A Rare Adult Primary Intracranial Sarcoma, DICER1-Mutant Identified by Epigenomic Profiling: A Case Report

Diagnoses of primary malignant mesenchymal brain tumors are a challenge for pathologists. Here, we report the case of a 52-year-old man with a primary brain tumor, histologically diagnosed as a high-grade glioma, not otherwise specified (NOS). The patient underwent two neurosurgeries in several months, followed by radiotherapy and chemotherapy. We re-examined the tumor samples by methylome profiling. Methylome analysis revealed an epi-signature typical of a primary intracranial sarcoma, DICER1-mutant, an extremely rare tumor. The diagnosis was confirmed by DNA sequencing that revealed a mutation in DICER1 exon 25. DICER1 mutations were not found in the patient’s blood cells, thus excluding an inherited DICER1 syndrome. The methylome profile of the DICER1 mutant sarcoma was then compared with that of a high-grade glioma, a morphologically similar tumor type. We found that several relevant regions were differentially methylated. Taken together, we report the morphological, epigenetic, and genetic characterization of the sixth described case of an adult primary intracranial sarcoma, DICER1-mutant to-date. Furthermore, this case report underscores the importance of methylome analysis to refine primary brain tumor diagnosis and to avoid misdiagnosis among morphologically similar subtypes.


Introduction
Primary malignant mesenchymal brain tumors are a diagnostic challenge because they are rare, similar in clinical presentation, and highly heterogeneous, at both the morphological and genomic levels [1]. Recently, a new "omic" approach based on whole-genome DNA methylation (methylome) analysis has allowed precise classification and subclassification of many tumors on the basis of their "epigenetic signatures" [2]. The methylation-based classifier of brain tumors, developed by Capper et al. in 2018 [3], is constantly updated and implemented by the addition of new entities, including sinonasal tumors and sarcomas [4,5].
Here, we report the case of a primary brain tumor, first diagnosed as a high-grade glioma, NOS (not otherwise specified) in our academic institution. The tumor DNA methylation profile revealed, instead, an epi-signature typical of a primary intracranial sarcoma, DICER1-mutant. The diagnosis was confirmed by tissue sample sequencing of the DICER1 gene. To date, in adults, only five other cases of primary intracranial sarcoma, DICER1-mutant, have been reported [5][6][7][8] (Table 1). In addition, this case presentation highlights the importance of methylome analysis to refine primary brain tumor diagnosis and to avoid misdiagnosis among morphologically similar subtypes.

Clinical and Radiological Description
In June 2020, a 52-year-old man received emergency surgery because of a cerebral hemorrhage in the left temporo-parietal lobe ( Figure 1A). Post-operative magnetic resonance imaging (MRI) revealed a pathological mass in the same left temporo-parietal lobe ( Figure 1B). Two months later, the patient experienced a new hemorrhagic episode, and he received a second emergency craniotomy for decompression and subtotal mass reduction ( Figure 1C). In December 2020, because of his worsening clinical condition, the patient underwent a new reductive subtotal surgery. In the patient's pathological history, a diagnosis of malignant histiocytosis was present. In fact, in 1987 he presented with axillary lymphadenopathy. An excisional lymph node biopsy revealed the abovementioned diagnosis. At that time, total-body computed tomography (CT) staging was negative for other localizations. DICER1-mutant, have been reported [5][6][7][8] (Table 1). In addition, this case presentation highlights the importance of methylome analysis to refine primary brain tumor diagnosis and to avoid misdiagnosis among morphologically similar subtypes.

