The chromosomal translocation t(1;6)(p35.3;p25.2), recurrent in chronic lymphocytic leukaemia, leads to RCC1::IRF4 fusion.

The chromosomal translocation t(1;6)(p35.3;p25.2) is a rare but recurrent aberration in chronic lymphocytic leukaemia (CLL). We report molecular characterization of 10 cases and show that this translocation juxtaposes interferon regulatory factor 4 (IRF4) on 6p25 with regulator of chromosome condensation 1 (RCC1) on 1p35. The breakpoints fell within the 5' untranslated regions of both genes, resulting in RCC1::IRF4 fusion transcripts without alterations of the protein-coding sequences. Levels of expression of both RCC1 and IRF4 proteins were not obviously deregulated. The cases showed other mutations typical of CLL and we confirm previously reported skewing towards the IGHV-unmutated subtype. RCC1::IRF4 fusion characterizes a rare subset of CLL.

Rhabdoid tumors in patients conceived following ART: is there an association?

STUDY QUESTION: In children affected by rhabdoid tumors (RT), are there clinical, therapeutic, and/or (epi-)genetic differences between those conceived following ART compared to those conceived without ART? SUMMARY ANSWER: We detected a significantly elevated female predominance, and a lower median age at diagnosis, of children with RT conceived following ART (RT_ART) as compared to other children with RT. WHAT IS KNOWN ALREADY: Anecdotal evidence suggests an association of ART with RT. STUDY DESIGN, SIZE, DURATION: This was a multi-institutional retrospective survey. Children with RT conceived by ART were identified in our EU-RHAB database (n = 11/311 children diagnosed between January 2010 and January 2018) and outside the EU-RHAB database (n = 3) from nine different countries. A population-representative German EU-RHAB control cohort of children with RTs conceived without ART (n = 211) (EU-RHAB control cohort) during the same time period was used as a control cohort for clinical, therapeutic, and survival analyses. The median follow-up time was 11.5 months (range 0-120 months) for children with RT_ART and 18.5 months (range 0-153 months) for the EU-RHAB control cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analyzed 14 children with RT_ART diagnosed from January 2010 to January 2018. We examined tumors and matching blood samples for SMARCB1 mutations and copy number alterations using FISH, multiplex ligation-dependent probe amplification, and DNA sequencing. DNA methylation profiling of tumor and/or blood samples was performed using DNA methylation arrays and compared to respective control cohorts of similar age (n = 53 tumors of children with RT conceived without ART, and n = 38 blood samples of children with no tumor born small for gestational age). MAIN RESULTS AND THE ROLE OF CHANCE: The median age at diagnosis of 14 individuals with RT_ART was 9 months (range 0-66 months), significantly lower than the median age of patients with RT (n = 211) in the EU-RHAB control cohort (16 months (range 0-253), P = 0.03). A significant female predominance was observed in the RT_ART cohort (M:F ratio: 2:12 versus 116:95 in EU-RHAB control cohort, P = 0.004). Eight of 14 RT_ART patients were diagnosed with atypical teratoid rhabdoid tumor, three with extracranial, extrarenal malignant rhabdoid tumor, one with rhabdoid tumor of the kidney and two with synchronous tumors. The location of primary tumors did not differ significantly in the EU-RHAB control cohort (P = 0.27). Six of 14 RT_ART patients presented with metastases at diagnosis. Metastatic stage was not significantly different from that within the EU-RHAB control cohort (6/14 vs 88/211, P = 1). The incidence of pathogenic germline variants was five of the 12 tested RT_ART patients and, thus, not significantly different from the EU-RHAB control cohort (5/12 versus 36/183 tested, P = 0.35). The 5-year overall survival (OS) and event free survival (EFS) rates of RT_ART patients were 42.9 ± 13.2% and 21.4 ± 11%, respectively, and thus comparable to the EU-RHAB control cohort (OS 41.1 ± 3.5% and EFS 32.1 ± 3.3). We did not find other clinical, therapeutic, outcome factors distinguishing patients with