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following in epidermolysis bullosa

Wound healing following surgery in epidermolysis bullosa

Adam George Harris

A thesis in fulfilment of the requirements for the degree of Master of Medicine (research)

University of New South Wales St George Hospital Clinical School of Medicine Faculty of Medicine Department of

Supervisors Professor Dedee F Murrell Professor Matthew Law

March 2017

2 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Harris

First name: Adam Other name/s: George Abbreviation for degree as given in the University calendar: MMed

School: St George and Sutherland Clinical School Faculty: Medicine

Title: following surgery in patients with epidermolysis bullosa

Epidermolysis Bullosa (EB) is heterogeneous group of rare heritable disorders characterised by blistering of the skin with minor trauma. Despite limited evidence to suggest patients with EB have more postoperative wound complications than the general population, we have noted reluctance among some surgeons to operate on these patients. We consequently designed this study to investigate postoperative wound healing in patients with EB.

Patients recruited from the Australian National Diagnostic Laboratory Database for EB and the Australasian EB Registry were posted the 'Surgical Wound and Healing in EB' questionnaire which contains twenty-two questions about a patients experience of having surgery.

Forty-six patients reported a total of 94 surgical procedures. The most frequent surgical procedure was the excision of a skin lesion, followed by musculoskeletal surgery, a or an . Five patients reported blistering at the surgical wound site after seven . Four patients reported four postoperative wound and one reported a postoperative wound dehiscence. Twenty patients had 34 postoperative which they felt healed slower than someone without EB and 30 patients had 55 postoperative wounds which they felt healed at the same rate as someone without EB. After 67 surgeries, the postoperative scar was reported to heal flat and after 18 it was reported to heal as a keloid scar.

It is unlikely that patients with localised EB simplex will develop blistering at the postoperative wound site, but about a quarter of patients with generalised EB may develop blistering which is likely to interfere with wound healing. Postoperative wound infections do not appear to occur any more frequently in patients with EB than in patients within the general population and wound dehiscence is uncommon in patients with EB. Postoperative wounds may heal at a similar or slower rate in patients with EB compared to those within the general population and patients with EB may have a propensity to develop keloid .

Despite the inherent limitations of a postal survey we feel clinicians should be more confident to refer patients with EB for surgery and surgeons reassured about postoperative wound healing and complications.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

Signature Witness Date 31/8/16 The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the aooroval of the Dean of Graduate Research.

FOR OFFICE USE ONLY Date of completion of requirements for Award: COPYRIGHT STATEMENT

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

Signed ......

26/3/17 Date ......

AUTHENTICITY STATEMENT

‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’

Signed ......

26/3/17 Date ...... Wound healing following surgery in epidermolysis bullosa

3 Wound healing following surgery in epidermolysis bullosa

Table of Contents

Title page - page 3 Table of contents - pages 5 & 6 List of acknowledgments - page 6 List of abbreviations - page 7 List of tables & figures - page 8

Chapter 1. Introduction - page 9 1.1 An overview of epidermolysis bullosa - page 9 1.2 The classification of epidermolysis bullosa - pages 9 & 10 1.2.1 Epidermolysis bullosa simplex - pages 10 -12 1.2.2 Junctional epidermolysis bullosa - pages 13 - 15 1.2.3 Dystrophic epidermolysis bullosa - pages 15 - 18 1.2.4 Kindler syndrome - page 19 1.3 The diagnosis of epidermolysis bullosa - pages 19 & 20 1.4 The management of epidermolysis bullosa - pages 20 & 22 1.5 Surgery and epidermolysis bullosa - page 22 1.5.1 Surgery for pseudosyndactyly and contractures - pages 22 & 23 1.5.2 Surgery of the - pages 23 1.5.3 Surgery of the genitourinary tract - page 23 & 24 1.5.4 Surgery of squamous cell carcinomas - page 24 1.6 Tattooing in epidermolysis bullosa - page 24 1.7 Surgical wound healing in epidermolysis bullosa - pages 24 & 25 1.8 Aims of the study - page 26 1.8.1 Primary aim - page 26 1.8.2 Secondary aims - pages 26

Chapter 2. Methods - page 27 2.1 The Australian National Diagnostic Laboratory Database for EB and the Australasian EB Registry - page 27 2.2 The ‘Surgical Wound and Scar Healing in EB’ questionnaire - pages 27 & 28 2.3 Sending and receiving the questionnaires - page 28

4 Wound healing following surgery in epidermolysis bullosa

Chapter 3. Results - page 30 3.1 Patient demographics - page 30 3.2 Surgical procedures - pages 30 & 31 3.3 Skin blistering - page 32 3.3.1 Postoperative wound blistering - page 32 3.3.2 Blistering at sites other than the postoperative wound - page 32 3.4 Postoperative wound complications not including blistering - pages 32 & 33 3.5 Rate of postoperative wound healing - page 33 3.6 Scar healing - page 33 3.7 Tattooing in patients with epidermolysis bullosa - page 34 3.8 Answers to the open-ended questions - page 34

Chapter 4. Discussion of findings - page 37 4.1 Response rates and patient demographics - page 37 4.2 Surgical procedures - page 38 4.3 Skin blistering - page 39 4.3.1 Postoperative wound blistering - page 39 4.3.2 Blistering at sites other than the postoperative wound - pages 39 & 40 4.4 Postoperative wound complications not including blistering - pages 40 & 41 4.5 Rate of postoperative wound healing - pages 41 & 42 4.6 Scar healing - page 42 4.7 Tattooing in patients with epidermolysis bullosa - page 43 4.8 Answers to the open-ended questions - page 43

Chapter 5. Limitations of the study - page 44 5.1 Study biases - page 44 5.2 Limitations of the data - pages 44 & 45

Chapter 6. Conclusions and further research - page 46

Relevant publications arising from the writing of this thesis - page 47 Appendix - pages 48 - 53 Bibliography - pages 53 - 61

5 Wound healing following surgery in epidermolysis bullosa

List of Acknowledgments

I would like to full-heartedly thank the following:

Professor Dedee Murrell and Professor Mathew Law, who acted as my supervisors and guided me in the development, implementation and completion of this thesis,

The Australasian Blistering Foundation for funding the ethics application for this thesis,

Margaret Norris, our esteemed EB nurse who manages the Australasian epidermolysis bullosa registry and helped liaise with our patients during this project,

Glendi McNab, our invaluable dermatology secretary who helped with sending and receiving the questionnaires,

Professor Dedee Murrell, Dr Gurpreet Singh and the patients who provided the photos for this thesis, and my mum, Sara Harris who helped to edit this thesis.

6 Wound healing following surgery in epidermolysis bullosa

List of Abbreviations

AEBR – Australasian epidermolysis bullosa registry DEB – dystrophic epidermolysis bullosa DEBRA – dystrophic epidermolysis bullosa research association DDEB – dominant dystrophic epidermolysis bullosa EB – epidermolysis bullosa EBS – epidermolysis bullosa simplex FTSG – full thickness skin graft IFM – antigenic mapping JEB – junctional epidermolysis bullosa K5 – Keratin 5 K14 – Keratin 14 KS – Kindler syndrome LOC syndrome – laryngo-onycho-cutaneous syndrome NGS – next generation sequencing PCR – polymerase chain reaction PTC – premature terminating codon QOL – quality of life RDEB – recessive dystrophic epidermolysis bullosa SCC – squamous cell carcinoma SS – Sanger sequencing SSG – split thickness skin graft TEM – transmission electron microscopy

7 Wound healing following surgery in epidermolysis bullosa

List of Tables and Figures

Table 1. Pages 35 & 36. Patient demographics and data from the questionnaire regarding postoperative wound healing.

