Case histories

Case 1 (XP12BE - XP-A)

The patient was a 44 year-old female with XP who had been followed at NIH since diagnosis at age 4 years [4, 8, 9, 14, 26, 35] (Figure 1A and B and Table 1). She experienced the first acute sun-sensitivity reaction at age 3 months with erythema and swelling after minimal sun exposure. Over time she developed freckle-like pigmentation on the sun exposed skin of her face, neck and dorsal upper extremities. Her first pathologically documented skin malignancy was a sclerosing basal cell carcinoma of the cheek at age 8 years. Subsequently, the patient developed more than two hundred skin cancers, which were mostly basal cell carcinomas (BCC) with 1 squamous cell carcinoma (SCC). At age 19 years, she was treated with oral 13-cis retinoic acid for skin cancer prevention [36]. She responded well with reduction in new skin cancer frequency from 43 BCC in two years before treatment to 3 new BCC after two years of treatment. Eighteen months after the discontinuation of 13-cis retinoic acid, she developed 18 new skin cancers. In the following years, she practiced increased protection from the sun with improved protective clothing and sunblocking agents. She experienced progressive neurologic decline, which further contributed to reduced outdoor exposure. Over time, fewer skin cancers developed and her overall XP-related skin pigmentation faded with only few lesions present.

While her early developmental milestones were within normal range, neurological signs were noted at age 7 years, with loss of deep tendon reflexes. At age 8 years, an audiogram revealed bilateral hearing loss at 8,000 Hertz, which progressed over the years [9, 14]. Learning difficulties and an IQ of 79 were noted at age 15 yr. Computerized tomography (CT) of the brain at age 20 showed no focal abnormalities and no appreciable atrophy (Figure 2A). At age 21 years, truncal ataxia became more prominent and tongue movement appeared uncoordinated. At the age of 22, she began to lose pain sensation, the Romberg test became positive and she exhibited child-like behavior [9]. By age 37 years, she was immobile and required assistance to walk. Because she was unable to swallow solid food without choking a gastrostomy tube (G-tube) was placed for nutritional support. Despite this therapeutic measure, she continued to become markedly cachectic. At age 41 years, there was evidence of prominent brain atrophy on imaging studies (Figure 2B). Neurological deterioration progressed inexorably and she became completely unresponsive and expired at the age of 44. An unrestricted autopsy was performed. Compound heterozygous mutations were detected in the XPA DNA repair gene (Table 1) with G>T intron 3 splice acceptor, and G>C splice donor of exon 4 [37, 38]. The post-UV DNA repair rate (unscheduled DNA synthesis (UDS)) of the cultured skin fibroblasts was about 1% of normal [4, 35, 39, 40]. Post-UV survival of cultured skin fibroblasts was markedly reduced [41].

Case 2 (XP18BE- XP-D)

This 45-year-old female patient with XP had a history of progressive neurologic deficits [14, 26] (Figure 1C and Table 1). She presented to the NIH at age 5 years with hearing difficulty and developmental delay. In addition, she had marked photosensitivity with blistering on minimal sun exposure. At age 8 years, SCC was removed from her nose. Subsequently, she practiced extensive early sun protection and only developed 9 skin cancers during her lifetime (2 BCC and 7 SCC). At age 13 years, she was first noted to have high frequency sensorineural hearing loss and at age 30 years, a repeat audiologic evaluation revealed profound sensorineural hearing loss [14]. At age 31 she developed ataxia with a broad-based gait, eventually requiring confinement to a wheelchair at age 36. By age 39 years, she was unable to care for herself and needed to read lips and write notes in order to communicate. She developed progressive dysphagia and at age 44 years a gastrostomy tube was placed for nutritional support that proved successful for weight maintenance. Her final admission was for pneumonia and sepsis. She expired 3 days later and an unrestricted autopsy was performed. Laboratory testing demonstrated XP-D complementation group (Table 1) with compound heterozygous mutations in the XPD (ERCC2) gene: R616P and R683W. Cultured skin fibroblasts (XPKABE) showed post-UV UDS of 25-50% of normal but markedly reduced post-UV survival [41].

