ORIGINAL ARTICLE
Clinical impact of TP53 alterations in adrenocortical carcinomas
Jens Waldmann . Nikolaos Patsalis . Volker Fendrich . Peter Langer .
Wolfgang Saeger . Brunhilde Chaloupka . Annette Ramaswamy . Martin Fassnacht .
Detlef K. Bartsch . Emily P. Slater
Received: 19 July 2011 / Accepted: 24 October 2011 /Published online: 29 December 2011 C Springer-Verlag 2011
Abstract
Background To evaluate the role of somatic TP53 muta- tions and to correlate somatic and germline mutations with results of immunostaining, a large cohort of ACC patients was analyzed.
Patients and methods Patients with ACC who underwent potential curative surgery at the authors’ department were screened for TP53 somatic and germline mutations in exons 5, 6, 7, 8, and 10 by DHPLC analysis. Aberrant samples were further analyzed by direct sequencing. Immunostain- ing was performed on corresponding paraffin sections in all
This work was presented at the ESES workshop in Lyon, France, May 2011.
J. Waldmann () . N. Patsalis . V. Fendrich . P. Langer .
B. Chaloupka · D. K. Bartsch · E. P. Slater
Department of Visceral, Thoracic and Vascular Surgery, University Hospital Giessen and Marburg, Baldingerstraße, 35043 Marburg, Germany e-mail: jwaldman@med.uni-marburg.de
N. Patsalis Department of Surgery, University of Cologne, Cologne, Germany
W. Saeger Department of Pathology, Marienkrankenhaus, Hamburg, Germany
A. Ramaswamy
Department of Pathology, University Hospital Giessen and Marburg, Marburg, Germany
M. Fassnacht
Department of Internal Medicine, Division of Diabetology and Endocrinology, University Hospital Wuerzburg, Wuerzburg, Germany
patients. Complete clinical and follow-up data were correlated with the status of TP53.
Results Thirty ACC patients were included. Four of 30 patients showed aberrant DHPLC configuration and direct sequencing confirmed 2 (7%) germline mutations (R337H, R248W), 1 (3%) somatic mutation (R213X), and 1 (3%) noncoding polymorphism (g.17708 A>T). The only patient with a positive family history harbored a TP53 mutation. Tumors of the three patients with mutations showed aberrant p53 expression in more than 10% of cells by immunostaining, compared to only 3 of 27 patients without mutations (p=0.009). Aberrant p53 expression (>5%) was detected in 12/30 (40%) ACCs. The latter was associated with an increased Ki67 and van Slooten index (p≤0.001; p= 0.020). Disease-free survival decreased significantly in patients with aberrant p53 IHC of more than 5% of cells (65.7±12.4 vs. 26.6±8.7 months; p=0.043 log rank test). Conclusions Patients with ACC revealed aberrant expres- sion of p53 in 40%, and mutations were identified in 25% of these patients. Therefore aberrant p53 expression should be considered an indicator for genetic testing. A subgroup of apparently sporadic ACC is caused by TP53 germline mutations, and family history is a strong indicator for p53 germline mutations.
Keywords Adrenocortical cancer . Adrenal cancer . TP53 . Adrenalectomy
Introduction
Adrenocortical carcinomas (ACC) arise from the adrenal cortex and are responsible for 0.2% of cancer-related deaths [1]. The prognosis varies considerably with an overall 5- year survival rate ranging from 16% to 60% [2, 3]. This
may reflect different biological entities and underlying affected pathways within ACC. The events in oncogenesis are still poorly understood, although progress has been made. Microarray analyses revealed multiple pathways which are differentially regulated in ACC compared to benign adrenocortical tumors [4-6]. Hereditary syndromes associated with ACC such as the Li-Fraumeni (LF) and the Beckwith Wiedemann syndrome have contributed to our understanding of the pathogenesis of ACC [7, 8]. TP53 mutations are identified in approximately 70% of patients with LF syndrome which predispose to certain malignancies (breast carcinoma, brain tumors, soft tissue sarcomas, osteosarcoma, leukemia, and ACC [9]). Furthermore, loss of heterozygosity of chromosomal region 17p13, including the TP53 gene, is observed in 40-85% of sporadic ACC [10, 11]. Somatic TP53 mutations occur in 25-70% of patients with sporadic ACC [11-13]. Most of the former studies lacked complete data on somatic and germline TP53 mutations and immunohistochem- ical analysis for aberrant p53 expression. Recently, TP53 mutations were reported to be associated with more aggressive disease and advanced tumor stage in a set of 29 ACC, although TP53 mutations did not affect overall survival in this patient cohort [11].
