High prognostic significance of Mdm2/p53 co-overexpression in soft tissue sarcomas of the extremities.

Soft tissue sarcomas are a heterogeneous group of neoplasms with various histological subtypes. Up to now, no individual causal molecular markers for prognosis and therapeutic success have been identified. A tumorigenic connection between the oncogene product Mdm2 and tumor suppressor p53 is generally accepted, but their possible clinical relevance has not yet been investigated sufficiently in soft tissue sarcoma. In 86 primary soft tissue sarcoma of the extremities (RO-resected, T1/2 N0 M0), Mdm2 and p53 overexpression were investigated by immunohistochemistry. The results were adjusted to clinico-pathological characteristics and evaluated for their prognostic relevance by multivariate analysis. In Cox's multivariate analysis with stratification of Mdm2 to p53 results, we determined four groups which had different prognostic values for relapse-free and overall survival (Mdm2-/p53- < Mdm2-/p53+ < Mdm2+/p53- < Mdm2+/p53+). The most striking finding was a relative risk (rr) for overall survival of 18.77 (P=0.006) for patients with Mdm2/p53 co-overexpression (n=40). It is noticeably higher than the additive risk from both factors. Coincident Mdm2/p53 overexpression is an independent molecular marker with the highest prognostic relevance described for soft tissue sarcoma. Thus, a high risk sarcoma group has been defined which we believe requires alternative therapeutic approaches.

Tumor hypoxia, p53, and prognosis in cervical cancers.

BACKGROUND: The p53 protein is involved in the regulation of initiation of apoptosis. In vitro, p53-deficient cells do not respond to hypoxia with apoptosis as do p53-normal cells, and this may lead to a relative growth advantage of cells without a functioning p53 under hypoxia. On the basis of this hypothesis, a selection of cells with a functionally inactive p53 may occur in hypoxic tumors. The development of uterine cervical carcinomas is closely associated with infections of human papilloma viruses, which may cause a degradation of the tumor suppressor gene p53, resulting in a restriction of apoptosis. Thus, cervical cancers have often a functionally inactive p53. The purpose of our clinical study was therefore to investigate the association between p53, hypoxia, and prognosis in cervical cancers in which the oxygenation status can be determined by clinical methods. MATERIAL AND METHODS: Seventy patients with locally advanced squamous cell cervical cancer Stages IIB (n = 14), IIIB (n = 49), and IVA (n = 7) were investigated in the period from 1996 through 1999. All were treated with definitive radiotherapy with curative intent by a combination of external radiotherapy plus high-dose-rate afterloading. Before therapy, tumor oxygenation was measured with a needle probe polarographically using the Eppendorf histograph. Hypoxic tumors were defined as those with pO(2) measurements below 5 mm Hg (HF5). Pretreatment biopsies were taken and analyzed immunohistologically for p53 protein expression with the DO-7 antibody. The DNA index was measured by flow cytometry. The statistical data analysis was done with SPSS 9.0 for Windows. RESULTS: The 3-year overall survival was 55% for the whole group of patients. Clinical prognostic factors in a multivariate analysis were pretreatment hemoglobin level (3-year survival 62% for patients with a pretreatment hemoglobin > or =11 g/dl vs. 27% for hemoglobin <11 g/dl, p = 0.006) and FIGO stage (Stage IIB: 65%; Stage IIIB: 60%; Stage IVA: 29%, p = 0.01). Sixty of the 70 tumors showed positive immunohistologic staining for p53 protein (transformed p53 = tp53), and 10/70 were negative (wild-type p53 = wtp53); p53 expression had no significant impact on survival (50% for tp53 vs. 79% for wtp53, p = 0.11). FIGO stage and anemia had no impact on p53 expression. Forty-nine of 70 tumors were hypoxic (HF5+), and 21 showed no hypoxia (HF5-). Hypoxic carcinomas were more frequently positive for p53 as compared to nonhypoxic tumors (27% vs. 13%, p = 0.011) and showed a trend toward a lower survival (48% vs. 70%, p = 0.07). In a further multivariate analysis, the impact of a combination of p53 expression and hypoxia on survival was examined. After adjusting for FIGO stage and pretreatment anemia, patients with wtp53 tumors had the best prognosis (3-year survival 79%) followed by tp53-HF5(-) patients (57%), and the most unfavorable prognosis was observed for tp53-HF5(+) patients (47%). The DNA index was higher in tp53 carcinomas compared to wtp53 tumors, 1.97 +/- 0.4 vs. 1.67 +/- 0.1, p = 0.05. The highest DNA index was found in hypoxic tumors with transformed p53 (2.2 +/- 3.1). CONCLUSIONS: Advanced stage and pretreatment hemoglobin level are independent prognostic factors in cervical carcinomas. The immunohistologic detection of (a functionally inactive) p53 and the presence of hypoxia had no prognostic impact, if analyzed as single parameters. However, the combination of both parameters was able to discriminate different prognostic subgroups. Moreover, hypoxic cancers were more often immunohistologically positive for tp53 protein and had a higher DNA index with the highest DNA index in tumors with both hypoxia and tp53 protein expression. These findings in summary support the theory that the tumor's microenvironment may influence the biologic behavior via hypoxia.