Clinical and Radiological Description
In June 2020, a 52-year-old man received emergency surgery because of a cerebral hemorrhage in the left temporo-parietal lobe ( Figure 1A). Post-operative magnetic resonance imaging (MRI) revealed a pathological mass in the same left temporo-parietal lobe ( Figure 1B). Two months later, the patient experienced a new hemorrhagic episode, and he received a second emergency craniotomy for decompression and subtotal mass reduction ( Figure 1C). In December 2020, because of his worsening clinical condition, the patient underwent a new reductive subtotal surgery. In the patient's pathological history, a diagnosis of malignant histiocytosis was present. In fact, in 1987 he presented with axillary lymphadenopathy. An excisional lymph node biopsy revealed the abovementioned diagnosis. At that time, total-body computed tomography (CT) staging was negative for other localizations.  After the second neurosurgery, the patient received 10 cycles of external-beam radiation therapy (EBRT) at 3 Gray, but radiotherapy was interrupted after 9 cycles because of the poor performance status of the patient. After the last neurosurgery, the patient received chemotherapy based on doxorubicin and dacarbazine. The therapy was interrupted after two cycles because the patient died.
The medical history of the patient is depicted in Figure 2.

Histopathology
As shown in Figure 3, hematoxylin-eosin staining of the lesion excised in December 2020 revealed a highly cellular neoplasm. The tumor cells were spindle-shaped to epithelioid, arranged in a patternless solid growth. There was brisk mitotic activity, also with atypical figures, and geographic necrosis with a "pseudo-palizading pattern". The tumor was highly hemorrhagic, with abundant foamy histiocytes infiltrating the mass. Upon immunohistochemistry, the tumor cells were found to be GFAP (gliofibrillary acid protein) negative, SMA (smooth muscle actin) negative, IDH1 (isocitrate dehydrogenase1) wild-type, and the cellular proliferative index Ki67 was high, near 70%. A diagnosis of high-grade glioma, NOS, was made.

Classification Based on DNA Methylation Profiling
To refine the histopathological characterization, we performed a DNA methylation profile on the same lesion using the Illumina Technologies EPIC array 850 k.
Formalin-fixed paraffin-embedded (FFPE) tumoral sample DNA was extracted using the FFPE DNA Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. The extracted DNA was converted using bisulfite (EZ gold Kit, Zymo Research, Irvine, CA, USA), following the manufacturer's instructions. The bisulfite-treated DNA was analyzed using the Illumina EPIC ARRAY 850 Bead-Chip (Infinium human methylation EPIC, Illumina, San Diego, CA, USA), as described in [9]. The array covered 850.000 CpG distributed across the genome. The resulting data (in IDAT format) were processed and analyzed using a previously published bioinformatic algorithm available from the German Cancer Research Center (DKFZ, Heidelberg, Germany) [3]. The DKFZ pipeline for methylome analysis calculated a "calibrated score", which provided information about the methylation classes and the presence and relative amounts of tumor cells. This score ranges between 0 and 1, where 1 indicates a very high confidence of tumor identity and classification. The generated methylation data were compared with the Heidelberg brain tumor classifier to assign a subgroup score for the tumor compared with 91 different brain tumor entities [10]. The methylation class family is predicted if the score is >0.9, but even a score of >0.5 is sufficient to identify predicted methylation subclasses.
Methylome analysis showed a match (scores > 0.9) with the new DICER1-mutant class: primary intracranial sarcoma, DICER1-mutant [11]. To further confirm the diagnosis, we also performed a methylation profile analysis of the tumor removed in June 2020. In this case, the methylome analysis showed the epi-signature typical of primary intracranial sarcomas, DICER1-mutant. In accordance with the frequent CNV alterations described in primary intracranial sarcomas, DICER1-mutant [6], and with the observed high Ki67 index (Figure 3), we found, in both cancer lesions, 1q and 9p chromosome deletions and high amplification of the CCND2 gene encoding for the D2-type cyclin protein, a strong regulator of cell cycle onset and, thus, of proliferation ( Figure 4A). MGMT promoter methylation analysis by Methylation Specific PCR (MSP) [12] showed that the MGMT promoter was not methylated. Methylome analysis showed a match (scores > 0.9) with the new DICER1-mutant class: primary intracranial sarcoma, DICER1-mutant [11]. To further confirm the diagnosis, we also performed a methylation profile analysis of the tumor removed in June 2020. In this case, the methylome analysis showed the epi-signature typical of primary intracranial sarcomas, DICER1-mutant. In accordance with the frequent CNV alterations described in primary intracranial sarcomas, DICER1-mutant [6], and with the observed high Ki67 index (Figure 3), we found, in both cancer lesions, 1q and 9p chromosome deletions and high amplification of the CCND2 gene encoding for the D2-type cyclin protein, a strong regulator of cell cycle onset and, thus, of proliferation ( Figure 4A). MGMT promoter methylation analysis by Methylation Specific PCR (MSP) [12] showed that the MGMT promoter was not methylated.

