252 Original article
Anatomic and pathological characterization of choroidal melanoma using multimodal imaging: what is practical, what is needed? Tobias Lindnera,*, Sönke Langnerf,*, Karen Falkeb, Uwe Walterc, Paul-Christian Krügerf, Andreas Pohlmanng, Annette Zimpferd, Thomas Stahnkeb, Stefan Hadlichf, Rudolf Guthoffe,g, Andreas Erbersdoblerd, Thoralf Niendorfg and Oliver Stachsb Choroidal melanoma is the most frequently occurring intraocular tumor in adults. The aim of this work is to assess the potential of state-of-the art in-vivo and ex-vivo imaging modalities for the characterization of choroidal melanoma. Multimodal imaging of a choroidal melanoma was performed in a 53-year-old male patient. In-vivo ophthalmoscopy, ultrasound microscopy, duplex ultrasound, and 7.0 T MRI were performed. Ex-vivo examination of the enucleated eye included 7.0 and 9.4 T magnetic resonance microscopy as well as histopathology with hematoxylin and eosin staining. Imaging of choroidal melanoma with ultrahigh field MRI and duplex sonography provides detailed morphologic and functional information of the eye. High-spatial-resolution MRI at 9.4 T shows details of the internal texture of melanoma and other structures of the eye with an in-plane spatial resolution of 32 μm. Ultrahigh field in-vivo MRI at 7.0 T and ex-vivo MRI at 7.0 and 9.4 T correlate well with histologic evaluation. In-vivo ultrahigh field MRI is an emerging technique for the characterization and staging of ocular tumors. The
Introduction Choroidal melanoma is the most frequent intraocular tumor in adults [1]. With an incidence of five to six per million per year, choroidal melanoma accounts for 3–4% of all melanomas [2]. The majority of choroidal melanomas are asymptomatic and detected incidentally during routine eye examinations with a slit lamp or an ophthalmoscope [3]. Symptomatic patients usually see flashes, notice distortions, or report loss of vision during an advanced stage of the disease [4].
combination of in-vivo ultrahigh-field MRI and duplex sonography has the potential to complement or even substitute complex and invasive biopsies. Melanoma Res 25:252–258 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Melanoma Research 2015, 25:252–258 Keywords: choroidal melanoma, histology, magnetic resonance imaging, multimodal imaging, ultrasound a
Core Facility Small Animal Imaging, bDepartment of Ophthalmology, cDepartment of Neurology, dInstitute of Pathology, eInstitute for Biomedical Engineering, University Medicine Rostock, Rostock, fInstitute for Diagnostic Radiology and Neuroradiology, University Medicine Greifswald, Greifswald and gBerlin Ultrahigh Field Facility (BUFF), Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Correspondence to Tobias Lindner, Dipl. Ing. (FH), Dr. rer. hum., Core Facility Small Animal Imaging, University Medicine Rostock, Schillingallee 69a, 18057 Rostock, Germany Tel: + 49 381 494 2505; fax: + 49 381 494 2502; e-mail: [email protected] *Tobias Lindner and Sönke Langner contributed equally to the writing of this article. Received 19 August 2014 Accepted 27 February 2015
by in-vivo color-coded duplex ultrasound (CDUS), in-vivo 7.0 T ultrahigh field MRI (UHF-MRI), ex-vivo magnetic resonance microscopy (MRM) at 7.1 T [5,6] and 9.4 T, and conventional histopathology.
Patients and methods Background
In individuals with an intraocular mass such as uveal melanoma, diagnostic information on the extent, shape, structure, and infiltration of surrounding tissue is of particular interest for treatment decisions. Different imaging modalities can be used to obtain this information.
A 53-year-old man presented to our ophthalmic clinic with a 6-month history of deteriorating visual acuity in his left eye. During clinical examination, a visual acuity of 1.0 (Snellen) was documented for the right eye and ‘hand movement’ for the left eye. The left eye showed a relative afferent pupillary defect. Fundoscopic examination indicated a malignant choroidal melanoma. Tumor extension was evaluated by ultrasonography. Because of the extent of the tumor and absence of metastases, enucleation was considered the primary treatment.
