Immunohistochemical Features of Lacrimal Gland Epithelial Tumors

Immunohistochemical Features of Lacrimal Gland Epithelial Tumors PIA R. MENDOZA, FREDERICK A. JAKOBIEC, AND JEFFREY F. KRANE
PURPOSE: To investigate the immunohistochemical features of ocular adnexal pleomorphic adenoma and adenoid cystic carcinoma.
DESIGN: Retrospective clinicopathologic study.
METHODS: Clinical records and microscopic slides of 7 cases of each tumor type were reviewed. Immunohistochemical probes for Ki-67 and p53, and newer nuclear markers MYB for adenoid cystic carcinoma and PLAG1 for pleomorphic adenoma, were employed.
RESULTS: Pleomorphic adenomas were asymptomatic, whereas adenoid cystic carcinomas were painful. No pleomorphic adenomas recurred; 4 adenoid cystic carcinomas recurred, resulting in 3 deaths. Unusual histopathologic variants for which immunohistochemistry proved useful included a myoepithelioma, an atypical pleomorphic adenoma, tubular and solid/basaloid variants of adenoid cystic carcinoma, and a morphologically heterogeneous adenoid cystic carcinoma of a Wolfring gland. For the pleomorphic adenomas, the average Ki-67 proliferation index was 3.8%; p53 was weakly staining, with an average positivity of 18.5%; PLAG1 was strongly positive in all cases; MYB was negative in 5 cases and weakly focally positive in 2 cases. For the adenoid cystic carcinomas, the average Ki-67 proliferation index was 29.1%; p53 stained positively and strongly with an average of 39%; none stained positively for PLAG1; and 6 out of 7 were MYB positive.
CONCLUSIONS: Between pleomorphic adenoma and adenoid cystic carcinoma, there was no overlap in Ki-67 positivity. Positivity for p53 showed overlap in only one lesion of each type. PLAG1 and MYB positivity were highly discriminating between pleomorphic adenoma and adenoid cystic carcinoma. Immunohistochemical analysis should be investigated further for its role in the evaluation of pleomorphic adenoma and adenoid cystic carcinoma. (Am J Ophthalmol 2013;156: 1147–1158. Ó 2013 by Elsevier Inc. All rights reserved.)

I

N COMPARISON WITH SALIVARY GLAND EPITHELIAL

tumors,1–3 the immunohistochemical study of those of the lacrimal glands (both major and accessory) is a neglected subject.4 The present article, based on the investigation of 14 benign and malignant lacrimal gland epithelial tumors, is intended to establish a baseline for a set of immunohistochemical markers that in certain circumstances could provide assistance in the diagnosis of difficult or controversial cases. Examples might include discrepant diagnostic opinions encountered with small piecemeal excisions, needle biopsies, recurrent tumors, mesenchymal and neurogenic spindle cell tumors distinct from myoepitheliomas, invading carcinomas from surrounding compartments, and metastases in the lacrimal fossa region. Benign and malignant epithelial tumors with ambiguous or aberrant morphologic features, such as an atypical pleomorphic adenoma displaying internal worrisome cytologic features; tumors with overlapping sclerotic stromas; adenoid cystic carcinoma or poorly differentiated carcinoma arising in situ within the capsular confines of a pleomorphic adenoma; infiltrative carcinoma arising in a pleomorphic adenoma (also called carcinoma ex pleomorphic adenoma or malignant mixed tumor); and adenoid cystic carcinoma with fields imitating a pleomorphic adenoma or a basaloid monomorphic tumor (adenoma) must also be considered separately.4–7 We have selected two well-established probes for estimating the degree of atypicality in lacrimal epithelial tumors: Ki-67 for DNA synthesis prior to mitosis and aberrant p53 suppressor gene protein; and two newer markers, PLAG1 for supporting the diagnosis of pleomorphic adenoma and MYB for adenoid cystic carcinoma. We preliminarily outline an immunopathologic approach based on our findings for the diagnosis of benign and malignant lacrimal gland epithelial tumors.

METHODS Accepted for publication June 24, 2013. From the David G. Cogan Laboratory of Ophthalmic Pathology, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts (P.R.M., F.A.J.); the Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts (J.F.K.); and the Harvard Medical School, Boston, Massachusetts (P.R.M., F.A.J., J.F.K.). Inquiries to Frederick A. Jakobiec, David G. Cogan Laboratory of Ophthalmic Pathology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Suite 328, Boston, MA 02114; e-mail: Fred_Jakobiec@ meei.harvard.edu 0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2013.06.034

Ó

2013 BY

THIS RETROSPECTIVE STUDY WAS CONDUCTED UNDER THE

auspices of the Massachusetts Eye and Ear Infirmary Institutional Review Board (protocol # 13-034) and in compliance with the rules and regulations of the Health Insurance Portability and Accountability Act and in adherence to the Declaration of Helsinki and all relevant federal and state laws. The cases were identified by searching through the records of the David G. Cogan Laboratory

ELSEVIER INC. ALL

RIGHTS RESERVED.

