DNA Mismatch Repair Defects and Microsatellite Instability Status in Periocular Sebaceous Carcinoma ANAND RAJAN KD, CHRISTOPHER BURRIS, NICHOLAS ILIFF, MICHAEL GRANT, JAMES R. ESHLEMAN, AND CHARLES G. EBERHART
PURPOSE: To characterize mismatch repair protein expression and the role of DNA repair abnormalities in sebaceous carcinomas of the ocular adnexa.
DESIGN: Retrospective case-series study.
METHODS: We reviewed 10 cases of sporadic sebaceous carcinoma and 1 case involving a patient with a family history consistent with Muir-Torre syndrome. Immunohistochemistry was used to analyze the presence of 4 mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) in these tumors. DNA was extracted from 7 of the larger tumors as well as from adjacent normal control tissue and microsatellite instability (MSI) analysis using 5 highly sensitive mononucleotides and 2 pentanucleotides was performed.
RESULTS: All 10 sporadic periocular sebaceous carcinomas maintained strong staining of the 4 mismatch repair genes, while tumor from the patient with MuirTorre syndrome showed loss of staining for the mismatch repair genes MSH2 and MSH6. MSI testing of 7 tumors identified no changes in sporadic cases and yielded results supporting presence of repeat sequence instability in the Muir-Torre–associated case.
CONCLUSIONS: Sporadic sebaceous carcinoma of the ocular adnexa is not commonly associated with a loss of mismatch repair genes or microsatellite instability. (Am J Ophthalmol 2014;157:640–647. Ó 2014 by Elsevier Inc. All rights reserved.)

They also have the capacity for more distant metastasis, making complete excision of great importance. Unfortunately, diffuse intraepithelial disease spread often poses a surgical problem, necessitating mapping biopsies and, on occasion, radical excision including orbital exenteration. Sebaceous tumors of the skin occurring in association with an internal malignancy characterize Muir-Torre syndrome, which has overlapping features with Lynch syndrome.5 These show germline loss-of-function mutations in genes encoding DNA mismatch repair proteins MLH1 or MSH2, and less commonly in MSH6, PMS1, and PMS2.6 This results in loss of immunohistochemical staining for these proteins, as well as microsatellite instability, which can be identified using sensitive molecular assays. Such changes can also be seen in sporadic tumors. However, the molecular alterations in periocular sebaceous carcinoma are poorly understood compared to extraocular ones, although data in one report suggested that lesions arising around the eyes are less likely to show immunohistochemical changes consistent with mutations.7 To address the role of mismatch repair defects in periocular sebaceous carcinoma, we examined microsatellite instability and expression of MSH1, MSH2, MSH6, and PMS2 in a series of tumors.

S

EBACEOUS CARCINOMAS ARE RARE BUT AGGRESSIVE

THIS WAS A RETROSPECTIVE, OBSERVATIONAL CASE-SERIES

neoplasms, comprising approximately 5% of malignant periocular tumors in large series.1–3 The eyelid is a common site as compared to the rest of the skin, and contains close to 40% of sebaceous carcinoma overall, presumably owing to the abundant sebaceous meibomian and Zeis glands at this location.4 Multicentricity and extensive intraepithelial spread are characteristic features of periocular sebaceous carcinoma, and the tumors show a propensity for dissemination to local lymph nodes.

study. We reviewed the files of the Division of Ophthalmic Pathology at the Wilmer Eye Institute and retrieved 11 cases of sebaceous carcinoma with full clinical history and adequate paraffin-embedded tissue for molecular analysis. Immunohistochemistry was performed in the Department of Pathology clinical laboratory using standard techniques with antibodies to MLH1 (BD Pharmingen, Franklin Lakes New Jersey, USA; clone G168-15, 1:50), MSH2 (Calbiochem, San Diego California, USA; clone FE11, 1:100), MSH6 (Transduction Labs, Franklin Lakes, New Jersey, USA; clone 44 B/BD, 1:100), and PMS2 (BD Pharmingen, Franklin Lakes, New Jersey, USA; clone A16-4, B, 1:100). Hematoxylin was employed as counterstain. For cases with sufficient well-demarcated tumor (7 of 11), DNA was extracted from lesional tissue in unstained slides identified in paired routine hematoxylin-eosinstained sections. Five mononucleotide markers (BAT-25,