How is the mutational status for tumor suppressors p53 and p16(INK4A) in MFH of the bone?

Both tumor suppressor genes p53 and p16(INK4A) play a crucial role in the control of cell cycle and tumor development. In this study 19 malignant fibrous histiocytomas of the bone (MFH-b), a very rare sarcoma entity, were investigated for mutations in p53 and p16 genes by a PCR-SSCP-sequencing analysis. In the tumor samples two p53 mutations and two polymorphisms (one in the p53 gene and one in the p16 gene) were found. The occurrence rate for p53 mutations and the absence of p16 mutations in MFH-b are comparable to the findings for MFH of soft tissues (MFH-st) and osteosarcomas, suggesting that p53 rather than p16 may play a role in tumorigenesis of MFH-b.

Clinical relevance of pRb and p53 co-overexpression in soft tissue sarcomas.

The goal of this study was to examine the relationship between immunohistochemical pRb detectability and p53 overexpression in 198 soft tissue sarcomas (STS) with regard to its clinical relevance. Distinct pRb detectability multivariately shows a correlation to survival rate (relative risk (RR)=1.59, P=0.037). p53 positivity was also multivariately correlated to poor prognosis (RR=2.17, P=0.0014). Stratification of pRb staining to p53 results shows a prognostical graduation. Patients with negativity for both proteins have the most favorable prognosis (projected 5-year survival rate (psr)=54.5%). In contrast to this, positivity for both antibodies has the highest risk (RR=2.48, P=0.02) and the poorest prognosis (psr=17.4%). To conclude, these results explain that the clinical relevance of immunohistochemical pRb positivity in STS is connected with p53 in the form of having an increasing effect on the known prognostic relevance of p53 overexpression.

Detection of numerical chromosomal changes in 20 malignant fibrous histiocytomas by FISH.

We investigated 20 malignant fibrous histiocytomas (MFHs) with the help of specific centromeric probes for chromosomes 1, 3, 4, 6, 8, 9, 12, 16, 17 and 18. The results show a broad variation in the number of signals per nucleus. However, tumors can be assigned into four groups: i) with mostly disomic clones, ii) with a high percentage of polysomic clones, iii) with a considerable amount of monosomic and nullisomic clones and iv) with a tendency in both directions. A gain of spots per nucleus takes place in 75-100% of the investigated tumors - the highest incidence occurring with respect to chromosome 3. A loss of spots per nucleus occurred in 20-60% of the tumors - predominantly with respect to chromosome 1.

Molecular characterization and liposomal transfection of a p53-mutated cell line established from a poorly differentiated leiomyosarcoma.

A human cell line LMS6-93 has been established from a leiomyosarcoma (LMS). Characteristics for ultrastructure, growth characteristics, cell cycle distribution, karyotype, protein expression detected by immunohistochemistry (IHC), p53 mutational status and liposomal transfection behaviour were studied and determined. The primary tumor was clearly positive for á-smooth muscle type actin and desmin in moderately differentiated areas and indicated a loss of myogenic differentiation in other regions and therefore was classified as a poorly differentiated LMS. The cell line LMS6-93 contains mainly polymorphic spindle shaped or polygonal tumor cells which possess the characteristics of primitive mesenchymal cells, based on their morphology and positive reaction with an antibody to vimentin. IHC staining for S100, synaptophysin A, NSE, neurofilament proteins and cytokeratins were negative. Cytogenetic analysis revealed in the cell line diploid karyotypes comparatively close to several structural and numerical aberrations for chromosomes 2, 5, 6, 9, 10, 12, 14, 17, 18, 20, 22, and Y. IHC positivity was found for the tumor suppressor protein Rb and the oncogene product MDM2. In a p53 mutational analysis a 1 bp insertional mutation in exon 6 (G insertion in codon 215) was detected and confirmed in the original primary tumor. The other p53 allele appears to be wild-type as indicated in Western hybridization. Using different cationic lipid formulations complexed with a reporter expression vector (GFP) successful transfection into LMS6-93 cells was observed. The highest transfection rates (20-30% GFP expression in the viable cell population) were obtained with lipofectin. These results suggest that LMS6-93 functions as a good in vitro model for transfection studies on an LMS cell line carrying a heterozygous p53-frameshift mutation.