RT_ART from children with RTs conceived without ART (EU-RHAB control cohort). DNA methylation analyses of 10 tumors (atypical teratoid RT = 6, extracranial, extrarenal malignant RT = 4) and six blood samples from RT_ART patients showed neither evidence of a general DNA methylation difference nor underlying imprinting defects, respectively, when compared to a control group (n = 53 RT samples of patients without ART, P = 0.51, n = 38 blood samples of patients born small for gestational age, P = 0.1205). LIMITATIONS, REASONS FOR CAUTION: RTs are very rare malignancies and our results are based on a small number of children with RT_ART. WIDER IMPLICATIONS OF THE FINDINGS: This cohort of patients with RT_ART demonstrated a marked female predominance, and a rather low median age at diagnosis even for RTs. Other clinical, treatment, outcome, and molecular factors did not differ from those conceived without ART (EU-RHAB control cohort) or reported in other series, and there was no evidence for imprinting defects. Long-term survival is achievable even in cases with pathogenic germline variants, metastatic disease at diagnosis, or relapse. The female preponderance among RT_ART patients is not yet understood and needs to be evaluated, ideally in larger international series. STUDY FUNDING/COMPETING INTEREST(S): M.C.F. is supported by the 'Deutsche Kinderkrebsstiftung' DKS 2020.10, by the 'Deutsche Forschungsgemeinschaft' DFG FR 1516/4-1 and by the Deutsche Krebshilfe 70113981. R.S. received grant support by Deutsche Krebshilfe 70114040 and for infrastructure by the KinderKrebsInitiative Buchholz/Holm-Seppensen. P.D.J. is supported by the Else-Kroener-Fresenius Stiftung and receives a Max-Eder scholarship from the Deutsche Krebshilfe. M.H. is supported by DFG (HA 3060/8-1) and IZKF Münster (Ha3/017/20). BB is supported by the 'Deutsche Kinderkrebsstiftung' DKS 2020.05. We declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Recurrent atypical teratoid/rhabdoid tumors (AT/RT) reveal discrete features of progression on histology, epigenetics, copy number profiling, and transcriptomics.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors manifesting in infancy. They split into four molecular types. The major three (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC) all carry mutations in SMARCB1, the fourth quantitatively smaller type is characterized by SMARCA4 mutations (AT/RT-SMARCA4). Molecular characteristics of disease recurrence or metastatic spread, which go along with a particularly dismal outcome, are currently unclear. Here, we investigated tumor tissue from 26 patients affected by AT/RT to identify signatures of recurrences in comparison with matched primary tumor samples. Microscopically, AT/RT recurrences demonstrated a loss of architecture and significantly enhanced mitotic activity as compared to their related primary tumors. Based on DNA methylation profiling, primary tumor and related recurrence were grossly similar, but three out of 26 tumors belonged to a different molecular type or subtype after second surgery compared to related primary lesions. Copy number variations (CNVs) differed in six cases, showing novel gains on chromosome 1q or losses of chromosome 10 in recurrences as the most frequent alterations. To consolidate these observations, our cohort was combined with a data set of unmatched primary and recurrent AT/RT, which demonstrated chromosome 1q gain and 10 loss in 18% (n = 7) and 11% (n = 4) of the recurrences (n = 38) as compared to 7% (n = 3) and 0% (n = 0) in the primary tumors (n = 44), respectively. Similar to the observations made by DNA methylation profiling, RNA sequencing of our cohort revealed AT/RT primary tumors and matched recurrences clustering closely together. However, a number of genes showed significantly altered expression in AT/RT-SHH recurrences. Many of them are known tumor driving growth factors, involved in embryonal development and tumorigenesis, or are cell-cycle-associated. Overall, our work identifies subtle molecular changes that occur in the course of the disease and that may help define novel therapeutic targets for AT/RT recurrences.

Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B-cell lymphomas.

Histone methylation-modifiers, such as EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%), also identified by prior exome or RNA-sequencing studies, we here found recurrent alterations to KDM4C in chromosome 9p24, encoding a histone demethylase. Focal structural variation was the main mechanism of KDM4C alterations, and was independent from 9p24 amplification. We also identified KDM4C alterations in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNA-sequencing and genome sequencing data we predict that KDM4C structural variants result in loss-offunction. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as a tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as a putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas.

T-cell prolymphocytic leukemia is associated with deregulation of oncogenic microRNAs on transcriptional and epigenetic level.

Deregulation of micro(mi)-RNAs is a common mechanism in tumorigenesis. We investigated the expression of 2083 miRNAs in T-cell prolymphocytic leukemia (T-PLL). Compared to physiologic CD4+ and CD8+ T-cell subsets, 111 miRNAs were differentially expressed in T-PLL. Of these, 33 belonged to miRNA gene clusters linked to cancer. Genomic variants affecting miRNAs were infrequent with the notable exception of copy number aberrations. Remarkably, we found strong upregulation of the miR-200c/-141 cluster in T-PLL to be associated with DNA hypomethylation and active promoter marks. Our findings suggest that copy number aberrations and epigenetic changes could contribute to miRNA deregulation in T-PLL.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant: A clinical and histopathological distinct entity showing epigenetic similarity with ATRT-MYC.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant, is a histopathological distinct low-grade lesion encountered in older children and young adults that shows epigenetic similarity with ATRT-MYC and has the potential for malignant progression.

ATRT-SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance.

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT-TYR, ATRT-MYC and ATRT-SHH. ATRT-SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT-SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT-SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT-SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT-SHH has prognostic relevance and might aid to stratify patients within future clinical trials.

Transposable element insertion as a mechanism of SMARCB1 inactivation in atypical teratoid/rhabdoid tumor.

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant brain tumor predominantly occurring in infants. Biallelic SMARCB1 mutations causing loss of nuclear SMARCB1/INI1 protein expression represent the characteristic genetic lesion. Pathogenic SMARCB1 mutations comprise single nucleotide variants, small insertions/deletions, large deletions, which may be also present in the germline (rhabdoid tumor predisposition syndrome 1), as well as somatic copy-number neutral loss of heterozygosity (LOH). In some SMARCB1-deficient AT/RT underlying biallelic mutations cannot be identified. Here we report the case of a 24-months-old girl diagnosed with a large brain tumor. The malignant rhabdoid tumor showed loss of nuclear SMARCB1/INI1 protein expression and the diagnosis of AT/RT was confirmed by DNA methylation profiling. While FISH, MLPA, Sanger sequencing and DNA methylation data-based imbalance analysis did not disclose alterations affecting SMARCB1, OncoScan array analysis revealed a 28.29 Mb sized region of copy-number neutral LOH on chromosome 22q involving the SMARCB1 locus. Targeted next-generation sequencing did also not detect a single nucleotide variant but instead revealed insertion of an AluY element into exon 2 of SMARCB1. Specific PCR-based Sanger sequencing verified the Alu insertion (SMARCB1 c.199_200 Alu ins) resulting in a frame-shift truncation not present in the patient's germline. In conclusion, transposable element insertion represents a hitherto not widely recognized mechanism of SMARCB1 disruption in AT/RT, which might not be detected by several widely applied conventional diagnostics assays. This finding has particular clinical implications, if rhabdoid predisposition syndrome 1 is suspected, but germline SMARCB1 alterations cannot be identified.

The mutational landscape of Burkitt-like lymphoma with 11q aberration is distinct from that of Burkitt lymphoma.

