Successful Multimodal Treatment of Intracranial Growing Teratoma Syndrome with Malignant Features.

Molecular analysis of the growing teratoma syndrome has not been extensively studied. Here, we report a 14-year-old boy with a growing mass during treatment for a mixed germ cell tumor of the pineal region. Tumor markers were negative; thus, growing teratoma syndrome was suspected. A radical resection via the occipital transtentorial approach was performed, and histopathological examination revealed a teratoma with malignant features. Methylation classifier analysis confirmed the diagnosis of teratoma, and DMRT1 loss and 12p gain were identified by copy number variation analysis, potentially elucidating the cause of growth and malignant transformation of the teratoma. The patient remains in remission after intense chemoradiation treatment as a high-risk germ cell tumor.

Implications of DNA Methylation Classification in Diagnosing Ependymoma.

BACKGROUND: Ependymoma is a central nervous system (CNS) tumor that arises from the ependymal cells of the brain's ventricles and spinal cord. The histopathology of ependymomas is indistinguishable regardless of the site of origin, and the prognosis varies. Recent studies have revealed that the development site and prognosis reflect the genetic background. In this study, we used genome-wide DNA methylation array analysis to investigate the epigenetic background of ependymomas from different locations treated at our hospital. METHODS: Four cases of posterior fossa ependymomas and 11 cases of spinal ependymomas were analyzed. RESULTS: DNA methylation profiling using the DKFZ methylation classifier showed that the methylation diagnoses of the 2 cases differed from the histopathological diagnoses, and 2 cases could not be classified. Tumor that spread from the brain to the spinal cord was molecularly distinguishable from other primary spinal tumors. CONCLUSIONS: Although adding DNA methylation classification to conventional diagnostic methods may be helpful, the diagnosis in some cases remains undetermined. This may affect decision-making regarding treatment strategies and follow-up. Further investigations are required to improve the diagnostic accuracy of these tumors.

Decoding the DNA methylome of central nervous system tumors: An emerging modality for integrated diagnosis.

The definitive diagnosis and classification of individual cancers are crucial for patient care and cancer research. To achieve a robust diagnosis of central nervous system (CNS) tumors, a genotype-phenotype integrated diagnostic approach was introduced in recent versions of the World Health Organization classification, followed by the incorporation of a genome-wide DNA methylome-based classification. Microarray-based platforms are widely used to obtain DNA methylome data, and the German Cancer Research Center (Deutsches Krebsforschungszentrum [DKFZ]) has a webtool for a DNA methylation-based classifier (DKFZ classifier). Integration of DNA methylome will further enhance the precision of CNS tumor classification, especially in diagnostically challenging cases. However, in the clinical application of DNA methylome-based classification, challenges related to data interpretation persist, in addition to technical caveats, regulations, and limited accessibility. Dimensionality reduction (DMR) can complement integrated diagnosis by visualizing a profile and comparing it with other known samples. Therefore, DNA methylome-based classification is a highly useful research tool for auxiliary analysis in challenging diagnostic and rare disease cases, and for establishing novel tumor concepts. Decoding the DNA methylome, especially by DMR in addition to DKFZ classifier, emphasizes the capability of grasping the fundamental biological principles that provide new perspectives on CNS tumors.

Myxoid liposarcoma with nuclear pleomorphism: a clinicopathological and molecular study.