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TABLE 1. Clinical Features of Patients with Lacrimal Gland Pleomorphic Adenomas

Case #

Age/Sex

Laterality

Visual Acuity

1

55/M

Left

20/20 Proptosis (2 mm); 6 mo

2

51/F

Right

3

18/F

Right

4

55/M

Right

5

69/F

Left

6

54/M

Left

7

28/F

Right

20/20 Eyelid mass and swelling, ptosis; 60 mo 20/20 Eyelid mass; 60 mo Well-demarcated, round mass 20/20 Eyelid mass; 12 mo Well-demarcated, round mass 20/25 Eyelid mass; 96 mo Well-demarcated, round, lobulated mass 20/25 Proptosis (2 mm); 24 mo Enlargement of palpebral lobe of lacrimal gland 20/20 Eyelid mass; 12 mo Well-demarcated, round, lobulated mass; bone indentation

Symptoms/Duration

Radiologic Findings

Treatment

Length of Follow-up (months)

Well-demarcated, round, lobulated mass with calcific foci; slightly enlarged orbit No imaging studies done

Tumor resection

37

No evidence of recurrence

Tumor resection

14

Tumor resection

4

Tumor resection

12

Tumor resection

13

Tumor resection

14

Tumor resection

8

No evidence of recurrence No evidence of recurrence No evidence of recurrence No evidence of recurrence No evidence of recurrence No evidence of recurrence

of Ophthalmic Pathology at the Massachusetts Eye and Ear Infirmary from 2007 to 2013 for lacrimal gland and orbital specimens with diagnoses of adenoid cystic carcinoma (7 cases) and pleomorphic adenoma or benign mixed tumor (7 cases). The clinical records, clinical photographs, radiologic imaging studies, and operative reports were reviewed. Archived tissue blocks with adequate remaining tissue for additional staining of slides were available for all cases selected for inclusion. Tumor sections were stained with hematoxylin and eosin, periodic acid Schiff, and mucicarmine. Formalin-fixed, paraffin-embedded specimens were used to prepare 5-mm sections for immunohistochemical staining. The immunoperoxidase method was applied with appropriate controls to validate antibody quality. The following probes were utilized: MYB (clone EP769Y; Epitomics, Burlingame, California, USA; 1:200); PLAG1 (mouse monoclonal; Abnova, Taipei, Taiwan; 1:150), both processed at the Department of Pathology, Brigham and Women’s Hospital, using a Dako autostainer; p53 (mouse monoclonal; Leica Biosystems, Newcastle, UK; prediluted); and Ki-67 (mouse monoclonal, Dako, Carpinteria, California, USA; 1:200), employing a Leica Bond III autostainer, both processed at the Diagnostic Immunopathology Laboratory, Massachusetts General Hospital, Boston. The chromogen diaminobenzidine was used and the tissues were counterstained with hematoxylin. MYB and PLAG1 staining were initially evaluated blindly by a single pathologist (J.F.K.) and confirmed by another (F.A.J.). Staining in >50% of tumor cells was scored as strongly positive, >5 and <50% of cells as weakly positive, and <5% of cells as negative. Specific cell counts for nuclear positivity were performed for Ki-67 and p53 in two high-power fields, and the results were averaged. 1148

Outcome

RESULTS
CLINICAL FINDINGS:

The clinical features of the cases involved in this study are summarized in Table 1 for the pleomorphic adenomas and Table 2 for the adenoid cystic carcinomas. Among the cases of pleomorphic adenoma, three were male and four were female (Figure 1, top left). The mean age at presentation was 47 years. Five patients presented with an upper eyelid mass, two had mild proptosis (2 mm), and one had lateral upper eyelid ptosis. The mean duration of symptoms was 39 months. Radiologic imaging showed a well-defined, rounded mass; one lesion displayed intralesional foci of calcification but without major bone changes (Figure 1, top right). Another tumor occurred as an enlargement of the palpebral lobe of the lacrimal gland, which was readily visible on physical examination with eyelid eversion. The first surgery in all cases was an attempt at complete excision, and an incisional biopsy was studiously avoided. No patient has experienced a recurrence, with an average follow-up of 15 months. However, only one of the seven cases had a follow-up of longer than 3 years (37 months). Five adenoid cystic carcinomas arose in the major lacrimal gland, one in an accessory lacrimal gland of Wolfring in the lower eyelid, and one in the maxillary sinus with secondary orbital extension. The American Joint Committee on Cancer (AJCC) TNM (tumor, node, metastasis) classification8 for each tumor is given in Table 2. Four patients were male and three were female, with a mean age at presentation of 46 years (Figure 1, middle left). Five patients manifested proptosis (2 to 7mm), three complained of pain, and two had diplopia. The mean duration of symptoms was 7 months. Radiologic imaging

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VOL. 156, NO. 6 TABLE 2. Clinical Features of Patients with Lacrimal Gland Adenoid Cystic Carcinomas

Case #

Age/Sex

Diagnosis/Laterality/TNM Staging

Visual Acuity

Symptoms/Duration

Radiologic Findings

Treatment

Length of Follow-up (months)

Outcome

IMMUNOHISTOCHEMISTRY OF LACRIMAL GLAND TUMORS

8

10/F

ACC of lacrimal gland, right (T2N0M0)a

20/20

Proptosis (2 mm), eyelid swelling; 5 mo

Irregular mass with calcific foci

Tumor resection þ radiotherapy

9

53/F

ACC of lacrimal gland, right (T4bN0M0)

20/20

Irregular mass with bony destruction

Tumor resection with bone removal þ radiotherapy

25

10

59/M

ACC of lacrimal gland, right (T4bN0M0); HIV infection

20/30

Proptosis (4 mm), pain, globe displacement, diplopia; 1 mo Pain, eyelid swelling, ptosis; 6 months

Irregular mass with bony erosion

53

Lung metastasis after 2 yrs; died of disease

11

89/M

ACC of lacrimal gland, left (T4bN0M0)

