ADRENOCORTICAL ADENOCARCINOMA IN AN MSH2 CARRIER: COINCIDENCE OR CAUSAL RELATION?
MARAN J.W. BERENDS, MD, ANNEMIEKE CATS, MD, PHD, HARRY HOLLEMA, MD, PHD, AREND KARRENBELD, MD, JOHN A.M. BEENTJES, MD, ROLF H. SIJMONS, MD, PHD, ROB G.J. MENSINK, ROBERT M.W. HOFSTRA, PHD, RENÉ C.J. VERSCHUEREN, MD, PHD, AND JAN H. KLEIBEUKER, MD, PHD
A woman is described who developed an ovarian adenocarci- noma, 3 metachronous colorectal adenocarcinomas, and a primary adrenocortical adenocarcinoma. Genetic investigation of the mis- match repair genes MLH1 and MSH2 showed a germline mutation in MSH2. Colorectal and ovarian carcinoma belong to the tumor spec- trum of hereditary nonpolyposis colorectal cancer (HNPCC). Adre- nocortical adenocarcinoma, however, has never been described as 1 of the HNPCC-associated tumors. To investigate whether the adre- nocortical adenocarcinoma in this patient was caused by the MSH2 germline mutation, determination of microsatellite instability (MSI) and immunohistochemical analysis were performed on 1 of the colo- rectal tumors and the adrenocortical adenocarcinoma. MSI and gen-
Hereditary nonpolyposis colorectal cancer (HNPCC) is a genetic disorder with an autosomal dominant inheritance. HNPCC is clinically defined by the recently revised Amster- dam criteria, the Amsterdam criteria II (ACII).1 The diagno- sis HNPCC is established when (1) at least 3 relatives have histologically verified cancer of the colorectum, endome- trium, small bowel, ureter or renal pelvis, 1 of whom is a first degree relative of the other 2; (2) at least 2 consecutive generations are affected; (3) at least 1 of the cases of cancer is diagnosed before the age of 50 years; and (4) familial
eral loss of MSH2 protein expression could be seen in the colorectal tumor but not in the adrenocortical adenocarcinoma. Therefore, it is highly unlikely that the adrenocortical adenocarcinoma found in this patient was due to her genetic predisposition for HNPCC. HUM PATHOL 31:1522-1527. Copyright @ 2000 by W.B. Saunders Company Key words: adrenocortical adenocarcinoma, HNPCC, microsatel- lite instability, MSH2 protein expression.
Abbreviations: HNPCC, hereditary nonpolyposis colorectal can- cer; MSI, microsatellite instability; ACII, revised Amsterdam criteria; MMR, mismatch repair; PAA, polyacrylamide; EtBr, ethidium bro- mide; UF, urea-formamide.
adenomatous polyposis has been excluded. In addition to the already mentioned cancers, cancer of the ovaries, stomach, pancreas, biliary tract, and the brain also belong to the HNPCC tumor spectrum.2-5 So far, germline mutations in 5 mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS1 and PMS2) have been identified in HNPCC patients. Muta- tions in MLH1 and MSH2 account for the majority of genetic alterations observed in HNPCC kindreds.6,7 Loss of function of both copies of 1 of the MMR genes leads to widespread alterations in short tandem repeat sequences, referred to as microsatellite instability (MSI). The MSI phenotype has been found in more than 90% of the tumors in HNPCC patients.8,9 The presence of MSI, however, does not indicate which of the MMR genes is involved. In contrast, immunohistochemistry can be used to check for the absence or presence of either the MLH1 or the MSH2 protein in tumor material.10,11 We report on a female MSH2 mutation carrier with 3 colorectal adeno- carcinomas and an adenocarcinoma of the left ovary. She also developed Cushing’s syndrome as a result of an adrenocorti- cal adenocarcinoma, a tumor type which so far has not been
From the Departments of Gastroenterology, Pathology, Endocri- nology, Medical Genetics, and Surgery, University Hospital Gro- ningen, The Netherlands.
Address correspondence and reprint requests to Jan H. Klei- beuker, MD, PhD, Department of Gastroenterology, University of Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands.
Copyright @ 2000 by W.B. Saunders Company 0046-8177/00/3112-0012$10.00/0 doi:10.1053/hupa.2000.20409
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FIGURE 1. Lower power view of the (A) adenocarcinoma in the cecum after staining with hematoxylin and eosin; (B) ad- enocarcinoma in the descending colon after staining with hematoxylin and eosin; and (C) adenocarcinoma in the rectosigmoid after staining with hematoxylin and eosin.
considered to be part of the known HNPCC tumor spectrum. To investigate if the adrenocortical adenocarcinoma in this woman was caused by the MSH2 germline mutation, we per- formed MSI analysis and immunohistochemistry on both the colorectal and adrenocortical adenocarcinoma.