Table 2. Page 36. Questionnaire results from patients who reported having a .

Figure 1. Page 10. The structure of the skin and the location of the proteins affected in the four major subtypes of EB.

Figure 2. Page 12. A patient with localised epidermolysis bullosa simplex exhibiting blistering of the palms and soles.

Figure 3. Page 15. Widespread blistering and erosions on the back of a patient with generalised intermediate junctional epidermolysis bullosa.

Figure 4. Page 17. Extensive blistering, erosions and scarring in a patient with recessive dystrophic epidermolysis bullosa.

Figure 5. Page 18. Pseudosyndactyly of the hands in a patient with recessive dystrophic epidermolysis bullosa.

Figure 6. Page 28. The Surgical Wound and Scar Healing in Epidermolysis Bullosa questionnaire.

Figure 7. Page 31. The number of each reported surgical procedure.

8 Wound healing following surgery in epidermolysis bullosa

Chapter 1: Introduction

1.1 An Overview of Epidermolysis Bullosa

Epidermolysis Bullosa (EB) is heterogeneous group of rare heritable disorders caused by mutations in eighteen different genes encoding structural proteins within epithelial tissues [1, 2]. The resultant absence, reduction, or presence of dysfunctional protein leads to tissue fragility, which typically presents as blistering of the skin with minor trauma. As well as the skin, other epithelial tissues can be affected within the gastrointestinal, genitourinary, ocular and respiratory systems leading to local and systemic complications [3, 4]. The prevalence of EB in the Australian population is about fourteen cases per million people, although, the real prevalence is likely higher than this as epidemiological estimates involving EB are fraught with difficulty and bias [5-7]. Unfortunately, despite a vast increase in the knowledge about EB in recent years, currently no cure exists and treatment is largely supportive with a focus on preventing secondary complications, although, various experimental treatments aimed at ameliorating symptoms and improving quality and length of life are in development [2].

1.2 The Classification of Epidermolysis Bullosa

Because of the heterogeneity of EB, a consensus of leading international EB experts has been meeting regularly since 1988 to continually classify the [8, 9]. The latest consensus was held in 2013, which resulted in the classification of EB into four major subtypes based on the ultrastructural level of cleavage within the skin (Figure 1.) [10]. These major subtypes include EB simplex (EBS), dystrophic EB (DEB), junctional EB (JEB) and Kindler syndrome (KS). Within each of these major subtypes a variety of specific subtypes exist, classified by the phenotype and genotype [10]. To define these more specific subtypes, nomenclature is first broken down into distribution and severity, then mode of inheritance, protein affected, level of protein expression within the skin, the gene implicated and the mutation involved is reported. It was also recommended to do away with previous

9 Wound healing following surgery in epidermolysis bullosa

eponyms used to describe the different subtypes of EB to allow for better comparison between non-EB specialists [10].

Figure 1. The structure of the skin and the location of the proteins affected in the four major subtypes of EB. EBS – epidermolysis bullosa simplex, JEB – junctional epidermolysis bullosa, DEB – dystrophic epidermolysis bullosa, KS – Kindler syndrome.

1.2.1 Epidermolysis Bullosa Simplex

EBS is the most common major subtype of EB with a prevalence ranging from one to twenty-nine cases per million people, as reported in various national epidemiological studies [5, 11]. It is most frequently caused by autosomal dominant mutations in the genes encoding the proteins Keratin 5 (K5) and Keratin

10 Wound healing following surgery in epidermolysis bullosa

14 (K14) which dimerise into intermediate filaments providing structure, strength and flexibility to the keratinocyte cytoskeleton [12]. When these proteins are compromised they are susceptible to mechanical stress leading to fragility of basal keratinocytes and subsequent blistering of the [13]. Clinically there exists a spectrum of EBS caused by mutations in KRT5 and KRT14, which differ due to the location and type of mutations [14]. Localised forms, the most common type of EBS, resemble the eponym Weber-Cockayne and are characterized by blistering of the palms and soles made worse by friction, trauma, heat and sweating (Figure 2.) [15]. tend to heal without scarring and tend to improve with age, although focal can become a feature [1]. EBS generalised intermediate, resembling the eponym EBS-Koebner presents with more widespread blistering, but in a form that is milder than EBS generalised severe [10]. EBS generalised severe, resembling the eponym EBS Dowling-Meara, is characterised by widespread blistering, erosions, scarring and milia and can involve the mucous membranes, nails, hair and the eyes [3, 10, 13]. Patients can develop diffuse keratoderma of the palms and soles and blistering of the gastrointestinal tract and respiratory system leading to systemic complications [3, 4, 10]. Other specific clinical syndromes exist under the umbrella of EBS include EBS Ogna and EBS with muscular dystrophy caused by mutations in PLEC encoding the protein Plectin, EBS with pyloric atresia caused by mutations in PLEC, autosomal recessive EBS caused by mutations in KRT14, EXPH5 encoding Exophilin 5 and DST encoding antigen 1, EBS with mottled pigmentation caused by mutations in KRT5, EBS with circinate caused by mutations in KRT14, acral peeling skin syndrome caused by mutations in TGM5 encoding Transglutaminase 5, acantholytic EBS caused by mutations in DSP and JUP encoding Desmoplakin and Plakoglobin respectively, EBS superficialis and other skin fragility syndromes cause by mutations in DSP and PKP1 encoding Plakophilin 1 [10, 16-18].

11 Wound healing following surgery in epidermolysis bullosa

Figure 2. A patient with localised epidermolysis bullosa simplex exhibiting blistering of the palms and soles.

12 Wound healing following surgery in epidermolysis bullosa

1.2.2 Junctional Epidermolysis Bullosa

JEB is the second rarest major EB subtype, after KS, with an estimated prevalence of two cases per million people within the Australian population [5]. It is most commonly caused by autosomal recessive inheritance of compound heterozygous or homozygous mutations in the genes LAMA3, LAMB3 and LAMC2 which encode the α3, β3 and γ2 subunits of Laminin-332 [10]. Laminin-332 is a linking protein which binds basal keratinocytes to the underlying and when reduced or absent can result in a split in the lamina lucida within the epidermal basement membrane zone leading to a subepidermal [19]. The latest EB consensus recognises three major subtypes of JEB, generalised severe JEB, generalised intermediate JEB and localised JEB [10]. The generalised severe form of JEB, resembling the eponym Herlitz JEB is typically caused by nonsense mutations resulting in premature stop codons (PTCs) in the genes encoding Lamnin-332 leading to absent or markedly reduced levels of Laminin-332 [7]. Generalised intermediate and localised forms of JEB, most akin to the eponym non-Herlitz JEB, tend to be caused by a combination of a missense and a nonsense mutation in the genes encoding Lamnin-332 which lead to a milder reduction in Laminin-332 [20]. In addition to mutations in Laminin-332, both intermediate and localised forms of JEB can be caused by mutations in COL17A1 coding for XVII [10]. Patients with generalised severe JEB tend to pass away in the first year of life because of widespread severe skin blistering leading to sepsis, dehydration, respiratory failure, pneumonia, anaemia and failure to thrive [5, 7, 21, 22]. Exuberant , enamel hypoplasia, hoarseness, stridor, and mucous membrane involvement are also early features [21]. Patients have been documented to live into their teens but tend to develop severe cutaneous scarring leading to pseudosyndactyly and musculoskeletal contractures. Patients can also develop milia, alopecia, dental caries and a multitude of direct and indirect complications resulting from the blistering of the ocular, respiratory, gastrointestinal and genitourinary epithelium [22-26]. Ocular complications can include corneal blistering and erosions leading to scarring, symblephron and ectropion [3]. Gastrointestinal tract blistering can lead to dysphagia, oesophageal

