Adrenocortical carcinoma, an unusual extracolonic tumor associated with Lynch II syndrome
V. Medina-Arana · L. Delgado · L. González ·
A. Bravo . H. Díaz . E. Salido . D. Riverol .
J. J. González-Aguilera · A. M. Fernández-Peralta
Published online: 12 January 2011 @ Springer Science+Business Media B.V. 2011
Abstract Lynch syndrome (LS) is an autosomal domi- nant condition that predisposes to colorectal cancer and specific other tumors. Extracolonic tumors occur mainly in the endometrium, stomach, ovary, small intestine and uri- nary tract. The presence of rare tumors in patients belonging to families who have Lynch syndrome is always interesting, because the question arises whether these tumors should be considered as a coincidence or are related with the syndrome. In this last case, they are also the result of the defect in the mismatch repair system, opening the possibility of extending the tumor spectrum associated with the syndrome. Here we describe a patient from a Lynch syndrome family with a germline mutation c.2063T>G (p.M688R) in the MSH2 gene, who developed an adrenal cortical carcinoma, a tumor not usually associated with LS. We analyzed the adrenocortical tumour for microsatellite instability (MSI), LOH and the presence of the germline c.2063T>G (M688R) mutation. The adrenal cortical car- cinoma showed the MSH2 mutation, loss of heterozygosity of the normal allele in the MSH2 gene and loss of immu- nohistochemical expression for MSH2 protein, but no microsatellite instability. Additionally, the adrenal cortical
V. Medina-Arana · L. Delgado · L. González ·
A. Bravo · H. Díaz
Servicio de Cirugía General y Digestiva, Hospital Universitario de Canarias, La Laguna, Tenerife, Spain
E. Salido · D. Riverol
Unidad de Investigación, Hospital Universitario de Canarias, Centre for Biomedical Research on Rare Diseases (CIBERER), Tenerife, Spain
J. J. González-Aguilera ☒ · A. M. Fernández-Peralta Unidad de Genética. Dpto. Biología, Universidad Autónoma de Madrid. Cantoblanco, 28049 Madrid, Spain
e-mail: jj.gonzalez@uam.es
carcinoma did not harbour a TP53 mutation. The molecular study indicates that this adrenal cortical cancer is probably due to the mismatch repair defect.
Keywords Adrenal cortical carcinoma · Lynch syndrome · MSH2 mutation · Tumor spectrum
Abbreviations
CRC Colorectal cancer.
LOH Loss of heterozygosity.
MMR Mismatch-repair.
MRI Magnetic resonance imaging.
MSI Microsatellite instability.
PCR Polymerase chain reaction.
PET Positron emissiont tomography.
RFLP Restriction fragment length polymorphism.
SSCP Single strand conformation polymorphism.
Introduction
Lynch syndrome is a hereditary syndrome that predisposes to different types of cancer. It shows an autosomal domi- nant heredity and is characterized by early onset and the development of neoplastic lesions in a variety of tissues, with a penetrance of approximately 80% for colorectal cancer (CRC), 60% for endometrial cancer, and well below 20% for the other cancers [1]. The manifestation of a colorectal cancer (usually in the right colon) at a young age is the guiding symptom [2]. The frequency of extracolonic cancers in Lynch syndrome ranges from 2 to 20% in dif- ferent series [3, 4]. The presence of these extracolonic tumors, mainly located in endometrium, stomach, ovary,
small intestine and urinary tract defines Lynch II syndrome [5]. Of these, the endometrial cancer is the most common, appearing 15 years earlier than in the general population. Small bowel cancer also has an increased risk, while the risk of lung, breast, prostate, bladder, larynx and brain cancer does not seem to increase [6-8]. Furthermore, other tumors have been reported to be associated with the syn- drome, so Sijmons et al. [9] have reported a malignant fibrous histiocytoma in a patient with a mutation in MSH2 , and we found a leiomyosarcoma in a patient from a Lynch syndrome family [10].
On the island of Tenerife (Spain) we found a founder mutation in exon 13 of the MSH2 gene (c.2063T>G) that affects to ten families, resulting in a nonconservative aminoacid change, M688R, at the ATPase domain of the Msh2 protein [11]. In the analysis of a family who had this mutation and met the Amsterdam criteria, we found a patient who developed adrenal cortical carcinoma whose molecular study shows a relationship to the syndrome that would broaden the spectrum of tumors appearing in the Lynch syndrome.