Prognostic value of immunohistochemistry for p53 in primary soft-tissue sarcomas: a multivariate analysis of five antibodies.

Most changes of tumor suppressor p53 and its pathway involve a protein with prolonged half-life that permits immunohistochemical detection. The goal of this study was to compare the prognostic relevance of five different p53 antibodies in primary soft-tissue sarcomas (STS) with known p53 mutation status, using a multivariate Cox regression model (adjusted to tumor grading, staging, localization, tumor type, and therapy). A group of 198 primary STS of six types were investigated for p53 overexpression, using p53 antibodies DO-1, DO-7, Pab1801, Pab240, and CM-1. A positive marker frequency between 36.2% and 62.6% was detected. Out of 65 patients whose primary tumor reacted positively to all five antibodies, 52 (80%) died within the study period. Only the N-terminal-binding monoclonal antibodies DO-1, DO-7 and Pab1801 showed a multivariate correlation with survival (P = 0.0014, 0.0048 and 0.02). CM-1 and Pab240 had a univariate, but not a multivariate correlation, with a confounding effect of grading. The prognostic relevance for the five p53 antibodies was: DO-1 > Pab1801 > DO-7 > CM-1 > Pab240. This is the first study that investigates multivariately the prognostic relevance of p53 immunostaining in STS. If monoclonal antibodies with an epitope in the N-terminal region of the p53 protein (DO-1, Pab1801, DO-7) are applied, p53 immunohistochemistry provides an independent prognostic marker in STS.

Regulation of proliferation and apoptosis in sporadic and hereditary medullary thyroid carcinomas and their putative precursor lesions.

C-cell hyperplasia (CCH) and medullary thyroid carcinoma (MTC) in patients affected by germline mutations of the RET oncogene represent an exceptional opportunity to study the regulation of proliferation and apoptosis during tumour initiation and progression. In 56 specimens [CCH, n=1; MTC with CCH, n=26; MTC, n=20; lymph-node metastasis (LNM), n=9] from 46 patients [multiple endocrine neoplasia type 2a (MEN2a), n=24; MEN2b, n=2; familiar MTC (FMTC), n=4; sporadic MTC, n-16] and 3 cases of non-neoplastic CCH, proliferation activity (MIB1), the rate of apoptosis [dUTP nick end labelling (TUNEL)] and expression of p53, bcl-2, bcl-x and bax were investigated and compared with clinical data. In MEN-associated CCH and small MTC, bcl-2 was strongly expressed, bcl-x was moderately expressed and bax was only weakly expressed. Advanced tumours and LNM did show a more heterogeneous bcl-2 staining accompanied by an increased bax expression and accelerated proliferation. The rate of apoptosis was extremely low in all investigated tumours. P53 was detectable in three patients with rapidly growing and extensively metastasising MTC. No somatic p53 mutations were found. Hereditary MTC with germline RET mutations at codon 918 (MEN2b) and codon 634 revealed a bias towards a higher proliferation activity at a younger age and are more frequently accompanied by LNM. CCH and MTC are characterised with a preponderance of bcl-2 as a factor blocking the programmed cell death. While MTC, in general, is a slowly growing tumour, a minority of tumours do progress rapidly with high proliferation. The factors leading to an accelerated tumour progression do not seem to take their effect via the regulation of apoptosis. Certain alterations of RET are supposed to have a direct or indirect implication on proliferation and, because of this, an effect on the clinical course.

Primary hereditary medullary thyroid carcinoma--C-cell morphology and correlation with preoperative calcitonin levels.