Accepted for publication Dec 2, 2013. From the Department of Pathology, Johns Hopkins University School of Medicine (A.R.K.D., C.B., J.R.E., C.G.E.), and Department of Ophthalmology, Johns Hopkins University School of Medicine Wilmer Eye Institute (N.I., M.G., C.G.E.), Baltimore, Maryland. Inquiries to Charles G. Eberhart, Johns Hopkins University School of Medicine, 720 Rutland Ave - Ross Building 558, Baltimore, MD 21205; e-mail: [email protected]

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METHODS

ELSEVIER INC. ALL

RIGHTS RESERVED.

0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2013.12.002

TABLE 1. Clinical and Molecular Characteristics, Including Mismatch Repair Protein Status and Microsatellite Instability Analysis, in Patients With Sporadic Sebaceous Carcinoma (n ¼ 10) and Muir-Torre Syndrome (n ¼ 1) Case

Patient Age

Sex

Race

MLH1

MSH2

MSH6

PMS2

MSI

Follow-up

1 2 3 4 5 6 7

61 71 27 70 71 81 77

F F F M F F M

W W W W W W W

þ þ þ þ þ þ þ

 þ þ þ þ þ þ

 þ þ þ þ þ þ

þ þ þ þ þ þ þ

E . I . I I I

8 years, multiple recurrences 4 years, no recurrence 2 years, DU 3 years, DOD

8 9 10 11

84 75 88 82

F F F F

W W W B

þ þ þ þ

þ þ þ þ

þ þ þ þ

þ þ þ þ

. . I I

18 months, no recurrence, DU 8 years, multiple recurrences 6 months, no recurrence -

Comments

Muir-Torre syndrome Colorectal carcinoma Bilateral retinoblastoma Basal cell carcinoma, right lower lid

Sebaceous carcinoma metastatic to parotid

þ ¼ immunostain positive;  ¼ immunostain negative; B ¼ black; DOD ¼ died of disease; DU ¼ died of unrelated cause; E ¼ equivocal result (see Discussion); I = intact; MSI ¼ microsatellite instability; W ¼ white.

BAT-26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide markers (Penta C and Penta D) were used for microsatellite instability determination. Products were analyzed in the Pathology Department Molecular Diagnostics Laboratory by capillary electrophoresis with an ABI 3100 Genetic Analyzer using the Promega microsatellite instability analysis system (Fitchburg, Wisconsin, USA). The clinical record in each case was examined for past history or present complaints indicating visceral malignancies. Immunohistochemical stains were reviewed in tumor and normal adjacent tissues by 2 anatomic pathologists (A.R.K.D. and C.G.E.), while DNA electropherograms were examined for microsatellite instability by a clinical pathologist (J.R.E.). Approval was obtained from the Johns Hopkins Hospital Institutional Review Board.

RESULTS
CLINICAL AND DEMOGRAPHIC DATA:

The patients ranged in age from 27-88 years. Most were female (9 female, 2 male); 10 patients were white while 1 was of African descent. The upper eyelid was involved in 8 cases, with 1 of these tumors extending into the orbit, while the lower eyelid was involved in the remaining 3 cases (Table 1). Complete surgical resection was attempted in all cases. Most tumors were poorly differentiated and composed of large pleomorphic cells with hyperchromatic nuclei and scant cytoplasm, which displayed minimal sebaceous differentiation. Clinical follow-up after surgery for the sebaceous carcinoma was available for 7 patients (Table 1). Although several patients suffered from recurrence of the tumor, only 1 (Case #7) is known to have died of his disease. One patient had a diagnosis of Muir-Torre syndrome in her clinical record prior to the operation, which generated the pathology specimen analyzed (Case #1 in Table 1).