Myxoid liposarcoma (MLS) is a common type of liposarcoma. It is characterized by variably lipogenic uniform cells in myxoid stroma with arborizing capillaries and DDIT3 fusion. Nuclear uniformity is the rule, which is maintained even in high-grade round cell examples. In this study, we conducted an in-depth investigation of four MLS tumors that demonstrated nuclear pleomorphism in three patients. These cases accounted for 2.1% of 142 patients with MLS. All patients were male aged 26, 33, and 49 years. Nuclear pleomorphism was observed in both primary and metastatic tumors in one patient, a primary tumor in one patient, and a metastatic tumor in another patient. Pleomorphism was severe in three tumors and moderate in one. Histology resembled that of dedifferentiated liposarcoma with myxoid features, pleomorphic liposarcoma with myxoid features, or myxoid pleomorphic liposarcoma in two tumors, pleomorphic sarcoma with focal cartilaginous and rhabdomyoblastic differentiation in one tumor, and epithelioid pleomorphic liposarcoma in one tumor. All tumors harbored FUS::DDIT3 fusions and immunohistochemically expressed DDIT3. All tumors had TP53 mutations, whereas previous specimens with uniform cytology from the same patients lacked TP53 mutations. One tumor showed RB1 deletion and complete loss of Rb expression, which was unclassifiable using DNA methylation-based methods. The rare occurrence of nuclear pleomorphism is underrecognized in MLS and increases the complexity to the diagnosis of liposarcoma. DDIT3 evaluation can be liberally considered in liposarcoma assessment even in the presence of nuclear pleomorphism.

Inflammatory Rhabdomyoblastic Tumor: Clinicopathologic and Molecular Analysis of 13 Cases.

Inflammatory rhabdomyoblastic tumors (IRMTs) are newly recognized skeletal muscle tumors with uncertain malignant potential. We investigated 13 IRMTs using clinicopathologic, genetic, and epigenetic methods. The cohort included 7 men and 6 women, aged 23 to 80 years (median, 50 years), of whom 2 had neurofibromatosis type 1. Most tumors occurred in the deep soft tissues of the lower limbs, head/neck, trunk wall, and retroperitoneum/pelvis. Two tumors involved the hypopharyngeal submucosa as polypoid masses. Eight tumors showed conventional histology of predominantly spindled cells with nuclear atypia, low mitotic activity, and massive inflammatory infiltrates. Three tumors showed atypical histology, including uniform epithelioid or plump cells and mitotically active histiocytes. The remaining 2 tumors demonstrated malignant progression to rhabdomyosarcoma; one had additional IRMT histology and the other was a pure sarcoma. All 11 IRMTs without malignant progression exhibited indolent behavior at a median follow-up of 43 months. One of the 2 patients with IRMTs with malignant progression died of lung metastases. All IRMTs were positive for desmin and PAX7, whereas myogenin and MyoD1 were expressed in a subset of cases. Targeted next-generation sequencing identified pathogenic mutations in NF1 (5/8) and TP53 (4/8). All TP53 mutations co-occurred with NF1 mutations. TP53 variant allele frequency was much lower than that of NF1 in 2 cases. These tumors showed geographic (subclonal) strong p53 immunoreactivity, suggesting the secondary emergence of a TP53-mutant clone. DNA methylation-based copy number analysis conducted in 11 tumors revealed characteristic flat patterns with relative gains, including chromosomes 5, 18, 20, 21, and/or 22 in most cases. Widespread loss of heterozygosity with retained biparental copies of these chromosomes was confirmed in 4 tumors analyzed via allele-specific profiling. Based on unsupervised DNA methylation analysis, none of the 11 tumors tested clustered with existing reference entities but formed a coherent group, although its specificity warrants further study.

Genetic alterations that deregulate RB and PDGFRA signaling pathways drive tumor progression in IDH2-mutant astrocytoma.