The new recently described provisional lymphoma category Burkitt-like lymphoma with 11q aberration comprises cases similar to Burkitt lymphoma (BL) on morphological, immunophenotypic and gene-expression levels but lacking the IG-MYC translocation. They are characterized by a peculiar imbalance pattern on chromosome 11, but the landscape of mutations is not yet described. Thus, we investigated 15 MYC-negative Burkitt-like lymphoma with 11q aberration (mnBLL,11q,) cases by copy-number analysis and whole-exome sequencing. We refined the regions of 11q imbalance and identified the INO80 complex-associated gene NFRKB as a positional candidate in 11q24.3. Next to recurrent gains in 12q13.11-q24.32 and 7q34-qter as well as losses in 13q32.3-q34, we identified 47 genes recurrently affected by protein-changing mutations (each ≥3 of 15 cases). Strikingly, we did not detect recurrent mutations in genes of the ID3-TCF3 axis or the SWI/SNF complex that are frequently altered in BL, or in genes frequently mutated in germinal center-derived B-cell lymphomas like KMT2D or CREBBP An exception is GNA13, which was mutated in 7 of 15 cases. We conclude that the genomic landscape of mnBLL,11q, differs from that of BL both at the chromosomal and mutational levels. Our findings implicate that mnBLL,11q, is a lymphoma category distinct from BL at the molecular level.

CCND2 and CCND3 hijack immunoglobulin light-chain enhancers in cyclin D1- mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1- MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1-/D2- MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1-/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1- MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1- MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1- MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1-/D2-/D3- MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1- MCL.

Inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1/INI1 protein in a molecular subset of atypical teratoid/rhabdoid tumors.

Loss of nuclear SMARCB1 (INI1/hSNF5/BAF47) protein expression due to biallelic mutations of the SMARCB1 tumor suppressor gene is a hallmark of atypical teratoid/rhabdoid tumors (ATRT), but the presence of cytoplasmic SMARCB1 protein in these tumors has not yet been described. In a series of 102 primary ATRT, distinct cytoplasmic SMARCB1 staining on immunohistochemistry was encountered in 19 cases (19%) and was highly over-represented in cases showing pathogenic sequence variants leading to truncation or mutation of the C-terminal part of SMARCB1 (15/19 vs. 4/83; Chi-square: 56.04, p = 1.0E-10) and, related to this, in tumors of the molecular subgroup ATRT-TYR (16/36 vs. 3/66; Chi-square: 24.47, p = 7.6E-7). Previous reports have indicated that while SMARCB1 lacks a bona fide nuclear localization signal, it harbors a masked nuclear export signal (NES) and that truncation of the C-terminal region results in unmasking of this NES leading to cytoplasmic localization. To determine if cytoplasmic localization found in ATRT is due to unmasking of NES, we generated GFP fusions of one of the SMARCB1 truncating mutations (p.Q318X) found in the tumors along with a p.L266A mutation, which was shown to disrupt the interaction of SMARCB1-NES with exportin-1. We found that while the GFP-SMARCB1(Q318X) mutant localized to the cytoplasm, the double mutant GFP-SMARCB1(Q318X;L266A) localized to the nucleus, confirming NES requirement for cytoplasmic localization. Furthermore, cytoplasmic SMARCB1(Q318X) was unable to cause senescence as determined by morphological observations and by senescence-associated ß-galactosidase assay, while nuclear SMARCB1(Q318X;L266A) mutant regained this function. Selinexor, a selective exportin-1 inhibitor, was effective in inhibiting the nuclear export of SMARCB1(Q318X) and caused rapid cell death in rhabdoid tumor cells. In conclusion, inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1. Therapies aimed at preventing nuclear export of mutant SMARCB1 protein may represent a promising targeted therapy in ATRT harboring truncating C-terminal SMARCB1 mutations.

