TP53 Germline Mutations in Adult Patients with Adrenocortical Carcinoma
Leonie J. M. Herrmann,* Britta Heinze,* Martin Fassnacht, Holger S. Willenberg, Marcus Quinkler, Nicole Reisch, Martina Zink, Bruno Allolio, and Stefanie Hahner
Endocrinology and Diabetes Unit (L.J.M.H., B.H., M.F., M.Z., B.A., S.H.), Department of Internal Medicine I, University Hospital of Wuerzburg, University of Wuerzburg, D-97080 Wuerzburg, Germany; Department of Endocrinology, Diabetes, and Rheumatology (H.S.W.), University Hospital Duesseldorf, D-40225 Duesseldorf, Germany; Clinical Endocrinology (M.Q.), Charité Campus Mitte, Charité University Medicine Berlin, D-10117 Berlin, Germany; and Department of Endocrinology (N.R.), Medizinische Klinik-Innenstadt, University Hospital Munich, D-80336 Munich, Germany
Context: Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with germline mutations in TP53. According to the Chompret criteria for LFS, any patient with adrenocortical cancer (ACC), irrespective of age and family history, is at high risk for a TP53 germline mutation. However, whereas such mutations have been detected with high frequency in childhood ACC, a large cohort of adult patients with ACC has never been investigated for TP53 germline mutations.
Objective: The aim of the study was to evaluate the prevalence of TP53 germline mutations in adult patients with ACC.
Subjects and Methods: In 103 adult Caucasian patients with ACC, TP53 germline mutation analysis was performed. In patients with a TP53 germline mutation, tumor tissue was analyzed for loss of heterozy- gosity of TP53 and p53 immunohistochemistry. Family history and clinical course were also evaluated.
Results: In four patients, a total of five TP53 germline mutations were found. Two mutations occurred in exon 10 (R337H and I332M, respectively), outside the hot spot region. Here, three mutations are described for the first time in ACC, and one, which occurred combined with a second mutation (R202C) on the same allele, has never been reported before in the context of LFS. This combined mutation was associated with a remarkable family history of ACC also affecting the mother and uncle of the index patient. In the 23 patients with ACC below the age of 40 yr, 13% (95% confidence interval, 3.7-32.9%) carried a TP53 germline mutation, whereas such mutations were rare in older patients with ACC.
Conclusion: Our findings indicate a need to revise the Chompret criteria. However, in younger adults (<40 yr old) with ACC, screening for TP53 germline mutations may be justified. (J Clin Endocrinol Metab 97: E476-E485, 2012)
L’ i-Fraumeni syndrome (LFS) is a rare autosomal dominant cancer predisposition syndrome associated with germ- line mutations in the tumor suppressor gene TP53 (1). Typ- ical LFS tumors comprise sarcoma, brain tumor, breast can- cer, leukemia, and adrenocortical cancer (ACC), diagnosed before the age of 45 yr. However, a multitude of other com- mon cancers such as lung cancer, prostate cancer, gastric
cancer, melanoma, colorectal cancer, ovarian cancer, and pancreatic cancer occur more frequently in some affected families. Furthermore, some rare cancers such as choroid plexus papilloma, malignant phyllodes tumor, and Wilms tumor are also considered part of the tumor spectrum (2, 3).
A recent study on the clinical characteristics of families with TP53 germline mutations has reported that ACC is a
Copyright @ 2012 by The Endocrine Society
doi: 10.1210/jc.2011-1982 Received July 7, 2011. Accepted November 17, 2011. First Published Online December 14, 2011
* L.J.M.H. and B.H. are joint first authors.
Abbreviations: ACC, Adrenocortical cancer; DBD, DNA binding domain; ENSAT, European Network for the Study of Adrenal Tumors; LFS, Li-Fraumeni syndrome; VNTR, variable number of tandem repeats.
particularly strong indicator of a TP53 germline mutation, with 67% of tested patients showing a mutation (4). In this series, patients diagnosed with ACC before the age of 18 yr had an 80% probability to carry a TP53 mutation, regardless of the family history (4). This high prevalence of TP53 germline mutations in childhood ACC is in accor- dance with previous studies demonstrating such muta- tions in 50-80% of children with ACC (5, 6). Mutations are predominantly located in the hot spot region in exons 4-8 affecting the p53 DNA binding domain (DBD) and the loops opposing the protein-DNA contact surface (7), as has also been demonstrated for somatic mutations in sporadic ACC (8). More recently, the observation of an exceptionally high incidence of childhood ACC in Brazil, which is about 10-15 times higher compared with other countries, led to the identification of a specific germline mutation outside the hot spot region in exon 10 of TP53 (R337H) in virtually all cases (9). The mutational defect is located in the tetramerization domain and has been shown to be functionally influenced by pH (10). The penetrance of ACC in carriers of the R337H mutation is low (9.9%), suggesting additional tumorigenic mechanisms (11). Re- cent data have provided convincing evidence that the high local prevalence of the R337H mutation in Brazil is related to a founder effect (12).
