of the lymphedematous state to the normal state with resolution of edema, hypercellularity, inflammatory changes, and microlymphatic dilation. Both lymphatic vessel number and cross-sectional area were reduced following exogenous administration of the recombinant VEGF-C (Cheung et al.,

In another mouse model of chronic obstructive lymphedema, treatment with VEGF-C also improved lymphedema in the studied animals. The authors injected a pcDNA3.1-VEGF-C plasmid into the tail of these mice. Subsequent overexpression of VEGF-C enhanced

Using a rat hind limb model of lymphedema, Liu *et al*. also observed significant improvement in lymphedema following focal transfection with VEGF-C DNA. Rats were treated with a plasmid DNA encoding human VEGF-C (pcDNA3.1-VEGF-C) and monitored for resolution of their surgically-induced lymphedema in comparison to controls. Lymphedema was quantitatively reduced at 2 and 4 weeks in the therapy group as documented by magnetic resonance imaging (MRI), B-scan ultrasound, and water displacement volumetry measurements. Furthermore, numerous newly formed lymphatic vessels were observed in treated mice on both histological and immunofluorescence analysis

Finally, Tammela *et al*. found a significant role for adenovirally-delivered VEGF-C in improving outcomes of lymph node dissection and transplantation in mice. In their study, lymph node dissection and transplantation in combination with adenovirally-delivered VEGF-C induced the formation of functional collecting lymphatic vessels and the

Despite the growing body of evidence in support of the efficacy of VEGF-C gene therapy, the exact mechanism by which VEGF-C-induced lymphangiogenesis facilitates resolution of lymphedema remains controversial. In an effort to elucidate the mechanisms by which VEGF-C induces lymphatic microvascular remodeling, Jin *et al*. examined the effects of anti-VEGFR-3 neutralizing antibodies in a mouse tail model of post-surgical lymphedema. This study demonstrated that VEGFR-3 plays a central mechanistic role in lymphedema remodeling. In the presence of the neutralizing antibody, lymphatic remodeling was greatly

In contrast, Uzarski *et al*. failed to observe inhibition of edema resolution across surgicallyinduced wounds in a mouse tail lymphedema model upon blockage of VEGFR-3. In their study, two mouse models were compared. In the first mouse model, scar-free lymphatic obstruction was simulated with dissection and removal of the superficial lymphatics from a mouse tail. Distal lymphedema was noted, and resolution was stimulated by VEGF-C. Edema resolution was not, however, inhibited by VEGFR-3 neutralizing antibodies, although lymphangiogenesis was reduced. In the second mouse model, scar-containing

lymphangiogenesis *in vivo* and improved lymphedema (Hu et al., 2008).

reconstitution of a functional immunological barrier (Tammela et al., 2007).

attenuated due to blockage of VEGF-C-induced signaling (Jin da et al., 2009).

2006).

(Liu et al., 2008).

**3.2 Caveats to VEGF-C Therapies** 

### **2. Nonspecific treatments**

To date, studies have been unable to confirm the effectiveness of nonspecific treatment strategies such as selenium and benzo-pyrones on lymphedema, despite their common use in clinical practice. Selenium is a drug that is used to prevent or minimize the adverse effects of radiotherapy, chemotherapy, or surgery in oncology patients; however, after rigorous testing of this therapy, Dennert and Horneber concluded that inadequate evidence exists to advocate for or against the use of selenium for lymphedema (Dennert & Horneber, 2006). Similarly, benzo-pyrones have been considered a plausible treatment strategy for lymphedema, as these molecules reduce vascular permeability and thereby, reduce subcutaneous fluid. Furthermore, benzo-pyrones also increase macrophage activity and encourage protein degradation, which, in turn, reduces the formation of fibrotic tissue in the lymphedematous limb. A recent review of 15 trials of benzo-pyrones in the treatment lymphedema, however, failed to uncover any conclusions due to the poor quality of the analyzed trials (Badger et al., 2004). The limited and questionable efficacy of current nonspecific treatments has led many researchers toward using molecular strategies for the development of newer targeted pharmacological therapies. Specific attention has been paid to factors responsible for lymphangiogenesis, such as VEGF-C. By stimulating lymphangiogenesis and regeneration of lost or damaged lymphatics, VEGF-C offers significant promise as a treatment for all-cause lymphedema.

#### **3. Targeted therapies**

Currently, a number of molecular strategies for the treatment of lymphedema are being studied in animal models. Promising results have been obtained in the treatment of mouse models of lymphedema via methods of promoting lymphangiogenesis; however, significant work is still required before clinical application of these therapies becomes a reality.

#### **3.1 Treatment with VEGF-C**

In particular, VEGF-C has been found to be a potent regulator of lymphangiogenesis through its actions on two receptor tyrosine kinases, VEGFR-2 and VEGFR-3 (Haiko et al., 2008; Tammela et al., 2011). Multiple studies have evaluated the efficacy of VEGF-C gene therapy and plasmid transfection and revealed surprising success in a variety of animal lymphedema models.

The first study to document improvement in the clinical and pathologic features of lymphedema by therapeutic enhancement of lymphatic drainage with human VEGF-C gene therapy was performed in 2003 by Yoon *et al*. In two animal models, a rabbit ear model and a mouse tail model, these authors treated lymphedema with naked plasmid DNA that encoded human VEGF-C (phVEGF-C) injected subcutaneously. Following treatment, improvements in both lymphedema and lymphatic function were noted (Yoon et al., 2003).

Cheung *et al*. similarly found improvement in surgically-induced lymphedema in a mouse tail model using recombinant human VEGF-C. Mice were treated with cautery ablation of the large collecting lymphatics of the tail after identification of the vessels with injected methylene blue. A lymphedematous state was then documented by evidence of dilated cutaneous lymphatics, acute inflammation and hypercellularity, and impairment of immune

To date, studies have been unable to confirm the effectiveness of nonspecific treatment strategies such as selenium and benzo-pyrones on lymphedema, despite their common use in clinical practice. Selenium is a drug that is used to prevent or minimize the adverse effects of radiotherapy, chemotherapy, or surgery in oncology patients; however, after rigorous testing of this therapy, Dennert and Horneber concluded that inadequate evidence exists to advocate for or against the use of selenium for lymphedema (Dennert & Horneber, 2006). Similarly, benzo-pyrones have been considered a plausible treatment strategy for lymphedema, as these molecules reduce vascular permeability and thereby, reduce subcutaneous fluid. Furthermore, benzo-pyrones also increase macrophage activity and encourage protein degradation, which, in turn, reduces the formation of fibrotic tissue in the lymphedematous limb. A recent review of 15 trials of benzo-pyrones in the treatment lymphedema, however, failed to uncover any conclusions due to the poor quality of the analyzed trials (Badger et al., 2004). The limited and questionable efficacy of current nonspecific treatments has led many researchers toward using molecular strategies for the development of newer targeted pharmacological therapies. Specific attention has been paid to factors responsible for lymphangiogenesis, such as VEGF-C. By stimulating lymphangiogenesis and regeneration of lost or damaged lymphatics, VEGF-C offers

Currently, a number of molecular strategies for the treatment of lymphedema are being studied in animal models. Promising results have been obtained in the treatment of mouse models of lymphedema via methods of promoting lymphangiogenesis; however, significant

In particular, VEGF-C has been found to be a potent regulator of lymphangiogenesis through its actions on two receptor tyrosine kinases, VEGFR-2 and VEGFR-3 (Haiko et al., 2008; Tammela et al., 2011). Multiple studies have evaluated the efficacy of VEGF-C gene therapy and plasmid transfection and revealed surprising success in a variety of animal

The first study to document improvement in the clinical and pathologic features of lymphedema by therapeutic enhancement of lymphatic drainage with human VEGF-C gene therapy was performed in 2003 by Yoon *et al*. In two animal models, a rabbit ear model and a mouse tail model, these authors treated lymphedema with naked plasmid DNA that encoded human VEGF-C (phVEGF-C) injected subcutaneously. Following treatment, improvements in both lymphedema and lymphatic function were noted (Yoon et al., 2003). Cheung *et al*. similarly found improvement in surgically-induced lymphedema in a mouse tail model using recombinant human VEGF-C. Mice were treated with cautery ablation of the large collecting lymphatics of the tail after identification of the vessels with injected methylene blue. A lymphedematous state was then documented by evidence of dilated cutaneous lymphatics, acute inflammation and hypercellularity, and impairment of immune

work is still required before clinical application of these therapies becomes a reality.

significant promise as a treatment for all-cause lymphedema.

**2. Nonspecific treatments** 

**3. Targeted therapies** 

**3.1 Treatment with VEGF-C** 

lymphedema models.

trafficking via *in vivo* bioluminescent imaging. Three days post-surgery, the animals were then treated with parenteral recombinant human VEGF-C generated from engineered DNA encoding the human VEGF homology domain (amino acid residues Thr103-Arg227) fused to a human CD33 signal peptide at the N-terminus and a 10x-histidine tag at the C-terminus. Upon examination, treated animals were found to have reversal of the lymphedematous state to the normal state with resolution of edema, hypercellularity, inflammatory changes, and microlymphatic dilation. Both lymphatic vessel number and cross-sectional area were reduced following exogenous administration of the recombinant VEGF-C (Cheung et al., 2006).

In another mouse model of chronic obstructive lymphedema, treatment with VEGF-C also improved lymphedema in the studied animals. The authors injected a pcDNA3.1-VEGF-C plasmid into the tail of these mice. Subsequent overexpression of VEGF-C enhanced lymphangiogenesis *in vivo* and improved lymphedema (Hu et al., 2008).

Using a rat hind limb model of lymphedema, Liu *et al*. also observed significant improvement in lymphedema following focal transfection with VEGF-C DNA. Rats were treated with a plasmid DNA encoding human VEGF-C (pcDNA3.1-VEGF-C) and monitored for resolution of their surgically-induced lymphedema in comparison to controls. Lymphedema was quantitatively reduced at 2 and 4 weeks in the therapy group as documented by magnetic resonance imaging (MRI), B-scan ultrasound, and water displacement volumetry measurements. Furthermore, numerous newly formed lymphatic vessels were observed in treated mice on both histological and immunofluorescence analysis (Liu et al., 2008).

Finally, Tammela *et al*. found a significant role for adenovirally-delivered VEGF-C in improving outcomes of lymph node dissection and transplantation in mice. In their study, lymph node dissection and transplantation in combination with adenovirally-delivered VEGF-C induced the formation of functional collecting lymphatic vessels and the reconstitution of a functional immunological barrier (Tammela et al., 2007).

#### **3.2 Caveats to VEGF-C Therapies**

Despite the growing body of evidence in support of the efficacy of VEGF-C gene therapy, the exact mechanism by which VEGF-C-induced lymphangiogenesis facilitates resolution of lymphedema remains controversial. In an effort to elucidate the mechanisms by which VEGF-C induces lymphatic microvascular remodeling, Jin *et al*. examined the effects of anti-VEGFR-3 neutralizing antibodies in a mouse tail model of post-surgical lymphedema. This study demonstrated that VEGFR-3 plays a central mechanistic role in lymphedema remodeling. In the presence of the neutralizing antibody, lymphatic remodeling was greatly attenuated due to blockage of VEGF-C-induced signaling (Jin da et al., 2009).

