Histopathology 2015, 66, 791–797. DOI: 10.1111/his.12526
Fluorescence in-situ hybridization identifies Mastermind-like 2 (MAML2) rearrangement in odontogenic cysts with mucous prosoplasia: a pilot study Prokopios P Argyris,1 Rebecca N Wehrs,2 Joaquın J Garcıa2 & Ioannis G Koutlas1 1
Division of Oral and Maxillofacial Pathology, School of Dentistry, University of Minnesota, Minneapolis, MN, USA, and 2Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Date of submission 10 June 2014 Accepted for publication 8 August 2014 Published online Article Accepted 14 August 2014
Argyris P P, Wehrs R N, Garcıa J J & Koutlas I G (2015) Histopathology 66, 791–797. DOI: 10.1111/his.12526
Fluorescence in-situ hybridization identifies Mastermind-like 2 (MAML2) rearrangement in odontogenic cysts with mucous prosoplasia: a pilot study Aims: The pathogenesis of intraosseous mucoepidermoid carcinoma (IMEC) remains unknown. Coexistence with odontogenic cysts (ODC) has been reported in 32–48% of IMEC. Furthermore, prosoplastic mucous cells are often seen in the epithelial lining of ODCs. MECT1–MAML2 fusion transcripts have been identified in >66% of salivary gland MEC cases. The aim of this study was to investigate the presence of MAML2 rearrangement in ODCs featuring mucous prosoplasia. Methods and results: Ten cases of ODC with a mucous cell component and three cases of IMEC were evaluated using fluorescence in-situ hybridization. All cases occurred in the mandible. The ODCs exhibited a M:F ratio of 4:1 (mean age 49.2 years), while all
IMECs occurred in women (mean age 68.3 years). All three IMECs demonstrated MAML2 rearrangement, in 26–61% of tumour cells. Successful hybridization was observed in nine of 10 cases of ODC. In two of these nine, there was MAML2 rearrangement in 12% and 24% of the lining epithelial cells, while three of the nine showed rearrangement in 7–8% of cells; the remaining four cases were negative. Conclusions: We identified MAML2 rearrangements in five of nine ODCs lined by mucus-secreting cells. This suggests that at least a subset of ODCs with mucous prosoplasia are characterized by molecular events considered diagnostic for intraosseous and extraosseous MEC.
Keywords: FISH, MAML2 rearrangement, mucoepidermoid carcinoma, mucous prosoplasia, odontogenic cyst
Introduction Mucoepidermoid carcinoma (MEC) represents the most common malignant salivary gland neoplasm, in both the paediatric and adult populations.1 Originally described by Nordkvist et al.2 in 1994 and defined by Tonon et al.3 in 2003, the recurrent reciprocal translocation of MECT1 (mucoepidermoid translocated-1,
Address for correspondence: Ioannis G Koutlas DDS, MS, Division of Oral and Maxillofacial Pathology, School of Dentistry, University of Minnesota, 515 Delaware Street SE 16-206B, 55455 Minneapolis, MN, USA. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
also known as CRTC1, TORC1 and WAMTP1) at 19p13, and MAML2 (Mastermind-like 2) at 11q21, have been confirmed as an underlying molecular event in 38–100% of MEC cases.1,4–12 The MECT1– MAML2 chimeric protein may activate both cAMP– CREB as well as Notch signalling targets, leading to disruption of cell cycle progression and dysregulation of cellular differentiation, thus orchestrating the early stages of tumorigenesis.13–15 Although infrequent, MEC may affect non-salivary gland sites such as the upper aerodigestive tract, the tracheo-bronchial tree, breast, thyroid, lacrimal glands and conjunctiva and the jaws.15–18 Primary
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intraosseous/central MEC (IMEC) of the jaws is a well-described entity,19–22 comprising 2–4.3% of all MEC cases and predominantly affecting the third molar–ramus area of the mandible.23,24 The aetiopathogenetic mechanism underlying the development of IMEC remains unknown. Theories attempting to explain the origin of IMEC include neoplastic transformation of (i) ectopic salivary gland tissue developmentally entrapped within the jaws, (ii) maxillary sinus or submucosal seromucinous glands extending into the jaws, and (iii) odontogenic epithelium that lines odontogenic cysts (ODC).24–26 The latter postulate is supported by (i) mucous prosoplastic phenomena occasionally present in the epithelial lining of ODCs, and (ii) the reported coexistence of IMEC and ODC in 32–48% of cases.27,28 Herein, we evaluate the prevalence of MAML2 gene rearrangement by fluorescence in-situ hybridization (FISH) in a group of ODCs featuring mucous prosoplasia, and discuss how the study results support a cytogenetic relationship between IMEC and a subset of ODCs.