We present a case in which clinical routine in-vivo imaging [ophthalmoscopy, slit lamp photography, and high-frequency ultrasound (HFUS)] was complemented
The 7.0 T MR study was approved by the local ethics committee (registration number: DE/CA73/5550/09 Landesamt für Arbeitsschutz, Gesundheitsschutz und
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DOI: 10.1097/CMR.0000000000000156
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Multimodal imaging of choroidal melanoma Lindner et al. 253
technische Sicherheit, Berlin, Germany), and the patient provided written informed consent for additional research-related in-vivo imaging, ex-vivo imaging, histologic work-up of tissues, and publication of his medical images. Fundus photography
Fundoscopic examination was performed using a standard slit lamp (900 R; Haag-Streit, Bern, Switzerland). In patients with malignant melanoma, slit lamp ophthalmoscopy will typically show a pigmented tumor when its size exceeds 2 mm [1]. Other typical findings suggesting a malignant eye tumor are collateral exudative retinal detachment, vascularization of the tumor (confirmed by fluorescence angiography and Doppler sonography), and orange-colored pigment deposits indicating lipofuscin accumulation. Progressive tumor growth and symptoms are other typical criteria of malignancy. High-frequency ultrasound
The patient underwent clinical HFUS (CineScan; Quantel Medical, Cournon-d'auvergne, France) examination with a 10.0 MHz transducer. HFUS is typically used to measure the size of choroidal melanoma, to evaluate internal tumor reflectivity, and to identify melanoma extension beyond the sclera into the orbit [7]. The majority of choroidal melanomas appear dome like and less commonly like a mushroom on ultrasound [8]. Ultrasound can also show retinal detachment associated with choroidal melanoma. On A-scan images, the tumor typically shows high reflectivity or, less commonly, medium reflectivity. B-scan images enable exact estimation of tumor diameters. Furthermore, B-scan images can be used to calculate the position of the ruthenium-106 plaque for irradiation therapy. The affected eye was imaged with the lid closed in the A-scan and B-scan mode. Duplex ultrasound
Orbital CDUS was performed using a high-end ultrasound system (Acuson Antares; Siemens Healthcare, Erlangen, Germany) with an 8.0-MHz linear-array transducer. According to the regulations of the Center for Devices and Radiological Health, the B-mode ultrasound energy output was set at a mechanical index less than 0.3 and a thermal index for cranial bone less than 1.0 [9]. CDUS was applied only for short periods of less than 5 min, with the energy output set at mechanical index less than 0.7. CDUS through the eye lens was avoided. In-vivo ultrahigh field MRI at 7.0 T
In-vivo MRI was performed on a whole-body 7.0 T MR system (Magnetom 7 T; Siemens Healthcare, Erlangen, Germany). A custom-made six-channel transceiver radiofrequency (RF) coil tailored for imaging the eye and the orbit was used for signal transmission and detection [10–13]. To improve image quality and reduce motion-related artifacts, a blink/nonblink algorithm with an optoacoustic trigger was used as described elsewhere [14]. The trigger protocol
consisted of an acquisition window, followed by a pause to allow for eye blinking. The acquisition window was indicated by an acoustic signal transferred to the subject by an MR-compatible stethoscope-style pneumatic headphone (easyACT; MRI.TOOLS GmbH, Berlin, Germany) [15,16]. During acquisition, the patient fixated a cross that was presented on screen for 3 s. The fixation period was followed by a rest period of 3 s. T1-weighted (T1w) 3D fast low-angle shot (FLASH) imaging [TE = 4 ms, TR = 10.3 ms, flip angle = 6°, field of view (FoV) = 103 × 73 mm, imaging matrix = 320 × 230 interpolated to 640 × 480] was performed in axial and sagittal planes across the eye. In-plane resolution was 160 × 160 μm with a slice thickness of 1 mm and 24 slices/ slab. The total acquisition time was ∼ 2 min. Ex-vivo magnetic resonance microscopy at 7.1 T
After therapeutic enucleation of the globe, ex-vivo MRM was performed on a 7.1 T MRI system (ClinScan 70/30; Bruker Biospin, Ettlingen, Germany) [5,6]. For transport to the imaging facility, the sample was wrapped in gauze soaked in a physiologic saline solution (0.9% NaCl) and placed in a small container at room temperature. The eye was fixated on a commercial small animal bed using a gauze cushion. For image acquisition, a 2 × 2 channel surface coil array was placed on top of the sample without compressing the anterior chamber. The mean time between enucleation and MRM was ∼ 90 min. Fast T2-weighted (T2w) imaging was used for localization. Next, high-spatial-resolution T2w images of the eye were acquired in three orthogonal planes. Imaging parameters were as follows: TE = 48 ms, TR = 4900 ms, flip angle = 80°, slice thickness = 0.7 mm, imaging matrix = 512 × 512 interpolated to 1024 × 1024, FoV = 38 × 38 mm, and nominal in-plane resolution = 37 μm. Acquisition time was 8:45 min/plane. Ex-vivo magnetic resonance microscopy at 9.4 T
High-spatial-resolution ex-vivo MRI was performed on a 9.4 T small animal MR system (Biospec 94/20; Bruker Biospin) equipped with a cryogenic transceive quadrature RF surface coil (CryoProbe; Bruker Biospin) of curved rectangular geometry (size = 20 × 27 mm2) operating at around 30 K (preamplifiers at 77 K) [17,18]. T2*weighted (T2*w) MRI was performed using a spoiled gradient echo technique (2D FLASH, TR = 120 ms, TE = 10.3 ms, flip angle = 40°, averages = 96) with a total acquisition time of 2:33 h. Three parallel slices were acquired with an isotropic spatial in-plane resolution of 32 μm (FoV = 25.6 × 25.6 mm, matrix size = 800 × 800) and a slice thickness of 170 μm. Following intensity inhomogeneity correction using a simple linear gradient, the images were interpolated (bilinear) to a matrix size of 2400 × 2400. To avoid dehydration during ex-vivo imaging, the sample was wrapped in cling film.
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Histopathology
After ex-vivo MRM, the enucleated bulb was fixed in a 4% formalin solution for at least 24 h and cut in an anteroposterior manner after cutting the resection margin of the optic nerve. Two- to three-micrometer tissue sections, which matched the slices used in 7.0 T ex-vivo MRI, were stained with hematoxylin and eosin and peroxidase Schiff reagent. To observe possible venous invasion, further tissue sections were added in the planes needed. Additional fluorescence in-situ hybridization (FISH) was performed to test for monosomy of chromosome 3 [19,20]. GNA11 and GNAQ mutation analysis was carried out [21].