In contrast, Uzarski *et al*. failed to observe inhibition of edema resolution across surgicallyinduced wounds in a mouse tail lymphedema model upon blockage of VEGFR-3. In their study, two mouse models were compared. In the first mouse model, scar-free lymphatic obstruction was simulated with dissection and removal of the superficial lymphatics from a mouse tail. Distal lymphedema was noted, and resolution was stimulated by VEGF-C. Edema resolution was not, however, inhibited by VEGFR-3 neutralizing antibodies, although lymphangiogenesis was reduced. In the second mouse model, scar-containing

Strategies in Modulating Lymphedema 17

Similarly, Zhou *et al*. treated rabbits with hind limb lymphedema using bone marrow stromal cells and/or VEGF-C. The rabbits that were treated with both stem cells and the growth factor exhibited a significant decrease in volume of edema in the limb as compared to rabbits treated with only one of the two agents. Vessel numbers increased in the dual treatment group, and VEGF-C expression was also higher in the dual therapy-treated animals. The authors concluded that the treatments enhanced the therapeutic effect of each other (Hu et al.). Therefore, stem cells may play a role in matrix remodeling and lymphangiogenesis, particularly in the setting of upregulated VEGF-C expression; however,

As our understanding of lymphatics has improved, additional therapies with effects on lymphangiogenesis have also been identified. One such therapy is extracorporeal shock wave therapy (ECT). ECT is used for the treatment of plantar fasciitis and tennis elbow. Repeated shock waves are localized to an area to produce neo-vascularization. This treatment effectively induces therapeutic angiogenesis and improves myocardial ischemia in pigs and humans as well as hind limb ischemia in rabbits via a mechanism involving upregulation of VEGF. Serizawa *et al*. created a rat tail model of lymphedema and subsequently subjected the animals to serial ECT therapy. Enhanced drainage of lymphatic fluid as well as upregulation of VEGF-C expression were found in the treatment group

Similarly, skin graft repairs to injuries in a mouse tail model have also been shown to stimulate lymphatic regeneration. In a recent study by Yan *et al*., ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics associated with VEGF-C upregulation was observed following skin grafting (Avraham et al.). In the future, a combination of such therapeutic strategies may be required for optimal management of

Clearly, VEGF-C plays an important role in the lymphatic system; however, it is also becoming increasingly clear that additional factors are involved in lymphedema. One such factor, hepatocyte growth factor (HGF), has been found to promote lymphatic vessel formation in mice. Furthermore, both HGF and its high affinity HGF receptor (MET) have recently been found to be expressed in lymphatic endothelial cells but not in blood endothelial cells. Finegold *et al*. examined these genes in women with secondary lymphedema following treatment for breast cancer and found that patients with both primary and secondary lymphedema had mutations in HGF/MET, suggesting that mutations in HGF/MET may be a significant risk factor for lymphedema. Thus, HGF may also become a potent therapeutic target for the treatment of lymphedema (Finegold et al.,

Another potential new target for lymphedema therapy is matrix metalloproteinase (MMP)- 9. Sustained swelling induced by lymphatic ligation leads to lymphatic hyperplasia and VEGF-C upregulation; however, mice lacking MMP-9 have a larger increase in tail volume with secondary lymphedema as compared to wild-type mice (Rutkowski et al., 2006).

further research is needed before concrete conclusions can be made.

**3.4 Alternative therapies** 

lymphedema.

2008).

compared to the controls (Serizawa et al.).

**3.5 Additional molecular targets** 

lymphatic obstruction was simulated with dissection and removal of the superficial lymphatics with cautery. Subsequent treatment with either VEGF-C or VEGFR-3 neutralizing antibodies resulted in no improvement in lymphedema. The authors concluded that interstitial flow dynamics and lymphedema may actually be more dependent on the extracellular matrix that reforms at the site of the injury than on lymphangiogenesis and that this effect may be impeded by the formation of scar tissue (Uzarski et al., 2008).

Following the finding that resolution of lymphedema may be more dependent on interstitial flow than on VEGFR-3 or VEGF-C, Ongstad *et al*. set out to clarify the role of VEGFR signaling during edema resolution and to probe the mechanism by which VEGF-C hastens resolution of edema. In their study, inhibition of VEGFR-3 or VEGFR-2 alone in mouse models did not significantly change the evolution of lymphedema relative to controls; however, inhibition of both VEGFR-2 and VEGFR-3 led to reduced tissue repair and reduced resolution of tail swelling at 40 and 50 days post surgery. Thus, tissue repair was crucial to the resolution of edema as this process provides a matrix bridge for fluid drainage. These authors then hypothesized that edema resolution in the mouse may be VEGFR signaling dependent, but lymphangiogenesis independent (Ongstad et al.).

Careful analysis by Jin *et al*. identified 120 mouse genes, many of which share homology with human genes, that are upregulated in the presence of lymphedema and normalized following therapeutic VEGF-C administration. Many of these genes were found to be involved in processes unrelated to lymphangiogenesis, suggesting an underlying, but incompletely understood, complexity to VEGF-C-induced lymphedema resolution. It is likely that numerous processes, including inflammation, immune response, wound healing, angiogenesis, oxidative stress response, and adipogenesis, play important roles in the pathogenesis and therapeutic resolution of the disease (Jin da et al., 2009). Additional research in this area is certainly warranted.

#### **3.3 VEGF-C and stem cell combined therapies**

Several recent studies further complicated our understanding of the role of VEGF-C and the optimal application of VEGF-C therapy in the treatment of lymphedema. In two papers, augmentation strategies using synthetic extracellular matrix material, such as gelatin and/or stem cells, co-administered with VEGF-C resulted in greater resolution of lymphedema compared to VEGF-C therapy alone. This further highlights the complexity of lymphedema and VEGF-C therapy.

Gelatin is a natural and abundant polymer used for tissue engineering. Gelatin-based hydrogels are biodegradable, non-immunogenic, and non-toxic and are able to mimic the properties of the extracellular matrix. This polymer can be used to distribute growth factors in a localized, sustained, controlled manner to obtain an effective dose response. Due to these properties and the success of VEGF-C treatment in lymphedema mouse models, Hwang *et al*. created a mouse hind limb model of lymphedema and applied a gelatin hydrogel system to the site of injury to obtain a controlled release of VEGF-C in combination with injection of human adipose-derived stem cells (hADSCs). Decreased dermal edema depth and increased lymphatic vessel density were observed at all time periods in the mice treated with both hydrogel and hADSCs as compared to mice treated with either hydrogel or hADSCs alone (Hwang et al.)

lymphatic obstruction was simulated with dissection and removal of the superficial lymphatics with cautery. Subsequent treatment with either VEGF-C or VEGFR-3 neutralizing antibodies resulted in no improvement in lymphedema. The authors concluded that interstitial flow dynamics and lymphedema may actually be more dependent on the extracellular matrix that reforms at the site of the injury than on lymphangiogenesis and that

Following the finding that resolution of lymphedema may be more dependent on interstitial flow than on VEGFR-3 or VEGF-C, Ongstad *et al*. set out to clarify the role of VEGFR signaling during edema resolution and to probe the mechanism by which VEGF-C hastens resolution of edema. In their study, inhibition of VEGFR-3 or VEGFR-2 alone in mouse models did not significantly change the evolution of lymphedema relative to controls; however, inhibition of both VEGFR-2 and VEGFR-3 led to reduced tissue repair and reduced resolution of tail swelling at 40 and 50 days post surgery. Thus, tissue repair was crucial to the resolution of edema as this process provides a matrix bridge for fluid drainage. These authors then hypothesized that edema resolution in the mouse may be VEGFR

Careful analysis by Jin *et al*. identified 120 mouse genes, many of which share homology with human genes, that are upregulated in the presence of lymphedema and normalized following therapeutic VEGF-C administration. Many of these genes were found to be involved in processes unrelated to lymphangiogenesis, suggesting an underlying, but incompletely understood, complexity to VEGF-C-induced lymphedema resolution. It is likely that numerous processes, including inflammation, immune response, wound healing, angiogenesis, oxidative stress response, and adipogenesis, play important roles in the pathogenesis and therapeutic resolution of the disease (Jin da et al., 2009). Additional

Several recent studies further complicated our understanding of the role of VEGF-C and the optimal application of VEGF-C therapy in the treatment of lymphedema. In two papers, augmentation strategies using synthetic extracellular matrix material, such as gelatin and/or stem cells, co-administered with VEGF-C resulted in greater resolution of lymphedema compared to VEGF-C therapy alone. This further highlights the complexity of lymphedema

Gelatin is a natural and abundant polymer used for tissue engineering. Gelatin-based hydrogels are biodegradable, non-immunogenic, and non-toxic and are able to mimic the properties of the extracellular matrix. This polymer can be used to distribute growth factors in a localized, sustained, controlled manner to obtain an effective dose response. Due to these properties and the success of VEGF-C treatment in lymphedema mouse models, Hwang *et al*. created a mouse hind limb model of lymphedema and applied a gelatin hydrogel system to the site of injury to obtain a controlled release of VEGF-C in combination with injection of human adipose-derived stem cells (hADSCs). Decreased dermal edema depth and increased lymphatic vessel density were observed at all time periods in the mice treated with both hydrogel and hADSCs as compared to mice treated with either hydrogel

this effect may be impeded by the formation of scar tissue (Uzarski et al., 2008).

signaling dependent, but lymphangiogenesis independent (Ongstad et al.).

research in this area is certainly warranted.

and VEGF-C therapy.

or hADSCs alone (Hwang et al.)

**3.3 VEGF-C and stem cell combined therapies** 

Similarly, Zhou *et al*. treated rabbits with hind limb lymphedema using bone marrow stromal cells and/or VEGF-C. The rabbits that were treated with both stem cells and the growth factor exhibited a significant decrease in volume of edema in the limb as compared to rabbits treated with only one of the two agents. Vessel numbers increased in the dual treatment group, and VEGF-C expression was also higher in the dual therapy-treated animals. The authors concluded that the treatments enhanced the therapeutic effect of each other (Hu et al.). Therefore, stem cells may play a role in matrix remodeling and lymphangiogenesis, particularly in the setting of upregulated VEGF-C expression; however, further research is needed before concrete conclusions can be made.

#### **3.4 Alternative therapies**

As our understanding of lymphatics has improved, additional therapies with effects on lymphangiogenesis have also been identified. One such therapy is extracorporeal shock wave therapy (ECT). ECT is used for the treatment of plantar fasciitis and tennis elbow. Repeated shock waves are localized to an area to produce neo-vascularization. This treatment effectively induces therapeutic angiogenesis and improves myocardial ischemia in pigs and humans as well as hind limb ischemia in rabbits via a mechanism involving upregulation of VEGF. Serizawa *et al*. created a rat tail model of lymphedema and subsequently subjected the animals to serial ECT therapy. Enhanced drainage of lymphatic fluid as well as upregulation of VEGF-C expression were found in the treatment group compared to the controls (Serizawa et al.).

Similarly, skin graft repairs to injuries in a mouse tail model have also been shown to stimulate lymphatic regeneration. In a recent study by Yan *et al*., ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics associated with VEGF-C upregulation was observed following skin grafting (Avraham et al.). In the future, a combination of such therapeutic strategies may be required for optimal management of lymphedema.

#### **3.5 Additional molecular targets**

Clearly, VEGF-C plays an important role in the lymphatic system; however, it is also becoming increasingly clear that additional factors are involved in lymphedema. One such factor, hepatocyte growth factor (HGF), has been found to promote lymphatic vessel formation in mice. Furthermore, both HGF and its high affinity HGF receptor (MET) have recently been found to be expressed in lymphatic endothelial cells but not in blood endothelial cells. Finegold *et al*. examined these genes in women with secondary lymphedema following treatment for breast cancer and found that patients with both primary and secondary lymphedema had mutations in HGF/MET, suggesting that mutations in HGF/MET may be a significant risk factor for lymphedema. Thus, HGF may also become a potent therapeutic target for the treatment of lymphedema (Finegold et al., 2008).

Another potential new target for lymphedema therapy is matrix metalloproteinase (MMP)- 9. Sustained swelling induced by lymphatic ligation leads to lymphatic hyperplasia and VEGF-C upregulation; however, mice lacking MMP-9 have a larger increase in tail volume with secondary lymphedema as compared to wild-type mice (Rutkowski et al., 2006).

Strategies in Modulating Lymphedema 19

In contrast, another study revealed that increased COX-2 expression was associated with recurrence of lymph flow in wound granulation tissues and with increased formation of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)-positive lymphatic-like structures. The authors suggest that these results differed from those of Nakamura *et al*. as a result of the important selectivity of the COX inhibitors (Kashiwagi et al., 2011). Thus, the role of inflammatory mediators, such as TNF-α and COX-2, in the treatment of lymphedema

Most animal models of lymphedema are mouse models due to the practicalities of establishing new treatment methods; however, utilizing mice carries significant limitations. The microlymphatics in the superficial dermis of mouse tails and the complex macrostructure of human lymphatics, which includes both larger collecting vessels and lymph nodes, have notable differences that preclude direct correlation. Extrapolation of the therapeutic successes achieved in mice to humans, therefore, is dangerous without further

To that end, Lahteenvuo *et al*. recently investigated the benefits of adenoviral vectorassisted VEGF-C gene therapy in the treatment of lymphedema in pigs. Lymphedema was induced in pigs by excising a 3 cm piece of the inguinal lymphatic vessels that drain distally and proximally from the inguinal lymph node. The pedicular lymph node was then reattached to the remaining tissue 4 cm laterally from its original position, thereby mimicking lymph node transfer in human patients. Adenoviral vectors encoding fulllength VEGF-C were then injected into the lymph node. Following injection, expression of VEGF-C was significantly increased. Furthermore, survival and functionality of the transferred lymph nodes was markedly improved in the injected animals as compared to the controls. Lymph node transfer has been used in humans with limited success (22- 31%), but in this model, the presence of VEGF-C resulted in better lymphatic vessel function, collecting vessel formation, and lymph node histology compared to controls

Similar studies have also been performed on other large animals such as sheep. Using such a model, Baker *et al*. tested the effect of lymphangiogenic growth factors delivered via slowrelease diffusion on the resolution of lymphedema after lymph node extraction. A single popliteal lymph node was extracted from the sheep to induce distal limb lymphedema. Hydrogel HAMC (a blend of hyaluronan and methylcellulose that facilitates slow protein diffusion) infused with VEGF-C and angiopoietin-2 was then injected into the excision site. The animals that received treatment displayed significantly reduced edema compared with

Though no formal testing of targeted therapies for lymphedema has been performed in humans to date, a few isolated case reports have been published. Sorafenib, a synthetic compound produced to block the enzyme RAF-kinase, was found to cause a dramatic reduction in chronic lymphedema in one human case study. As part of a clinical study, the patient took 400 mg of sorafenib twice daily, and the lymphedema was dramatically reduced within a few days of starting treatment. The effect was directly proportional to the dose and was not sustained when the drug was discontinued due to other side

remains both complex and controversial.