In 2001, Chompret et al. (13) developed modified LFS criteria for identifying patients likely to carry TP53 mu- tations, even in the absence of a suggestive family history. According to these criteria, a subject with ACC is likely to carry a germline TP53 mutation regardless of age at di- agnosis or family history (13). However, TP53 germline mutations in adult ACC patients have so far been de- scribed only in small series (14, 15), as single case reports (16), or in the context of families connected to LFS (17- 19). The largest series in adult patients with ACC came again from southern Brazil, including 37 patients from the above-mentioned area of high ACC prevalence due the R337H founder mutation (20). In this series, 13.5% of adults carried the R337H mutation, compared with 77.7% of chil- dren with either benign or malignant sporadic adrenocortical tumors (20). However, larger cohorts of adult patients with ACC outside the Brazilian area are still missing for validation of the Chompret criterion concerning ACC.
We therefore investigated adult patients with ACC (>18 yr of age) derived from the German ACC registry (21, 22) for TP53 germline mutations.
Patients and Methods
Patients and samples
We studied 103 patients with ACC registered with the German Adrenocortical Carcinoma Registry (www.nebennierenkarzinom.de), which was established in 2003. At the time of the final analysis,
the registry comprised more than 400 patients. All data were collected by trained medical personnel as described previously (23). Screening for secondary malignancies is part of the regular follow-up imaging (including detection of local recurrence, me- tastasis, and secondary malignancies) of our patients, which is documented in the patient registry. Furthermore, patient histo- ries recording malignancies before primary diagnosis of ACC are documented within the registry. Contact with the registry is made by the local attending physician, by the patient, or by his/ her relatives. Patients had been recruited to the German ACC Registry from all over Germany and, to a small extent (n = 11), from other European countries also. All patients were Cauca- sians. Most patients had undergone clinical, radiological, and hormonal examination followed by surgery. Histopathological evaluation of adrenal tissue, including assessment of the Weiss score (24), was used to establish the diagnosis of ACC. Clinical stage was determined by ENSAT (European Network for the Study of Adrenal Tumors) classification (23). In one case of a patient affected by a TP53 germline mutation, tumor material from the deceased mother and a blood sample from the sister were also secured.
Ethical approval (no. 93/02) was obtained from the local eth- ics committee at the University of Wuerzburg, and patients gave written informed consent. Information from hospital records, blood samples, and paraffin-embedded tumor tissue were pro- vided by the respective supervising clinics.
DNA extraction
Peripheral blood (1-5 ml) was collected from all patients, and genomic DNA was extracted from blood samples using the FlexiGene DNA Extraction Kit (QIAGEN, Hilden, Germany).
DNA extraction from paraffin-embedded tissue was carried out following a modified protocol by Turbett et al. (25) (1996). Briefly, after dewaxing five 20-um sections of paraffin-embed- ded tissue by xylene at 45 C for 15 min, sections were treated with 100, 95, and 70% ethanol washes and then allowed to air-dry. Digestion was performed in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.5% Tween 20, and 2 mg/ml proteinase K (Sigma, Steinheim, Germany) overnight at 55 C. Samples were heated to 100 C for 20 min, and tissue debris was removed by low-speed centrifugation. The collected supernatant was centri- fuged once more and stored at -20 C until used.
PCR and sequence analysis
For sequence analysis of the TP53 exons from genomic DNA, samples were amplified by PCR using primers according to the design of BioGlobe GmbH, Hamburg (Metabion International AG, Martinsried, Germany) with 1 X Hotstar buffer (QIAGEN), 1.5 mM MgCl2, 200 µM of each dNTP (Fermentas Life Sci- ences, St. Leon-Rot, Germany), 2 U Hotstar DNA polymerase (QIAGEN), and 0.4 µM of each primer. PCR comprised 35 cycles (94 C for 60 sec, 63 C for 60 sec, and 72 C for 60 sec) with 25 ng of genomic DNA, and final extension was at 72 C for 10 min. Before cycle sequencing, PCR products were purified using Agencourt Ampure (Beckman Coulter, Krefeld, Germany). For cycle sequencing of TP53 exon PCR products, BigDye Termi- nator v3.1 Cycle Sequencing Kit (Applied Biosystems, Darm- stadt, Germany) was used. Each 10-ul reaction contained 1 pl BigDye reaction mix and 5 pmol forward or reverse primer (ac- cording to Bioglobe GmbH, Hamburg), respectively. Cycle se-
quencing comprised 35 cycles (96 C for 10 sec, 55 C for 5 sec, and 60 ℃ for 4 min). Sequence reactions were purified using Agencourt CleanSeq (Beckman Coulter). Samples were subjected to capillary- electrophoresis on a MegaBACE 1000 DNA Analyzer (GE Health- care, Munich, Germany). Electrokinetic injection was 2 kV for 12 sec, and electrophoresis was continued at 8 kV for 110 min.
