- Email: [email protected]
Mutational status of IDH1 in uveal melanoma
Experimental and Molecular Pathology 100 (2016) 476–481
Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Mutational status of IDH1 in uveal melanoma☆ Patrick J. Cimino a,⁎, Yungtai Kung b, Joshua I. Warrick c, Shu-Hong Chang b, C. Dirk Keene a a b c
Department of Pathology, Division of Neuropathology, University of Washington School of Medicine and Harborview Medical Center, Seattle, WA, United States Department of Ophthalmology, University of Washington School of Medicine and Harborview Medical Center, Seattle, WA, United States Department of Pathology, Penn State University School of Medicine and Milton S. Hershey Medical Center, Hershey, PA, United States
a r t i c l e
i n f o
Article history: Received 17 February 2016 and in revised form 26 April 2016 Accepted 2 May 2016 Available online 04 May 2016 Keywords: Melanoma IDH1 Isocitrate dehydrogenase Uveal melanoma Ophthalmic pathology
a b s t r a c t Uveal (intraocular) melanoma is an uncommon malignancy that comprises a small percentage of all melanoma cases. While many uveal melanomas harbor mutations in the BRCA-Associated Protein 1 (BAP1) gene, the genetics of non-BAP1 associated tumors are not completely understood. Recent studies have shown that a small subset of non-uveal melanomas hold mutations in isocitrate dehydrogenase (IDH), but the mutational status of IDH in uveal melanoma is unclear. Mutations in IDH are strongly prognostic and predictive of tumor behavior in other cancers, mainly diffuse gliomas, which commonly contain the IDH1-R132H mutation. For this study, we hypothesized that uveal melanoma may contain the IDH1-R132H mutation, similar to non-uveal melanoma and other cancers. A search of our institutional pathology files identified 50 consecutive cases of uveal melanoma with additional material utilized for retrospective IDH1-R132H immunohistochemical testing. The demographics of these patients included similar ages, gender distributions, and other clinical characteristics as described in previous studies. Similarly, histological subtype distributions and the presence of high risk pathologic features were consistent with other reports. All 50 of the uveal melanoma cases demonstrated negativity for IDH1-R132H by immunohistochemistry. This rate is unlike that of non-uveal melanoma and further supports their distinct molecular oncogenic profile. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Uveal melanomas are the most common primary malignant intraocular tumors and are associated with high morbidity and mortality (Schoenfield, 2014). Uveal melanomas comprise approximately 2.9% of all melanomas, with a reported incidence of 4.3 cases per million persons in the United States (Singh and Topham, 2003). There is slight male predominance (4.9 per million) for increased risk when compared to female patients (3.7 per million) (Singh and Topham, 2003). The mean age at diagnosis is around 61.4 years, with spindle cell histology tumors occurring more frequently in younger patients (mean age 60 years) than those with epithelioid cell variants (mean age 65 years) (Andreoli et al., 2015). Treatment varies amongst cases, however most management strategies for primary uveal melanoma incorporate conservative plaque radiotherapy and/or enucleation, depending upon several clinical characteristics, including maximal tumor size (Shields and Shields, 2015). In some centers, external beam radiotherapy is substituted for plaque radiotherapy as a means for ocular conservation therapy (Andreoli et al., 2015). Systemic therapy may be offered to
☆ Grant Numbers/Source of Support: This work was supported with funds provided by the University of Washington Departments of Ophthalmology and Pathology. ⁎ Corresponding author at: Department of Pathology, Division of Neuropathology, Box 359791, Address: 325 9th Avenue, Seattle, WA 98104-2499, United States. E-mail address: [email protected] (P.J. Cimino).
http://dx.doi.org/10.1016/j.yexmp.2016.05.002 0014-4800/© 2016 Elsevier Inc. All rights reserved.
patients who demonstrate high risk clinical characteristics or genetic features via testing of Fine Needle Aspiration (FNA) biopsy or enucleation surgical samples (Shields and Shields, 2015). Several prognostic factors have been investigated over the years, however prognosis is strongly predicted by the classification and staging system set forth by the American Joint Committee on Cancer Classification (AJCC) for uveal melanoma, which is based upon tumor characteristics such as size, ciliary body involvement, and extrascleral extension (Andreoli et al., 2015; Bagger et al., 2015; Force, 2015; Shields et al., 2015). In conjunction with the clinical and pathological based AJCC classification, molecular genetic diagnostics are increasingly being incorporated to provide important prognostic information. Gene expression profiles can segregate tumors into highly prognostic categorical groups, the Class 1 (low metastatic risk) and Class 2 (high metastatic risk) tumors (Correa and Augsburger, 2016; Harbour, 2012, 2014; Onken et al., 2004; van Gils et al., 2008; Worley et al., 2007). The poor prognostic class 2 tumors correspond to those cases with the cytogenetic abnormality of monosomy chromosome 1, which occurs in approximately half of all uveal melanoma cases (Schoenfield, 2014; Tschentscher et al., 2003). At the gene level, mutations in the BRCA-Associated Protein 1 (BAP1) gene located on chromosome 3p21.1 have been frequently found and are strongly associated with the class 2 tumors (Harbour, 2014; Harbour et al., 2010). BAP1 mutational status can also be detected by immunohistochemical staining of surgical samples, which can serve as an adjunct prognostic tool in uveal melanoma (Kalirai et al., 2014;
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
Koopmans et al., 2014; van Essen et al., 2014). While somatic BAP1 mutations are frequently identified in sporadic primary and metastated uveal melanomas, persons with germline BAP1 mutations are at increased risk for multiple cancers, including uveal melanoma (Gupta et al., 2015; Harbour et al., 2010; Klebe et al., 2015; Testa et al., 2011; Wiesner et al., 2011). In addition to BAP1, other mutated recurrent genes associated with uveal melanoma include PLCB4, GNAQ, GNA11, EIF1AX, and SF3B1 (Harbour et al., 2013; Johansson et al., 2015; Martin et al., 2013; Van Raamsdonk et al., 2009, 2010). Although recently there have been significant advances in our knowledge of the molecular drivers involved in uveal melanoma oncogenesis, the molecular pathogenesis and profile of these tumors is not completely understood, especially of uveal melanomas with disomy chromosome 3 and/or wild type BAP1 gene status. In a 2011 series of non-uveal melanoma, four cases were identified to have mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes, including the mutation encoding for the mutant IDH1-R132H protein (Shibata et al., 2011). More recently, a large scale genomic study by The Cancer Genome Atlas (TCGA) reported IDH1 as being mutated in 6.2% of cutaneous melanoma cases (Cancer Genome Atlas, 2015). Furthermore, mutations of IDH1 in these TCGA cases of cutaneous melanoma are associated with a CpG island methylator phenotype (CIMP) as in other cancers. Additionally, experimental models have shown that mutant IDH1 confers growth advantage of melanoma cells in vitro and IDH2 expression influences tumor-free survival in in vivo zebrafish animal models (Lian et al., 2012; Shibata et al., 2011). The status of IDH mutations in uveal melanoma, however, has not yet been described. IDH1, and less frequently IDH2, is commonly mutated early the oncogenesis of diffuse gliomas, where it has strong prognostic and therapeutic implications (Bleeker et al., 2010; Cancer Genome Atlas Research et al., 2015; Eckel-Passow et al., 2015; Gorovets et al., 2012; Houillier et al., 2010; Jha et al., 2011; Metellus et al., 2010; Olar et al., 2015; Reuss et al., 2015a; Turcan et al., 2012; Yan et al., 2009). The most common mutation of IDH seen in gliomas, IDH1-R132H, is easily and routinely tested for in clinical neuropathology practice by immunohistochemistry (Capper et al., 2010a, 2010b; Horbinski et al., 2009; Reuss et al., 2015b). IDH mutational status has been shown in some studies to impart prognostic and predictive value in a small subset of acute myeloid leukemia (Emadi et al., 2015; Im et al., 2014; Patel et al., 2012). In a large clinical series of 2119 cases of myeloid neoplasms, IDH1 mutational status was found to have the opposite prognostic value of that of gliomas, as a subset IDH1 mutant leukemias is associated with a decreased overall survival, especially in cases with early (ancestral) gene mutations (Molenaar et al., 2015). In addition to glioma, acute myeloid leukemia, and melanoma, other cancers are emerging to also demonstrate IDH mutations, including angioblastic T-cell lymphoma, chondrosarcoma, chondroma, and thyroid carcinoma (Amary et al., 2011; Cairns et al., 2012; Murugan et al., 2010). With the discovery of IDH mutations in a small subset of non-uveal melanoma, along with the strong clinical implications of IDH mutations in gliomas and other cancers, we sought to determine if uveal melanoma, like cutaneous melanoma, harbors IDH1 mutations.