**4. Larger animal models** 

experimentation in larger animals.

(Lahteenvuo et al., 2011).

the untreated animals (Baker et al.).

G-protein-coupled receptors are expressed during lymphatic development and function, and thus, these molecules are also potential targets for pharmacological treatment. Genetically engineered mouse models deficient in specific G-protein-coupled receptors have been used to identify several specific G-proteins, such as the adrenomedullin receptor, that are important for lymphatic vascular development and function (Dunworth & Caron, 2009).

Radiation therapy, infections, or extensive surgical resection promote scarring and fibrosis and are, thus, also risk factors for lymphedema. Based on this finding, Avraham *et al*. examined the specific impact of fibrosis, defined as the excessive deposition of extracellular matrix products, on the abnormal regeneration of lymphatic vessels. Inhibition of fibrosis via treatment of the mouse tails with collagen type 1 gel and a moist dressing accelerated lymphatic regeneration and reduced post-surgical acute lymphedema in this animal model. Lymphatic endothelial cell proliferation was also enhanced and lymphatic function was improved. These results were independent of VEGF-C expression (Avraham et al., 2009).

After discovering that fibrosis impairs lymphatic regeneration and function (Avraham et al., 2009), Avraham *et al*. then searched for factors that modulate fibrosis in lymphedemous tissue. Transforming growth factor (TGF)-β is a well-known regulator of extracellular matrix synthesis. Inhibition of TGF-β causes both decreased fibrosis in virtually every organ system and increased lymphatic endothelial cell proliferation, migration, and tubule formation. To study the role of TGF-β, the investigators compared biopsies from lymphedemous limbs of patients to biopsies of the normal contralateral limb. The limbs with lymphedema exhibited a 3-fold increase in the number of TGF-β1-positive cells as compared to the normal limbs. Similarly, a mouse tail model of lymphedema was studied to determine the effect of anti-TGF-β1 treatment on lymphedema. Application of a TGF-β1 antibody (TGFmab) induced a 50-60% decrease in tail volume (a surrogate measurement for lymphedema) in treated mice compared to control mice. The treatment was well tolerated with no evidence of toxicity or wound healing complications. In the future, blockage of TGF-β may lead to further therapeutic options for augmentation of lymphatic regeneration. (Avraham et al., 2010).

Lymphedema has also been found to arise from destructive tissue injury independent of lymph stasis. Based on this observation, inflammatory mediators such as tumor necrosis factor (TNF)-α and cyclooxygenase (COX) have also come under recent scrutiny. TNF-α is prominently expressed in lymphedematous tissue in mouse models and is a known inducer of VEGF-C expression. In a mouse tail model of lymphedema, Nakamura *et al*. examined the anti-inflammatory and possible lymphangiogenic potential of a non-steroidal antiinflammatory drug (NSAID) and a modified soluble form of a TNF-α receptor R1 (sTNF-R1) on TNF-α expression. Subcutaneous injections of the NSAID ketoprofen, which reduces inflammation by inhibiting COX but increases TNF-α levels, resulted in marked improvement in inflammation, normalization of histological changes, and disappearance of dilated microlymphatics with associated up-regulation TNF-α expression. Treatment with the NSAID also led to upregulation of VEGF-C, VEGFR-3, and Prox1, all factors associated with lymphangiogenesis. Treatment of mice with sTNF-R1, which directly inactivates TNF-α and downregulates its expression, did not result in improvement in lymphedema, and epidermal thickness actually increased in treated mice compared to untreated mice. In these treated mice, both VEGF-C and VEGFR-3 expression decreased as well. Though more evidence is needed, TNF-α activity and its downstream effects on VEGF-C may actually be a protective response to injury-induced lymphedema (Nakamura et al., 2009).

G-protein-coupled receptors are expressed during lymphatic development and function, and thus, these molecules are also potential targets for pharmacological treatment. Genetically engineered mouse models deficient in specific G-protein-coupled receptors have been used to identify several specific G-proteins, such as the adrenomedullin receptor, that are important for lymphatic vascular development and function (Dunworth & Caron, 2009). Radiation therapy, infections, or extensive surgical resection promote scarring and fibrosis and are, thus, also risk factors for lymphedema. Based on this finding, Avraham *et al*. examined the specific impact of fibrosis, defined as the excessive deposition of extracellular matrix products, on the abnormal regeneration of lymphatic vessels. Inhibition of fibrosis via treatment of the mouse tails with collagen type 1 gel and a moist dressing accelerated lymphatic regeneration and reduced post-surgical acute lymphedema in this animal model. Lymphatic endothelial cell proliferation was also enhanced and lymphatic function was improved. These results were independent of VEGF-C expression (Avraham et al., 2009).

After discovering that fibrosis impairs lymphatic regeneration and function (Avraham et al., 2009), Avraham *et al*. then searched for factors that modulate fibrosis in lymphedemous tissue. Transforming growth factor (TGF)-β is a well-known regulator of extracellular matrix synthesis. Inhibition of TGF-β causes both decreased fibrosis in virtually every organ system and increased lymphatic endothelial cell proliferation, migration, and tubule formation. To study the role of TGF-β, the investigators compared biopsies from lymphedemous limbs of patients to biopsies of the normal contralateral limb. The limbs with lymphedema exhibited a 3-fold increase in the number of TGF-β1-positive cells as compared to the normal limbs. Similarly, a mouse tail model of lymphedema was studied to determine the effect of anti-TGF-β1 treatment on lymphedema. Application of a TGF-β1 antibody (TGFmab) induced a 50-60% decrease in tail volume (a surrogate measurement for lymphedema) in treated mice compared to control mice. The treatment was well tolerated with no evidence of toxicity or wound healing complications. In the future, blockage of TGF-β may lead to further therapeutic options for augmentation of lymphatic regeneration. (Avraham et al., 2010).

Lymphedema has also been found to arise from destructive tissue injury independent of lymph stasis. Based on this observation, inflammatory mediators such as tumor necrosis factor (TNF)-α and cyclooxygenase (COX) have also come under recent scrutiny. TNF-α is prominently expressed in lymphedematous tissue in mouse models and is a known inducer of VEGF-C expression. In a mouse tail model of lymphedema, Nakamura *et al*. examined the anti-inflammatory and possible lymphangiogenic potential of a non-steroidal antiinflammatory drug (NSAID) and a modified soluble form of a TNF-α receptor R1 (sTNF-R1) on TNF-α expression. Subcutaneous injections of the NSAID ketoprofen, which reduces inflammation by inhibiting COX but increases TNF-α levels, resulted in marked improvement in inflammation, normalization of histological changes, and disappearance of dilated microlymphatics with associated up-regulation TNF-α expression. Treatment with the NSAID also led to upregulation of VEGF-C, VEGFR-3, and Prox1, all factors associated with lymphangiogenesis. Treatment of mice with sTNF-R1, which directly inactivates TNF-α and downregulates its expression, did not result in improvement in lymphedema, and epidermal thickness actually increased in treated mice compared to untreated mice. In these treated mice, both VEGF-C and VEGFR-3 expression decreased as well. Though more evidence is needed, TNF-α activity and its downstream effects on VEGF-C may actually be a protective response to

injury-induced lymphedema (Nakamura et al., 2009).

In contrast, another study revealed that increased COX-2 expression was associated with recurrence of lymph flow in wound granulation tissues and with increased formation of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)-positive lymphatic-like structures. The authors suggest that these results differed from those of Nakamura *et al*. as a result of the important selectivity of the COX inhibitors (Kashiwagi et al., 2011). Thus, the role of inflammatory mediators, such as TNF-α and COX-2, in the treatment of lymphedema remains both complex and controversial.

#### **4. Larger animal models**

Most animal models of lymphedema are mouse models due to the practicalities of establishing new treatment methods; however, utilizing mice carries significant limitations. The microlymphatics in the superficial dermis of mouse tails and the complex macrostructure of human lymphatics, which includes both larger collecting vessels and lymph nodes, have notable differences that preclude direct correlation. Extrapolation of the therapeutic successes achieved in mice to humans, therefore, is dangerous without further experimentation in larger animals.

To that end, Lahteenvuo *et al*. recently investigated the benefits of adenoviral vectorassisted VEGF-C gene therapy in the treatment of lymphedema in pigs. Lymphedema was induced in pigs by excising a 3 cm piece of the inguinal lymphatic vessels that drain distally and proximally from the inguinal lymph node. The pedicular lymph node was then reattached to the remaining tissue 4 cm laterally from its original position, thereby mimicking lymph node transfer in human patients. Adenoviral vectors encoding fulllength VEGF-C were then injected into the lymph node. Following injection, expression of VEGF-C was significantly increased. Furthermore, survival and functionality of the transferred lymph nodes was markedly improved in the injected animals as compared to the controls. Lymph node transfer has been used in humans with limited success (22- 31%), but in this model, the presence of VEGF-C resulted in better lymphatic vessel function, collecting vessel formation, and lymph node histology compared to controls (Lahteenvuo et al., 2011).

Similar studies have also been performed on other large animals such as sheep. Using such a model, Baker *et al*. tested the effect of lymphangiogenic growth factors delivered via slowrelease diffusion on the resolution of lymphedema after lymph node extraction. A single popliteal lymph node was extracted from the sheep to induce distal limb lymphedema. Hydrogel HAMC (a blend of hyaluronan and methylcellulose that facilitates slow protein diffusion) infused with VEGF-C and angiopoietin-2 was then injected into the excision site. The animals that received treatment displayed significantly reduced edema compared with the untreated animals (Baker et al.).

Though no formal testing of targeted therapies for lymphedema has been performed in humans to date, a few isolated case reports have been published. Sorafenib, a synthetic compound produced to block the enzyme RAF-kinase, was found to cause a dramatic reduction in chronic lymphedema in one human case study. As part of a clinical study, the patient took 400 mg of sorafenib twice daily, and the lymphedema was dramatically reduced within a few days of starting treatment. The effect was directly proportional to the dose and was not sustained when the drug was discontinued due to other side

Strategies in Modulating Lymphedema 21

Avraham, T., Clavin, N.W., Daluvoy, S.V., Fernandez, J., Soares, M.A., Cordeiro, A.P., &

Avraham, T., Daluvoy, S., Zampell, J., Yan, A., Haviv, Y.S., Rockson, S.G., & Mehrara, B.J.

Badger, C., Preston, N., Seers, K., & Mortimer, P. (2004). Benzo-pyrones for reducing and

Baker, A., Kim, H., Semple, J.L., Dumont, D., Shoichet, M., Tobbia, D., & Johnston, M. (2010).

Cheung, L., Han, J., Beilhack, A., Joshi, S., Wilburn, P., Dua, A., An, A., & Rockson, S.G.

Dunworth, W.P., & Caron, K.M. (2009). G protein-coupled receptors as potential drug

Finegold, D.N., Schacht, V., Kimak, M.A., Lawrence, E.C., Foeldi, E., Karlsson, J.M., Baty,

mouse embryos. *Molecular and Cellular Biology*, Vol.28, No.15, pp. 4843-4850. Hu, X.Q., Jiang, Z.H., & Liu, N.F. (2008). Experimental studies of VEGF-C gene for the

Hwang, J.H., Kim, I.G., Lee, J.Y., Piao, S., Lee, D.S., Lee, T.S., & Ra, J.C. (2011). Therapeutic

Jin da, P., An, A., Liu, J., Nakamura, K., & Rockson, S.G. (2009). Therapeutic responses to

Kashiwagi, S., Hosono, K., Suzuki, T., Takeda, A., Uchinuma, E., & Majima, M. (2011). Role

lymphedema. *Laboratory Investigation*, Vol.91, No.9, pp. 1314-1325.

lymphedema. *Lymphatic Research and Biology*, Vol.*6*, No.2, pp. 65-68. Haiko, P., Makinen, T., Keskitalo, S., Taipale, J., Karkkainen, M.J., Baldwin, M.E., Stacker,

therapeutic lymphangiogenesis. *BioDrugs,* Vol.20, No.6, pp. 363-370. Dennert, G., & Horneber, M. (2006). Selenium for alleviating the side effects of

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*of Plastic Surgery*, Vol.24, No.3, pp. 207-211.