All sequence data were analyzed using GENtle (M. Manske, University of Cologne, Cologne, Germany; http://gentle. magnusmanske.de/) and 4Peaks (Mekentosj, Aalsmeer, The Netherlands; http://mekentosj.com/4peaks).
Verification of found germline mutations and corresponding somatic mutation site analysis was performed by cycle sequence analysis of the relevant TP53 exons. Samples were amplified by PCR in a volume of 50 ul containing 1× Pfx amplification buf- fer, 1 mM MgSO4 (Invitrogen, Darmstadt, Germany), 10 mM of each dNTP (Peqlab, Erlangen, Germany), 1 U Platinum Pfx DNA polymerase (Invitrogen), and 20 pM of each primer (Biomers, Ulm, Germany). PCR comprised 40 cycles [94 C for 45 sec, “x” C (for x, see Supplemental Data, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals. org) for 30 sec, and 68 C for 30 sec] with 400 ng genomic or somatic DNA. Final extension was at 68 C for 10 min. Before cycle sequencing, PCR products were purified by peqGOLD Gel Extraction Kit (Peqlab) or, depending on purity of sample, by PCR purification using MinElute Kit (QIAGEN). DNA sequenc- ing of PCR products was performed by GATC Biotech (Kon- stanz, Germany).
PCR and loss of heterozygosity (LOH) analysis
PCR for LOH analysis was performed with 400 ng somatic DNA and microsatellite repeat primers (see Supplemental Data) for TP53 variable number of tandem repeats (VNTR) 1 and 4 (26, 27). We had to use two different markers because VNTR1 was not conclusive in patients 1 and 2 (data not shown). The markers were chosen because they are highly informative and closely located to the mutated genomic sequence.
PCR mix contained 1× Pfx amplification buffer, 1 mM MgSO4 (Invitrogen), 10 mM of each dNTP (Peqlab), 1 U Plati- num Pfx DNA polymerase (Invitrogen), and 20 pM of each primer (Biomers). After denaturation for 5 min at 95 C, PCR comprised 35 cycles (93 C for 1 min, 64 C for 2 min, and 68 C for 1 min) and final extension for 10 min at 68 C for VNTR1 primer. PCR with VNTR4 primer was performed by denatur- ation for 3 min at 94 C, followed by 35 cycles (94 C for 1 min, 60 C for 2 min, and 72 C for 45 sec) and final extension at 72 C for 5 min.
LOH analysis was conducted by Seq-IT (Kaiserslautern, Ger- many) through electrophoretic separation of DNA fragments in
the 3130xl Genetic Analyzer (Applied Biosystems). Data were analyzed by Genemapper v3.2 software (Applied Biosystems).
Haplotype analysis
Haplotype analysis was conducted according to Garritano et al. (12) by PCR containing 200 ng genomic DNA and primers (see Supplemental Data) encompassing SNP179 (rs ID no., rs9894946). PCR mix of 50 ul contained 1× Pfx amplification buffer, 1 mM MgSO4 (Invitrogen), 10 mM of each dNTP (Peqlab), 1 U Platinum Pfx DNA polymerase (Invitrogen), and 20 PM of each primer (Biomers). After denaturation for 5 min at 94 C, PCR comprised 40 cycles (94 C for 30 sec, 53 C for 45 sec, and 68 C for 45 sec) and final extension at 68 C for 10 min. DNA sequencing of PCR products was performed by GATC Biotech.