20/25

Proptosis (2 mm), ptosis; 7 months

Irregular mass with bony erosion

FNABb þ tumor resection þ radiotherapy þ chemotherapy Tumor resection þ radiotherapy

84

12

50/F

ACC of lacrimal gland, left (T4bN0M0)

20/20

Proptosis (3 mm); 4 mo

Irregular mass with bony destruction

Tumor resection with bone removal þ radiotherapy

13

37/M

ACC of accessory lacrimal gland, right (T1N0M0)

20/20

Eyelid mass and swelling; 1 month

Well-demarcated mass in inferolateral orbit

14

27/M

ACC of maxillary sinus and orbit, right (T4bN2cM0)

20/20

Pain, proptosis (7 mm) diplopia; 23 months

Tumor in maxillary sinus and orbit with bony destruction

Incisional biopsy þ tumor resection þ radiotherapy Incisional biopsy þ radiotherapy

Recurrence after 6 yrs treated with exenteration; died of disease Recurrence after 9 years treated with tumor resection þ proton beam radiotherapy; alive No evidence of recurrence; Alive

132

178

9

15

Recurrence after 10 yrs treated with surgery þ brachytherapy (placement of radiation catheters); alive Recurrence after 1 yr; alive

Brain metastasis after 1 year; died of disease

a ACC, adenoid cystic carcinoma; T, tumor, N, node, M, metastasis; T, size and extent of tumor; N, regional lymph node metastasis; M, distant metastasis (Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A. American Joint Committee on Cancer Staging Manual, 7th ed. New York: Springer, 2010:569-576). b FNAB, fine-needle aspiration biopsy.

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FIGURE 1. Clinical, radiographic, and histopathologic features of lacrimal gland epithelial tumors. Top left: A 55-year-old man with 20/20 vision OU developed 2 mm of relative left proptosis without any pain, epibulbar injection or congestion, or diplopia. (Courtesy of Dr. Aaron Fay). Top right: Sagittal computed tomographic view of a pleomorphic adenoma. The lesion is well circumscribed without causing any bone erosion or destruction. There are foci of intratumoral calcification (arrow). Middle left: A 53-year-old woman developed 4 mm of right proptosis over 1 month. Visual acuity was 20/20 OU. Double vision, pain, and pressure behind the globe were reported. (Courtesy of Dr. Francis Sutula). Middle right: Coronal computed tomographic view of an adenoid cystic carcinoma. There is some elevation of the superolateral orbital bone (arrow) without destruction. Bottom left: Ductules of a pleomorphic adenoma are shown on the left. They are lined by a double row of cells. On the right there is a bluish myxoid stroma populated by delaminating outer myoepithelial cells of the ductules. Bottom right: A focus within a pleomorphic adenoma of densely eosinophilic, sclerotic islands of collagen (C) placed among variably wide tracts of blue staining ovoid- to spindle-shaped myoepithelial cells devoid of lumen formation. (Hematoxylin and eosin: bottom left, 3200; bottom right, 3100)

showed bony destruction, erosion or remodeling of the orbital roof in four of the five lesions arising in the lacrimal gland (Figure 1, middle right). The orbital floor and medial wall in the primary maxillary tumor invading the orbit were partially destroyed. There were no bony changes in the tumor arising in the accessory gland of Wolfring. One tumor contained intralesional calcification without adjacent bony changes. The patient with the adenoid cystic carcinoma primarily in the maxillary sinus with orbital extension was the only patient who had regional lymph 1150

node involvement at the time of initial presentation. In all cases, a malignancy was strongly suspected based on the clinical and radiologic findings, and the widest excision possible was undertaken in the face of infiltrative margins. Average follow-up has been 71 months. Tumor recurrence has been found in four patients. Four patients are alive with disease and three have died as the result of their disease.
HISTOPATHOLOGIC FINDINGS:

The diameters of the pleomorphic adenomas ranged from 1.2 cm to 2.6 cm,

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FIGURE 2. Histopathology of lacrimal gland pleomorphic adenoma and adenoid cystic carcinoma. Top left: An atypical pleomorphic adenoma displays spindle cells with atypical hyperchromatic nuclei. Mitotic figures were not observed. Top right: Small glandular (microacinar) units, in atypical pleomorphic adenoma, also shown in inset, are composed of low cuboidal cells with scattered large hyperchromatic nuclei possessing prominent nucleoli. Middle left: Classical sievelike, Swiss cheese or cribriform pattern of adenoid cystic carcinoma. The inset demonstrates perineural invasion. Middle right: The adenoidal spaces are not true lumens because they are rimmed by periodic acid Schiff–positive basement membrane material (arrows) and filled with a delicate extracellular fibrillar matrix and not mucin. Bottom left: Left panel. Hyaline stroma has compressed the cribriform lobules into smaller cellular clusters that still manifest small pseudoglandular formations. Right panel. Advanced hyalinization has reduced the cellular component to narrow curvilinear strands. Bottom right: Tubular variant of an adenoid cystic carcinoma with eosinophilic secretory material in the lumens, shown in the inset to be intensely periodic acid Schiff–positive. Note that the outer cells of the glandular units do not splay off into the nonmyxoid stroma, unlike the feathery ablumenal cellular pattern in pleomorphic adenoma. (Hematoxylin and eosin: top left, 3200; top right, 3200; inset, 3600; middle left, 3200; inset, 3400; Periodic acid Schiff: middle right, 3400; Hematoxylin and eosin: bottom left, left panel 3100; bottom left, right panel 3100; bottom right, 3100; periodic acid Schiff: inset, 3600).

