DNA Mismatch Repair Defects and Microsatellite Instability Status in Periocular Sebaceous Carcinoma




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 Muir-Torre 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.


Sebaceous carcinomas are rare but aggressive neoplasms, comprising approximately 5% of malignant periocular tumors in large series. 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. Multicentricity and extensive intraepithelial spread are characteristic features of periocular sebaceous carcinoma, and the tumors show a propensity for dissemination to local lymph nodes. 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. 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. 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. 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.


Methods


This was a retrospective, observational case-series 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-eosin-stained sections. Five mononucleotide markers (BAT-25, 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.



Table 1

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




















































































































































Case Patient Age Sex Race MLH1 MSH2 MSH6 PMS2 MSI Follow-up Comments
1 61 F W + + E 8 years, multiple recurrences Muir-Torre syndrome
2 71 F W + + + + . 4 years, no recurrence Colorectal carcinoma
3 27 F W + + + + I 2 years, DU Bilateral retinoblastoma
4 70 M W + + + + . Basal cell carcinoma, right lower lid
5 71 F W + + + + I
6 81 F W + + + + I
7 77 M W + + + + I 3 years, DOD Sebaceous carcinoma metastatic to parotid
8 84 F W + + + + . 18 months, no recurrence, DU
9 75 F W + + + + . 8 years, multiple recurrences
10 88 F W + + + + I 6 months, no recurrence
11 82 F B + + + + I

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


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 ). 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. 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.




Figure 1


Eyelid resection from patient with Muir-Torre syndrome showing (Left) meibomian gland involved by sebaceous carcinoma (arrows) (4× original magnification); (Right) higher-power view of tumor showing pleomorphic nuclei and scattered cytoplasmic vacuoles indicating sebaceous differentiation (100× original magnification).


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 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).




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, 200× original magnification.)



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, 200× original magnification.)


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 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.




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).




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.



Table 1

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




















































































































































Case Patient Age Sex Race MLH1 MSH2 MSH6 PMS2 MSI Follow-up Comments
1 61 F W + + E 8 years, multiple recurrences Muir-Torre syndrome
2 71 F W + + + + . 4 years, no recurrence Colorectal carcinoma
3 27 F W + + + + I 2 years, DU Bilateral retinoblastoma
4 70 M W + + + + . Basal cell carcinoma, right lower lid
5 71 F W + + + + I
6 81 F W + + + + I
7 77 M W + + + + I 3 years, DOD Sebaceous carcinoma metastatic to parotid
8 84 F W + + + + . 18 months, no recurrence, DU
9 75 F W + + + + . 8 years, multiple recurrences
10 88 F W + + + + I 6 months, no recurrence
11 82 F B + + + + I

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Jan 8, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on DNA Mismatch Repair Defects and Microsatellite Instability Status in Periocular Sebaceous Carcinoma
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