Materials and methods The study was approved by the University of Minnesota Institutional Review Board (IRB#1308M41721). Three IMECs and 10 ODCs with mucous cells were evaluated. Demographic and clinical characteristics were collected and tabulated (Tables 1 and 2). Details of administered treatment and clinical outcome (recurrence, metastasis and disease-free survival), when available, were also recorded. DETECTION OF MAML2 GENE BREAK BY FISH
Formalin-fixed paraffin-embedded (FFPE) tissue sections mounted on positively charged slides were subjected to heating in a 90°C oven (15 min) followed by a series of two xylene (15 min) washes and one 100% ethanol (5 min) wash. Slides were air-dried, boiled (10 min) in 10 mmol/l citric acid solution and treated in 92 sodium chloride–sodium citrate buffer (5 min). Proteins were digested using Digest-All3 (Invitrogen, Carlsbad, CA, USA) at two 20-min intervals followed by a phosphate-buffered saline wash (1 min),
Table 1. Clinicopathological characteristics and MAML2 rearrangement status of odontogenic cysts
No.
Histopathogic diagnosis
Age/ Sex
Site
Size (cm)
Associated tooth
1
Dentigerous cyst
42/M
Mandible
1.0
2
Dentigerous cyst
67/F
Mandible
3
Dentigerous cyst
51/M
4
Dentigerous cyst
5
MAML2 status (%)
Treatment
Follow up (months)
Recurrence
Impacted 3rd molar, R
Marsupialization
14
No
7
1.7
Impacted 3rd molar, R
Surgical excision
14
No
0
Mandible
1.5
Impacted 3rd molar, R
Surgical excision
12
No
0
34/M
Mandible
4.2
Impacted 3rd molar, R
Surgical excision
12
No
24
Dentigerous cyst
49/M
Mandible
2.2
Impacted 3rd molar, L
Surgical excision
12
No
12
6
Radicular cyst
52/F
Mandible
1.3
Canine, 1st premolar, R
Surgical excision
11
No
7
7
Dentigerous cyst
40/M
Mandible
2.3
Impacted 3rd molar, R
Surgical excision
10
No
8
8
Glandular odontogenic cyst
40/M
Mandible
2.0
No association
Surgical excision
77
No
Failed
9
Dentigerous cyst
55/M
Mandible
2.8
Impacted 3rd molar, L
Surgical excision
4
No
0
10
Dentigerous cyst
62/M
Mandible
2.5
Impacted 3rd molar, L
Surgical excision
4
No
0
© 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
MAML2 rearrangement in odontogenic cysts
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Table 2. Clinicopathological characteristics and MAML2 rearrangement status of intraosseous mucoepidermoid carcinomas
No
Age/Sex
Site
Size (cm)
Treatment
Follow up (months)
Outcome
MAML2 status (%)
1
20/F
Mandible
7.0
En-bloc resection
14
NED
47
2
60/F
Mandible
1.0
En-bloc resection
75
NED
26
3
85/F
Mandible
2.5
NA
NA
NA
61
NED, no evidence of disease; NA, non available.
graded alcohols (70%, 85% and 100%; 2 min each), and air-dried. Dual-colour FISH was performed using a MAML2 break-apart probe (BAC clone RP111056O10-, CTD-2325K3- and RP11-8N17-labelled Spectrum Orange; and CTD-2554I7-, CTD-2252L1-, RP11-1123F20-, RP11-936C10- and RP11-7D4labelled Spectrum Green). Slides were hybridized at 80°C for 5 min, and held at 37°C overnight. The next day, slides were washed in 76°C 0.1% Nonidet-P40 in 92 sodium chloride–sodium citrate buffer (3 min) and counterstained with Vectashield mounting medium containing 40 ,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Analysis was performed using a Leica DM6000 microscope (Leica Microsystems, Buffalo Grove, IL, USA), and images were captured using CytoVision software (Genetix, Sunnyvale, CA, USA). FISH INTERPRETATION
For each case, approximately 100 cells were analysed in the targeted region. Only individual and well-delineated cells were scored. Significantly overlapping cells were excluded from the analysis. Normal cells without MAML2 rearrangement show a pair of green and orange signals in juxtaposition, typically resulting in two yellow conglomerate signals. Cells with the MAML2 gene rearrangement show one pair of signals (green and orange) in juxtaposition and one green and one orange signal separated. A previous case of major salivary gland MEC originating from the parotid gland with confirmed MAML2 rearrangement was used as a positive control.
2.15 cm (range 1.0–4.2 cm) and 3.5 cm (range 1.0– 7.0 cm), respectively. All lesions were confined to the mandible. Regarding ODCs, simple surgical excision was the treatment of choice in nine of 10 cases, while in one case marsupialization was performed. No recurrences were reported in a follow-up period ranging between 4 and 77 months (mean 17 months). Information on treatment and clinical outcome was available in two of the three IMEC cases; both were treated with en-bloc mandibulectomy, and the patients demonstrated no evidence of disease after 14 and 75 months, respectively. Among ODCs with mucous prosoplasia (Figures 1B, and 2A,B), eight were classified as dentigerous cysts, one as radicular and one as glandular odontogenic cyst. The three examples of IMECs featured prominent cystic architecture, increased numbers of mucous cells compared to their epidermoid neoplastic component, and a limited degree of cellular atypia with absence of mitotic figures. Hence, they were each classified as lowgrade MEC (Figure 1A). Invasive nests were identified in the associated connective tissue of all IMECs. FISH FINDINGS
All three IMECs exhibited MAML2 rearrangement by FISH, in 26–61% of tumour cells (Table 2, Figure 1C). Successful hybridization was observed in nine of 10 cases of ODCs with mucous prosoplasia (Table 1). Among these, two exhibited a MAML2 rearrangement in 12% and 24% of the lining epithelial cells (Figure 1D), respectively, and three showed rearrangement in 7–8% of cells (Figure 2C). The remaining four cases were negative (Figure 2D).