In IDH-mutant astrocytoma, IDH2 mutation is quite rare and biological mechanisms underlying tumor progression in IDH2-mutant astrocytoma remain elusive. Here, we report a unique case of IDH2 mutant astrocytoma, CNS WHO grade 3 that developed tumor progression. We performed a comprehensive genomic and epigenomic analysis for primary and recurrent tumors and found that both tumors harbored recurrent IDH2R172K and TP53R248W mutation with CDKN2A/B hemizygous deletion. We also found amplifications of CDK4 and MDM2 with PDGFRA gain in the recurrent tumor and upregulated protein expressions of these genes. We further developed, for the first time, a xenograft mouse model of IDH2R172K and TP53R248W mutant astrocytoma from the recurrent tumor, but not from the primary tumor. Consistent with parent recurrent tumor cells, amplifications of CDK4 and MDM2 and PDGFRA gain were found, while CDKN2A/B was identified as homozygous deletion in the xenografts, qualifying for integrated diagnosis of astrocytoma, IDH2-mutant, CNS WHO grade 4. Cell viability assay found that CDK4/6 inhibitor and PDGFR inhibitor potently decreased cell viability in recurrent tumor cells, as compared to primary tumor cells. These findings suggest that gene alterations that activate retinoblastoma (RB) signaling pathways and PDGFR may drive tumor progression and xenograft formation in IDH2-mutant astrocytoma, which is equivalent to progressive IDH1-mutant astrocytoma. Also, our findings suggest that these genomic alterations may represent therapeutic targets in IDH2-mutant astrocytoma.

Distinct patterns of copy number alterations may predict poor outcome in central nervous system germ cell tumors.

We have previously reported that 12p gain may predict the presence of malignant components and poor prognosis for CNS germ cell tumor (GCT). Recently, 3p25.3 gain was identified as an independent predictor of poor prognosis for testicular GCT. Eighty-one CNS GCTs were analyzed. Copy number was calculated using methylation arrays. Five cases (6.2%) showed 3p25.3 gain, but only among the 40 non-germinomatous GCTs (NGGCTs) (5/40, 12.5%; p = 0.03). Among NGGCTs, those with a yolk sac tumor component showed a significantly higher frequency of 3p25.3 gain (18.2%) than those without (1.5%; p = 0.048). NGGCTs with gain showed significantly shorter progression-free survival (PFS) than those without (p = 0.047). The 3p25.3 gain and 12p gain were independent from each other. The combination of 3p25.3 gain and/or 12p gain was more frequent among NGGCTs with malignant components (69%) than among those without (29%; p = 0.02). Germinomas containing a higher number of copy number alterations showed shorter PFS than those with fewer (p = 0.03). Taken together, a finding of 3p25.3 gain may be a copy number alteration specific to NGGCTs and in combination with 12p gain could serve as a marker of negative prognosis or treatment resistance. Germinoma with frequent chromosomal instability may constitute an unfavorable subgroup.

Pulmonary inflammatory leiomyosarcoma represents a potential diagnostic pitfall of DNA methylation-based classification of sarcomas: a case report.

BACKGROUND: Pulmonary inflammatory leiomyosarcoma (PILMS) is a rare type of myogenic tumor with prominent lymphohistiocytic infiltration. Despite their histological similarities, PILMS is immunohistochemically and genetically distinct from soft tissue inflammatory leiomyosarcoma, and its clinicopathological picture including DNA methylome data remains still unknown. CASE PRESENTATION: Here we present a case of PILMS in an 18-year-old male who underwent lobectomy. As reported previously, the current case demonstrated spindle myoid cell proliferation with smooth muscle differentiation within a prominent lymphohistiocytic infiltration and a diploid genome with a MUC3A gene alteration. DNA methylation analysis predicted this case to be an "inflammatory myofibroblastic tumor" (IMT) according to the Deutsches Krebsforschungszentrum (DKFZ) classifier. The data was analyzed by t-distributed stochastic neighbor embedding, which plotted the case tumor in the vicinity of IMT, however, there were no IMT histological features. These discordant results could be due to background non-neoplastic inflammatory cells. CONCLUSIONS: As the DNA methylation classification of PILMS might be a potential diagnostic pitfall, an integrative histological and genetic approach is required for its accurate diagnosis.

KDM2B-Rearranged Soft Tissue Sarcomas Expand the Concept of BCOR-Associated Sarcoma.