477
immunohistochemistry. Chart review was performed to obtain demographics and pertinent clinical information when possible. 2.2. IDH1-R132H immunohistochemistry Formalin-fixed paraffin-embedded (FFPE) tissue slides were prepared and immunohistochemistry (IHC) was performed by a CAP/CLIA-certified pathology core IHC lab within the University of Washington Department of Pathology. The primary antibody used was the anti-IDH1-R132H mouse monoclonal antibody clone H09 at a dilution of 1:100 (Dianova, Hamburg, Germany). Staining was performed using a Leica Bond III Fully Automated IHC and ISH Staining System (Leica Biosystems Inc., IL, USA). Antigen retrieval was performed using citric acid at pH 6.0. The final step included the Bond Polymer Refine Red Detection Kit (catalog #DS9390). Oligodendroglioma samples were used as positive external immunostaining controls. IDH1-R132H IHC stained slides were reviewed and interpreted independently by two experienced neuropathologists, PJC and CDK. 2.3. Public database query Possible somatic variants in IDH1 and IDH2 genes were queried in published datasets from the Catalog Of Somatic Mutations In Cancer (COSMIC); accessed on January 18, 2016 (http://cancer.sanger.ac.uk/ cancergenome/projects/cosmic/) (Forbes et al., 2015). Using the Cancer Browser tool, ‘eye’ tissue was selected with the subtissue selection of ‘uveal tract’. Then ‘malignant melanoma’ was chosen from the histology selection with ‘all’ subhistology cases chosen. Both IDH1 and IDH2 genes were then searched for somatic variants. 2.4. Statistics Comparisons made between groups were performed using the R statistics package (R, version 3.2.5, RProject for Statistical Computing, http://www.r-project.org/). P-values were determined by the use of the binomial test; binom.test(0,106). 3. Results 3.1. Demographics and clinical parameters
2. Material and methods
Here we report the clinical and pathologic findings for 50 consecutive cases of uveal melanoma enucleation specimens (findings summarized in Table 1). Patient age varied widely from 23 to 94 years (average age of 60.9 years). A slight male predominance in distribution was observed with 60% (30 of 50) occurring in male and 40% (20 of 50) in female patients. The laterality of melanoma cases was nearly equally represented, with involvement of the right globe in 46% (23 of 50) and the involvement of the left globe in 54% (27 of 50) of cases. The basal tumor width ranged from 0.6 to 3.0 cm in greatest dimension, with an average width of 1.4 cm. The tumor height, or thickness, ranged from 0.1 to 2.1 cm in greatest dimension, with an average height of 0.8 cm. Nineteen of the patients had long term follow up documented. Seven of these 19 patients demonstrated distant metastases at an average of 28.3 months (range of 7–60 months) post-enucleation.
2.1. Tissue and case selection
3.2. Pathology and IDH1 mutational status
Approval for the use of human subject material was granted by the Institutional Review Board for the University of Washington. A search of our institutional files identified a total of 51 consecutive enucleation specimens containing ocular melanoma spanning the years 2009 to 2012. Hematoxylin and eosin (H&E) stains performed during routine clinical work up were reviewed when available, and the remaining information was assessed from finalized pathology reports. Of these 51 patients, 50 had additional material to be used for IDH1-R132H
Gross and microscopic pathologic evaluation was performed for all 50 enucleation specimens (Table 1). A majority of tumors were centered in the choroid (Figs. 1A, B, 2A, and B), with 34% (17 of 50) involving the ciliary body (Figs. 1C and 2C). Hematoxylin and eosin (H&E)-stained sections showed that cytologically, a 58% (29 of 50) majority of the melanomas exhibited mixed epithelioid and spindle cytomorphologies. Predominantly epithelioid or predominantly spindle morphologies were seen less frequently, at rates of 18 (9 of 50) and 24% (12 of 50),
478
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
Table 1 Clinical and pathological characteristics of uveal melanoma. Age is given in years. Mitoses listed are per 40 high powered fields. Case
Age
Sex
Laterality
Location
Cytology
W (cm)
H (cm)
ESE
ONI
NVL
Mitoses
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
23 25 29 34 36 37 45 49 49 51 51 51 51 51 51 52 52 54 55 57 57 58 58 61 61 62 63 63 64 66 67 68 69 69 72 72 72 73 74 74 75 77 79 79 80 81 82 85 87 94
F F M M M M M F M F F F F F M F M F F M M M M M M M F M M M M F F M M M M M F M M F F M M M F F M F
Right Right Left Right Left Right Right Left Left Left Left Left Left Left Right Right Right Left Left Left Left Left Left Left Right Left Left Left Right Right Right Left Left Right Left Left Right Right Left Left Right Right Left Right Right Right Right Left Right Right
Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Ciliary body Ciliary body Ciliary body Choroidal Ciliochoroidal Ciliochoroidal Ciliochoroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Ciliary body Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Choroidal Choroidal Ciliochoroidal Ciliochoroidal Choroidal Ciliochoroidal Ciliochoroidal Choroidal Ciliary body Ciliochoroidal Choroidal Choroidal Choroidal Choroidal
E MES E MES S MES MES S S MES MES MES MES MES MES MES MES MES MES S MES S MES MES MES E E S MES S MES S S S S E MES MES MES MES S E MES MES E E E MES MES MES
1.0 1.1 1.1 0.6 1.5 1.5 0.5 1.5 1.4 0.9 1.0 1.1 1.2 1.5 0.8 1.1 1.6 1.4 1.6 1.3 2.5 0.8 1.5 0.8 2.5 1.2 0.6 0.9 1.7 1.8 0.7 1.3 3.0 1.0 1.4 1.5 2.4 1.3 0.8 1.3 0.9 1.5 1.5 1.5 0.6 2.9 1.0 2.7 1.0 1.5
0.5 0.6 0.9 0.2 0.7 1.2 0.3 1.1 1.0 0.6 0.5 0.5 0.8 1.3 0.6 0.6 0.6 0.6 1.6 1.6 2.0 0.4 0.6 0.4 2.1 1.2 0.2 0.2 1.0 0.8 0.1 0.6 1.5 0.5 1.0 0.9 1.7 0.4 0.8 0.4 0.5 1.5 0.7 1.5 0.4 0.4 0.1 1.5 0.2 1.0
No No No No No No No No No No No Yes No No No Yes No No No No No No No No Yes No No Yes No No No No Yes No No Yes No No No No No No No No No Yes No No No No
No No No No No No No No No No No No No No No No No No No No Yes No No No Yes No No No No No No No No No No No No No No No No No No No No No No No No Yes
No No Yes No No No No No No No No No No Yes No Yes No No No No No No No No No No No No No No No No Yes No No No Yes Yes No Yes No No No No No No Yes Yes No Yes
5 4 17 0 14 2 7 6 18 4 5 8 29 34 7 9 11 5 1 5 6 3 19 3 1 11 1 1 14 4 2 22 21 83 5 14 3 0 2 14 4 5 10 3 4 15 4 4 7 18
Abbreviations: F = Female, M = Male, E = Epithelioid, S—spindle, W = Width, H = Height, ESE = Extrascleral Extension, ONI = Optic Nerve Invasion, NVL = Nested Vascular Loops.
respectively (Fig. 2D–E). Mitotic activity was quite variable, ranging from 0 to 83 mitotic figures per 40 high powered fields, with an average of 10 mitoses per 40 high powered fields (Fig. 2F). Optic nerve invasion by neoplastic cells was rarely (6%; 3 of 50) identified microscopically,
and was never identified at the optic nerve margin of resection (Fig. 2G). Geographic necrosis was occasionally seen (Fig. 2H). Extrascleral extension of melanoma was identified in 14% (7 of 50) of enucleation specimens, with one tumor demonstrating extensive
Fig. 1. Gross pathology of uveal melanoma. Enucleation specimens demonstrating intraocular A) pigmented and B) amelanotic choroidal melanoma. C) There is occasional involvement of the ciliary body and extrascleral extension.