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This 61-year-old woman had a history of multiple sebaceous carcinomas of the eyelids over a 15-year period. Interestingly, this tumor appeared in most areas to be better differentiated than the other sebaceous carcinomas in our series, with cells containing more abundant vacuolated cytoplasm (Figure 1). No osteomas or bony growths were noted, but several benign polyps had been removed during routine colonoscopy. Multiple second-degree relatives had a history of colon cancer on her paternal side, which was thought to satisfy the Amsterdam criteria for Lynch syndrome.4 Two other patients without a recorded clinical diagnosis of Muir-Torre syndrome had a history of other malignancies. Case #2 involved a 71-year-old woman with colonic carcinoma resected a decade before presentation to the eye clinic. Case #3, representing hereditary retinoblastoma, involved a 27-year-old woman, with bilateral retinoblastoma diagnosed at 10 years of age, who had undergone enucleation of the right eye and received radiation to the left eye. Subsequently she developed a tumor associated with the optic nerve, diagnosed as ‘‘spindle cell sarcoma’’ at an outside institution, for which she received radiation and chemotherapy; and a melanoma on her right arm, which was excised. Her daughter developed unilateral retinoblastoma at the age of 3.
IMMUNOHISTOCHEMICAL

AND

DNA

ANALYSIS:

Diffusely positive immunostaining for mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) was observed in all sporadic sebaceous carcinomas (Cases #2-11, Figure 2). The staining was predominantly nuclear and was present in both tumor cells and surrounding nonneoplastic stromal elements, although it was stronger in the former. Several sporadic cases showed clear intraepithelial spread of tumor, and the neoplastic cells were strongly positive in these regions as well. In contrast, essentially complete absence of MSH2 and MSH6 protein was observed in the tumor

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FIGURE 1. Eyelid resection from patient with Muir-Torre syndrome showing (Left) meibomian gland involved by sebaceous carcinoma (arrows) (43 original magnification); (Right) higher-power view of tumor showing pleomorphic nuclei and scattered cytoplasmic vacuoles indicating sebaceous differentiation (1003 original magnification).

FIGURE 2. Immunostaining for mismatch repair proteins in patient with sporadic sebaceous carcinoma. (Top left) MLH1, (Top right) MSH2, (Bottom left) MSH6, and (Bottom right) PMS2 (Case #9, Table 1). Strong positivity is noted in tumor and is observed with all mismatch proteins, including intraepithelial (pagetoid) tumor (arrows). (All images, 2003 original magnification.)

resected from the patient in Case #1, with adjacent immunoreactive stromal elements serving as a positive control (Figure 3, Top right and Bottom left). MLH1 and PMS2 immunostaining remained strongly positive in this case (Figure 3, Top left and Bottom right). 642

We used mononucleotide and pentanucleotide markers to examine microsatellite instability (MSI) in 7 patients from whom sufficient lesional DNA could be extracted. Molecular changes were identified in Case #1, while no signs of microsatellite instability were detected in the

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FIGURE 3. Immunostaining for mismatch repair proteins in patient with Muir-Torre syndrome (Case #1, Table 1). (Top left) MLH1, (Top right) MSH2, (Bottom left) MSH6, and (Bottom right) PMS2. Complete absence of tumor immunostaining is observed with MSH2 and MSH6 (Top right and Bottom left, respectively). (All images, 2003 original magnification.)

remaining 6 tumors examined (Figure 4, Table 1). In Case #1, a shift was noted with 1 pentanucleotide (germline) microsatellite marker (Penta D) (Figure 4, Top right: tumor; Bottom right: control). These results are abnormal, and they suggest the presence of microsatellite instability but are not diagnostic of high microsatellite instability (termed ‘‘MSI-high’’) under present criteria (see Discussion). Analysis of DNA from tumor in Case #3, involving a patient with sebaceous carcinoma, optic nerve tumor, and retinoblastoma, showed no microsatellite instability. Microsatellite analysis could not be performed in Case #2 owing to inadequate remaining tumor tissue.

