It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.
Basal cell cancer (BCC) is the most common type of cancer among Caucasian populations (Green et al., 1996). Tumors often develop in chronically sun-exposed skin in elderly patients and exposure to ultraviolet radiation (UV) is considered a major risk factor (Gallagher et al., 1995). BCC exhibits unique growth characteristics, including lack of precursor lesions and tumor progression, dependency on specific connective tissue stroma and virtual inability to metastasize. In addition to prevalent sporadic BCC, numerous BCCs can be found at early age in patients with Gorlin syndrome (nevoid basal cell carcinoma syndrome).
Gorlin syndrome is an autosomal dominantly inherited disorder, where the underlying genetic abnormality is a mutation of the patched tumor suppressor gene (PTCH), located on chromosome 9q 22.3 (Gailani and Bale, 1997; Hahn et al., 1996; Johnson et al., 1996). Accordingly, loss of heterozygosity (LOH) in 9q 22.3 and/or PTCH mutations are frequently found in tumors from these patients. Alterations in the PTCH gene are also common in sporadic BCCs. Approximately 50% of all tumors have a mutated PTCH gene and 70% display LOH in the PTCH locus (Gailani et al., 1996; Taipale et al., 2001). The PTCH gene encodes a large transmembrane protein which functions as receptor for the diffusible hedgehog protein in the hedgehog signaling pathway (Toftgard, 2000). Loss of normal PTCH function due to mutations results in constitutive hedgehog signaling, promoting proliferation rather than differentiation. It has also been shown that normal PTCH protein down-regulates its own transcription, and thus a mutation of the PTCH gene results in overexpression of mutant PTCH mRNA (Undén et al., 1997). Alterations in the hedgehog-PTCH signaling pathway resulting in over-expression of GLI-1 is proposed a necessary step for development of BCC (Nilsson et al., 2000).
The most common genetic alteration detected in human cancer is mutation of the p53 gene (Greenblatt et al., 1994; Hollstein et al., 1991). Approximately 50% of BCCs show a p53 mutation (Ziegler et al., 1993). Most p53 mutations found in skin display a typical UV signature, i.e. CC to TT or C to T transitions at dipyrimidine sites (Brash et al., 1991; Moles et al., 1993). In contrast to the combination of a point mutation and LOH common in internal cancer, two or more point mutations are often found in both p53 alleles in BCC (Pontén et al., 1997). In a recent study of UV-exposed PTCH heterozygous knockout mice, p53 mutations were detected in 2/5 BCCs (Aszterbaum et al., 1999). p53 mutations are also frequent in non-neoplastic, morphologically normal keratinocytes from chronically sun-exposed skin. Keratinocytes over-expressing p53 protein can be found as dispersed positive cells or clustered as p53 positive clones (Jonason et al., 1996; Pontén et al., 1995; Ren et al., 1996). p53 mutations are common in both single, dispersed keratinocytes (Ling et al., 2001) and in epidermal p53 clones. At least 70% of epidermal p53 clones show a mutation in the p53 gene (Pontén et al., 1997; Ren et al., 1997; Williams et al., 1998). Epidermal p53 clones are often found adjacent to BCCs and squamous cell cancers (SCCs) in skin, and may represent a very early step in skin carcinogenesis (Rebel et al., 2001; Tabata et al., 1999).
Skin cancer, e.g. BCC, provides an excellent model for studying events important in carcinogenesis. Tumors often display characteristic mutations caused by a known carcinogen (UV) and BCCs exist both in a sporadic and hereditary setting. Furthermore, tumors are easily detected, diagnosed and can be sampled also as early lesions. Compelling evidence shows that dysregulation of PTCH and p53 pathways are deeply involved the pathogenesis of BCC (Brash and Pontén, 1998; Sarasin, 1999; Zhang et al., 2001). In a previous study we analysed microdissected tumor cells from 11 cases of sporadic BCC with respect to p53 mutations (Pontén et al., 1997). In the present study we have analysed PTCH gene alterations in microdissected fractions of sporadic BCCs as well as p53 mutations and PTCH gene alterations in BCCs from patients with Gorlin syndrome. LOH in 9q 22.3 appears more frequent in BCCs from Gorlin syndrome patients compared to sporadic BCCs. Furthermore p53 mutations appear different and less common in hereditary BCC. The unique power of microdissection enabled the detection of subclones containing cells with different p53 and PTCH genotypes within individual tumors. Analogous to what has been shown for p53 gene mutations in BCC (Pontén et al., 1997), subclones with a deleted PTCH gene can also be selected for during tumor development.