Case 3 (XP24BE- XP-C)

Case 3 was a 35-year-old female diagnosed with XP at age 6 years (Figures 1D and 3 and Table 1). During her disease course, she developed almost 200 skin cancers including BCC, SCC, 11 melanomas in situ (Figure 3G and H), and one invasive melanoma (0.65 mm). At age 11 years, she participated in a study with oral 13-cis retinoic acid (isotretinoin) for skin cancer chemoprevention with a good response [36]. She developed premature menopause before age 30 years.

At age 29 years, she was found to have an asymptomatic left frontal lobe, non-enhancing brain mass lesion (Figure 3A). The patient elected follow-up with serial MRIs. However, two years later, the lesion enlarged and became symptomatic. She underwent craniotomy with subtotal resection of the mass. Pathologic examination revealed a low grade astrocytoma (Figure 3C and D). She was treated postoperatively with external beam radiation therapy with a total dose of 5,940 cGy. As with other XP patients receiving radiotherapy [42], she had a normal skin response. The patient was stable until age of 34 years, when she developed expressive aphasia and fatigue. Imaging revealed recurrent tumor with abnormal increase of enhancement on the post-contrast scan (Figure 3B), a feature considered indicative of malignant transformation. Resection showed that the tumor had progressed to glioblastoma (Figure 3E and F). Worsening aphasia and right-sided hemiparesis complicated her postoperative course. She began temozolomide therapy. She did not have any unusual side effects to standard doses of temozolomide which was well tolerated. The patient’s condition continued to deteriorate, and she expired at home. A partially restricted autopsy was performed. Laboratory testing showed compound heterozygous mutations in the XPC gene (Table 1): A155X and 83bp insertion of intron 5.1 with stop codon 34 codons downstream [43].

Case 4 (XP1BE- XP-C)

Case 4 was a 49-year-old Caucasian female with XP, who had been followed at the NIH since age 24 years [4, 10, 14, 44, 45] (Figure 1E and F and Table 1). She had more than 200 biopsy-proven BCC and SCC and developed approximately 38 separate primary melanomas, half of which were in-situ lesions [46]. A number of these lesions responded to intra-lesional alpha interferon [47]. Bilateral orbital exenterations were performed for recurrent squamous cell carcinomas of the cornea and globe at age 32 and 36 years, followed by 5,000 rads of irradiation with a normal response [48]. In addition, she was found to have subclinical, asymptomatic neurologic disease, including right peroneal nerve weakness and subclinical hearing loss, without mental deterioration [10, 14, 44]. She had multiple urinary tract infections, chronic glomerulonephritis following left nephrectomy at age 40 years for staghorn calculi, premature menopause secondary to hypogonadotropic hypogonadism, and fatty liver. Just prior to death, she experienced shortness of breath and developed a superaventricular tachycardia. An unrestricted autopsy was performed [10]. She had mutations in the XPC gene (Table 1): homozygous deletion of AA Exon 8 del AA 1396–7, fs188 - stop 5 codons downstream [49]. Cultured skin fibroblasts had about 10% of normal post-UV UDS [4, 35, 39]. Post-UV skin fibroblast cell survival was reduced [41].

Case 1 (XP12BE- XP-A)

Neuropathology

Gross description: The brain weighed 660 g (normal for ~ 6 month old infant) and was bisected fresh (Figure 2C and Table 1). The leptomeninges were smooth and transparent. There was no venous congestion or evidence of acute subarachnoid hemorrhage. Generalized brain atrophy with marked dilatation of lateral ventricles and thinning of the corpus callosum was noted. The gyri/sulci were grossly within normal limits. Examination of the basal cerebral vasculature revealed no dilatation, thrombosis, atheroma, or other abnormalities. Coronal sectioning of the right cerebral hemispheres confirmed diffuse atrophy (Figure 2C) with marked ventricular dilatation. While the cortical ribbon was thin throughout the hemispheres, the loss of brain substance seemed greater in the subjacent white matter. Deep gray structures including the mamillary body, thalami, subthalamic nuclei, basal ganglia, and the hippocampus were grossly unremarkable. Serial sectioning of the right brainstem and cerebellum revealed no grossly significant abnormalities. The spinal leptomeninges were thin and translucent and the subarachnoid space was free of exudates. The spinal arteries and veins were unremarkable. Significant eye findings included exposure keratopathy and bilateral optic atrophy as previously described [26].

