Loss of the DNA Repair Gene RNase H2 Identifies a Unique Subset of DDR-Deficient Leiomyosarcomas.

Targeting the DNA damage response (DDR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS), and loss of RNase H2, a DDR pathway member, is a potentially actionable alteration for DDR targeted treatments. Therefore, we designed a protein and genomic based RNase H2 screening assay to determine its prevalence and prognostic significance. Using a selective RNase H2 antibody on a pan-tumor tissue microarray (TMA), RNase H2 loss was more common in LMS (11.5%, 9/78) than across all tumors (3.8%, 32/843). In a separate LMS cohort, RNase H2 deficiency was confirmed in uterine LMS (U-LMS, 21%, 23/108) and soft-tissue LMS (ST-LMS) (30%, 39/102). In the TCGA database, RNASEH2B homozygous deletions (HomDels) were found in 6% (5/80) of LMS cases, with a higher proportion in U-LMS (15%; 4/27) compared to ST-LMS (2%; 1/53). Using the SNiPDx targeted-NGS sequencing assay to detect biallelic loss of function in select DDR related genes, we found RNASEH2B HomDels in 54% (19/35) of U-LMS cases with RNase H2 loss by IHC, and 7% (3/43) HomDels in RNase H2 intact cases. No RNASEH2B HomDels were detected in ST-LMS. In U-LMS patient cohort (n = 109), no significant overall survival difference was seen in patients with RNase H2 loss versus intact, or RNASEH2B HomDel (n=12) vs Non-HomDel (n=37). The overall diagnostic accuracy, sensitivity, and specificity of RNase H2 IHC for detecting RNASEH2B HomDels in U-LMS was 76%, 93% and 71% respectively, and it is being developed for future predictive biomarker driven clinical trials targeting DDR in U-LMS.

Patterns of structural variants within TP53 introns and relocation of the TP53 promoter: a commentary†.

Gene disruption from double-strand DNA breaks within introns is a mechanism of inactivating the tumor suppressor TP53. This occurs more frequently in osteosarcoma and biliary adenocarcinoma compared with other cancer types. The patterns of intron breakpoints within TP53 do not correlate with prevalence, intron length, or overall genome-wide levels of rearrangements. Therefore, these breakpoints appear to be selected for reasons other than to disrupt TP53. A recent article published by Saba et al in The Journal of Pathology illustrates a benefit to having breakpoints within intron 1 using high-quality matched genomic and transcriptomic osteosarcoma sequencing data as well as in vitro validation. The authors describe how the rearrangement results in relocation of the TP53 promoter region to regions upstream of genes that encode members of cartilage, growth plate development, osteoclast formation, and other TP53-related pathways. The upregulation of these genes by the TP53 promoter are gain-of-function events that are likely to promote tumor development and growth. Therefore, this article presents a potential new paradigm in which a single mutation would result in both the loss of a tumor suppressor and the gain of an oncogenic program. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Complete loss of TP53 and RB1 is associated with complex genome and low immune infiltrate in pleomorphic rhabdomyosarcoma.

Rhabdomyosarcoma accounts for roughly 1% of adult sarcomas, with pleomorphic rhabdomyosarcoma (PRMS) as the most common subtype. Survival outcomes remain poor for patients with PRMS, and little is known about the molecular drivers of this disease. To better characterize PRMS, we performed a broad array of genomic and immunostaining analyses on 25 patient samples. In terms of gene expression and methylation, PRMS clustered more closely with other complex karyotype sarcomas than with pediatric alveolar and embryonal rhabdomyosarcoma. Immune infiltrate levels in PRMS were among the highest observed in multiple sarcoma types and contrasted with low levels in other rhabdomyosarcoma subtypes. Lower immune infiltrate was associated with complete loss of both TP53 and RB1. This comprehensive characterization of the genetic, epigenetic, and immune landscape of PRMS provides a roadmap for improved prognostications and therapeutic exploration.

Comparative Tumor Microenvironment Analysis of Primary and Recurrent Ovarian Granulosa Cell Tumors.