A summary of results is shown in Table 1. A total of 27 tumor samples from 9 BCCs previously partly characterized (Pontén et al., 1997) were analysed. In summary 7/9 tumors showed p53 protein over-expression in 10–100% of the tumor cells. Six tumors had two p53 mutations, two tumors only had one p53 mutation and one BCC had four different mutations in the p53 gene. At least one p53 mutation was common in different parts of the individual tumors. Four BCCs showed a partly different genotype with respect to p53 mutations (U-6, 8, 9 and 11) (Figure 1). A typical UV-signature was found in 12/18 mutations (Table 3). LOH in the p53 gene locus was found in 1/9 tumors. Analysis of LOH in PTCH locus in the 27 tumor samples showed allelic loss in 6/8 informative tumors. Four of these showed consistent LOH in all different parts of the individual tumors, whereas in the two remaining tumors (U-4 and U-11) certain portions of the BCCs did not show LOH in the PTCH locus. In one BCC (U-4) part of the tumor displayed a p53 mutation (codon 135) and no LOH in PTCH locus, whereas two other regions of the same tumor showed the same p53 mutation accompanied by LOH in PTCH. A similar result was found in U-11, where two p53 mutations without loss of PTCH allele was evident in one region of the tumor, whereas other areas showed the same p53 mutations with two different additional mutations in p53 and LOH in PTCH locus (Figure 2). In situ hybridization analysis of PTCH mRNA showed increased expression in all nine tumors (Figure 3). When combining p53 and PTCH data we found subclones with partially different genotypes in 5/9 BCCs (Figure 1). Analysis of normal epidermis and epidermal p53 clones from the nine patients did not show LOH in p53 or PTCH loci. In 6/9 epidermal p53 clones we found a missense p53 mutation, different from the mutations in adjacent tumors. p53 mutations were not detected in normal epidermis, except in U-5 where a germline mutation in p53 exon 7 (codon 235) was present in all samples including blood lymphocytes. No silent p53 mutations were detected in the nine tumors.
BCC from Gorlin syndrome patients
A summary of results is shown in Table 2. Twenty different BCCs from three patients were included. LOH in the PTCH locus was found in 17/19 tumors. In two BCCs (G5 and G9) we found no alterations in PTCH or p53 loci. Analysis of LOH in the p53 locus showed allelic loss in 1/12 informative BCCs. Immunohistochemistry showed p53 over-expression in 5% or less of the tumor cells in five tumors. A total of nine different p53 alterations, seven compatible with UV as mutagen, were found in 7/20 analysed tumors (Table 3). In patient I, 5/8 tumors showed a mutation in the p53 gene. One tumor (G1) showed a single nucleotide deletion in exon 4, whereas another BCC (G3) showed both a C-T transition and a single nucleotide deletion in codon 297 (exon 8). In G4 a double substitution was found (CC-TA) and in G7 a double-base substitution (GG-AC) in the intronic sequence upstream of exon 4 was detected. In patient II, 2/4 tumors showed p53 mutations with typical UV-signature. One BCC showed two different mutations, one double substitution (GG-AA) in a splice site in intron 4 in combination with a transition (C-T) in exon 9. The other tumor showed a double substitution (CC-TT) in exon 5. No p53 mutations were found in BCCs from patient III. One epidermal p53 clone was detected in close proximity to one BCC (G4). Microdissected keratinocytes from this p53 clone showed wild type p53 sequence.
The power of microdissection to obtain morphologically defined cell populations, combined with robust techniques for gene amplification and sequencing has enabled characterization of similarities and differences between hereditary and sporadic basal cell cancer. Independent of genetic background, BCC shows a fairly invariable histological picture as well as an indolent clinical behavior. Tumors grow slowly and without progression to metastatic disease despite sustained carcinogenic challenge of UV-radiation. The crucial importance of a disrupted normal hedgehog signaling pathway for development of BCC is well accepted. It is also well known that p53 gene mutations are common in these tumors, suggesting that dysregulation of the p53 pathway is involved in BCC carcinogenesis. Although both genes often are affected, the dominating finding relates to the hedgehog-PTCH pathway. An earlier analysis of BCCs from six xeroderma pigmentosum patients revealed PTCH mutations to occur in 90% of tumors compared to less common p53 mutations (38%) (D'Errico et al., 2000). In the present study LOH in the PTCH locus was very prevalent in both sporadic and hereditary BCC. These findings are in concert with previous studies and in agreement with BCC developing as a result of a first mutation in one PTCH allele accompanied by the frequent loss of one PTCH allele (Gailani et al., 1996; Taipale et al., 2001).