Results Fundus photography
Slit lamp ophthalmoscopy (Fig. 1a) showed a large retinal detachment in the lower circumference with an underlying tumor mass in the 6 o’clock position. The malignant melanoma protruded prominently from the surface, giving rise to the typical mushroom shape, and had a pigmented surface. However, detailed assessment was limited by the surrounding retinal detachment.
The sclera showed uniform hypointensity on T2w images with no evidence of infiltration. Anteriorly, the tumor extended to the ciliary body, which appeared distorted (Fig. 3). Signal intensity of the ciliary body was comparable with that of the nonaffected eye, making tumor infiltration unlikely. Within the tumor, multiple tubular structures of low T2 signal intensity were detected. The subretinal fluid collection appeared hypointense, consistent with recent hemorrhage. Ex-vivo magnetic resonance microscopy at 9.4 T
T2*w imaging at 9.4 T delineated the microstructure of the mass (Fig. 4) with excellent correlation to conventional histology (Fig. 5a and b). The sclera showed a uniform low signal intensity. The outer margin of the sclera appeared slightly distorted because of susceptibility artifacts. The retina was clearly differentiated from the tumor and the two layers of the retina could be appreciated. The ciliary body was distorted, but there were no signs of infiltration as shown at 7.0 T. Compared with ex-vivo MRM at 7.0 T, the ultrastructure of the tumor was delineated in more detail on the T2*w images. Histopathology
High-frequency ultrasound
HFUS showed a dome-shaped mass of low reflectivity with a highly reflective surface. The detached retina appeared hyper-reflective with subretinal hypoechogenic effusions (Fig. 1b). The mass height was 10 mm and the basal diameter was 13 mm. There was no evidence of extraocular growth. Duplex ultrasound
CDUS showed intense vascularization of the domeshaped mass with several arteries originating from the base of the tumor (Fig. 1c) and normal vasculature in the adjacent choroid and retina. In-vivo ultrahigh field MRI at 7.0 T
The tumor showed high signal intensity in T1w MRI and presented as a dome-shaped mass with smooth margins and a size of 8 × 15 mm (Fig. 1d and Fig. 2). The tumor was clearly delineated from the hypointense sclera and retina. There was no evidence of extraocular growth. The retina was attached to the optic nerve head ending. Retinal detachment had a wing-shaped appearance. Subretinal fluid collections appeared slightly hyperintense compared with the vitreous body, indicating protein-rich fluid or concomitant hemorrhage, which was confirmed by conventional histology. Ex-vivo magnetic resonance microscopy at 7.0 T
On T2w images the tumor appeared hypointense compared with the vitreous body (Fig. 1g and Fig. 3). The tumor showed a clearly defined, strongly hypointense margin clearly demarcating the tumor from the retina.
The gross specimen showed a brown tumor 16 mm size at the base and 8.5 mm in the prominence was found at the lower pole of the vitreous body. Macroscopically, the retina was detached and the sclera was intact. No infiltration of the ciliary body was observed. The tumor showed no contact with the optic nerve. Microscopically, at × 10 magnification, prominent ectatic tumor vessels were noticeable. The tumor was composed of a mixture of malignant spindle A and spindle B cells in a typical fascicular pattern (Fig. 5c) and showed initial scleral and venous invasion (Fig. 5d). Immunohistochemistry with a primary antibody against melan A (Dako, Hamburg, Germany) was strongly positive (Fig. 5e). Only a few mitotic figures were visible in hematoxylin and eosin stains. Tumor growth toward the ciliary body was observed, but no certain invasion (Fig. 5a and b). FISH analysis for chromosomes 3 and 8 showed disomy 3 and 8 (Fig. 5f). In the additional mutation analysis of exons 4 and 5 of GNA11 and GNAQ, exon 5 point mutation Q209L of the GNA11 gene was found.
Discussion Depending on the extent of the tumor, choroidal melanoma can be treated with curative intent. Pretreatment assessment of precise tumor extension is mandatory because knowledge of tumor size and location is essential to choose the appropriate treatment that governs the overall prognosis [1]. Yet, clinical routine invivo imaging is limited in early detection of small tumors because of inherent limitations – for example, optical distortions [22].
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Multimodal imaging of choroidal melanoma Lindner et al. 255
Fig. 1
Fundus photography
Duplex ultrasound
HF ultrasound (b)
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MRI at 7 T
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Multimodal imaging of choroidal melanoma. (a–d) In-vivo results derived from conventional fundus photography, high-frequency (HF) and duplex ultrasound, and MRI. (e–h) Multimodal imaging results obtained after enucleation of the eye [scale bar in (e) also applies to (f)–(h)].
Fig. 2
# ∗
10 mm Sagittal T1-weighted in-vivo MR image (7.0 T) of the human bulb. Strongly hyperintense mass (*) with the corresponding retinal detachment (#).