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
479
Fig. 2. Histopathologic characteristics of uveal melanoma. A) Low power magnification of hematoxylin and eosin-stained sections show solid growing intraocular neoplasms with a variable amount of brown melanin pigment (20× original magnification). Most of the tumors are centered in the B) choroid, with a minority involving the C) ciliary body (100× original magnification). Neoplastic cellular morphology is seen as D) epithelioid, E) spindled, or more commonly as mixed epithelioid and spindled (600× original magnification). F) Mitotic figures are variably present but may be frequent (600× original magnification). G) Melanoma invasion into the optic nerve can occur (100× original magnification). H) Bland geographic necrosis may be present (200× original magnification). I) Extrascleral extension is rarely identified and can involve the extraocular soft tissue of the orbit (100× original magnification). J) Periodic Acid-Schiff staining highlights occasional nested vascular loops (40× original magnification). K) All uveal melanomas tested are negative for the IDH1-R132H mutation by immunohistochemistry (400× original magnification). L) Representative appropriate oligodendroglioma immunohistochemistry positive control (400× original magnification).
involvement of the orbital soft tissue (Fig. 2I). Periodic Acid-Schiff (PAS) staining highlighted nested vascular loops in only 20% (10 of 50) of cases (Fig. 2J). Fifty patients had sufficient material available for IDH1R132H immunohistochemistry, and all cases were negative for the IDH1-R132H mutation (Fig. 2K). Oligodendroglioma samples served as appropriate external positive staining controls (Fig. 2L). As a validation set, gene level somatic mutations in IDH1, and IDH2, were queried in the publically available COSMIC database (Forbes et al., 2015). There were no somatic mutations found in either IDH1 (56 samples) or IDH2 (54 samples) for any of the reported uveal melanoma samples. This sequencing data is concordant with the lack of IDH1-R132H immunohistochemical reactivity in our 50 separate uveal melanoma cases. The absence of IDH1 mutations in the combined 106 total cases of uveal melanoma was statistically significant compared to cutaneous melanoma, utilizing 6.2% (the percentage of cutaneous melanomas with IDH1 mutation in the TCGA study set (Cancer Genome Atlas, 2015)) as the hypothesized probability (p = 0.0046, confidence interval 0.0–0.035; binomial test). 4. Discussion In our series of adult uveal melanoma, the demographics reflect those of several other studies. There was a slight male to female predominance (3:2) with an average age of 60.9 years at time of
enucleation. Microscopically, a majority of the tumors had mixed epithelioid and spindle histomorphology. A small subset of the cases demonstrated poor prognostic clinical features including large size and extrascleral extension. Similarly, a small proportion of cases contained poor prognostic cytoarchitectural characteristics including predominantly epithelioid cytolmorphology, high mitotic activity, and nested vascular loops. Overall, the clinical and pathological features were representative of typical uveal melanoma cohorts. As previously discussed, a majority of uveal melanoma cases are characterized by mutations in the BAP1 gene, whereas many non-uveal melanomas are characterized by BRAF-V600E mutation (Davies et al., 2002). Although the BRAF-V600E mutation is not observed in primary uveal melanoma, there is activation of the downstream MEK/ERK signaling pathway (Cohen et al., 2003; Cruz et al., 2003; Edmunds et al., 2003; Rimoldi et al., 2003). Conversely, BAP1 germline mutations have been reported to be associated with cutaneous melanoma in certain familial cases (Njauw et al., 2012; Wadt et al., 2012). While this may indicate exceedingly rare overlaps in the genetic profiles of uveal and non-uveal melanoma, they largely appear as molecularly distinct entities. For the first time, we demonstrate that all uveal melanoma tumors tested are negative for the IDH1-R132H mutation by immunohistochemistry, further supporting a distinctive molecular background when compared to non-uveal melanoma.
480
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
A possible shortcoming of this study is an absence of pediatric uveal melanoma cases in our series. Pediatric uveal melanoma is exceedingly rare, however these tumors demonstrate favorable outcomes when compared to adult cases (Al-Jamal and Kivela, 2014; Blasi et al., 2015; Dimaras et al., 2013; Kaliki et al., 2013; Pukrushpan et al., 2014; Singh et al., 2000; Sivalingam et al., 2014; Sondak and Messina, 2014; Yousef and Alkilany, 2015). Little is known about the molecular drivers of pediatric uveal melanoma, but limited data indicate unique cytogenetic profiles (Blasi et al., 2015). Given the distinctive clinical characteristics and cytogenetic profiles of pediatric uveal melanoma, it is possible that these rare tumors harbor distinct genetic mutations and may serve for future IDH-related mutational studies. Although the IDH1-R132H mutation is the most common IDH1 mutation present in diffuse gliomas, this is not the case for several other cancers. Other cancers with recurrent IDH1/2 mutations have other commonly found specific mutations including IDH2-R140Q (acute myeloid leukemia), IDH1-R132C (chondrosarcoma and cholangiocarcinoma), IDH2-G145R (gastric cancer), IDH2-R172S (Osteosarcoma), and IDH2-R172K (angioimmunoblastic T-cell lymphoma) (Molenaar et al., 2014). In our series of 50 uveal melanoma cases tested for IDH1R132H mutation by immunohistochemistry, we cannot exclude the possibility that one of these other commonly found IDH1/2 mutations is present. However, we do note that the DNA sequencing data available in COSMIC for the separate validation set does not report any of these mutations in IDH1 (n = 56) or IDH2 (n = 54). This retrospective case series study of uveal melanoma has demonstrated similar histological and clinical characteristics that have been well-established and described in several other case series and case reports. Importantly, we show a lack of IDH1-R132H mutations within this tumor, implicating unique alternative genetic events leading to tumorigenesis when compared to subsets of other non-uveal melanoma. This also suggests that uveal melanoma may not respond to IDH targeted therapies (Krell et al., 2013). In the era of genomic and precision medicine, it is hoped that this information can serve as an important negative mutational study and allow focus on other genetic molecular events for clinical risk stratification and therapeutic decision making. Disclosure/Conflict of Interest The authors declare that there are no conflicts of interest. References Al-Jamal, R.T., Kivela, T., 2014. Uveal melanoma among Finnish children and young adults. J. AAPOS 18, 61–66. Amary, M.F., et al., 2011. IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours. J. Pathol. 224, 334–343. Andreoli, M.T., et al., 2015. Epidemiological trends in uveal melanoma. Br. J. Ophthalmol. 99, 1550–1553. Bagger, M., et al., 2015. The prognostic effect of American Joint Committee on Cancer staging and genetic status in patients with choroidal and ciliary body melanoma. Invest. Ophthalmol. Vis. Sci. 56, 438–444. Blasi, M.A., et al., 2015. Unique genomic profile associated with pediatric uveal melanoma. Eur. J. Ophthalmol. 25, e31–e34. Bleeker, F.E., et al., 2010. The prognostic IDH1(R132) mutation is associated with reduced NADP+-dependent IDH activity in glioblastoma. Acta Neuropathol. 119, 487–494. Cairns, R.A., et al., 2012. IDH2 mutations are frequent in angioimmunoblastic T-cell lymphoma. Blood 119, 1901–1903. Cancer Genome Atlas, N., 2015. Genomic classification of cutaneous melanoma. Cell 161, 1681–1696. Cancer Genome Atlas Research, N., et al., 2015. Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N. Engl. J. Med. 372, 2481–2498. Capper, D., et al., 2010a. Application of mutant IDH1 antibody to differentiate diffuse glioma from nonneoplastic central nervous system lesions and therapy-induced changes. Am. J. Surg. Pathol. 34, 1199–1204. Capper, D., et al., 2010b. Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors. Brain Pathol. 20, 245–254. Cohen, Y., et al., 2003. Lack of BRAF mutation in primary uveal melanoma. Invest. Ophthalmol. Vis. Sci. 44, 2876–2878.