DISCUSSION ALTHOUGH SEBACEOUS CARCINOMA IS A FAIRLY UNCOM-

mon tumor, the periocular region is the most frequent site of occurrence. Owing to this, and given the potential for aggressive clinical behavior, sebaceous carcinoma represents an important part of the differential diagnosis of VOL. 157, NO. 3

eyelid neoplasms.8 Most sebaceous carcinomas are sporadic, but they can occur as part of Muir-Torre syndrome, which is defined clinically by the co-occurrence of a sebaceous neoplasm or keratoacanthoma with a visceral neoplasm. Mismatch repair defects, as evidenced by microsatellite instability and the loss of DNA repair protein expression, are considered an integral feature of MuirTorre syndrome.6 However, relatively little is known about the prevalence of mismatch repair abnormalities in periocular sebaceous carcinoma. Prior studies have examined mismatch repair protein status and microsatellite instability in periocular sebaceous neoplasms in patients with Muir-Torre syndrome and sporadic cases. None, however, have to our knowledge examined expression of all 4 mismatch repair proteins and a large panel of markers for microsatellite instability. Entius and associates reported 8 cases of sporadic sebaceous carcinoma; of these, 3 involved the eyelid and none exhibited mismatch repair protein loss (MLH1 and MSH2 were studied) or microsatellite instability (Bethesda markers).9 Singh and associates evaluated 35 cases, including 10 periocular sebaceous carcinomas, for expression of MLH1,

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FIGURE 4. DNA electropherographs from microsatellite instability analysis in patient with Muir-Torre syndrome (Case #1, Table 1). (Top row) Tumor; (Bottom row) control normal tissue. Left and middle panels at Top and Bottom are BAT25 and MONO27, respectively, both of which are normal. An extra peak (asterisk) and shifts are observed with pentanucleotide marker Penta D (Top right), which is not seen in the control (Bottom right).

MSH2, and MSH6.7 None of the 10 cases displayed protein loss, but history and clinical Muir-Torre syndrome status were unavailable in their series. A review of the published reports examining mismatch repair protein expression and microsatellite instability status in sporadic sebaceous carcinoma and Muir-Torre syndrome is presented in Table 2. A total of 18 periocular sebaceous carcinoma cases with molecular data have been reported in the literature. If the cases presented by Singh and associates are presumed 644

to be sporadic, none of these exhibit mismatch repair protein loss. Microsatellite instability was examined in 3 tumors but was not identified. In our series, in Case #1, despite the absence of proteins MSH2 and MSH6 on immunohistochemical analysis and a clinical history of Muir-Torre syndrome, the molecular changes exhibited features of microsatellite instability but did not meet criteria for high microsatellite instability (instability in 2 or more mononucleotide markers). Several

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TABLE 2. Published Literature on Mismatch Repair Proteins and Microsatellite Instability in Sporadic Sebaceous Carcinoma and MuirTorre Syndrome

Paper

Sebaceous

Sebaceous

Periocular Sebaceous

Neoplasia

Carcinoma

Carcinoma

10

Machin et al 2002 Popnikolov et al 200311 Mathiak et al 200212 Singh et al 20087 Kruse et al 199813 Kruse et al 200314 Mangold et al 20046 Morales-Burgos et al 200815 Entius et al 20009 Harwood et al 200116 Orta et al 200917 Cesinaro et al 200718 Mojtahed et al 201119 Plocharczyk et al 201320

