Histopathology 2015, 67, 410–415. DOI: 10.1111/his.12655
CASE REPORT
Genomic copy number alterations of primary and secondary metastasizing pleomorphic adenomas Fernanda Viviane Mariano, Rogerio de Oliveira Gondak,1 Antonio Santos Martins,2 Ricardo Della Coletta,3 Oslei Paes de Almeida,3 Luiz Paulo Kowalski,4 Ana Cristina Victorino Krepischi5 & Albina Altemani Pathology Department, Faculty of Medicine, State University of Campinas (UNICAMP), Campinas, Brazil, 1Pathology Department, Faculty of Santa Catarina (UFSC), Florianopolis, Brazil, 2Head and Neck Surgery Department, Faculty of Medicine, UNICAMP, Campinas, Brazil, 3Oral Pathology Department, Piracicaba Dental School, UNICAMP, Piracicaba, Brazil, 4Head and Neck Surgery Department, AC Camargo Cancer Center, S~ao Paulo, Brazil, and 5 Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of S~ao Paulo, S~ao Paulo, Brazil Date of submission 27 October 2014 Accepted for publication 13 January 2015 Published online Article Accepted 20 January 2015
Mariano F V, Gondak R O, Martins A S, Coletta R D, Almeida O P, Kowalski L P, Krepischi A C V & Altemani A (2015) Histopathology 67, 410–415. DOI: 10.1111/his.12655
Genomic copy number alterations of primary and secondary metastasizing pleomorphic adenomas Aims: Metastasizing pleomorphic adenoma (MPA) is a rare tumour, and its mechanism of metastasis still is unknown. To date, there has been no study on MPA genomics. We analysed primary and secondary MPAs with array comparative genomic hybridization to identify somatic copy number alterations and affected genes. Methods and results: Tumour DNA samples from primary (parotid salivary gland) and secondary (scalp skin) MPAs were subjected to array comparative genomic hybridization investigation, and the data were analysed with NEXUS COPY NUMBER DISCOVERY. The primary MPA showed copy number losses affecting 3p22.2p14.3 and 19p13.3p123, and a complex pattern of four different deletions at chromosome
6. The 3p deletion encompassed several genes: CTNNB1, SETD2, BAP1, and PBRM1, among others. The secondary MPA showed a genomic profile similar to that of the primary MPA, with acquisition of additional copy number changes affecting 9p24.3p13.1 (loss), 19q11q13.43 (gain), and 22q11.1q13.33 (gain). Conclusion: Our findings indicated a clonal origin of the secondary MPA, as both tumours shared a common profile of genomic copy number alterations. Furthermore, we were able to detect in the primary tumour a specific pattern of copy number alterations that could explain the metastasizing characteristic, whereas the secondary MPA showed a more unbalanced genome.
Keywords: array comparative genomic hybridization, metastasizing pleomorphic adenoma, somatic copy number alterations
Introduction *Address for correspondence: F V Mariano, Departamento de Anatomia Patol ogica, Faculdade de Ci^encias Medicas, Universidade Estadual de Campinas, Rua Tessalia Vieira de Camargo, 126, Campinas, S~ ao Paulo, Brazil. e-mail: [email protected] © 2015 John Wiley & Sons Ltd.
Three types of malignancy have been reported to be associated with pleomorphic adenoma (PA): carcinoma ex-pleomorphic adenoma (CXPA), carcinosarcoma, and metastasizing pleomorphic adenoma
Metastasizing pleomorphic adenoma
(MPA). The last of these is the least common, and to date only 50 MPA cases have been reported.1–3 The mechanism of the metastatic behaviour of MPA is still unknown, and it is speculated that the accumulation of key genetic alterations drives cancer progression, which is not always accompanied by histological changes.4 Chromosomal rearrangements and genomic copy number alterations have been reported in CXAP and PA,2,5–11 but there are few studies on genomic alterations in MPAs, and they have failed to provide additional criteria that could predict MPA development. Herein, we present an array comparative genomic hybridization analysis of two MPAs affecting the same patient, one from the parotid gland and another in scalp skin, with the aim of finding relevant somatic copy number alterations.
411
presence of PA. The tumour was excised and diagnosed as PA (Figure 1A,B). Three years later, the patient was readmitted to the hospital with a nodule (20 9 20 mm) in the scalp skin. The patient was subjected to a lesion resection with a diagnosis of PA (Figure 1C,D). After histopathological review, the final diagnosis of the parotid gland tumour was primary MPA and secondary MPA (scalp skin tumour). The DNA tumour was extracted from paraffinembedded tissue with an extraction kit (Qiagen, Hilden, Germany). Five hundred nanograms of test and reference DNA were co-hybridized with a 180 000oligonucleotide array (SurePrint G3 Human CGH, design 22060; Agilent Technologies, Palo Alto, CA, USA). The current study was carried out in accordance with the ethical guidelines of our institution (CEP/ FCM-1155/2011; 22 November 2011).
Patients and methods A 65-year-old woman was referred to the Clinics Hospital of the State University of Campinas for evaluation of a nodule in the left parotid gland (35 9 20 mm), with no palpable cervical lymph nodes, and an absence of oral lesions. A fine needle aspiration biopsy was performed, and showed the
Results All detected chromosomal alterations are described in Table 1, and affected known cancer genes according to Cancer Gene Census Sanger are indicated. The primary MPA showed five copy number losses, affecting chromosomes 3 and 6, and a gain affecting
A
B
C
D
Figure 1. A, B, Pleomophic adenoma in the parotid gland or primary metastasizing pleomorphic adenoma (MPA). A, A well-demarcated lesion showing an admixture of epithelial, myoepithelial and stromal components [haematoxylin and eosin (H&E)]. B, The tumour shows a variety of growth patterns, including ductal structures composed of epithelial and myoepithelial cells without atypia cellular and myxochondroid stroma (H&E). C, D, Pleomorphic adenoma in the scalp skin or secondary MPA. C, The tumour in the scalp is surrounded by hairy follicles, and shows a similar appearance to the tumour of the parotid gland (H&E). D, Ductal structures with epithelial and myoepithelial cells showing benign features (H&E). © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
412
F V Mariano et al.
