Relationship of Sun Exposure to Skin Cancer
Epidemiologic studies in the normal population have been used as evidence to support a role for sunlight as a cause of skin cancer. For example, a higher frequency of skin cancer reported in a) Caucasians with light colored skin and eyes and frequent sunburns, b) individuals with large outdoor exposures such as sunbathers and outdoor laborers, c) association with latitudes closer to the equator, and d) exposed areas of the body compared to covered areas. However, this relationship is most clearly and powerfully demonstrated in XP patients, where UV damage leads to an early onset and increased frequency of both non-melanoma skin cancer (NMSC) and melanoma. In XP patients, the median age of first NMSC was 9 years (Bradford et al. 2011
) compared to 67 years in the general population (). Similarly, the median age of first XP melanoma was 22 years, compared to 55 yr in the general population (). This highlights the profound role of an intact DNA repair system in providing protection against skin cancer, in effect, giving the average Caucasian individual more than half a century delay in the onset of skin cancer.
XP patients can develop hundreds of skin cancers. Compared to the general population, XP patients under age 20 years have a 10,000- fold increase in the frequency of NMSC, 2000-fold increase in melanomas, a 1000-fold increase in cancer of the sun exposed tissues of the eye and 100,000-fold increase tongue cancers (Kraemer et al. 1994
; Bradford et al. 2011
). The anatomic distribution of NMSC in XP patients is similar to that in the general population with over 80% occurring on the face, head and neck (Kraemer et al. 1994
). The distribution of melanomas is different from that of NMSC in XP patients and in the general population. Melanoma occurs more commonly on the extremities and in both groups more than 45% of melanomas were found on the extremities. This suggests that there are different mechanisms involved in the generation of melanoma versus NMSC. The similarity of the distribution of both melanomas and NMSC in both groups suggests that the mechanism of carcinogenesis in XP patients mirrors that in the general population. However, the reversal in median age of onset of NMSC and melanomas in XP patients (9 years and 22 years) in comparison to the general population (67 years and 55 years) () points to a greater role of sun exposure/ DNA repair in induction of NMSC.
The UV exposed areas of the skin, tongue and eye have a high cancer risk (). While there is an increased risk to the anterior portion of the eye (Ramkumar et al. 2011
), the lens is a barrier to UV penetration and acts as protection to deeper eye structures. Similarly, the UV exposed areas of the lips and tongue have an increased cancer risk compared to deeper, more shielded mucous membrane surfaces. The observation that covered areas of the skin and other tissues are highly protected in XP patients (), with lower cancer frequencies, demonstrates the benefit of UV protection in the prevention of sun induced malignancy.
XP patients under age 20 years have an approximately 50-fold increase in cancers of the brain and other central nervous system (Kraemer et al. 1994
). These include brain medulloblastoma (Giannelli et al. 1981
), glioblastoma, spinal cord astrocytoma (DiGiovanna et al. 1998
) and Schwannoma. These are not sunlight exposed tissues and the relationship of these cancers to DNA damage is not known. On the other hand, carcinogens in cigarette smoke bind to DNA and cause the type of damage that would be repaired by the NER system in normal cells (Maher et al. 1977
; Maher et al. 1987
). Thus XP patients are at greater risk of smoking induced cancer. A 34 year old smoker with XP died of lung cancer (Kraemer et al. 1994
). In a study of 106 XP patients followed at the NIH for almost 4 decades, the median age at death of the XP patients was 32 years, a significant reduction compared to the general population () (Bradford et al. 2011
). The median age at death of XP patients with neurologic degeneration (29 years) was younger than those patients who had no neurological degeneration (37 years) (). Neurological degeneration was second only to cancer in cause of death in XP patients.