13 Wound healing following surgery in epidermolysis bullosa

strictures and stenosis, constipation and malabsorption which can contribute to anaemia, delayed puberty, growth delay and osteoporosis [3, 4, 10, 27]. Blistering of the genitourinary tract can lead to strictures, stenoses and obstruction leading to infections and renal failure [24]. The most severe and feared complication of JEB generalised severe is partial or complete occlusion of the upper airway from acute or chronic consequences of blistering causing death [3, 26]. Some EB specialists therefore advocate for an elective tracheostomy if early symptoms of airway compromise are apparent [26, 28]. Patients with generalised intermediate JEB can have similar phenotypes and complications as patients with the severe subtype, but these tend to be less extensive, frequent and severe (Figure 3.) [10, 20]. Mortality is increased compared to the general population, but patients can live a normal life span [22]. Squamous cell carcinomas (SCCs) are a feature and rates are higher than in those with generalised severe JEB, likely because patients tend to live longer than those with severe disease [29-31]. Patients with localised JEB have less severe and extensive blistering than the generalised forms and very rarely suffer from systemic complications, although patients can suffer from nail dystrophy, milia, mucous membrane involvement, enamel hypoplasia and excessive dental caries [10, 20]. Other specific clinical syndromes of JEB include laryngo-onycho-cutaneous (LOC) syndrome caused by specific mutations in LAMA3A occurring in Punjabi Muslims, JEB localised inversa caused by mutations in Laminin-332, JEB with pyloric atresia [32, 33], JEB late onset, JEB with respiratory and renal involvement caused by mutations in ITGB4 and ITGA6 coding for Integrin α6β4, COL17A1 and ITGA3 coding for the α3 integrin subunit respectively [10].

14 Wound healing following surgery in epidermolysis bullosa

Figure 3. Widespread blistering and erosions on the back of a patient with generalised intermediate junctional epidermolysis bullosa.

1.2.3 Dystrophic Epidermolysis Bullosa

DEB is the second commonest form of EB with just over one-hundred cases documented in Australia and an estimated worldwide prevalence of about two to six cases per million [5, 6, 34]. DEB is caused by mutations in the COL7A1 gene encoding collagen VII, a major component of anchoring fibrils located in the upper most portion of the dermis helping to anchor it to the overlying epidermis [35, 36]. Fragility of the anchoring fibrils leads to splitting within the sublamina densa resulting in subepidermal blistering of epithelial tissues [37]. Two major subtypes of DEB exist, dominant DEB (DDEB) and recessive DEB (RDEB), which differ in their inheritance and severity [10]. DDEB, resembling the

15 Wound healing following surgery in epidermolysis bullosa

eponyms Cockayne-Touraine and Pasini are typically caused by dominantly inherited missense mutations in one allele of COL7A1 [34]. This leads to normal or reduced collagen VII within the skin, causing blistering that is less severe than the recessively inherited form, RDEB [10]. RDEB is typically caused by inheritance of two PTC causing mutations in COL7A1 on separate alleles, resulting in absent or severely reduced collagen VII [37]. Two subtypes of RDEB were recognised at latest EB consensus, RDEB generalised severe, resembling the eponym Hallopeau- Siemens RDEB and RDEB generalised intermediate resembling the eponym non- Hallopeau-Siemens RDEB [10]. Patients with DDEB develop blistering at birth or soon after affecting any area of the skin but preferentially acral sites and sites of repeated trauma [37]. Patients can develop scarring, milia, dystrophic nails, mucous membrane and scalp involvement that can lead to alopecia [10, 38]. Systemic complications are rare, but the disease can involve the gastrointestinal and ocular systems akin to RDEB, but unlike RDEB there is no increased risk of developing SCCs [10]. RDEB generalised severe is characterised by extensive and severe blistering of epithelial tissues (Figure 4.) [37]. One of the most striking features is the resultant cutaneous scarring leading to pseudosyndactyly, mitten deformities and limb contractures (Figure 5.) [4, 23]. Patients have increased rates of mortality and high rates of SCC [31, 39, 40]. Recurrent cutaneous infections and sepsis are features, which can contribute to glomerulonephritis, renal amyloidosis and IgA nephropathy causing renal impairment and failure necessitating dialysis or renal transplant [37, 41]. In addition to the complications found in DDEB, patients can develop exuberant granulation tissue, excessive dental caries as well as gastrointestinal and ocular involvement [10]. Gastrointestinal complications can include microstoma, ankyloglossia, dysphagia, oesophageal webs, strictures and stenosis, constipation, anal stenosis and malabsorption exacerbating and leading to failure to thrive, , growth retardation, delayed puberty, immunocompromise, anaemia, osteoporosis and vitamin deficiencies [3, 4, 42]. Patients may also have a propensity to develop cardiomyopathy [43]. Ocular complications are similar to what can be found in severe forms of JEB [3].

16 Wound healing following surgery in epidermolysis bullosa

Patients with RDEB generalised intermediate has similar signs and symptoms as RDEB generalised severe but are much less severe and extensive [10]. Other rare specific subtypes of DDEB and RDEB include pretibial DDEB and RDEB, DDEB and RDEB pruriginosa, DDEB nails only, RDEB inversa, RDEB centripetalis and DDEB and RDEB bullous dermolysis of the newborn [10].

Figure 4. Extensive blistering, erosions and scarring in a patient with recessive dystrophic epidermolysis bullosa.

17 Wound healing following surgery in epidermolysis bullosa

Figure 5. Pseudosyndactyly of the hands in a patient with recessive dystrophic epidermolysis bullosa.

18 Wound healing following surgery in epidermolysis bullosa

1.2.4 Kindler Syndrome

KS is the rarest form of EB with only one documented case in Australia [5]. This subtype typically involves autosomal recessive inheritance of two PTC mutations in the FERMT1 gene [44]. FERMT1 codes for the protein Kindlin-1, which helps to link intracellular proteins to cell membrane proteins, and abnormalities lead to disruption of keratinocyte cell architecture, adhesion to the dermis and abnormal [45]. Clinically this results in generalised, trauma induced blistering within multiple levels of the epidermis and beneath the basement membrane zone [10]. Blistering tends to improve with age but severe scarring can develop causing webbing of the fingers, toes and pseudoainhum [10, 44]. Patients can also develop milia, dystrophic nails and scalp involvement leading to alopecia [10]. Blistering can affect the gastrointestinal tract, particularly the oral mucosa and the ocular and urothelial epithelium leading to systemic complications such as anaemia [10]. As well as blistering, cardinal symptoms include photosensitivity, progressive poikiloderma, skin and a propensity to develop SCCs [44, 46].