Table 1. Somatic copy number alterations identified in the primary and secondary metastasizing pleomorphic adenomas (MPAs) Gene (n)
Cancer genes according to CGCS
3p22.2p14.3
302
CTNNB1, SETD2, BAP1, PBRM1
2.8
6p25.3p25.2
13
Loss
1.4
6p12.1p11.2
9
–
chr6: 61 000 000–67 633 594
Loss
6.6
6q11.1q12
7
–
chr6: 73 197 185–171 115 067
Loss
97.9
6q13q27
489
PRDM1, ROS1, GOPC, STL, MYB, TNFAIP3, ECT2L, EZR, FGFR1OP, MLLT4
chr19: 242 133–24 358 623
Gain
24
19p13.3p11
657
FSTL3, STK11, TCF3, GNA11, SH3GL1, MLLT1, DNM2, SMARCA4, LYL1, BRD4, TPM4, JAK3, ELL
chr6: 0–2 851 685
Loss
2.8
6p25.3p25.2
14
chr6: 55 741 179–57 188 081
Loss
1.4
6p12.1p11.2
9
–
chr6: 61 000 000–67 633 594
Loss
6.6
6q11.1q12
7
–
chr6: 73 942 580–171 115 067
Loss
97
6q13q27
485
PRDM1, ROS1, GOPC, STL, MYB, TNFAIP3, ECT2L, EZR, FGFR1OP, MLLT4
chr9: 115 654–38 628 408
Loss
38
9p24.3p13.1
238
JAK2, CD274, NFIB, MLLT3, FANCG, PAX5
chr19: 145 280–24 407 049
Gain
24
19p13.3p11
658
FSTL3, STK11, TCF3, GNA11, SH3GL1, MLLT1, DNM2, SMARCA4, LYL1, BRD4, TPM4, JAK3, ELL
chr19: 28 184 331–59 128 983
Gain
30.9
19q11q13.43
1078
chr22: 17 353 633–51 262 548
Gain
33
22q11.1q13.33
Tumour site
Chromosome coordinates (Hg19)
CN event
Size (Mb)
Cytoband
Primary MPA (salivary gland)
chr3: 38 709 079–58 440 513
Loss
19.7
chr6: 0–2 801 338
Loss
chr6: 55 741 179–57 188 081
Secondary MPA (scalp)
542
IRF4
IRF4
CCNE1, CEBPA, AKT2, CD79A, CIC, BCL3, CBLC, ERCC2, KLK2, PPP2R1A, ZNF331, TFPT CLTCL1, BCR, SMARCB1, MN1, CHEK2, EWSR1, NF2, MYH9, PDGFB, MKL1, EP300
ch, Chromosome; CN, Copy number; CGCS, Cancer Gene Census Sanger (https://www.sanger.ac.uk/research/projects/ cancergenome/census.html). The chromosomal changes exclusively detected in the secondary MPA are highlighted in bold.
chromosome 19p (Figure 2A). The 19.7-Mb 3p22.2p14.3 deletion encompassed several genes, among them CTNNB1, SETD2, BAP1, and PBRM1. Chromosome 6 showed a complex pattern of deletions affecting the short and long arms (three of the four deletions were small, submicroscopic alterations, and
one was a large 97-Mb deletion at 6q13q27) (Figure 2C). The secondary MPA showed the chromosome 6 rearrangements and the 19p gain identified in the primary tumour, and additional copy number changes affecting chromosomes 9, 19, and 22. The © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
Metastasizing pleomorphic adenoma
A
B
C
D
413
Figure 2. A, B, Array comparative genomic hybridization genomic profiles showing somatic copy number alterations identified in primary (A) and secondary (B) metastasizing pleomorphic adenomas derived from the same patient. Images are adapted from the software NEXUS COPY NUMBER 7.0, BIODISCOVERY. All 24 human chromosomes are represented by ideograms depicting the detected copy number alterations in colour (genomic losses in red). On the right, log2 ratios of all oligoprobes are ordered along the chromosomes, with deviation to the left indicating genomic losses (deletions), and deviation to the right indicating gains. Copy number alterations common to both tumour samples are indicated by red boxes; deletions identified exclusively either in the primary or in the secondary tumour are marked by arrows and blue boxes, respectively. C, D, Chromosome 6 array comparative genomic hybridization profile. C, Chromosome 6 copy number deletions identified in the primary MPA, showing the pattern of four genomic losses: 2.8 Mb at 6p25.3p25.2, 1.4 Mb at 6p12.1p11.2, 6.6 Mb at 6q11.1q12, and 97 Mb at 6q13q27. D, Chromosome 6 profile of the secondary MPA showing the same pattern of genomic losses.
newly acquired large 38-Mb deletion at 9p24.3p13.1 involved several cancer genes, and chromosomes 19 and 22 showed aneuploidy (Figure 2B). Chromosome 6 of the secondary MPA showed the same complex pattern of alterations identified in the primary tumour (Figure 2D).