Immunohistochemical analysis
Immunohistochemical detection of p53 was performed in ACC tissue samples from patients 2, 3 and 4 using an indirect immunoperoxidase technique. Sections were deparaffinized twice in 100% xylene, followed by rehydration in 100, 90, 80, and 70% ethanol and an extensive washing step in distilled wa- ter. After high temperature and pressure, antigen retrieval was conducted in 10 mM citric acid monohydrate buffer (pH 6.5) for 13 min. This step was followed by inhibition of endogenous peroxidase activity with 3% hydrogen peroxide/methanol solu- tion for 10 min. Blocking of unspecific protein-antibody inter- actions was performed with 20% AB human serum (Invitrogen) in Dulbecco’s PBS for 1 h at room temperature. p53 was detected by incubation with monoclonal mouse antibody against p53 pro- tein (clone DO-7; Dako, Glostrup, Denmark), 1:150 in Dulbec-
co’s PBS for 1 h at room temperature, with the reaction con- trolled with N-Universal Negative Control Mouse (Dako, Glostrup, Denmark). Signal amplification was achieved by per- oxidase-based ADVANCE kit (Polymer-HRP anti-mouse; Dako, Hamburg, Germany) for 2 × 20 min and washed in PBS before visualization was enabled using DAB (3,3’-diaminoben- zidine tetrahydrochloride; Sigma) chromogen containing 0.05% hydrogen peroxide for 10 min. After washing in PBS and H2O, the slides were counterstained with hematoxylin. Results were analyzed using a light microscope at a magnification of 40X.
p53 staining was assessed by the reference pathologist of the ACC study group (Prof. Wolfgang Saeger, Hamburg, Germany).
Results
Patients
Clinical data of the 103 Caucasian patients from the German ACC Registry are given in Table 1. Median age at
| Men (n = 41) | Women (n = 62) | |
|---|---|---|
| Age at PD (yr), median (range) | 49 (18-77) | 51 (20-78) |
| Survival after PD (yr), median (range) | 5.8 (0.5-27.2) | 4.1 (0.1-28.2) |
| ENSAT stage at PDª | I = 7%, Il = 37%, III = 24%, IV = 32% | I = 5%, Il = 22%, III = 32%, IV = 27% |
| Tumor size (cm), mean (range) | 12.2 (4.5-30) | 10.0 (3-20) |
| Hormone activity | Yes, 52.9%; no, 47.1% | Yes, 75.0%; no, 25.0% |
| Secondary malignancy | Yes, 17.1%; no, 82.9% | Yes, 9.7%; no, 90.3% |
PD, Primary diagnosis.
a Ref. 23.
primary diagnosis was 50 yr (range, 18-78 yr); 41 (39.8%) were male, and 62 (60.2%) were female. Fol- low-up information was available for all patients, with a mean follow-up of 58 months.
Identification of TP53 mutations, LOH analysis, immunohistochemical staining, and clinical presentation of patients with germline TP53 mutation
Analysis of TP53 coding exons 4-11 in genomic DNA of all patients revealed four patients with a germline TP53 mutation. No further TP53 germline mutations were found by additional analysis of exons 2 and 3 in 80 of these patients.
In all patients with a TP53 germline mutation, paraffin- embedded tumor tissue was analyzed for corresponding somatic mutations, LOH, and immunohistochemical staining of p53.
Patient 1 was diagnosed with ACC at the age of 21 yr (see Table 3). He presented with abdominal discomfort and a left-sided palpable mass. Before surgery, no hor- mone analyses were performed. Clinical examination and hormonal evaluation after surgery showed no signs of hormonal disequilibrium. No family history was available due to the adoption status of the patient. After removal of a 15-cm tumor, oral mitotane was initiated. Bone metastases located in the hip were diagnosed 6 months after surgery and were treated by radiotherapy. Later, the patient was included in the FIRM-ACT trial (www.firm-act.org; www.ClinicalTrials.gov identifier, NCT00094497), initially receiving streptozotocin. At progression of bone metastases, a crossover to etoposide- doxorubicin-cisplatin was performed. The disease re- mained stable until brain metastases developed. The pa- tient succumbed to ACC 26 months after primary diagnosis.
Patient 1 had a heterozygous germline mutation in exon 4 (Table 2 and Fig. 1A) within the DBD. The mutation affects the third nucleotide position of codon 125, leading to an exchange of ACG to ACA (both encoding for ty- rosine). Although encoding a silent mutation on the amino acid level (Thr→Thr), the mutation has been reported to affect splicing and leads to abnormal transcripts of p53
(17, 28). Moreover, the germline mutation has been shown to be causative for the cancer predisposition in a family including a member affected by ACC (17). The mutation has been described in germline and somatic tis- sue from ACC patients (17, 28).