Role of Sun Burning in the Development of UV Induced Skin Damage and Skin Cancer
Studies in the general population have dissociated the role of acute burning versus chronic lower exposures in the causation of skin cancer. The chronic exposure of light skinned, outdoor workers has been associated with the development of multiple basal cell carcinomas and squamous cell carcinomas. In contrast, acute blistering burns in childhood have been implicated as a cause of melanoma. However, experience with XP demonstrates that this relationship is more complex. XP typically presents as either of two diverse clinical scenarios. A young child of 1–2 years of age outdoors in a relatively shady environment can develop an alarming blistering or oozing eruption (). The onset of the eruption may be delayed for a day or so and may be misdiagnosed as impetigo. After repeated occurrences the parents learn to rigorously protect the child. Other children with XP do not burn after minimal sun exposure (). However, most XP patients do develop early onset freckling before the age of 2 years. XP children who do not burn but only freckle, may not utilize rigorous sun avoidance and paradoxically may accumulate more sun exposure and often develop skin cancers in early childhood (Bradford et al. 2011
). It is somewhat surprising that in the general population, blistering burns are associated with earlier onset of melanoma, while in XP, this is reversed. XP patients who never burned on minimal sun exposure were found to be significantly more likely to develop skin cancer at an earlier age than those who always or sometimes burned on minimal sun exposure (Bradford et al. 2011
XP patients with defects in complementation groups A, B, D and G tend to have blistering burns on minimal sun exposure; while those in groups C, E and variant do not ( and and ). However, all are at high risk to develop early onset freckling, lentigines and skin cancers. These observations dissociate the acute burning from the mechanism of UV carcinogenesis raise important unanswered questions about how the different abnormalities of DNA repair lead to increased cancer risk in all, but acute photosensitivity only in some. Clearly, the inflammatory reaction of acute burning is not necessary for the development of skin cancer in XP patients.
A Model of Photoaging?
Photoaging, or dermatoheliosis, describes changes to the skin from chronic exposure to the sun or UV radiation. This includes pigmentary changes and alterations in texture and color. Early damage to melanocytes appears as freckling (tan, symmetrical, round macules) and later as lentigines (colored variable intensity of brown with irregular shapes, sizes and borders). In contrast, solar elastosis gives the skin a bumpy, yellowed appearance and is thought to be secondary to damage to structural components of the dermis including elastic and collagen fibers. Telangiectasias are the vascular components of UV damage and atrophy becomes noticeable when the full spectrum of poikilodermatous changes are present. These changes are common in the sun exposed areas of light skinned Caucasians in the general population who have sustained excessive, chronic UV damage, where skin laxity, sagging, and wrinkles are prominent. In contrast, while patients with XP develop freckling at an early age followed by the development of large numbers of lentigos and telangiectasias, they do not develop the skin laxity, sagging, wrinkles or cutis rhomboidalis of the posterior neck. As described by Kaposi in 1874 (Hebra and Kaposi 1874
), they actually develop skin tightening-the opposite of wrinkling. This contrast dissociates the mechanisms causing the pigmentary and vascular changes (DNA damage in the epidermis and upper dermis) from the causes of damage to structural components of dermal elastic and collagen fibers. This may be explained in part by the absorption of shorter wavelength, DNA damaging, UVB by the epidermis and greater penetration of UVA into the deeper dermis with a direct damaging effect on protein. The degree of elastosis may serve as a “dosimeter” of the amount of UV reaching the dermis (Robbins et al. 1974
). Thus XP patients demonstrate severe epidermal changes with minimal UV exposure to the proteins in the dermis because of their defective repair of epidermal DNA damage.