In the present study, a cohort of 30 patients with ACC was tested for somatic and germline mutations of the TP53 gene. Corresponding paraffin sections of all patients were then assessed for aberrant expression of p53 by immunos- taining, and this was correlated to mutation status, clinical pattern, and outcome.
Patients and methods
Patients
All patients with adrenocortical carcinoma who underwent potential curative surgery (R0 resection) and two patients with palliative cytoreductive surgery at the authors depart- ment were included if fresh frozen tumor tissue and blood were available. This study was performed according to the guidelines of the local ethics committee. Patients gave their informed consent in written form.
Diagnostic work-up and surgery
Preoperatively all patients underwent CT scan or MRI of the abdomen and were screened for elevated hormone levels associated with ACC (estrogen, estradiol, testoster- one, cortisol, ACTH, aldosterone). In a subgroup of patients, DHEAS was also measured. In case of a suspicious tumor thrombus, an MRI phlebography was performed. Renal scintigraphy was only performed if creatinine levels were elevated, and infiltration of the
kidney was suspected. The standard procedure was an open adrenalectomy with lymphadenectomy through an abdom- inal or abdominothoracical approach. However in two patients with small tumors (P13, P15), a laparoscopic adrenalectomy was performed as no malignant tumors was suspected preoperatively. If infiltration was apparent at surgery, a nephrectomy was performed to ensure complete tumor removal. Distant metastases were resected to achieve complete tumor removal or to milden a severe hormonal syndrome (n=6, see Table 1). More specifically three patients underwent a resection to achieve complete tumor removal [one liver metastasis (P16), one lung metastasis (P19), one metastasis within the major omentum (P29)], two patients with a severe Cushing syndrome had multiple lung metastases (P3, P18), and in one patient (P21), minimal (<5 mm) lesions in the lung were not yet been proven to be metastases as there were small and nonprogressing in the follow-up.
Histology and immunostaining
All specimens were independently reviewed and assessed by two experienced pathologists (WS and AR). Tumors were scored according to Weiss and van Sloten [14, 15]. A tumor cellularity of at least 85% was confirmed based on HE staining before DNA was extracted from fresh frozen tumor tissue. Formalin-fixed and paraffin-embedded ar- chived tumor samples were stained as previously described [16]. Briefly, slides from archived PC were heated to 60℃ for 1 h, deparaffinized using xylene, and hydrated by a graded series of ethanol washes. Antigen retrieval was accomplished by microwave heating in 10 mM sodium citrate buffer, pH 6.0 for 30 min (Ki67 20 min in EDTA, 1 mM pH= 8). For immunohistochemistry (IHC), endogenous peroxidase activity was quenched by 10 min incubation in 3% H2O2. Nonspecific binding was blocked with 10% bovine serum. Sections were then incubated with a primary antibody against Ki67 (1:100; M7240, Dako, Glostrup, Denmark) or p53 (1:50; NCL-p53-D07, Menarini, Florence, Italy) over- night at 4℃. Bound antibodies were detected using the avidin-biotin complex (ABC) peroxidase method (ABC Elite Kit; Vector Labs, Burlingame, CA). Final staining was developed with the Sigma FAST DAB peroxidase substrate kit (Sigma, Deisenhofen, Germany).
Analysis of sequence variations
DNA was extracted from frozen tumor tissue as well as from whole blood leukocytes in all patients using standard procedures. Tumor tissues were obtained during surgery and stored in liquid nitrogen until DNA isolation. Primers were selected, and polymerase chain reaction (PCR) conditions for each amplicon were established (Table 2).