Sequencing of DNA from tumor tissue of patient 1 showed the mutation to be homozygous in codon 125 (Fig. 1A), and analysis of LOH revealed loss of the wild-type TP53 allele in tumor tissue (Fig. 1B). p53 immunohisto- chemistry showed no positive staining in tumor tissue (Fig. 2). However, as previously described (29-31), mutations in TP53 are not necessarily connected with accumulation of p53.
Patient 2 was diagnosed with ACC at the age of 39 yr after he presented with symptoms of hypercortisolism (Ta- ble 3). The patient had been diagnosed with testicular can- cer at the age of 20 and had been successfully treated by surgery. He had a family history of cancer because both grandfathers died from malignancies at about 70 yr of age (leukemia and lung cancer, respectively) (Table 3). In ad- dition, his paternal uncle died of leukemia at age 73.
Adrenalectomy (R0 resection status, complete resec- tion, no residue) of the left adrenal gland with removal of a 12-cm tumor was performed, with subsequent radiation treatment of the tumor bed (50 Gy). Adjuvant treatment with mitotane was started 4 months after the primary di- agnosis and discontinued 21 months later. After 12 months of tumor-free survival, a metastasis in the liver was treated by radiofrequency ablation, followed by radiation therapy of a local recurrence 4 months later. Nine months later, new metastases in the liver were treated by surgery. A solitary intraabdominal lymph node metastasis was re- moved by surgery 28 months after diagnosis of ACC. The patient was also included later in the FIRM-ACT trial. He died from progressive disease 32 months after he was di- agnosed with ACC.
Patient 2 had a heterozygous CGC→TGC exchange in codon 158 within exon 5 leading to an amino acid sub- stitution of arginine with cysteine in the DBD (Table 2 and Fig. 1A).
Functional analysis of this mutation has been shown to change function and temperature sensitivity of p53 in
| Patient no. | Exon | Codon | Base exchange | Amino acid change | Domain | Mutation effect | LOH |
|---|---|---|---|---|---|---|---|
| 1 | 4 | 125 | ACG>ACA | Thr het. splicing | DBD | Splice site | Yes |
| 2 | 5 | 158 | CGC>TGC | Arg>Cys | DBD | Missense | Yes |
| 3 | 6 | 202 | CGT>TGT | Arg>Cys | DBD | Missense | Yes |
| 10 | 332 | ATC>ATG | lle>Met | TD | Missense | Yes | |
| 4 | 10 | 337 | CGC>CAC | Arg>His | TD | Missense | Yes |
TD, Tetramerization domain; het., heterozygous.
A
Exon
4
5
6
10
Codon
125
158
202
332
TGCACGGTC
TTGCGTGTG
CAGATCCGT
Blood
GTCCGCGCC V
V
A
L
M
R
V
MON I R
0
wt sequence
TGCACNGTC
TT
GN
GT
GT
G
CAGATECGT
Blood
GTCNGCGCC V
-
V
DAN A
L
-
V
Q
-
R
MON
TGCACAG TC di
GTCTGCGCC V
TTGTGTGTG
CAGATGCGT M
T
V
A
I
V
La O
Q
R
Tumor
Patient 1
Patient 2
Patient 3
Patient 3
B
110 bp
128 bp
132 bp
127 bp 137 bp
127
1
VA
1
A
1
128 bp
127 bp
110
N
Patient 1
Patient 2
Patient 3
Patient 4
yeast cell lines (32-35). To our knowledge, this mutation has not yet been described in ACC but has been observed in several other cancers like head and neck carcinoma, basal cell carcinoma, or colorectal carcinoma (36-38).
Analysis of the mutation in codon 158 in tumor DNA of patient 2 revealed LOH with loss of the wild-type allele (Fig. 1B). Immunohistochemistry of tumor tissue showed negative staining for p53 (Fig. 2).