Microscopic description of sections from the midbrain, pons, medulla and hippocampus revealed neuronal loss and gliosis. In addition increased capillary density was seen in the thalamus. Sections from the right frontal and temporal cortex (Figure 2D) showed neuronal loss and gliosis accompanied by microglial activation. In addition, patchy white matter rarefaction was seen and highlighted by a LFB stain (Figure 2E). There were no neuritic plaques, tangles or neuronal inclusions. Sections from the right cerebellum demonstrated overall atrophy of the folia. Focal loss of Purkinje cells with increased numbers of torpedoes (Purkinje cell axonal swellings) and associated Bergmann astrocytosis was seen at the junction of the granular and molecular layers. The granular layer was mostly preserved with only mild rarefaction. A section from the dura was unremarkable. Electron microscopy of the radial nerve showed increased interstitial collagen with reduced density of large and myelinated fibers, axonal atrophy and occasional regeneration clusters consistent with axonal neuropathy (Figure 4A). Rare thinly myelinated fibers were seen. Denervation atrophy (esterase positive angular atrophic fibers, pyknotic nuclear clumps) of the skeletal muscles was present. Fiber type grouping, however, was not a feature. Temporal bone histology revealed marked atrophy of the cochlear sensory epithelium and neurons [14].

Other findings: The indicated cause of death was XP-related neurologic degeneration. Hemangioma of the lip, seborrheic keratosis, uterine leiomyoma and fibrocystic change in bilateral breasts were also identified (Table 1).

Case 2 (XP18BE- XP-D)

Neuropathology

Gross description of the skull was notable for a substantially thickened calvarium, measuring 2.3 cm (frontal) (Figure 5A), 1.7 cm (occipital) and 1.0 cm (temporal). There were two boney excrescences measuring 1.4x1.2x0.4 cm and 1.2x1.0x0.6 cm in the right middle fossa of the skull base. The brain weighed 760 g (normal for ~ 1 yr old child) and showed diffuse widening of the sulci consistent with global atrophy (Table 1). The cerebral gyri/sulci were unremarkable and the hemispheres were symmetric. Coronal sections of the hemispheres confirmed the diffuse cerebral atrophy with dilated ventricles and a thin corpus callosum, but no focal lesions were seen (Figure 5B). Cerebellar hemispheres and vermis also appeared atrophic. Examination of peripheral nerves including both brachial plexuses, and radial, median, ulnar, femoral, sural and vagal nerves was unremarkable.

Microscopic description: Sections from the right frontal and parietal cortex showed thinning of the cortical ribbon with extensive neuronal loss widespread vacuolization of the neuropil (Figure 5C and 5D). Vacuolization was most prominent in the deep cortical layers (resembling the pattern of coarse vacuolation termed status spongiosis); neuronal loss and gliosis were accompanying features. Neurofibrillary tangles, plaques or other inclusions or storage products were not identified. Sections from the hippocampus demonstrated atrophy, gliosis, and pyramidal cell loss and vacuolization of the neuropil. Hippocampal neuronal loss most prominently involved the CA2 and CA3 sectors. Sections from the right basal ganglia and mid brain-pontine junction revealed extensive neuronal loss and gliosis, Figure 6 (A-C). Sections from the right cerebellum showed focal loss of Purkinje cells and accompanying Bergmann gliosis (Figure 5E and 5F) with patchy myelin pallor of the cerebellar white matter (Figure 5G). A section from the grossly described boney excrescence in the right middle fossa showed cortical sclerosis with no evidence of tumor. Immunohistochemistry and special stains for GFAP and LFB were performed on sections from the cortex, hippocampus and cerebellum. The GFAP stain highlighted reactive astrocytes (Figure 5H).

Histology of the peripheral nerves (Figure 7A and 7B), pharyngeal muscle and esophageal muscle were unremarkable. Examination of the esophagus showed infiltration of the ganglia of Auerbach’s plexus by CD3 positive T-lymphocytes (Figure 7C and 7D).

Other findings: The immediate cause of death was aspiration pneumonia. Focal patchy consolidation, bronchial mucus plugging and a cluster of plant (vegetable) cells with alveolar and interstitial infiltration by neutrophils and lymphocytes were seen in both lungs. A small (0.2 cm), hemangioma was present on the patient’s back. Laryngeal ulceration, bilateral breast fibrocystic changes, mild renal glomerulosclerosis, subserosal uterine adenomyosis, and cholelithisasis were also present (Table 1). There was no ocular neoplasm identified. The eye findings were reported previously [26], and consisted of bilateral pinguecula, corneal pannus, exposure keratopathy, retinal gliosis and bilateral peripheral retinal pigmentary degeneration.