Molecular Investigation: DICER1 and p53 Gene Sequencing
To verify the presence of the DICER1 mutations predicted by methylome analysis, we performed DNA sequencing of the DICER1 gene utilizing DNA extracted from the tumor removed in December 2020. As shown in Figure 4B, we found a homozygous, hotspot mutation in DICER1 exon 25, resulting in the aminoacidic substitution Glu1813Ala. The same mutation was reported in other cases [13]. Thus, DICER1 DNA sequencing confirmed the diagnosis predicted by the methylome profile. The same Glu1813Ala mutation was also found at 50% in the primary intracranial lesion removed in June 2020. In this case, however, we could not definitively determine the zygosity status because of the heterogeneity in tumor cell composition. DICER1 variants have been described not only in sporadic tumors but also in familial cancers, patients with germline DICER1 mutations being predisposed to multiple cancer types [14]. Thus, to address this matter, we sequenced the DNA extracted from the peripheral blood of the patient and from the axillary lymph node eradicated 35 years before at the time of the malignant histiocytosis onset. Sequencing analyses of both blood ( Figure 4C) and lymph node did not detect any mutation in DICER1. In light of these data, we excluded a germinal mutation in DICER1, demonstrating that tumor DICER1 alterations were caused by two somatic mutational events.
In primary intracranial sarcoma, DICER1-mutant, p53 loss-of-function mutations have frequently been described [5]. However, no p53 mutations were detected in the two tumor samples we analyzed.

Methylome Profiling of Primary Intracranial Sarcoma, DICER1 Mutant versus Mesenchymal Glioblastoma
Primary intracranial sarcomas, DICER1-mutant, are morphologically similar to highgrade gliomas, and thus, the histopathological differential diagnosis may be challenging. To conduct an explorative analysis to determine the extent of the methylation differences, the methylome of the primary intracranial sarcoma, DICER1-mutant (surgically removed on December 2020), was compared with the methylome profile of a high-grade glioma. Bioinformatic analyses were performed using RnBeads scripts, as described in [15,16]. Only the CpG sites with a difference of at least 25% in DNA methylation between the primary intracranial sarcoma, DICER1-mutant, and the high-grade glioma were filtered and retained. As shown in Figure 5, we identified 20.022 CpGs with a lower degree of DNA methylation in the primary intracranial sarcoma, DICER1-mutant, while 8.254 CpG sites were found more methylated in the primary intracranial sarcoma, DICER1-mutant, compared with the high-grade glioma. We then associated the differentially methylated CpG sites with the respective genes using the R-based library "GenomicRanges" [17]. Only genes with at least five differentially methylated CpG sites were considered. In this way, as shown in Figure 5