BCC displays an unusually low degree of genetic instability and perhaps only few genetic hits are required to transform a potential progenitor cell to a basal cell cancer cell. However, previous studies have shown that genetic progression with development of subclones containing additional p53 mutations exist within a tumor (Pontén et al., 1997). In the present study we show that also subclones with different genotype with respect to LOH in the PTCH locus also develop. Patients with Gorlins syndrome carry a germline mutation in the PTCH gene as a pre-requisite for early onset of BCC, in accordance with the two-hit hypothesis for tumor suppressor genes (Knudson et al., 1975). If knocking out the function of PTCH gene is necessary and perhaps sufficient for a BCC to develop it is expected that both alleles in all tumor cells are affected and indeed in situ hybridization results and the frequent finding of LOH in PTCH locus is consistent with this notion. Surprisingly, however, we found clones within sporadic BCCs with different LOH status in the PTCH locus. In tumors U-4 and U-11 we found areas of the tumors lacking LOH, whereas in other areas tumor cells had acquired LOH in PTCH locus. In tumor cells without LOH, it is still possible that both PTCH alleles are altered by mutations or small deletions not detectable by analysis of the three microsatellites in the 9q 22.3 region. This indicates that additional hits affecting PTCH gene are selected for during growth of BCC, analogous with what is evident for p53 mutations (Pontén et al., 1997). It is possible that PTCH gene function is already abrogated by mutations and, although improbable, additional LOH in the PTCH locus is a random event without consequence. The alternative explanation would imply that BCCs occasionally develop with only one hit in the PTCH gene.
Our findings suggest that some BCCs develop with p53 mutations preceding PTCH alterations (U-4) while others develop with PTCH alterations preceding a p53 mutations (U-9). The tumor U-11 appears to progress from a clone with two p53 mutations (codon 130 and 285) and without LOH in PTCH, into different subclones with selection for both loss of one PTCH allele as well as a selection for additional p53 mutations. Perhaps growth advantage can be achieved from additional disruption of both the p53 and PTCH gene and that the timing of these events is of lesser importance.
The p53 pathway plays a key role in human carcinogenesis and p53 mutations have been detected in a variety of skin tumors including precursor lesions. The vast majority of p53 mutations in sporadic BCCs were typical missense mutations and often showed a UV-signature. Another major finding in this study was that p53 mutations in BCCs from Gorlin patients were partly different compared to sporadic BCC. There was also a difference in p53 protein expression where only very few tumor cells showed p53 immunoreactivity in the hereditary tumors. In BCCs from Gorlin syndrome patients we found eight mutations in 12 tumors from two of the three patients with Gorlins syndrome. The detected p53 mutations differed to certain extent from mutations found in sporadic BCC. Two tumors showed a single nucleotide deletion, not earlier reported in BCCs, SCCs, actinic keratosis or epidermal p53 clones. In addition, two other BCCs showed double-base substitutions of an unusual type (CC-TA and GG-AC). The reason for finding such rare mutations in hereditary BCC is unclear. There is no obvious explanation why a cell with a germline PTCH mutation would achieve different type of mutations when challenged with UV-radiation. In a recent study novel p53 mutations as well as UV-specific PTCH mutations were detected in a sporadic BCC (Ratner et al., 2001). One possible explanation could be that patients with Gorlin syndrome avoid solar radiation due to propensity for skin cancer and that several mutations are secondary to other mutagenic events than UVB-induced DNA damage, e.g. oxidative stress (Kawanishi et al., 2001).