Sarcomas with BCOR genetic alterations (BCOR-associated sarcomas) represent a recently recognized family of soft tissue and bone tumors characterized by BCOR fusion, BCOR internal tandem duplication, or YWHAE::NUTM2B fusion. Histologically, the tumors demonstrate oval to spindle cell proliferation in a variably vascular stroma and overexpression of BCOR and SATB2. Herein, we describe 3 soft tissue sarcomas with KDM2B fusions that phenotypically and epigenetically match BCOR-associated sarcomas. The cases included 1 infant, 1 adolescent, and 1 older patient. All tumors showed histologic findings indistinguishable from those of BCOR-associated sarcomas and were originally diagnosed as such based on the phenotype. However, none of the tumors had BCOR or YWHAE genetic alterations. Instead, targeted RNA sequencing identified in-frame KDM2B::NUTM2B, KDM2B::CREBBP, and KDM2B::DUX4 fusions. KDM2B fusions were validated using reverse-transcription PCR, Sanger sequencing, and in situ hybridization assays. Genome-wide DNA methylation analysis matched all 3 tumors with BCOR-associated sarcomas using the Deutsches Krebsforschungszentrum (DKFZ) classifier and t-distributed stochastic neighbor embedding analysis. One localized tumor showed a flat genome-wide copy number profile, and the patient remained disease-free after treatment. The other tumors showed multiple copy number alterations, including MDM2/CDK4 amplification and/or CDKN2A/B loss, and both tumors metastasized, leading to the patient's death in one of the cases. When tested using KDM2B immunohistochemistry, all 3 KDM2B-rearranged sarcomas showed diffuse strong staining, and all 13 sarcomas with BCOR genetic alterations also demonstrated diffuse, strong, or weak staining. By contrast, among 72 mimicking tumors, only a subset of synovial sarcomas showed focal or diffuse weak KDM2B expression. In conclusion, our study suggests that KDM2B-rearranged soft tissue sarcomas belong to the BCOR-associated sarcoma family and expand its molecular spectrum. This may be related to the known molecular relationship between KDM2B and BCOR in the polycomb repressive complex 1.1. Immunohistochemical analysis of KDM2B is a potentially valuable diagnostic tool for BCOR-associated sarcomas, including those with KDM2B rearrangement.

Utility of genome-wide DNA methylation profiling for pediatric-type diffuse gliomas.

Despite the current progress of treatment, pediatric-type diffuse glioma is one of the most lethal primary malignant tumors in the central nervous system (CNS). Since pediatric-type CNS tumors are rare disease entities and highly heterogeneous, the diagnosis is challenging. An accurate diagnosis is essential for the choice of optimal treatment, which leads to precision oncology and improvement of the patient's outcome. Genome-wide DNA methylation profiling recently emerged as one of the most important tools for the diagnosis of CNS tumors, and the utility of this novel assay has been reported in both pediatric and adult patients. In the current World Health Organization classification published in 2021, several new entities are recognized in pediatric-type diffuse gliomas, some of which require methylation profiling. In this review, we investigated the utility of genome-wide DNA methylation profiling in pediatric-type diffuse glioma, as well as issues in the clinical application of this assay. Furthermore, the combination of genome-wide DNA methylation profiling and other comprehensive genomic assays, which may improve diagnostic accuracy and detection of the actionable target, will be discussed.

A prediction model for distant metastasis after isolated locoregional recurrence of breast cancer.

PURPOSE: The impact of progesterone receptor (PR) status on the prognosis of breast cancer after isolated locoregional recurrence (ILRR) remains unclear. This study evaluated the impact of clinicopathologic factors, including PR status of ILRR, on distant metastasis (DM) after ILRR. METHODS: We retrospectively identified 306 patients with ILRR diagnosed at the National Cancer Center Hospital between 1993 and 2021 from the database. Cox proportional hazards analysis was performed to examine factors associated with DM after ILRR. We developed a risk prediction model based on the number of detected risk factors and estimated survival curves using the Kaplan-Meier method. RESULTS: During a median follow-up time of 4.7 years after ILRR diagnosis, 86 patients developed DM, and 50 died. Multivariate analysis revealed that seven risk factors were associated with poor distant metastasis-free survival (DMFS): estrogen receptor-positive/PR-negative/human epidermal growth factor receptor 2-negative ILRR, short disease-free interval, recurrence site other than ipsilateral breast, no-resection of ILRR tumor, chemotherapy for the primary tumor, nodal stage in the primary tumor, and no endocrine therapy for ILRR. The predictive model classified patients into 4 groups based on the number of risk factors: low-, intermediate-, high-, and the highest-risk groups with 0 to 1, 2, 3 to 4, and 5 to 7 factors, respectively. This revealed significant variation in DMFS among the groups. A higher number of the risk factors was associated with poorer DMFS. CONCLUSION: Our prediction model, which considered the ILRR receptor status, may contribute to the development of a treatment strategy for ILRR.