Reconstruction of rearranged T-cell receptor loci by whole genome and transcriptome sequencing gives insights into the initial steps of T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive tumor with leukemic presentation of mature T-lymphocytes. Here, we aimed at characterizing the initial events in the molecular pathogenesis of T-PLL and particularly, at determining the point in T-cell differentiation when the hallmark oncogenic events, that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) occur. To this end, we mined whole genome and transcriptome sequencing data of 17 and 11 T-PLL cases, respectively. Mapping of the 14q32.1 locus breakpoints identified only TCL1A, which was moreover significantly overexpressed in T-PLL as compared to benign CD4+ and CD8+ T-cells, as the only common oncogenic target of aberrations. In cases with t(14;14), the breakpoints mapped telomeric and in cases with inv(14) centromeric or in the 3'-untranslated region of TCL1A. Regarding the T-cell receptor alpha (TRA) locus-TCL1A breakpoint junctions, all 17 breakpoints involved recombination signal sequences and 15 junctions contained nontemplated (N-) nucleotides. All T-PLL cases studied carried in-frame TRA rearrangements on the intact allele, which skewed significantly toward usage of distal/central TRAV/TRAJ gene segments as compared to the illegitimate TRA rearrangements. Our findings suggest that the oncogenic TRA-TCL1A/MTCP1 rearrangements in T-PLL occur during opening of the TRA locus, that is, during the progression from CD4+ immature single positive to early double positive thymocyte stage, just before physiologic TCL1A expression is silenced. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Thereafter, it switches off RAG and DNTT expression in line with the mature T-cell phenotype at presentation of T-PLL.

Desmoplastic myxoid tumor, SMARCB1-mutant: clinical, histopathological and molecular characterization of a pineal region tumor encountered in adolescents and adults.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly occurring in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. SMARCB1-mutant tumors in adolescents and adults are rare and may show uncommon histopathological and clinical features. Here we report seven SMARCB1-deficient intracranial tumors sharing distinct clinical, histopathological and molecular features. Median age of the four females and three males was 40 years (range 15-61 years). All tumors were located in the pineal region. Histopathologically, these tumors displayed spindled and epithelioid cells embedded in a desmoplastic stroma alternating with a variable extent of a loose myxoid matrix. All cases showed loss of nuclear SMARCB1/INI1 protein expression, expression of EMA and CD34 was frequent and the Ki67/MIB1 proliferation index was low in the majority of cases (median 3%). Three cases displayed heterozygous SMARCB1 deletions and two cases a homozygous SMARCB1 deletion. On sequencing, one tumor showed a 2 bp deletion in exon 4 (c.369_370del) and one a short duplication in exon 3 (c.237_276dup) both resulting in frameshift mutations. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-SNE analysis and hierarchical clustering analysis, however, all tumors grouped closely together and showed similarities with ATRT-MYC. After a median observation period of 48 months, three patients were alive with stable disease, whereas one patient experienced tumor progression and three patients had succumbed to disease. In conclusion, our series represents an entity with distinct clinical, histopathological and molecular features showing epigenetic similarities with ATRT-MYC. We propose the designation desmoplastic myxoid tumor (DMT), SMARCB1-mutant, for these tumors.

Spinal cord atypical teratoid/rhabdoid tumors in children: Clinical, genetic, and outcome characteristics in a representative European cohort.