Discussion
Primary intracranial sarcomas, DICER1-mutant, are rare neoplasms identified and typified through analysis of the whole-genome DNA methylation profile. To date, very few cases have been described, and these were predominantly primary intracranial and pediatric [5][6][7][8]. In general, primary intracranial sarcomas, DICER1-mutant, are invariably associated with a poor prognosis. In contrast, the prognosis for extra-cranial DICER1 sarcomas with mutations in DICER1 correlates with the histopathological grade. Here we report a new case of primary intracranial sarcomas, DICER1-mutant that to the best of our knowledge, is the sixth ever described in an adult.
Histopathological diagnosis of primary intracranial sarcomas, DICER1-mutant, is problematic because they have several morphological characteristics in common with other tumor entities. A considerable morphological overlap exits between primary intracranial sarcomas, DICER1-mutant, and high-grade gliomas, both tumor types being characterized by high cellularity, necrosis, vascular proliferation, a high-mitotic index, and high-grade cytological features. For this reason, the first interpretation, purely morphological, can lead to a diagnosis of a high-grade glial lesion. Even a negative immunostaining for the glial fibrillary acidic protein (GFAP) cannot exclude high-grade glioma diagnosis, as GFAP-negative glioblastomas do exist [19]. A further complication is that gliosarcoma with a diffuse mesenchymal metaplasia and without residual glial component should be considered in the differential diagnosis. The main differential diagnoses include not only gliosarcoma, but also synovial sarcoma (CD99-, CD56-positive), malignant peripheral nerve sheath tumor (usually S100-and SOX10-positive), rhabdoid tumor (desmin-and cytokeratin-positive), malignant solitary fibrous tumor (STAT6-positive, variable CD34), rabdomyosarcoma (myogenin-, muscle-specific actin-, and desmin-positive) and metastatic undifferentiated carcinomas [6,20] We then performed KEGG analysis using DAVID [18] to identify the enriched pathways for the differentially methylated genes. In particular, 48 demethylated genes were assigned to the MAPK signaling pathway, already described as frequently activated in primary intracranial sarcomas, DICER1-mutant, and associated with a high tumor proliferative cellular rate and aggressiveness [6]. Because of the rarity of these tumor entities, the data available so far, it is not possible to obtain robust results; however, the above data suggest that the differences between the two entities are well reflected by the methylation signatures.

Discussion
Primary intracranial sarcomas, DICER1-mutant, are rare neoplasms identified and typified through analysis of the whole-genome DNA methylation profile. To date, very few cases have been described, and these were predominantly primary intracranial and pediatric [5][6][7][8]. In general, primary intracranial sarcomas, DICER1-mutant, are invariably associated with a poor prognosis. In contrast, the prognosis for extra-cranial DICER1 sarcomas with mutations in DICER1 correlates with the histopathological grade. Here we report a new case of primary intracranial sarcomas, DICER1-mutant that to the best of our knowledge, is the sixth ever described in an adult.
Histopathological diagnosis of primary intracranial sarcomas, DICER1-mutant, is problematic because they have several morphological characteristics in common with other tumor entities. A considerable morphological overlap exits between primary intracranial sarcomas, DICER1-mutant, and high-grade gliomas, both tumor types being characterized by high cellularity, necrosis, vascular proliferation, a high-mitotic index, and high-grade cytological features. For this reason, the first interpretation, purely morphological, can lead to a diagnosis of a high-grade glial lesion. Even a negative immunostaining for the glial fibrillary acidic protein (GFAP) cannot exclude high-grade glioma diagnosis, as GFAP-negative glioblastomas do exist [19]. A further complication is that gliosarcoma with a diffuse mesenchymal metaplasia and without residual glial component should be considered in the differential diagnosis. The main differential diagnoses include not only gliosarcoma, but also synovial sarcoma (CD99-, CD56-positive), malignant peripheral nerve sheath tumor (usually S100-and SOX10-positive), rhabdoid tumor (desmin-and cytokeratin-positive), malignant solitary fibrous tumor (STAT6-positive, variable CD34), rabdomyosarcoma (myogenin-, muscle-specific actin-, and desmin-positive) and metastatic undifferentiated carcinomas [6,20]. To date, primary intracranial sarcomas, DICER1-mutant, are a molecularly defined tumor type, the diagnosis being impossible without the detection