Local recurrence of submucosal invasive colorectal cancer after endoscopic submucosal dissection revealed by copy number variation.

We report a case in which analysis of copy number variation revealed local recurrence of submucosal invasive colorectal cancer after curative endoscopic submucosal dissection (ESD). An 86-year-old man with a history of abdominoperineal resection of the rectum for rectal cancer underwent resection with ESD for early-stage sigmoid cancer 5 cm away from the stoma opening. At the same time, ileocecal resection was performed for advanced cecal cancer. Twelve months after ESD, advanced cancer occurred in the area of the ESD lesion. It was unclear if the cancer was a local recurrence after ESD, implantation of cecal cancer, or a new lesion. Copy number variation analysis performed for the three lesions revealed that the new lesion originated from residual tumor cells from ESD and was unlikely to be cecal cancer.

Utility of real-time polymerase chain reaction for the assessment of CDKN2A homozygous deletion in adult-type IDH-mutant astrocytoma.

The World Health Organization Classification of Tumors of the Central Nervous System 5th Edition (WHO CNS5) introduced a newly defined astrocytoma, IDH-mutant grade 4, for adult diffuse glioma classification. One of the diagnostic criteria is the presence of a CDKN2A/B homozygous deletion (HD). Here, we report a robust and cost-effective quantitative polymerase chain reaction (qPCR)-based test for assessing CDKN2A HD. A TaqMan copy number assay was performed using a probe located within CDKN2A. The linear correlation between the Ct values and relative CDKN2A copy number was confirmed using a serial mixture of DNA from normal blood and U87MG cells. The qPCR assay was performed in 109 IDH-mutant astrocytomas, including 14 tumors with CDKN2A HD, verified either by multiplex ligation-dependent probe amplification (MLPA) or CytoScan HD microarray platforms. Receiver operating characteristic curve analysis indicated that a cutoff value of 0.85 yielded optimal sensitivity (100%) and specificity (99.0%) for determining CDKN2A HD. The assay applies to DNA extracted from frozen or formalin-fixed paraffin-embedded tissue samples. Survival was significantly shorter in patients with than in those without CDKN2A HD, assessed by either MLPA/CytoScan or qPCR. Thus, our qPCR method is clinically applicable for astrocytoma grading and prognostication, compatible with the WHO CNS5.

Keratin-positive fibrotic extraskeletal myxoid chondrosarcoma: a close mimic of myoepithelial tumour.

AIMS: Extraskeletal myxoid chondrosarcoma (EMC) is a rare form of adult sarcoma with distinct histology and NR4A3 gene fusion. Immunohistochemically, EMCs are variably positive for S100 protein and neuroendocrine markers. Unlike histologically similar soft-tissue myoepithelial tumours, keratin expression is rare. Prompted by two recent EMC cases with diffuse keratin expression, we investigated the expression of epithelial markers in a molecularly confirmed cohort of EMC and identified two additional similar cases. METHODS AND RESULTS: Four keratin-positive EMCs occurred in one man and three women aged 46-59 years. All tumours displayed nonclassic histology with prominent stromal fibrosis, and keratin AE1/AE3 was expressed either diffusely (N = 2) or focally (N = 2). In one tumour, keratin expression was limited to the sclerotic area. All tumours coexpressed epithelial membrane antigen and two additionally expressed S100 protein or glial fibrillary acidic protein. All tumours harboured NR4A3 fusions, including TAF15::NR4A3 (N = 1) and EWSR1::NR4A3 (N = 3). Two cases were initially considered as most consistent with myoepithelial tumours based on widespread stromal fibrosis and keratin expression. DNA methylation analysis classified two tumours tested as EMCs. CONCLUSIONS: We identified a small subset of EMCs characterised by keratin expression and prominent stromal fibrosis. This histological pattern must be recognised in the differential diagnosis of myoepithelial tumours because misclassification may lead to the erroneous prediction of tumour behaviour and may alter patient management. NR4A3 genetic analysis should be considered even in the face of keratin expression and prominent stromal fibrosis.