Materials and methods
Case report
The patient was a 60 year old woman from a Lynch syn- drome family (Fig. 1) that meets the criteria [12, 13]. with a history of endometrial adenocarcinoma (stage III-A) diagnosed in 2000, being operated by hysterectomy, oophorectomy and double lymphadenectomy. Subsequent follow-up revealed no evidence of disease. In 2008 she was diagnosed with breast infiltrating ductal carcinoma, grade III of Bloom-Richardson, which was surgically resected. In
May 2009, in an MRI performed in a follow-up appoint- ment to monitor breast cancer, a right adrenal nodule 5.1 × 4.3 cm was found, intermediate signal on T2 and low signal on T1 was described. Given the neoplastic history of the patient, the most likely diagnosis would be metastasis but cannot be ruled out a primary neoplasm. Afterward is performed a PET to confirm the diagnosis of neoplastic lesion in right adrenal gland. The patient was evaluated by the Endocrinology Service ruling out a right adrenal pheochromocytoma. She was operated on in July 2009 performing a laparoscopic right adrenalectomy. The histological examination of the surgical specimen con- firmed the diagnosis of adrenal cortical carcinoma, and now the patient has recovered well from surgery and evaluated by the oncology department has not received adjuvant therapy.
Amplification and analysis by PCR-SSCP of the exon 13 region of the MSH2 gene
DNA from patient was obtained from 3 ml peripheral blood and purified by the standard phenol-chloroform method. Also, DNA was purified from adrenal cancer samples, either fresh tissue or paraffin embedded when necessary. Tissue blocks with mostly tumoral tissue (more than 70%) were chosen. When this was not possible, tissue sections were microdissected to select tumor-specific samples. The initial screening was performed by poly- merase chain reaction (PCR) amplification of each MSH2 exon followed by SSCP (single strand conformation polymorphism) using 10% acrylamide gels (99:1 acryl- amide:bisacrylamide), in 0.5 x TBE buffer for 6 h at 200 V. PCR was carried out in a final volume of 50 ul containing 0.5 mM of each oligonucleotide, 0.1 mM of dNTPs, 1 U of Taq DNA polymerase. The PCR cycles
Colorectal ca.
☒ Ovary ca.
Endometrium ca.
Brain ca.
Lung ca.
K Adrenal ca.
Breast ca.
☒ Leiomyosarcoma
consisted of denaturing at 95℃ for 3 min followed by 30 cycles of 94℃ for 1 min, 54℃ for 1 min and 72ºC for 1 min. The amplified fragment was analyzed by SSCP and visualized by silver staining.
Sequence analysis
The PCR products showing aberrant electrophoretic mobility were cycle sequenced using fluorochrome-labeled dideoxynucleotides (Applied Byosystems and Beckman) and either the forward or reverse primer. Reaction products were separated by capillary electrophoresis and analyzed with automatic sequencing software (ABI310, Applied Byosystems, and CEQ 2000 XL DNA Analysis System, Beckman). BLAST software (http://www.ncbi.nlm.nih. gov/BLAST/) was used to compare the sequence with MSH2 genomic and cDNA data in GenBank (http://www. ncbi.nlm.nih.gov/genbank, accession numbers: U41218 and NM_000251).
LOH analysis and diagnostic PCR-RFLP test of the c.2063T>G (M688R) mutation
Typing of the tumor was done by PCR-RFLP (restriction fragment length polymorphism), by amplifying with a pair of oligonucleotides flanking the mutation found: MSH2-1: 5’-CGCGATTAATCATCAGTG-3’ and MSH2-2:5’-AT- GGCACAAAACACCCAA- 3’ (we used also this region for determining LOH). The PCR conditions were: 95℃, 3 min; 1 min/47°C, 1 min/72°C, 1 min, for 30 cycles. The 183 bp PCR product was cut with Hsp92II restriction enzyme, whose recognition site (CATG’) is present in the wild type allele, coding for methionine. The digestion products were analyzed in 5% acrylamide gels and stained with ethidium bromide. The allele coding for arginine remains as a 183 bp fragment, while the allele coding for methionine produces two fragments of 158 and 25 bp after enzymatic digestion of the PCR product. Positive and negative controls were included to check for the presence of partial digestions and PCR contaminations. The results were also confirmed by direct sequencing of the PCR product.