| Patient ID | Age | Sex | P53 mutation (G/S) | IHC P35 | TNM | Ki67 (%) | Scoring (W/VS) | Sec mal | Follow-up (months) | Distant metastases |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 50 | f | – | 1 | II | 1 | 5/9 | N | DOD (17) | Y |
| 2 | 15 | m | – | 1 | II | 1 | 5/19.1 | N | DOD (23) | Y |
| 3 | 43 | f | – | 50 | IV | 10 | 6/20 | N | DOD (14) | Y |
| 4 | 42 | m | – | 1 | III | 10 | 5/18 | N | DOD (41) | Y |
| 5 | 50 | f | – | 0 | II | 2 | 4/8 | N | NED (153) | N |
| 6 | 53 | f | – | 5 | III | 20 | 4/23.5 | N | DOD (17) | N |
| 7 | 72 | m | R337H (G) | 20 | II | 5 | 6/26.8 | Y | AWD(119) | Y |
| 8 | 73 | f | – | 0 | III | 1 | 5/12.3 | N | DOD (15) | Y |
| 9 | 39 | f | – | 0 | II | 10 | 2/5.7 | N | NED (115) | N |
| 10 | 37 | f | – | 1 | III | 30 | 5/14.7 | N | DOD (46) | Y |
| 11 | 83 | f | – | 10 | II | 30 | 2/5.7 | N | DOD (61) | N |
| 12 | 41 | m | – | 0 | II | 1 | 7/17.8 | N | DOD (11) | Y |
| 13 | 46 | f | IPM | 1 | I | 10 | 4/9 | N | NED (51) | N |
| 14 | 17 | f | – | 5 | III | ND | 7/17.8 | N | NED (78) | N |
| 15 | 67 | f | – | 10 | I | 1 | 4/9 | Y | AWD (120) | N |
| 16 | 41 | f | – | 1 | IV | 20 | 5/13.7 | N | DOD (48) | N |
| 17 | 58 | f | – | 1 | II | 2 | 4/14.5 | N | NED (75) | N |
| 18 | 34 | f | R248W (G) | 100 | IV | 40 | 6/26.8 | N | DOD (58) | Y |
| 19 | 55 | f | R213X (S) | 70 | IV | 30 | 7/17.8 | N | DOD (35) | Y |
| 20 | 49 | f | – | 0 | II | 2 | 5/13.7 | N | NED (51) | N |
| 21 | 72 | f | – | 1 | IV | 5 | 7/17.8 | N | AWD (8) | Y |
| 22 | 21 | f | – | 1 | II | 3 | 3/8 | N | NED (36) | N |
| 23 | 72 | m | – | 5 | II | 5 | 4/10.4 | N | NED (5) | N |
| 24 | 22 | m | – | 5 | III | 30 | 7/13.4 | N | DOD (64) | Y |
| 25 | 40 | m | – | 20 | III | 30 | 5/9 | Y | DOD (75) | Y |
| 26 | 43 | m | – | 5 | III | 20 | 7/22.7 | N | DOD (33) | Y |
| 27 | 43 | f | – | 0 | II | 20 | 2/5.7 | N | NED (44) | N |
| 28 | 45 | f | – | 0 | III | ND | 2/5.7 | Y | AWD (45) | Y |
| 29 | 69 | m | – | 50 | IV | 10 | ND | N | DURC (38) | N |
| 30 | 26 | m | – | 1 | III | 5 | 7/20.1 | N | NED (24) | N |
f female, m male, G germline, S somatic, IHC immunohistochemistry; W Weiss; VS van Slooten, N no, Y yes, DOD dead of disease, NED no evidence of disease, AWD alive with disease, DURC dead of unrelated cause, Sec mal second malignancy
Exons 5, 6, 7, 8, and 10 of the TP53 gene including exon-intron boundaries were amplified. These exons include the hot spots for ACC-associated TP53 muta- tions. As recommended the melting profile of each amplicon was predicted using the computer program WAVE Maker 5.1 (Transgenomic, Omaha, Nebraska, USA). Exons 5, 7, and 10 were amplified with GC- clamped primers to prevent complete melting of the amplicons at high temperatures needed to unfold the secondary structure of dsDNA. Occasionally, if the predicted conditions failed to produce well-defined peaks, they were empirically modified. Analysis conditions are displayed in Table 2.
Prior to WAVE analysis, heteroduplex formation of an equal amount of the patients sample and a wild-type sample occurred in a PCR system: 95℃ for 5 min, followed by a declining temperature T-1℃ for 1 min until the sample reaches a temperature of 60℃. Samples with aberrant wave profiles were subjected to direct sequencing with the ABI 310 sequencing analyzer according to standard procedures.