Discussion The first case of MPA was described by Foote and Frazel,12 and 49 cases have since been added to the literature.11,13–15 There is no predictive factor available to determine which PA may have the potential to metastasize. According to some studies, recurrence may constitute the first step in the dissemination and further metastasis of this tumour.16,17 In fact, MPA usually presents with multiple local recurrences (at least two) before the development of metastatic disease, but some MPAs do not show prior evidence of local recurrences,1,2 as in the current case. The time between the diagnosis of a primary PA and metastasis has ranged from 3 to 22 years.14,18 Our case showed an interval of 3 years between the primary and secondary MPAs. The exact mechanism of metastatis from MPA is not clear. It has been postulated that metastasis occurs especially when there is a rupture of the capsule during enucleation or an incomplete excision is © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
performed.18 Additionally, it has been suggested that the surgical manipulation can cause iatrogenic dislodgement of tumour cells to vascular spaces, which then metastasize;11,19–21 however, a recent study by Skalova et al.22 showed that intravascular tumour in PA is an innocuous and rare phenomenon that might be related to artefactual spillage caused by fine needle aspiration or intraoperative trauma rather than true angioinvasion, but the biological significance is not clear. In fact, no association between the intravascular tumour deposits and benign metastasizing PA has been demonstrated, and nor does there appear to be any relationship with aggressive behaviour or tumour recurrences.1,22 It has also been speculated that aspiration during removal of intraoral PA causes implantation metastatic foci in the lungs.1 Atypical features (hypercellularity, capsular violation, hyalinization, necrosis, cellular anaplasia, and mitotic rate) were indicative of initial malignant transformation to CXPA, but not in MPA.23 Even so, El-Naggar et al.13 have suggested that MPA may represent an unrecognized true malignancy. In fact, the biological behaviour (mortality rate, and the development of metastatic lesions with a delay of many years after the primary tumour) seems to justify the inclusion of MPA in the group of low-grade malignant salivary tumours.20,24
414
F V Mariano et al.
Some authors have suggested that MPA could be an intermediate link in the malignant transformation of PA to CXPA.25 Czader et al.25 pointed out that MPA and CXPA could represent different stages along a common biological pathway. They hypothesize that the metastatic capability of MPA more likely occurs secondary to the accumulation of genetic mutations. Weissferdt and Langman4 reported an intracapsular CXPA with lung metastases composed exclusively of benign elements, reinforcing the histological evidence for the continuum between MPA and CXPA. Flow-cytometric analysis revealed DNA aneuploidy in two 11 MPAs,11 but no conclusion about the predictors of metastasis was drawn. Jin et al.2 found clonal chromosomal aberrations in three MPAs in the same patient, two of them with identical cytogenetic findings: a hypodiploid clone with an unbalanced translocation involving chromosomes 1 and 13, leading to 1q gain; an unknown segment attached to 3p11; chromosome 9 and 21 translocation, resulting in loss of the two p-arms; and monosomy for chromosome 22. The third MPA was a metastasis with a similar karyotype but with additional loss of a large segment from chromosome 3, with the formation of a small marker chromosome. Our MPA samples also showed rearrangements of 3p and 9p. Deletions of 3p are frequently found in a large variety of malignant epithelial neoplasms,26 and it is possible that the loss of one or more genes associated with cancer and metastasis may be important in the metastatic process of MPA. Our case of primary MPA showed exclusively a 3p22.2p14.3 loss involving relevant cancer genes such as CTNNB1, SETD2, BAP1, and PBRM1. A similar complex pattern at chromosome 6 of four deletions in different genomic regions was detected in both primary and secondary MPAs. These losses encompassed the following cancer genes: IRF4, PRDM1, ROS1, GOPC, STL, MYB, TNFAIP3, ECT2L, EZR, FGFR1OP, and MLLT4. Additionally, both MPAs showed the same loss at 19p13.3p11 involving the cancer genes FSTL3, STK11, TCF3, GNA11, SH3GL1, MLLT1, DNM2, SMARCA4, LYL1, BRD4, TPM4, JAK3, and ELL. In the light of these findings, these deleted cancer genes might be important for PA tumorigenesis, and demonstrate the same clonal origin. Three copy number alterations were exclusive to secondary MPA: loss at 9p24.3p13.1, gain at 19q11.q13.43, and gain at 22q11.1q13.33. These alterations are associated with several cancer genes. Among the deleted genes at 9p24, we identified
NFIB, which is considered to be a recurrent translocation partner of HMGA in PA. HMGIC was identified as the target gene affected by the 12q13–15 aberrations, and ~12% of PAs harbour this event.27 Rearrangements of HMGA are characteristic of PA and most benign human mesenchymal tumours, whereas unrearranged HMGA overexpression is a feature of malignant tumours.28 This fact is in line with our findings, as no copy number alteration was found involving chromosome 12. Interestingly, gain of 8q12.1 encompassing PLAG1 was not found in the current cases, as gain or amplification of PLAG1 is a hallmark of PA.8–10 The gains of 19q11 and 22q11 include genes associated with several biological mechanisms, such as cell cycle control (CCNE1, CEBPA, TFPT, CHEK2, and PDGFB), phosphorylation of proteins (AKT2 and BRC), transcriptional mechanisms (BCL3, KLK2, ZNF331, SMARCB1, MN1, EWSR1, and EP300), DNA damage repair mechanisms (ERCC2), signalling pathways (PPP2R1A, EWSR1, and MKL1), and RNA processing and transport (EWSR1). Most of these genes are oncogenes, and all are correlated with some type of neoplasm. Thus, all findings made exclusively in secondary MPA may contribute to its tumorigenesis in this anatomical site, as well as tumour maintenance and survival. Our findings indicated a clonal origin of the secondary MPA, as the two tumours showed common genomic alterations. In addition, we detected in the primary tumour a specific pattern of copy number alterations (3p22.2p14.3 loss and a complex pattern of chromosome 6 deletions) that could explain the ability to metastasize. Additional methodological approaches should be used to provide more knowledge about this metastatic process.
Acknowledgements This study was supported by Processo FAPESP: 2011/23204-5 and Processo FAPESP: 2011/233665.