Although it is easy to focus on the physical consequences of EB, it is prudent to remember the pronounced and reduction in the quality of life (QOL) these patients have as well as the impact the disease has on family and carers [47-49].

1.3 The Diagnosis of Epidermolysis Bullosa

When a case of EB is suspected from the history and examination, invasive tests such a skin biopsy and a blood test are required to confirm the diagnosis [1, 10]. If there are classical signs and symptoms, and a family history of confirmed EB, no further investigations may be needed to make the diagnosis [1]. EB was first classified by transmission electron microscope (TEM) findings on skin biopsy in 1962. Since then there have been numerous improvements in the diagnosis of the disease including the advent of immunofluorescence antigenic mapping (IFM) and genetic testing [50]. TEM is still used visualise pathogenic ultrastructural changes to confirm IFM results, as well when IFM results are

19 Wound healing following surgery in epidermolysis bullosa

inconclusive, although, in most circumstances IFM is recommended as the first line investigation to diagnosis EB [1, 51]. IFM is comparatively cheaper than TEM, more widely available and more sensitive and specific [10, 51, 52]. Like TEM, IFM requires a skin biopsy of a freshly induced blister which is targeted with fluorescent antibodies against proteins known to cause EB [52, 53]. The location of the fluorescence under the microscope helps to define the level of blistering and the quantity of the fluorescence is used to determine if protein expression is normal, reduced or absent, helping to diagnose the subtype of EB [52]. Mutational analysis is performed by amplifying individual exons and flanking introns of genes known to cause EB and then analysing the polymerase chain reaction (PCR) products via Sanger sequencing (SS) [54]. Readouts give specific information about the site and type of inherited mutations which is useful for diagnosis, predicting prognosis, genetic counselling, prenatal testing and for targets of future molecular treatments [52]. Next generation sequencing (NGS) is a relatively new technology, which sequences the whole genome or exome (protein component of genome) [55]. NGS may save time, cost and personnel compared to SS and is an exciting new technology used in the diagnosis of EB [56, 57].

1.4 The Management of Epidermolysis Bullosa

Unfortunately, despite the current advancements in the knowledge of the and diagnosis of EB, a cure for the disease does not exist [2]. Management is largely supportive with various strategies used to minimise the amount of blistering and erosions such as protective dressings and padding [58- 60]. Surveillance and treatment of secondary complications in severe disease is important, as well as management by a multidisciplinary team [58, 61, 62]. Numerous topical therapies are available, but there is a paucity of large-scale trials showing benefit and regular topical antibiotics tend to be discouraged because of the risk of developing resistant bacteria. Oral medications including tetracycline’s and sulforaphane have showed some benefit in EBS, colchicine has improved granulation tissue in JEB (published by us), thalidomide has led to improvement in DEB pruriginosa and LOC syndrome and phenytoin has shown to decrease

20 Wound healing following surgery in epidermolysis bullosa

blistering in JEB and RDEB [13, 63-72]. Despite these studies, no oral treatment is routinely recommended in EB. There are a multitude of experimental treatments which have been studied and which are currently in development for EB [2]. Cell based therapies include intradermal injections of allogeneic , which when used in patients with RDEB showed some transient wound healing [73, 74]; systemic injections of bone marrow non-hematopoietic stem cells helped to decrease blistering and improve healing in patients with RDEB [75]; injections of mesenchymal stem cells in children with RDEB helped with wound and healing [76]; and application of autologous revertant mosaic cells in patients with JEB helped improve wound healing and QOL [77, 78]. Bone marrow transplantation in patients with RDEB showed a marked reduction in blistering and an improvement in QOL, but unfortunately two participants out of the six who underwent this treatment passed away from complications of the therapy [79, 80]. Gene therapy with ex-vivo correction of fibroblasts and keratinocytes with viral or non-viral vectors is established but expensive and not without the inherent problems and limitations associated with this technology [2]. New emerging integrating strategies such as CRISP (clustered regularly interspaced short palindromic repeats)/cas editing, RNA trans-splicing and PTC read-through techniques have exciting promise and may improve the utility of this technology [2]. Protein replacement therapy with intravenous collagen VII for the treatment of RDEB has shown some promise in animals but human trials have been delayed because of the fear of toxicity [81, 82]. While these treatments are experimental, considerable progress has been made within this area with the help of funding from DEBRA (Dystrophic EB Research Association) giving promise of an exciting future for patients with EB. Despite this range of therapeutic strategies, surgery is still a large part of patient care in treating complications of the disease.

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1.5 Surgery and Epidermolysis Bullosa

Patients with EB require surgery for problems encountered within the general population as well problems directly and indirectly related to the disease. The majority of the surgical literature focuses on corrective surgery for common problems arising as a direct consequence of severe EB including pseudosyndactyly, cutaneous and musculoskeletal contractures, gastrointestinal tract and genitourinary tract strictures, SCCs, dental and ocular . This literature comprises of case series or case reports and tends to focus on clinician experience regarding differing surgical techniques and their functional outcomes for patients [83-89]. A small number of case series with low patient numbers and case reports detail surgical procedures in patients with EB for pathology not related to the disease such as caesarean sections, breast surgery and repairs [90-97].

1.5.1 Surgery for Pseudosyndactyly and Contractures

Repeated blistering causing cutaneous scarring is a feature of severe EB, particularly those with RDEB generalised severe. This can lead to pseudosyndactyly of the hand and foot, as well as cutaneous and musculoskeletal contractures necessitating corrective surgery [23]. Advanced scarring can cause a mitten deformity of the hand with fusion of adjacent digits and coverage by an epithelial ‘cocoon’ (Figure 5.) [98]. By far, most of the surgical literature is concerned with the corrective surgery of pseudosyndactyly and mitten deformities of the hand in patients with RDEB [83, 98]. Although this problem indefinitely reoccurs to some degree, patients can have a substantial postoperative improvement in function and QOL [23, 99-103]. This procedure typically involves degloving of the epidermal cocoon, if present, dissection of the underlying connective tissue to separate the digits, incision of contractures and closure of the defect with a split skin graft (SSG) [87, 100, 103, 104]. Some surgeons choose not to remove the epidermal cocoon and others may use K-wires to splint the fingers, but these may lead to , joint stiffness and joint damage [98, 101, 105]. As well as SSGs, other options described to close the cutaneous defect include cadaveric dermal grafts, composite cultured

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skin grafts, skin substitutes, skin flaps, full thickness skin grafts (FTSGs) and healing by secondary intent, although there are no studies comparing these different techniques [84, 99, 104, 106-110]. Intense postoperative splinting and rehabilitation is important and can lead to improved outcomes [105]. Areas affected by contractures may include the lower and upper limbs, axilla and the skin in and around the anus, mouth and eye [84, 87, 98]. Operations to improve these typically involve incision or lengthening of connective tissues and musculotendinous structures.

1.5.2 Surgery of the Gastrointestinal Tract

Patients with severe forms of EB can develop gastrointestinal tract complications including oesophageal strictures and stenosis or be born with pyloric atresia frequently necessitating surgical intervention [111, 112]. Open surgery and bougienage for the treatment of oesophageal strictures and stenosis has now been largely replaced by endoscopically guided balloon dilatation and fluoroscopy guided hydrostatic balloon dilatation [88, 102, 113]. Surgical techniques to correct pyloric atresia differ depending on the type of atresia but tend to require a , although laparoscopic corrections have been performed [114, 115]. Postoperative mortality for this complication of EB is high, likely in part to the severity of the disease in this subset of patients [114, 116- 118]. In addition to these procedures, tubes are commonplace in severe EB to improve nutrition and facilitate administration of medications in young patients. These may be inserted in an open or percutaneous fashion [88, 113, 119, 120].