Early thyroidectomy offers an opportunity of preventing the development of medullary thyroid carcinoma (MTC) in patients at risk for hereditary MTC. We investigated the thyroid glands of 32 patients with hereditary MTC to identify the changes in C-cell morphology and to correlate these with plasma calcitonin (CT) levels and with clinical data. The entire thyroid gland was processed for histological examination including immunostaining for CT. All glands revealed C-cell hyperplasia (CCH), and MTC was found in 21 patients (66% of 32, youngest patient 6 years, youngest with lymph node metastases [LNM] 17 years). The transition from CCH to MTC was characterized by destruction of the follicular basement membrane and by diminished intensity of CT immunostaining. Normal plasma CT levels after provocation with pentagastrin were found only in patients with CCH. Basally elevated plasma CT levels were restricted to MTC. LNM were only found in multifocal tumours at least 4 mm in diameter. It is not yet clear whether or not CCH in patients at risk for hereditary MTC is a neoplastic change, but in these patients the term 'C-cell hyperplasia' is of doubtful value. All MEN gene carriers reveal CCH, and almost all of them will develop multifocal MTC, so that CCH is probably a precursor lesion of an indubitably malignant tumour. Prophylactic thyroidectomy is justified at the age of 6 to anticipate development of a MTC. Lymphadenectomy is necessary in children if they are older than 10 years or have elevated plasma CT levels.

Immunohistochemical and clinical evaluation of cathepsin expression in soft tissue sarcomas.

Lysosomal proteases are known to enhance the spread of epithelial tumour cells, but little is known of the possible role of proteases in the growth of soft tissue sarcomas (STS). We investigated the expression of cathepsins D, B, S, H, L and procathepsin L in frozen sections of 34 STS from 34 patients by immunohistochemistry (IHC). Cathepsins D, B and H were relatively highly expressed in STS (77-91%). The expression rate of cathepsins S and L and of procathepsin L was lower (40-66%). Cathepsin S and L expression showed a moderate (P = 0.078 and P = 0.019) and procathepsin L a strong (P = 0.00001) correlation with the survival rate of STS patients. Cathepsin S expression is also correlated with the local recurrence rate (P < 0.01). Lysosomal proteases may play a role in STS progression, and cathepsin expression may also have significance as a prognostic factor in STS.

Prognostic relevance of C-terminal Mdm2 detection is enhanced by p53 positivity in soft tissue sarcomas.

We examined the clinical value of immunohistochemical (IHC) Mdm2 detection by an N-terminal (IF2) and a C-terminal (19E3) binding monoclonal antibody (Ab) in soft tissue sarcomas (STSs) with regard to the p53 status. Therefore, we investigated a cohort of 198 patients with STSs of six entities with known p53 IHC by using a multivariate Cox regression model to determine the prognostic value of Mdm2 staining. Only positivity with the 19E3 Ab correlated multivariately significantly with survival (RR = 2.32, p = 0.0035). We stratified the C-terminal Mdm2 staining (19E3) according to p53 IHC (DO-1) and found patients could be divided into three groups with an increasing risk: (a) patients with Mdm2 (19E3)-negative as well as p53 (DO-1)-negative tumors, (b) patients with tumors that were either Mdm2 (19E3) or p53 (DO-1) positive, and (c) patients with tumors that were Mdm2 (19E3) as well as p53 (DO-1) positive. Positive staining for both Mdm2 and p53 meant a very poor prognosis with a relative risk of 4.63 (p = 0.00001). This points to the possibility that--in addition to the p53-dependent pathway--Mdm2 could have an effect through a p53-independent pathway. Thus, our results indicate that C-terminal Mdm2 staining (19E3) constitutes an independent prognostic marker in STS.

Influence of high density lipoprotein (HDL), prepared from human blood, on prostanoid formation, serum and tissue lipids and development of arteriosclerosis in cholesterol rich fed rabbits.

The i.v. administration of high density lipoprotein (HDL) into cholesterol fed rabbits decreased statistically significantly the serum level of total cholesterol and of low density lipoprotein cholesterol after a feeding period of 8 weeks. These diminished levels of cholesterol were associated with a statistically significant reduction in the levels of cholesterol esters in kidneys and platelets but not in hepatic tissue or in aorta. Macroscopically detectable arteriosclerosis was not statistically significantly diminished. The formation of prostanoids by the aorta remained unchanged. The atherogenic role of immunologic factors acting against the heterologous HDL may have compensated for the antiatherogenic HDL action on plasma and tissue lipids.

Cytogenetic analysis of a benzpyrene induced osteosarcoma in the rat (Rattus norvegicus).