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Mehrara, B.J. (2009). Fibrosis is a key inhibitor of lymphatic regeneration. *Plastic and* 

(2010). Blockade of transforming growth factor-beta1 accelerates lymphatic regeneration during wound repair. *The American Journal of Pathology,* Vol.177, No.6,

controlling lymphoedema of the limbs. *Cochrane Database Syst Rev,* Vol.2,

Experimental assessment of pro-lymphangiogenic growth factors in the treatment of post-surgical lymphedema following lymphadenectomy. *Breast Cancer Research*,

(2006). An experimental model for the study of lymphedema and its response to

chemotherapy, radiotherapy and surgery in cancer patients. *Cochrane Database Syst* 

targets for lymphangiogenesis and lymphatic vascular diseases. *Arteriosclerosis,* 

C.J., & Ferrell, R.E. (2008). HGF and MET mutations in primary and secondary

S.A., Achen, M.G., & Alitalo, K. (2008). Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in

treatment of chronic obstructive lymphedema in mouse tail model. *Chinese Journal* 

lymphangiogenesis using stem cell and VEGF-C hydrogel. *Biomaterials*, Vol.32,

exogenous VEGF-C administration in experimental lymphedema: immunohistochemical and molecular characterization. *Lymphatic Research and* 

of COX-2 in lymphangiogenesis and restoration of lymphatic flow in secondary

**7. References** 

pp. 3202-3214.

Vol.12, No.5, R70.

*Rev,* Vol.3, CD005037.

No.19, pp. 4415-4423.

*Biology*, Vol.7, No.1, pp. 47-57.

CD003140.

effects. The authors hypothesized that the reduction of lymphedema was due to VEGFR-2 blockade, which reduced vascular permeability but did not affect the VEGFR-3 pathway involved in the proliferation of lymphatic endothelial cells (Moncrieff et al., 2008).

#### **5. Limitations**

Though research into targeted therapies for lymphedema is progressing rapidly, significant work is still required. As stated above, there are a number of limitations with the animal models that are currently available. First, the models are based on acute lymphatic damage and not on chronic lymphedema. Lymphedema in humans is slowly progressive and does not recover naturally, whereas the models that are studied progress quickly and often regress naturally. Rats, for example, heal very quickly, and tail lymphedema heals itself. In fact, one of the problems in studying lymphedema treatment strategies is the difficulty in developing a method to sustain lymphedema long enough to study the outcomes of the therapies (Yoon et al., 2003). In addition, the hydrostatic conditions of humans differ from those of small animals, such as mice, rats, and rabbits, that are usually used to study lymphedema. The absolute lymphatic area damaged in humans is greater than in these small animals, and the regenerating lymphatic vessels must span a longer distance. Models using pigs, which are closer in size to humans, will likely be useful in addressing some of these issues. Another major concern is that VEGF-C and VEGFR-3 are known to promote metastasis. Since a large portion of lymphedema patients are breast cancer survivors, an understanding of the metastatic risks associated with VEGF-C treatment of lymphedema is of paramount importance. However, these potentially damaging side effects of VEGF-C treatment are difficult to study in animal models.

#### **6. Conclusion**

Lymphangiogenesis is a complex process that involves the interplay of many molecules with redundant mechanisms. VEGF-C and VEGFR-3 are known to be the primary players, but these molecules work in sync with other factors. Currently, no specific molecular treatment options are available for clinical use; however, promising results from animal trials suggest a role for VEGF-C gene therapy in treatment of lymphedema. Other results indicate that other factors such as COX-2, MMP-9, and interstitial flow dynamics may also be important in future management of lymphedema. Combination therapies such as stem cell implantation, skin grafting, and lymph node transfer in conjunction with VEGF-C therapy may further expand the effectiveness of future therapies as well. Design of a drug to treat lymphedema will require an effective animal model that accurately mimics lymphedema in humans. And further evaluation of the metastatic risk of inducing lymphangiogenesis in cancer patients is also needed. However, dramatic progress has been made towards effective targeted molecular therapies for lymphedema. Due to the proliferative efforts of researchers over the last decade, effective treatments for lymphedema in humans may soon be a reality. However, though a solid body of evidence now exists in support of targeted therapies for lymphedema, significant research is ultimately still needed.

#### **7. References**

20 Novel Strategies in Lymphedema

effects. The authors hypothesized that the reduction of lymphedema was due to VEGFR-2 blockade, which reduced vascular permeability but did not affect the VEGFR-3 pathway involved in the proliferation of lymphatic endothelial cells (Moncrieff et al.,

Though research into targeted therapies for lymphedema is progressing rapidly, significant work is still required. As stated above, there are a number of limitations with the animal models that are currently available. First, the models are based on acute lymphatic damage and not on chronic lymphedema. Lymphedema in humans is slowly progressive and does not recover naturally, whereas the models that are studied progress quickly and often regress naturally. Rats, for example, heal very quickly, and tail lymphedema heals itself. In fact, one of the problems in studying lymphedema treatment strategies is the difficulty in developing a method to sustain lymphedema long enough to study the outcomes of the therapies (Yoon et al., 2003). In addition, the hydrostatic conditions of humans differ from those of small animals, such as mice, rats, and rabbits, that are usually used to study lymphedema. The absolute lymphatic area damaged in humans is greater than in these small animals, and the regenerating lymphatic vessels must span a longer distance. Models using pigs, which are closer in size to humans, will likely be useful in addressing some of these issues. Another major concern is that VEGF-C and VEGFR-3 are known to promote metastasis. Since a large portion of lymphedema patients are breast cancer survivors, an understanding of the metastatic risks associated with VEGF-C treatment of lymphedema is of paramount importance. However, these potentially damaging side effects of VEGF-C treatment are difficult to study in animal

Lymphangiogenesis is a complex process that involves the interplay of many molecules with redundant mechanisms. VEGF-C and VEGFR-3 are known to be the primary players, but these molecules work in sync with other factors. Currently, no specific molecular treatment options are available for clinical use; however, promising results from animal trials suggest a role for VEGF-C gene therapy in treatment of lymphedema. Other results indicate that other factors such as COX-2, MMP-9, and interstitial flow dynamics may also be important in future management of lymphedema. Combination therapies such as stem cell implantation, skin grafting, and lymph node transfer in conjunction with VEGF-C therapy may further expand the effectiveness of future therapies as well. Design of a drug to treat lymphedema will require an effective animal model that accurately mimics lymphedema in humans. And further evaluation of the metastatic risk of inducing lymphangiogenesis in cancer patients is also needed. However, dramatic progress has been made towards effective targeted molecular therapies for lymphedema. Due to the proliferative efforts of researchers over the last decade, effective treatments for lymphedema in humans may soon be a reality. However, though a solid body of evidence now exists in support of targeted therapies for lymphedema, significant research is

2008).

models.

**6. Conclusion** 

ultimately still needed.

**5. Limitations** 


**2** 

*Sweden* 

Fredrik Berglund

**Titanium and Yellow Nail Syndrome** 

Yellow nail syndrome was defined in 1964 as a combination of slow growing, discoloured nails and edema (Samman & White, 1964). The rate of growth of finger nails was less than 0.2 mm per week compared with the normal 0.5-1.2 mm per week. The nails were thickened and the cuticles were deficient. The color varied from pale yellow to slightly greenish. Onycholysis (separation of the nail from its bed) and shedding of one or more nails was mentioned. - Of the 13 patients with yellow nails 10 presented with edema, mainly in the

Subsequently, several symptoms were described and included. *Sinusitis* was reported by Wells (Wells,1966), although clearly he did not recognize it as part of the syndrome. Chronic intermittent *cough* with sputum production was reported by Zerfas (Zerfas, 1966), and in combination with *sinusitis* and *bronchitis* by Dilley et al (Dilley et al.,1968). Hiller et al. (1972)

*Pleural effusion* in combination with lymphedema was reported in three patients by Emerson (Emerson,1966). The pleural fluid was clear, with a protein content of 4-9 mg/100 ml. The predominant cell was the lymphocyte. The effusion always re-accumulated after drainage. *Pericardial effusion* in addition to pleural effusions and lymphedema was reported by

In 78 papers listed in PubMed from 1964 to 2009 there were 185 patients diagnosed with yellow nail syndrome: 18 % with yellow nails only, 42 % together with lymphedema, 21 % together with pleural effusion, and 19 % with the complete triad yellow nails-lymphedemapleural effusion. Thus lymphedema was diagnosed totally in 61 %. Cough and sinusitis

Among several authors reporting sinusitis, some mention radiography revealing mucoid thickening of one or more sinuses (Nakielna et al, 1976, Hassard et al,1984, Camilleri, 1990, Varney et al, 1994, Cebecci et al, 2009). Varney et al. — at a Nose Clinic in London reported that 14 out of 17 patients with yellow nail syndrome suffered severe rhinosinusitis, which predated nail changes in four, coincided with yellow nails in six, and occurred later

emphasized that chronic cough was a persistent finding in all their patients.

**1. Introduction** 

Wakasa et al. (1987).

were diagnosed in 32 %.

in the remaining seven patients.

**1.1 Symptom frequency in published papers** 

ankles.

*Swedish Society of Dental Amalgam Patients, Trollhättan,* 


## **Titanium and Yellow Nail Syndrome**

### Fredrik Berglund

*Swedish Society of Dental Amalgam Patients, Trollhättan, Sweden* 

#### **1. Introduction**

22 Novel Strategies in Lymphedema

Kligman, L., Wong, R.K., Johnston, M., & Laetsch, N.S. (2004). The treatment of

Lahteenvuo, M., Honkonen, K., Tervala, T., Tammela, T., Suominen, E., Lahteenvuo, J.,

Liu, Y., Fang, Y., Dong, P., Gao, J., Liu, R., Tian, H., Ding, Z., Bi, Y., & Liu, Z. (2008). Effect of

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Rutkowski, J.M., Moya, M., Johannes, J., Goldman, J., & Swartz, M.A. (2006). Secondary

Tammela, T., Saaristo, A., Holopainen, T., Lyytikka, J., Kotronen, A., Pitkonen, M., Abo-

Tammela, T., Zarkada, G., Nurmi, H., Jakobsson, L., Heinolainen, K., Tvorogov, D., Zheng,

Uzarski, J., Drelles, M.B., Gibbs, S.E., Ongstad, E.L., Goral, J.C., McKeown, K.K., Raehl,

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lymphedema related to breast cancer: a systematic review and evidence summary.

Kholova, I., Alitalo, K., Yla-Herttuala, S., & Saaristo, A. (2011). Growth factor therapy and autologous lymph node transfer in lymphedema. *Circulation*, Vol.123,

vascular endothelial growth factor C (VEGF-C) gene transfer in rat model of

of chronic lymphoedema of the lower limb with sorafenib therapy. *Melanoma* 

pharmacotherapy with ketoprofen ameliorates experimental lymphatic vascular

in lymphatic dysfunction and disease. *Lymphatic Research and Biology*, Vol.6, No.3-4,

V.M., Roberts, M.A., & Goldman, J. (2010). Lymphangiogenesis-independent resolution of experimental edema. *American Journal of Physiology Heart and* 

lymphedema in the mouse tail: Lymphatic hyperplasia, VEGF-C upregulation, and the protective role of MMP-9. *Microvascular Research*, Vol.72, No.3, pp. 161-171. Serizawa, F., Ito, K., Matsubara, M., Sato, A., Shimokawa, H., & Satomi, S. (2011).

Extracorporeal shock wave therapy induces therapeutic lymphangiogenesis in a rat model of secondary lymphoedema. *European Journal of Vascular and Endovascular* 

Ramadan, U., Yla-Herttuala, S., Petrova, T.V., & Alitalo, K. (2007). Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection

W., Franco, C.A., Murtomaki, A., Aranda, E.*, et al.* (2011). VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling. *Nature Cell* 

A.M., Roberts, M.A., Pytowski, B., Smith, M.R.*, et al.* (2008). The resolution of lymphedema by interstitial flow in the mouse tail skin. *American Journal of* 

Kirchmair, R., Hu, C.S., Kearney, M.*, et al.* (2003). VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema. *Journal of*  Yellow nail syndrome was defined in 1964 as a combination of slow growing, discoloured nails and edema (Samman & White, 1964). The rate of growth of finger nails was less than 0.2 mm per week compared with the normal 0.5-1.2 mm per week. The nails were thickened and the cuticles were deficient. The color varied from pale yellow to slightly greenish. Onycholysis (separation of the nail from its bed) and shedding of one or more nails was mentioned. - Of the 13 patients with yellow nails 10 presented with edema, mainly in the ankles.

Subsequently, several symptoms were described and included. *Sinusitis* was reported by Wells (Wells,1966), although clearly he did not recognize it as part of the syndrome. Chronic intermittent *cough* with sputum production was reported by Zerfas (Zerfas, 1966), and in combination with *sinusitis* and *bronchitis* by Dilley et al (Dilley et al.,1968). Hiller et al. (1972) emphasized that chronic cough was a persistent finding in all their patients.