1.5.3 Surgery of the Genitourinary Tract

Patients with JEB and RDEB have a high incidence of urological complications compared to the other forms of EB and may require urological intervention [24]. Common procedures include the dilatation or incision of urinary tract and genital strictures [121]. Ureteral and urethral stenoses often reoccur or form as result of

23 Wound healing following surgery in epidermolysis bullosa

instrumentation and may require long-term catheterisation, nephrostomy tube placement, stents or an ureterosigmoidostomy [122]. A circumcision may also be required in males due to recurrent scarring of the prepuce causing phimosis [84]. With renal impairment being a frequent feature of severe EB, patients may require haemo or necessitating a fistula to be fashioned or an indwelling peritoneal catheter inserted [123]. There is one documented case of a patient with JEB who had a successful kidney transplant [124].

1.5.4 Surgery of Squamous Cell Carcinomas

SCCs in patients with EB are particularly aggressive and tend to require wide local excision [40]. Unfortunately, amputation sometimes cannot be avoided [29, 125]. Mohs micrographic surgery may be beneficial, although sentinel lymph node biopsies are controversial [39, 126]. There are a variety of options to close the surgical defect such as those used in the surgery of the hand described above [84, 125].

1.6 Tattooing in Epidermolysis Bullosa

In our clinical experience, we have noted that some patients with EB, who have had their skin tattooed, have ceased developing blisters within the areas which were tattooed. Despite this clinical observation, there is no literature detailing any tattooing performed in patients with EB.

1.7 Surgical Wound Healing in Epidermolysis Bullosa

There are no comparative studies within the medical literature comparing surgical wound healing in patients with EB to the general population, although, various case series and case reports do comment on the postoperative surgical wound in patients with EB, with the majority detailing satisfactory healing [83, 87, 92, 103, 104, 114, 121, 124, 127, 128]. In the two largest series, one detailed 25 patients

24 Wound healing following surgery in epidermolysis bullosa

with DEB and JEB who had 110 surgical procedures and ‘all patients who tolerated surgery had satisfactory healing of wounds’ [87]; in the other, 45 patients with DEB had hand surgery, 41 hands had SSGs with greater than 90% take, 44 had 70- 90% take and nine had less than 70% take. 5% required a repeat graft [103]. In two smaller series, four patients with DEB who required SSGs for hand surgery had no postoperative complications [101], and seven patients with RDEB requiring FTSGs for hand surgery all healed successfully with no wound infections [104]. However, it is likely that the literature is subject to publication bias, with positive outcomes such as good healing being preferentially published. Whilst this is likely the case, some authors did report negative outcomes including one patient with RDEB who required a SSG for hand surgery which resulted in an infection and delayed wound healing [105], two patients experienced postoperative wound infections after caesarean sections [95] and one patient with DEB developed blisters at the site of a midline abdominal incision [129]. Despite limited evidence to suggest patients with EB have more postoperative wound complications compared to the general population, we have noted reluctance among some surgeons to operate on patients with EB due to this thus far unfounded concern. Interestingly in one published case report, a patient with JEB who had a mastectomy for breast cancer and who was originally refused reconstruction because of her EB, later had a reconstruction with no blistering, infection or wound dehiscence [96]. In our experience, postoperative wounds in patients with EB do heal well and do not frequently become infected or dehisce. In some patients with RDEB we have seen surgical scars fade very quickly and one author described after operating on patients with RDEB that ‘the wound heals very quickly because of the sensitized healing process and rapid turnover of the epidermis’ [104]. Although there is little doubt that anaesthesia for patients with EB is complicated and dealing with EB skin is challenging, we are worried that the reluctance of some surgeons to operate on patients with EB may be having a detrimental effect on this population [89, 130]. We have therefore consequently designed this study to further investigate surgical wound healing in patients with EB.

25 Wound healing following surgery in epidermolysis bullosa

1.8 Study Aims

1.8.1 Primary Aim

• Investigate postoperative wound healing in patients with EB by assessing postoperative wound complications and scar healing.

1.8.1 Secondary Aims

• Investigate problems patients with EB encounter in hospital, not related to the postoperative wound.

• Investigate whether tattooing of EB skin can help improve blistering.

26 Wound healing following surgery in epidermolysis bullosa

Chapter 2. Methods

2.1 The Australian National Diagnostic Laboratory Database for EB and the Australasian EB Registry

Male and female patients with confirmed EB living in Australia and New Zealand were included in this study. Participants were recruited from the Australian National Diagnostic Laboratory Database for EB (ANDLDEB) and the Australasian EB Registry (AEBR). The ANDLAEB was developed in 1996 and includes patients with EB who have had skin biopsies reviewed at the EB national diagnostic lab at St George Hospital, Sydney, Australia [61]. The AEBR was developed in 2006 to aid in EB research and includes patients with EB who have agreed to take part in the registry. The AEBR includes the majority of patients within the ANDLDEB, as well as patients who have been diagnosed with EB through family history or genetic testing [5]. Both the ANDLAEB and AEBR were created by Professor Dedee Murrell, consultant dermatologist at St George Hospital, Sydney and continue to prospectively enrol patients. There is a considerable overlap of patients within the ANDLDEB and the AEBR and patient data for this project was collated to remove any duplicates.

2.2 The ‘Surgical Wound and Scar Healing in EB’ questionnaire

The ‘Surgical Wound and Scar Healing in EB’ questionnaire contains twenty-two questions in a one-page document about a patient with EB’s experience of having surgery (Figure 6.). This questionnaire was specifically designed by the investigators for this thesis and has not yet been validated. The first question within the questionnaire asks the participant what type of EB they have. The next question asks the participant if they have had more than one surgical procedure, and if they have, to fill out a separate questionnaire for each surgical procedure. Surgery involving the eye and inside of the mouth were asked not to be included, as we specifically wanted to look at the healing of the

27 Wound healing following surgery in epidermolysis bullosa

skin. Questions three to eight were designed to gather more details about the surgery. Questions nine to eleven were developed to look at complications of the surgery. Questions twelve to sixteen assessed how the surgical wound healed. Question seventeen asks about any advice the participant would like to give to doctors looking after patients with EB after having surgery. Questions eighteen to twenty-one gathered information about any the patient has had. Question twenty-two was an open-ended question asking about anything else the patient would like to share with the investigators.

2.3. Sending and Receiving the Questionnaires

Postal addresses gained from the ANDLDEB and the AEBR were used to post copies of the questionnaire, as well as a patient information sheet, informed consent form and a revocation of consent form (appendix). If the patient was under eighteen there was an option for the patient’s legal guardian to sign the consent form and fill out the questionnaire with the patient. A return addressed envelope was also included so the questionnaire and consent form could be posted back to the Department of Dermatology, St George Hospital, Sydney without the patient incurring a financial cost. If an email address was listed within the ANDLAEB or AEBR, electronic versions of the above documents were emailed to that email address. Data from questionnaires was collected throughout 2015 and were collated in a tabulated form. This was then analysed both qualitatively and quantitatively with ranges and medians when data was discrete. This study was approved by a full board review by the Bellberry human research ethics committee, application number 2015-05-369.