with a mean of 1.6 cm. All were invested with a variably thick pseudocapsule and all but one of the lesions were composed of a mixture of double-layered, epitheliumlined glandular structures with small to patulous lumens (Figure 1, bottom left) and spindly outer myoepithelial cells. The lumens commonly contained amorphous, eosinophilic secretory material that was positive for periodic acid Schiff (PAS) and mucicarmine. Basophilic mucoid VOL. 156, NO. 6

stromal material surrounded the ductlike units. The outer myoepithelial cells of the ductular structures delaminated into the stroma. One myoepitheliomatous tumor predominantly displayed non-lumen–forming oval and spindleshaped cells. This variant manifested stromal regions of compacted collagen (Figure 1, bottom right). One lesion was remarkable because of its scattered, atypical, hyperchromatic spindle cells (Figure 2, top left) and atypical

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FIGURE 3. Histopathology of varieties of lacrimal gland adenoid cystic carcinoma. Top left: Aggregates of small blue cells, representing the solid/basaloid variant of adenoid cystic carcinoma, are separated by a collagenous stroma. The bottom left inset reveals the regular, bland, round character of the tumor cell nuclei. A small pseudolumen with fibrillar material is shown in the bottom right inset. Top right: Solid/basaloid lobules of tumor display bone invasion (arrow). There are scattered cribriform formations within the small lobules in the upper left portion of the field. Middle left: Multiple, suspicious, different-sized nodules (arrows) are present subconjunctivally, having originated in an accessory tarsal gland of Wolfring. Some pathologists thought that the lesion was an ‘‘atypical’’ pleomorphic adenoma. Middle right: The arrangement of the cells with small lumens in a loose stroma looks deceptively like a pleomorphic adenoma. The inset shows a classical cribriform lobule at a deeper level of sectioning. Bottom left: Myriad back-toback tubules with secretory material were also discovered. Bottom right: Cords of small basaloid cells are imitative of a monomorphic adenoma in this field. (Hematoxylin and eosin: top left, 3100; bottom left inset, 3600; bottom right inset, 3400; top right, 3200; middle left, 340; middle right, 3200; inset, 3100; bottom left, 3200; bottom right, 3100.)

low cuboidal cells, which created micro-acinar units (Figure 2, top right and inset). Another tumor exhibited foci of dystrophic calcification. Of the five adenoid cystic carcinomas arising primarily in the main lacrimal gland, the widest diameters of the lesions ranged from 1.4 cm to 3.1 cm, with a mean of 2 cm. The lesions were removed only exceptionally as single specimens and more typically fragmentarily. The outer perimeters of some lesional fragments possessed only thin partial pseudocapsules that were commonly infiltrated by small 1152

islands of tumor cells. Five tumors had a combination of cribriform (Figure 2, middle left and middle right) and tubular patterns; one cribriform lesion was reduced to small tumor cell clusters with pseudolumens or curvilinear strands by a prominent enveloping and compressive hyaline stroma (Figure 2, bottom left, left and right panels); one lesion in the sinus had a trabecular pattern occasionally punctuated by minute lumens; one had a predominantly tubular pattern (Figure 2, bottom right); and one had a predominantly solid/basaloid pattern (Figure 3, top left

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TABLE 3. Immunohistochemical Results for Lacrimal Gland Pleomorphic Adenoma and Adenoid Cystic Carcinoma of the Ocular Adnexa

Pleomorphic Adenoma (n ¼ 7) Adenoid Cystic Carcinoma (n ¼ 7)

Ki-67 Proliferation Index (Mean [Range])

P53 Positivity (Mean [Range])

PLAG1 (Positive/Negative)

MYB (Positive/Negative)

3.8% [2.6  5.4%] 29.1% [23.8 – 42.5%]

18.5% [11.9 – 28.9%] 39% [24.2 – 50.3%]a

7b/0 0/7b

2c/5 6b/1

a

Two specimens (basaloid type lacrimal gland adenoid cystic carcinoma and adenoid cystic carcinoma from maxillary sinus) did not stain for p53. b Strongly positive: staining in >50% of cells. c Weakly positive: staining in >5% and <50% of cells.

and top right). A final tumor manifested a mixture of the most unusual features: subconjunctival multinodularity due to its origin in an accessory gland of Wolfring in the lower lid tarsus (Figure 3, middle left); small lumenforming cellular clusters suggestive of a pleomorphic adenoma (Figure 3, middle right); tightly packed tubular structures (Figure 3, bottom left); and interanastomosing cords of small, banal, basaloid cells encountered more typically in extremely rare monomorphic adenomas (Figure 3, bottom right). In lesions with a tubular pattern, the true lumens focally contained PAS and mucicarmine-positive eosinophilic secretions (Figure 2, bottom right inset). Evidence of perineural invasion was seen in three cases (Figure 2, middle left inset).
IMMUNOHISTOCHEMICAL FINDINGS:

The immunohistochemistry results of nuclear staining with the four probes are summarized in Table 3. The Ki-67 proliferation index for the pleomorphic adenomas (Figure 4, top left) was relatively low with an average of 3.8% (range of 2.6% to 5.4%). The average Ki-67 proliferation index for adenoid cystic carcinomas (Figure 4, top right) was 29.1% (range of 23.8% to 42.5%). The basaloid variant had the highest Ki-67 positivity of 42.5% (Figure 4, top right inset). p53 in the pleomorphic adenomas (Figure 4, middle left) displayed an average positivity of 18.5% (range, 11.9% to 28.9%). The average p53 positivity in the adenoid cystic carcinomas (Figure 4, middle right) was 39% (range, 24.2% to 50.3%). Two cases (the solid/basaloid adenoid cystic carcinoma and the adenoid cystic carcinoma originating in the maxillary sinus) were totally negative for p53, possibly an artifactual finding. In all specimens of pleomorphic adenoma, PLAG1 stained strongly positive in the outer ductular myoepithelial cells and spindled stromal cells (Figure 4, bottom left and bottom right). The adenoid cystic carcinomas were all negative for PLAG1 (Figure 5, top left). None of the pleomorphic adenomas stained strongly positive for MYB: two cases stained focally and weakly positive for MYB, and five cases were totally negative, including the myoepitheliomatous variant (Figure 5, top right and middle left). In six cases of adenoid cystic carcinoma, tumor cells stained strongly positive for MYB (Figure 5, middle right, bottom left and bottom right), VOL. 156, NO. 6

whereas one case was negative. Positive staining was seen in both the outer myoepithelial cells and the inner luminal cells in cases with mixed tubular-cribriform and predominantly tubular patterns. MYB staining was diffuse and uniform within the solid lobules of cells of the basaloid tumor (Figure 5, bottom right).

DISCUSSION FROM

THE

HISTOPATHOLOGIC

POINT

OF

VIEW,1–4

pleomorphic adenoma is a pseudoencapsulated benign neoplasm composed of bland, lumen-forming as well as spindled myoepitheliomatous cells, both possessing generally oval, uniform, finely stippled nuclei chromatin without a nucleolus or with only a minute, punctate one. Lumens encountered in pleomorphic adenoma had double-layered cellular walls and manifest splaying-off or feathering of the outer myoepithelial cells into the stroma, where they underwent various modifications and transformations into hyaline, myxoid or chondroid foci. Such lesions are highly distinctive and do not require the aid of immunohistochemistry for their diagnosis because standard microscopy is adequate. Adenoid cystic carcinoma has infiltrative margins and typically displays perineural invasion, which accounts for the frequent clinical complaint of pain. A useful adjunct for identifying perineural invasion is immunohistochemical staining for the presence of neurofilaments, which can be detected in axons in the centrally located nerve surrounded by the malignant cells. Adenoid cystic carcinoma is composed, paradoxically for a malignancy, of small, bland cells, with a 1-to-1 nuclear-tocytoplasmic ratio. The nuclei are regular, round or slightly angulated, uniformly hematoxylinophilic and generally lacking a nucleolus and mitotic activity. The cytoplasm is delicately eosinophilic, amphophilic or clear. The tumor cells of adenoid cystic carcinoma characteristically display arrangements described as being sievelike, cribriform or ‘‘Swiss cheese’’ patterns composed of small holes simulating lumens (which are actually entrapped collections of extracellular stroma). Tumors exhibiting this feature are readily diagnosed by standard microscopy.

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FIGURE 4. Immunohistochemical staining results of lacrimal gland epithelial tumors. Top left: Low Ki-67 nuclear positivity in a pleomorphic adenoma (average of 3.8% in the series). Top right: High Ki-67 proliferation index in an adenoid cystic carcinoma (average of 29.1% in the series). The inset shows that the solid/basaloid variant of adenoid cystic carcinoma had the highest Ki-67 proliferation index (42.5%). Middle left: p53-positive nuclear staining in a pleomorphic adenoma is weak to moderate with an average of 18.5% of cells. Middle right: p53-positive nuclear staining in 49% of cells in an adenoid cystic carcinoma is strong and the nuclei appear larger than those in a pleomorphic adenoma (compare to middle left). Bottom left: In pleomorphic adenoma, PLAG1 reacts with virtually all of the nuclei of the outer myoepithelial cells. None of the inner adlumenal cells are positively staining. Bottom right: Non–lumen-forming myoepithelial cells are uniformly PLAG1-positive. (Immunoperoxidase reaction, diaminobenzidine chromogen, hematoxylin counterstain: top left, 3400; top right, 3200; inset, 3100; middle left, 3400; middle right, 3400; bottom left, 3200; bottom right, 3200.)

Adenoid cystic carcinoma can also be substantially constituted by true lumen-forming tubules, which in microscopic sectioning may appear like adenomatous or adenocarcinomatous units.5 The tubules, whose contents stain positively with PAS and mucicarmine, exhibit multiple cellular layers and harbor a myoepithelial population that is sharply defined with a straight border devoid of any cellular detachments, irregularities or delaminations where the adenomatous units abut the stroma, as occurs in pleomorphic adenoma. Overlapping of morphologic features of pleomorphic adenoma and adenoid cystic carcinoma are caused 1154

primarily by the shared feature of focal sclerosis or hyalinization, but cords of monomorphic basaloid cells may be encountered rarely. Adenoid cystic carcinoma can also assume a primitive basaloid character with small, undifferentiated primitive cells that are organized into solid lobules; more typical differentiated elements can often be found in step sections. In the histopathologic specimens of pleomorphic adenoma and adenoid cystic carcinoma reviewed for the current study, five tumor types received some diagnostic assistance from the immunohistochemical evaluations: an atypical pleomorphic adenoma (Table 1,

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FIGURE 5. Immunohistochemical staining results of lacrimal gland epithelial tumors. Top left: Cribriform cells of an adenoid cystic carcinoma fail to stain for PLAG1. Top right: MYB does not stain the nuclei of the inner ductular or outer myoepithelial cells of pleomorphic adenoma. Middle left: Pure myoepitheliomatous population of a pleomorphic adenoma is MYB-negative. Middle right: All of the nuclei are MYB-positive in the cribriform cell formations of an adenoid cystic carcinoma. Bottom left: Uniform nuclear MYB-positivity of inner and outer cells in a tubular variant of adenoid cystic carcinoma. Bottom right: Solid/basaloid adenoid cystic carcinoma is intensely MYB-positive. (Immunoperoxidase reaction, diaminobenzidine chromogen, hematoxylin counterstain: top left, 3200; top right, 3200; middle left, 3200; middle right, 3200; inset, 3600; bottom left, 3200; bottom right, 3200.)