Statistical analysis was performed using SPSS 15.0. Cate- gorical data were tested for significance linked to the TP53 status with Fisher’s exact test if appropriate. All continuous variables were reported as mean and standard deviation and were tested for significance with two-tailed T test and ANOVA. p<0.05 was considered to be statistically significant.
| P53 exon | Primer sequence (5'-3') | GCT clamp | ||||
|---|---|---|---|---|---|---|
| 5 | tgt tca ctt gtg ccc tga ct cag ccc tgt cgt ctc tec ag | N | ||||
| 6 | gtc ccc agg cct ctg att cet c cgc ccg ccg cgc ccc geg ccc gtc ccg ccg ccc ccg tta acc cct cct ccc aga | Y | ||||
| 7 | gtg tta tct cct agg ttg gc cgc ccg ccg cgc ccc geg ccc gte ccg ccg ccc ccg cac agc agg cca gtg tgc agg | Y | ||||
| 8 | ctg att tcc tta ctg cct ctt gc cc get tet tgt cct get tgc | N | ||||
| 10 | ctc agg tac tgt gta tat ac cgc ccg ccg cgc ccc geg ccc gtc ccg ccg ccc ccg tec tat gge ttt cca acc | Y | ||||
| Amplicon | DN 96°℃ | DN 96°C | Annealing | Ext 72°℃ | Ext 72°C | Cycles |
| P53 Ex 5 | 10 | 0.5 | 56°C/0.5 | 0.5 | 10 | 35 |
| P53 Ex 6 | 10 | 1 | 54°C/1 | 1 | 10 | 35 |
| P53 Ex 7 | 10 | 1 | 54°C/1 | 1 | 10 | 35 |
| P53 Ex 8 | 10 | 0.5 | 63/0.5 | 0.5 | 10 | 35 |
| P53 Ex 10 | 10 | 0.5 | 50/0.5 | 0.5 | 10 | 35 |
| Amplicon | Temp (C) DHPLC | Runtime | Time-shift | |||
| P53 Ex 5 | 64.7 | 2.5 | 0 | |||
| P53 Ex 6 | 64.1 | 2.5 | 0 | |||
| P53 Ex 7 | 62.3 | 2.5 | 0 | |||
| P53 Ex 8 | 62.0/65.8 | 2.5 | -0.5/+1.5 | |||
| P53 Ex 10 | 58.6 | 2.5 | 0 | |||
DN denaturation, Ext extension, Temp temperature, DHPLC denaturated high-pressure liquid chromatography
Results
Patients
Thirty ACC patients who underwent surgery at the authors department were included in the present study. Twenty female and 10 male patients were diagnosed by median age of 47±17 years with a mean tumor diameter of 9.86±4.33 cm. At diagnosis 2 (6%) were TNM stage I, 12(40%) stage II, 10 (34%) stage III, and 6 (20%) stage IV of disease. After a median follow-up of 49± 37 months, 4 (13%) patients were alive with disease, 10 (33%) patients were alive without evidence of disease, 15 (50%) patients succumbed to disease, and 1 (3%) patient died of unrelated cause. Patients who were alive at final follow-up were followed for a mean of 66± 45 months. Further details on the present patient cohort including histopathological scoring of all tumors accord- ing to Weiss, van Slooten, and TNM stage are shown in Table 1 [14, 15].
Postoperatively 14 patients received no adjuvant treat- ment, whereas 16 were treated either with Mitotane alone (5) or in combination with radio-and/or chemotherapy (10). One patient solely underwent radiation of the formerly tumor-bearing region.
TP53 mutations
A TP53 mutation was identified in 3/30 (10%) patients: two germline mutations (7%) and one somatic mutation (3%). Germline mutations were located in exons 7 and 10, and the somatic mutation was detected within exon 6. The mutation in exon 6 was a nonsense mutation (R213X) leading to a premature stop codon, while mutations in exon 7 (R248W) and exon 10 (R337H) were missense mutations (see also Table 3). One polymorphism was detected in a noncoding region of intron 10, 30 bp following the donor splice site. Mutation analysis was performed in all patients on genomic and tumor DNA.
p53 immunostaining
Eighteen tumors (60%) showed no aberrant TP53 expres- sion (≤1%) by IHC while 12 tumors (40%) displayed increased aberrant expression (>5%). In five tumors aberrant p53 expression was detected in more than 5% of cells and in seven tumors in more than 10% of cells. Aberrant p53 expression in positive tumor samples ranged between 5% and 100% of stained cells (see also Table 1). A typical staining pattern is shown in Fig. 1.