*Pleural effusion* in combination with lymphedema was reported in three patients by Emerson (Emerson,1966). The pleural fluid was clear, with a protein content of 4-9 mg/100 ml. The predominant cell was the lymphocyte. The effusion always re-accumulated after drainage.

*Pericardial effusion* in addition to pleural effusions and lymphedema was reported by Wakasa et al. (1987).

#### **1.1 Symptom frequency in published papers**

In 78 papers listed in PubMed from 1964 to 2009 there were 185 patients diagnosed with yellow nail syndrome: 18 % with yellow nails only, 42 % together with lymphedema, 21 % together with pleural effusion, and 19 % with the complete triad yellow nails-lymphedemapleural effusion. Thus lymphedema was diagnosed totally in 61 %. Cough and sinusitis were diagnosed in 32 %.

Among several authors reporting sinusitis, some mention radiography revealing mucoid thickening of one or more sinuses (Nakielna et al, 1976, Hassard et al,1984, Camilleri, 1990, Varney et al, 1994, Cebecci et al, 2009). Varney et al. — at a Nose Clinic in London reported that 14 out of 17 patients with yellow nail syndrome suffered severe rhinosinusitis, which predated nail changes in four, coincided with yellow nails in six, and occurred later in the remaining seven patients.

Titanium and Yellow Nail Syndrome 25

sensation in the ear while holding a coca-cola can for a while in his hand). Ions of different sorts can be introduced through the skin by means of electric current, so-called

**Reaction Potential (Volts)** 

My first patient got a titanium and a cobalt-chromium implant in her right knee. Half a year later she developed a persistent cough. Later her nails became thick and yellow, and a few nails were shed. She had gold in many of her teeth. Analysis for metals in nail clippings revealed high levels of titanium (Berglund & Carlmark, 2011). Three years later she had normal nails and no cough, but her knee implant had loosened. She got a new implant, and

The dominant cause of yellow nail syndrome in my patients was the galvanic interaction between titanium implants and gold. There were 23 females and 4 males, aged 15-86 years at onset of symptoms. Most titanium implants were in the teeth (pins or crowns) or in the jaw bones, but also in the knees and hips, or in the abdomen (clips and staples) after laparoscopic surgery. In one patient there was iontophoresis of titanium ions from titanium

The gold electrodes were present mostly as dental inlays and crowns, but in two patients as wedding or engagement rings. Some patients reported intolerance to gold jewelry. Patch tests with gold cannot be done in titanium implant patients. In three patients amalgam, which contains silver and mercury, formed the electric circuit with titanium. I always advise patients with amalgam or titanium not to wear metal jewelry. In three patients the local application of fluoride gel caused release of titanium from titanium inlays in the teeth. In vitro, the oxidative release of titanium ions from titanium increases sharply in the presence of fluoride (Reclaru & Meyer, 1998, Strietzel et al., 1998, Schiff et

Most patients with titanium implants suspected metal involvement and contacted me through the Swedish Society of Dental Amalgam Patients. However, in published papers

Titanium dioxide, TiO2, is insoluble in water and acid but soluble in alkali. Because of its brightness and high refractive index it is the most widely used white pigment. Titanium dioxide is widely used in the food and drug industry as a whitening agent and is given the

Ti+2 + 2e<sup>−</sup> ⟺ Ti −1.63 TiO2 + 4H+ + 4e<sup>−</sup> ⟺ Ti + 2H2O −0.86 Ag+ + e<sup>−</sup> ⟺ Ag +0.80 Hg+2 + 2e<sup>−</sup> ⟺ Hg +0.85 Au+ + e<sup>−</sup> ⟺ Au +1.69 F2 + 2e<sup>−</sup> ⟺ 2F− +2.87

iontophoresis.

**2.3 Titanium implant patients** 

spectacles to gold in the teeth.

al., 2002).

a year later she had her cough and yellow nails again.

most patients were probably exposed to titanium dioxide.

**2.4 Titanium dioxide: Uses and intestinal uptake** 

European food additive number E171.

Among my patients, sinusitis and cough were the most common symptoms (Berglund & Carlmark, 2011). Several patients spontaneously mention postnasal drip and a "strange cough" as a main feature, often starting half a year after a titanium implant or start of medication with drugs containing titanium dioxide.

#### **2. Etiology: Titanium and titanium dioxide**

Since 1997 I have seen 35 patients with one or more of the symptoms or signs mentioned above. Twenty-seven patients had titanium implants, whereas eight were exposed to titanium dioxide.

Titanium in nail clippings or shed nails from the patients was analyzed by energy dispersive x-ray fluorescence (Forsell et al., 1997). It was present in the nails in concentrations varying between 1 and 170 µg/g, with a median level of 5 µg/g (Berglund & Carlmark, 2011). Shed nails had high levels (46, 41, 22 and 6 µg/g), but some nail clippings had even higher levels (170, 120 and 111 µg/g). Titanium levels do not seem to correlate with yellowness or thickness of nails. Titanium was not found in nails from healthy subjects, not even if exposed to titanium + gold (one subject) or to drugs with titanium dioxide.

In two patients titanium was analyzed in separate nail clippings of the left hand. The thumbnail had higher level (5.6 and 4.1 µg/g) than the other nails (2.8 – 3.2 and 3.1 - 4.1 µg/g, respectively). In two patients the levels in clippings from thumb and big toe were identical (1.7 µg/g) or nearly identical (1.7 versus 1.6 µg/g). In one patient the titanium level was 48 µg/g in a shed nail, but only 6.7 µg/g in fresh clippings. Nowadays I ask for clippings from the left thumbnail, except when shed nails are available.

#### **2.1 Titanium metal**

Titanium (Ti): atomic weight 47.9, atomic number 22, specific gravity 4.54. When pure, titanium is a lustrous, white metal. It is 60 % heavier than aluminum, but twice as strong. Titanium is a much harder metal than aluminum and approaches the high hardness possessed by some of the heat-treated steels, which causes some difficulties in dentistry. Its modulus of rigidity falls between that of aluminum and that of steel. The high ductility enables the use of titanium in cochlear implant electrodes. Since it is nonferromagnetic, patients with titanium implants can be safely examined with magnetic resonance imaging.

Titanium is quite sensitive to galvanic corrosion, e.g. by other metals and fluorine. This is to be expected when comparing their reduction potentials.

#### **2.2 Reduction potentials (Hunsburger, 1976)**

Our body shows high electrical conductivity. This enables our recording of the electrocardiogram through electrodes applied to the skin. The higher resistance in the skin is abolished by perspiration (hypotonic saline). The presence of metals with different reduction potentials may give rise to galvanic phenomena within the body or across the skin. (A young man with a gold ring in one ear experienced a buzzing

Among my patients, sinusitis and cough were the most common symptoms (Berglund & Carlmark, 2011). Several patients spontaneously mention postnasal drip and a "strange cough" as a main feature, often starting half a year after a titanium implant or start of

Since 1997 I have seen 35 patients with one or more of the symptoms or signs mentioned above. Twenty-seven patients had titanium implants, whereas eight were exposed to

Titanium in nail clippings or shed nails from the patients was analyzed by energy dispersive x-ray fluorescence (Forsell et al., 1997). It was present in the nails in concentrations varying between 1 and 170 µg/g, with a median level of 5 µg/g (Berglund & Carlmark, 2011). Shed nails had high levels (46, 41, 22 and 6 µg/g), but some nail clippings had even higher levels (170, 120 and 111 µg/g). Titanium levels do not seem to correlate with yellowness or thickness of nails. Titanium was not found in nails from healthy subjects, not even if exposed to titanium + gold (one subject) or to drugs with

In two patients titanium was analyzed in separate nail clippings of the left hand. The thumbnail had higher level (5.6 and 4.1 µg/g) than the other nails (2.8 – 3.2 and 3.1 - 4.1 µg/g, respectively). In two patients the levels in clippings from thumb and big toe were identical (1.7 µg/g) or nearly identical (1.7 versus 1.6 µg/g). In one patient the titanium level was 48 µg/g in a shed nail, but only 6.7 µg/g in fresh clippings. Nowadays I ask for

Titanium (Ti): atomic weight 47.9, atomic number 22, specific gravity 4.54. When pure, titanium is a lustrous, white metal. It is 60 % heavier than aluminum, but twice as strong. Titanium is a much harder metal than aluminum and approaches the high hardness possessed by some of the heat-treated steels, which causes some difficulties in dentistry. Its modulus of rigidity falls between that of aluminum and that of steel. The high ductility enables the use of titanium in cochlear implant electrodes. Since it is nonferromagnetic, patients with titanium implants can be safely examined with magnetic

Titanium is quite sensitive to galvanic corrosion, e.g. by other metals and fluorine. This is to

Our body shows high electrical conductivity. This enables our recording of the electrocardiogram through electrodes applied to the skin. The higher resistance in the skin is abolished by perspiration (hypotonic saline). The presence of metals with different reduction potentials may give rise to galvanic phenomena within the body or across the skin. (A young man with a gold ring in one ear experienced a buzzing

clippings from the left thumbnail, except when shed nails are available.

be expected when comparing their reduction potentials.

**2.2 Reduction potentials (Hunsburger, 1976)** 

medication with drugs containing titanium dioxide.

**2. Etiology: Titanium and titanium dioxide** 

titanium dioxide.

titanium dioxide.

**2.1 Titanium metal** 

resonance imaging.

sensation in the ear while holding a coca-cola can for a while in his hand). Ions of different sorts can be introduced through the skin by means of electric current, so-called iontophoresis.


#### **2.3 Titanium implant patients**

My first patient got a titanium and a cobalt-chromium implant in her right knee. Half a year later she developed a persistent cough. Later her nails became thick and yellow, and a few nails were shed. She had gold in many of her teeth. Analysis for metals in nail clippings revealed high levels of titanium (Berglund & Carlmark, 2011). Three years later she had normal nails and no cough, but her knee implant had loosened. She got a new implant, and a year later she had her cough and yellow nails again.

The dominant cause of yellow nail syndrome in my patients was the galvanic interaction between titanium implants and gold. There were 23 females and 4 males, aged 15-86 years at onset of symptoms. Most titanium implants were in the teeth (pins or crowns) or in the jaw bones, but also in the knees and hips, or in the abdomen (clips and staples) after laparoscopic surgery. In one patient there was iontophoresis of titanium ions from titanium spectacles to gold in the teeth.

The gold electrodes were present mostly as dental inlays and crowns, but in two patients as wedding or engagement rings. Some patients reported intolerance to gold jewelry. Patch tests with gold cannot be done in titanium implant patients. In three patients amalgam, which contains silver and mercury, formed the electric circuit with titanium. I always advise patients with amalgam or titanium not to wear metal jewelry. In three patients the local application of fluoride gel caused release of titanium from titanium inlays in the teeth. In vitro, the oxidative release of titanium ions from titanium increases sharply in the presence of fluoride (Reclaru & Meyer, 1998, Strietzel et al., 1998, Schiff et al., 2002).

Most patients with titanium implants suspected metal involvement and contacted me through the Swedish Society of Dental Amalgam Patients. However, in published papers most patients were probably exposed to titanium dioxide.

#### **2.4 Titanium dioxide: Uses and intestinal uptake**

Titanium dioxide, TiO2, is insoluble in water and acid but soluble in alkali. Because of its brightness and high refractive index it is the most widely used white pigment. Titanium dioxide is widely used in the food and drug industry as a whitening agent and is given the European food additive number E171.

Titanium and Yellow Nail Syndrome 27

increased lymph formation, e.g. after some infection. Protein-losing enteropathy in patients with pleural effusions, in some cases together with ascites, has also been reported (Duhra et al., 1985, Battaglia et al., 1985, Malek et al., 1996). In a patient with edema, plural effusion, hypoalbuminaemia and yellow nails, D'Allessandro et al. (2001) found a 10-fold increase in normal albumin enteric loss. Since in most cases of yellow nail syndrome the pleural fluid has a very high protein content, this reflects increased protein permeability of systemic capillaries. D'Allessandro et al claimed that "the theory of pure lymphatic block is not sufficient to explain all the clinical manifestations of yellow nail syndrome", and suggested that microangiopathy and increased microvascular filtration at different sites (pleura, liver, limbs, intestine) due to an alteration in the interstitial matrix, could play a role in addition to

These reports indicate that increased vascular permeability rather than lymphatic

There are two reports on pregnancy in women with yellow nails and bronchitis or productive cough (Govaert et al., 1992, Slee et al., 2000). Ultrasonography demonstrated polyhydramnios and bilateral pleural effusion in the fetuses at 23 and 29 weeks of gestation, respectively. At delivery both infants were hydropic and had bilateral pleural effusions, which were treated by thoracic drainage. Following initial recovery, pleural effusion recurred 2 days after starting enteral feeding at 4 weeks of age in the first infant, but not in the second. Our interpretation is that both mothers were exposed to titanium or titanium dioxide, and that titanium was transferred across the placenta, and possibly also via

There are a few reports of familiar occurrence of yellow nail syndrome (Lambert et al., 2006). They might be explained by several family members consuming drugs or chewing gum

Yellow discoloration of the nails is often a late sign of the syndrome and therefore not necessary for the diagnosis of yellow nail syndrome; sinusitis and cough, lymphedema and pleural effusion occurring alone or in combination may represent the same syndrome (Varney et al., 1994, Cebecci et al., 2009). The diagnosis is supported by history or evidence of exposure to titanium implants or to titanium dioxide, and can be confirmed (if necessary)

Most often it is impossible or extremely difficult to remove titanium implants. It is usually much easier to remove the gold (in teeth, jewelry or rings). In a few patients gold removal (Berglund & Carlmark, 2011) has led to recovery after a period of several months. Because of the hardness of titanium, drilling of dental inlays should not be attempted! Even if removed, titanium may have migrated into gold inlays and will slowly be released over months or

lymphatic abnormality.

maternal milk.