A Model for Clinical Research: Chemoprevention of Skin Cancers
Because of the high frequency of skin cancers in XP patients and the associated morbidity, effective chemoprevention approaches would convey enormous benefit. In fact, XP has been used as a model for skin cancer chemoprevention studies. Since each patient may develop large numbers of new skin cancers significant differences may be observed with small numbers of patients. A trial of oral isotretinon conducted with only 7 XP patients demonstrated a statistically significant (63%) reduction in new skin cancers compared to the 2-year interval before treatment (Kraemer et al. 1988
). This controlled clinical trial was one of the first to conclusively demonstrate effective chemoprevention of any cancer in humans. Today isotretinoin and the related retinoid acitretin are widely used in patients at high risk of developing new skin cancers who have other predisposing conditions including post-transplantation and the nevoid basal cell carcinoma syndrome. T4 endonuclease V, a bacterial DNA repair enzyme, was also tested in a double-blind study of 20 XP patients and found to lower the rate of actinic keratoses and basal cell carcinoma (Yarosh et al. 2001
Specificity in Sensitivity to Damaging Agents
One of the lessons learned from XP is that patient hypersensitivity to damaging agents is specific. While XP cells are hypersensitive to killing by UV they have normal killing after x-rays. In normal cells the bulky DNA damage caused by UV is repaired by the NER system which is defective in XP patients (). Most of the X-ray damage is different and the X-ray repair systems are normal in XP cells. In fact, patients with XP who develop inoperable eye or internal tumors such as brain or spinal cord tumors have been treated with high dose x-irradiation as therapy and tolerated the treatment well (Grier 1919
; Giannelli et al. 1981
; DiGiovanna et al. 1998
). This is in contrast to patients who are hypersensitive to x-irradiation, such as patients with the nevoid basal cell carcinoma syndrome (NBCC). NBCC patients have a germline mutation in the PATCH gene, and their cells retain only one of the two normally present functional alleles. Basal cell carcinomas result when NBCC cells sustain a second hit, which can be the result of x-irradiation. NBCC patients who develop neuroblastoma at a young age and receive treatment with radiation therapy frequently develop large number of basal cell carcinomas in the radiation port, where they may also be at risk for development of additional central nervous system tumors (Kleinerman 2009
). These observations clearly highlight the differences in mechanisms of repair of UV induced versus x-irradiation induced DNA damage.
DNA Repair - Molecular Mechanisms of Carcinogenesis
The skin of XP patients is hypersensitive to sun exposure and this is reflected in a hypersensitivity of cultured skin fibroblasts following exposure to UV radiation (Ruenger et al. 2008
; Kraemer and Ruenger 2008
). Thus examination of cultured cells from XP patients provides an opportunity to obtain insights into detailed mechanism of the relationship of UV damage to carcinogenesis. For example, cells from XP patients are hypersensitive to killing by UV and by UV-mimetic chemical compounds such as benzo-a-pyrene in cigarette smoke (Maher et al. 1977
; Maher et al. 1987
; Kraemer and Ruenger 2008
). In addition, XP cells are hypermutable following UV exposure thereby linking sun exposure to somatic mutations.
UV exposure of DNA produces several types of stable dipyrimidine nucleotide photoproducts (Kraemer and Ruenger 2008
). The major photoproduct is the cyclobutane pyrimidine dimer (CPD) of adjacent thymines (T), cytosines (C) or mixed T and C. Also formed are 6-4 pyrimidine-pyrimodone TC photoproducts (6-4PP). These DNA lesions serve as substrates for the nucleotide excision repair (NER) pathway (). In normal cells the DNA distorting 6-4PP are repaired more rapidly (within 6 h) than the CPD (about 50% removed by 12 h). Neither photoproduct is repaired by XP cells. Unrepaired photoproducts are pre-mutagenic lesions. During replication, a DNA polymerase meeting an unrepaired photoproduct can stop replication – leading to cell death. Since the photoproduct distorts the nucleotides they do not code properly. Polymerases that bypass the photoproducts frequently incorporate the incorrect nucleotide (for example incorporating a T in place of a C that is involved in a TC photoproduct) (Lange et al. 2011