Patient 3 was diagnosed with ACC at the age of 30 yr (Table 3). Symptoms of hypercortisolism including hyper- tension, weight gain, and a moon-shaped face were rec- ognized, and hormone excess of cortisol, aldosterone, and androgens was ascertained. Imaging revealed a tumor of the right adrenal gland and a lesion in the inferior right lung, indicating metastasis. Adrenalectomy was per-
formed with additional resection of a 10 large tumor thrombus in the vena cava 337 inferior. In addition, resection of the in- ferior lobe of the right lung was per- GAGCGCTTC formed. The tumor measured 14.5 cm H R F and showed 30% Ki-67-positive tumor cells. Oral therapy with mitotane was begun and was followed 2 months later by radiation therapy of the tumor bed GAGCNCTTC 0 (35 Gy). Liver metastases occurred 6 - F months after diagnosis and were re- moved by surgery, followed by consec- utive resection of a tumor relapse and of GAGCACTTC H E newly developed metastases of the right and left lung 1 month later. At this point, the patient was referred to the University Hospital of Wuerzburg to Patient 4 participate in the FIRM-ACT trial. The patient died 20 months after diagnosis from progressive ACC. The patient had no second malignancy, but there was a 122 bp 127 bp remarkable family history (Table 3). Of note, both the mother of the patient and a half-brother of the mother died from adrenal malignancies. Furthermore, the grandfather from the maternal side 127 bp died of gastric cancer. The patient’s mother was diagnosed with an adrenal tumor (diameter, 24 cm) at the age of 32 yr. Adrenalectomy was performed, and the tumor was histologically classified as a pheochromocytoma by the local pathologist. No increased catechol- amine secretion was documented. In contrast, elevated 17-ketosteroids in urine were detected, indicating ACC. Tissue to reassess the diagnosis was only available from a tumor metastasis leading to a revised diagnosis of the lesion as a metastasis of an ACC.
Patient 3 showed two heterozygous germline mutations (Table 2 and Fig. 1A). One mutation was located in the DBD in exon 6 codon 202, leading to an amino acid ex- change of arginine to cysteine encoded by a CGT→TGT substitution. A further mutation was found in exon 10 located in the tetramerization domain in codon 332. The nucleotide change of ATC→ATG leads to an amino acid substitution of isoleucine by methionine. To our knowl- edge, both mutations have never been described in ACC so far (7).
Analysis of tumor DNA in patient 3 demonstrated both mutations in tumor tissue with loss of the wild-type allele
1
2
3
4
(Fig. 1B). Immunohistochemistry of tumor tissue demon- strated strong staining of nuclei (Fig. 2), indicating high expression of p53.
Tissue from a metastasis of ACC diagnosed in the mother 20 yr earlier was also analyzed. The mutation in codon 332 was found to occur homozygously (Fig. 3). Analysis of exon 6 could not be performed because no material was left. Shortly after primary diagnosis in 1988, the mother had succumbed to her disease.
Analysis of germline DNA from the patient’s healthy sister, who was 30 yr old at time of evaluation, also showed both mutations to occur heterozygously (Fig. 3).
Patient 4 was diagnosed with ACC at the age of 71 yr when a routine ultrasound examination as part of colon cancer surveillance showed a right adrenal mass (Table 3). He had been diagnosed with colon cancer at the age of 65 yr and was later diagnosed with prostate cancer at the age
of 74 yr. The father of the patient had been diagnosed with prostate cancer at the age of 54 yr, and his daughter had died from breast cancer at the age of 37 yr.
At diagnosis, elevated cortisol, androgen, and estrogen levels were found. The patient had undergone breast reduc- tion mammoplasty for gynecomastia 2 yr earlier. There were no metastases at primary diagnosis. The patient underwent surgery, and a 14-cm tumor was removed (R0 resection).
After surgery, the patient received adjuvant radiotherapy of the tumor bed (51 Gy), and adjuvant oral therapy with mitotane was begun but was abandoned after 8 months because of side effects (weight loss, fatigue, and disgeusia).
Two years after diagnosis, a metastasis in the lung was diagnosed. The patient refused surgery or any other treat- ment. Surveillance of the lung metastasis showed only slow progression. No other metastases or local recur- rences are detectable to date. Because he was diagnosed with ACC 10 yr ago, the patient is followed regularly and is not affected in his daily activities by the tumor.
Patient 4 carries the well-characterized R337H hotspot germline mutation (Table 2 and Fig. 1A) related to the high prevalence of ACC in Brazilian children and affecting the tetramerization domain in a pH-sensitive fashion (10). This mutation has been extensively characterized in germ- line and tumor tissue of ACC patients (39).
Corresponding analysis of the tumor DNA showed the mu- tation homozygously in tumor tissue, and LOH analysis dem- onstrated loss of the wild-type allele. Immunohistochemistry showed positive p53 staining of nuclei in ACC tissue.