**4. Diagnosis** 

**5. Treatment** 

**5.1 Titanium implants** 

containing titanium.

abnormality is a key factor in yellow nail syndrome.

by the presence of titanium in nail clippings (>1 µg/g).

**3.3 Prenatal and neonatal manifestations** 

After oral administration of titanium dioxide to rats, particles of titanium dioxide were present in the gut associated lymphoid tissue, but also in the liver, spleen, lungs and peritoneal tissues, but were not detected in the heart or the kidney (Jani et al., 1994).

Titanium, together with aluminum, has consistently been found in lymphoid tissue of the ileum and in mesenteric lymph glands in patients with intestinal disease and in postmortem cases with no evidence of gastrointestinal disease (Shepherd et al., 1987). After administration of gelatin capsules with 23 mg titanium dioxide (mean particle size 0.16 µm) to five male subjects, blood levels of titanium rose from 12 µg/L to 43 µg/L at 4-12 hours (Böckmann et al., 2000). The concentration/time curves were considered to be characteristic for a persorption mechanism (absorption in pores only slightly wider than the diameter of absorbed molecules).

#### **2.5 Titanium dioxide patients**

Several papers mention the exposure to drugs preceding the development of yellow nails and also return to normal conditions after withdrawal of the drugs (David-Vaudey et al., 2004). It turns out that that all the drug tablets mentioned contain titanium dioxide.

I had eight patients (4 male, 4 female, age 15-79 years at onset of symptoms) exposed to titanium dioxide, six via drug tablets, one via confectionary, and one via chewing gum. Seven had more than ten amalgam restorations in their teeth and had gastrointestinal symptoms, mostly diarrhea, that may have facilitated the absorption of titanium dioxide. A lactulose test in one patient showed increased intestinal permeability. This might explain why relatively few patients develop the syndrome. Also, some patients told me that their strange cough started only after 6 months medication. Most patients don't take their medicines (e.g. antibiotics) that long.

#### **3. Pathogenesis**

#### **3.1 Nail changes**

Samman and White (1964) suggested that nail changes and defective lymph drainage are related. Emerson (1966) even considered that "nail changes are believed to be the result of defective lymph drainage". Since titanium is always present in the nails of these patients, it is more tempting to consider the nail changes as a toxic reaction to titanium.

#### **3.2 Lymphedema, pleural effusions and ascites; defective lymph drainage or increased vascular leakage?**

Lymphangiograms, as performed in four patients with lymphedema in the legs, were interpreted as showing defective lymph drainage (Samman & White, 1964) Similar findings have been reported later (Müller et al., 1979). More recently Danielsson et al. (2006), using lymphoscintigraphy in a patient with edema, pleural effusions, hypoalbuminemia and yellow nails, found normal lymph flow and no signs of lymphatic obstruction in the lower extremities!

Emerson (1966) noted high protein content (40-90 g/L) in pleural effusion, but still considered the primary event to be defective lymph drainage, which could not handle

After oral administration of titanium dioxide to rats, particles of titanium dioxide were present in the gut associated lymphoid tissue, but also in the liver, spleen, lungs and

Titanium, together with aluminum, has consistently been found in lymphoid tissue of the ileum and in mesenteric lymph glands in patients with intestinal disease and in postmortem cases with no evidence of gastrointestinal disease (Shepherd et al., 1987). After administration of gelatin capsules with 23 mg titanium dioxide (mean particle size 0.16 µm) to five male subjects, blood levels of titanium rose from 12 µg/L to 43 µg/L at 4-12 hours (Böckmann et al., 2000). The concentration/time curves were considered to be characteristic for a persorption mechanism (absorption in pores only slightly wider than the diameter of

Several papers mention the exposure to drugs preceding the development of yellow nails and also return to normal conditions after withdrawal of the drugs (David-Vaudey et al.,

I had eight patients (4 male, 4 female, age 15-79 years at onset of symptoms) exposed to titanium dioxide, six via drug tablets, one via confectionary, and one via chewing gum. Seven had more than ten amalgam restorations in their teeth and had gastrointestinal symptoms, mostly diarrhea, that may have facilitated the absorption of titanium dioxide. A lactulose test in one patient showed increased intestinal permeability. This might explain why relatively few patients develop the syndrome. Also, some patients told me that their strange cough started only after 6 months medication. Most patients don't take their

Samman and White (1964) suggested that nail changes and defective lymph drainage are related. Emerson (1966) even considered that "nail changes are believed to be the result of defective lymph drainage". Since titanium is always present in the nails of these patients, it

Lymphangiograms, as performed in four patients with lymphedema in the legs, were interpreted as showing defective lymph drainage (Samman & White, 1964) Similar findings have been reported later (Müller et al., 1979). More recently Danielsson et al. (2006), using lymphoscintigraphy in a patient with edema, pleural effusions, hypoalbuminemia and yellow nails, found normal lymph flow and no signs of lymphatic obstruction in the lower

Emerson (1966) noted high protein content (40-90 g/L) in pleural effusion, but still considered the primary event to be defective lymph drainage, which could not handle

is more tempting to consider the nail changes as a toxic reaction to titanium.

**3.2 Lymphedema, pleural effusions and ascites; defective lymph drainage or** 

2004). It turns out that that all the drug tablets mentioned contain titanium dioxide.

peritoneal tissues, but were not detected in the heart or the kidney (Jani et al., 1994).

absorbed molecules).

**2.5 Titanium dioxide patients** 

medicines (e.g. antibiotics) that long.

**increased vascular leakage?** 

**3. Pathogenesis 3.1 Nail changes** 

extremities!

increased lymph formation, e.g. after some infection. Protein-losing enteropathy in patients with pleural effusions, in some cases together with ascites, has also been reported (Duhra et al., 1985, Battaglia et al., 1985, Malek et al., 1996). In a patient with edema, plural effusion, hypoalbuminaemia and yellow nails, D'Allessandro et al. (2001) found a 10-fold increase in normal albumin enteric loss. Since in most cases of yellow nail syndrome the pleural fluid has a very high protein content, this reflects increased protein permeability of systemic capillaries. D'Allessandro et al claimed that "the theory of pure lymphatic block is not sufficient to explain all the clinical manifestations of yellow nail syndrome", and suggested that microangiopathy and increased microvascular filtration at different sites (pleura, liver, limbs, intestine) due to an alteration in the interstitial matrix, could play a role in addition to lymphatic abnormality.

These reports indicate that increased vascular permeability rather than lymphatic abnormality is a key factor in yellow nail syndrome.

#### **3.3 Prenatal and neonatal manifestations**

There are two reports on pregnancy in women with yellow nails and bronchitis or productive cough (Govaert et al., 1992, Slee et al., 2000). Ultrasonography demonstrated polyhydramnios and bilateral pleural effusion in the fetuses at 23 and 29 weeks of gestation, respectively. At delivery both infants were hydropic and had bilateral pleural effusions, which were treated by thoracic drainage. Following initial recovery, pleural effusion recurred 2 days after starting enteral feeding at 4 weeks of age in the first infant, but not in the second. Our interpretation is that both mothers were exposed to titanium or titanium dioxide, and that titanium was transferred across the placenta, and possibly also via maternal milk.

There are a few reports of familiar occurrence of yellow nail syndrome (Lambert et al., 2006). They might be explained by several family members consuming drugs or chewing gum containing titanium.

#### **4. Diagnosis**

Yellow discoloration of the nails is often a late sign of the syndrome and therefore not necessary for the diagnosis of yellow nail syndrome; sinusitis and cough, lymphedema and pleural effusion occurring alone or in combination may represent the same syndrome (Varney et al., 1994, Cebecci et al., 2009). The diagnosis is supported by history or evidence of exposure to titanium implants or to titanium dioxide, and can be confirmed (if necessary) by the presence of titanium in nail clippings (>1 µg/g).

#### **5. Treatment**

#### **5.1 Titanium implants**

Most often it is impossible or extremely difficult to remove titanium implants. It is usually much easier to remove the gold (in teeth, jewelry or rings). In a few patients gold removal (Berglund & Carlmark, 2011) has led to recovery after a period of several months. Because of the hardness of titanium, drilling of dental inlays should not be attempted! Even if removed, titanium may have migrated into gold inlays and will slowly be released over months or

Titanium and Yellow Nail Syndrome 29

D'Allessandro A, Muzi G, Monaco A, Filiberto S, Barboni A & Abbritti G (2001). Yellow nail syndrome: does protein leakage play a role? *Eur Respir J* 17: 149-152. Danielsson Å, Toth E & Thorlacius H (2006). Capsule endoscopy in the management of a

David-Vaudey E, Jamard B, Hermant C & Cantagrel A (2004). Yellow nail syndrome in rheumatoid arthritis: a drug-induced disease? *Clin Rheumatol* 23: 376-378. Dilley JJ, Kierland RR, Randall RV & Shick RM (1968). Primary lymphedema associated with

Duhra PM, Quigley EMM & Marsh MN (1985). Chylous ascites, intestinal lymphoangiectasia and the yellow nail syndrome. *Gut* 26:1266-1269 Emerson PA (1966). Yellow nails, lymphoedema, and pleural effusions. *Thorax* 21: 247-253. Forsell M, Marcusson JA, Carlmark B & Johansson O (1997). Analysis of the metal content

Govaert P, Leroy JG, Pauwels R, Vanhaesebrouck P, De Praeter C, Van Kets H & Goeteyn M

Hassard AD, Martin J & Ross JB (1984). Yellow nail syndrome and chronic sinusitis. *J* 

Hiller E, Rosenow *III* EC & Olsen AM (1972). Pulmonary manifestations of the yellow nail

Hunsberger JF (1976). Electrochemical series Table I. In: Weast RC (ed) *Handbook of chemistry* 

Jani PU, McCarthy DE & Florence AT (1994). Titanium dioxide (rutile) particle uptake from

Kawano T, Matsuse H, Shigematsu K, Miyazaki M, Taguchi T & Kohno S (2003). Chemical

Malek NP, Ocran K, Tietge UJ, Maschek H, Gratz KF, Trautwein C, Wagner S & Manns MP

Müller R-P, Peters PE, Echternacht-Happle K & Happle R (1979). Roentgenographic and

Nakielna EM, Wilson J & Ballon HS (1976). Yellow nail syndrome: report of three cases. *Can* 

Reclaru L & Meyer J-M (1998). Effects of fluoride on titanium and other dental alloys in

Slee J, Nelson J, Dickinson J, Kendall P & Halbert A (2000). Yellow nail syndrome presenting as non-immune hydrops: Second case report. *Am J Med Genet* 93:1-4

Samman PD & White WF (1964). The "yellow nail" syndrome. *Br J Dermatol* 76:153-157 Schiff N, Grosgogeat B, Lissac M & Dalard F (2002). Influence of fluoride content and pH on the corrosion resistance of titanium and its alloys. *Biomaterials* 23: 1995-2002 Shepherd NA, Crocker PR, Smith AP & Levison DA (1987). Exogenous pigment in Peyer's

the rat GI tract and translocation to systemic organs after oral administration. *Int J* 

pleurodesis could exacerbate lymphedema of yellow nail syndrome. *Acta Med* 

(1996). A case of the yellow nail syndrome associated with massive chylous ascites,

*and physics*; 57th edn. CRC Press, Boca Raton, pp D141–D143

pleural and pericardial effusions. *Z Gastroenterol* 34:763-766.

clinical signs in yellow nail syndrome. *Lymphology* 12:257-261

of in vivo fixed dental alloys by means of a simple office procedure. *Swed Dent J* 21:

(1992). Perinatal manifestations of maternal yellow nail syndrome. *Pediatrics*

patient with a rare syndrome. *Gut* 55: 196

161-168

89:1016-1018

*Otolaryngol* 13:318–320

*Pharm* 105: 157-168.