Author contributions R. Gondak: array comparative genomic hybridization reaction. D. Coletta and A. Krepischi: genomic findings review. O. Almeida and A. Altemani: histopathology diagnosis review. A. Martins and L. Kowalski: surgical resection. A. Altemani and A. Krepischi: review of the manuscript. © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
Metastasizing pleomorphic adenoma
References 1. Akiba J, Harada H, Kawahara A et al. A case of metastasizing pleomorphic adenoma in the maxillary bone appearing twenty years after initial resection of pleomorphic adenoma of the hard palate. Pathol. Int. 2013; 63; 463–468. 2. Jin Y, Jin C, Arheden K et al. Unbalanced chromosomal rearrangements in a metastasizing salivary gland tumor with benign histology. Cancer Genet. Cytogenet. 1998; 102; 59–64. 3. Gnepp DR. Metastasizing pleomorphic adenoma. In Barnes L, Eveson JW, Reichart P, Sidransky D eds. World Health Organization classification of tumours, pathology & genetics, head and neck tumours, 2nd edn. Lyon: IARC Press, 2005; 245-1. 4. Weissferdt A, Langman G. An intracapsular carcinoma ex pleomorphic adenoma with lung metastases composed exclusively of benign elements: histological evidence of a continuum between metastasizing pleomorphic adenoma and carcinoma ex-pleomorphic adenoma. Pathol. Res. Pract. 2010; 206; 480–483. 5. Voz ML, Van de Ven WJ, Kas K. First insights into the molecular basis of pleomorphic adenoma of the salivary glands. Adv. Dent. Res. 2000; 14; 81–83. 6. Tsang YT, Chang YM, Lu X et al. Amplification of MGC2177, PLAG1, PSMC6P, and LYN in a malignant mixed tumor of salivary gland detected by cDNA microarray with tyramide signal amplification. Cancer Genet. Cytogenet. 2004; 152; 124– 128. 7. Martins C, Fonseca I, Roque L et al. PLAG1 gene alterations in salivary gland pleomorphic adenoma and carcinoma ex-pleomorphic adenoma: a combined study using chromosome banding, in situ hybridization and immunocytochemistry. Mod. Pathol. 2005; 18; 1048–1055. 8. Persson F, Andren Y, Winnes M et al. High-resolution genomic profiling of adenomas and carcinomas of the salivary glands reveals amplification, rearrangement, and fusion of HMGA2. Genes Chromosom. Cancer 2009; 48; 69–82. 9. Matsuyama A, Hisaoka M, Nagao Y et al. Aberrant PLAG1 expression in pleomorphic adenomas of the salivary gland: a molecular genetic and immunohistochemical study. Virchows Arch. 2011; 458; 583–592. 10. Bahrami A, Dalton JD, Shivakumar B et al. PLAG1 alteration in carcinoma ex pleomorphic adenoma immunohistochemical and fluorescence in situ hybridization studies of 22 cases. Head Neck Pathol. 2012; 6; 328–335. 11. Wenig BM, Hitchcock CL, Ellis GL et al. Metastasizing mixed tumor of salivary glands. A clinicopathologic and flow cytometric analysis. Am. J. Surg. Pathol. 1992; 16; 845–858. 12. Foote FW, Frezell EL. Tumors of the major salivary glands. Cancer 1953; 6; 1065–1133. 13. El-Naggar AK, Batsakis JG, Kessler S. Benign metastatic mixed tumours or unrecognized salivary carcinomas? J. Laryngol. Otol. 1988; 102; 810–812.
© 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
415
14. Bradley PJ. Metastasizing pleomorphic salivary adenoma should now be considered a low-grade malignancy with a lethal potential. Curr. Opin. Otolaryngol. Head Neck Surg. 2005; 13; 123–126. 15. Nouraei SA, Ferguson MS, Clarke PM et al. Metastasizing pleomorphic salivary adenoma. Arch. Otolaryngol. Head Neck Surg. 2006; 132; 788–793. 16. Rodrıguez Fern andez J, Mateos Micas M, Martınez Tello FJ et al. Metastatic benign pleomorphic adenoma. Report of a case and review of the literature. Med. Oral Patol. Oral Cir. Bucal. 2008; 13; 193–196. 17. Santaliz-Ruiz LE, Morales G, Santini H et al. Metastasizing pleomorphic adenoma: a fascinating enigma. Case Rep. Med. 2012; 2012; 148103 5 pages. 18. Bae CH, Kim YD, Song SY. Benign pleomorphic adenoma of the soft palate metastasizing to the sphenoid sinus. Clin. Exp. Otorhinolaryngol. 2010; 3; 172–175. 19. Morrison PD, McMullin JP. A case of metastasizing benign pleomorphic adenoma of the parotid. Clin. Oncol. 1984; 10; 173–176. 20. Klijanienko J, El-Naggar AK, Servois V et al. Clinically aggressive metastasizing pleomorphic adenoma: report of two cases. Head Neck 1997; 19; 629–633. 21. Marioni G, Marino F, Stramare R et al. Benign metastasizing pleomorphic adenoma of the parotid gland: a clinicopathologic puzzle. Head Neck 2003; 25; 1071–1076. 22. Skalova A, Altemani A, Di Palma S et al. Pleomorphic adenoma of the salivary glands with intravascular tumor deposits: a diagnostic pitfall. Am. J. Surg. Pathol. 2012; 36; 167–182. 23. Auclair PL, Ellis GL. Atypical features in salivary gland mixed tumors: their relationship to malignant transformation. Mod. Pathol. 1996; 9; 652–657. 24. Tarsitano A, Foschini MP, Farneti P et al. Metastasizing benign pleomorphic salivary adenoma: a dramatic case report and literature review. J. Craniomaxillofac. Surg. 2014; 42; 1562– 1565. E-pub ahead of print. 25. Czader M, Eberhart CG, Bhatti N et al. Metastasizing mixed tumor of the parotid: initial presentation as a solitary kidney tumor and ultimate carcinomatous transformation at the primary site. Am. J. Surg. Pathol. 2000; 24; 1159–1164. 26. Mertens F, Johansson B, H€ oglund M et al. Chromosomal imbalance maps of malignant solid tumors: a cytogenetic survey of 3,185 neoplasms. Cancer Res. 1997; 57; 2765–2780. 27. Geurts JM, Schoenmakers EF, Roijer E et al. Identification of NFIB as recurrent translocation partner gene of HMGIC in pleomorphic adenomas. Oncogene 1998; 16; 865–872. 28. Fusco A, Fedele M. Roles of HMGA proteins in cancer. Nat. Rev. Cancer 2007; 7; 899–910.