Case 3 (XP24BE- XP-C)

Case 4 (XP1BE- XP-C)

Neuropathology

Gross description: The brain weighed 1300 gm (normal for an adult) (Table 1) and was bisected fresh. The dura, cerebral hemispheres, and infratentorial structures were externally unremarkable. The optic nerves and chiasm were atrophic. There was patchy non-occlusive atherosclerosis involving the basilar artery and circle of Willis. Sequential sections through the hemispheres, brain stem, and cerebellum revealed no focal lesions. The spinal arteries and veins were unremarkable. Peripheral nerves including median, sural nerves and celiac plexus revealed no gross abnormalities.

Microscopic description: The cerebellum showed focal loss of Purkinje cells (Figure 8A) with accompanying Bergmann astrocyte proliferation, and gliosis of the molecular layer. The hemispheric cortical grey matter and subcortical white matter were unremarkable. The pituitary was unremarkable. Sections of optic nerves and geniculate nuclei revealed degenerative changes (Figure 8B and 8C) including marked axonal loss with gliosis and resultant fibrosis of the fibro-vascular bundles and trans-synaptic degeneration in the lateral geniculate body with gliosis, consistent with the patient’s 10-year history of bilateral orbital exenterations [48]. Sections of spinal cord revealed no pathologic changes; however, the dorsal root ganglia showed severe neuronal loss with sclerotic foci occupying previous neuronal sites (Figure 8D) as reported previously [10]. Peripheral ganglia (celiac) were unremarkable.

Multiple pulmonary emboli were the immediate cause of her death. The underlying cause of the pulmonary emboli was a clinically unsuspected well differentiated mucinous adenocarcinoma of endocervical origin (Figure 9A), which directly invaded the surrounding pelvic organs including ovaries and sigmoid colon, causing diffuse abdominal carcinomatosis that involved the diaphragm (Figure 9B), omentum, appendix, large and small bowel, and metastasized to multiple splenic hilar lymph nodes, spleen and lungs.

Other findings included obesity, glomerulonephritis, multiple simple renal parenchymal cysts, steatohepatitis, nodular regenerative hyperplasia of liver, right ventricular dilatation and left ventricular hypertrophy, and thyroid follicular adenomas. In addition, there was mild to moderate atherosclerosis of the left anterior descending artery, and moderate to severe atherosclerosis of the right coronary artery.

Neurodegeneration in XP patients

The present study represents the first detailed description of the postmortem neuropathologic findings in an XP-D patient. These features have been systematically compared to an XP-A and two XP-C patients (Table 1). Of note, all four patients were included in a large series of XP patients followed at the NIH for many years permitting close observation of disease progression and the effects of treatment [1, 14]. Major clinical neurologic abnormalities in XP patients include sensorineural hearing loss, peripheral neuropathy, microcephaly and dementia. The XP-A and XP-D patients were diagnosed with XP at age 4 and 5 years, respectively, and developed high frequency sensorineural hearing loss at a young age. Their neurologic status showed progressive decline with loss of ability to walk and talk leading to inability to care for themselves and death at age 44 and 45 years of age (Table 1).

The core neuropathologic findings of this study are profound global cerebral and pancerebellar atrophy in the XP-A and XP-D cases. The most striking gross finding was generalized brain atrophy (660 and 760 g) with brain size similar to that of a 6 to 12 month old infant [50]. Consistent with what has been described previously in XP-A patients [4–6, 11], we found brain atrophy involving all lobes. There was diffuse neurodegeneration with neuronal loss and gliosis of both hemispheres, similar changes in the cerebellum, basal ganglia, and corticospinal tracts. Retinal gliosis was identified in both cases [26]. In addition, the XP-D patient had a thickened calvarium, vacuolization of the deep cortical neuropil, patchy Purkinje cell loss, and infiltration of the ganglia of esophageal Auerbach’s plexus by T-lymphocytes. Intriguingly, calvarial thickening which has been described in XP-A patients from Finland [5] was not observed in the XP-A patient.