*Nagasaki* 48:71-72

*Med Assoc J* 115:46–48

dentistry. *Biomaterials* 19: 85-92

patches. *Hum Pathol* 18: 50-54.

syndrome. *Chest* 61:452-458.

yellow nails and pleural effusions. *JAMA* 204:122-125

years. I know of two instances where titanium inlays were successfully removed by the use of ultrasonic sound.

Removal of dental amalgam fillings has as yet not been encouraging. Some mercury will remain in the surrounding dental tissue and in various organs in the body. Mercury enters the enterohepatic circulation, often maintaining diarrhea or constipation.

Pleural effusions may require serial thoracocentesis. Pleurodesis relieves pleural effusion but may exacerbate lymphedema of the lower limbs (Kawano et al., 2003). In other patients a pleuroperitoneal (Brofman et al., 1990) or a pleurovenous shunt has been applied (Tanaka et al., 2005).

#### **5.2 Titanium dioxide**

Patients taking drugs containing titanium dioxide may have difficulties finding titaniumfree replacements. They may have to resort to fluid preparations designed for children, or have titanium-free capsules made. Almost all chewing gums contain titanium dioxide, but not Stimorol senses (except the one with peppermint taste).

#### **6. Summary**

Yellow nail syndrome was defined in 1964 to include thick yellow nails and lymphedema. A number of symptoms have later been included, most importantly perhaps sinusitis and chronic cough, because they are easily overlooked by the doctor. Most serious are pleural effusions and protein-losing enteritis. The syndrome is caused by titanium or titanium dioxide.

#### **7. Acknowledgement**

All costs for this study were defrayed by the Swedish Society of Dental Amalgam Patients, Trollhättan,Sweden.

#### **8. References**


years. I know of two instances where titanium inlays were successfully removed by the use

Removal of dental amalgam fillings has as yet not been encouraging. Some mercury will remain in the surrounding dental tissue and in various organs in the body. Mercury enters

Pleural effusions may require serial thoracocentesis. Pleurodesis relieves pleural effusion but may exacerbate lymphedema of the lower limbs (Kawano et al., 2003). In other patients a pleuroperitoneal (Brofman et al., 1990) or a pleurovenous shunt has been applied (Tanaka et

Patients taking drugs containing titanium dioxide may have difficulties finding titaniumfree replacements. They may have to resort to fluid preparations designed for children, or have titanium-free capsules made. Almost all chewing gums contain titanium dioxide, but

Yellow nail syndrome was defined in 1964 to include thick yellow nails and lymphedema. A number of symptoms have later been included, most importantly perhaps sinusitis and chronic cough, because they are easily overlooked by the doctor. Most serious are pleural effusions and protein-losing enteritis. The syndrome is caused by titanium or titanium

All costs for this study were defrayed by the Swedish Society of Dental Amalgam Patients,

Battaglia A, Di Ricco G, Mariani G & Giuntini C (1985). Pleural effusion and recurrent

Berglund F & Carlmark B (2011). Titanium, sinusitis, and the yellow nail syndrome *Biol* 

Böckmann J, Lahl H, Eckert T & Unterhalt B (2000). Titan-Blutspiegel vor und nach

Brofman JD, Hall JB, Scott W & Little AG (1990). Yellow nails, lymphedema and pleural

Camilleri AE (1990). Chronic sinusitis and the yellow nail syndrome. *J Laryngol Otol* 104:811–

Cebeci F, Celebi M & Onsun N (2009). Nonclassical yellow nail syndrome in six-year-old

Belastungsversuchen mit Titandioxid. *Pharmazie* 55:140-143.

bronchopneumonia with lymphedema, yellow nails and protein losing

effusion. Treatment of chronic pleural effusion with pleuroperitoneal shunting.

the enterohepatic circulation, often maintaining diarrhea or constipation.

not Stimorol senses (except the one with peppermint taste).

enteropathy. *Eur J Respir Dis* 66: 65-69.

girl: a case report. *Cases J* 2:165–169

*Trace Elem Res* 143:1-7.

of ultrasonic sound.

**5.2 Titanium dioxide** 

**6. Summary** 

dioxide.

**7. Acknowledgement** 

97:743-5

813

Trollhättan,Sweden.

**8. References** 

al., 2005).


**3** 

*United States* 

**Arm Lymphedema as a Consequence of** 

Lymphedema is the result of an abnormality of the lymphatic system. It is caused by an excessive accumulation of lymphatic fluid, known as interstitial fluid, in the interstitial tissue, particularly in the subcutaneous fat. Ultimately, this leads to swelling of affected tissues due to a build up of and inadequate lymph drainage, known as lymphedema

Primary lymphedema occurs when people are born with abnormalities in the lymphatic system, such as missing or impaired lymphatic vessels (Farrow, 2010b). The severity of the

**1. Introduction** 

(Farrow, 2010a) [Figure 1].

Fig. 1. Right arm lymphedema

*Weill Cornell Medical College, Department of Radiation Oncology* 

**Breast Cancer Therapy** 

A. Gabriella Wernicke, Yevgeniya Goltser, Michael Shamis and Alexander J. Swistel


## **Arm Lymphedema as a Consequence of Breast Cancer Therapy**

A. Gabriella Wernicke, Yevgeniya Goltser, Michael Shamis and Alexander J. Swistel *Weill Cornell Medical College, Department of Radiation Oncology United States* 

#### **1. Introduction**

30 Novel Strategies in Lymphedema

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60:254-255.

*Soc Med* 59: 448.

Lymphedema is the result of an abnormality of the lymphatic system. It is caused by an excessive accumulation of lymphatic fluid, known as interstitial fluid, in the interstitial tissue, particularly in the subcutaneous fat. Ultimately, this leads to swelling of affected tissues due to a build up of and inadequate lymph drainage, known as lymphedema (Farrow, 2010a) [Figure 1].

Fig. 1. Right arm lymphedema

Primary lymphedema occurs when people are born with abnormalities in the lymphatic system, such as missing or impaired lymphatic vessels (Farrow, 2010b). The severity of the

Arm Lymphedema as a Consequence of Breast Cancer Therapy 33

Both primary and secondary lymphedemas possess characteristic features that can be distinguished over time. History should include information such as age of onset, location(s) of swelling, pain and other symptoms of discomfort, medications that instigate swelling, the course of progression of swelling, and factors prompting swelling such as cancer, injury, or infection. Family history is important to diagnose inherited forms of lymphedema (Gupta, 2006). A physical examination assesses the vascular system, the soft tissue and skin surrounding the swollen body parts, palpation of lymph nodes, and looks for changes in the body systems in accordance with inherited lymphedemas (Wang, 2005; Moseley et al., 2008). Further diagnostic tests and imagining coupled with history, family history, and physical examination are used to correctly diagnose a patient (Lewin et al., 2010; Smith et al., 2010). Lympehedema is a type of side effect requiring attention to diagnosis and management by a number of specialists taking care of a patient with breast cancer. It is critical for each discipline to pay particular attention to the patients' subjective reporting of their symptoms suggestive of lymphedema. Therefore, a multidisciplinary approach to diagnosis and management of lympehedema is essential for the routine surveillance after treatment by all the involved physicians, including the surgeon, radiation oncologist, and medical oncologist. A lymphedema specialist's consultation is often necessary, especially when the patient is at risk for developing lymphedema or has an evidence of this diagnosis on

Physical examination includes placing hands on location of lymphedema and feeling palpation as well as the surrounding area of the affected limb. This procedure is called subjective palpation. Upon physical examination, the standard way of detecting lymphedema is by taking measures of limb volume (Chen et al., 2008; Cheville et al., 2003; Hayes et al., 2005). An enlargement, or increase in volume, of the limb is the result of fluid build up in the tissues. Volume is measured by 3 methods including tape measurements, perometry, and water displacement. These measurements of volume illustrate the presence and severity of the condition (Unno et al., 2008). Tape measurements are most accurate when done at precisely defined intervals and when taken by the same individual, ultimately

Perometry uses an infra-red optical electronic scanner to calculate volume by precisely positioning the body part exactly the same each time and calibrating the machine (Rockson et al., 2007). This method can detect volume changes in breast cancer survivors as little as 3% (Czerneic et al., 2010). Water displacement measurements are taken by immersing the limb in a large cylinder and determining the volume of water displaced, or pushed out of the cylinder. However, measuring volume cannot differentiate lymphedema from other types of edema and is a technique best used as follow up for treatment of lymphedema rather then

A radiological technique, that detects slow or absent lymph flow and areas of reflux or backup of lymph node and lymph vessel imaging due to lymphedema is called a lymphoscintigraphy. Normally, technetium labeled sulfur is the radio-labeled particle of protein injected directly under the skin to detect and image the affected area (Piller, 2009; Szuba et al., 2003; Hayes et al., 2008; Bellini et al., 2005). The procedure identifies lymphatic

physical examination.

**2. Diagnosis of lymphedema** 

diagnosis (Unno et al., 2008).

using geometric formulas to determine the total volume.

condition is able to assess whether swelling is present at birth or develops at the onset of puberty or in adulthood. It can affect from one up to four limbs and/or body parts (Farrow, 2010a). Secondary lymphedema is more common and occurs from damage to the lymphatic system that occurs as a result of cancer and its treatment, due to the resection of lymph nodes. Trauma to the skin, such as burns or infections, can also cause secondary lymphedema (Gordon, 2007; Piller, 2009).

Signs and symptoms of progressive lymphedema include discomfort and pain associated with full sensation in the limb(s) and the skin feeling tight, as well as difficulty with daily tasks due to a decreased flexibility in the hand, wrist, or ankle and the inability to fit into clothing and jewelry in a certain area. Progressive lymphedema is complicated by recurrent infections, non-healing wounds, as well as emotional and social distress (Ridner, 2009; Ahmed, 2008; Shih, 2009).

Lymphedema is prevalent and most often studied as a consequence of breast cancer. However, it has been studied in cases of melanoma, gynecological cancer, head and neck cancer as well as sarcoma (Lewin et al., 2010; Murphy et al., 2010; Smith et al., 2010; Cormier et al., 2010; Lacomba, 2010). Research shows that there is a lifelong risk of developing lymphedema due to cancer and the overall risk has been reported to be 15.5% (Chang et al., 2010) [Figure 2].

Fig. 2. A woman with locally advanced breast cancer status post left modified radical mastectomy and lymph node dissection and radiotherapy to the left chest wall, supraclavicular area and axillary lymph nodes. **A.** Left arm at 3 months after treatment. **B.** Left arm demonstrates marked lymphedema at 3 years after treatment.

condition is able to assess whether swelling is present at birth or develops at the onset of puberty or in adulthood. It can affect from one up to four limbs and/or body parts (Farrow, 2010a). Secondary lymphedema is more common and occurs from damage to the lymphatic system that occurs as a result of cancer and its treatment, due to the resection of lymph nodes. Trauma to the skin, such as burns or infections, can also cause secondary

Signs and symptoms of progressive lymphedema include discomfort and pain associated with full sensation in the limb(s) and the skin feeling tight, as well as difficulty with daily tasks due to a decreased flexibility in the hand, wrist, or ankle and the inability to fit into clothing and jewelry in a certain area. Progressive lymphedema is complicated by recurrent infections, non-healing wounds, as well as emotional and social distress (Ridner, 2009;

Lymphedema is prevalent and most often studied as a consequence of breast cancer. However, it has been studied in cases of melanoma, gynecological cancer, head and neck cancer as well as sarcoma (Lewin et al., 2010; Murphy et al., 2010; Smith et al., 2010; Cormier et al., 2010; Lacomba, 2010). Research shows that there is a lifelong risk of developing lymphedema due to cancer and the overall risk has been reported to be 15.5% (Chang et al.,

Fig. 2. A woman with locally advanced breast cancer status post left modified radical mastectomy and lymph node dissection and radiotherapy to the left chest wall,

Left arm demonstrates marked lymphedema at 3 years after treatment.

supraclavicular area and axillary lymph nodes. **A.** Left arm at 3 months after treatment. **B.**

lymphedema (Gordon, 2007; Piller, 2009).

**A B**

Ahmed, 2008; Shih, 2009).

2010) [Figure 2].

Both primary and secondary lymphedemas possess characteristic features that can be distinguished over time. History should include information such as age of onset, location(s) of swelling, pain and other symptoms of discomfort, medications that instigate swelling, the course of progression of swelling, and factors prompting swelling such as cancer, injury, or infection. Family history is important to diagnose inherited forms of lymphedema (Gupta, 2006). A physical examination assesses the vascular system, the soft tissue and skin surrounding the swollen body parts, palpation of lymph nodes, and looks for changes in the body systems in accordance with inherited lymphedemas (Wang, 2005; Moseley et al., 2008). Further diagnostic tests and imagining coupled with history, family history, and physical examination are used to correctly diagnose a patient (Lewin et al., 2010; Smith et al., 2010).