Results CLINICOPATHOLOGICAL CHARACTERISTICS
Discussion
Among the 10 ODCs included in the present study, eight occurred in men and two in women, resulting in a M:F ratio of 4:1; the mean age was 49.2 years (range 34–67 years). All IMECs occurred in female patients with mean age of 68.3 years (range 20– 85 years). The average size of ODCs and IMECs was
Mucoepidermoid carcinoma is the most frequent malignant neoplasm of salivary glands. Molecular studies have identified MECT1–MAML2 t(11;19)(q21; p13) as an underlying pathogenetic signature in 38– 100% of MECs1,4–12 as well as in IMECs,19,29 including all three cases in the present study. The MECT1–
© 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
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A
B
C
D
Figure 1. A, Histopathological characteristics of intraosseous mucoepidermoid carcinoma. Photomicrograph of case 1 (Table 2) demonstrating aggregates of neoplastic epidermoid and mucus-secreting cells forming cystic-like spaces and solid islands embedded in a fibrocollagenous stroma (H&E). B, Histopathological features of an odontogenic cyst positive for MAML2 rearrangements. Photomicrograph of case 4 (Table 1) depicting the stratified squamous epithelium lining a dentigerous cyst and featuring foci of mucous prosoplasia. Microcilia formation is also present (H&E). C,D, FISH analyses of (A) and (B), respectively. In (C), break-apart FISH revealed MAML2 rearrangements in 47% of the neoplastic cells of this IMEC, as confirmed by the numerous split red–green signals indicative of disruption. In (D) MAML2 rearrangements were observed in 24% of the cystic lining epithelial cells.
MAML2 fusion gene is composed of the N-terminal CREB protein-binding domain (exon 1) of MECT1 juxtaposed to the C-terminal transcriptional activation domain (exons 2–5) of the Notch coactivator MAML2.3,30,31 Hence, the MECT1–MAML2 chimeric gene may function as an oncogene through the constitutive activation of both cAMP–CREB and Notch signalling targets.13–15 In fact, MAML2 rearrangements are considered to be driver alterations with a pivotal role in the initiation and maintenance of MEC tumorigenesis.29 Herein, we investigated the presence of MAML2 rearrangement by FISH in ODCs characterized by mucous prosoplasia of the lining epithelium, aiming to assess the possibility of ODCs being precursor
lesions for IMEC development. In the current study, MAML2 rearrangement was identified in more than half of ODCs. IMECs demonstrated a higher percentage of MAML2 rearrangement when compared to MAML2-positive ODCs. Interestingly, in one example of the latter, an otherwise histologically typical dentigerous cyst (case 4), MAML2 status was essentially similar to IMEC case 2. In all ODC cases included in the present study, mucous cells represented only a minority of the epithelial cells lining the cystic cavity. With the exception of one case (case 1), mucous prosoplasia was observed only focally, which is a frequently encountered and well-known phenomenon in ODCs. In case 1, the number of mucus-secreting cells was increased. This case, however, demonstrated © 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
MAML2 rearrangement in odontogenic cysts
A
B
C
D
795
Figure 2. A,B, Histopathological characteristics of cases 6 and 3 (Table 1), respectively. A radicular (A) and dentigerous (B) cyst featuring mucous prosoplasia with numerous mucous cells (H&E). C,D, FISH analyses of (A) and (B), respectively. In (C), break-apart FISH revealed MAML2 rearrangements in 7% of the epithelial cells in this ODC. In (D), however, total absence of MAML2 rearrangement was observed (0%).