28 Wound healing following surgery in epidermolysis bullosa

Figure 6. The Surgical Wound and Scar Healing in Epidermolysis Bullosa questionnaire

29 Wound healing following surgery in epidermolysis bullosa

Chapter 3. Results

A total of 351 living patients with EB are included in the ANDLDEB and the AEBR. Of these patients, 238 had postal or email addresses listed and were sent a paper or electronic copy of the questionnaire, information and consent forms. Sixty-two patients returned the questionnaire, a total of 26%. Forty-three patients reported having surgery, three reported having surgery and tattoos and one patient reported having tattoos alone. Fifteen patients reported having no surgery or tattoos. Of the 46 patients who reported having surgery, 94 questionnaires were completed for a total of 94 surgical procedures. A summary of the results of the questionnaires can be found in table 1 & 2.

3.1 Patient Demographics

The age range of the patients who returned the questionnaire was eight to 87 years with a median age of 46 years. Twenty patients were male and 27 were female. Twenty-nine patients had been diagnosed with EBS, 19 with EBS localised, six EBS generalised intermediate and three with EBS generalised severe; four had been diagnosed with JEB, two with JEB generalised intermediate and one with LOC syndrome; and 11 had been diagnosed with DDEB and three with RDEB. No patients had been diagnosed with KS.

3.2 Surgical Procedures

The number of surgeries per patient ranged from one to six surgeries with a median of one surgery per patient. The most frequent surgery reported was the excision of a skin lesion, followed by musculoskeletal surgery, caesarean section, appendectomy, inguinal , varicose vein surgery, skin biopsy, hysterectomy with or without a salpingo-oophorectomy, insertion and removal of a gastrostomy tube, , breast augmentation, , circumcision, carpal tunnel release, repair of pyloric stenosis, vasectomy, radical

30 Wound healing following surgery in epidermolysis bullosa

prostatectomy, drainage of an , peritoneal dialysis catheter insertion and removal, suprapubic catheter insertion and removal and tracheostomy. Musculoskeletal surgeries included repair of a bony deformity, tendinoplasty, repair of a skin defect with a skin graft, spinal fusion, surgery for pseudosyndactyly, toe joint fusion, contracture release of the hand, finger amputation, bone graft, nerve repair and jaw reconstruction. The number of each reported surgical procedure is represented in figure 7.

Figure 7. The number of each reported surgical procedure

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3.3 Skin Blistering

3.3.1 Postoperative Wound Blistering

Out of the 94 surgical procedures performed, five patients reported blistering at the surgical wound site after seven surgeries, totalling just over seven percent. In all except two, the patient felt that the blistering impaired wound healing. Three out of these five patients had EBS generalised severe, one had EBS generalised intermediate and the other had DDEB, none had EBS localised. The surgeries in which blistering occurred, were skin biopsies, caesarean section, cholecystectomy, hysterectomy and repair of a bony deformity.

3.3.2 Blistering at sites other than the Postoperative Wound

Eleven patients while in the hospital for 27 surgical procedures developed blistering not directly related to the postoperative surgical wound, totalling just over 28%. The majority of these reported blistering with bandages and tapes. Others reported blistering related to intravenous lines and under a plaster of Paris cast. One patient with RDEB reported a twelve by eight centimetre wound caused by a diathermy plate being removed after surgery, as well as blistering from manual transfers. One patient reported pruritus from bandages leading to blistering. As well as RDEB, patients who reported blistering under these circumstances had EBS generalised severe, EBS generalised intermediate and EBS localised.

3.4 Postoperative Wound Complications not including Blistering

Five participants reported postoperative wound complications not including blistering, after five surgeries. This included four postoperative wound infections requiring antibiotics occurring in three patients with EBS localised and one patient with DDEB. These

32 Wound healing following surgery in epidermolysis bullosa

postoperative infections occurred after the removal of a skin lesion, caesarean section, toe joint fusion and repair of a bony deformity. One seventy-year-old female patient with DDEB, reported dehiscence of the postoperative wound after an open appendectomy at age seven which caused a delay in wound healing.

3.5 Rate of Postoperative Wound Healing

Twenty patients had 34 postoperative wounds which they felt healed slower than someone without EB, 30 patients had 55 postoperative wounds which they felt healed at the same rate as someone without EB and three patients had five postoperative wounds which they felt healed faster than someone without EB. No specific EB subtype appeared to be associated with any particular rate of healing and each rate of healing did not have a particular EB subtype overrepresented in it.

3.6 Scar Healing

After 67 surgeries, the postoperative scar was reported to heal flat, after 18 it was reported to heal with a keloid scar and after seven it was reported heal as a . There were two reports of atrophic postoperative scars. There were 54 reports of the scar healing as the same colour as the surrounding skin, 20 reports of it being lighter, eight reports of it being darker and 12 reports of it being erythematous. The patients feeling about the appearance of the scar was gauged on a linear scale with zero representing very ugly to ten being perfect. No patient rated their scar as a zero, three rated it as a one, four as a two, three as a three, two as a four, 18 as a five, one as a six, 17 as an eight, 18 as a nine, 24 as a ten. The median rating was eight.

33 Wound healing following surgery in epidermolysis bullosa

3.7 Tattooing in patients with Epidermolysis Bullosa

Four patients reported a total of eight tattoos. These were located on the arms, ankle, lower back, leg and breast. Tattooing on both arms in a patient with EBS generalised intermediate caused blistering. One patient with EBS generalised intermediate, who had blistering occur on the wrist before the tattoo, had the blistering improve after the tattoo. The other two patients who had tattooing performed had EBS localised and did not have blistering at the sites of the tattoo before or after having the tattoo.

3.8 Answers to the Open-Ended Questions

The most common answer to the open-ended questions, reiterated the frustration of patients not being listened to, when they have asked healthcare workers specifically not to have tapes and bandages applied to their skin. Other comments included two patients remarking that EB patients take longer to heal, two patients felt wounds may heal better when they are left uncovered, two patients said to be wary about the use of plaster of Paris casts, two patients felt it was useful to bring their own dressings and involve healthcare workers familiar with EB, one patient pointed out that tape used over eyelids during anaesthesia can cause significant harm and one patient felt EB patients are more likely to get postoperative infections.

34 Wound healing following surgery in epidermolysis bullosa

Table 1. Patient demographics and data from the questionnaire regarding postoperative wound healing

35 Wound healing following surgery in epidermolysis bullosa

Table 2. Questionnaire results from patients who reported having a tattoo

36 Wound healing following surgery in epidermolysis bullosa

Chapter 4. Discussion of Findings

4.1 Response Rate and Participant Demographics

The low response rate may be due to a variety of factors. Although 15 patients who returned the questionnaire had not had surgery or tattoos, our feeling is that the majority of those who had not had surgery or tattoos did not return the questionnaire as there was little incentive for them to do so. In addition to this, patient details within the ANDLDEB and the AEBR are not routinely updated, as these are both maintained by a charity and securing funding is an ongoing problem. Therefore contact details may have changed leading to misdirected questionnaires [6]. The response rate for patients with each subtype of EB was comparatively proportional to the frequency of each subtype within the Australian population, with patients with EBS returning the most surveys [6]. There was a slightly higher representation of patients with RDEB, which may represent an increase in rates of surgery in this severe form of the disease or other factors such as a vested interest in EB research or population mobility, with severe patients moving house less frequently [6]. We also noted a low response rate from by patients in rural areas and consequently we completed a side project looking at the geographical distribution of patients with EB which was published in the Australian Journal of Dermatology (see appendix) [6]. The average age of the patients who returned the questionnaire was higher than the average age of the patients within the AEBR [5]. This may be because the cumulative incidence of surgery increases with age, but may also reflect the lower likelihood of younger people to return postal surveys [131]. Overall, there were a slightly higher number of females compared to males who returned the survey. Because of this low response rate, it would therefore be difficult to make a comment using this data on the incidence of surgery and the different types of surgery within the EB population. In the future, to increase response rates when targeting this population with surveys, these factors may need to be addressed as

37 Wound healing following surgery in epidermolysis bullosa

well as the method of contact, as postal surveys tend to have low response rates, especially when compared to telephone surveys [131].