Garritano et al. (12) recently demonstrated that indi- viduals with the R337H mutation were all carriers of the same genotype in SNP179 (rs ID no. rs9894946). They implied that this proves the founder effect that had been assumed for the Brazilian individuals previously (40). The analysis of SNP179 in our patient (see Supplemental Data) did not reveal the same genotype described in Brazil and in one family of Portuguese origin (12), indicating indepen- dent occurrence of the germline mutation.
| Patient no. | Sex | Age at PD (yr) | Survival after PD (yr) | Weiss score | ENSAT stage at PDª | Tumor size (cm) | Hormone activity | Secondary malignancy | Family history of cancer (age at death in yr) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | M | 21 | 2.1+ | 6 | IV | 15 | ND | None | NI |
| 2 | M | 39 | 2.6+ | 5 | III | 12 | Cortisol | Teratocarcinoma of the testis | Uncle, paternal; leukemia (73+) Grandfather, maternal; leukemia (<70+) Grandfather, paternal; lung cancer (>70+) |
| 3 | F | 30 | 1.7+ | 8 | IV | 14.5 | Cortisol, androgen, aldosterone | None | Mother; ACC (<40+) Grandfather, maternal; gastric cancer (+) Uncle, maternal; ACC (+) |
| 4 | M | 71 | 10 | 6 | II | 12 | Cortisol, estrogen, androgen | Colon cancer, prostate cancer | Father; prostate cancer (62+) Daughter; breast cancer (37+) |
PD, Primary diagnosis; M, male; F, female; t, deceased; NI, not informative due to adoption status of patient; ND, not determined before surgery. a Ref. 23.
Codon
202
332
Blood
TTGCGTGTG L V
CAGATCCGT
R
d
I
R
Panely
wt sequence
Tumor
CAGATGCGT
Q
M
R
Mother of Patient 3
Blood
TTGNGTGTG
CAGATNCGT
L
-
V
Q
-
R
Sister of Patient 3
Discussion
Our study is the first to investigate a large cohort of adult patients with ACC for TP53 germline mutations demon- strating a mutation prevalence of 3.9% (95% confidence interval, 1.2-9.9%) in the Caucasian population. This prevalence is substantially lower than has been observed for childhood ACC (6, 9) and is comparable to the fre- quency of TP53 germline mutations in young women with breast cancer (41). The much lower prevalence of TP53 mutations in our series may be related to some ascertain- ment bias in previous investigations because mainly fam- ilies who were referred to a specialized center and had been chosen for TP53 testing were included. In contrast, in our series patients were contacted via the German ACC Reg- istry, and all patients who were asked agreed to partici- pate. Because we did not perform deletion and duplication analysis in addition to direct sequencing, we may have missed patients exhibiting such kind of sequence altera- tions. However, because this has been detected in a very small proportion of mutation carriers (19, 42), we do not believe that additional analysis would significantly alter the results of our study.
The TP53 germline mutations in our series were found in exons 4, 5, 6, and 10, respectively. To our knowledge, three of the mutations have not been described in ACC previously, and one of these mutations (I332M) has not been described in any tumor before, according to the In- ternational Agency for Research on Cancer (IARC) TP53
database (7). The patient carrying the newly described mutation (I332M) in exon 10 not only had a second mu- tation that was found in exon 6 (R202C) but was also particularly remarkable because her mother presented with ACC and a maternal uncle with an adrenal malig- nancy, probably also an ACC. Further analysis detected the exon 10 mutation also in the tissue derived from the ACC of the mother. These findings suggest that these mu- tations are specifically linked to the development of ACC, although the relative contribution of the two mutations to the clinical phenotype remains currently unknown. It is tempting to speculate that the mutation in exon 6 is linked to the high penetrance of the disease, whereas the mutation in exon 10 may play a role in the adrenal phenotype.
In all patients with a germline TP53 mutation, LOH at the TP53 locus was demonstrated in tumor tissue, and LOH analysis revealed loss of the wild-type TP53 allele. Two of the four patients in our series carry a mutational change in exon 10. In fact, from the germline mutations located in exon 10 that are published in the IARC data- base, more than 80% were associated with adrenal cancer (7). Although this is mainly due to the high frequency of the R337H variant in Brazil, further mutational changes in exon 10 have been described in non-Brazilian patients with ACC. In one of our patients, we observed the R337H mutation that is highly prevalent in southern Brazil (9, 20). Intriguingly, the history of this patient indicates that this mutant was associated with other tumors in the patient (colonic cancer, prostate cancer). Furthermore, the clini- cal course of this patient highlights that TP53 germline mutations leading to only subtle functional changes may manifest also at a higher age with a less aggressive behav- ior of ACC.
The clinical course of our patients with germline TP53 mutations was highly variable, with one patient surviving for more than 10 yr despite evidence of metastatic spread of one of his malignancies. The patient with the R337H mutant survived his disease for several years so far. There- fore, the clinical phenotype and the prognostic impact of a mutation largely depend on the kind of mutation and possibly on further confounders.