UHF-MRI and MRM are emerging techniques for the characterization of ocular masses [22,23]. Unlike ultrasound, which depends on the operator’s experience [24], image quality of MRM and UHF-MRI primarily relies on the sensitivity at high magnetic fields and on the uniformity of the transmission and reception fields [25]. Previous studies have reported an excellent correlation between MRM and conventional histology with respect to tumor size and ultrastructure [17]. These findings are supported by our study. In addition, it is possible to clearly distinguish between tumor and surrounding subretinal fluid collections on both in-vivo and ex-vivo images. Our study also shows that, because of different signal intensities, hemorrhage can be differentiated from subretinal effusion on T1w and T2w images. UHF-MRI and MRM showed an excellent correlation with conventional histology for tumor size and shape. Furthermore, both imaging techniques enable
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MRM enables both detection of tumor-feeding vessels [27] and assessment of the extent of vascularity [28]. The latter is a possible biomarker of the degree of malignancy and can serve to monitor the response to therapy.
Fig. 3
Furthermore, changes in tumor microstructure can be used as a potential surrogate parameter for treatment response.
# ∗ 10 mm Sagittal T2-weighted ex-vivo MR image (7.0 T) of the human bulb showing an inhomogeneous hypointense mass (*) with the corresponding retinal detachment (#). Potential infiltration of the ciliary body (arrow).
Therefore, UHF-MRI may facilitate initial diagnostic assessment in patients first presenting with an intraocular mass. Although UHF-MRI cannot yet replace biopsy for the final confirmation of the diagnosis before treatment [3], our results are promising and the diagnostic accuracy of UHF-MRI and MRM should be evaluated in comparison with conventional histology in a prospective study of a larger patient population.
assessment of the ultrastructure of the tumor. The hypointense structures shown on T2w images in ex-vivo MR histology at 7.1 T correlate well with histopathologically proven intratumoral hemorrhages and melanin deposits. With MR histology at 9.4 T, the ultrastructure can be appreciated in more detail and the tubular structures correlate well with the course of collagen fibrils and melanin deposits. On UHF-MRI, the tumor appears hyperintense on T1w images because of the paramagnetic effects of melanin.
Despite providing an important multimodality comparison, our study has some limitations. First, MRM can be time consuming, which increases the risk of motionrelated artifacts. However, the occurrence of motionrelated artifacts can be reduced by improving patient comfort with the use of dedicated RF coils in conjunction with dedicated acquisition schemes [14]. Second, UHFMRI allows an in-plane spatial resolution as good as 100 μm. In comparison, MRM provides a spatial resolution of up to 30 μm. Although this is inferior to conventional histology, the major advantage of UHF-MRI is its noninvasiveness. Because of the limited spatial resolution, early scleral and venous infiltration will be missed. How this will affect clinical outcome has to be evaluated in a larger patient cohort. Spatial resolution of MRI is also inferior to optical coherence tomography. However, MRI enables undistorted simultaneous assessment of the eye and the orbit. Further improvements in MR technology, RF coil design, and optimization of MRI protocols tailored to ophthalmic imaging may overcome the current limitations – for example, RF power deposition constraints, ultimately allowing in-vivo imaging with a spatial resolution currently only available for ex-vivo imaging [29–31]. Although imaging protocols for UHF-MRI are tailored to clinical imaging with acquisition of T1w and T2w sequences and the use of contrast agent, MRM imaging protocols are optimized for providing highspatial-resolution and eliminating artifacts related to magnetic field strength. Therefore, imaging protocols differ between the different modalities. Finally, although UHF-MRI and MRM may facilitate the diagnosis of intraocular masses and may therefore reduce the need for biopsies, invasive tissue sampling will still be necessary for genetic analysis.
Doppler ultrasound enables assessment of the vascularity of an intraocular lesion and is a valuable tool for the diagnosis of recurrent melanoma [26]. Contrast-enhanced
In conclusion, UHF-MRI and MRM are promising tools for imaging intraocular lesions. With wider availability, MRI may help to overcome some of the limitations of
Fig. 4
10 mm T2*-weighted image of the tumor derived from ex-vivo magnetic resonance microscopy at 9.4 T.
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Multimodal imaging of choroidal melanoma Lindner et al. 257
Fig. 5
(a)
(b)
500 μm (c)
500 μm (d)
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#
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100 μm (f)
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Choroidal melanoma of a left eye. (a) Overview, of pigmented choroidal melanoma lying close to the ciliary body [hematoxylin and eosin staining (H&E) × 2]. (b) T2*-weighted image acquired ex-vivo at 9.4 T. (c) Choroidal melanoma spindle cell type A and B with pigmentation and noticeable ectatic venous blood vessel (arrow) (H&E, × 20). (d) Initial scleral invasion (#) and venous invasion with ectatic capillary vein (*) within melanoma tissue (peroxidase Schiff reaction, × 10). (e) Spindle-cell choroidal melanoma with strong melan A immunoreactivity and a fascicular growing pattern (melan A, × 20). (f) Disomy of chromosomes 3 and 8 (fluorescence in-situ hybridization, × 100).
current clinical in-vivo imaging techniques. Beyond anatomic imaging, MRM enables insights into the microstructure of ocular tumors. This capability may improve the diagnostic characterization of ocular tumors and might serve as a biomarker to monitor treatment response.
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Conflicts of interest
Thoralf Niendorf is founder and CEO of MRI.TOOLS GmbH, Berlin, Germany. The remaining authors have no conflicts of interest.