). This leads to a C to T mutation which is characteristic of UV mutagenesis. In cultured XP cells and UV treated plasmids grown in XP cells these C to T or CC to TT “UV signature mutations” are frequently found after UV exposure (Bredberg et al. 1986
; Gozukara et al. 1994
If purified plasmid DNA is exposed to UV and transfected (introduced) into cells, the plasmid DNA is subject to repair by the DNA repair processes of the cell. The efficiency of repair can be assessed by use of a plasmid that codes for a marker gene. The DNA damage in the plasmid can be measured or modified before introduction into the cells and then the effect measured (Emmert et al. 2002
). We used this assay to demonstrate that one UV photoproduct in the coding strand of the marker gene was sufficient to block its transcription in sensitive XP cells (Protic-Sabljic and Kraemer 1985
) thereby demonstrating the importance of DNA repair in removal of DNA damage that blocks transcription. Similarly, replicating plasmid coding for a suppressor tRNA marker that is assessed in bacteria revealed that the XP cells introduce a high frequency of mutations into the plasmids. Sequence analysis of the recovered plasmids showed that the mutations following UV exposure of the plasmid are frequently at sites of dipyrimidine photoproducts and lead to C to T mutations (Bredberg et al. 1986
). This is strong direct evidence of the role of UV in mutagenesis. These cellular studies have provided a molecular foundation for demonstration the UV induced origin of mutations found in non-melanoma skin cancer (Giglia et al. 1998
; Couve-Privat et al. 2004
) and melanomas (Daya-Grosjean and Sarasin 2005
; Wang et al. 2009
) in cancer suppressing genes [p53, PTCH and PTEN] in XP patients. This CC to TT UV “signature” has been used to link sun exposure to mutations in many other cancer related genes in melanomas and other skin cancers in the general population (Prickett et al. 2009
; Pleasance et al. 2010
; Wei et al. 2011
XP cells are defective in NER () (Van Steeg and Kraemer 1999
). This system serves to recognize DNA damage, excise the damage and replace the damaged region with undamaged DNA. Global genome repair (GGR) serves to identify DNA damage in the 99% of the DNA that is not involved in transcription. Transcription coupled repair (TCR) is triggered by a stalled RNA polymerase that contacts DNA damage in actively transcribed genes comprising the remaining 1% of the DNA. DNA photoproducts in the global genome are recognized by several proteins acting in tandem including double strand DNA binding protein 2 (DDB2) and XPC. TCR related proteins include Cockayne syndrome A and B. After recognition the DNA is unwound by XPB and XPD helicases which are part of the 10 subunit basal transcription factor IIH (TFIIH). These proteins are thus involved in both DNA repair and transcription of many other genes. The XPA protein maintains the open DNA region containing the damage which is then cut out by XPF /ERCC1 and XPG endonucleases as part of an approximately 30 nucleotide single stranded fragment. The resulting gap is filled in by DNA polymerase and ligase. The TCR pathway acts more rapidly than the GGR pathway and in fact shows strand specific repair with preferential repair of the transcribed strand. The NER pathway is closely coordinated so that if one of the proteins is defective then the entire pathway does not function correctly. Thus mutations in any of the above proteins lead to clinical diseases ( and ). The “XP variant” form of XP has normal NER. These patients have clinical XP with increased skin cancer susceptibility. Their cells are deficient in an error-prone DNA polymerase, polymerase eta, which normally serves to permit DNA replication past unrepaired photoproducts. Identification of this class of bypass polymerases provides insights into the varied mechanisms that organisms have developed to cope with DNA damage (Lange et al. 2011
XP Neurologic Degeneration
About 25% of the XP patients have progressive neurological degeneration (Bradford et al. 2011
). These patients often have defects in the XPA, XPB
gene ( and ). They are usually born with normal size and weight. The earliest clinical abnormalities are frequently absent deep tendon reflexes and high frequency hearing loss and these can act as screening tests. Affected individuals may have delayed developmental milestones. The age of onset and rate of progression of the neurological abnormalities is variable among patients. Typical involvement includes sensorineural hearing loss, progressive intellectual impairment which may progress in severe cases to slurred speech, loss of ability to walk, difficulty swallowing and requirement for use of a feeding gastrostomy. Imaging studies show thinning of the cortex of the brain with concomitant dilation of the ventricles, and thickening of the skull bones. The pathology is a primary neuronal degeneration without evidence of inflammation or infiltration by other cells. XP patients with neurological degeneration have a high mortality () (Bradford et al. 2011