Both XP-A and XP-D patients developed dysphagia, which poses difficult challenges for clinical management. In addition to CNS dysfunction, we carefully evaluated the peripheral neuromuscular system, and found axonal neuropathy and chronic denervation atrophy of skeletal muscle including those that related to swallowing in the XP-A patient. The peripheral neuropathy has been previously reported and involves both motor and sensory nerves. The underlying pathogenic mechanism is thought to be a neuronopathy [7, 51]. In contrast, significant peripheral neuronopathy was not identified in the XP-D patient where section of the esophagus showed T-cell infiltrates of Auerbach’s plexus. This finding suggests an immune-mediated mechanism may be associated with acute autonomic and sensory neuropathy leading to impairment of both superficial and deep sensation in the XP-D patient.

Both the XP-A and the XP-D patients had severe progressive neurodegeneration. However, while the swallowing difficulty led to the placement of a G-tube for feeding of both patients, only the XP-A patient developed cachexia. The XP-D patient retained her weight, which suggests that cachexia is not directly related to the extent of neurodegeneration or the inability to swallow.

A patient with XP/Cockayne syndrome complex was described with a defect in the XP-G gene (XP20BE) [27]. He died at age 6 yr and had marked growth retardation with short stature and severe cachexia. However the autopsy findings of XP20BE were different from the XP-A and XP-D cases. His brain appeared proportionally small and not atrophic with a grossly normal cortical ribbon and an unusual tigroid pattern of demyelination. In addition, the patient had cataracts, and retinal pigmentary atrophy typical of Cockayne syndrome.

With respect to the two XP-C cases, one case showed unexpected carcinomatosis with endocervical adenocarcinoma. The other patient developed a glioblastoma, which arose from a pre-existing low-grade, diffuse astrocytoma. This observation is furthered supported by the IDH1 labeling of the tumor cells. It has been reported that as high as 90% of diffuse and anaplastic gliomas and secondary glioblastomas (arising from a pre-existing low-grade astrocytoma) are IDH1-positive [52]. The generalized neurodegenerative changes seen in the XP-A and XP-D cases were not observed in the XP-C cases although the older patient had focal loss of Purkinje cells and, neuronal loss in the dorsal root ganglion.

There was a clinical correlation between the skin abnormalities and the presence of progressive neurological degeneration. In a longitudinal study of 73 XP patients with audiograms examined at the NIH, all 17 patients who developed sensorineural hearing loss and progressive neurological degeneration had a history of acute burning on minimal sun exposure, including the XP-A and XP-D patients in this report [14]. This burning history was not present in the XP-C patients and they did not show progressive neurological degeneration. Further, cultured skin cells from XP patients with a history of burning on minimal sun exposure and neurological degeneration were hypersensitive to killing by ultraviolet radiation [41, 53].

Cancer in XP patients

Our previous data have shown that 65% (69/106) of XP patients developed sunlight-induced skin cancers. Non-melanoma skin cancer was increased 10,000-fold and melanoma was increased 2,000-fold in patients under age 20 [1, 2]. The XP-A, and XP-C patients developed hundreds of skin cancers (Table 1). The XP-A patient and XP-C patient XP24BE were treated with oral 13-cis-retinoic acid for chemoprevention of skin cancer [36]. They both had a good response, with no further skin malignancies identified at autopsy. The XP-D patient was sun protected from early childhood and developed only 9 skin cancers.

The XP-C patients developed internal cancers with a glioma in XP24BE and a cervical adenocarcinoma in XP1BE. XP patients have been estimated to have an 11-fold increase in internal neoplasms and a 50-fold increase in cancers of the central nervous system [2]. The reported tumors include Schwannoma in a 73 y/o [1], spinal cord astrocytoma in a 24 y/o, gastric cancer in a 50 y/o and lung cancer in a 37 y/o smoker [42]. Some carcinogenic components of cigarette smoke bind to DNA forming adducts that are repaired by the NER system. It is thought that the lung cancers in the young XP patients are related to mutations resulting from this unrepaired damage. The underlying mechanism of induction of the CNS neoplasms is not known.