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Anatomic and pathological characterization of choroidal melanoma using multimodal imaging: what is practical, what is needed? Tobias Lindnera,*, Sönke Langnerf,*, Karen Falkeb, Uwe Walterc, Paul-Christian Krügerf, Andreas Pohlmanng, Annette Zimpferd, Thomas Stahnkeb, Stefan Hadlichf, Rudolf Guthoffe,g, Andreas Erbersdoblerd, Thoralf Niendorfg and Oliver Stachsb Choroidal melanoma is the most frequently occurring intraocular tumor in adults. The aim of this work is to assess the potential of state-of-the art in-vivo and ex-vivo imaging modalities for the characterization of choroidal melanoma. Multimodal imaging of a choroidal melanoma was performed in a 53-year-old male patient. In-vivo ophthalmoscopy, ultrasound microscopy, duplex ultrasound, and 7.0 T MRI were performed. Ex-vivo examination of the enucleated eye included 7.0 and 9.4 T magnetic resonance microscopy as well as histopathology with hematoxylin and eosin staining. Imaging of choroidal melanoma with ultrahigh field MRI and duplex sonography provides detailed morphologic and functional information of the eye. High-spatial-resolution MRI at 9.4 T shows details of the internal texture of melanoma and other structures of the eye with an in-plane spatial resolution of 32 μm. Ultrahigh field in-vivo MRI at 7.0 T and ex-vivo MRI at 7.0 and 9.4 T correlate well with histologic evaluation. In-vivo ultrahigh field MRI is an emerging technique for the characterization and staging of ocular tumors. The
Introduction Choroidal melanoma is the most frequent intraocular tumor in adults [1]. With an incidence of five to six per million per year, choroidal melanoma accounts for 3–4% of all melanomas [2]. The majority of choroidal melanomas are asymptomatic and detected incidentally during routine eye examinations with a slit lamp or an ophthalmoscope [3]. Symptomatic patients usually see flashes, notice distortions, or report loss of vision during an advanced stage of the disease [4].
combination of in-vivo ultrahigh-field MRI and duplex sonography has the potential to complement or even substitute complex and invasive biopsies. Melanoma Res 25:252–258 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Melanoma Research 2015, 25:252–258 Keywords: choroidal melanoma, histology, magnetic resonance imaging, multimodal imaging, ultrasound a
Core Facility Small Animal Imaging, bDepartment of Ophthalmology, cDepartment of Neurology, dInstitute of Pathology, eInstitute for Biomedical Engineering, University Medicine Rostock, Rostock, fInstitute for Diagnostic Radiology and Neuroradiology, University Medicine Greifswald, Greifswald and gBerlin Ultrahigh Field Facility (BUFF), Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Correspondence to Tobias Lindner, Dipl. Ing. (FH), Dr. rer. hum., Core Facility Small Animal Imaging, University Medicine Rostock, Schillingallee 69a, 18057 Rostock, Germany Tel: + 49 381 494 2505; fax: + 49 381 494 2502; e-mail: [email protected] *Tobias Lindner and Sönke Langner contributed equally to the writing of this article. Received 19 August 2014 Accepted 27 February 2015
by in-vivo color-coded duplex ultrasound (CDUS), in-vivo 7.0 T ultrahigh field MRI (UHF-MRI), ex-vivo magnetic resonance microscopy (MRM) at 7.1 T [5,6] and 9.4 T, and conventional histopathology.
Patients and methods Background
In individuals with an intraocular mass such as uveal melanoma, diagnostic information on the extent, shape, structure, and infiltration of surrounding tissue is of particular interest for treatment decisions. Different imaging modalities can be used to obtain this information.
A 53-year-old man presented to our ophthalmic clinic with a 6-month history of deteriorating visual acuity in his left eye. During clinical examination, a visual acuity of 1.0 (Snellen) was documented for the right eye and ‘hand movement’ for the left eye. The left eye showed a relative afferent pupillary defect. Fundoscopic examination indicated a malignant choroidal melanoma. Tumor extension was evaluated by ultrasonography. Because of the extent of the tumor and absence of metastases, enucleation was considered the primary treatment.