| Patient ID | Exon/intron | G/S | Codon | Consequence of mutation |
|---|---|---|---|---|
| 7 | Exon 10 | G | 337 | c.1010 G>A, missense R337H |
| 18 | Exon 7 | G | 248 | c.742 C>T, missense R248W |
| 19 | Exon 6 | S | 213 | c.637 C>T, nonsense R213X |
| 13 | Intron 4 | G | I10+30 | g.17708 A>T |
G germline, S somatic
Correlation of TP53 mutations and p53 immunostaining
All three patients (P7, 18, 19) with mutations showed increased aberrant expression in more than 10% of cells by immunostaining, compared to only 3/27 patients without mutation (p=0.009). When resetting the p53 threshold to 5%, the difference failed to reach statistical significance (3/12 vs. 0/18, p=0.054).
Correlation of TP53 mutation, immunostaining, and clinical parameters
TP53 mutations were associated with higher percentages of aberrant p53-stained cells (63.33±40.41 vs. 6.34±13.54; p< 0.001) and with an increased van Slooten score (21.9±8.48 vs. 13.24±5.56; p=0.020). All three patients with TP53 mutations showed p53 staining of more than 10%, whereas
WT aaacactttt cgaca P19 aaacacttttt/cgaca
Wwwww WT t gtcgaaaagtgttt
P19 t gtcgaaaagtgttt
M/ minho
a)
b)
WT gagegett
P7 gagegiactt
MAM
WT cgaagcgct
P7 cg aagc/tg c t
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c)
d)
e)
only 3/27 patients without mutations demonstrated aberrant p53 staining of more than 10% (3/3 vs. 3/27; p=0.009).
Aberrant staining of p53 in more than 5% of cells was associated with an increased Ki67 index (21.2± 12.44 vs. 8.92±9.18; p=0.015) and a higher van Slooten score (17.1±7.40 vs. 12.32±4.94; p=0.046). Interestingly, Ki67 and aberrant TP53-IHC correlated significantly (Pearson correlation, p=0.024). An advanced tumor stage (III and IV vs. I and II) tended to have a higher amount of aberrant p53-positive cells (19.68±30.77% vs. 3.57±5.98%; p=0.064). A bivariate analysis showed a significant correla- tion between tumor stage and p53 IHC (Spearman correla- tion; p=0.036, r=0.385). Gender, frequency of secondary malignancies age at diagnosis, and Weiss score did not correlate with either TP53 mutation status or aberrant p53 immunostaining (see Table 1).
Correlation of TP53 status and outcome
Patients with a TP53 mutation did not have a higher risk of succumbing to disease than patients without a mutation (2/3 vs. 13/27; p=0.552). Patients with a staining of more than 5% of cells for aberrant p53 by IHC tended to have an increased risk of death (9/12 vs. 7/18; p=0.072) when compared to patients without aberrant p53 staining. That was also true for patients with a staining of more than 10% of cells (6/7 vs. 10/23; p=0.061), although both fail to reach statistical significance.
The overall survival was not statistically different between aberrant p53 IHC-positive and negative patients (p=0.254, log rank test), whereas an advanced tumor stage was associated with decreased overall survival (109.8±17.8 vs. 43.8±5.8, p=0.017, log rank test). However, immuno- histochemical staining of more than 5% of cells was associated with decreased disease-free survival (65.7±12.4 vs. 26.6±8.7 months; p=0.043 log rank test) (see Figure 2).
Discussion
Previous studies reported a frequency of somatic TP53 mutations in patients with sporadic ACC between 25% and 35% [11, 12, 17-20]. Most of these studies were small case series with incomplete data on immunohistochemistry or germline and somatic TP53 mutations. More recently, two studies assessed 36 and 31 adrenocortical tumors, including 29 and 13 ACC, respectively [11, 18]. However, we found a higher rate of TP53 germline mutations (7%), but only 3% showed somatic mutations in the present cohort of 30 ACC which is intriguingly less than in the previously published cohorts (33% and 25%). Despite the low mutation rate in the present study, about 40% of the ACCs showed aberrant p53 expression by immunohistochemistry, which is similar to previous reports [11, 13, 21, 22]. Especially in light of the relatively high incidence of TP53 germline mutations in patients with ACC, it should be
0.0
* p=0.043
-0.5
Log Survival
-1.0
-1.5
-2.0
+
-2.5
0.0000
20.0000
40.0000
60.0000
80.0000
100.0000
120.0000
DFS [months]
Survival Function
IHC P53
<5%
… >5%
+
.00-censored
+ 1.00-censored
mandatory that patients with aberrant p53-positive tumors by IHC should be referred for genetic testing. Both patients with TP53 germline mutations in the present cohort displayed no clinically distinct phenotype and only one revealed a second (and third) malignancy in their medical history (P7). In that particular patient (P7), the family history was positive, while the second patient (P18) had an unremarkable family history. However the early onset in P18 by age 37 might be considered suspicious for a hereditary background. Second malignancies occurred in a total of 4 (13%) patients.