A cytogenetic comparison of primary and transplant tumor cell-lines, both originating from a benzpyrene induced osteosarcoma, with normal rat cell-lines (Rattus norvegicus) is presented here. In all tumor cell-lines tested, the number of chromosomes was increased by one or two. Using Giemsa-banding, structural chromosomal changes, i.e. a Robertsonian translocation t(4;4)(q10;q10) and an interstitial deletion del(6)(q11q16) could be recorded. Furthermore, staining of nucleolus organizer regions (NORs) revealed a shift in NOR activity from chromosome number 11 to 12 and a decrease in NOR activity at chromosome number 3.

Cytogenetic characterization of ten malignant fibrous histiocytomas.

We examined 10 malignant fibrous histiocytomas (MFHs) using metaphase preparations. Six tumors showed clonal structural and/or numerical chromosomal aberrations, and four tumors had normal karyotypes. For the most part, chromosomes 1, 3, 6, 9, 12, 16, 18, and 20 were involved in structural aberrations. The breakpoint regions most frequently were in 1p32, 3p25, and the centromeric region of chromosomes 1 and 16. There was a conspicuous loss in chromosome 18. We detected ring chromosomes in two tumors. One tumor showed a high percentage of near-haploid cells. Our results show many parallels to data which have already been published. MFHs include a broad spectrum of tumors of widely different histology and clinical course. So it is not surprising to find a cytogenetic diversity of chromosomal aberrations in this study.

Radiosensitization in sarcoma cell lines with a p53 missense mutation correlates with prevention of irradiation G2/M arrest but not with induction of apoptosis.

We analysed the effects of caffeine and taxol on the radiobiological behaviour of two human sarcoma cell lines (RD, SK-LMS-1) each with a p53 missense mutation. Treatment with 2 mM caffeine resulted in an inhibition of the irradiation induced G2/M arrest in both cell lines. This effect was coupled with a radiosensitization in cell line SK-LMS-1 after an irradiation with 6 Gy (enhancement factor of 5.0). However, the effect of radiosensitization was not correlated with an induction of apoptosis. Incubation with 20 nM taxol increased the irradiation induced apoptosis almost 3-fold in cell line SK-LMS-1, but not in cell line RD. However, taxol had no effect on the irradiation induced G2/M arrest or radiosensitivity in either cell line. The results support the hypothesis that the prevention of irradiation induced G2/M arrest but not the induction of apoptosis plays a critical role in determining radiosensitivity in sarcoma cell lines with p53 mutations.

Assessment of genomic imbalances in malignant fibrous histiocytomas by comparative genomic hybridization.

In order to investigate genomic imbalances, comparative genomic hybridization was applied to 20 malignant fibrous histiocytomas. Deletions were rare and found mainly in chromosomes 2q33-35, 4q32-qter, 8p, 9p21-pter, 12p and 19p, whereas, over-representations frequently affected chromosomes 3, 4q31, 5p, 6, 7, 14q22-ter, 18p, as well as, five distinct amplifications within the regions 12q12-15 and 15q24-qter. The total number of genetic imbalances per tumor was slightly increased in primary tumors when compared to relapses. No relationship was found between the patterns of gain and loss when compared to the histological subtype, tumor grading, the clinical outcome and the p53 mutation status.

A multifactorial prognostic model for adult soft tissue sarcoma considering clinical, histopathological and molecular data.

Soft tissue sarcomas (STS) are malignant mesenchymal lesions with a high degree of prognostic variability. Different prognostic markers such as grading, staging, tumour type and localisation are known. The establishment of these markers was based on the evaluation at results of extensive cohorts of patients. Therefore, only the established markers provide us with information about probabilities in relation to other qualities. Considering as many different markers as possible in one prognostic statement should increase the value of the resultant information. Therefore, we developed a model involving known prognostic markers to formulate an individual prognostic index. In a retrospective analysis, different prognostic factors of 198 adult STS patients with histological tumour free resection margins were evaluated using a multifactorial analysis. On the basis of a Cox-Regression-Model with proportional hazards, the prognostic factors (tumour type, staging, localisation and type of surgical resection) were selected using previous knowledge and a statistical step backward selection procedure adjusting the immunohistochemical status of p53/Mdm2 expression. On the basis of the baseline survival function of our cohort (S0 (t)), the cumulative probability of survival for two S (2) and five S (5) years was estimated. As a result of our analysis the equations S (2) = (e-00393)P and S (5) = (e-00869)P can be used to estimate the individual two and five-year probability of survival in our cohort. Here p is the result of the amount of the estimated regression-coefficients of the exact variables of the respective individual patient. This model makes it possible to include all the evaluated prognostic factors which, in turn, increases the accuracy of the prognostic information for individual patients underlining the proportional hazards assumption.