Adult-type granulosa cell tumors (aGCT) are rare ovarian sex cord tumors with few effective treatments for recurrent disease. The objective of this study was to characterize the tumor microenvironment (TME) of primary and recurrent aGCTs and to identify correlates of disease recurrence. Total RNA sequencing (RNA-seq) was performed on 24 pathologically confirmed, cryopreserved aGCT samples, including 8 primary and 16 recurrent tumors. After read alignment and quality-control filtering, DESeq2 was used to identify differentially expressed genes (DEG) between primary and recurrent tumors. Functional enrichment pathway analysis and gene set enrichment analysis was performed using "clusterProfiler" and "GSVA" R packages. TME composition was investigated through the analysis and integration of multiple published RNA-seq deconvolution algorithms. TME analysis results were externally validated using data from independent previously published RNA-seq datasets. A total of 31 DEGs were identified between primary and recurrent aGCTs. These included genes with known function in hormone signaling such as LHCGR and INSL3 (more abundant in primary tumors) and CYP19A1 (more abundant in recurrent tumors). Gene set enrichment analysis revealed that primarily immune-related and hormone-regulated gene sets expression was increased in recurrent tumors. Integrative TME analysis demonstrated statistically significant depletion of cancer-associated fibroblasts in recurrent tumors. This finding was confirmed in multiple independent datasets. IMPLICATIONS: Recurrent aGCTs exhibit alterations in hormone pathway gene expression as well as decreased infiltration of cancer-associated fibroblasts, suggesting dual roles for hormonal signaling and TME remodeling underpinning disease relapse.

Osteosarcoma.

Osteosarcoma is the most common primary malignant tumour of the bone. Osteosarcoma incidence is bimodal, peaking at 18 and 60 years of age, and is slightly more common in males. The key pathophysiological mechanism involves several possible genetic drivers of disease linked to bone formation, causing malignant progression and metastasis. While there have been significant improvements in the outcome of patients with localized disease, with event-free survival outcomes exceeding 60%, in patients with metastatic disease, event-free survival outcomes remain poor at less than 30%. The suspicion of osteosarcoma based on radiographs still requires pathological evaluation of a bone biopsy specimen for definitive diagnosis and CT imaging of the chest should be performed to identify lung nodules. So far, population-based screening and surveillance strategies have not been implemented due to the rarity of osteosarcoma and the lack of reliable markers. Current screening focuses only on groups at high risk such as patients with genetic cancer predisposition syndromes. Management of osteosarcoma requires a multidisciplinary team of paediatric and medical oncologists, orthopaedic and general surgeons, pathologists, radiologists and specialist nurses. Survivors of osteosarcoma require specialized medical follow-up, as curative treatment consisting of chemotherapy and surgery has long-term adverse effects, which also affect the quality of life of patients. The development of osteosarcoma model systems and related research as well as the evaluation of new treatment approaches are ongoing to improve disease outcomes, especially for patients with metastases.

PPP1R7 Is a Novel Translocation Partner of CBFB via t(2;16)(q37;q22) in Acute Myeloid Leukemia.

In a subset of acute myeloid leukemia (AML) cases, the core binding factor beta subunit gene (CBFB) was rearranged via inv(16)(p13.1q22) or t(16;16)(p13.1;q22), in which the smooth muscle myosin heavy chain 11 gene (MYH11) was the partner (CBFB::MYH11). Rare variants of CBFB rearrangement occurring via non-classic chromosomal aberrations have been reported, such as t(1;16), t(2;16), t(3;16), t(5;16), and t(16;19), but the partners of CBFB have not been characterized. We report a case of AML with a complex karyotype, including t(2;16)(q37;q22), in which the protein phosphatase 1 regulatory subunit 7 gene (PPP1R7) at chromosome 2q37 was rearranged with CBFB (CBFB::PPP1R7). This abnormality was inconspicuous by conventional karyotype and interphase fluorescence in situ hybridization (FISH), thus leading to an initial interpretation of inv(16)(p13.1q22); however, metaphase FISH showed that the CBFB rearrangement involved chromosome 2. Using whole genome and Sanger sequencing, the breakpoints were identified as being located in intron 5 of CBFB and intron 7 of PPP1R7. A microhomology of CAG was found in the break and reconnection sites of CBFB and PPP1R7, thus supporting the formation of CBFB::PPP1R7 by microhomology-mediated end joining.

The androgen receptor is a therapeutic target in desmoplastic small round cell sarcoma.

Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block 5α-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including WT1 (the C-terminal partner of the pathognomonic fusion protein) and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway, neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell lineage commitment. Our findings have direct clinical implications given the widespread availability of FDA-approved androgen-targeted agents used for prostate cancer.

Multi-site desmoplastic small round cell tumors are genetically related and immune-cold.