Clusters of p53 immunoreactive keratinocytes are abundant in chronically sun-exposed skin (Brash and Pontén, 1998; Jonason et al., 1996). Tumor risk in mouse skin has recently been shown to correlate well with frequency of epidermal p53 clones (Rebel et al., 2001). Several studies have also analysed p53 clones in human skin, however, no study has shown a direct link between BCC or SCC and epidermal p53 clones (Pontén et al., 1997; Ren et al., 1997; Williams et al., 1998). Epidermal p53 clones may well represent an expansion of undifferentiated keratinocytes harboring a p53 mutation. Continuous solar radiation would favor expansion of such cells due to a resistance to UV induced apoptosis. Such p53 clones could thus be an initial step in BCC or SCC development. In one study, LOH in 9q was found in 30% of examined p53 clones suggesting a possible role as forerunners of BCC (Tabata et al., 1999). In skin adjacent to the BCCs, we found a point mutation in 6/9 p53 clones. These mutations were never the same as the mutations found in the BCCs. In skin adjacent to the BCCs in Gorlin patients p53 immunoreactivity was extremely sparse and we could only detect one epidermal p53 clone in one case (G4). This discrepancy between patients with sporadic BCCs and Gorlin patients is unclear. It is possible that it reflects a difference in life-time sun exposure despite occurrence of UV-signature mutations also in Gorlin patient tumors. There is no other obvious reason why patients with germline mutations in the PTCH gene should have a lesser amount of epidermal p53 clones. Another possible explanation is that Gorlin patients develop ‘invisible’ p53 clones due to p53 mutations not resulting in p53 protein overexpression, analogous to the low p53 expression found in BCCs from these patients. ‘Invisible p53 clones’ have previously been detected in skin from a patient with xeroderma pigmentosum (Williams et al., 1998).
Microdissection of morphologically defined tumor cells from sporadic and hereditary BCCs have revealed a high frequency of LOH in PTCH locus as well as frequent mutations in the p53 gene. In conclusion, the pattern of PTCH alterations appears similar in both type of tumors, whereas the type of p53 mutations and p53 protein expression shows disparity. Furthermore we found that sporadic BCCs often consisted of subclones with genetically linked tumor cells. In such subclones additional alterations in the p53 gene and/or the PTCH gene had been selected for during tumor development.
Materials and methods
Patients and tumors
Nine BCCs, seven from chronically sun-exposed skin, from eight patients (age 49–85 years) were investigated (Table 1). Excised tumors were obtained from the Department of Plastic Surgery, Uppsala University Hospital. Morphologically, four tumors were of solid type, three were superficial and two were mixed solid/superficial. A central slice from the tumor, including adjacent normal skin, was snap frozen and stored at −70°C prior to immunohistochemistry and microdissection. The remaining part of the excised tumor was fixed in buffered formalin and hematoxylin-eosin stained for routine diagnosis.
BCCs from Gorlin patients
Twenty BCCs from three patients (age 37–72 years) with Gorlin syndrome were included (Table 2). None of the patients had a history of radiation treatment. The patients were diagnosed by clinical manifestations and genetic analysis of the PTCH gene (Undén et al., 1997). Patient I was a 72-year-old male with a 5 base pair duplication in exon 16 (nucleotide position 2788), patient II was a 37-year-old male with a 23 base pair deletion in exon 2 (nucleotide position 253) and patient III was a 61-year-old female with a 4 base pair deletion in exon 3 (nucleotide position 416–419) of the PTCH gene. Biopsies, obtained from the Department of Dermatology, Karolinska Hospital, were snap frozen on dry ice and stored at −70°C subsequent to analysis. One section was stained with hematoxylin-eosin for diagnosis.
Sixteen μm thick cryosections were made and air dried at room temperature for 1 h. Immunostaining was performed essentially as previously described (Pontén et al., 1997). Tissue sections were incubated with a primary monoclonal anti-p53 antibody DO-7 (DAKO, code M7001) and biotinylated rabbit anti-mouse antibody (DAKO, code E354) was used as secondary antibody. The reaction was visualized by avidin/biotin (DAKO code K355), using diaminobenzidine (DAB) as chromogen. Mayer's hematoxylin was used for counter-staining.