Correa, Z.M., Augsburger, J.J., 2016. Independent prognostic significance of gene expression profile class and largest basal diameter of posterior uveal melanomas. Am J. Ophthalmol. 162 (20–27), e1. Cruz 3rd, F., et al., 2003. Absence of BRAF and NRAS mutations in uveal melanoma. Cancer Res. 63, 5761–5766. Davies, H., et al., 2002. Mutations of the BRAF gene in human cancer. Nature 417, 949–954. Dimaras, H., et al., 2013. Molecular testing prognostic of low risk in epithelioid uveal melanoma in a child. Br. J. Ophthalmol. 97, 323–326. Eckel-Passow, J.E., et al., 2015. Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors. N. Engl. J. Med. 372, 2499–2508. Edmunds, S.C., et al., 2003. Absence of BRAF gene mutations in uveal melanomas in contrast to cutaneous melanomas. Br. J. Cancer 88, 1403–1405. Emadi, A., et al., 2015. Presence of isocitrate dehydrogenase mutations may predict clinical response to hypomethylating agents in patients with acute myeloid leukemia. Am. J. Hematol. 90, E77–E79. Forbes, S.A., et al., 2015. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res. 43, D805–D811. Force, A.O.O.T., 2015. International Validation of the American Joint Committee on Cancer's 7th Edition Classification of Uveal Melanoma. JAMA Ophthalmol. 133, 376–383. Gorovets, D., et al., 2012. IDH mutation and neuroglial developmental features define clinically distinct subclasses of lower grade diffuse astrocytic glioma. Clin. Cancer Res. 18, 2490–2501. Gupta, M.P., et al., 2015. Clinical characteristics of uveal melanoma in patients with germline BAP1 mutations. JAMA Ophthalmol. 133, 881–887. Harbour, J.W., 2012. The genetics of uveal melanoma: an emerging framework for targeted therapy. Pigment Cell Melanoma Res. 25, 171–181. Harbour, J.W., 2014. A prognostic test to predict the risk of metastasis in uveal melanoma based on a 15-gene expression profile. Methods Mol. Biol. 1102, 427–440. Harbour, J.W., et al., 2010. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 330, 1410–1413. Harbour, J.W., et al., 2013. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat. Genet. 45, 133–135. Horbinski, C., et al., 2009. Diagnostic use of IDH1/2 mutation analysis in routine clinical testing of formalin-fixed, paraffin-embedded glioma tissues. J. Neuropathol. Exp. Neurol. 68, 1319–1325. Houillier, C., et al., 2010. IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas. Neurology 75, 1560–1566. Im, A.P., et al., 2014. DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies. Leukemia 28, 1774–1783. Jha, P., et al., 2011. IDH1 mutations in gliomas: first series from a tertiary care centre in India with comprehensive review of literature. Exp. Mol. Pathol. 91, 385–393. Johansson, P., et al., 2015. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4. Oncotarget. Kaliki, S., et al., 2013. Influence of age on prognosis of young patients with uveal melanoma: a matched retrospective cohort study. Eur. J. Ophthalmol. 23, 208–216. Kalirai, H., et al., 2014. Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing. Br. J. Cancer 111, 1373–1380. Klebe, S., et al., 2015. BAP1 hereditary cancer predisposition syndrome: a case report and review of literature. Biomark. Res. 3, 14. Koopmans, A.E., et al., 2014. Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma. Mod. Pathol. 27, 1321–1330. Krell, D., et al., 2013. IDH mutations in tumorigenesis and their potential role as novel therapeutic targets. Future Oncol. 9, 1923–1935. Lian, C.G., et al., 2012. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell 150, 1135–1146. Martin, M., et al., 2013. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat. Genet. 45, 933–936. Metellus, P., et al., 2010. Absence of IDH mutation identifies a novel radiologic and molecular subtype of WHO grade II gliomas with dismal prognosis. Acta Neuropathol. 120, 719–729. Molenaar, R.J., et al., 2014. The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation. Biochim. Biophys. Acta 1846, 326–341. Molenaar, R.J., et al., 2015. Clinical and biological implications of ancestral and non-ancestral IDH1 and IDH2 mutations in myeloid neoplasms. Leukemia 29, 2134–2142. Murugan, A.K., et al., 2010. Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer. Biochem. Biophys. Res. Commun. 393, 555–559. Njauw, C.N., et al., 2012. Germline BAP1 inactivation is preferentially associated with metastatic ocular melanoma and cutaneous-ocular melanoma families. PLoS One 7, e35295. Olar, A., et al., 2015. IDH mutation status and role of WHO grade and mitotic index in overall survival in grade II–III diffuse gliomas. Acta Neuropathol. 129, 585–596. Onken, M.D., et al., 2004. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res. 64, 7205–7209. Patel, J.P., et al., 2012. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N. Engl. J. Med. 366, 1079–1089. Pukrushpan, P., et al., 2014. Congenital uveal malignant melanoma. J. AAPOS 18, 199–201. Reuss, D.E., et al., 2015a. IDH mutant diffuse and anaplastic astrocytomas have similar age at presentation and little difference in survival: a grading problem for WHO. Acta Neuropathol. 129, 867–873.
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481 Reuss, D.E., et al., 2015b. ATRX and IDH1-R132H immunohistochemistry with subsequent copy number analysis and IDH sequencing as a basis for an “integrated” diagnostic approach for adult astrocytoma, oligodendroglioma and glioblastoma. Acta Neuropathol. 129, 133–146. Rimoldi, D., et al., 2003. Lack of BRAF mutations in uveal melanoma. Cancer Res. 63, 5712–5715. Schoenfield, L., 2014. Uveal melanoma: a pathologist's perspective and review of translational developments. Adv. Anat. Pathol. 21, 138–143. Shibata, T., et al., 2011. Mutant IDH1 confers an in vivo growth in a melanoma cell line with BRAF mutation. Am. J. Pathol. 178, 1395–1402. Shields, J.A., Shields, C.L., 2015. Management of posterior uveal melanoma: past, present, and future: the 2014 Charles L. Schepens lecture. Ophthalmology 122, 414–428. Shields, C.L., et al., 2015. American Joint Committee on Cancer Classification of Uveal Melanoma (Anatomic Stage) predicts prognosis in 7731 patients: the 2013 Zimmerman Lecture. Ophthalmology 122, 1180–1186. Singh, A.D., Topham, A., 2003. Incidence of uveal melanoma in the United States: 1973–1997. Ophthalmology 110, 956–961. Singh, A.D., et al., 2000. Uveal melanoma in young patients. Arch. Ophthalmol. 118, 918–923. Sivalingam, M.D., et al., 2014. Choroidal melanoma in children: be aware of risks. J. Pediatr. Ophthalmol. Strabismus 51, e85–e88 (Online). Sondak, V.K., Messina, J.L., 2014. Unusual presentations of melanoma: melanoma of unknown primary site, melanoma arising in childhood, and melanoma arising in the eye and on mucosal surfaces. Surg. Clin. North Am. 94, 1059–1073 (ix).
481
Testa, J.R., et al., 2011. Germline BAP1 mutations predispose to malignant mesothelioma. Nat. Genet. 43, 1022–1025. Tschentscher, F., et al., 2003. Tumor classification based on gene expression profiling shows that uveal melanomas with and without monosomy 3 represent two distinct entities. Cancer Res. 63, 2578–2584. Turcan, S., et al., 2012. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479–483. van Essen, T.H., et al., 2014. Prognostic parameters in uveal melanoma and their association with BAP1 expression. Br. J. Ophthalmol. 98, 1738–1743. van Gils, W., et al., 2008. Gene expression profiling in uveal melanoma: two regions on 3p related to prognosis. Invest. Ophthalmol. Vis. Sci. 49, 4254–4262. Van Raamsdonk, C.D., et al., 2009. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 457, 599–602. Van Raamsdonk, C.D., et al., 2010. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363, 2191–2199. Wadt, K., et al., 2012. A cryptic BAP1 splice mutation in a family with uveal and cutaneous melanoma, and paraganglioma. Pigment Cell Melanoma Res. 25, 815–818. Wiesner, T., et al., 2011. Germline mutations in BAP1 predispose to melanocytic tumors. Nat. Genet. 43, 1018–1021. Worley, L.A., et al., 2007. Transcriptomic versus chromosomal prognostic markers and clinical outcome in uveal melanoma. Clin. Cancer Res. 13, 1466–1471. Yan, H., et al., 2009. IDH1 and IDH2 mutations in gliomas. N. Engl. J. Med. 360, 765–773. Yousef, Y.A., Alkilany, M., 2015. Characterization, treatment, and outcome of uveal melanoma in the first two years of life. Hematol. Oncol. Stem Cell Ther. 8, 1–5.
Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Mutational status of IDH1 in uveal melanoma☆ Patrick J. Cimino a,⁎, Yungtai Kung b, Joshua I. Warrick c, Shu-Hong Chang b, C. Dirk Keene a a b c
Department of Pathology, Division of Neuropathology, University of Washington School of Medicine and Harborview Medical Center, Seattle, WA, United States Department of Ophthalmology, University of Washington School of Medicine and Harborview Medical Center, Seattle, WA, United States Department of Pathology, Penn State University School of Medicine and Milton S. Hershey Medical Center, Hershey, PA, United States
a r t i c l e
i n f o
Article history: Received 17 February 2016 and in revised form 26 April 2016 Accepted 2 May 2016 Available online 04 May 2016 Keywords: Melanoma IDH1 Isocitrate dehydrogenase Uveal melanoma Ophthalmic pathology
a b s t r a c t Uveal (intraocular) melanoma is an uncommon malignancy that comprises a small percentage of all melanoma cases. While many uveal melanomas harbor mutations in the BRCA-Associated Protein 1 (BAP1) gene, the genetics of non-BAP1 associated tumors are not completely understood. Recent studies have shown that a small subset of non-uveal melanomas hold mutations in isocitrate dehydrogenase (IDH), but the mutational status of IDH in uveal melanoma is unclear. Mutations in IDH are strongly prognostic and predictive of tumor behavior in other cancers, mainly diffuse gliomas, which commonly contain the IDH1-R132H mutation. For this study, we hypothesized that uveal melanoma may contain the IDH1-R132H mutation, similar to non-uveal melanoma and other cancers. A search of our institutional pathology files identified 50 consecutive cases of uveal melanoma with additional material utilized for retrospective IDH1-R132H immunohistochemical testing. The demographics of these patients included similar ages, gender distributions, and other clinical characteristics as described in previous studies. Similarly, histological subtype distributions and the presence of high risk pathologic features were consistent with other reports. All 50 of the uveal melanoma cases demonstrated negativity for IDH1-R132H by immunohistochemistry. This rate is unlike that of non-uveal melanoma and further supports their distinct molecular oncogenic profile. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Uveal melanomas are the most common primary malignant intraocular tumors and are associated with high morbidity and mortality (Schoenfield, 2014). Uveal melanomas comprise approximately 2.9% of all melanomas, with a reported incidence of 4.3 cases per million persons in the United States (Singh and Topham, 2003). There is slight male predominance (4.9 per million) for increased risk when compared to female patients (3.7 per million) (Singh and Topham, 2003). The mean age at diagnosis is around 61.4 years, with spindle cell histology tumors occurring more frequently in younger patients (mean age 60 years) than those with epithelioid cell variants (mean age 65 years) (Andreoli et al., 2015). Treatment varies amongst cases, however most management strategies for primary uveal melanoma incorporate conservative plaque radiotherapy and/or enucleation, depending upon several clinical characteristics, including maximal tumor size (Shields and Shields, 2015). In some centers, external beam radiotherapy is substituted for plaque radiotherapy as a means for ocular conservation therapy (Andreoli et al., 2015). Systemic therapy may be offered to
☆ Grant Numbers/Source of Support: This work was supported with funds provided by the University of Washington Departments of Ophthalmology and Pathology. ⁎ Corresponding author at: Department of Pathology, Division of Neuropathology, Box 359791, Address: 325 9th Avenue, Seattle, WA 98104-2499, United States. E-mail address: [email protected] (P.J. Cimino).
http://dx.doi.org/10.1016/j.yexmp.2016.05.002 0014-4800/© 2016 Elsevier Inc. All rights reserved.
patients who demonstrate high risk clinical characteristics or genetic features via testing of Fine Needle Aspiration (FNA) biopsy or enucleation surgical samples (Shields and Shields, 2015). Several prognostic factors have been investigated over the years, however prognosis is strongly predicted by the classification and staging system set forth by the American Joint Committee on Cancer Classification (AJCC) for uveal melanoma, which is based upon tumor characteristics such as size, ciliary body involvement, and extrascleral extension (Andreoli et al., 2015; Bagger et al., 2015; Force, 2015; Shields et al., 2015). In conjunction with the clinical and pathological based AJCC classification, molecular genetic diagnostics are increasingly being incorporated to provide important prognostic information. Gene expression profiles can segregate tumors into highly prognostic categorical groups, the Class 1 (low metastatic risk) and Class 2 (high metastatic risk) tumors (Correa and Augsburger, 2016; Harbour, 2012, 2014; Onken et al., 2004; van Gils et al., 2008; Worley et al., 2007). The poor prognostic class 2 tumors correspond to those cases with the cytogenetic abnormality of monosomy chromosome 1, which occurs in approximately half of all uveal melanoma cases (Schoenfield, 2014; Tschentscher et al., 2003). At the gene level, mutations in the BRCA-Associated Protein 1 (BAP1) gene located on chromosome 3p21.1 have been frequently found and are strongly associated with the class 2 tumors (Harbour, 2014; Harbour et al., 2010). BAP1 mutational status can also be detected by immunohistochemical staining of surgical samples, which can serve as an adjunct prognostic tool in uveal melanoma (Kalirai et al., 2014;
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
Koopmans et al., 2014; van Essen et al., 2014). While somatic BAP1 mutations are frequently identified in sporadic primary and metastated uveal melanomas, persons with germline BAP1 mutations are at increased risk for multiple cancers, including uveal melanoma (Gupta et al., 2015; Harbour et al., 2010; Klebe et al., 2015; Testa et al., 2011; Wiesner et al., 2011). In addition to BAP1, other mutated recurrent genes associated with uveal melanoma include PLCB4, GNAQ, GNA11, EIF1AX, and SF3B1 (Harbour et al., 2013; Johansson et al., 2015; Martin et al., 2013; Van Raamsdonk et al., 2009, 2010). Although recently there have been significant advances in our knowledge of the molecular drivers involved in uveal melanoma oncogenesis, the molecular pathogenesis and profile of these tumors is not completely understood, especially of uveal melanomas with disomy chromosome 3 and/or wild type BAP1 gene status. In a 2011 series of non-uveal melanoma, four cases were identified to have mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes, including the mutation encoding for the mutant IDH1-R132H protein (Shibata et al., 2011). More recently, a large scale genomic study by The Cancer Genome Atlas (TCGA) reported IDH1 as being mutated in 6.2% of cutaneous melanoma cases (Cancer Genome Atlas, 2015). Furthermore, mutations of IDH1 in these TCGA cases of cutaneous melanoma are associated with a CpG island methylator phenotype (CIMP) as in other cancers. Additionally, experimental models have shown that mutant IDH1 confers growth advantage of melanoma cells in vitro and IDH2 expression influences tumor-free survival in in vivo zebrafish animal models (Lian et al., 2012; Shibata et al., 2011). The status of IDH mutations in uveal melanoma, however, has not yet been described. IDH1, and less frequently IDH2, is commonly mutated early the oncogenesis of diffuse gliomas, where it has strong prognostic and therapeutic implications (Bleeker et al., 2010; Cancer Genome Atlas Research et al., 2015; Eckel-Passow et al., 2015; Gorovets et al., 2012; Houillier et al., 2010; Jha et al., 2011; Metellus et al., 2010; Olar et al., 2015; Reuss et al., 2015a; Turcan et al., 2012; Yan et al., 2009). The most common mutation of IDH seen in gliomas, IDH1-R132H, is easily and routinely tested for in clinical neuropathology practice by immunohistochemistry (Capper et al., 2010a, 2010b; Horbinski et al., 2009; Reuss et al., 2015b). IDH mutational status has been shown in some studies to impart prognostic and predictive value in a small subset of acute myeloid leukemia (Emadi et al., 2015; Im et al., 2014; Patel et al., 2012). In a large clinical series of 2119 cases of myeloid neoplasms, IDH1 mutational status was found to have the opposite prognostic value of that of gliomas, as a subset IDH1 mutant leukemias is associated with a decreased overall survival, especially in cases with early (ancestral) gene mutations (Molenaar et al., 2015). In addition to glioma, acute myeloid leukemia, and melanoma, other cancers are emerging to also demonstrate IDH mutations, including angioblastic T-cell lymphoma, chondrosarcoma, chondroma, and thyroid carcinoma (Amary et al., 2011; Cairns et al., 2012; Murugan et al., 2010). With the discovery of IDH mutations in a small subset of non-uveal melanoma, along with the strong clinical implications of IDH mutations in gliomas and other cancers, we sought to determine if uveal melanoma, like cutaneous melanoma, harbors IDH1 mutations.