39 8 92 19 58 26 15 9 10 53 57 49 43

1 7 2 35 1 2 5 4 9 10 34 21 12 15

N/A 0 N/A 10 0 N/A N/A 3 0 5 N/A N/A N/A

Clinical MLH1

MSH2

MSH6

PMS2

MSI

S S S S -

S S S S -

S -

-

S S S

S S S S S S S

S S S S S S S

S S S

S S S

S S S S S -

Panel

History a

Bethesda Expandedb Expandedb Expandedb Bethesdaa Expandedb Bethesdaa Bethesdaa -

A N/A A N/A A A A A A A A* A A* A

A ¼ available; A* ¼ not available in all cases; MSI ¼ microsatellite instability; N/A ¼ not available; S ¼ examined in study. a ‘‘Bethesda’’ indicates microsatellite markers BAT25, BAT26, D2S123, D127S250, D5S346 were analyzed. b Kruse et al 1998 and Mangold et al 2004 used an expanded panel consisting of markers BAT25, BAT26, D2S123, D2S136, D3S1298, D5S346, D6S470, D16S663, D18S35, D18S37, and D21S171. Mathiak et al 2002 employed BAT40 in addition. Harwood et al 2001 used a panel consisting of markers D1S201, D1S214, D9S160, D9S176, D9S162, D9S171, D10S185, D13S155, D17S520, D17S796, and D17S785. Kruse et al 2003 used the Bethesda panel and 5 additional markers: BAT40, D3S1298, D3S1611, D6S470, and D18S69.

explanations are possible. First, the distinctive location of the BAT26 mononucleotide repeat (intragenic in hMSH2) can affect test results in a small number of cases. In patients with biallelic hMSH2 deletion, MSH2 is lost and immunohistochemistry for MSH2 is negative but at the same time no appreciable shifts in BAT26 are seen.21 In a study examining microsatellite instability in 462 colon carcinoma cases, 1.5% (n ¼ 7) showed loss of MSH2 by immunohistochemistry but were classified as microsatellite stable by pentaplex PCR.22 The tumor in Case #1 showed a shift in the pentanucleotide marker Penta D, a finding substantiating instability.23,24 Tumor size and mitotic history affect accumulation of subclones with repeat sequence length changes, and molecular analysis could be affected by tumor volume.21 Supporting this notion, a study examining colorectal adenomas in 34 patients with established Muir-Torre syndrome found significant dependence of high microsatellite instability (‘‘MSI-H’’) detection on polyp size, using the same assay as our present work.25 It is interesting to note that though our Case #1 patient was from a family with multiple first- and second-degree relatives with colon cancer, she never developed colorectal carcinoma herself. This suggests variable expressivity in individuals with clinical criteria and family history supporting diagnosis of Muir-Torre syndrome. Two other patients had malignancies in addition to their periocular sebaceous carcinoma. In reviewing medical records, we discovered the patient in Case #2 had a history VOL. 157, NO. 3

of colorectal carcinoma in addition to the eyelid tumor. But she lacked a family history of cancer, did not exhibit mismatch protein loss by immunohistochemistry, and was microsatellite stable. We therefore classify this case as sporadic Muir-Torre syndrome. The sporadic association between sebaceous carcinoma and colorectal carcinoma has been described.26 Ponti and associates estimated that over a third of patients with clinically defined Muir-Torre syndrome may not display microsatellite instability.27 In Case #3, development of sebaceous carcinoma was preceded by a long interval (17 years) following irradiation to the affected eye owing to bilateral retinoblastoma. Patients with hereditary retinoblastoma have been previously shown to be at risk for development of sebaceous carcinoma in the field of radiation,28 and these may develop even in the absence of radiation therapy.29 A number of tests can be used to search for evidence of DNA repair defects in sebaceous neoplasms. In general, immunohistochemistry for mismatch repair–associated proteins MLH1, MSH2, MSH6, and PMS2 is adequate to make the distinction between sporadic and Muir-Torre– associated sebaceous carcinoma. Microsatellite instability testing of the type we used represents another common approach. In our small series, immunohistochemical analysis identified absence of mismatch repair proteins in the Muir-Torre–associated tumor, even though the full spectrum of repeat sequence alterations was not present. Other methods that have been employed to test for mismatch