Cell samples were acquired from multiple areas within the same tumors as well as from epidermal p53 clones and normal skin. In brief, p53 immunostained sections were immersed in 1×PCR buffer (10 mM Tris-HCL pH 8.3, 50 mM KCl) and microdissection was conducted at the microscope using a small scalpel (Alcon Ophthalmic knife 15°). The amount of cells in each sample ranged from 50 to >1000. Forty-five samples were obtained from nine sporadic BCCs (nine from normal epidermis, nine from epidermal p53 clones and 27 from tumor cells). A total of 25 samples were collected from the three patients with Gorlin syndrome (four from normal epidermis, one from a p53 clone and 20 from BCC tumors). Microdissected cells were transferred to tubes containing 50 or 25 μl PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl). Cells were incubated at 56°C and lysed by addition of 2 μl freshly prepared proteinase K solution (20 mg/ml, dissolved in re-distilled water) for 1 h. Proteinase K was heat inactivated (95°C, 10 min) and samples were stored in −20°C prior to PCR.
Amplification and DNA sequencing
PCR amplifiction and DNA sequencing was essentially performed as previously described (Berg et al., 1995). In summary, exons 2–11 of the human p53 gene were amplified in a multiplex/nested configuration. Outer multiplex amplification was performed in one tube with 12 primers located in intronic sequences flanking the six exons (4–9), and in a separate tube with six primers located in intronic sequences flanking four exons (2, 3, 10 and 11). The outer PCR was performed in a mixture (50 μl) containing sample (5 μl cell lysate), 10 μl Tris-HCL (pH 8.3), 2.0 μM MgCl2, 50 μM KCl, 1 ml/l Tween 20, 0.2 mM dNTPs, 0.1 μM of each primer, 1.0 U of AmpliTaq DNA polymerase and 2.0 U of Amplitaq DNA polymerase, Stoffel Fragments (both from applied Biosystems, CA, USA) for 30 cycles. After dilution of the reaction mixture (25-fold for p53 exons 2, 3, 4, 5, 7, 8, 10, 11 and 100-fold for exon 6, 9), the inner region specific amplifications were performed for p53 exons 2–11. The amount of template used for inner PCR was 2 μl for all exons except 6 and 9 where 0.5 μl was used. For all exons except exon 5 inner PCR (50 μl reaction) was performed using the same reagents as in the outer PCR except that only 1.0 U of AmpliTaq DNA polymerase was used. For exon 5, 1.0 U of Stoffel Fragments was used as well as different concentrations of KCl (10 μM) and MgCl2 (2.5 μM). The temperature cycles consisted of denaturation at 94°C 0.5 min (98°C 0.25 min for inner PCR exon 5), annealing at 50°C for outer PCR, 63°C for inner PCRs for 0.5 min and extension at 72°C for 1 min. Each PCR was initiated by a 5 min denaturation at 94°C (3 min 98°C for inner PCR exon 5), and the final cycle was followed by a 10 min extension at 72°C. Perkin Elmer 8600 thermocycler was used for all amplifications.
The p53 gene sequencing analysis was carried out using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems). The DNA sequence was determined by direct sequencing on the ABI PRISM 377 (Applied Biosystems). Each sample was sequenced in both directions and an independent amplification and DNA sequencing starting from the original cell lysate confirmed exons containing mutations.
Loss of heterozygosity
Two p53 microsatellites consisting of an intronic (AAAAT)-repeat in intron 1 (Futreal et al., 1991) and a (CA)-repeat located downstream of exon 11 (Jones and Nakamura, 1992) were co-amplified with three microsatellites (D9S280, D9287, D9S180) in the 9q22.3 region (Gyapay et al., 1994; Holmberg et al., 1996). One primer in each pair was fluorescent labeled. For D9S280 and D9S280 and D9S180 amplifications the HEX dye label was used while the remaining microsatellites were labeled with 6-FAM dye label. The amplification mixture comprised 10 mM Tris-HCl (pH 8.3), 2.25 mM MgCl2, 50 mM KCL, 0.1% Tween 20, 0.2 mM dNTPs, 1.6 μM of each p53 microsatellite primers, 3.3 μM of D9S280 and D9S180 primers, 0.8 μM of D9S287 primers and 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems) in a total volume of 30 μl. We used 8 μl of the cell lysate as a template.
Multiplex PCR was initiated by a 12 min denaturation at 94°C followed by a cycling profile (35 cycles) comprised of denaturation at 94°C, annealing at 60°C and extension at 72°C, with 1 min duration at each temperature. The final cycle was followed by a 10 min extension at 72°C. The amplicons were diluted 1 : 10 with deionized formamide. An internal, labeled marker (Tamra 500) was used as size standard. A 4% denaturing polyacrylamide gel for ABI PRISM 377 (Applied Biosystems) was used and GeneScan software (Applied Biosystems) was employed for quantification and interpretation of raw data output.