477
immunohistochemistry. Chart review was performed to obtain demographics and pertinent clinical information when possible. 2.2. IDH1-R132H immunohistochemistry Formalin-fixed paraffin-embedded (FFPE) tissue slides were prepared and immunohistochemistry (IHC) was performed by a CAP/CLIA-certified pathology core IHC lab within the University of Washington Department of Pathology. The primary antibody used was the anti-IDH1-R132H mouse monoclonal antibody clone H09 at a dilution of 1:100 (Dianova, Hamburg, Germany). Staining was performed using a Leica Bond III Fully Automated IHC and ISH Staining System (Leica Biosystems Inc., IL, USA). Antigen retrieval was performed using citric acid at pH 6.0. The final step included the Bond Polymer Refine Red Detection Kit (catalog #DS9390). Oligodendroglioma samples were used as positive external immunostaining controls. IDH1-R132H IHC stained slides were reviewed and interpreted independently by two experienced neuropathologists, PJC and CDK. 2.3. Public database query Possible somatic variants in IDH1 and IDH2 genes were queried in published datasets from the Catalog Of Somatic Mutations In Cancer (COSMIC); accessed on January 18, 2016 (http://cancer.sanger.ac.uk/ cancergenome/projects/cosmic/) (Forbes et al., 2015). Using the Cancer Browser tool, ‘eye’ tissue was selected with the subtissue selection of ‘uveal tract’. Then ‘malignant melanoma’ was chosen from the histology selection with ‘all’ subhistology cases chosen. Both IDH1 and IDH2 genes were then searched for somatic variants. 2.4. Statistics Comparisons made between groups were performed using the R statistics package (R, version 3.2.5, RProject for Statistical Computing, http://www.r-project.org/). P-values were determined by the use of the binomial test; binom.test(0,106). 3. Results 3.1. Demographics and clinical parameters
2. Material and methods
Here we report the clinical and pathologic findings for 50 consecutive cases of uveal melanoma enucleation specimens (findings summarized in Table 1). Patient age varied widely from 23 to 94 years (average age of 60.9 years). A slight male predominance in distribution was observed with 60% (30 of 50) occurring in male and 40% (20 of 50) in female patients. The laterality of melanoma cases was nearly equally represented, with involvement of the right globe in 46% (23 of 50) and the involvement of the left globe in 54% (27 of 50) of cases. The basal tumor width ranged from 0.6 to 3.0 cm in greatest dimension, with an average width of 1.4 cm. The tumor height, or thickness, ranged from 0.1 to 2.1 cm in greatest dimension, with an average height of 0.8 cm. Nineteen of the patients had long term follow up documented. Seven of these 19 patients demonstrated distant metastases at an average of 28.3 months (range of 7–60 months) post-enucleation.
2.1. Tissue and case selection
3.2. Pathology and IDH1 mutational status
Approval for the use of human subject material was granted by the Institutional Review Board for the University of Washington. A search of our institutional files identified a total of 51 consecutive enucleation specimens containing ocular melanoma spanning the years 2009 to 2012. Hematoxylin and eosin (H&E) stains performed during routine clinical work up were reviewed when available, and the remaining information was assessed from finalized pathology reports. Of these 51 patients, 50 had additional material to be used for IDH1-R132H
Gross and microscopic pathologic evaluation was performed for all 50 enucleation specimens (Table 1). A majority of tumors were centered in the choroid (Figs. 1A, B, 2A, and B), with 34% (17 of 50) involving the ciliary body (Figs. 1C and 2C). Hematoxylin and eosin (H&E)-stained sections showed that cytologically, a 58% (29 of 50) majority of the melanomas exhibited mixed epithelioid and spindle cytomorphologies. Predominantly epithelioid or predominantly spindle morphologies were seen less frequently, at rates of 18 (9 of 50) and 24% (12 of 50),
478
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
Table 1 Clinical and pathological characteristics of uveal melanoma. Age is given in years. Mitoses listed are per 40 high powered fields. Case
Age
Sex
Laterality
Location
Cytology
W (cm)
H (cm)
ESE
ONI
NVL
Mitoses
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
23 25 29 34 36 37 45 49 49 51 51 51 51 51 51 52 52 54 55 57 57 58 58 61 61 62 63 63 64 66 67 68 69 69 72 72 72 73 74 74 75 77 79 79 80 81 82 85 87 94
F F M M M M M F M F F F F F M F M F F M M M M M M M F M M M M F F M M M M M F M M F F M M M F F M F
Right Right Left Right Left Right Right Left Left Left Left Left Left Left Right Right Right Left Left Left Left Left Left Left Right Left Left Left Right Right Right Left Left Right Left Left Right Right Left Left Right Right Left Right Right Right Right Left Right Right
Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Ciliary body Ciliary body Ciliary body Choroidal Ciliochoroidal Ciliochoroidal Ciliochoroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Ciliary body Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Choroidal Ciliochoroidal Choroidal Choroidal Ciliochoroidal Ciliochoroidal Choroidal Ciliochoroidal Ciliochoroidal Choroidal Ciliary body Ciliochoroidal Choroidal Choroidal Choroidal Choroidal
E MES E MES S MES MES S S MES MES MES MES MES MES MES MES MES MES S MES S MES MES MES E E S MES S MES S S S S E MES MES MES MES S E MES MES E E E MES MES MES
1.0 1.1 1.1 0.6 1.5 1.5 0.5 1.5 1.4 0.9 1.0 1.1 1.2 1.5 0.8 1.1 1.6 1.4 1.6 1.3 2.5 0.8 1.5 0.8 2.5 1.2 0.6 0.9 1.7 1.8 0.7 1.3 3.0 1.0 1.4 1.5 2.4 1.3 0.8 1.3 0.9 1.5 1.5 1.5 0.6 2.9 1.0 2.7 1.0 1.5
0.5 0.6 0.9 0.2 0.7 1.2 0.3 1.1 1.0 0.6 0.5 0.5 0.8 1.3 0.6 0.6 0.6 0.6 1.6 1.6 2.0 0.4 0.6 0.4 2.1 1.2 0.2 0.2 1.0 0.8 0.1 0.6 1.5 0.5 1.0 0.9 1.7 0.4 0.8 0.4 0.5 1.5 0.7 1.5 0.4 0.4 0.1 1.5 0.2 1.0
No No No No No No No No No No No Yes No No No Yes No No No No No No No No Yes No No Yes No No No No Yes No No Yes No No No No No No No No No Yes No No No No
No No No No No No No No No No No No No No No No No No No No Yes No No No Yes No No No No No No No No No No No No No No No No No No No No No No No No Yes
No No Yes No No No No No No No No No No Yes No Yes No No No No No No No No No No No No No No No No Yes No No No Yes Yes No Yes No No No No No No Yes Yes No Yes
5 4 17 0 14 2 7 6 18 4 5 8 29 34 7 9 11 5 1 5 6 3 19 3 1 11 1 1 14 4 2 22 21 83 5 14 3 0 2 14 4 5 10 3 4 15 4 4 7 18
Abbreviations: F = Female, M = Male, E = Epithelioid, S—spindle, W = Width, H = Height, ESE = Extrascleral Extension, ONI = Optic Nerve Invasion, NVL = Nested Vascular Loops.
respectively (Fig. 2D–E). Mitotic activity was quite variable, ranging from 0 to 83 mitotic figures per 40 high powered fields, with an average of 10 mitoses per 40 high powered fields (Fig. 2F). Optic nerve invasion by neoplastic cells was rarely (6%; 3 of 50) identified microscopically,
and was never identified at the optic nerve margin of resection (Fig. 2G). Geographic necrosis was occasionally seen (Fig. 2H). Extrascleral extension of melanoma was identified in 14% (7 of 50) of enucleation specimens, with one tumor demonstrating extensive
Fig. 1. Gross pathology of uveal melanoma. Enucleation specimens demonstrating intraocular A) pigmented and B) amelanotic choroidal melanoma. C) There is occasional involvement of the ciliary body and extrascleral extension.