BACKGROUND: Case reports have portrayed spinal cord atypical teratoid/rhabdoid tumor (spATRT) as an aggressive form of ATRT. We conducted a retrospective European survey to collect data on clinical characteristics, molecular biology, treatment, and outcome of children with intramedullary spATRT. METHODS: Scrutinizing a French national series and the European Rhabdoid Registry database, we identified 13 patients (median age 32 months; metastatic disease at diagnosis, n = 6). Systemic postoperative chemotherapy was administered to all patients; three received intrathecal therapy and six were irradiated (craniospinal, n = 3; local, n = 3). RESULTS: Median observation time was 8 (range, 1-93) months. Progression-free and overall survival rates at 1 and (2 years) were 35.2% ± 13.9% (26.4% ± 12.9%) and 38.5% ± 13.5% (23.1% ± 11.7%). Four patients (ATRT-SHH, n = 2; ATRT-MYC, n = 1; DNA methylation subgroup not available, n = 1) achieved complete remission (CR); two of them are alive in CR 69 and 72 months from diagnosis. One patient relapsed after CR and is alive with progressive disease (PD) and one died of the disease. Three patients (ATRT-MYC, n = 2; subgroup not available, n = 1) died after 7 to 22 months due to PD after having achieved a partial remission (n = 1) or stabilization (n = 2). Five patients (ATRT-MYC, n = 2; subgroup not available, n = 3) developed early PD and died. One patient (ATRT-MYC) died of intracerebral hemorrhage prior to response evaluation. CONCLUSIONS: Long-term survival is achievable in selected patients with spATRT using aggressive multimodality treatment. Larger case series and detailed molecular analyses are needed to understand differences between spATRT and their inracranial counterparts and the group of extradural malignant rhabdoid tumors.

Non-leukemic pediatric mixed phenotype acute leukemia/lymphoma: Genomic characterization and clinical outcome in a prospective trial for pediatric lymphoblastic lymphoma.

Rare cases of hematological precursor neoplasms fulfill the diagnostic criteria of mixed phenotype acute leukemia (MPAL), characterized by expression patterns of at least two hematopoietic lineages, for which a highly aggressive behavior was reported. We present a series of 11 pediatric non-leukemic MPAL identified among 146 precursor lymphoblastic lymphomas included in the prospective trial Euro-LBL 02. Paraffin-embedded biopsies of 10 cases were suitable for molecular analyses using OncoScan assay (n = 7), fluorescence in situ hybridization (FISH; n = 7) or both (n = 5). Except for one case with biallelic KMT2A (MLL) breaks, all cases analyzed by FISH lacked the most common translocations defining molecular subsets of lymphoblastic leukemia/lymphomas. Two non-leukemic B-myeloid MPALs showed the typical genomic profile of hyperdiploid precursor B-cell lymphoblastic leukemia with gains of chromosomes 4, 6, 10, 14, 18, and 21. One B-T MPAL showed typical aberrations of T-cell lymphoblastic lymphoma, such as copy number neutral loss of heterozygosity (CNN-LOH) at 9p targeting a 9p21.3 deletion of CDKN2A and 11q12.2-qter affecting the ATM gene. ATM was also mutated in a T-myeloid MPAL case with additional loss at 7q21.2-q36.3 and mutation of NRAS, two alterations common in myeloid disorders. No recurrent regions of CNN-LOH were observed. The outcome under treatment was good with all patients being alive in first complete remission after treatment according to a protocol for precursor lymphoblastic lymphoma (follow-up 3-10 years, median: 4.9 years). In summary, the present series of non-leukemic MPALs widely lacked recurrently reported translocations in lymphoid/myeloid neoplasias and showed heterogeneous spectrum of chromosomal imbalances.

Multiple DICER1-related tumors in a child with a large interstitial 14q32 deletion.

Germ-line interstitial deletions involving the 14q32 chromosomal region, resulting in 14q32 deletion syndrome, are rare. DICER1 is a recently described cancer-predisposition gene located at 14q32.13. We report the case of a male child with a ∼5.8 Mbp 14q32.13q32.2 germ-line deletion, which included the full DICER1 locus. We reviewed available clinical and pathological material, and conducted genetic analyses. In addition to having congenital dysmorphic features, the child developed multiple DICER1 syndrome-related tumors before age 5 y: a pediatric cystic nephroma (pCN), a ciliary body medulloepithelioma (CBME), and a small lung cyst (consistent with occult pleuropulmonary blastoma Type I/Ir cysts seen in DICER1 mutation carriers). He also developed a cerebral spindle-cell sarcoma with myogenous differentiation. Our investigations revealed that the deletion encompassed 31 protein-coding genes. In addition to the germ-line DICER1 deletion, somatic DICER1 RNase IIIb mutations were found in the CBME (c.5437G > A, p.E1813K), pCN (c.5425G > A, p.G1809R), and sarcoma (c.5125G > A, p.D1709N). The sarcoma also harbored a somatic TP53 mutation: c.844C > T, p.R282W. Additional copy number alterations were identified in the CBME and sarcoma using an OncoScan array. Among the 8 cases with molecularly-defined 14q32 deletions involving DICER1 and for whom phenotypic information is available, our patient and one other developed DICER1-related tumors. Biallelic DICER1 mutations have not previously been reported to cause cerebral sarcoma, which now may be considered a rare manifestation of the DICER1 syndrome. Our study shows that DICER1-related tumors can occur in children with 14q32 deletions and suggests surveillance for such tumors may be warranted.