MAML2 rearrangements in only 7% of epithelial cells. Hence, the results of our study suggest that distinction between ODCs harbouring MAML2 rearrangements and ODCs negative for such genetic aberrations cannot be made on histopathological grounds, because an absence of correlation was noticed between microscopic features, such as number and distribution of mucous cells, and the MAML2 status of the lesions. In a recently published study investigating the presence of MAML2 rearrangement in 21 cases of glandular odontogenic cyst (GOC), a rare type of ODC, and five cases of IMEC, MAML2 rearrangement was identified by FISH in all malignant tumours but in none of the GOCs.32 Based on these findings, it was concluded that GOC and IMEC represent independent mechanisms of disease and that GOC should not be considered a precursor to IMEC.32 Our study pro© 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
vides evidence of MAML2 rearrangement in some ODCs. Based on these findings, one may hypothesize that (1) a subset of ODCs can harbour MAML2 rearrangement in the epithelial lining and can transform towards IMEC without obvious phenotypical change, or (2) ODC with MAML2 rearrangement is best regarded as cystic IMEC. The latter has therapeutic implications; i.e. additional surgical treatment for an apparently totally excised lesion. Herein, we do not advocate the performance of FISH to assess the MAML2 rearrangement status in ODCs with mucous cells in their epithelial lining. This would be impractical, given the current state of laboratory affairs. It is also well known that mucous prosoplasia in what appear to be conventional ODCs is far more frequent than IMECs. Furthermore, it is plausible that a gradual increase in genomic instability in the epithelial lining of ODCs, either through additional MAML2
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rearrangements or alterations in various tumour suppressor genes such as DCC, SMAD4, GALR1 and CDKN2A/B, may lead to malignant transformation and acquisition of the MEC phenotype.10 In summary, the presence of MAML2 rearrangements in the lining of ODCs demonstrates the importance of using cytogenetic and molecular techniques to shed light on the pathogenesis of IMEC. It appears that at least a subset of cases that qualify as ODC using conventional histopathological criteria are characterized by early molecular events leading towards neoplastic transformation. Our results should be confirmed by additional larger-scale studies that include the investigation of other genes, such as DCC, SMAD4, GALR1 and CDKN2A/B.
9.
10.
11.
12. 13.
14.
Acknowledgements The authors are indebted to Mr Brian Dunnette (University of Minnesota) for his assistance with the illustrations.
Conflict of interest
15.
16.
17.
The authors declare no potential conflicts of interest.
References 1. Seethala RR, Dacic S, Cieply K, Kelly LM, Nikiforova MN. A reappraisal of the MECT1/MAML2 translocation in salivary mucoepidermoid carcinomas. Am. J. Surg. Pathol. 2010; 34; 1106–1121. 2. Nordkvist A, Gustafsson H, Juberg-Ode M, Stenman G. Recurrent rearrangements of 11q14-22 in mucoepidermoid carcinoma. Cancer Genet. Cytogenet. 1994; 74; 77–83. 3. Tonon G, Modi S, Wu L et al. t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway. Nat. Genet. 2003; 33; 208–213. 4. Martins C, Cavaco B, Tonon G, Kaye FJ, Soares J, Fonseca I. A study of MECT1–MAML2 in mucoepidermoid carcinoma and Warthin’s tumor of salivary glands. J. Mol. Diagn. 2004; 6; 205–210. 5. Behboudi A, Enlund F, Winnes M et al. Molecular classification of mucoepidermoid carcinomas-prognostic significance of the MECT1–MAML2 fusion oncogene. Genes Chromosom. Cancer 2006; 45; 470–481. 6. Okabe M, Miyabe S, Nagatsuka H et al. MECT1–MAML2 fusion transcript defines a favorable subset of mucoepidermoid carcinoma. Clin. Cancer Res. 2006; 12; 3902–3907. 7. Tirado Y, Williams MD, Hanna EY, Kaye FJ, Batsakis JG, ElNaggar AK. CRTC1/MAML2 fusion transcript in high grade mucoepidermoid carcinomas of salivary and thyroid glands and Warthin’s tumors: implications for histogenesis and biologic behavior. Genes Chromosom. Cancer 2007; 46; 708–715. 8. Chiosea SI, Dacic S, Nikiforova MN, Seethala RR. Prospective testing of mucoepidermoid carcinoma for the MAML2 translo-
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cation: clinical implications. Laryngoscope 2012; 122; 1690– 1694. Skalova A, Vanecek T, Simpson RH et al. CRTC1–MAML2 and CRTC3–MAML2 fusions were not detected in metaplastic Warthin tumor and metaplastic pleomorphic adenoma of salivary glands. Am. J. Surg. Pathol. 2013; 37; 1743–1750. Jee KJ, Persson M, Heikinheimo K et al. Genomic profiles and CRTC1–MAML2 fusion distinguish different subtypes of mucoepidermoid carcinoma. Mod. Pathol. 2013; 26; 213–222. Garcıa JJ, Hunt JL, Weinreb I et al. Fluorescence in situ hybridization for detection of MAML2 rearrangements in oncocytic mucoepidermoid carcinomas: utility as a diagnostic test. Hum. Pathol. 2011; 42; 2001–2009. Chenevert J, Barnes LE, Chiosea SI. Mucoepidermoid carcinoma: a five-decade journey. Virchows Arch. 2011; 458; 133–140. Wu L, Liu J, Gao P et al. Transforming activity of MECT1– MAML2 fusion oncoprotein is mediated by constitutive CREB activation. EMBO J. 2005; 24; 2391–2402. Coxon A, Rozenblum E, Park YS et al. Mect1–Maml2 fusion oncogene linked to the aberrant activation of cyclic AMP/CREB regulated genes. Cancer Res. 2005; 65; 7137–7144. O’Neill ID. t(11;19) Translocation and CRTC1–MAML2 fusion oncogene in mucoepidermoid carcinoma [Review]. Oral Oncol. 