In the context of an increasing availability of genetic diagnostics, a clinical phenotype which suggests a germline TP53 mutation in the background of ACC would be helpful to select for patients with a higher personal risk of TP53 mutations. Clinical and histopathological parameters in the present cohort could be identified that were associated with a higher individual risk for TP53 germline mutations. Accordingly, patients with aberrant p53 staining of more than 10% of cells by IHC and a higher VS index showed an increased risk for TP53 germline mutations. The present data emphasize the referral of such patients for genetic testing for TP53 germline mutation after genetic counseling despite a negative family history.
Reviewing the IARC database for TP53 mutations, 159 cases of TP53 mutations that were associated with ACC were identified [23]. Ninety-seven percent of mutations were located in exons 5, 6, 7, 8, and 10, while only 3% were found in exon 4. Interestingly, 5% were de novo mutations and 28% had no family history of TP53 associated cancers, which support the results of the present study [23]. All three mutations identified in the present cohort are published pathogenic mutations of the TP53 gene and associated with ACC [24]. One of two patients with TP53 germline mutations revealed a positive family history.
In the present study, we focused on exons 5, 6, 7, 8, and 10 which are known hot spots for TP53 mutations in ACC and other malignancies. Recently, Libe and colleagues reported 2/29 patients with ACC harboring somatic TP53 mutations in exon 4 [11]. Moreover IHC-p53-positive tumors displayed unexclusively somatic TP53 mutations. By contrast, in the present cohort, aberrant p53 staining was not restricted to tumors of patients with TP53 mutations. In the Libe study, immunostaining was only available in 20/36 presented cases [11].
Limitations of the study are the limited number although the rarity of the disease and the single-center nature must be taken into consideration. Furthermore not all coding exons of the TP53 gene were analyzed due to the compelling 97% of ACC-associated TP53 mutations in the herein assessed exons (5, 6, 7, 8, and 10) according to the IARCC database [23].
Overall survival was not significantly decreased, neither in patients with TP53 mutations nor with positive IHC, which is in line with previously published reports. However, both Libe and Sidhu reported a shorter disease- free survival in patients with TP53 mutations [11, 13]. In the present cohort, aberrant p53 staining of more than 5% was also associated with decreased disease-free survival (see Fig. 2), but the inheritance of a TP53 mutation was not.
Nuclear accumulation of aberrant p53 in other solid tumors such as hepatocellular carcinoma, prostate cancer, and breast cancer is caused by somatic TP53 mutations only in approximately 50% of cases [24-26]. The present study indicates that there may be alternative mechanisms also in ACCs involved such as epigenetic modifications or protein interactions, which leads to a nuclear accumulation of p53, as only 25% of p53-positive tumors (≥5%) showed TP53 mutations. Previous studies were not conclusive regarding this very issue. Libe found immune reactivity in all tumors with somatic TP53 mutations, but in none of the patients with wild-type TP53. Although Barzon detected somatic mutations more often (6/7 ACCs), aberrant p53 was more abundantly expressed in only 1/6 ACC with somatic mutations [11, 17].
ACCs with somatic TP53 mutations or p53-positive IHC might represent a subgroup with an unfavorable outcome, although a decrease in overall survival cannot yet be proven. Therefore, as recommended for patients with a Ki67 index of more than 10% or R1 status after surgery, adjuvant mitotane therapy might be beneficial for patients with aberrant p53 IHC-positive tumors. The decreased relapse-free survival of p53 ICH-positive ACCs and the strong correlation of Ki67 and p53 in the present cohort both support this hypothesis.
Acknowledgments We thank Prof. Domann, PD, Dr. Imirzalioglu, and Prof. Chakraborty for the technical support with the WAVE analysis and the use of the WAVE DHPLC system.
Conflicts of interest The authors hereby declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding The present research did not receive any specific grant from any funding agency in the public, commercial, or nonprofit sector.
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