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
479
Fig. 2. Histopathologic characteristics of uveal melanoma. A) Low power magnification of hematoxylin and eosin-stained sections show solid growing intraocular neoplasms with a variable amount of brown melanin pigment (20× original magnification). Most of the tumors are centered in the B) choroid, with a minority involving the C) ciliary body (100× original magnification). Neoplastic cellular morphology is seen as D) epithelioid, E) spindled, or more commonly as mixed epithelioid and spindled (600× original magnification). F) Mitotic figures are variably present but may be frequent (600× original magnification). G) Melanoma invasion into the optic nerve can occur (100× original magnification). H) Bland geographic necrosis may be present (200× original magnification). I) Extrascleral extension is rarely identified and can involve the extraocular soft tissue of the orbit (100× original magnification). J) Periodic Acid-Schiff staining highlights occasional nested vascular loops (40× original magnification). K) All uveal melanomas tested are negative for the IDH1-R132H mutation by immunohistochemistry (400× original magnification). L) Representative appropriate oligodendroglioma immunohistochemistry positive control (400× original magnification).
involvement of the orbital soft tissue (Fig. 2I). Periodic Acid-Schiff (PAS) staining highlighted nested vascular loops in only 20% (10 of 50) of cases (Fig. 2J). Fifty patients had sufficient material available for IDH1R132H immunohistochemistry, and all cases were negative for the IDH1-R132H mutation (Fig. 2K). Oligodendroglioma samples served as appropriate external positive staining controls (Fig. 2L). As a validation set, gene level somatic mutations in IDH1, and IDH2, were queried in the publically available COSMIC database (Forbes et al., 2015). There were no somatic mutations found in either IDH1 (56 samples) or IDH2 (54 samples) for any of the reported uveal melanoma samples. This sequencing data is concordant with the lack of IDH1-R132H immunohistochemical reactivity in our 50 separate uveal melanoma cases. The absence of IDH1 mutations in the combined 106 total cases of uveal melanoma was statistically significant compared to cutaneous melanoma, utilizing 6.2% (the percentage of cutaneous melanomas with IDH1 mutation in the TCGA study set (Cancer Genome Atlas, 2015)) as the hypothesized probability (p = 0.0046, confidence interval 0.0–0.035; binomial test). 4. Discussion In our series of adult uveal melanoma, the demographics reflect those of several other studies. There was a slight male to female predominance (3:2) with an average age of 60.9 years at time of
enucleation. Microscopically, a majority of the tumors had mixed epithelioid and spindle histomorphology. A small subset of the cases demonstrated poor prognostic clinical features including large size and extrascleral extension. Similarly, a small proportion of cases contained poor prognostic cytoarchitectural characteristics including predominantly epithelioid cytolmorphology, high mitotic activity, and nested vascular loops. Overall, the clinical and pathological features were representative of typical uveal melanoma cohorts. As previously discussed, a majority of uveal melanoma cases are characterized by mutations in the BAP1 gene, whereas many non-uveal melanomas are characterized by BRAF-V600E mutation (Davies et al., 2002). Although the BRAF-V600E mutation is not observed in primary uveal melanoma, there is activation of the downstream MEK/ERK signaling pathway (Cohen et al., 2003; Cruz et al., 2003; Edmunds et al., 2003; Rimoldi et al., 2003). Conversely, BAP1 germline mutations have been reported to be associated with cutaneous melanoma in certain familial cases (Njauw et al., 2012; Wadt et al., 2012). While this may indicate exceedingly rare overlaps in the genetic profiles of uveal and non-uveal melanoma, they largely appear as molecularly distinct entities. For the first time, we demonstrate that all uveal melanoma tumors tested are negative for the IDH1-R132H mutation by immunohistochemistry, further supporting a distinctive molecular background when compared to non-uveal melanoma.
480
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481
A possible shortcoming of this study is an absence of pediatric uveal melanoma cases in our series. Pediatric uveal melanoma is exceedingly rare, however these tumors demonstrate favorable outcomes when compared to adult cases (Al-Jamal and Kivela, 2014; Blasi et al., 2015; Dimaras et al., 2013; Kaliki et al., 2013; Pukrushpan et al., 2014; Singh et al., 2000; Sivalingam et al., 2014; Sondak and Messina, 2014; Yousef and Alkilany, 2015). Little is known about the molecular drivers of pediatric uveal melanoma, but limited data indicate unique cytogenetic profiles (Blasi et al., 2015). Given the distinctive clinical characteristics and cytogenetic profiles of pediatric uveal melanoma, it is possible that these rare tumors harbor distinct genetic mutations and may serve for future IDH-related mutational studies. Although the IDH1-R132H mutation is the most common IDH1 mutation present in diffuse gliomas, this is not the case for several other cancers. Other cancers with recurrent IDH1/2 mutations have other commonly found specific mutations including IDH2-R140Q (acute myeloid leukemia), IDH1-R132C (chondrosarcoma and cholangiocarcinoma), IDH2-G145R (gastric cancer), IDH2-R172S (Osteosarcoma), and IDH2-R172K (angioimmunoblastic T-cell lymphoma) (Molenaar et al., 2014). In our series of 50 uveal melanoma cases tested for IDH1R132H mutation by immunohistochemistry, we cannot exclude the possibility that one of these other commonly found IDH1/2 mutations is present. However, we do note that the DNA sequencing data available in COSMIC for the separate validation set does not report any of these mutations in IDH1 (n = 56) or IDH2 (n = 54). This retrospective case series study of uveal melanoma has demonstrated similar histological and clinical characteristics that have been well-established and described in several other case series and case reports. Importantly, we show a lack of IDH1-R132H mutations within this tumor, implicating unique alternative genetic events leading to tumorigenesis when compared to subsets of other non-uveal melanoma. This also suggests that uveal melanoma may not respond to IDH targeted therapies (Krell et al., 2013). In the era of genomic and precision medicine, it is hoped that this information can serve as an important negative mutational study and allow focus on other genetic molecular events for clinical risk stratification and therapeutic decision making. Disclosure/Conflict of Interest The authors declare that there are no conflicts of interest. References Al-Jamal, R.T., Kivela, T., 2014. Uveal melanoma among Finnish children and young adults. J. AAPOS 18, 61–66. Amary, M.F., et al., 2011. IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours. J. Pathol. 224, 334–343. Andreoli, M.T., et al., 2015. Epidemiological trends in uveal melanoma. Br. J. Ophthalmol. 99, 1550–1553. Bagger, M., et al., 2015. The prognostic effect of American Joint Committee on Cancer staging and genetic status in patients with choroidal and ciliary body melanoma. Invest. Ophthalmol. Vis. Sci. 56, 438–444. Blasi, M.A., et al., 2015. Unique genomic profile associated with pediatric uveal melanoma. Eur. J. Ophthalmol. 25, e31–e34. Bleeker, F.E., et al., 2010. The prognostic IDH1(R132) mutation is associated with reduced NADP+-dependent IDH activity in glioblastoma. Acta Neuropathol. 119, 487–494. Cairns, R.A., et al., 2012. IDH2 mutations are frequent in angioimmunoblastic T-cell lymphoma. Blood 119, 1901–1903. Cancer Genome Atlas, N., 2015. Genomic classification of cutaneous melanoma. Cell 161, 1681–1696. Cancer Genome Atlas Research, N., et al., 2015. Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N. Engl. J. Med. 372, 2481–2498. Capper, D., et al., 2010a. Application of mutant IDH1 antibody to differentiate diffuse glioma from nonneoplastic central nervous system lesions and therapy-induced changes. Am. J. Surg. Pathol. 34, 1199–1204. Capper, D., et al., 2010b. Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors. Brain Pathol. 20, 245–254. Cohen, Y., et al., 2003. Lack of BRAF mutation in primary uveal melanoma. Invest. Ophthalmol. Vis. Sci. 44, 2876–2878.
