Herein, we described a nine-month-old male with a sporadic, isolated, intracranial, sellar-suprasellar region retinoblastoma (i.e., without evidence of preceding, concomitant, or subsequent development of retinal or pineal gland neoplasia). DNA array-based methylation classified the tumor with high confidence as pineoblastoma group A/intracranial retinoblastoma.
Given the diffuse and strong staining essentially limited to synaptophysin, following performance of an extensive IHC stain panel, many CNS and extra-CNS small cell embryonal tumors were considered in our differential diagnosis. There was no C19MC locus amplification or Lin28a IHC reactivity, making embryonal tumor with multilayered rosettes (ETMR) or related tumor unlikely. Additionally, neither IHC nor FISH detected alterations in BCOR, CIC, or NUTM1, making newly described pediatric embryonal tumors such as the CNS high-grade neuroepithelial tumor with BCOR alteration and CNS Ewing sarcoma family tumor with CIC alteration unlikely. The absence of EWSR1 rearrangement by FISH helped exclude intra- or extra-cranial Ewing’s sarcoma. Retained nuclear expression of INI-1 and Brg-1 excluded atypical teratoid/rhabdoid tumors. Comprehensive germ cell markers, including SALL4, were negative, ruling out poorly differentiated malignant germ cell tumor. Negative staining for Olig-2 and GFAP essentially ruled out a poorly differentiated high-grade glioma/astrocytoma. Negative staining for EMA and GFAP made a poorly differentiated ependymoma unlikely, and the negative Olig-2 made CNS neuroblastoma with FOXR2 activation unlikely. Although synaptophysin was positive, negative chromogranin staining made a poorly differentiated neuroendocrine tumor unlikely. The lack of glandular elements and lack of DICER1 alteration made pituitary blastoma unlikely. Other negative IHC markers (e.g. myogenin, HMB-45, Neu-N, neurofilament, Oct 3/4, Glypican 3, CD1a, and Langerin) essentially excluded rhabdomyosarcoma, melanoma, malignant neuronal/glio-neuronal neoplasms, and Langerhans cell histiocytosis. Instead, the overall findings of diffuse synaptophysin reactivity, presence of Flexner-Wintersteiner-like rosettes, DNA array-based methylation classification, detected RB1 alterations, and complete loss of RB1 expression by IHC in tumor cells, allowed a confident diagnosis of intracranial retinoblastoma.
Along with the tumor location, overall histopathology and molecular characteristics, the methylation-based classification allowed for an essentially definitive diagnosis of intracranial retinoblastoma in what would otherwise have been a difficult to classify embryonal neoplasm. Further molecular workup elucidated the mechanism of somatic RB1 inactivation in this tumor in the absence of germline RB1 or DICER1 alterations. As part of a clinical trial for medulloblastomas and other CNS embryonal tumors, our patient was treated with intensive chemotherapy followed by three tandem stem-cell rescues and with no evidence of residual tumor at completion of therapy. Similar regimens have been described in the treatment of other intracranial retinoblastomas, obviating the use of radiotherapy .
The integration of classification by genome-wide DNA array-based methylation profiling into the clinical diagnostic paradigm for CNS tumors with challenging histopathology has aided in refining both diagnosis and clinical management . The utility of this methodology in combination with comprehensive genomic profiling was evident in this patient, as standard histopathological work-up originally resulted in a less-definitive diagnosis of CNS embryonal neoplasm, NOS, for this infant.
Given the differential diagnoses of intracranial retinoblastoma and pituitary blastoma, potential germline alterations of RB1 and DICER1 were investigated but not found. Of clinical importance, children with hereditary RB1 alterations are at increased risk for developing midline intracranial tumors, including pineal and sellar region tumors . This phenomenon can be attributed to a common embryogenesis between the retinae and pineal gland, explaining the co-occurrence of retinoblastomas and pineoblastoma in patients with “trilateral” retinoblastoma. Moreover, the presence of ectopic photoreceptor cells along intracranial portions of the optic nerve system provides an explanation for the development of midline, intracranial, sellar region retinoblastoma [26, 27, 31]. Therefore, accurately identifying individuals with germline RB1 alterations is critical for appropriate genetic counseling and clinical management (e.g., surveillance strategies and cancer risk assessment) . Notably, diagnosis of intracranial retinoblastoma in an individual without hereditary retinoblastoma, incorporating DNA array-based methylation profiling, has previously been described , though this was in a patient with a concomitant pineal lesion. This report and the aforementioned prior study  highlight the utility of this methodology for this rare subset of non-heritable intracranial retinoblastoma.