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repair defects include functional studies and direct sequencing of relevant genes.30 However, it is not clear that these methods are more sensitive than those we used for initial analysis.30 Since sebaceous carcinoma can be the presenting clinical marker of syndromes predisposing to visceral malignancy, a thorough physical examination (including endoscopic studies where warranted) and detailed family history of malignancy should be carried out in all cases. It has been recommended that a full 4-protein immunohistochemistry screening and diagnostic evaluation of all patients with cutaneous sebaceous carcinoma be performed.31 Immunohistochemical analysis is relatively easy to implement, but staining may not always be synonymous with lack of microsatellite instability owing to nonfunctional repair proteins from missense mutations.32 Some recent guidelines for colorectal carcinoma therefore include both microsatellite testing and mismatch repair protein immunohistochemistry when a clinical or family history supporting Lynch/MuirTorre syndrome is present.33 Where positive (as in Case #1), direct sequencing might also be undertaken as a confirmatory test and to enable screening of family members. Although our study suggests that periocular tumors are

less likely to be associated with systemic disease, given the relatively small number of cases examined at this site it seems prudent for now to follow the more general guidelines—that is, immunohistochemical analysis in patients without a significant clinical and/or family history supporting Lynch syndrome, particularly in those whose tumors appear better differentiated, and adding microsatellite testing in patients with a positive history. In summary, we found that only 1 of 11 cases of periocular sebaceous carcinoma showed loss of mismatch repair protein expression, and this was in a patient with a clinical diagnosis of Muir-Torre syndrome. Interestingly, the tumor in this case was better differentiated than most periocular sebaceous carcinomas, and resembled those often seen on skin away from the eyes where an association with MuirTorre syndrome is more frequent. None of the sporadic cases showed unequivocal evidence of microsatellite instability. This suggests that microsatellite instability is not commonly a pathogenic contributor in eyelid sebaceous carcinoma, although these results should be further corroborated by analysis of additional cases and possibly other types of testing, such as direct mismatch repair gene sequencing and/or repair protein functional assays.

ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST and none were reported. Funding support was provided by departmental funds from Pathology, Johns Hopkins Hospital. Contributions of authors: design of study (C.G.E.); conduct of study (C.B., A.R.K.D., J.R.E., N.I., M.G.); interpretation of data (A.R.K.D., C.G.E., J.R.E.); preparation of manuscript (A.R.K.D., C.G.E., J.R.E.).

REFERENCES 1. Chhibber V, Dresser K, Mahalingam M. MSH-6: extending the reliability of immunohistochemistry as a screening tool in Muir-Torre syndrome. Mod Pathol 2008;21(2):159–164. 2. Shields JA, Demirci H, Marr BP, Eagle RC Jr, Shields CL. Sebaceous carcinoma of the ocular region: a review. Surv Ophthalmol 2005;50(2):103–122. 3. Deprez M, Uffer S. Clinicopathological features of eyelid skin tumors. A retrospective study of 5504 cases and review of literature. Am J Dermatopathol 2009;31(3):256–262. 4. Dasgupta T, Wilson LD, Yu JB. A retrospective review of 1349 cases of sebaceous carcinoma. Cancer 2009;115(1): 158–165. 5. Lynch HT, Lynch PM, Lanspa SJ, Snyder CL, Lynch JF, Boland CR. Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications. Clin Genet 2009;76(1):1–18. 6. Mangold E, Pagenstecher C, Leister M, et al. A genotypephenotype correlation in HNPCC: strong predominance of msh2 mutations in 41 patients with Muir-Torre syndrome. J Med Genet 2004;41(7):567–572. 7. Singh RS, Grayson W, Redston M, et al. Site and tumor type predicts DNA mismatch repair status in cutaneous sebaceous neoplasia. Am J Surg Pathol 2008;32(6):936–942. 8. Dores GM, Curtis RE, Toro JR, Devesa SS, Fraumeni JF Jr. Incidence of cutaneous sebaceous carcinoma and risk of