Lympehedema is a type of side effect requiring attention to diagnosis and management by a number of specialists taking care of a patient with breast cancer. It is critical for each discipline to pay particular attention to the patients' subjective reporting of their symptoms suggestive of lymphedema. Therefore, a multidisciplinary approach to diagnosis and management of lympehedema is essential for the routine surveillance after treatment by all the involved physicians, including the surgeon, radiation oncologist, and medical oncologist. A lymphedema specialist's consultation is often necessary, especially when the patient is at risk for developing lymphedema or has an evidence of this diagnosis on physical examination.

#### **2. Diagnosis of lymphedema**

Physical examination includes placing hands on location of lymphedema and feeling palpation as well as the surrounding area of the affected limb. This procedure is called subjective palpation. Upon physical examination, the standard way of detecting lymphedema is by taking measures of limb volume (Chen et al., 2008; Cheville et al., 2003; Hayes et al., 2005). An enlargement, or increase in volume, of the limb is the result of fluid build up in the tissues. Volume is measured by 3 methods including tape measurements, perometry, and water displacement. These measurements of volume illustrate the presence and severity of the condition (Unno et al., 2008). Tape measurements are most accurate when done at precisely defined intervals and when taken by the same individual, ultimately using geometric formulas to determine the total volume.

Perometry uses an infra-red optical electronic scanner to calculate volume by precisely positioning the body part exactly the same each time and calibrating the machine (Rockson et al., 2007). This method can detect volume changes in breast cancer survivors as little as 3% (Czerneic et al., 2010). Water displacement measurements are taken by immersing the limb in a large cylinder and determining the volume of water displaced, or pushed out of the cylinder. However, measuring volume cannot differentiate lymphedema from other types of edema and is a technique best used as follow up for treatment of lymphedema rather then diagnosis (Unno et al., 2008).

A radiological technique, that detects slow or absent lymph flow and areas of reflux or backup of lymph node and lymph vessel imaging due to lymphedema is called a lymphoscintigraphy. Normally, technetium labeled sulfur is the radio-labeled particle of protein injected directly under the skin to detect and image the affected area (Piller, 2009; Szuba et al., 2003; Hayes et al., 2008; Bellini et al., 2005). The procedure identifies lymphatic

Arm Lymphedema as a Consequence of Breast Cancer Therapy 35

Fig. 3. A tonometer is used to assess the firmness of a tissue by measuring how much force

Cardiovascular diseases or abnormalities may also serve as a trigger for many forms of edema (Schumacher et al., 2008). For those given a diagnosis of primary lymphedema, it is important to determine if congestive heart failure, deep venous thrombosis, damaged valves in the vein or any arterial conditions account for the swelling or are an adjunct to existent lymphedema (Szuba, 2000; Bellini, 2005). Secondary lymphedema, as a result of cancer, can be studied by taking images of the heart, veins and valves to determine the case, severity, and treatment options of the edema. Cardiovascular studies usually ordered for edema studies include echocardiogram, venous ultrasound, and arterial ultrasound with ankle brachial index (ABI). It is best to do ultrasounds in a standing up position to test for incompetency of the valves. More advanced forms of imaging for insufficiency of blood vessels is by means of a computed tomography, venogram, and arteiogram. These are normally used to assess conditions in the chest, abdomen, or pelvis

Blood tests are unavailable for diagnosis of lymphedema. However, conditions that mimic lymphedema's symptoms such as hypothyroidism or hypoproteinemia may cause swelling and need to be assessed by means of a blood test. For some lymphedemas that are genetically inherited x-rays are important to detect orthopedic abnormalities (Bellini et al.,

There are recommended guidelines to follow for optimal prevention, screening and measurement for early detection of breast cancer related lymphedema (Farrow, 2010c). There should be a pro-active approach pre-operatively and post-operatively for arm measurements taken by patients and physicians. Patients should receive risk‐reduction strategies prior to treatment (NLN Position Paper, 2011; Fu et al., 2010). Weight and height should be accurately measured during each visit to a specialist in order to

**3. Lymphedema as a long-term sequelae of breast cancer therapy** 

is needed to indent the tissue sample.

(Farrow, 2010).

2009; Gupta et al., 2006).

problems at late stages of lymphedema and shows the basics of the peripheral lymphatic system and larger more prominent lymph nodes and vessels. Radiology dpartments are apt at performing lymphoscintigraphy studies aimed at identifying the sentinel lymph node for cancers of the breast and ultimately further studies for the diagnosis of lymphedema Cornish et al., 2007; Szuba et al., 2000, 2003, 2007)

A more recent technique of lymph vessel imaging uses indocyanine green (ICG) injections into the skin and an infrared fluorescence camera to detect the function of even the smallest lymphatic vessels. This is called Near Infra‐Red Florescence Imaging (NIR). NIR-ICG can pick up early stages of lymphedema and diagnose diseased non-contracting lymphatics even before swelling occurs (Farrow, 2010; Adams et al.; Rasmussen et al., 2009, 2010; Unno et al., 2010; Maus, 2010)

There is a variety of diagnostic tests that can be performed in order to classify and detect lymphedema. These include soft tissue imaging, bioimpedance spectroscopy, tonometry, genetic testing, various forms of vascular imaging, as well as blood tests (Farrow, 2010).

Soft tissue imaging like MRIs (magnetic resonance imaging), CTs (computed tomography scans, and US (ultrasounds) detect excess fluid in the tissues. Since lymphedema is the result of interstitial fluid build up these imaging techniques are often used to determine the cause of the condition as well as lymphedema that is a result of an untreated cancer (Astrom et al., 2001; Deltombe et al., 2007; Unno et al., 2008)

Bioimpedance Spectroscopy (BIS) measures water content in tissues by passing a small, harmless, electrical current through the limb in order to measure the impedance to current flow. The higher the water content in the area the lower the resistance. BIS assesses the condition by comparing the resistance of electrical flow in the intracellular and interstitial fluid of a whole limb, because calculations are performed to the length of the body part (Gergich et al., 2008; Ward, 2006; Rockson et al., 2007).

Lymphedema is graded according to increased size as well as staging of the progression in the change of the skin texture. As a consequence lymphedema the skin and subcutaneous tissue become harder and denser (Executive Committee of International Society of Lymphology, 2009). Upon physical examination, tissue texture, pitting, larger skin folds, wounds or papillomas are noted. Current examinations to determine skin texture and resistance are tissue dielectric constant and tonometry. The tissue dielectric constant measures tissue water content and uses a specific frequency of an electrical current to measure the reflected return wave in order to indicate how much water is present in the tissue (Mayrovitz, 2009; Corica et al., 2006; Mirnajafi et al., 2004; Ridner et al., 2007). Tonometry determines how firm a tissue is by measuring how much force is needed to indent the tissue sample (Mayrovitz, 2009) [Figure 3].

Young patients diagnosed with primary lymphedema should undergo genetic testing and counseling and have a karyotype test performed in order to detect abnormalities. Turner's syndrome has been linked to lymphedema and can be determined from a karyotype. Specific genes are also associated with lymphedema (Ferrell, 2008). These include FOXC2 an SOX18 (Connell et al., 2009; Brice et al, 2002). However, inherited lymphedema is not detectible on gene or chromosome tests and genetic testing for late-onset lymphedema does not prove to have benefits (Farrow, 2010b).

problems at late stages of lymphedema and shows the basics of the peripheral lymphatic system and larger more prominent lymph nodes and vessels. Radiology dpartments are apt at performing lymphoscintigraphy studies aimed at identifying the sentinel lymph node for cancers of the breast and ultimately further studies for the diagnosis of lymphedema

A more recent technique of lymph vessel imaging uses indocyanine green (ICG) injections into the skin and an infrared fluorescence camera to detect the function of even the smallest lymphatic vessels. This is called Near Infra‐Red Florescence Imaging (NIR). NIR-ICG can pick up early stages of lymphedema and diagnose diseased non-contracting lymphatics even before swelling occurs (Farrow, 2010; Adams et al.; Rasmussen et al., 2009, 2010; Unno

There is a variety of diagnostic tests that can be performed in order to classify and detect lymphedema. These include soft tissue imaging, bioimpedance spectroscopy, tonometry, genetic testing, various forms of vascular imaging, as well as blood tests (Farrow, 2010).

Soft tissue imaging like MRIs (magnetic resonance imaging), CTs (computed tomography scans, and US (ultrasounds) detect excess fluid in the tissues. Since lymphedema is the result of interstitial fluid build up these imaging techniques are often used to determine the cause of the condition as well as lymphedema that is a result of an untreated cancer (Astrom et al.,

Bioimpedance Spectroscopy (BIS) measures water content in tissues by passing a small, harmless, electrical current through the limb in order to measure the impedance to current flow. The higher the water content in the area the lower the resistance. BIS assesses the condition by comparing the resistance of electrical flow in the intracellular and interstitial fluid of a whole limb, because calculations are performed to the length of the body part

Lymphedema is graded according to increased size as well as staging of the progression in the change of the skin texture. As a consequence lymphedema the skin and subcutaneous tissue become harder and denser (Executive Committee of International Society of Lymphology, 2009). Upon physical examination, tissue texture, pitting, larger skin folds, wounds or papillomas are noted. Current examinations to determine skin texture and resistance are tissue dielectric constant and tonometry. The tissue dielectric constant measures tissue water content and uses a specific frequency of an electrical current to measure the reflected return wave in order to indicate how much water is present in the tissue (Mayrovitz, 2009; Corica et al., 2006; Mirnajafi et al., 2004; Ridner et al., 2007). Tonometry determines how firm a tissue is by measuring how much force is needed to

Young patients diagnosed with primary lymphedema should undergo genetic testing and counseling and have a karyotype test performed in order to detect abnormalities. Turner's syndrome has been linked to lymphedema and can be determined from a karyotype. Specific genes are also associated with lymphedema (Ferrell, 2008). These include FOXC2 an SOX18 (Connell et al., 2009; Brice et al, 2002). However, inherited lymphedema is not detectible on gene or chromosome tests and genetic testing for late-onset lymphedema does

Cornish et al., 2007; Szuba et al., 2000, 2003, 2007)

2001; Deltombe et al., 2007; Unno et al., 2008)

(Gergich et al., 2008; Ward, 2006; Rockson et al., 2007).

indent the tissue sample (Mayrovitz, 2009) [Figure 3].

not prove to have benefits (Farrow, 2010b).

et al., 2010; Maus, 2010)

Fig. 3. A tonometer is used to assess the firmness of a tissue by measuring how much force is needed to indent the tissue sample.

Cardiovascular diseases or abnormalities may also serve as a trigger for many forms of edema (Schumacher et al., 2008). For those given a diagnosis of primary lymphedema, it is important to determine if congestive heart failure, deep venous thrombosis, damaged valves in the vein or any arterial conditions account for the swelling or are an adjunct to existent lymphedema (Szuba, 2000; Bellini, 2005). Secondary lymphedema, as a result of cancer, can be studied by taking images of the heart, veins and valves to determine the case, severity, and treatment options of the edema. Cardiovascular studies usually ordered for edema studies include echocardiogram, venous ultrasound, and arterial ultrasound with ankle brachial index (ABI). It is best to do ultrasounds in a standing up position to test for incompetency of the valves. More advanced forms of imaging for insufficiency of blood vessels is by means of a computed tomography, venogram, and arteiogram. These are normally used to assess conditions in the chest, abdomen, or pelvis (Farrow, 2010).

Blood tests are unavailable for diagnosis of lymphedema. However, conditions that mimic lymphedema's symptoms such as hypothyroidism or hypoproteinemia may cause swelling and need to be assessed by means of a blood test. For some lymphedemas that are genetically inherited x-rays are important to detect orthopedic abnormalities (Bellini et al., 2009; Gupta et al., 2006).

#### **3. Lymphedema as a long-term sequelae of breast cancer therapy**

There are recommended guidelines to follow for optimal prevention, screening and measurement for early detection of breast cancer related lymphedema (Farrow, 2010c). There should be a pro-active approach pre-operatively and post-operatively for arm measurements taken by patients and physicians. Patients should receive risk‐reduction strategies prior to treatment (NLN Position Paper, 2011; Fu et al., 2010). Weight and height should be accurately measured during each visit to a specialist in order to

Arm Lymphedema as a Consequence of Breast Cancer Therapy 37

<2cm, L only (wide excision or quadrantectomy)

<2, 2-5,>5cm, L, MRM, axillary RT

1-2 +SLN

≤2.0, 2.1-4.0, >4.0 cm, L or M

≤2.0, 2.1-4.0, ≥4.1 cm, L or M

10 265 <5.0cm, L \*6/111 (5.4)

**Lymphedema by medical professionals (%)**

\* 7/100 (7)

L,MRM 0.522 N/A

\* 14/226 (6) # 26/242 (11)

\*303/1459 (20.8)

#21/115 (18.3)