Although the genomic landscape of retinoblastoma is diverse, large structural rearrangements are infrequently reported and, when described, are typically associated with chromothripsis [2, 15, 21, 22, 30]. Herein, the identification of the RB1-SIAH3 and ZC3H13-KLHL1 fusions by RNA-sequencing led us to further investigate the possibility of a larger chromosomal event. RB1 and SIAH3 reside in close proximity (~ 2 Mb apart) to one another on chromosome 13 but are transcribed in alternate directions. The chimeric protein product of the RB1-SIAH3 fusion is predicted to truncate the linker domain and pocket domain B of RB1. The interaction of the RB1 pocket domains A and B with the linker domain is critical to tumor suppression and the E2F regulatory function associated with RB1 . Through SMRT sequencing technologies, we identified a second RB1 fusion with non-genic chromosome 13 sequence expected to disrupt RB1 protein expression. Both identified fusions demonstrated RB1 breakpoints within intron 17. Although structural rearrangements in RB1 are exceedingly rare, intron 17 breakpoints have been reported in multiple tumors with complex structural rearrangement in two independent studies, suggesting this may be a recurrent region for rearrangement [2, 20]. However, larger studies are necessary to confirm this finding. These fusions are predicted to result in complete loss-of-function, as evidenced by the absence of RB1 protein expression as demonstrated by IHC herein. Through multiple methodologies, we determined that the copy number state of chromosome 13 remained neutral, despite the presence of complex structural rearrangements.
Utilization of NGS methodologies to fully elucidate the complex biological mechanism of this intracranial retinoblastoma proved challenging. However, through the use of multiple sequencing methodologies and algorithms, we identified significant overlap in the high-confidence SVs. SMRT sequencing technologies overcome many of the limitations of NGS approaches which rely on short reads, including the ability to interrogate reads > 10 kb in length from a single DNA molecule for SV detection . These approaches are more sensitive to larger chromosomal events than NGS technologies; the reads are more likely to encompass the breakpoint or the entire structural rearrangement and can provide the advantage of predicting phased genomic variation [19, 25]. Through SMRT sequencing methodologies, we were able to confirm two somatic rearrangements within RB1, predicted to be in trans, and supported by the absence of RB1 staining by IHC.
Our patient was treated on Head Start 4, a clinical trial for pediatric patients with medulloblastoma or other CNS embryonal tumors and randomized to undergo tandem autologous hematopoietic stem-cell rescues with three cycles of carboplatin and thiotepa. A prior report used a similar regimen of tandem high-dose chemotherapy with carboplatin, etoposide, cyclophosphamide, and thiotepa followed by autologous hematopoietic stem cell transplant for an infant with intracranial retinoblastoma . In both our described patient and the prior case , a partial resection with a stable response was achieved, obviating the need for ancillary radiation therapy and its associated morbidity in this age group. Retrospective studies and case reports have shown the utilization of high-dose chemotherapy in the setting of trilateral retinoblastoma improves survival, although a standardized regimen has yet to be determined [4, 34, 35].
In summary, we report on an infant with an isolated, intracranial, sellar-suprasellar region retinoblastoma in the absence of a germline RB1 alteration. A comprehensive approach to molecular profiling, including DNA array-based methylation profiling, Illumina NGS, and Pacific Bioscience SMRT sequencing, were critical for providing a definitive diagnosis and understanding the biological mechanism of RB1 inactivation. The patient was treated using high-dose chemotherapy with autologous hematopoietic stem cell transplant, a regimen that has demonstrated benefit in small studies of trilateral retinoblastoma [4, 34, 35].