We present a case in which clinical routine in-vivo imaging [ophthalmoscopy, slit lamp photography, and high-frequency ultrasound (HFUS)] was complemented
The 7.0 T MR study was approved by the local ethics committee (registration number: DE/CA73/5550/09 Landesamt für Arbeitsschutz, Gesundheitsschutz und
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DOI: 10.1097/CMR.0000000000000156
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Multimodal imaging of choroidal melanoma Lindner et al. 253
technische Sicherheit, Berlin, Germany), and the patient provided written informed consent for additional research-related in-vivo imaging, ex-vivo imaging, histologic work-up of tissues, and publication of his medical images. Fundus photography
Fundoscopic examination was performed using a standard slit lamp (900 R; Haag-Streit, Bern, Switzerland). In patients with malignant melanoma, slit lamp ophthalmoscopy will typically show a pigmented tumor when its size exceeds 2 mm [1]. Other typical findings suggesting a malignant eye tumor are collateral exudative retinal detachment, vascularization of the tumor (confirmed by fluorescence angiography and Doppler sonography), and orange-colored pigment deposits indicating lipofuscin accumulation. Progressive tumor growth and symptoms are other typical criteria of malignancy. High-frequency ultrasound
The patient underwent clinical HFUS (CineScan; Quantel Medical, Cournon-d'auvergne, France) examination with a 10.0 MHz transducer. HFUS is typically used to measure the size of choroidal melanoma, to evaluate internal tumor reflectivity, and to identify melanoma extension beyond the sclera into the orbit [7]. The majority of choroidal melanomas appear dome like and less commonly like a mushroom on ultrasound [8]. Ultrasound can also show retinal detachment associated with choroidal melanoma. On A-scan images, the tumor typically shows high reflectivity or, less commonly, medium reflectivity. B-scan images enable exact estimation of tumor diameters. Furthermore, B-scan images can be used to calculate the position of the ruthenium-106 plaque for irradiation therapy. The affected eye was imaged with the lid closed in the A-scan and B-scan mode. Duplex ultrasound
Orbital CDUS was performed using a high-end ultrasound system (Acuson Antares; Siemens Healthcare, Erlangen, Germany) with an 8.0-MHz linear-array transducer. According to the regulations of the Center for Devices and Radiological Health, the B-mode ultrasound energy output was set at a mechanical index less than 0.3 and a thermal index for cranial bone less than 1.0 [9]. CDUS was applied only for short periods of less than 5 min, with the energy output set at mechanical index less than 0.7. CDUS through the eye lens was avoided. In-vivo ultrahigh field MRI at 7.0 T
In-vivo MRI was performed on a whole-body 7.0 T MR system (Magnetom 7 T; Siemens Healthcare, Erlangen, Germany). A custom-made six-channel transceiver radiofrequency (RF) coil tailored for imaging the eye and the orbit was used for signal transmission and detection [10–13]. To improve image quality and reduce motion-related artifacts, a blink/nonblink algorithm with an optoacoustic trigger was used as described elsewhere [14]. The trigger protocol
consisted of an acquisition window, followed by a pause to allow for eye blinking. The acquisition window was indicated by an acoustic signal transferred to the subject by an MR-compatible stethoscope-style pneumatic headphone (easyACT; MRI.TOOLS GmbH, Berlin, Germany) [15,16]. During acquisition, the patient fixated a cross that was presented on screen for 3 s. The fixation period was followed by a rest period of 3 s. T1-weighted (T1w) 3D fast low-angle shot (FLASH) imaging [TE = 4 ms, TR = 10.3 ms, flip angle = 6°, field of view (FoV) = 103 × 73 mm, imaging matrix = 320 × 230 interpolated to 640 × 480] was performed in axial and sagittal planes across the eye. In-plane resolution was 160 × 160 μm with a slice thickness of 1 mm and 24 slices/ slab. The total acquisition time was ∼ 2 min. Ex-vivo magnetic resonance microscopy at 7.1 T
After therapeutic enucleation of the globe, ex-vivo MRM was performed on a 7.1 T MRI system (ClinScan 70/30; Bruker Biospin, Ettlingen, Germany) [5,6]. For transport to the imaging facility, the sample was wrapped in gauze soaked in a physiologic saline solution (0.9% NaCl) and placed in a small container at room temperature. The eye was fixated on a commercial small animal bed using a gauze cushion. For image acquisition, a 2 × 2 channel surface coil array was placed on top of the sample without compressing the anterior chamber. The mean time between enucleation and MRM was ∼ 90 min. Fast T2-weighted (T2w) imaging was used for localization. Next, high-spatial-resolution T2w images of the eye were acquired in three orthogonal planes. Imaging parameters were as follows: TE = 48 ms, TR = 4900 ms, flip angle = 80°, slice thickness = 0.7 mm, imaging matrix = 512 × 512 interpolated to 1024 × 1024, FoV = 38 × 38 mm, and nominal in-plane resolution = 37 μm. Acquisition time was 8:45 min/plane. Ex-vivo magnetic resonance microscopy at 9.4 T
High-spatial-resolution ex-vivo MRI was performed on a 9.4 T small animal MR system (Biospec 94/20; Bruker Biospin) equipped with a cryogenic transceive quadrature RF surface coil (CryoProbe; Bruker Biospin) of curved rectangular geometry (size = 20 × 27 mm2) operating at around 30 K (preamplifiers at 77 K) [17,18]. T2*weighted (T2*w) MRI was performed using a spoiled gradient echo technique (2D FLASH, TR = 120 ms, TE = 10.3 ms, flip angle = 40°, averages = 96) with a total acquisition time of 2:33 h. Three parallel slices were acquired with an isotropic spatial in-plane resolution of 32 μm (FoV = 25.6 × 25.6 mm, matrix size = 800 × 800) and a slice thickness of 170 μm. Following intensity inhomogeneity correction using a simple linear gradient, the images were interpolated (bilinear) to a matrix size of 2400 × 2400. To avoid dehydration during ex-vivo imaging, the sample was wrapped in cling film.
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Histopathology
After ex-vivo MRM, the enucleated bulb was fixed in a 4% formalin solution for at least 24 h and cut in an anteroposterior manner after cutting the resection margin of the optic nerve. Two- to three-micrometer tissue sections, which matched the slices used in 7.0 T ex-vivo MRI, were stained with hematoxylin and eosin and peroxidase Schiff reagent. To observe possible venous invasion, further tissue sections were added in the planes needed. Additional fluorescence in-situ hybridization (FISH) was performed to test for monosomy of chromosome 3 [19,20]. GNA11 and GNAQ mutation analysis was carried out [21].