CASE REPORT
A white female patient is presented with an ovarian tu- mor in the left ovary at age 42 for which a hysterectomy and bilateral ovariectomy were performed. Histological examina- tion showed an adenocarcinoma of the left ovary with squa- mous cell metaplasia. After surgery she received high-dose external radiotherapy. At age 48 the patient developed an adenocarcinoma of the cecum (T2NOM0, grade I adenocar- cinoma, intestinal type) (Fig 1A) which required a hemico- lectomy of the right colon with ileotransversostomy. A limited hemicolectomy of the left colon was performed at age 62 because of an adenocarcinoma in the descending colon (T3NOMO, grade II adenocarcinoma, intestinal type) (Fig 1B). A tubular adenoma in the rectosigmoid, found at the same time, was left in situ to be removed during colonoscopy. That procedure, however, was not successful. A wait-and-see policy was pursued, considering the difficulties encountered
| BAT25 | BAT26 | D2S123 | D5S346 | D17S250 | Others | |
|---|---|---|---|---|---|---|
| Colorectal tumor | positive | positive | positive | positive | positive | D1S158 positive BAT40 positive |
| Adrenal tumor | negative | negative | loss | negative | negative | D1S158 negative BAT40 loss |
during the previous operations due to many adhesions and radiation damage. At age 65 biopsy samples of the polyp showed an adenocarcinoma (T3NOMO, grade II adenocarci- noma, intestinal type) (Fig 1C), and a surgical resection was performed. That same year, the patient was referred because of a plethoric face, weight gain, lethargy, and proximal mus- cular weakness. Physical examination showed clear stigmata of hypercortisolism. Additional laboratory tests established the diagnosis of ACTH-independent Cushing’s syndrome. Ul- trasonography showed a tumor in the right adrenal cortex. The adrenal tumor was surgically removed. The tumor weight was 190 g. Histological examination showed extensive necro- sis, a diffuse growth pattern, partly trabecular, partly tubular, incidentally giant cells, and a mitotic rate of 130 per 50 high-power fields. Venous invasion was not seen. The diagno- sis adrenocortical adenocarcinoma was made (Fig 2). The appearance of an adenocarcinoma of the ovary and 3 meta- chronous colorectal adenocarcinomas in the same patient, 2 of them occurring before the age of 50, raised the suspicion of HNPCC. The patient was referred to the clinical geneticist. The family history showed 3 first degree relatives (father and two half-sisters) with tumors, among these possibly a brain and a lung tumor. A third degree relative possibly developed colorectal cancer at the age of 36. The medical histories of all these relatives could not be verified. Based on the available data, the family did not meet the original Amsterdam criteria nor the new ACII. About a year after removal of the adrenal tumor, pulmonary and extensive skeletal metastases were seen on a chest radiograph and skeletal scan, respectively. An arterial digital substraction angiography showed a mass be- tween the liver and the right kidney, vascularisized by the renal arteries, indicating a local recurrence. Apart from pal- liative measures, no therapeutic options were left. She died at the age of 66. No autopsy was performed.
METHODS
Mutation Analysis
Mutation analysis of MLH1 and MSH2 was carried out using two-dimensional DNA electrophoresis as previously de- scribed.12 The entire coding sequence of the genes was am- plified by PCR. The PCR products of each gene were mixed in equal amounts, ethanol-precipitated with tRNA, and redis- solved. A 30 ul mixture of the amplified exons was first subjected to size separation in a 0.75-mm thick 9% polyacryl- amide (PAA) gel (acrylamide: bisacrylamide = 37.5:1) in 0.5 X TAE (1 X TAE = 40 mM Tris, HAC pH 8.0; 20 mM NaAc; 1 mM Na2EDTA) at 200 V/19 cm and 45°℃ for 5 hours. The separation pattern was visualized by ethidium bromide (EtBr) staining for 10 minutes and UV transillumination of the gel, which was lying on a glass plate to protect the DNA fragments from damage by UV light. The 100 base pair (bp) to 500 bp region in the middle part of the lane was cut out and applied to a 1-mm thick 9% PAA gel containing a de- naturing gradient of 30% to 65% ureaformamide (100% urea-formamide [UF] contains 7 M urea and 40% deionized formamide) and a 0% to 10% glycerol gradient. Two-dimen-
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CASE STUDIES
sional DNA electrophoresis was performed at 110 V/19 cm and 56℃ for 17 hours. After staining the DNA with EtBr, the gel pattern was documented. Variants were confirmed by direct sequencing. Sequencing was performed with an ABI PRISM 377 DNA sequencer (PE Biosystems, Foster City, CA).