The criterion for loss of heterozygosity was based on allelic imbalance in the tumor (T1 : T2) divided by the allelic imbalance in the normal (N1 : N2). An allele ratio (T1 : T2)/(N1 : N2) of less than 0.6 was scored as LOH. Allele ratio 1/(T1 : T2)/(N1 : N2) was used when the expression (T1 : T2)/(N1 : N2) was above 1.00. Loss of heterozygosity was interpreted as negative when the allele ratio was more than 0.6. LOH results were confirmed by a second PCR amplification and fragment analysis using the starting sample material.
In situ hybridization
Preparation of PATCHED–RNA probes and in situ hybridization were performed as previously described (Undén et al., 1997) with the exception that two PTCH anti-sense probes from different parts of the cDNA were used. Briefly, two human cDNA fragments (bases 190–628 and 3625–4269) were cloned into PGEM5, appropriately linearized and in vitro transcribed to obtain a sense and two different anti-sense probes. Paraffin-embedded sections of 11 mm were treated with proteinase K (Sigma Chemical Co) and washed in 0.1 M triethanolamine buffer containing 0.25% acetic anhydride. Subsequently, sections were hybridized overnight with 2.5×106 c.p.m. of 35S labeled anti-sense or sense probe at 55°C. Autoradiography was performed for 14 days. After development of the photographic emulsion, slides were stained with H&E.
Aszterbaum M, Epstein J, Oro A, Douglas V, LeBoit PE, Scott MP, Epstein Jr EH . 1999 Nat. Med. 5: 1285–1291
Berg C, Hedrum A, Holmberg A, Pontén F, Uhlen M, Lundeberg J . 1995 Clin. Chem. 41: 1461–1466
Brash DE, Pontén J . 1998 Cancer Surv. 32: 69–113
Brash DE, Rudolph JA, Simon JA, Lin A, Mckenna GJ, Baden HP, Halperin AJ, Pontén J . 1991 Proc. Natl. Acad. Sci. USA 88: 10124–10128
D'Errico M, Calcagnile A, Canzona F, Didona B, Posteraro P, Cavalieri R, Corona R, Vorechovsky I, Nardo T, Stefanini M, Dogliotti E, Rebel H, Mosnier LO, Berg RJ, Westerman-de Vries A, van Steeg H, van Kranen HJ, de Gruijl FR . 2000 Oncogene 19: 463–467
Futreal PA, Barrett JC, Wiseman RW . 1991 Nucleic Acids Res. 19: 6977
Gailani MR, Bale AE . 1997 J. Natl. Cancer Inst. 89: 1103–1108
Gailani MR, Leffell DJ, Ziegler A, Gross EG, Brash DE, Bale AE . 1996 J. Natl. Cancer Inst. 88: 349–354
Gallagher RP, Hill GB, Bajdik CD, Fincham S, Coldman AJ, McLean DI, Threlfall WJ . 1995 Arch. Dermatol. 131: 157–163
Green A, Battistutta D, Hart V, Leslie D, Weedon D . 1996 Am. J. Epidemiol. 144: 1034–1040
Greenblatt MS, Bennett WP, Hollstein M, Harris CC . 1994 Cancer Res. 54: 4855–4878
Gyapay G, Morissette J, Vignal A, Dib C, Fizames C, Millasseau P, Marc S, Bernardi G, Lathrop M, Weissenbach J . 1994 Nat. Genet. 7: 246–339
Hahn H, Wiking C, Zaphiropoulos PG, Gailani MR, Shanley S, Chidambaram A, Vorechovsky I, Holmberg E, Unden AB, Gillies S, Negus K, Smyth I, Pressman C, Leffell DJ, Gerrard B, Goldstein AM, Dean M, Toftgard R, Chenevix-Trench G, Wainwright B, Bale AE . 1996 Cell 85: 841–851
Hollstein M, Sidransky D, Vogelstein B, Harris CC . 1991 Science 253: 49–53