2009; 45; 2–9. Gnepp DR, Henley JD, Simpson RH, Eveson J. Salivary and lacrimal glands. In Gnepp DR ed. Diagnostic surgical pathology of the head and neck. 2nd edn. Philadelphia: Saunders Elsevier, 2009; 471–476. Baloch ZW, Solomon AC, LiVolsi VA. Primary mucoepidermoid carcinoma and sclerosing mucoepidermoid carcinoma with eosinophilia of the thyroid gland: a report of nine cases. Mod. Pathol. 2000; 13; 802–807. Di Tommaso L, Foschini MP, Ragazzini T et al. Mucoepidermoid carcinoma of the breast. Virchows Arch. 2004; 444; 13–19. Khan HA, Loya A, Azhar R, Din NU, Bell D. Central mucoepidermoid carcinoma, a case report with molecular analysis of the TORC1/MAML2 gene fusion. Head Neck Pathol. 2010; 4; 261–264. Raut D, Khedkar S. Primary intraosseous mucoepidermoid carcinoma of the maxilla: a case report and review of literature. Dentomaxillofac. Radiol. 2009; 38; 163–168. Pires FR, Paes de Almeida O, Lopes MA, Elias da Cruz Perez D, Kowalski LP. Central mucoepidermoid carcinoma of the mandible: report of four cases with long-term follow-up. Int. J. Oral Maxillofac. Surg. 2003; 32; 378–382. Tapia JL, Aguirre A, Garvey M, Zeid M. Mandibular unilocular radiolucency with ill-defined borders. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2004; 97; 301–306. Ellis GL, Auclair PL. Malignant epithelial neoplasms. In Silverberg SG and Sobin LG ed. AFIP atlas of tumor pathology, fourth series, fascicle 9. Tumors of the salivary glands. Washington DC: ARP Press, 2008; 193–195. Neville BW, Damm DD, Allen CM, Bouquot JE. Salivary gland pathology. In Silverberg SG and Sobin LG ed. Oral and maxillofacial pathology. 3rd edn. Philadelphia: Saunders Elsevier, 2009; 490–491. He Y, Wang J, Fu HH, Zhang ZY, Zhuang QW. Intraosseous mucoepidermoid carcinoma of jaws: report of 24 cases. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2012; 114; 424–429. Johnson B, Velez I. Central mucoepidermoid carcinoma with an atypical radiographic appearance. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2008; 106; 51–53. © 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
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27. Brookstone MS, Huvos AG. Central salivary gland tumors of the maxilla and mandible: a clinicopathologic study of 11 cases with an analysis of the literature. J. Oral Maxillofac. Surg. 1992; 50; 229–236. 28. Eversole LR, Sabes WR, Rovin S. Aggressive growth and neoplastic potential of odontogenic cysts: with special reference to central epidermoid and mucoepidermoid carcinomas [Review]. Cancer 1975; 35; 270–282. 29. Bell D, Holsinger CF, El-Naggar AK. CRTC1/MAML2 fusion transcript in central mucoepidermoid carcinoma of mandible – diagnostic and histogenetic implications. Ann. Diagn. Pathol. 2010; 14; 396–401.
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30. Stenman G. Fusion oncogenes in salivary gland tumors: molecular and clinical consequences [Review]. Head Neck Pathol. 2013; 7; 12–19. 31. Bell D, El-Naggar AK. Molecular heterogeneity in mucoepidermoid carcinoma: conceptual and practical implications. Head Neck Pathol. 2013; 7; 23–27. 32. Bishop JA, Yonescu R, Batista D, Warnock GR, Westra WH. Glandular odontogenic cysts (GOCs) lack MAML2 rearrangements: a finding to discredit the putative nature of GOC as a precursor to central mucoepidermoid carcinoma. Head Neck Pathol. 2014; 8; 298–302.
Fluorescence in-situ hybridization identifies Mastermind-like 2 (MAML2) rearrangement in odontogenic cysts with mucous prosoplasia: a pilot study Prokopios P Argyris,1 Rebecca N Wehrs,2 Joaquın J Garcıa2 & Ioannis G Koutlas1 1
Division of Oral and Maxillofacial Pathology, School of Dentistry, University of Minnesota, Minneapolis, MN, USA, and 2Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Date of submission 10 June 2014 Accepted for publication 8 August 2014 Published online Article Accepted 14 August 2014
Argyris P P, Wehrs R N, Garcıa J J & Koutlas I G (2015) Histopathology 66, 791–797. DOI: 10.1111/his.12526
Fluorescence in-situ hybridization identifies Mastermind-like 2 (MAML2) rearrangement in odontogenic cysts with mucous prosoplasia: a pilot study Aims: The pathogenesis of intraosseous mucoepidermoid carcinoma (IMEC) remains unknown. Coexistence with odontogenic cysts (ODC) has been reported in 32–48% of IMEC. Furthermore, prosoplastic mucous cells are often seen in the epithelial lining of ODCs. MECT1–MAML2 fusion transcripts have been identified in >66% of salivary gland MEC cases. The aim of this study was to investigate the presence of MAML2 rearrangement in ODCs featuring mucous prosoplasia. Methods and results: Ten cases of ODC with a mucous cell component and three cases of IMEC were evaluated using fluorescence in-situ hybridization. All cases occurred in the mandible. The ODCs exhibited a M:F ratio of 4:1 (mean age 49.2 years), while all
IMECs occurred in women (mean age 68.3 years). All three IMECs demonstrated MAML2 rearrangement, in 26–61% of tumour cells. Successful hybridization was observed in nine of 10 cases of ODC. In two of these nine, there was MAML2 rearrangement in 12% and 24% of the lining epithelial cells, while three of the nine showed rearrangement in 7–8% of cells; the remaining four cases were negative. Conclusions: We identified MAML2 rearrangements in five of nine ODCs lined by mucus-secreting cells. This suggests that at least a subset of ODCs with mucous prosoplasia are characterized by molecular events considered diagnostic for intraosseous and extraosseous MEC.