Inmunohistochemistry
Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections of tumor, after antigen retrieval in high pH solution (Dako), at 123ºC for 1 min. Sections were treated with 0.1% Triton X-100 in phosphate buffered saline (PBS), and then incubated with mouse monoclonal antibody, diluted 1:50 in 3% bovine serum albumin/PBS, against human MSH2 (MSH2-AB2, Calbiochem) or mouse anti-p53 (Dako). After blocking endogenous peroxidase
with 0.3% H2O2 in methanol, for 15 min, slides were washed three times, 5 min each, with PBS and then incu- bated for 30 min with HRP-conjugated anti-mouse IgG serum (Dako). After another three PBS washes, a DAB- H2O2 solution was used as chromogen, and sections were counterstained with haematoxylin. Adjacent normal tissue and surrounding tissue lymphocytes served as internal positive controls for each case. Nuclear staining of the tumor was scored as either present or absent compared to the corresponding internal control.
Analysis of Microsatellite Instability (MSI)
In all cases, DNA was amplified in a 25 ul volume con- taining 200 ng of tumor DNA, 0.5 uM of each primer, 1 x buffer, 1.5 mM MgCl2, 200 µM dNTPs, 1U Tth DNA polymerase (Pacisa-Giralt). To assess microsatellite insta- bility we utilized the MSI Analysis System v 1.2 (Pro- mega), that meets the recommendations proposed by the National Cancer Institute [14]. Products were amplified in a Perkin Elmer Cetus 480.0 termocycler and electrophore- sed in nondenaturing 8-15% polyacrylamide gels and Ag-stained.
Results
We knew previously that the patient carried a germinal mutation c.2063T>G (M688R) in the MSH2 gene [11], and the adrenal tumor also presented the mutation (Fig. 2). The results of MSI analysis in the family show that colorectal tumors display instability (allelic shift at two or more markers compared with normal mucosae, thus they are considered as microsatellite instability high (MSI-H) tumors. In contrast, the adrenal cortical carcinoma shows no allelic shift at any microsatellite marker and was clas- sified as microsatellite stable (MSS), according to the established National Cancer Institute criteria [14] (Fig. 4). Furthermore, this adrenal cortical cancer showed loss of heterozygosity of the normal allele in the MSH2 gene (Fig. 3), and complete loss of immunohistochemical expression for MSH2 protein (data not show). The breast cancer developed by of the same patient was MSI-H and the endometrial carcinoma was MSI-L. Additionally, the adrenal cortical carcinoma does not have mutation in TP53 (data not show).
Discussion
The presence of rare tumors in patients belonging to fam- ilies who have Lynch syndrome is always interesting, because the question arises whether these tumors should be
120 130 ACTCA KGGCCCAAATTGGGT
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considered as a coincidence or are related with the syn- drome arising as the result of the defect in the mismatch repair system. This last finding opens the possibility of extending the tumor spectrum associated with the syn- drome. We have analyzed an adrenal cortical carcinoma in a patient belonging to a Lynch syndrome family with a germline mutation in the MSH2 gene [11].
Berends et al. [15] described a patient with a germinal mutation in exon 4 of MSH2 who, in addition to ovarian cancer and three metachronous colorectal tumors, pre- sented an adrenocortical carcinoma. The family of the patient did not meet the Amsterdam criteria. The colorectal tumors showed MSI and loss of Msh2 protein expression but the adrenocortical carcinoma was MSI-stable, showed only loss of the allele containing the MSH2 mutation (LOH) and expressed both the Mlh1 and Msh2 protein. In view of these results the authors concluded that the development of this tumor should not be considered to be associated with the germline MSH2 mutation. Therefore, the occurrence of this rare tumor in this Lynch patient should be considered as a coincidence. Later, Broaddus et al. [16] described an adrenal carcinoma in a family that
meets the Amsterdam criteria. The patient was probably a carrier of a germline mutation in MSH2 (his father had the mutation). This mutation, located in intron 12 of the gene, affects a splice site which results in skipping of exon 11 and exon 12 and a truncated inactive protein. The adrenal tumor showed complete loss of immunohistochemical expression for Msh2 protein, but was MSI-stable. Probably In this case the tumor was related with the mutation in MSH2 gene.