Case 1); a myoepithelioma with unusual foci of collagen hyalinization (Table 1, Case 2); a tubular adenoid cystic carcinoma arising from a sinus (Table 2, Case 14); a solid/basaloid adenoid cystic carcinoma (Table 2, Case 9); and an adenoid cystic carcinoma with large areas reminiscent of a basaloid monomorphic tumor (adenoma) that arose in an accessory lacrimal gland of Wolfring in the tarsus of the lower eyelid and was initially diagnosed as a benign tumor (Table 2, Case 13). Although many markers are available and have been studied for their expression in salivary gland tumors,9,10 we chose Ki-67 and p53, two widely applied diagnostic immunohistochemical markers directed at the identification VOL. 156, NO. 6

of intranuclear factors, to test their ability to additionally delineate useful overall diagnostic differences between pleomorphic adenoma and adenoid cystic carcinoma of the lacrimal gland and orbit. Two newer nuclear markers, PLAG1 and MYB, have only recently been introduced into the immunohistochemical armamentarium. The preceding four probes in combination, to the best of our knowledge, have had no applications to lacrimal gland tumors up to the present. Compared to nucleic acid–based approaches such as reverse transcriptase-polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH), immunohistochemical methods may be less specific but nonetheless still have some practical advantages. They

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include the avoidance of contamination by non-neoplastic cells, which constantly affects PCR-based techniques, as well as the familiarity of all pathology laboratories with performing such procedures, making them suitable for routine evaluations. Ki-67, a marker for cells undergoing DNA synthesis prior to mitosis, has been studied extensively in numerous malignancies and is associated with cellular proliferation. The Ki-67 and MIB-1 (not to be confused with MYB) monoclonal antibodies are directed against different epitopes of the same antigen. These antibodies recognize a nuclear protein involved in the active premitotic phases (G1, S, G2 and M) of the cell cycle. This protein can be used as a measure of the growth fraction by dividing the number of positive cells by all cells present (Ki-67 proliferation index, or PI). Among salivary-gland tumors, it has been shown that adenoid cystic carcinoma has a higher mean Ki-67 PI (21.4%, range 11.3% to 56.7%) compared to polymorphous low-grade adenocarcinoma (2.4%; range, 0.2% to 6.4%).11,12 High proliferative activity measured by Ki-67 expression has been strongly correlated with decreased patient survival.13,14 Our series demonstrated a markedly low proliferation index in pleomorphic adenoma (average 3.8%; range, 2.6% to 5.4%), whereas adenoid cystic carcinoma manifested much higher replicative DNA activity (average 29.1%; range, 23.8% to 42.5%). The tumor suppressor gene p53 is one of the most commonly mutated genes identified in human neoplasms. It codes for a transcription factor protein that normally induces apoptosis or cell cycle arrest in cells containing damaged DNA, preventing them from proliferating. Mutation of the gene causes overexpression of a deranged p53 protein detectable in the nucleus by immunohistochemistry. Based on prior reports, the average rate of p53 positivity in pleomorphic adenomas and polymorphous low-grade adenocarcinoma of salivary glands was reported as 1.2%, vs 4.3% to 24% positivity in adenoid cystic carcinoma.15–17 In our current study, p53 positivity was high in adenoid cystic carcinoma (average 39%; range, 24.2% to 50.3%) while significantly lower in pleomorphic adenoma (average 18.5%; range, 11.9% to 28.9%). Additionally, from a qualitative perspective, the nuclear staining in adenoid cystic carcinoma was strong, and the nuclei appeared large, probably the result of the excessive accumulation of the abnormal p53 protein. In contrast, in pleomorphic adenoma the p53 immunostaining was weaker, and the nuclei remained small because there is less accumulation of the abnormal p53 protein. Previous studies suggest that lower-grade adenoid cystic carcinoma may progress to higher-grade tumors through mutations in p53,16 and that progressive alterations in p53 are associated with adenoid cystic carcinoma tumors in cases of recurrence and metastasis.18 Cytogenetic analysis of pleomorphic adenomas have demonstrated a recurrent translocation t(5;8)(p13;q12), resulting in upregulation of PLAG1 (pleomorphic adenoma 1156