Clinical significance of tumor cell seeding associated with needle biopsy in patients with breast cancer.

BACKGROUND/OBJECTIVE: The occurrence of iatrogenic tumor cell seeding (seeding) in needle tract scars formed by core needle biopsy (CNB) or vacuum-assisted biopsy (VAB) is well known. Some risk factors for seeding have been reported, but the clinicopathological risk factors and its prognosis have not been fully investigated. We evaluated the clinical features and prognosis of seeding. METHODS: We included 4405 patients who had undergone surgery (lumpectomy or mastectomy) with a diagnosis of breast cancer by preoperative CNB or VAB at our hospital between January 2012 and February 2021. Data of patients with confirmed presence of seeding in resected specimens were collected from pathological records. We analyzed the risk factors of seeding using logistic regression analysis and compared the ipsilateral breast tumor recurrence (IBTR) rate between cases based on the presence or absence of seeding in the lumpectomy group. RESULTS: Of the 4405 patients, 133 (3.0%) had confirmed seeding. Univariate analysis revealed the association of clinicopathological features of seeding with lower nuclear grade (NG1 vs NG2-3; p = 0.043), lower Ki-67 (<30 vs. ≥30; p = 0.049), estrogen receptor (ER) positivity (positive vs negative; p<0.01), and human epidermal growth factor receptor 2 (HER2) negativity (negative vs positive; p = 0.016). Multivariate analysis showed ER positivity (odds ratio, 5.23; p<0.05) as an independent risk factor of seeding. The IBTR rate was not significantly different between the seeding and non-seeding groups. CONCLUSIONS: Seeding was more likely to occur in ER positive, HER2 negative carcinomas with less aggressive features, and may remain subclinical if adequate adjuvant treatments are administered.

High-grade neuroepithelial tumor with EP300::BCOR fusion and negative BCOR immunohistochemical expression: a case report.

In the World Health Organization tumor classification (fifth edition), central nervous system (CNS) tumors with BCOR internal tandem duplications have been recognized as a new tumor type. Some recent studies have reported CNS tumors with EP300::BCOR fusions, predominantly in children and young adults, expanding the spectrum of BCOR-altered CNS tumors. This study reports a new case of high-grade neuroepithelial tumor (HGNET) with an EP300::BCOR fusion in the occipital lobe of a 32-year-old female. The tumor displayed anaplastic ependymoma-like morphologies characterized by a relatively well-circumscribed solid growth with perivascular pseudorosettes and branching capillaries. Immunohistochemically, OLIG2 was focally positive and BCOR was negative. RNA sequencing revealed an EP300::BCOR fusion. The Deutsches Krebsforschungszentrum DNA methylation classifier (v12.5) classified the tumor as CNS tumor with BCOR/BCORL1 fusion. The t-distributed stochastic neighbor embedding analysis plotted the tumor close to the HGNET with BCOR alteration reference samples. BCOR/BCORL1-altered tumors should be included in the differential diagnosis of supratentorial CNS tumors with ependymoma-like histological features, especially when they lack ZFTA fusion or express OLIG2 even in the absence of BCOR expression. Analysis of published CNS tumors with BCOR/BCORL1 fusions revealed partly overlapping but not identical phenotypes. Further studies of additional cases are required to establish their classification.