Microsatellite Instability
The 5 microsatellite repeat markers as used were recently defined at an international workshop on HNPCC in Bethesda, MD.13 These include 2 mononucleotide repeats (BAT25 and BAT26) and 3 dinucleotide repeats (D2S123, D5S346, D17S250). Two additional markers, BAT40 and DIS158, were also used. DNA was extracted from formalin-fixed paraffin- embedded sections of one of the colorectal carcinomas (CRCs) and of the adrenocortical adenocarcinoma. Sample DNA from peripheral blood lymphocytes and normal colon
tissue served as control. Tumors were classified as MSI-high when at least 2 markers were instable, as MSI-low on finding 1 marker instable, and as MSI-stable when no instability was encountered. Loss of heterozygosity (LOH) was determined using 2 markers (D2S2298 and D2S391) on both sides of the MSH2 gene.
Immunohistochemical Analysis
MLH1 and MSH2 staining was performed on 3-um thick sections of deparaffinized colorectal and adrenal tissue. An- tigens were retrieved by autoclave heating.
The slides were incubated with mouse monoclonal anti- bodies to the MLH1 protein (1:500) (PharMingen, San Di- ego, CA) or MSH2 protein (1:100) (Calbiochem, San Diego, CA). The slides were successively treated with rabbit-anti- mouse peroxidase and with goat-anti-rabbit peroxidase. The
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chromogen was diaminobenzidine and counterstaining was performed with hematoxylin. Normal tissue next to the tu- mor served as internal positive control.
RESULTS
Mutation analysis of the mismatch repair genes MSH2 and MLH1 showed a deletion (delG759) in exon 4 of the MSH2 gene. This deletion causes a premature stopcode up- stream. The results of the MSI-analyses are shown in Table 1. The colorectal tumor turned out to be MSI-high, and the adrenocortical tumor was MSI-stable. The marker D2S391 was not informative in determining LOH in both tumors. In the colorectal tumor, LOH could be determined with the marker D2S2298. The adrenal tumor showed only loss of the allele containing the MSH2 mutation (Fig 3). Immunohistochem- istry showed lack of MSH2 protein expression in the colorec- tal tumor. A positive control was present (Figs 4A, B). The adrenal tumor expressed both the MLH1 and MSH2 protein (Figs 5A, B, C).
DISCUSSION
Tumors of the colorectum, endometrium, ovary, stom- ach, small bowel, pancreas, biliary tract, urinary tract, and probably also brain tumors have been associated with HNPCC.3,5,14 Primary adrenocortical carcinoma is rare with an incidence in the Netherlands of 0.12 per 100,000 person- years (data obtained from the Comprehensive Cancer Centre, North Netherlands, 1989-1998). To our knowledge, the rela- tion between adrenocortical carcinoma and a mutation in
one of the MMR genes has never been described. Lynch et al15 described a patient with a strong family history of colo- rectal and endometrial cancer, very suspicious for HNPCC, who died of adrenocortical carcinoma at age 44. Mutation analysis or immunohistochemistry has not been reported so far in this patient and his tumor.
Although the family of our patient did not meet the Amsterdam Criteria, HNPCC was nevertheless suspected, be- cause of the development of primary adenocarcinomas at a relatively young age. The finding of an MSH2 germline mu- tation confirmed the diagnosis. Due to the very low incidence of adrenocortical carcinoma, it was suspected that in this present patient, the MSH2 mutation was not only the cause of the ovarian and the colorectal tumors but of the adrenocor- tical tumor as well. However, microsatellite analysis of the adrenocortical tumor showed an MSI-stable tumor, whereas 1 of the colorectal tumors was MSI-high. Additional immuno- histochemical analysis showed lack of MSH2 expression in that colorectal tumor, whereas MSH2 protein expression was present in the adrenal tumor.
In conclusion, because the adrenal tumor was MSI-stable and no general loss of MSH2 protein in the adrenal tumor could be shown, the development of this tumor should not be considered to be associated with the germline MSH2 muta- tion. The occurrence of this rare tumor in this HNPCC- patient with an ovarian adenocarcinoma and 3 metachronous colorectal tumors should therefore be considered as a coin- cidence. Based on the findings in this patient, there is no reason to include adrenocortical adenocarcinoma in the HNPCC-tumor spectrum.
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