LIN28A immunoreactivity is a potent diagnostic marker of embryonal tumor with multilayered rosettes (ETMR)
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- Korshunov, A., Ryzhova, M., Jones, D.T.W. et al. Acta Neuropathol (2012) 124: 875. doi:10.1007/s00401-012-1068-3
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Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR.
KeywordsETMRPediatric brain tumorLIN28ADiagnostic marker
Embryonal tumor with multilayered rosettes (ETMR), also previously reported as embryonal tumor with abundant neuropil and true rosettes (ETANTR) is a rare, albeit likely underdiagnosed embryonal CNS neoplasm with fewer than 100 cases reported in the literature since its initial description in 2000 [3, 5, 19]. In the 2007 WHO classification, ETANTR was only discussed as a possibly unique variant of CNS tumors . Supratentorial localization has mainly been reported, but it may also occur in the brain stem and cerebellum [3, 5, 11]. ETMR predominantly affects children under the age of 3–4 years and is associated with a highly aggressive disease course with reported overall survival times ranging from 5 to 30 months. The histopathological appearance is distinctive in its mixture of poorly differentiated small cell areas with prominent multilayered (ependymoblastic) rosettes, and areas with focal neuronal differentiation, thus combining features of CNS neuroblastoma and ependymoblastoma [3, 5, 11]. On the other hand, a wide spectrum of morphological patterns may be encountered, with some features being considerably less specific. For this reason, misdiagnosis is not uncommon, with common considerations including “small round blue cell tumor, not otherwise specified”, i.e., central nervous system primitive neuroectodermal tumors (CNS PNET) including controversial variants (e.g., ependymoblastoma, central neuroblastoma), medulloblastoma, and anaplastic ependymoma [9, 13, 25]. Moreover, similar rosettes can sometimes occur in other embryonal CNS tumors (e.g., CNS PNET, AT/RT) . Nevertheless, accurate pathological verification of ETMR is of important clinical relevance, given its poor response to current CNS PNET treatment protocols and thus universally fatal outcome [5, 11, 13]. Amplification of a miRNA cluster at 19q13.42 has been identified as a genetic hallmark of ETMR, affecting up to 95 % of samples tested and it is considered a unifying molecular diagnostic marker for these tumors [11, 13, 16, 25].
However, the requirement of non-commercial custom made BAC probes for FISH testing renders this assay virtually inaccessible to nearly every pathology laboratory around the globe. In contrast, the use of immunohistochemistry is universally utilized in neuropathology centers everywhere. As such, ETMR is a highly attractive target for developing tumor-specific immunohistochemistry. For this purpose, we herein performed a screen for ETMR-specific genes based on gene expression profiles collected for a large cohort of various brain tumors (n = 1,404). We selected the most specifically expressed gene, namely LIN28A, and subsequently tested its diagnostic specificity for ETMR with a high-quality commercial antibody on a large cohort (n = 816) of paraffin-embedded brain tumors. Our results show that LIN28A expression can be used as a specific diagnostic biomarker to detect ETMR.
Materials and methods
Distribution of LIN28A immunoexpression in various pediatric brain tumors
LIN28A + diffuse
LIN28A + focal
37 (100 %)
36 (97 %)
6 (12 %)
50 (100 %)
Results and discussion
Therefore, this study represents a report of LIN28A as a highly specific and sensitive diagnostic marker for the distinct pathological verification of ETMR and, correspondingly, as a new important diagnostic tool to be routinely applied in all malignant childhood brain tumors where ETMR may be considered a possible diagnosis. Variability of LIN28A gene expression in ETMR samples correlated closely with the intratumoral heterogeneity observed immunohistochemically. Focal positivity of LIN28A in a small subset of AT/RTs is not considered problematic when taking the simultaneous loss of INI1 expression into account, with the latter representing a fairly specific molecular marker for AT/RT. On the other hand, LIN28A immunoexpression has also been detected recently in CNS germ-cell tumors with diffuse staining patterns found in germinomas, embryonal carcinomas and yolk sac tumors but being only focal in immature teratomas . Nevertheless, these tumors usually affect older children (apart from the some immature teratomas of infancy) than those at risk for ETMRs, are typically not situated in those locations commonly involved by ETMRs, and do not enter the differential diagnosis on histopathologic and immunohistochemical grounds.
Our current data also confirmed that ETMR is a unique clinico-pathologic entity from a molecular perspective, exhibiting 19q13.42 amplification and high levels of LIN28A expression accompanied by particularly aggressive clinical behavior. It will now be important to understand the biological consequence of overexpression of such universal and direct miRNA translation regulators as LIN28A and LIN28B in ETMR, taking into account the prototypic amplification of the oncogenic miRNA cluster at 19q13.42 in these tumors.
Andrea Wittmann, Laura Sieber, and Linda Linke are acknowledged for excellent technical assistance and Dominik Sturm for help with the Figures. This study was supported by grants from the Dutch Cancer Foundations KWF (2010-4713) and KIKA to MK.
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