4.2 Surgical Procedures

The most frequent surgical procedure was the excision of a skin lesion. Three patients with the highest total number of surgical procedures had multiple excisions of skin lesions among other procedures. These three patients were aged 77, 52 and 77 respectively and each had EBS localised. This is not surprising, since Australia has a high incidence of skin cancer and patients with EBS localised tend to live a normal life span [132]. As a whole, musculoskeletal procedures were particularly frequent, but variable in nature, with some surgeries being fairly specific for EB, such as surgery for the correction of pseudosyndactyly and others less so, such as spinal fusions. Most of the other frequent surgical procedures are fairly common within the general population including caesarean sections, , inguinal hernia repairs, varicose vein surgery and hysterectomies. We expected more patients to report having skin biopsies as these are often required for the diagnosis of EB [1]. Reasons for the low number of skin biopsies being reported may be because patients do not think of this is a significant surgery or patients may have been diagnosed with EB by other means, such as a typical clinical presentation with a family history of EB or genetic testing. We were surprised that two patients underwent breast augmentation given that this is typically a cosmetic procedure with significant risks and there is limited literature regarding this type of surgery in patients with EB. These two patients both had EBS localised. Overall from this data, it seems that the majority of the surgical procedures in patients with EB tend to not be related to their EB.

38 Wound healing following surgery in epidermolysis bullosa

4.3 Skin Blistering

4.3.1 Postoperative Wound Blistering

Interestingly, less than 10% of the surgical procedures resulted in blistering at the surgical site. This is less than we expected as trauma induced blistering is a common feature of EB. All of the patients who experienced blistering at the wound site had generalised forms of EB and this equated to about 20% of all patients with generalised forms of EB who completed the questionnaire. No patient with EBS localised, the most common form of EB, developed blistering at the surgical wound site. Because patients with EBS localised do not tend to get skin fragility at sites other than the hands and feet this was not unexpected. The distinction between localised and generalised EB is therefore for important in preoperative planning for patients with EB. Reasons for a lower rate of blistering than expected, may be because of patients not recalling blistering, as it may not have been significant, or that the dressing applied after the procedure may have obscured the blistering. This may be a true result and represent a low rate of surgical wound blistering in patients with EB. The rate of blistering at the surgical wound site in patients with EB is difficult to gauge from the surgical literature, as it is not typically commented on, and again may be subject to under reporting, although, blistering at the surgical site has been reported in at least one patient with DEB after abdominal surgery and another with RDEB after a caesarean section [10, 94, 129]. Half of the patients who experienced blistering at the postoperative wound site felt it interfered with wound healing. Although we did not ask these patients specifically if they experienced increased levels of discomfort or reduced QOL, it appears that postoperative wound site blistering may lead to negative outcomes for the patient. Careful handling of the skin during surgery should therefore be practiced to minimise this potential complication particularly in those with generalised forms of EB.

39 Wound healing following surgery in epidermolysis bullosa

4.3.2 Blistering at sites other than the Postoperative Wound

While in the hospital, a large number of patients developed blistering of the skin indirectly related to the surgery itself. This was mainly caused by extraneous adhesive medical devices being applied to the patient and when removed led to traumatisation of the skin. Being that this type of blistering is potentially avoidable, it reinforces the need to take upmost care of these patients while in hospital. Certain measures outlined in the anaesthetic literature detail ways to minimise blistering, such as cushioned blood pressure cuffs and suturing in intravenous lines instead of using adhesive bandages [86]. It is also suggested to treat some patients in specialised units with clinicians familiar with EB [130]. It is important to note that pruritus resulting from pharmacological and physical therapies may lead to blistering as it did in one patient who returned a questionnaire and that this should be monitored for and managed appropriately.

4.4 Postoperative Wound Complications not including Blistering

There was a fairly low rate of postoperative wound complications, with postoperative wound infections being the most common complication. One postoperative wound infection was reported after a caesarean section, giving a rate of about 14% from the seven reported caesarean sections within our study. Rates of postoperative wound infections after a caesarean section have been reported to be between five and 23%, which is comparable to the rate we found in our patients with EB [133-135]. At least one postoperative wound infection has been reported previously in the literature after a caesarean section in a patient with EB [95]. Out of the 23 excisions of skin cancers, one postoperative wound infection was reported, giving a rate of about four percent. This is again equal or lower than rates reported in patients within the general population [136]. With a low number of patients within this study reporting toe joint fusions and repair of a bony deformities, as well as limited literature regarding these surgical procedures and postoperative wound infection rates, it is difficult to

40 Wound healing following surgery in epidermolysis bullosa

compare postoperative infection rates for these operations in patients with EB to the general population. It is also difficult to make a definitive comparison on the rate of postoperative wound infections as a whole, compared to the general population, as multiple variables would first need to be controlled for, such as patient specific factors. We were surprised that there were no postoperative wound infections reported in patients with RDEB, which may result from patients with this severe form of EB having resilience towards skin infections. Although, this may also represent a sampling error as only three patients with RDEB were included in this study. Arguably the most important finding of this study was the low rate of postoperative wound dehiscence, with only one patient reporting this complication. Our feeling is that this is due to the simple properties of the skin, with the greatest tensile strength being within the dermis, which apart from in KS tends not to be affected in EB (figure 1.) [137]. Given that sutures are placed within the dermis after a surgical procedure, patients with EB therefore should have a similar strength postoperative surgical wound than someone without EB.

4.5 Rate of Postoperative Wound Healing

The majority of patients felt that the postoperative surgical wound healed at the same rate as someone without EB. Although a significant portion of patients did feel that the wound healed slower than someone without EB and a small fraction felt the wound healed faster than someone without EB. We were somewhat surprised that there was no pattern of a particular EB subtype having a particular rate of healing. We thought we might find that patients with EBS localised reported normal rates of healing, as only the palms and soles tend to be affected and the majority of the surgical procedures reported were not performed in these areas [15]. We also thought that we might see a pattern of either slower or faster healing in patients with generalised severe EB. Exuberant granulation tissue, poor nutrition, physiologic stress, chronic disease and more medical comorbidities are known to affect wound healing and are frequent in

41 Wound healing following surgery in epidermolysis bullosa

patients with generalised severe EB [138]. On the contrary, because of frequent wound healing in patients with severe generalised EB, ‘sensitized healing’ may lead to patients healing faster than patients without EB [104]. Despite this, we did not find a particular trend, although our findings may represent a combination of factors leading to both an increased or decreased rate of healing in different patients, negating an overall measured effect.