CASE REPORT
Genomic copy number alterations of primary and secondary metastasizing pleomorphic adenomas Fernanda Viviane Mariano, Rogerio de Oliveira Gondak,1 Antonio Santos Martins,2 Ricardo Della Coletta,3 Oslei Paes de Almeida,3 Luiz Paulo Kowalski,4 Ana Cristina Victorino Krepischi5 & Albina Altemani Pathology Department, Faculty of Medicine, State University of Campinas (UNICAMP), Campinas, Brazil, 1Pathology Department, Faculty of Santa Catarina (UFSC), Florianopolis, Brazil, 2Head and Neck Surgery Department, Faculty of Medicine, UNICAMP, Campinas, Brazil, 3Oral Pathology Department, Piracicaba Dental School, UNICAMP, Piracicaba, Brazil, 4Head and Neck Surgery Department, AC Camargo Cancer Center, S~ao Paulo, Brazil, and 5 Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of S~ao Paulo, S~ao Paulo, Brazil Date of submission 27 October 2014 Accepted for publication 13 January 2015 Published online Article Accepted 20 January 2015
Mariano F V, Gondak R O, Martins A S, Coletta R D, Almeida O P, Kowalski L P, Krepischi A C V & Altemani A (2015) Histopathology 67, 410–415. DOI: 10.1111/his.12655
Genomic copy number alterations of primary and secondary metastasizing pleomorphic adenomas Aims: Metastasizing pleomorphic adenoma (MPA) is a rare tumour, and its mechanism of metastasis still is unknown. To date, there has been no study on MPA genomics. We analysed primary and secondary MPAs with array comparative genomic hybridization to identify somatic copy number alterations and affected genes. Methods and results: Tumour DNA samples from primary (parotid salivary gland) and secondary (scalp skin) MPAs were subjected to array comparative genomic hybridization investigation, and the data were analysed with NEXUS COPY NUMBER DISCOVERY. The primary MPA showed copy number losses affecting 3p22.2p14.3 and 19p13.3p123, and a complex pattern of four different deletions at chromosome
6. The 3p deletion encompassed several genes: CTNNB1, SETD2, BAP1, and PBRM1, among others. The secondary MPA showed a genomic profile similar to that of the primary MPA, with acquisition of additional copy number changes affecting 9p24.3p13.1 (loss), 19q11q13.43 (gain), and 22q11.1q13.33 (gain). Conclusion: Our findings indicated a clonal origin of the secondary MPA, as both tumours shared a common profile of genomic copy number alterations. Furthermore, we were able to detect in the primary tumour a specific pattern of copy number alterations that could explain the metastasizing characteristic, whereas the secondary MPA showed a more unbalanced genome.
Keywords: array comparative genomic hybridization, metastasizing pleomorphic adenoma, somatic copy number alterations
Introduction *Address for correspondence: F V Mariano, Departamento de Anatomia Patol ogica, Faculdade de Ci^encias Medicas, Universidade Estadual de Campinas, Rua Tessalia Vieira de Camargo, 126, Campinas, S~ ao Paulo, Brazil. e-mail: [email protected] © 2015 John Wiley & Sons Ltd.
Three types of malignancy have been reported to be associated with pleomorphic adenoma (PA): carcinoma ex-pleomorphic adenoma (CXPA), carcinosarcoma, and metastasizing pleomorphic adenoma
Metastasizing pleomorphic adenoma
(MPA). The last of these is the least common, and to date only 50 MPA cases have been reported.1–3 The mechanism of the metastatic behaviour of MPA is still unknown, and it is speculated that the accumulation of key genetic alterations drives cancer progression, which is not always accompanied by histological changes.4 Chromosomal rearrangements and genomic copy number alterations have been reported in CXAP and PA,2,5–11 but there are few studies on genomic alterations in MPAs, and they have failed to provide additional criteria that could predict MPA development. Herein, we present an array comparative genomic hybridization analysis of two MPAs affecting the same patient, one from the parotid gland and another in scalp skin, with the aim of finding relevant somatic copy number alterations.
411
presence of PA. The tumour was excised and diagnosed as PA (Figure 1A,B). Three years later, the patient was readmitted to the hospital with a nodule (20 9 20 mm) in the scalp skin. The patient was subjected to a lesion resection with a diagnosis of PA (Figure 1C,D). After histopathological review, the final diagnosis of the parotid gland tumour was primary MPA and secondary MPA (scalp skin tumour). The DNA tumour was extracted from paraffinembedded tissue with an extraction kit (Qiagen, Hilden, Germany). Five hundred nanograms of test and reference DNA were co-hybridized with a 180 000oligonucleotide array (SurePrint G3 Human CGH, design 22060; Agilent Technologies, Palo Alto, CA, USA). The current study was carried out in accordance with the ethical guidelines of our institution (CEP/ FCM-1155/2011; 22 November 2011).
Patients and methods A 65-year-old woman was referred to the Clinics Hospital of the State University of Campinas for evaluation of a nodule in the left parotid gland (35 9 20 mm), with no palpable cervical lymph nodes, and an absence of oral lesions. A fine needle aspiration biopsy was performed, and showed the
Results All detected chromosomal alterations are described in Table 1, and affected known cancer genes according to Cancer Gene Census Sanger are indicated. The primary MPA showed five copy number losses, affecting chromosomes 3 and 6, and a gain affecting
A
B
C
D
Figure 1. A, B, Pleomophic adenoma in the parotid gland or primary metastasizing pleomorphic adenoma (MPA). A, A well-demarcated lesion showing an admixture of epithelial, myoepithelial and stromal components [haematoxylin and eosin (H&E)]. B, The tumour shows a variety of growth patterns, including ductal structures composed of epithelial and myoepithelial cells without atypia cellular and myxochondroid stroma (H&E). C, D, Pleomorphic adenoma in the scalp skin or secondary MPA. C, The tumour in the scalp is surrounded by hairy follicles, and shows a similar appearance to the tumour of the parotid gland (H&E). D, Ductal structures with epithelial and myoepithelial cells showing benign features (H&E). © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
412
F V Mariano et al.