Of note, three patients had received adjuvant radiation of the tumor bed, with two patients suffering from tumor recurrence in the radiation field, suggesting radioresis- tance of the tumor. However, none of the patients devel- oped secondary malignancies in the radiation field, pos- sibly due to the short survival in two of the three patients. Similarly, radiotherapy for bone metastases was of limited efficacy in the remaining patient, whereas in general, ra- diotherapy for treatment of ACC has been shown to be efficacious (43, 44). Evidence of somatic TP53 mutations in ACC has been associated with poor outcome (8, 45). As
previously described, approximately 25-44% of ACC ex- hibit somatic mutations (8, 46, 47). We suspect a similar distribution of somatic mutations in our group of patients, which might further have affected their survival. Formal analysis is hampered by the small number of all patients. However, we could not detect a poorer outcome than is generally observed in stage IV ACC with a median survival of 15 months (23).
The functional consequences of TP53 mutations lead- ing to later disease onset may be different in older patient populations. The penetrance of the mutations has been relatively late, indicating a less severe functional impair- ment of TP53 function in our patients. A low penetrance has already been extensively described for the R337H mu- tant (11). However, in the remaining three cases, patient age was still relatively low and the course of the disease was aggressive, arguing against a less severe phenotype of these mutations. Two of these mutations have also been described in younger patients with ACC (28, 39), indicat- ing that additional factors seem to be involved in the de- termination of the clinical manifestation of the TP53 mu- tation (48).
Regarding systematic screening of adult ACC patients for TP53 mutations, the clinical relevance of a systematic screening for TP53 mutations outside the LFS is difficult to establish, and the occurrence of mutations appears to be too low to recommend screening in all adult patients. However, whereas in most other cancer entities only 1% of patients carry a TP53 germline mutation (49), the fre- quency of 3.9% in our study indicates the specific rele- vance of TP53 for ACC pathogenesis.
Furthermore, the even higher prevalence of 13% of TP53 germline mutations in the 23 adult ACC patients below the age of 40 yr raises the question of whether such patients should be systematically screened for TP53 germ- line mutations in the future. Obviously, genetic counseling is indicated before genetic testing to discuss the implica- tions for both the patient and his or her family members. Furthermore, management of LFS is not yet well standard- ized and remains controversial because the effectiveness of structured supervision remains to be demonstrated.
Moreover, the psychological burden of the diagnosis LFS may be substantial (50). On the other hand, the pa- tient may directly benefit concerning the management of ACC. It is well known that TP53 mutant cells are ex- tremely sensitive to DNA damage. Cytotoxic chemother- apy and radiation therapy can cause second malignancies in these patients (51-53). Accordingly, adjuvant radiation therapy of the tumor bed may be counterproductive in carriers of TP53 germline mutations, and the use of mi- totane and modern targeted therapies (e.g. IGF-I receptor antagonists) could be advantageous in these patients. Fur-
thermore, affected family members may benefit from in- creased tumor surveillance (54). Thus, in our view, in adult patients with ACC below the age of 40 yr, analysis for TP53 germline mutations should be considered on an in- dividual basis. It is important to note that our study was performed in a Caucasian population, and it remains to be shown that these results can be transferred to other populations.
In conclusion, our findings argue against the use of ACC as a case-finding tool for LFS in adult patients be- yond the age of 40 yr. However, in younger adults with ACC from the Caucasian population, screening for TP53 germline mutations may be justified and may even affect management of the disease.
Acknowledgments
We are grateful to all colleagues and patients who provided clin- ical data and tissue material for the German Adrenocortical Car- cinoma (ACC) Registry. We also appreciate the support of Prof. W. Saeger (Marienkrankenhaus, Hamburg, Germany) and the members of the German ACC Registry Group (especially Mi- chaela Haaf and Luitgard Kraus).
Address all correspondence and requests for reprints to: PD Dr. Stefanie Hahner, M.D., Endocrinology and Diabetes Unit, Department of Medicine I, University of Wuerzburg, Oberdu- errbacher Strasse 6, D-97080 Wuerzburg, Germany. E-mail: hahner_s@medizin.uni-wuerzburg.de.
This work was supported by the Deutsche Krebshilfe (Grant 107111, to M.F.), the Deutsche Forschungsgemeinschaft (Grant FA 466/3-1, to M.F.), the Interdisziplinaeres Zentrum fuer Klinische Forschung Wuerzburg (Grant F-124, to S.H.), and the Else-Kroener-Fresenius-Stiftung.
Disclosure Summary: The authors have nothing to disclose.
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