4.6 Scar Healing

Although the majority of the scars healed flat, a significant amount healed with a keloid scar. This was more than expected, as rates of keloid scarring in the general population tend to be less than we found [139]. The majority of the keloid scars reported in this study did not occur on sites typically associated with keloid scarring such as the chest, shoulder, back and ears [140]. Although low patient numbers may have influenced this result and patients may have misinterpreted the term keloid scarring, overall, we think this is an interesting observation and may point towards patients with EB having an exaggerated wound healing response. The majority of the patients reported postoperative scars that healed as the same colour as the surrounding skin, but a significant amount reported a scar that was lighter than the surrounding skin. These tended to be scars which were flat and may represent post inflammatory hypopigmentation. Due to lack of research into this area, low patient numbers and multiple possible contributing factors it is difficult to interpret the relevance of this. Despite there being a high reported rate of keloid scarring, we were surprised that the majority of the patients were very pleased about the appearance of the scar. This may reflect that patients with EB are used to having wounds and scarring present and may not bothered particularly by them, but may also represent overall that postsurgical scars tend to heal well in patients with EB.

42 Wound healing following surgery in epidermolysis bullosa

4.7 Tattooing in patients with Epidermolysis Bullosa

We were excited to find that one patient who had a tattoo at a site that previously blistered, no longer developed blistering at that site after the tattoo. We wonder if the tattooing process has some action of recruiting epidermal and dermal proteins which improve skin strength much the same as microneedling skin therapy does in the treatment of fine lines and scarring [141]. We consequently reviewed the microneedling procedure in preparation for another project investigating microneedling skin therapy in EB which was published in the International Journal of Women’s Dermatology (see appendix) [141]. It is important to point out that only one patient of the four had blistering at a tattoo site prior to having the tattoo which significantly limits the generalisability of our data. One reason for this may be that patients wanted to limit blistering with the tattoo and therefore preferentially choose sites that did not blister.

4.8 Answers to the Open-Ended Questions

Overall the answers to this question detailed the importance of listening to patients with EB and of healthcare workers being educated about the disease. This is important to move forward in the understanding of the disease and improve patient outcomes.

43 Wound healing following surgery in epidermolysis bullosa

Chapter 5. Limitations of the study

5.1 Study Biases

The data in this thesis, being that it is gained from a postal and email survey, is inherently affected by reporting, recall and selection bias. There is likely an element of reporting bias, with patients being more likely to respond to the survey if they had an experience that significantly impacted on them, or an abnormal outcome, rather than a surgical procedure that was uneventful. This may well be the case with the reporting of skin biopsies, as there was much lower amount reported than we would expect. We also feel that there may be a bias towards increased reporting of negative outcomes such as blistering, postoperative complications, slower and abnormal wound and scar healing as well as positive outcomes such as an improvement in blistering with tattooing. Being that the data relies on the recollection past experiences, patients may recall something that is different than what actually happened. Patients may not recall minor adverse events, such as mild blistering or infections. Also, as some patients had multiple procedures over a long period of time, negative or positive outcomes may have also been generalised to all surgical procedures reported. With the method of recruiting live patients by mail and email we have preferentially selected against patients who may be too unwell to complete the survey, patients who are in hospital having a surgical procedure or patients who have passed away recently. We have also selected towards patients with particular demographics who are motivated to fill out surveys and who have reduced population mobility, as the contact details in the ANDLDEB and the AEBR are not updated regularly.

5.2 Limitations of the Data

EB is a rare disease and our sample size was small, meaning the data gained from these patients may not represent the true characteristics of this population. It also means that the conclusions we have made may lack generalisability, particularly as

44 Wound healing following surgery in epidermolysis bullosa

there is much heterogeneity between the different subtypes of EB. A larger sample would help to better to depict subtype specific factors and if gave similar findings would provide a firmer base to lay more definitive conclusions on. Another major limitation of the study data is that there is no controlled comparison group to compare the study participants too. We therefore can only reliably state what we have found and attempt to compare this to our clinical experience and other published data. In doing so, we are at risk making conclusions which are subject to a large amount of confounding.

45 Wound healing following surgery in epidermolysis bullosa

Chapter 6. Conclusions and Future Research

Despite the limitations of our data, we feel we can draw some useful conclusions, which may be beneficial to patients with EB and the clinicians who look after them.

• It is unlikely that patients with localised EBS will develop blistering at the postoperative wound site, but about a quarter of patients with generalised EB may develop blistering which may interfere with wound healing.

• Postoperative wound infections do not appear to occur any more frequently in patients with EB than in patients within the general population, particularly so with excisions of skin lesions and caesarean sections and in those with EBS.

• Postoperative wound dehiscence is uncommon in patients with EB, especially in those with EBS.

• Postoperative wounds may heal at a similar or slower rate in patients with EB. The postoperative scar tends to heal flat and the same colour as the surrounding scar, but patients may have a propensity to develop keloid scars.

• It is fairly common for patients with all types of EB to develop skin blistering while in hospital at sites other than the surgical wound. Healthcare workers should be aware of this and educated about how to reduce the chances of this occurring.

• Tattooing may help to improve blistering in patients with EB, although this was based on an observation in only one patient.

With this information, we feel clinicians should be more confident to refer patients with EB for surgery and surgeons reassured about postoperative wound healing and complications. To make more definitive conclusions a study with similar aims could be designed with larger patient numbers and a comparison group without EB.

46 Wound healing following surgery in epidermolysis bullosa

Relevant publication arising from the writing of this thesis

The Rurality and Remoteness of Patients with Epidermolysis Bullosa in Australia. AG Harris, N Todes-Taylor, N Petrovic, DF Murrell. 2015. Australasian Journal of Dermatology

Pigmented hair thickening fibers, a camouflage technique for alopecia in patients with Epidermolysis Bullosa. AG Harris, M Kim, DF Murrell. 2015. Skin Appendage Disorders

Skin needling as a treatment for scarring: An up to date review of the literature. AG Harris, C Naidoo, DF Murrell. 2015. International Journal of Women's Dermatology

An analysis and correction of increased plantar pressures in a patient with epidermolysis bullosa – a step in the right direction. AG Harris, DF Murrell, MT Khan. Journal of the American Podiatric Medical Association. (accepted for publication)

Colchicine may assist in reducing granulation tissue in junctional epidermolysis bullosa. M Kim, S Jain, AG Harris, DF Murrell. 2016. International Journal of Women's Dermatology

Botulinum toxin A injection for chronic anal fissures and anal sphincter spasm improves quality of life in recessive dystrophic epidermolysis bullosa. C Chaptini, G Casey, AG Harris, D Wattchow, L Gordon, DF Murrell DF. 2015. International Journal of Women's Dermatology

Recessive dystrophic epidermolysis bullosa (RDEB) complicated by secondary hepatic amyloidosis. C Chaptini, G Casey, AG Harris, DF Murrell, L Gordon. 2015. Journal of the American Academy of Dermatology Case Reports

47 Wound healing following surgery in epidermolysis bullosa

Appendix

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49 Wound healing following surgery in epidermolysis bullosa

50 Wound healing following surgery in epidermolysis bullosa

51 Wound healing following surgery in epidermolysis bullosa

52 Wound healing following surgery in epidermolysis bullosa

53 Wound healing following surgery in epidermolysis bullosa

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