Keywords: FISH, MAML2 rearrangement, mucoepidermoid carcinoma, mucous prosoplasia, odontogenic cyst
Introduction Mucoepidermoid carcinoma (MEC) represents the most common malignant salivary gland neoplasm, in both the paediatric and adult populations.1 Originally described by Nordkvist et al.2 in 1994 and defined by Tonon et al.3 in 2003, the recurrent reciprocal translocation of MECT1 (mucoepidermoid translocated-1,
Address for correspondence: Ioannis G Koutlas DDS, MS, Division of Oral and Maxillofacial Pathology, School of Dentistry, University of Minnesota, 515 Delaware Street SE 16-206B, 55455 Minneapolis, MN, USA. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
also known as CRTC1, TORC1 and WAMTP1) at 19p13, and MAML2 (Mastermind-like 2) at 11q21, have been confirmed as an underlying molecular event in 38–100% of MEC cases.1,4–12 The MECT1– MAML2 chimeric protein may activate both cAMP– CREB as well as Notch signalling targets, leading to disruption of cell cycle progression and dysregulation of cellular differentiation, thus orchestrating the early stages of tumorigenesis.13–15 Although infrequent, MEC may affect non-salivary gland sites such as the upper aerodigestive tract, the tracheo-bronchial tree, breast, thyroid, lacrimal glands and conjunctiva and the jaws.15–18 Primary
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intraosseous/central MEC (IMEC) of the jaws is a well-described entity,19–22 comprising 2–4.3% of all MEC cases and predominantly affecting the third molar–ramus area of the mandible.23,24 The aetiopathogenetic mechanism underlying the development of IMEC remains unknown. Theories attempting to explain the origin of IMEC include neoplastic transformation of (i) ectopic salivary gland tissue developmentally entrapped within the jaws, (ii) maxillary sinus or submucosal seromucinous glands extending into the jaws, and (iii) odontogenic epithelium that lines odontogenic cysts (ODC).24–26 The latter postulate is supported by (i) mucous prosoplastic phenomena occasionally present in the epithelial lining of ODCs, and (ii) the reported coexistence of IMEC and ODC in 32–48% of cases.27,28 Herein, we evaluate the prevalence of MAML2 gene rearrangement by fluorescence in-situ hybridization (FISH) in a group of ODCs featuring mucous prosoplasia, and discuss how the study results support a cytogenetic relationship between IMEC and a subset of ODCs.
Materials and methods The study was approved by the University of Minnesota Institutional Review Board (IRB#1308M41721). Three IMECs and 10 ODCs with mucous cells were evaluated. Demographic and clinical characteristics were collected and tabulated (Tables 1 and 2). Details of administered treatment and clinical outcome (recurrence, metastasis and disease-free survival), when available, were also recorded. DETECTION OF MAML2 GENE BREAK BY FISH
Formalin-fixed paraffin-embedded (FFPE) tissue sections mounted on positively charged slides were subjected to heating in a 90°C oven (15 min) followed by a series of two xylene (15 min) washes and one 100% ethanol (5 min) wash. Slides were air-dried, boiled (10 min) in 10 mmol/l citric acid solution and treated in 92 sodium chloride–sodium citrate buffer (5 min). Proteins were digested using Digest-All3 (Invitrogen, Carlsbad, CA, USA) at two 20-min intervals followed by a phosphate-buffered saline wash (1 min),
Table 1. Clinicopathological characteristics and MAML2 rearrangement status of odontogenic cysts
No.