Results Fundus photography
Slit lamp ophthalmoscopy (Fig. 1a) showed a large retinal detachment in the lower circumference with an underlying tumor mass in the 6 o’clock position. The malignant melanoma protruded prominently from the surface, giving rise to the typical mushroom shape, and had a pigmented surface. However, detailed assessment was limited by the surrounding retinal detachment.
The sclera showed uniform hypointensity on T2w images with no evidence of infiltration. Anteriorly, the tumor extended to the ciliary body, which appeared distorted (Fig. 3). Signal intensity of the ciliary body was comparable with that of the nonaffected eye, making tumor infiltration unlikely. Within the tumor, multiple tubular structures of low T2 signal intensity were detected. The subretinal fluid collection appeared hypointense, consistent with recent hemorrhage. Ex-vivo magnetic resonance microscopy at 9.4 T
T2*w imaging at 9.4 T delineated the microstructure of the mass (Fig. 4) with excellent correlation to conventional histology (Fig. 5a and b). The sclera showed a uniform low signal intensity. The outer margin of the sclera appeared slightly distorted because of susceptibility artifacts. The retina was clearly differentiated from the tumor and the two layers of the retina could be appreciated. The ciliary body was distorted, but there were no signs of infiltration as shown at 7.0 T. Compared with ex-vivo MRM at 7.0 T, the ultrastructure of the tumor was delineated in more detail on the T2*w images. Histopathology
High-frequency ultrasound
HFUS showed a dome-shaped mass of low reflectivity with a highly reflective surface. The detached retina appeared hyper-reflective with subretinal hypoechogenic effusions (Fig. 1b). The mass height was 10 mm and the basal diameter was 13 mm. There was no evidence of extraocular growth. Duplex ultrasound
CDUS showed intense vascularization of the domeshaped mass with several arteries originating from the base of the tumor (Fig. 1c) and normal vasculature in the adjacent choroid and retina. In-vivo ultrahigh field MRI at 7.0 T
The tumor showed high signal intensity in T1w MRI and presented as a dome-shaped mass with smooth margins and a size of 8 × 15 mm (Fig. 1d and Fig. 2). The tumor was clearly delineated from the hypointense sclera and retina. There was no evidence of extraocular growth. The retina was attached to the optic nerve head ending. Retinal detachment had a wing-shaped appearance. Subretinal fluid collections appeared slightly hyperintense compared with the vitreous body, indicating protein-rich fluid or concomitant hemorrhage, which was confirmed by conventional histology. Ex-vivo magnetic resonance microscopy at 7.0 T
On T2w images the tumor appeared hypointense compared with the vitreous body (Fig. 1g and Fig. 3). The tumor showed a clearly defined, strongly hypointense margin clearly demarcating the tumor from the retina.
The gross specimen showed a brown tumor 16 mm size at the base and 8.5 mm in the prominence was found at the lower pole of the vitreous body. Macroscopically, the retina was detached and the sclera was intact. No infiltration of the ciliary body was observed. The tumor showed no contact with the optic nerve. Microscopically, at × 10 magnification, prominent ectatic tumor vessels were noticeable. The tumor was composed of a mixture of malignant spindle A and spindle B cells in a typical fascicular pattern (Fig. 5c) and showed initial scleral and venous invasion (Fig. 5d). Immunohistochemistry with a primary antibody against melan A (Dako, Hamburg, Germany) was strongly positive (Fig. 5e). Only a few mitotic figures were visible in hematoxylin and eosin stains. Tumor growth toward the ciliary body was observed, but no certain invasion (Fig. 5a and b). FISH analysis for chromosomes 3 and 8 showed disomy 3 and 8 (Fig. 5f). In the additional mutation analysis of exons 4 and 5 of GNA11 and GNAQ, exon 5 point mutation Q209L of the GNA11 gene was found.
Discussion Depending on the extent of the tumor, choroidal melanoma can be treated with curative intent. Pretreatment assessment of precise tumor extension is mandatory because knowledge of tumor size and location is essential to choose the appropriate treatment that governs the overall prognosis [1]. Yet, clinical routine invivo imaging is limited in early detection of small tumors because of inherent limitations – for example, optical distortions [22].
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Multimodal imaging of choroidal melanoma Lindner et al. 255
Fig. 1
Fundus photography
Duplex ultrasound
HF ultrasound (b)
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MRI at 7 T
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MRI at 9.4 T
Multimodal imaging of choroidal melanoma. (a–d) In-vivo results derived from conventional fundus photography, high-frequency (HF) and duplex ultrasound, and MRI. (e–h) Multimodal imaging results obtained after enucleation of the eye [scale bar in (e) also applies to (f)–(h)].
Fig. 2
# ∗
10 mm Sagittal T1-weighted in-vivo MR image (7.0 T) of the human bulb. Strongly hyperintense mass (*) with the corresponding retinal detachment (#).