gene 1) located on chromosome 8q12. This leads to overexpression of its protein PLAG1, a zinc finger transcription factor, due to swapping of the promoter region.19,20 The PLAG1 gene translocation has so far been identified to be highly specific for pleomorphic adenoma, making it a potentially powerful diagnostic marker. However, the same translocation is also closely associated with lipoblastomas21,22 and PLAG1 protein expression has been demonstrated in assorted mesenchymal tumors,23 acute myeloid leukemia24 and hepatoblastoma.25 In one study, immunohistochemistry and FISH targeting PLAG1 was shown to aid in discriminating pleomorphic adenoma from other salivary gland neoplasms and in distinguishing carcinoma ex pleomorphic adenoma from de novo carcinomas. The PLAG1 immunostain was deemed specific for carcinoma ex pleomorphic adenoma in comparison other carcinomas (salivary gland adenoid cystic carcinoma and mucoepidermoid carcinoma, among others, and various nonsalivary neoplasms), but its use alone was limited by variable expression. Therefore, the recommendation has been made that it be interpreted in combination with FISH.26 In the current series, all of the pleomorphic adenoma cases stained strongly positive for PLAG1, and all of the adenoid cystic carcinoma cases were negative. The introduction of the latest epithelial neoplastic nuclear marker MYB represents a discriminating genetic diagnostic tool for adenoid cystic carcinoma.27–32 It involves a translocation between the 6q and 9p chromosomal regions, specifically t(6;9)(q22-23, p2324).28 This translocation results in the fusion of the oncogene MYB and the transcription factor gene NFIB leading to a high expression of the MYB protein. Mutations in the MYB gene have been shown to affect downstream target genes involved in salivary adenoid cystic carcinoma such as BCL-2, C-Kit (CD117), and COX-2.30,33 It is associated with several other malignancies such as leukemia,34 breast cancer35 and colorectal carcinoma.36 The loss of MYB regulation may lead to overactivation of critical MYB targets that include genes involved in apoptosis, cell cycle control, cell growth, and cell adhesion. Analyses of the MYB-NFIB fusion transcript by RT-PCR consistently showed that the fusion transcript was present in samples of adenoid cystic carcinoma and was not found in non–adenoid cystic carcinoma neoplasms, thereby proving the high specificity of this technique.29,31,32,37 An indirect and efficient way of identifying abnormalities in the MYB gene is by demonstrating the presence of the MYB protein with immunohistochemistry. In one study31 employing immunohistochemistry, 82% of adenoid cystic carcinoma cases stained positively for the MYB protein; however, 14% of non–adenoid cystic carcinoma neoplasms also stained positively. Sensitivity and specificity can be substantially improved by assessing clinicopathologic correlations between MYB expression using immunohistochemistry and identification of the MYB translocation by means of FISH.27,32 In our present series, 6 of 7 cases of

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adenoid cystic carcinomas exhibiting a spectrum of morphologic features were strongly positive for MYB evaluated immunohistochemically; we did not resort to FISH, which is capable of demonstrating the MYB translocation in adenoid cystic carcinoma of the lacrimal gland32 but is more cumbersome to perform than immunohistochemistry. Among our cases of pleomorphic adenoma, two of seven stained weakly positive for MYB. Focal, weak staining in a small subset of non–adenoid cystic carcinoma salivary gland tumors (pleomorphic adenoma, atypical pleomorphic adenoma, polymorphous adenocarcinoma, and myoepithelial carcinoma) can cause a diagnostic problem in small surgical samples or fine needle aspiration biopsy.27 In this study, Ki-67, p53, and MYB immunostaining were all far more pronounced in adenoid cystic carcinoma than in pleomorphic adenoma. PLAG1 immunostaining clearly

distinguished pleomorphic adenoma from adenoid cystic carcinoma. The strong MYB staining in our lacrimal adenoid cystic carcinoma cases supports the theory that MYB translocation and overexpression are common driving mechanisms for adenoid cystic carcinoma arising from a variety of sites (as shown by others27,31,37), and that this also holds true for lacrimal adenoid cystic carcinoma.32 Similarly, for pleomorphic adenoma, overexpression of PLAG1 is commonly seen in tumors of many different salivary gland sites19,23,26 (most but not all through translocation); our lacrimal tumors thus seem to share the same pathogenic mechanism. We believe that a combined histopathologic and immunohistochemical approach for the diagnosis of lacrimal gland epithelial tumors holds promise but deserves further investigation before it is widely accepted. In the meantime, a judicious approach can offer diagnostic benefits in selected cases.

ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST. Design of study (P.R.M., F.A.J., J.F.K.); Conduct of study (P.R.M., F.A.J., J.F.K.); Collection, management, analysis, and interpretation of the data (P.R.M., F.A.J., J.F.K.); Preparation, review, or approval of the manuscript (P.R.M., F.A.J., J.F.K.). Each of the coauthors has seen and agrees with each of the changes made to this manuscript in the revision and with the way his or her name is listed.

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10. Eveson JW, Kusafuka K, Stenman G, Nagao T. Pleomorphic adenoma. In: Barnes L, Eveson JW, Reichart P, Sidransky D, eds. World Health Organization classification of tumours: pathology and genetics of head and neck tumours. Lyon, France: IARC Press, 2005:254–262. 11. Skalova A, Simpson RH, Lehtonen H, Leivo I. Assessment of proliferative activity using the MIB1 antibody help to distinguish polymorphous low grade adenocarcinoma from adenoid cystic carcinoma of salivary glands. Pathol Res Pract 1997; 193(10):695–703. 12. Beltran D, Faquin WC, Gallagher G, August M. Selective immunohistochemical comparison of polymorphous lowgrade adenocarcinoma and adenoid cystic carcinoma. J Oral Maxillofac Surg 2006;64(3):415–423. 13. Ettl T, Schwarz S, Kleinsasser N, Hartmann A, Reichert TE, Driemel O. Overexpression of EGFR and absence of C-KIT expression correlate with poor prognosis in salivary gland carcinomas. Histopathology 2008;53(5):567–577. 14. Ben-Izhak O, Akrish S, Nagler RM. Ki-67 and salivary cancer. Cancer Invest 2008;26(10):1015–1023. 15. Vargas HSD, Kaplan MJ, Regezi JA, Weidner N. Mixed tumor, polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma of the salivary gland: pathogenic implication and differential diagnosis by Ki-67 (MIB 1), BCL 2 and S-100 immunohistochemistry. Appl Immunol Mol Morphol 1997;5(1):8–16. 16. Yamamoto Y, Wistuba II, Kishimoto Y, et al. DNA analysis at p53 locus in adenoid cystic carcinoma: comparison of molecular study and p53 immunostaining. Pathol Int 1998; 48(4):273–280. 17. Karja VJ, Syrjanen KJ, Kurvinen AK, Syrjanen SM. Expression and mutations of p53 in salivary gland tumours. J Oral Pathol Med 1997;26(5):217–223.