Histopathogic diagnosis
Age/ Sex
Site
Size (cm)
Associated tooth
1
Dentigerous cyst
42/M
Mandible
1.0
2
Dentigerous cyst
67/F
Mandible
3
Dentigerous cyst
51/M
4
Dentigerous cyst
5
MAML2 status (%)
Treatment
Follow up (months)
Recurrence
Impacted 3rd molar, R
Marsupialization
14
No
7
1.7
Impacted 3rd molar, R
Surgical excision
14
No
0
Mandible
1.5
Impacted 3rd molar, R
Surgical excision
12
No
0
34/M
Mandible
4.2
Impacted 3rd molar, R
Surgical excision
12
No
24
Dentigerous cyst
49/M
Mandible
2.2
Impacted 3rd molar, L
Surgical excision
12
No
12
6
Radicular cyst
52/F
Mandible
1.3
Canine, 1st premolar, R
Surgical excision
11
No
7
7
Dentigerous cyst
40/M
Mandible
2.3
Impacted 3rd molar, R
Surgical excision
10
No
8
8
Glandular odontogenic cyst
40/M
Mandible
2.0
No association
Surgical excision
77
No
Failed
9
Dentigerous cyst
55/M
Mandible
2.8
Impacted 3rd molar, L
Surgical excision
4
No
0
10
Dentigerous cyst
62/M
Mandible
2.5
Impacted 3rd molar, L
Surgical excision
4
No
0
© 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
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Table 2. Clinicopathological characteristics and MAML2 rearrangement status of intraosseous mucoepidermoid carcinomas
No
Age/Sex
Site
Size (cm)
Treatment
Follow up (months)
Outcome
MAML2 status (%)
1
20/F
Mandible
7.0
En-bloc resection
14
NED
47
2
60/F
Mandible
1.0
En-bloc resection
75
NED
26
3
85/F
Mandible
2.5
NA
NA
NA
61
NED, no evidence of disease; NA, non available.
graded alcohols (70%, 85% and 100%; 2 min each), and air-dried. Dual-colour FISH was performed using a MAML2 break-apart probe (BAC clone RP111056O10-, CTD-2325K3- and RP11-8N17-labelled Spectrum Orange; and CTD-2554I7-, CTD-2252L1-, RP11-1123F20-, RP11-936C10- and RP11-7D4labelled Spectrum Green). Slides were hybridized at 80°C for 5 min, and held at 37°C overnight. The next day, slides were washed in 76°C 0.1% Nonidet-P40 in 92 sodium chloride–sodium citrate buffer (3 min) and counterstained with Vectashield mounting medium containing 40 ,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Analysis was performed using a Leica DM6000 microscope (Leica Microsystems, Buffalo Grove, IL, USA), and images were captured using CytoVision software (Genetix, Sunnyvale, CA, USA). FISH INTERPRETATION
For each case, approximately 100 cells were analysed in the targeted region. Only individual and well-delineated cells were scored. Significantly overlapping cells were excluded from the analysis. Normal cells without MAML2 rearrangement show a pair of green and orange signals in juxtaposition, typically resulting in two yellow conglomerate signals. Cells with the MAML2 gene rearrangement show one pair of signals (green and orange) in juxtaposition and one green and one orange signal separated. A previous case of major salivary gland MEC originating from the parotid gland with confirmed MAML2 rearrangement was used as a positive control.
2.15 cm (range 1.0–4.2 cm) and 3.5 cm (range 1.0– 7.0 cm), respectively. All lesions were confined to the mandible. Regarding ODCs, simple surgical excision was the treatment of choice in nine of 10 cases, while in one case marsupialization was performed. No recurrences were reported in a follow-up period ranging between 4 and 77 months (mean 17 months). Information on treatment and clinical outcome was available in two of the three IMEC cases; both were treated with en-bloc mandibulectomy, and the patients demonstrated no evidence of disease after 14 and 75 months, respectively. Among ODCs with mucous prosoplasia (Figures 1B, and 2A,B), eight were classified as dentigerous cysts, one as radicular and one as glandular odontogenic cyst. The three examples of IMECs featured prominent cystic architecture, increased numbers of mucous cells compared to their epidermoid neoplastic component, and a limited degree of cellular atypia with absence of mitotic figures. Hence, they were each classified as lowgrade MEC (Figure 1A). Invasive nests were identified in the associated connective tissue of all IMECs. FISH FINDINGS
All three IMECs exhibited MAML2 rearrangement by FISH, in 26–61% of tumour cells (Table 2, Figure 1C). Successful hybridization was observed in nine of 10 cases of ODCs with mucous prosoplasia (Table 1). Among these, two exhibited a MAML2 rearrangement in 12% and 24% of the lining epithelial cells (Figure 1D), respectively, and three showed rearrangement in 7–8% of cells (Figure 2C). The remaining four cases were negative (Figure 2D).
Results CLINICOPATHOLOGICAL CHARACTERISTICS
Discussion
Among the 10 ODCs included in the present study, eight occurred in men and two in women, resulting in a M:F ratio of 4:1; the mean age was 49.2 years (range 34–67 years). All IMECs occurred in female patients with mean age of 68.3 years (range 20– 85 years). The average size of ODCs and IMECs was
Mucoepidermoid carcinoma is the most frequent malignant neoplasm of salivary glands. Molecular studies have identified MECT1–MAML2 t(11;19)(q21; p13) as an underlying pathogenetic signature in 38– 100% of MECs1,4–12 as well as in IMECs,19,29 including all three cases in the present study. The MECT1–
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A
B
C
D
Figure 1. A, Histopathological characteristics of intraosseous mucoepidermoid carcinoma. Photomicrograph of case 1 (Table 2) demonstrating aggregates of neoplastic epidermoid and mucus-secreting cells forming cystic-like spaces and solid islands embedded in a fibrocollagenous stroma (H&E). B, Histopathological features of an odontogenic cyst positive for MAML2 rearrangements. Photomicrograph of case 4 (Table 1) depicting the stratified squamous epithelium lining a dentigerous cyst and featuring foci of mucous prosoplasia. Microcilia formation is also present (H&E). C,D, FISH analyses of (A) and (B), respectively. In (C), break-apart FISH revealed MAML2 rearrangements in 47% of the neoplastic cells of this IMEC, as confirmed by the numerous split red–green signals indicative of disruption. In (D) MAML2 rearrangements were observed in 24% of the cystic lining epithelial cells.