Table 1. Somatic copy number alterations identified in the primary and secondary metastasizing pleomorphic adenomas (MPAs) Gene (n)
Cancer genes according to CGCS
3p22.2p14.3
302
CTNNB1, SETD2, BAP1, PBRM1
2.8
6p25.3p25.2
13
Loss
1.4
6p12.1p11.2
9
–
chr6: 61 000 000–67 633 594
Loss
6.6
6q11.1q12
7
–
chr6: 73 197 185–171 115 067
Loss
97.9
6q13q27
489
PRDM1, ROS1, GOPC, STL, MYB, TNFAIP3, ECT2L, EZR, FGFR1OP, MLLT4
chr19: 242 133–24 358 623
Gain
24
19p13.3p11
657
FSTL3, STK11, TCF3, GNA11, SH3GL1, MLLT1, DNM2, SMARCA4, LYL1, BRD4, TPM4, JAK3, ELL
chr6: 0–2 851 685
Loss
2.8
6p25.3p25.2
14
chr6: 55 741 179–57 188 081
Loss
1.4
6p12.1p11.2
9
–
chr6: 61 000 000–67 633 594
Loss
6.6
6q11.1q12
7
–
chr6: 73 942 580–171 115 067
Loss
97
6q13q27
485
PRDM1, ROS1, GOPC, STL, MYB, TNFAIP3, ECT2L, EZR, FGFR1OP, MLLT4
chr9: 115 654–38 628 408
Loss
38
9p24.3p13.1
238
JAK2, CD274, NFIB, MLLT3, FANCG, PAX5
chr19: 145 280–24 407 049
Gain
24
19p13.3p11
658
FSTL3, STK11, TCF3, GNA11, SH3GL1, MLLT1, DNM2, SMARCA4, LYL1, BRD4, TPM4, JAK3, ELL
chr19: 28 184 331–59 128 983
Gain
30.9
19q11q13.43
1078
chr22: 17 353 633–51 262 548
Gain
33
22q11.1q13.33
Tumour site
Chromosome coordinates (Hg19)
CN event
Size (Mb)
Cytoband
Primary MPA (salivary gland)
chr3: 38 709 079–58 440 513
Loss
19.7
chr6: 0–2 801 338
Loss
chr6: 55 741 179–57 188 081
Secondary MPA (scalp)
542
IRF4
IRF4
CCNE1, CEBPA, AKT2, CD79A, CIC, BCL3, CBLC, ERCC2, KLK2, PPP2R1A, ZNF331, TFPT CLTCL1, BCR, SMARCB1, MN1, CHEK2, EWSR1, NF2, MYH9, PDGFB, MKL1, EP300
ch, Chromosome; CN, Copy number; CGCS, Cancer Gene Census Sanger (https://www.sanger.ac.uk/research/projects/ cancergenome/census.html). The chromosomal changes exclusively detected in the secondary MPA are highlighted in bold.
chromosome 19p (Figure 2A). The 19.7-Mb 3p22.2p14.3 deletion encompassed several genes, among them CTNNB1, SETD2, BAP1, and PBRM1. Chromosome 6 showed a complex pattern of deletions affecting the short and long arms (three of the four deletions were small, submicroscopic alterations, and
one was a large 97-Mb deletion at 6q13q27) (Figure 2C). The secondary MPA showed the chromosome 6 rearrangements and the 19p gain identified in the primary tumour, and additional copy number changes affecting chromosomes 9, 19, and 22. The © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
Metastasizing pleomorphic adenoma
A
B
C
D
413
Figure 2. A, B, Array comparative genomic hybridization genomic profiles showing somatic copy number alterations identified in primary (A) and secondary (B) metastasizing pleomorphic adenomas derived from the same patient. Images are adapted from the software NEXUS COPY NUMBER 7.0, BIODISCOVERY. All 24 human chromosomes are represented by ideograms depicting the detected copy number alterations in colour (genomic losses in red). On the right, log2 ratios of all oligoprobes are ordered along the chromosomes, with deviation to the left indicating genomic losses (deletions), and deviation to the right indicating gains. Copy number alterations common to both tumour samples are indicated by red boxes; deletions identified exclusively either in the primary or in the secondary tumour are marked by arrows and blue boxes, respectively. C, D, Chromosome 6 array comparative genomic hybridization profile. C, Chromosome 6 copy number deletions identified in the primary MPA, showing the pattern of four genomic losses: 2.8 Mb at 6p25.3p25.2, 1.4 Mb at 6p12.1p11.2, 6.6 Mb at 6q11.1q12, and 97 Mb at 6q13q27. D, Chromosome 6 profile of the secondary MPA showing the same pattern of genomic losses.
newly acquired large 38-Mb deletion at 9p24.3p13.1 involved several cancer genes, and chromosomes 19 and 22 showed aneuploidy (Figure 2B). Chromosome 6 of the secondary MPA showed the same complex pattern of alterations identified in the primary tumour (Figure 2D).