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18. Kiyoshima T, Shima K, Kobayashi I, et al. Expression of p53 tumor suppressor gene in adenoid cystic and mucoepidermoid carcinomas of the salivary glands. Oral Oncol 2001;37(3): 315–322. 19. Voz ML, Astrom AK, Kas K, Mark J, Stenman G, Van de Ven WJ. The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in upregulation of PLAG1 gene expression under control of the LIFR promoter. Oncogene 1998;16(11):1409–1416. 20. Kas K, Voz ML, Hensen K, Meyen E, Van de Van WY. Transcriptional activation capacity of the novel PLAG family of zinc finger proteins. J Biol Chem 1998;273(36):23026–23032. 21. Gisselsson D, Hibbard MK, Dal Cin P, et al. PLAG1 alterations in lipoblastoma: involvement in varied mesenchymal cell types and evidence for alternative oncogenic mechanisms. Am J Pathol 2001;159(3):955–962. 22. Hibbard MK, Kozakewich HP, Dal Cin P, et al. PLAG1 fusion oncogenes in lipoblastoma. Cancer Res 2000;60(17):4869–4872. 23. Matsuyama A, Hisaoka M, Hashimoto H. PLAG1 expression in mesenchymal tumors: an immunohistochemical study with special emphasis on the pathogenetical distinction between soft tissue myoepithelioma and pleomorphic adenoma of the salivary gland. Pathol Int 2012;62(1):1–7. 24. Landrette SF, Kuo YH, Hensen K, et al. PLAG1 and PLAG2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11. Blood 2005;105(7):2900–2907. 25. Zatkova A, Rouillard JM, Hartmann W, et al. Amplification and overexpression of the IGF2 regulator PLAG1 in hepatoblastoma. Genes Chromosomes Cancer 2004;39(2):126–137. 26. Bahrami A, Dalton JD, Shivakumar B, Krane JF. PLAG1 alteration in carcinoma ex pleomorphic adenoma: immunohistochemical and fluorescence in situ hybridization studies of 22 cases. Head Neck Pathol 2012;6(3):328–335. 27. West RB, Kong C, Clarke N, et al. MYB expression and translocation in adenoid cystic carcinomas and other salivary gland tumors with clinicopathologic correlation. Am J Surg Pathol 2011;35(1):92–99.

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28. Nordkvist A, Mark J, Gustafsson H, Bang G, Stenman G. Non-random chromosome rearrangements in adenoid cystic carcinoma of the salivary glands. Genes Chromosomes Cancer 1994;10(2):115–121. 29. Persson M, Andren Y, Mark J, Horlings HM, Persson F, Stenman G. Recurrent fusion of MYB and NFIB transcription factor genes in carcinomas of the breast and head and neck. Proc Natl Acad Sci 2009;106(44):18740–18744. 30. Bell D, Roberts D, Karpowicz M, Hanna EY, Weber RS, ElNaggar AK. Clinical significance of Myb protein and downstream target genes in salivary adenoid cystic carcinoma. Cancer Biol Ther 2011;12(7):569–573. 31. Brill LB 2nd, Kanner WA, Fehr A, et al. Analysis of MYB expression and MYB-NFIB gene fusions in adenoid cystic carcinoma and other salivary neoplasms. Mod Pathol 2011; 24(9):1169–1176. 32. von Holstein SL, Fehr A, Persson M, et al. Adenoid cystic carcinoma of the lacrimal gland: MYB gene activation, genomic imbalances, and clinical characteristics. Ophthalmology 2013 May 30. 33. Oh IH, Reddy EP. The myb gene family in cell growth, differentiation and apoptosis. Oncogene 1999;18(19):3017–3033. 34. Slamon DJ, Boone TC, Murdock DC, et al. Studies of the human c-myb gene and its product in human acute leukemias. Science 1986;233(4761):347–351. 35. Drabsch Y, Robert RG, Gonda TJ. MYB suppresses differentiation and apoptosis of human breast cancer cell. Breast Cancer Res 2010;12(4):R55. 36. Wilkins HR, Doucet K, Duke V, Morra A, Johnson N. Estrogen prevents sustained COLO-205 human colon cancer cell growth by inducing apoptosis, decreasing c-myb protein, and decreasing transcription of the anti-apoptotic protein bcl-2. Tumour Biol 2010;31(1):16–22. 37. Mitani Y, Li J, Rao PH, et al. Comprehensive analysis of the MYB-NFIB gene fusion in salivary adenoid cystic carcinoma: incidence, variability, and clinicopathologic significance. Clin Cancer Res 2010;16(19):4722–4731.

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Biosketch Pia R. Mendoza, MD, finished her Ophthalmology residency at St. Luke’s Medical Center, Quezon City, Philippines. She is a research fellow in Ophthalmic Pathology at the David G. Cogan Laboratory of Ophthalmic Pathology at the Massachusetts Eye and Ear Infirmary in Boston, Massachusetts.

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