MAML2 fusion gene is composed of the N-terminal CREB protein-binding domain (exon 1) of MECT1 juxtaposed to the C-terminal transcriptional activation domain (exons 2–5) of the Notch coactivator MAML2.3,30,31 Hence, the MECT1–MAML2 chimeric gene may function as an oncogene through the constitutive activation of both cAMP–CREB and Notch signalling targets.13–15 In fact, MAML2 rearrangements are considered to be driver alterations with a pivotal role in the initiation and maintenance of MEC tumorigenesis.29 Herein, we investigated the presence of MAML2 rearrangement by FISH in ODCs characterized by mucous prosoplasia of the lining epithelium, aiming to assess the possibility of ODCs being precursor
lesions for IMEC development. In the current study, MAML2 rearrangement was identified in more than half of ODCs. IMECs demonstrated a higher percentage of MAML2 rearrangement when compared to MAML2-positive ODCs. Interestingly, in one example of the latter, an otherwise histologically typical dentigerous cyst (case 4), MAML2 status was essentially similar to IMEC case 2. In all ODC cases included in the present study, mucous cells represented only a minority of the epithelial cells lining the cystic cavity. With the exception of one case (case 1), mucous prosoplasia was observed only focally, which is a frequently encountered and well-known phenomenon in ODCs. In case 1, the number of mucus-secreting cells was increased. This case, however, demonstrated © 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
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A
B
C
D
795
Figure 2. A,B, Histopathological characteristics of cases 6 and 3 (Table 1), respectively. A radicular (A) and dentigerous (B) cyst featuring mucous prosoplasia with numerous mucous cells (H&E). C,D, FISH analyses of (A) and (B), respectively. In (C), break-apart FISH revealed MAML2 rearrangements in 7% of the epithelial cells in this ODC. In (D), however, total absence of MAML2 rearrangement was observed (0%).
MAML2 rearrangements in only 7% of epithelial cells. Hence, the results of our study suggest that distinction between ODCs harbouring MAML2 rearrangements and ODCs negative for such genetic aberrations cannot be made on histopathological grounds, because an absence of correlation was noticed between microscopic features, such as number and distribution of mucous cells, and the MAML2 status of the lesions. In a recently published study investigating the presence of MAML2 rearrangement in 21 cases of glandular odontogenic cyst (GOC), a rare type of ODC, and five cases of IMEC, MAML2 rearrangement was identified by FISH in all malignant tumours but in none of the GOCs.32 Based on these findings, it was concluded that GOC and IMEC represent independent mechanisms of disease and that GOC should not be considered a precursor to IMEC.32 Our study pro© 2014 John Wiley & Sons Ltd, Histopathology, 66, 791–797.
vides evidence of MAML2 rearrangement in some ODCs. Based on these findings, one may hypothesize that (1) a subset of ODCs can harbour MAML2 rearrangement in the epithelial lining and can transform towards IMEC without obvious phenotypical change, or (2) ODC with MAML2 rearrangement is best regarded as cystic IMEC. The latter has therapeutic implications; i.e. additional surgical treatment for an apparently totally excised lesion. Herein, we do not advocate the performance of FISH to assess the MAML2 rearrangement status in ODCs with mucous cells in their epithelial lining. This would be impractical, given the current state of laboratory affairs. It is also well known that mucous prosoplasia in what appear to be conventional ODCs is far more frequent than IMECs. Furthermore, it is plausible that a gradual increase in genomic instability in the epithelial lining of ODCs, either through additional MAML2
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rearrangements or alterations in various tumour suppressor genes such as DCC, SMAD4, GALR1 and CDKN2A/B, may lead to malignant transformation and acquisition of the MEC phenotype.10 In summary, the presence of MAML2 rearrangements in the lining of ODCs demonstrates the importance of using cytogenetic and molecular techniques to shed light on the pathogenesis of IMEC. It appears that at least a subset of cases that qualify as ODC using conventional histopathological criteria are characterized by early molecular events leading towards neoplastic transformation. Our results should be confirmed by additional larger-scale studies that include the investigation of other genes, such as DCC, SMAD4, GALR1 and CDKN2A/B.
9.
10.
11.
12. 13.
14.
Acknowledgements The authors are indebted to Mr Brian Dunnette (University of Minnesota) for his assistance with the illustrations.
Conflict of interest
15.
16.
17.
The authors declare no potential conflicts of interest.
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