Discussion The first case of MPA was described by Foote and Frazel,12 and 49 cases have since been added to the literature.11,13–15 There is no predictive factor available to determine which PA may have the potential to metastasize. According to some studies, recurrence may constitute the first step in the dissemination and further metastasis of this tumour.16,17 In fact, MPA usually presents with multiple local recurrences (at least two) before the development of metastatic disease, but some MPAs do not show prior evidence of local recurrences,1,2 as in the current case. The time between the diagnosis of a primary PA and metastasis has ranged from 3 to 22 years.14,18 Our case showed an interval of 3 years between the primary and secondary MPAs. The exact mechanism of metastatis from MPA is not clear. It has been postulated that metastasis occurs especially when there is a rupture of the capsule during enucleation or an incomplete excision is © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
performed.18 Additionally, it has been suggested that the surgical manipulation can cause iatrogenic dislodgement of tumour cells to vascular spaces, which then metastasize;11,19–21 however, a recent study by Skalova et al.22 showed that intravascular tumour in PA is an innocuous and rare phenomenon that might be related to artefactual spillage caused by fine needle aspiration or intraoperative trauma rather than true angioinvasion, but the biological significance is not clear. In fact, no association between the intravascular tumour deposits and benign metastasizing PA has been demonstrated, and nor does there appear to be any relationship with aggressive behaviour or tumour recurrences.1,22 It has also been speculated that aspiration during removal of intraoral PA causes implantation metastatic foci in the lungs.1 Atypical features (hypercellularity, capsular violation, hyalinization, necrosis, cellular anaplasia, and mitotic rate) were indicative of initial malignant transformation to CXPA, but not in MPA.23 Even so, El-Naggar et al.13 have suggested that MPA may represent an unrecognized true malignancy. In fact, the biological behaviour (mortality rate, and the development of metastatic lesions with a delay of many years after the primary tumour) seems to justify the inclusion of MPA in the group of low-grade malignant salivary tumours.20,24
414
F V Mariano et al.
Some authors have suggested that MPA could be an intermediate link in the malignant transformation of PA to CXPA.25 Czader et al.25 pointed out that MPA and CXPA could represent different stages along a common biological pathway. They hypothesize that the metastatic capability of MPA more likely occurs secondary to the accumulation of genetic mutations. Weissferdt and Langman4 reported an intracapsular CXPA with lung metastases composed exclusively of benign elements, reinforcing the histological evidence for the continuum between MPA and CXPA. Flow-cytometric analysis revealed DNA aneuploidy in two 11 MPAs,11 but no conclusion about the predictors of metastasis was drawn. Jin et al.2 found clonal chromosomal aberrations in three MPAs in the same patient, two of them with identical cytogenetic findings: a hypodiploid clone with an unbalanced translocation involving chromosomes 1 and 13, leading to 1q gain; an unknown segment attached to 3p11; chromosome 9 and 21 translocation, resulting in loss of the two p-arms; and monosomy for chromosome 22. The third MPA was a metastasis with a similar karyotype but with additional loss of a large segment from chromosome 3, with the formation of a small marker chromosome. Our MPA samples also showed rearrangements of 3p and 9p. Deletions of 3p are frequently found in a large variety of malignant epithelial neoplasms,26 and it is possible that the loss of one or more genes associated with cancer and metastasis may be important in the metastatic process of MPA. Our case of primary MPA showed exclusively a 3p22.2p14.3 loss involving relevant cancer genes such as CTNNB1, SETD2, BAP1, and PBRM1. A similar complex pattern at chromosome 6 of four deletions in different genomic regions was detected in both primary and secondary MPAs. These losses encompassed the following cancer genes: IRF4, PRDM1, ROS1, GOPC, STL, MYB, TNFAIP3, ECT2L, EZR, FGFR1OP, and MLLT4. Additionally, both MPAs showed the same loss at 19p13.3p11 involving the cancer genes FSTL3, STK11, TCF3, GNA11, SH3GL1, MLLT1, DNM2, SMARCA4, LYL1, BRD4, TPM4, JAK3, and ELL. In the light of these findings, these deleted cancer genes might be important for PA tumorigenesis, and demonstrate the same clonal origin. Three copy number alterations were exclusive to secondary MPA: loss at 9p24.3p13.1, gain at 19q11.q13.43, and gain at 22q11.1q13.33. These alterations are associated with several cancer genes. Among the deleted genes at 9p24, we identified
NFIB, which is considered to be a recurrent translocation partner of HMGA in PA. HMGIC was identified as the target gene affected by the 12q13–15 aberrations, and ~12% of PAs harbour this event.27 Rearrangements of HMGA are characteristic of PA and most benign human mesenchymal tumours, whereas unrearranged HMGA overexpression is a feature of malignant tumours.28 This fact is in line with our findings, as no copy number alteration was found involving chromosome 12. Interestingly, gain of 8q12.1 encompassing PLAG1 was not found in the current cases, as gain or amplification of PLAG1 is a hallmark of PA.8–10 The gains of 19q11 and 22q11 include genes associated with several biological mechanisms, such as cell cycle control (CCNE1, CEBPA, TFPT, CHEK2, and PDGFB), phosphorylation of proteins (AKT2 and BRC), transcriptional mechanisms (BCL3, KLK2, ZNF331, SMARCB1, MN1, EWSR1, and EP300), DNA damage repair mechanisms (ERCC2), signalling pathways (PPP2R1A, EWSR1, and MKL1), and RNA processing and transport (EWSR1). Most of these genes are oncogenes, and all are correlated with some type of neoplasm. Thus, all findings made exclusively in secondary MPA may contribute to its tumorigenesis in this anatomical site, as well as tumour maintenance and survival. Our findings indicated a clonal origin of the secondary MPA, as the two tumours showed common genomic alterations. In addition, we detected in the primary tumour a specific pattern of copy number alterations (3p22.2p14.3 loss and a complex pattern of chromosome 6 deletions) that could explain the ability to metastasize. Additional methodological approaches should be used to provide more knowledge about this metastatic process.
Acknowledgements This study was supported by Processo FAPESP: 2011/23204-5 and Processo FAPESP: 2011/233665.