Relationships Within the Family of DNA Repair Disorders
There are three related, clinically defined disorders of DNA repair that can be used as archetypes to understand the spectrum of genotype/phenotype relationships within this group ( and )(Kraemer et al. 2007
). Photosensitivity, neurologic/developmental abnormalities and skin cancer are important pathological features which can be used to distinguish between these three archetypes: XP, trichothiodystrophy (TTD) and Cockayne syndrome (CS). In addition, there are several related or overlapping disorders with similar features that form a family of syndromes involving neural, oncologic, cutaneous, developmental and other abnormalities. lists detailed clinical features which may be useful in distinguishing between XP, XP with neurologic disease, TTD, CS and XP/CS complex. While photophobia and skin sun sensitivity may be seen in all of these conditions, lentiginous hyperpigmentation is seen in XP but not TTD nor CS. Pigmentary retinal degeneration is seen in CS, but not in XP or TTD. More precise clinical delineation has permitted the identification of subtle overlap syndromes in patients with features of two of these disorders. diagrams the current state of the evolving genotype/phenotype relationships within this group. Phenotypes representing the clinical disorders are shown in red and molecular defects are shown in grey with the overlapping patterns showing the underlying molecular defect identified in patients within each phenotypic group. XP can be diagnosed on clinical criteria based on the presence of acute burning on minimal sun exposure, early onset freckling before the age of 2 years and skin cancer. While XP patients usually do not have developmental abnormalities, about 25% of XP patients develop progressive neurologic degeneration. TTD is a disorder characterized by short, brittle hair, and multisystem abnormalities (). TTD developmental abnormalities may be evident in the pregnant mother carrying a TTD affected fetus. These pregnancy abnormalities may include abnormal triple screen test results, preterm delivery, preeclampsia, placental abnormalities or HELLP syndrome (Moslehi et al. 2010
; Tamura et al. 2011
). The newborn may present with a collodion membrane, short stature, micrognathia, and have increased risk of infections, growth and developmental delay, congenital cataracts and other abnormalities. While patients frequently have photosensitivity, they do not develop skin cancer or the freckle-like pigmentary abnormalities of XP. In contrast to XP, the neurologic involvement in TTD patients is usually not one of progressive decline. CS has features of both disorders with photosensitivity and both developmental delay and progressive growth and neurologic decline, but not skin cancer. Cerebro-oculo-facial-skeletal syndrome is a severe variant of CS with abnormalities beginning in utero. Infants are born with contractures (arthrogryposis), thought to be due to decreased fetal movement, extreme microcephaly, congenital cataracts and facial dysmorphism (Laugel et al. 2008
; Laugel et al. 2010
). Careful and precise assessment of clinical features of each disorder has led to the identification of patients with several overlap syndromes. Patients with XP/TTD have features of both diseases, albeit mildly attenuated. They have tiger tail banding and hair shafts defects, but less prominent than occurs in TTD leading to longer hair. They are at risk for skin and possibly internal malignancies, but at a lower frequency than seen in XP. Similarly, overlap syndromes of XP/CS, CS/TTD and COSF/TTD have been described (). Different mutations in the XPD gene have led to the greatest heterogeneity in clinical phenotype. Patients with the rare UV-sensitive syndrome have mild photosensitivity without pigmentary abnormalities or apparent CNS defects (Itoh et al. 1996
). Their cells have the same transcription defects as CS cells and have been reported to have defects in the CS-A or CS-B genes (Horibata et al. 2004
; Nardo et al. 2009
) (). This suggests that the CNS defect in CS patients may be related to an additional property of the CS proteins.