UHF-MRI and MRM are emerging techniques for the characterization of ocular masses [22,23]. Unlike ultrasound, which depends on the operator’s experience [24], image quality of MRM and UHF-MRI primarily relies on the sensitivity at high magnetic fields and on the uniformity of the transmission and reception fields [25]. Previous studies have reported an excellent correlation between MRM and conventional histology with respect to tumor size and ultrastructure [17]. These findings are supported by our study. In addition, it is possible to clearly distinguish between tumor and surrounding subretinal fluid collections on both in-vivo and ex-vivo images. Our study also shows that, because of different signal intensities, hemorrhage can be differentiated from subretinal effusion on T1w and T2w images. UHF-MRI and MRM showed an excellent correlation with conventional histology for tumor size and shape. Furthermore, both imaging techniques enable
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MRM enables both detection of tumor-feeding vessels [27] and assessment of the extent of vascularity [28]. The latter is a possible biomarker of the degree of malignancy and can serve to monitor the response to therapy.
Fig. 3
Furthermore, changes in tumor microstructure can be used as a potential surrogate parameter for treatment response.
# ∗ 10 mm Sagittal T2-weighted ex-vivo MR image (7.0 T) of the human bulb showing an inhomogeneous hypointense mass (*) with the corresponding retinal detachment (#). Potential infiltration of the ciliary body (arrow).
Therefore, UHF-MRI may facilitate initial diagnostic assessment in patients first presenting with an intraocular mass. Although UHF-MRI cannot yet replace biopsy for the final confirmation of the diagnosis before treatment [3], our results are promising and the diagnostic accuracy of UHF-MRI and MRM should be evaluated in comparison with conventional histology in a prospective study of a larger patient population.
assessment of the ultrastructure of the tumor. The hypointense structures shown on T2w images in ex-vivo MR histology at 7.1 T correlate well with histopathologically proven intratumoral hemorrhages and melanin deposits. With MR histology at 9.4 T, the ultrastructure can be appreciated in more detail and the tubular structures correlate well with the course of collagen fibrils and melanin deposits. On UHF-MRI, the tumor appears hyperintense on T1w images because of the paramagnetic effects of melanin.
Despite providing an important multimodality comparison, our study has some limitations. First, MRM can be time consuming, which increases the risk of motionrelated artifacts. However, the occurrence of motionrelated artifacts can be reduced by improving patient comfort with the use of dedicated RF coils in conjunction with dedicated acquisition schemes [14]. Second, UHFMRI allows an in-plane spatial resolution as good as 100 μm. In comparison, MRM provides a spatial resolution of up to 30 μm. Although this is inferior to conventional histology, the major advantage of UHF-MRI is its noninvasiveness. Because of the limited spatial resolution, early scleral and venous infiltration will be missed. How this will affect clinical outcome has to be evaluated in a larger patient cohort. Spatial resolution of MRI is also inferior to optical coherence tomography. However, MRI enables undistorted simultaneous assessment of the eye and the orbit. Further improvements in MR technology, RF coil design, and optimization of MRI protocols tailored to ophthalmic imaging may overcome the current limitations – for example, RF power deposition constraints, ultimately allowing in-vivo imaging with a spatial resolution currently only available for ex-vivo imaging [29–31]. Although imaging protocols for UHF-MRI are tailored to clinical imaging with acquisition of T1w and T2w sequences and the use of contrast agent, MRM imaging protocols are optimized for providing highspatial-resolution and eliminating artifacts related to magnetic field strength. Therefore, imaging protocols differ between the different modalities. Finally, although UHF-MRI and MRM may facilitate the diagnosis of intraocular masses and may therefore reduce the need for biopsies, invasive tissue sampling will still be necessary for genetic analysis.
Doppler ultrasound enables assessment of the vascularity of an intraocular lesion and is a valuable tool for the diagnosis of recurrent melanoma [26]. Contrast-enhanced
In conclusion, UHF-MRI and MRM are promising tools for imaging intraocular lesions. With wider availability, MRI may help to overcome some of the limitations of
Fig. 4
10 mm T2*-weighted image of the tumor derived from ex-vivo magnetic resonance microscopy at 9.4 T.
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Multimodal imaging of choroidal melanoma Lindner et al. 257
Fig. 5
(a)
(b)
500 μm (c)
500 μm (d)
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#
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100 μm (f)
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Choroidal melanoma of a left eye. (a) Overview, of pigmented choroidal melanoma lying close to the ciliary body [hematoxylin and eosin staining (H&E) × 2]. (b) T2*-weighted image acquired ex-vivo at 9.4 T. (c) Choroidal melanoma spindle cell type A and B with pigmentation and noticeable ectatic venous blood vessel (arrow) (H&E, × 20). (d) Initial scleral invasion (#) and venous invasion with ectatic capillary vein (*) within melanoma tissue (peroxidase Schiff reaction, × 10). (e) Spindle-cell choroidal melanoma with strong melan A immunoreactivity and a fascicular growing pattern (melan A, × 20). (f) Disomy of chromosomes 3 and 8 (fluorescence in-situ hybridization, × 100).
current clinical in-vivo imaging techniques. Beyond anatomic imaging, MRM enables insights into the microstructure of ocular tumors. This capability may improve the diagnostic characterization of ocular tumors and might serve as a biomarker to monitor treatment response.
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Conflicts of interest
Thoralf Niendorf is founder and CEO of MRI.TOOLS GmbH, Berlin, Germany. The remaining authors have no conflicts of interest.
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