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associated neoplasms: insight into Muir-Torre syndrome. Cancer 2008;113(12):3372–3381. Entius MM, Keller JJ, Drillenburg P, Kuypers KC, Giardiello FM, Offerhaus GJ. Microsatellite instability and expression of hMLH-1 and hMSH-2 in sebaceous gland carcinomas as markers for Muir-Torre syndrome. Clin Cancer Res 2000;6:1784–1789. Machin P, Catasus L, Pons C, et al. Microsatellite instability and immunostaining for MSH-2 and MLH-1 in cutaneous and internal tumors from patients with the Muir-Torre syndrome. J Cutan Pathol 2002;29(7):415–420. Popnikolov NK, Gatalica Z, Colome-Grimmer MI, Sanchez RL. Loss of mismatch repair proteins in sebaceous gland tumors. J Cutan Pathol 2003;30(3):178–184. Mathiak M, Rutten A, Mangold E, et al. Loss of DNA mismatch repair proteins in skin tumors from patients with Muir-Torre syndrome and MSH2 or MLH1 germline mutations: establishment of immunohistochemical analysis as a screening test. Am J Surg Pathol 2002;26(3):338–343. Kruse R, Rutten A, Lamberti C, et al. Muir-Torre phenotype has a frequency of DNA mismatch-repair-gene mutations similar to that in hereditary nonpolyposis colorectal cancer families defined by the Amsterdam criteria. Am J Hum Genet 1998;63(1):63–70. Kruse R, Rutten A, Schweiger N, et al. Frequency of microsatellite instability in unselected sebaceous gland neoplasias and hyperplasias. J Invest Dermatol 2003;120(5):858–864.

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15. Morales-Burgos A, Sanchez JL, Figueroa LD, et al. MSH-2 and MLH-1 protein expression in Muir Torre syndromerelated and sporadic sebaceous neoplasms. P R Health Sci J 2008;27(4):322–327. 16. Harwood CA, Swale VJ, Bataille VA, et al. An association between sebaceous carcinoma and microsatellite instability in immunosuppressed organ transplant recipients. J Invest Dermatol 2001;116(2):246–253. 17. Orta L, Klimstra DS, Qin J, et al. Towards identification of hereditary DNA mismatch repair deficiency: sebaceous neoplasm warrants routine immunohistochemical screening regardless of patient’s age or other clinical characteristics. Am J Surg Pathol 2009;33(6):934–944. 18. Cesinaro AM, Ubiali A, Sighinolfi P, Trentini GP, Gentili F, Facchetti F. Mismatch repair proteins expression and microsatellite instability in skin lesions with sebaceous differentiation: a study in different clinical subgroups with and without extracutaneous cancer. Am J Dermatopathol 2007;29(4): 351–358. 19. Mojtahed A, Schrijver I, Ford JM, Longacre TA, Pai RK. A two-antibody mismatch repair protein immunohistochemistry screening approach for colorectal carcinomas, skin sebaceous tumors, and gynecologic tract carcinomas. Mod Pathol 2011;24(7):1004–1014. 20. Plocharczyk EF, Frankel WL, Hampel H, Peters S. Mismatch repair protein deficiency is common in sebaceous neoplasms and suggests the importance of screening for Lynch syndrome. Am J Dermatopathol 2013;35(2):191–195. 21. Laghi L, Bianchi P, Malesci A. Differences and evolution of the methods for the assessment of microsatellite instability. Oncogene 2008;27(49):6313–6321. 22. Xicola RM, Llor X, Pons E, et al. Performance of different microsatellite marker panels for detection of mismatch repair-deficient colorectal tumors. J Natl Cancer Inst 2007; 99(3):244–252. 23. Bacher JW, Flanagan LA, Smalley RL, et al. Development of a fluorescent multiplex assay for detection of

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Biosketch Dr Anand Rajan KD MD, received his clinical training in Pathology at the All India Institute of Medical Sciences, New Delhi. He completed a two-year fellowship in Ophthalmic Pathology at Wilmer Eye Institute at the Johns Hopkins Hospital and is currently resident in Anatomic and Clinical Pathology at Washington University at St Louis. His research interests include investigation of pathogenesis of neoplasia, using uncommon and rare tumors as models.

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Biosketch Dr Charles Eberhart MD, PhD, received post-graduate clinical training in Anatomical Pathology and Neuropathology at Johns Hopkins Hospital, and has been a member of the Johns Hopkins School of Medicine faculty since 2001. He currently directs the divisions of Neuropathology and Ophthalmic Pathology, and works as both a diagnostic pathologist and a researcher studying how brain and eye diseases can be better classified and treated.

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