The extraordinary challenge of treating patients with congenital rhabdoid tumors-a collaborative European effort.

BACKGROUND: Congenital rhabdoid tumors are rare and highly aggressive malignancies. In general, patients are considered to be incurable and are often treated using an exclusive, primarily palliative approach. METHODS: A prospective and retrospective collection of 42 patients from the European Rhabdoid Registry (EU-RHAB), France and Moscow (2006-2016) diagnosed within the first 28 days of life was evaluated. Genetic and clinical reference evaluation included SMARCB1 and/or SMARCA4 (fluorescence-in-situ-hybridization, multiplex ligation-dependent probe amplification, and sequencing) mutation analysis and immunohistochemistry. Forty-eight percent (20/42) of patients were treated according to the EU-RHAB therapy, 7% (3/42) according to the pilot approach Rhabdoid 2007, 33% (14/42) with individual schedules, and 12% (5/42) received no chemotherapy at all. RESULTS: Forty point five percent (17/42) of patients presented with extracranial rhabdoid tumors, 33.5% (14/42) with rhabdoid tumors of the central nervous system (atypical teratoid/rhabdoid tumor), and the remainder 26% (11/42) demonstrated synchronous tumors. Metastases at diagnosis were present in 52% (22/42) of patients. A germline mutation was detected in 66% (25/38) and was associated with a poor prognosis (4.2 ± 4.1% vs. 48 ± 16.4%, P < 0.00005). A gross total resection (GTR) was realized in 17%. A GTR (42.9 ± 18.7% vs. 4.9 ± 4.3%, P = 0.04), therapy according to a standardized approach (20.9 ± 8.7% vs. 7.1 ± 6.9 %, P = 0.0018), and a complete remission (CR) (23.6 ± 9.8% vs. 1.3 ± 3.6%, P = 0.04) were significant prognostic factors. CONCLUSIONS: The management of patients with congenital rhabdoid tumors requires a major multidisciplinary effort. In many instances, cure is not possible and a palliative approach is warranted. Our data indicate a positive impact of standardized therapeutic approaches on survival, making a tailored approach toward affected patients and their families mandatory.

MYC expression and translocation analyses in low-grade and transformed follicular lymphoma.