The p53 gene in soft tissue sarcomas: prognostic value of DNA sequencing versus immunohistochemistry.

BACKGROUND: The aim of this study was to compare sequencing and immunohistochemistry (IHC) for their ability to detect p53 mutations and to assess their prognostic value in soft tissue sarcomas (STS). MATERIAL AND METHODS: 146 STS samples were investigated by single strand DNA conformation polymorphism (SSCP)-sequencing for p53 mutations. Additionally, IHC with five p53 antibodies (CM-1, Pab1801, Pab240, DO1, DO-7) was performed. RESULTS: In 65 to 131 tumor samples (44.5% to 90.4%) elevated levels of p53 protein were recorded by IHC, depending on the antibody applied. Sixteen out of 146 STS tumor samples (11%) had p53 gene mutations. 13 cases (81.2%) of the 16 tumors with p53 mutations could be detected by DO-1. However, of the 93 DO-1 positive tumors 79 were negative by SSCP-sequencing analysis. Using multivariate regression analysis (Cox proportional hazards model) both p53 mutations (non-frameshift mutations) and IHC detection of p53 protein overexpression were prognostic predictors for poor survival. CONCLUSIONS: This study suggests that IHC is valuable for assessing p53 mutations in STS, but sequencing provides additional important information on the molecular characteristics of the alterations.

G2/M checkpoint is p53-dependent and independent after irradiation in five human sarcoma cell lines.

To determine the role of p53 in G2/M arrest, G2/M transition and apoptosis, we investigated five human sarcoma cell lines with different p53 gene status in their response to X-rays. The p53 status of the cell lines was mutant (US 8-93, LMS 6-93 and RD), null (SAOS-2) and wildtype (A-204). Clonogenic survival of the cell lines varied as the survival fraction at 2 Gy (SF2) ranged from 0.28 to 0.79. Compared with the mutated p53 cell lines (SF2 with a range from 0.46 to 0.79) the clonogenic survival of the wildtype p53 (wt-p53) cell line A-204 (SF2 = 0.34) was lower. The p53 null cell line (SAOS-2) was also sensitive to X-rays (SF2 = 0.28). We detected, in all cell lines a similar behavior in their response to irradiation with G2/M arrest and apoptosis. However, the maximal rate of apoptosis with a range from 7.0 to 18.0% was rather small. The decrease of G2/M cells was coupled with an increased percentage of apoptotic cells. However, a different delay in G2/M did not result in a change of radiation sensitivity. Western analyses showed an increased P53 level only for the cell line A-204 (wt-p53) after irradiation. Our results point out that there is not always a simple relationship between p53 gene status and radiation sensitivity. We suggest, that wt-p53 plays an active role in G2/M arrest and in decreasing the number of G2/M cells as a response to apoptosis. Therefore, p53-dependent regulation in G2/M may be as important as p53-independent mechanisms.

Simultaneously occurring liver metastases of pheochromocytoma and medullary thyroid carcinoma--a diagnostic pitfall with clinical implications for patients with multiple endocrine neoplasia type 2a.

Malignant pheochromocytoma is an exceptional complication in patients with Multiple Endocrine Neoplasia Type 2a (MEN2a). In this paper, we report on a 53-year-old male patient with an evident RET gene germline mutation, who simultaneously developed hepatic metastases of medullary thyroid carcinoma (MTC) and pheochromocytoma. Comprehensive immunohistochemical investigations were performed to elaborate markers which could be useful for differentiating between MTC metastases and pheochromocytoma, respectively. Calcitonin and CEA, in particular cytokeratins and trefoil factor family 1 (TFF1), were detectable exclusively in MTC, whereas all the other markers revealed a comparable expression in both MTC and pheochromocytoma. The only clues that could indicate a potential malignant course were size, a lack of sustentacular cells and hyaline globules, and a focal spindle cell pattern in pheochromocytoma. Owing to a wide agreement in cellular differentiation and a lack of specific, routinely applicable markers for pheochromocytomas, a comprehensive and goal-directed immunohistochemistry is required to rule out pheochromocytoma metastasis in patients with MEN2a. A misinterpretation could lead to harmful clinical complications, as shown in the present case.