The patient that we investigate belongs to a family with Lynch syndrome and has a relative (with the same germinal mutation) that developed other infrequent cancer, a lei- omysoarcoma (Fig. 1) that was MSI-H. That discovery allowed us increase the range of extracolonic cancers related with hereditary non-polyposis cancer [10, 17]. As another member of the family suffered from leiomysarco- ma, could be that the patient had a Li Fraumeni syndrome. However, the tumor has no TP53 gene mutations that characterize the syndrome. The patient is a carrier of the germline mutation c.2063T>G (p.M688R) in MSH2 [11] and had undergone an endometrial carcinoma and a breast cancer. The mutation c.2063T>G (p.M688R) is located in exon 13 of MSH2, in the V structural domain of the Msh2 protein. This domain contains the Walker A motif required for repair activity, structurally similar to the ATPase domain of ABC transporters [18, 19]. It has been shown that non-conservative amino acid substitutions in this motif (as is the case of the mutation M688R) result in mutator phenotypes in E. coli [20] and yeast [21], that are charac- teristic of loss of mismatch repair activity. This motif interacts with the & and ß phosphates of ATP, and the mutations in this motif confers a dramatic decrease in both ATP binding and hydrolysis of purified yeast and human Msh2-Msh6 complexes, but this does not abolish ATPase activity completely [22, 23]. The adrenal carcinoma
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presents LOH of the normal allele of MSH2 (Fig. 3), thus only has the mutant allele, and this has very low repair capacities.
This situation is similar to found in the colorectal can- cers of the family, and would provide evidence that the defect in MMR system is related to the etiology of this adrenal cortical tumor. The germline MSH2 mutation and adrenal cortical tumor are causally linked because the
tumor showed loss of MSH2 protein by immunohisto- chemistry expression.
The adrenal cancer, despite the defect in the MMR system, does not present microsatellite instability, in con- trast to colorectal tumors in the family that display high microsatellite instability (MSI-H). It is noteworthy that the adrenal carcinoma analyzed by Broaddus et al. [16] was also microsatellite stable (MSS). Furthermore, we
described a family with Lynch Syndrome, which presented cases of colorectal cancers, extracolonic tumors (endome- trial and ureter), and other tumors (breast and bladder) not described in the Lynch spectrum. In that family the tumors exhibited MSI-H, irrespective of their location and regardless whether they were primary, synchronous, or metachronous, with the exception of the endometrial tumors (two) that showed low MSI (MSI-L) [24]. This circumstance seems to be frequent in this tumor type associated with the syndrome [16]. In the present study, the breast cancer and the endometrial carcinoma developed by of the same patient were MSI-H and MSI-L, respectively. The reasons for the differences in microsatellite instability for some extracolonic cancers associated to the syndrome are unclear. As we mentioned above, the mutation does not eliminate ATPase activity completely [22, 23], and the possibility of having a slight level of repair, taking into account also that the rate of cell renewal of this tissue is much lower than that found in the colonic mucosae, could explain the behavior of the adrenal cortical carcinoma as MSS. There are evidences indicating that tissue-specific factors, close to the defects in the mismatch repair genes, influence the levels of microsatellite instability and might be responsible for the differences found in tumors of dif- ferent tissues in the same patient [25, 26]. This finding in a rare tumor associated to the syndrome suggests the that microsatellite instability analysis using the National Cancer Institute guideline [14] may not detect microsatellite instability in tumors that fall outside the usual tumor spectrum of this syndrome.
It is very interesting the occurrence of two rare tumors in the same family; is tempting to think that the demo- graphic history of isolation in these populations since they first became on the island in 1500, has allowed a high degree of inbreeding. The subsequent consanguinity might produce an increasing homozygosity, and a more intense manifestation of mutated genes, in the absence of an exchange with wild-type genes. The same effect could occur in other low-penetrance genes associated with cancer risk, and this would contribute to rare tumors associated with the syndrome.
In conclusion, our results suggest the possibility of extending the tumor spectrum in Lynch syndrome to adrenal cortical adenocarcinoma. Because these tumors are infrequent in the general population, the risk of developing this type of tumors must be also low in families with Lynch syndrome, though in the family of this study it is the second rare tumor found. Anyhow, the incorporation to the tumoral spectrum of adrenal cortical carcinoma can contribute to the most correct application of the criteria of Amsterdam and Bethesda and surveil- lance of the patients.
Acknowledgments This work was supported by grant from the Fondo de Investigación Sanitaria FIS (04/0236).
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