DNA repair defects in XP patients

Defects in the repair-enzyme genes XPA through XPG are known to cause XP [12, 13, 54]. Cells from XP patients with mutations in the XPC DNA repair gene have defective global genome DNA repair and normal transcription coupled repair. In contrast, cells from patients with mutations in the CSA or CSB genes have defects only in transcription coupled repair. These patients have clinical Cockayne syndrome with severe progressive neurological degeneration with short stature and retinal degeneration but without an increase in cancer [12, 54]. However, cells from XP patients with mutations in the XPA and XPD genes have defective global genome repair and in addition, defective transcription coupled DNA repair. This additional transcription coupled DNA repair defect is associated with progressive neurological degeneration in humans (see for example [5]) and in mouse model systems [55]. Patients with trichothiodystrophy have mutations in the XPD gene in association with developmental abnormalities (including absent myelin in the brain) as well as sulfur deficient brittle hair, cataracts, bone abnormalities and increased susceptibility to infections, but no increase in cancer [12, 54, 56]. The level of DNA repair does not correlate with the clinical phenotype. Thus, while cells from XP-A patients with severe neurological degeneration generally have <2% of normal post-UV unscheduled DNA synthesis (UDS) in cultured skin fibroblasts, cells from XP-D patients, who also have severe neurological degeneration, have 25-50% UDS and cells from Cockayne syndrome patients have normal UDS. Further, cells from XP-C patients with no neurological abnormalities have 10% UDS [4, 35, 39, 41, 56]. Thus the relationship of mutations in the DNA repair genes to clinical phenotypes is complex and the precise mechanisms of these differences are not understood.

XP patients have defective repair of UV induced DNA damage to skin and eyes. Unrepaired DNA damage can lead to increased cell death and to a high frequency of somatic mutations in the surviving, dividing cells. This process is believed to underlie the skin sun sensitivity and high frequency of sunlight-induced cancers in XP patients. Skin tumors from XP patients frequently have evidence of UV type mutations [57, 58]. There is a good correlation between post-UV killing and neurological degeneration in cultured cells from XP patients. Thus, skin fibroblasts from XP-A and XP-D patients with severe neurological degeneration are more sensitive to post-UV killing than are cells from XP-C patients who have no neurological abnormalities [41]. However, UV radiation does not reach the brain because of shielding from the scalp and bones of the skull. It has been hypothesized that oxidative DNA damage generated through metabolism or other sources might produce DNA damage that is not repaired in the non-dividing cells of the nervous system leading to progressive neuronal death [8]. Most types of oxidative DNA damage are repaired by base excision repair, a process that is not defective in XP cells. Indeed, both XP-C patients received therapeutic doses of x-radiation (which produces oxidative damage) for treatment (brain tumor in XP24BE and orbital tumors in XP1BE and spinal cord astrocytoma in another XP-C patient [42]) (Table 1) with a normal response. However, certain types of products of oxidative DNA damage, such as cyclopurine deoxynucleosides, are repaired by the NER system. Impaired repair of this type of oxidative DNA damage has been proposed as a mechanism of the progressive neurodegeneration seen in these XP patients [59, 60].

Neuropathological examination

The patients were examined at the NIH under protocols approved by the Institutional Review Board of the National Cancer Institute. They gave their permission for use of identifying images. The families of the patients have given their consent for performance of the autopsies. The brain, peripheral nerves, muscles were fixed with 10% buffered formalin and embedded in paraffin. Histological examinations were performed on 5 μm thick sections using hematoxylin–eosin (HE). Selected sections were evaluated with special stains (luxol fast blue (LFB), Bielschowsky, Masson’s trichrome) and immunostains (glial fibrillary acidic protein (GFAP), synaptophysin, NeuN, neurofilament proteins, KP-1, beta-amyloid, Isocitrate dehydrogenase 1 (IDH1), p53, EGFR, MIB-1). For immunohistochemical analyses, 5 μm thick sections were serially cut in selected brain regions and brain tumors. The control subjects had no neurological abnormalities and showed no morphological changes in the brain. Slides were incubated in Tris with 3% goat serum for 15 min and then incubated for 1–2 h at room temperature with primary antibody. Detection was carried out using an automated slide stainer (DAKO; Autostainer) with a horseradish peroxidase/3,3^′-diaminobenzidine polymer-based detection system (DAKO; Envision+). No staining was observed in the sections incubated in the absence of either antibody.