AIMS: Low-grade follicular lymphoma (FL) (grade 1/2, FL1/2) has an annual risk of transformation of ≈3%, which is associated with aberrations in CDKN2A/B, TP53, and MYC. As in diffuse large B-cell lymphoma, high MYC expression in transformed FL (tFL) might predict a MYC breakpoint. METHODS AND RESULTS: We quantified MYC expression by immunohistochemistry and digital analysis in 41 paired biopsies from 20 patients with FL1/2 with subsequent transformation and in four isolated biopsies of tFL. As controls, 28 biopsies of FL1/2 without transformation (median follow-up of 105 months) and nine biopsies of FL3A/B were analysed. In the 20 FL1/2-tFL pairs, MYC expression was significantly higher in tFL than in the initial FL1/2 biopsies (median 54% versus 6%; 7% in FL3A, and 35% in FL3B). MYC breaks (MYC-R) were detected in eight of 21 (38%) tFLs analysed by fluorescence in-situ hybridization (FISH), with a median MYC score of 86%. In two of the analysed tFL cases, the translocation was already detected in antecedent FL1/2. MYC partners were immunoglobulin (IG) loci in three of eight cases (one IGL, one IGH, and one IGK) and non-IG in five of eight cases (two PAX5, one BCL6, and two unknown). Of the eight MYC-R+ cases, six were BCL2+/MYC+ double-hit, one was BCL2+/BCL6+/MYC+ triple-hit, and one was MYC+ single-hit. All three IG-MYC+ cases showed a MYC expression level of >85%, whereas the five cases with a non-IG MYC partner had a wider range of expression (median 68%, range 13-86%). Among the 13 MYC-R- tFLs, two groups with almost dichotomous MYC expression could be observed (three cases showed ≥90% MYC expression), suggesting alternative mechanisms of MYC activation. CONCLUSIONS: we show an increase in MYC expression from FL1/2 to tFL. MYC breakpoints were present in ≈40% of the cases, which is markedly higher than in de novo DLBCL. MYC expression was uniformly high in cases with an IG-MYC translocation but much more heterogeneous and in part independent of the presence of a MYC break in non-IG-MYC and MYC-negative cases.

Atypical teratoid/rhabdoid tumor arising in a malignant glioma.

Atypical teratoid/rhabdoid tumor (AT/RT), a highly malignant brain tumor in young children, usually arises de novo and has only rarely been described as a secondary malignancy. Here, we present a case of a child with glioblastoma, who was treated postoperatively by a combination of temozolomide, irradiation, and bevacizumab. AT/RT was diagnosed as a secondary tumor, 2.5 years following primary diagnosis. The child died 13 months after the diagnosis of AT/RT. This case demonstrates that malignant gliomas may give rise to AT/RT. It also emphasizes the diagnostic value of a repeated tumor biopsy in the recurrence setting.

Molecular characterization of acute lymphoblastic leukemia with high CRLF2 gene expression in childhood.

BACKGROUND: A high-level expression of the CRLF2 gene is frequent in precursor B-cell acute lymphoblastic leukemia (pB-ALL) and can be caused by different genetic aberrations. The presence of the most frequent alteration, the P2RY8/CRLF2 fusion, was shown to be associated with a high relapse incidence in children treated according to ALL-Berlin-Frankfurt-Münster (BFM) protocols, which is poorly understood. Moreover, the frequency of other alterations has not been systematically analyzed yet. PROCEDURE: CRLF2 mRNA expression and potential genetic aberrations causing a CRLF2 high expression were prospectively assessed in 1,105 patients treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP)-BFM ALL 2009 protocol. Additionally, we determined copy number alterations in selected B-cell differentiation genes for all CRLF2 high-expressing pB-ALL cases, as well as JAK2 and CRLF2 mutations. RESULTS: A CRLF2 high expression was detected in 26/178 (15%) T-cell acute lymphoblastic leukemia (T-ALL) cases, 21 of them (81%) had been stratified as high-risk patients by treatment response. In pB-ALL, a CRLF2 high expression was determined in 91/927 (10%) cases; the P2RY8/CRLF2 rearrangement in 44/91 (48%) of them, supernumerary copies of CRLF2 in 18/91 (20%), and, notably, the IGH/CRLF2 translocation was detected in 16/91 (18%). Remarkably, 7 of 16 (44%) patients with IGH/CRLF2 translocation had already relapsed. P2RY8/CRLF2- and IGH/CRLF2-positive samples (70 and 94%, respectively) were characterized by a high frequency of additional deletions in B-cell differentiation genes such as IKZF1 or PAX5. CONCLUSION: Our data suggest that this high frequency of genetic aberrations in the context of a high CRLF2 expression could contribute to the high risk of relapse in P2RY8/CRLF2- and IGH/CRLF2-positive ALL.