Holmberg E, Rozell BL, Toftgard R, Rebel H, Mosnier LO, Berg RJ, Westerman-de Vries A, van Steeg H, van Kranen HJ, de Gruijl FR . 1996 Br. J. Cancer 74: 246–250
Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM, Quinn AG, Myers RM, Cox DR, Epstein Jr EH, Scott MP . 1996 Science 272: 1668–1671
Jonason AS, Kunala S, Price GJ, Restifo RJ, Spinelli HM, Persing JA, Leffell DJ, Tarone RE, Brash DE . 1996 Proc. Natl. Acad. Sci. USA 93: 14025–14029
Jones MH, Nakamura Y . 1992 Genes Chrom. Cancer 5: 89–90
Kawanishi S, Hiraku Y, Oikawa S . 2001 Mutat. Res. 488: 65–76
Knudson AG, Hethcote HW, Brown BW, Levine AJ . 1975 Proc. Natl. Acad. Sci. USA 72: 5116–5120
Ling G, Persson Å, Berne B, Lundeberg J, Pontén F . 2001 Am. J. Pathol. 159: 1247–1253
Moles JP, Moyret C, Guillot B, Jeanteur P, Guilhou JJ, Theillet C, Basset-Seguin N . 1993 Oncogene 8: 583–588
Nilsson M, Unden AB, Krause D, Malmqwist U, Raza K, Zaphiropoulos PG, Toftgard R . 2000 Proc. Natl. Acad. Sci. USA 97: 3438–3443
Pontén F, Berg C, Ahmadian A, Ren Z-P, Nistér M, Lundeberg J, Uhlén M, Pontén J . 1997 Oncogene 15: 1059–1067
Pontén F, Berne B, Ren Z, Nistér M, Pontén J . 1995 J. Invest. Dermatol. 105: 402–406
Ratner D, Peacocke M, Zhang H, Ping XL, Tsou HC . 2001 J. Am. Acad. Dermatol. 44: 293–297
Rebel H, Mosnier LO, Berg RJ, Westerman-de Vries A, van Steeg H, van Kranen HJ, de Gruijl FR . 2001 Cancer Res. 61: 977–983
Ren Z-P, Ahmadian A, Pontén F, Nistér M, Berg C, Lundeberg J, Uhlén M, Pontén J . 1997 Am. J. Pathol. 150: 1791–1803
Ren Z-P, Hedrum A, Pontén F, Nistér M, Ahmadian A, Lundeberg J, Uhlén M, Pontén J . 1996 Oncogene 12: 765–773
Sarasin A . 1999 Mutat. Res. 428: 5–10
Tabata H, Nagano T, Ray A, Flanagan N, Birch-MacHin M, Rees J . 1999 J. Invest. Dermatol. 113: 972–976
Taipale J, Beachy PA, Levine AJ . 2001 Nature 411: 349–354
Toftgard R . 2000 Cell. Mol. Life Sci. 57: 1720–1731
Undén AB, Zaphiropoulos PG, Bruce K, Toftgård R, Ståhle-Bäckdahl M . 1997 Cancer Res. 57: 2336–2340
Williams C, Pontén F, Ahmadian A, Ren ZP, Ling G, Rollman O, Ljung A, Jaspers NG, Uhlen M, Lundeberg J, Pontén J . 1998 Cancer Res. 58: 2449–2455
Zhang H, Ping XL, Lee PK, Wu XL, Yao YJ, Zhang MJ, Silvers DN, Ratner D, Malhotra R, Peacocke M, Tsou HC . 2001 Am. J. Pathol. 158: 381–385
Ziegler A-M, Leffell DJ, Kunala S, Sharma HW, Gailani M, Simon JA, Halperin AJ, Baden HP, Shapiro PE, Bale AE, Brash DE . 1993 Proc. Natl. Acad. Sci. USA 90: 4216–4220
This study was supported by grants from The Swedish Cancer Foundation and The Foundation for Strategic Research.
About this article
Der Ophthalmologe (2017)
Relation between sonic hedgehog pathway gene polymorphisms and basal cell carcinoma development in the Polish population
Archives of Dermatological Research (2016)
Journal of Pharmaceutical Innovation (2016)
Population pharmacokinetics of sonidegib (LDE225), an oral inhibitor of hedgehog pathway signaling, in healthy subjects and in patients with advanced solid tumors
Cancer Chemotherapy and Pharmacology (2016)
Modern Pathology (2014)