Correa, Z.M., Augsburger, J.J., 2016. Independent prognostic significance of gene expression profile class and largest basal diameter of posterior uveal melanomas. Am J. Ophthalmol. 162 (20–27), e1. Cruz 3rd, F., et al., 2003. Absence of BRAF and NRAS mutations in uveal melanoma. Cancer Res. 63, 5761–5766. Davies, H., et al., 2002. Mutations of the BRAF gene in human cancer. Nature 417, 949–954. Dimaras, H., et al., 2013. Molecular testing prognostic of low risk in epithelioid uveal melanoma in a child. Br. J. Ophthalmol. 97, 323–326. Eckel-Passow, J.E., et al., 2015. Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors. N. Engl. J. Med. 372, 2499–2508. Edmunds, S.C., et al., 2003. Absence of BRAF gene mutations in uveal melanomas in contrast to cutaneous melanomas. Br. J. Cancer 88, 1403–1405. Emadi, A., et al., 2015. Presence of isocitrate dehydrogenase mutations may predict clinical response to hypomethylating agents in patients with acute myeloid leukemia. Am. J. Hematol. 90, E77–E79. Forbes, S.A., et al., 2015. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res. 43, D805–D811. Force, A.O.O.T., 2015. International Validation of the American Joint Committee on Cancer's 7th Edition Classification of Uveal Melanoma. JAMA Ophthalmol. 133, 376–383. Gorovets, D., et al., 2012. IDH mutation and neuroglial developmental features define clinically distinct subclasses of lower grade diffuse astrocytic glioma. Clin. Cancer Res. 18, 2490–2501. Gupta, M.P., et al., 2015. Clinical characteristics of uveal melanoma in patients with germline BAP1 mutations. JAMA Ophthalmol. 133, 881–887. Harbour, J.W., 2012. The genetics of uveal melanoma: an emerging framework for targeted therapy. Pigment Cell Melanoma Res. 25, 171–181. Harbour, J.W., 2014. A prognostic test to predict the risk of metastasis in uveal melanoma based on a 15-gene expression profile. Methods Mol. Biol. 1102, 427–440. Harbour, J.W., et al., 2010. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 330, 1410–1413. Harbour, J.W., et al., 2013. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat. Genet. 45, 133–135. Horbinski, C., et al., 2009. Diagnostic use of IDH1/2 mutation analysis in routine clinical testing of formalin-fixed, paraffin-embedded glioma tissues. J. Neuropathol. Exp. Neurol. 68, 1319–1325. Houillier, C., et al., 2010. IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas. Neurology 75, 1560–1566. Im, A.P., et al., 2014. DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies. Leukemia 28, 1774–1783. Jha, P., et al., 2011. IDH1 mutations in gliomas: first series from a tertiary care centre in India with comprehensive review of literature. Exp. Mol. Pathol. 91, 385–393. Johansson, P., et al., 2015. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4. Oncotarget. Kaliki, S., et al., 2013. Influence of age on prognosis of young patients with uveal melanoma: a matched retrospective cohort study. Eur. J. Ophthalmol. 23, 208–216. Kalirai, H., et al., 2014. Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing. Br. J. Cancer 111, 1373–1380. Klebe, S., et al., 2015. BAP1 hereditary cancer predisposition syndrome: a case report and review of literature. Biomark. Res. 3, 14. Koopmans, A.E., et al., 2014. Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma. Mod. Pathol. 27, 1321–1330. Krell, D., et al., 2013. IDH mutations in tumorigenesis and their potential role as novel therapeutic targets. Future Oncol. 9, 1923–1935. Lian, C.G., et al., 2012. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell 150, 1135–1146. Martin, M., et al., 2013. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat. Genet. 45, 933–936. Metellus, P., et al., 2010. Absence of IDH mutation identifies a novel radiologic and molecular subtype of WHO grade II gliomas with dismal prognosis. Acta Neuropathol. 120, 719–729. Molenaar, R.J., et al., 2014. The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation. Biochim. Biophys. Acta 1846, 326–341. Molenaar, R.J., et al., 2015. Clinical and biological implications of ancestral and non-ancestral IDH1 and IDH2 mutations in myeloid neoplasms. Leukemia 29, 2134–2142. Murugan, A.K., et al., 2010. Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer. Biochem. Biophys. Res. Commun. 393, 555–559. Njauw, C.N., et al., 2012. Germline BAP1 inactivation is preferentially associated with metastatic ocular melanoma and cutaneous-ocular melanoma families. PLoS One 7, e35295. Olar, A., et al., 2015. IDH mutation status and role of WHO grade and mitotic index in overall survival in grade II–III diffuse gliomas. Acta Neuropathol. 129, 585–596. Onken, M.D., et al., 2004. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res. 64, 7205–7209. Patel, J.P., et al., 2012. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N. Engl. J. Med. 366, 1079–1089. Pukrushpan, P., et al., 2014. Congenital uveal malignant melanoma. J. AAPOS 18, 199–201. Reuss, D.E., et al., 2015a. IDH mutant diffuse and anaplastic astrocytomas have similar age at presentation and little difference in survival: a grading problem for WHO. Acta Neuropathol. 129, 867–873.
P.J. Cimino et al. / Experimental and Molecular Pathology 100 (2016) 476–481 Reuss, D.E., et al., 2015b. ATRX and IDH1-R132H immunohistochemistry with subsequent copy number analysis and IDH sequencing as a basis for an “integrated” diagnostic approach for adult astrocytoma, oligodendroglioma and glioblastoma. Acta Neuropathol. 129, 133–146. Rimoldi, D., et al., 2003. Lack of BRAF mutations in uveal melanoma. Cancer Res. 63, 5712–5715. Schoenfield, L., 2014. Uveal melanoma: a pathologist's perspective and review of translational developments. Adv. Anat. Pathol. 21, 138–143. Shibata, T., et al., 2011. Mutant IDH1 confers an in vivo growth in a melanoma cell line with BRAF mutation. Am. J. Pathol. 178, 1395–1402. Shields, J.A., Shields, C.L., 2015. Management of posterior uveal melanoma: past, present, and future: the 2014 Charles L. Schepens lecture. Ophthalmology 122, 414–428. Shields, C.L., et al., 2015. American Joint Committee on Cancer Classification of Uveal Melanoma (Anatomic Stage) predicts prognosis in 7731 patients: the 2013 Zimmerman Lecture. Ophthalmology 122, 1180–1186. Singh, A.D., Topham, A., 2003. Incidence of uveal melanoma in the United States: 1973–1997. Ophthalmology 110, 956–961. Singh, A.D., et al., 2000. Uveal melanoma in young patients. Arch. Ophthalmol. 118, 918–923. Sivalingam, M.D., et al., 2014. Choroidal melanoma in children: be aware of risks. J. Pediatr. Ophthalmol. Strabismus 51, e85–e88 (Online). Sondak, V.K., Messina, J.L., 2014. Unusual presentations of melanoma: melanoma of unknown primary site, melanoma arising in childhood, and melanoma arising in the eye and on mucosal surfaces. Surg. Clin. North Am. 94, 1059–1073 (ix).
481
Testa, J.R., et al., 2011. Germline BAP1 mutations predispose to malignant mesothelioma. Nat. Genet. 43, 1022–1025. Tschentscher, F., et al., 2003. Tumor classification based on gene expression profiling shows that uveal melanomas with and without monosomy 3 represent two distinct entities. Cancer Res. 63, 2578–2584. Turcan, S., et al., 2012. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479–483. van Essen, T.H., et al., 2014. Prognostic parameters in uveal melanoma and their association with BAP1 expression. Br. J. Ophthalmol. 98, 1738–1743. van Gils, W., et al., 2008. Gene expression profiling in uveal melanoma: two regions on 3p related to prognosis. Invest. Ophthalmol. Vis. Sci. 49, 4254–4262. Van Raamsdonk, C.D., et al., 2009. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 457, 599–602. Van Raamsdonk, C.D., et al., 2010. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363, 2191–2199. Wadt, K., et al., 2012. A cryptic BAP1 splice mutation in a family with uveal and cutaneous melanoma, and paraganglioma. Pigment Cell Melanoma Res. 25, 815–818. Wiesner, T., et al., 2011. Germline mutations in BAP1 predispose to melanocytic tumors. Nat. Genet. 43, 1018–1021. Worley, L.A., et al., 2007. Transcriptomic versus chromosomal prognostic markers and clinical outcome in uveal melanoma. Clin. Cancer Res. 13, 1466–1471. Yan, H., et al., 2009. IDH1 and IDH2 mutations in gliomas. N. Engl. J. Med. 360, 765–773. Yousef, Y.A., Alkilany, M., 2015. Characterization, treatment, and outcome of uveal melanoma in the first two years of life. Hematol. Oncol. Stem Cell Ther. 8, 1–5.