Author contributions R. Gondak: array comparative genomic hybridization reaction. D. Coletta and A. Krepischi: genomic findings review. O. Almeida and A. Altemani: histopathology diagnosis review. A. Martins and L. Kowalski: surgical resection. A. Altemani and A. Krepischi: review of the manuscript. © 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
Metastasizing pleomorphic adenoma
References 1. Akiba J, Harada H, Kawahara A et al. A case of metastasizing pleomorphic adenoma in the maxillary bone appearing twenty years after initial resection of pleomorphic adenoma of the hard palate. Pathol. Int. 2013; 63; 463–468. 2. Jin Y, Jin C, Arheden K et al. Unbalanced chromosomal rearrangements in a metastasizing salivary gland tumor with benign histology. Cancer Genet. Cytogenet. 1998; 102; 59–64. 3. Gnepp DR. Metastasizing pleomorphic adenoma. In Barnes L, Eveson JW, Reichart P, Sidransky D eds. World Health Organization classification of tumours, pathology & genetics, head and neck tumours, 2nd edn. Lyon: IARC Press, 2005; 245-1. 4. Weissferdt A, Langman G. An intracapsular carcinoma ex pleomorphic adenoma with lung metastases composed exclusively of benign elements: histological evidence of a continuum between metastasizing pleomorphic adenoma and carcinoma ex-pleomorphic adenoma. Pathol. Res. Pract. 2010; 206; 480–483. 5. Voz ML, Van de Ven WJ, Kas K. First insights into the molecular basis of pleomorphic adenoma of the salivary glands. Adv. Dent. Res. 2000; 14; 81–83. 6. Tsang YT, Chang YM, Lu X et al. Amplification of MGC2177, PLAG1, PSMC6P, and LYN in a malignant mixed tumor of salivary gland detected by cDNA microarray with tyramide signal amplification. Cancer Genet. Cytogenet. 2004; 152; 124– 128. 7. Martins C, Fonseca I, Roque L et al. PLAG1 gene alterations in salivary gland pleomorphic adenoma and carcinoma ex-pleomorphic adenoma: a combined study using chromosome banding, in situ hybridization and immunocytochemistry. Mod. Pathol. 2005; 18; 1048–1055. 8. Persson F, Andren Y, Winnes M et al. High-resolution genomic profiling of adenomas and carcinomas of the salivary glands reveals amplification, rearrangement, and fusion of HMGA2. Genes Chromosom. Cancer 2009; 48; 69–82. 9. Matsuyama A, Hisaoka M, Nagao Y et al. Aberrant PLAG1 expression in pleomorphic adenomas of the salivary gland: a molecular genetic and immunohistochemical study. Virchows Arch. 2011; 458; 583–592. 10. Bahrami A, Dalton JD, Shivakumar B et al. PLAG1 alteration in carcinoma ex pleomorphic adenoma immunohistochemical and fluorescence in situ hybridization studies of 22 cases. Head Neck Pathol. 2012; 6; 328–335. 11. Wenig BM, Hitchcock CL, Ellis GL et al. Metastasizing mixed tumor of salivary glands. A clinicopathologic and flow cytometric analysis. Am. J. Surg. Pathol. 1992; 16; 845–858. 12. Foote FW, Frezell EL. Tumors of the major salivary glands. Cancer 1953; 6; 1065–1133. 13. El-Naggar AK, Batsakis JG, Kessler S. Benign metastatic mixed tumours or unrecognized salivary carcinomas? J. Laryngol. Otol. 1988; 102; 810–812.
© 2015 John Wiley & Sons Ltd, Histopathology, 67, 410–415.
415
14. Bradley PJ. Metastasizing pleomorphic salivary adenoma should now be considered a low-grade malignancy with a lethal potential. Curr. Opin. Otolaryngol. Head Neck Surg. 2005; 13; 123–126. 15. Nouraei SA, Ferguson MS, Clarke PM et al. Metastasizing pleomorphic salivary adenoma. Arch. Otolaryngol. Head Neck Surg. 2006; 132; 788–793. 16. Rodrıguez Fern andez J, Mateos Micas M, Martınez Tello FJ et al. Metastatic benign pleomorphic adenoma. Report of a case and review of the literature. Med. Oral Patol. Oral Cir. Bucal. 2008; 13; 193–196. 17. Santaliz-Ruiz LE, Morales G, Santini H et al. Metastasizing pleomorphic adenoma: a fascinating enigma. Case Rep. Med. 2012; 2012; 148103 5 pages. 18. Bae CH, Kim YD, Song SY. Benign pleomorphic adenoma of the soft palate metastasizing to the sphenoid sinus. Clin. Exp. Otorhinolaryngol. 2010; 3; 172–175. 19. Morrison PD, McMullin JP. A case of metastasizing benign pleomorphic adenoma of the parotid. Clin. Oncol. 1984; 10; 173–176. 20. Klijanienko J, El-Naggar AK, Servois V et al. Clinically aggressive metastasizing pleomorphic adenoma: report of two cases. Head Neck 1997; 19; 629–633. 21. Marioni G, Marino F, Stramare R et al. Benign metastasizing pleomorphic adenoma of the parotid gland: a clinicopathologic puzzle. Head Neck 2003; 25; 1071–1076. 22. Skalova A, Altemani A, Di Palma S et al. Pleomorphic adenoma of the salivary glands with intravascular tumor deposits: a diagnostic pitfall. Am. J. Surg. Pathol. 2012; 36; 167–182. 23. Auclair PL, Ellis GL. Atypical features in salivary gland mixed tumors: their relationship to malignant transformation. Mod. Pathol. 1996; 9; 652–657. 24. Tarsitano A, Foschini MP, Farneti P et al. Metastasizing benign pleomorphic salivary adenoma: a dramatic case report and literature review. J. Craniomaxillofac. Surg. 2014; 42; 1562– 1565. E-pub ahead of print. 25. Czader M, Eberhart CG, Bhatti N et al. Metastasizing mixed tumor of the parotid: initial presentation as a solitary kidney tumor and ultimate carcinomatous transformation at the primary site. Am. J. Surg. Pathol. 2000; 24; 1159–1164. 26. Mertens F, Johansson B, H€ oglund M et al. Chromosomal imbalance maps of malignant solid tumors: a cytogenetic survey of 3,185 neoplasms. Cancer Res. 1997; 57; 2765–2780. 27. Geurts JM, Schoenmakers EF, Roijer E et al. Identification of NFIB as recurrent translocation partner gene of HMGIC in pleomorphic adenomas. Oncogene 1998; 16; 865–872. 28. Fusco A, Fedele M. Roles of HMGA proteins in cancer. Nat. Rev. Cancer 2007; 7; 899–910.