Desmoplastic small round cell tumor (DSRCT) is a highly aggressive soft tissue sarcoma that is characterized by the EWSR1-WT1 fusion protein. Patients present with hundreds of tumor implants in their abdominal cavity at various sites. To determine the genetic relatedness among these sites, exome and RNA sequencing were performed on 22 DSRCT specimens from 14 patients, four of whom had specimens from various tissue sites. Multi-site tumors from individual DSRCT patients had a shared origin and were highly related. Other than the EWSR1-WT1 fusion, very few secondary cancer gene mutations were shared among the sites. Among these, ARID1A, was recurrently mutated, which corroborates findings by others in DSRCT patients. Knocking out ARID1A in JN-DSRCT cells using CRISPR/CAS9 resulted in significantly lower cell proliferation and increased drug sensitivity. The transcriptome data were integrated using network analysis and drug target database information to identify potential therapeutic opportunities in EWSR1-WT1-associated pathways, such as PI3K and mTOR pathways. Treatment of JN-DSRCT cells with the PI3K inhibitor alpelisib and mTOR inhibitor temsirolimus reduced cell proliferation. In addition, the low mutation burden was associated with an immune-cold state in DSRCT. Together, these data reveal multiple genomic and immune features of DSRCT and suggest therapeutic opportunities in patients.

Single cell T cell landscape and T cell receptor repertoire profiling of AML in context of PD-1 blockade therapy.

In contrast to the curative effect of allogenic stem cell transplantation in acute myeloid leukemia via T cell activity, only modest responses are achieved with checkpoint-blockade therapy, which might be explained by T cell phenotypes and T cell receptor (TCR) repertoires. Here, we show by paired single-cell RNA analysis and TCR repertoire profiling of bone marrow cells in relapsed/refractory acute myeloid leukemia patients pre/post azacytidine+nivolumab treatment that the disease-related T cell subsets are highly heterogeneous, and their abundance changes following PD-1 blockade-based treatment. TCR repertoires expand and primarily emerge from CD8+ cells in patients responding to treatment or having a stable disease, while TCR repertoires contract in therapy-resistant patients. Trajectory analysis reveals a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B and a bone marrow-residing memory CD8+ T cell subset, in which a population with stem-like properties expressing granzyme K is enriched in responders. Chromosome 7/7q loss, on the other hand, is a cancer-intrinsic genomic marker of PD-1 blockade resistance in AML. In summary, our study reveals that adaptive T cell plasticity and genomic alterations determine responses to PD-1 blockade in acute myeloid leukemia.

B-cell lymphoma/leukaemia 11B (BCL11B) expression status helps distinguish early T-cell precursor acute lymphoblastic leukaemia/lymphoma (ETP-ALL/LBL) from other subtypes of T-cell ALL/LBL.

B-cell lymphoma/leukaemia 11B (BCL11B) is an essential transcription factor for T-cell lineage commitment and maturation. We investigated BCL11B expression by immunohistochemistry in T-lymphoblastic leukaemia/lymphoma (T-ALL/LBL) (n = 115). The majority (83%) of early T-cell precursor T-ALL/LBL (ETP-ALL) cases showed negative BCL11B expression, while most (84%) of non-ETP-ALL/LBL were positive for BCL11B. A simplified three-marker [BCL11B, cluster of differentiation 5 (CD5), CD13] immunophenotypic score discriminated reliably between ETP-ALL and non-ETP-ALL/LBL. In ETP-ALL, patients with positive BCL11B expression had a better overall survival than those with negative BCL11B (P = 0·009). In summary, BCL11B is a valuable marker for T-ALL/LBL subtyping and serves as a potential prognostic marker in patients with ETP-ALL.

Enhancer reprogramming in PRC2-deficient malignant peripheral nerve sheath tumors induces a targetable de-differentiated state.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that frequently harbor genetic alterations in polycomb repressor complex 2 (PRC2) components-SUZ12 and EED. Here, we show that PRC2 loss confers a dedifferentiated early neural-crest phenotype which is exclusive to PRC2-mutant MPNSTs and not a feature of neurofibromas. Neural crest phenotype in PRC2 mutant MPNSTs was validated via cross-species comparative analysis using spontaneous and transgenic MPNST models. Systematic chromatin state profiling of the MPNST cells showed extensive epigenomic reprogramming or chromatin states associated with PRC2 loss and identified gains of active enhancer states/super-enhancers on early neural crest regulators in PRC2-mutant conditions around genomic loci that harbored repressed/poised states in PRC2-WT MPNST cells. Consistently, inverse correlation between H3K27me3 loss and H3K27Ac gain was noted in MPNSTs. Epigenetic editing experiments established functional roles for enhancer gains on DLX5-a key regulator of neural crest phenotype. Consistently, blockade of enhancer activity by bromodomain inhibitors specifically suppressed this neural crest phenotype and tumor burden in PRC2-mutant PDXs. Together, these findings reveal accumulation of dedifferentiated neural crest like state in PRC2-mutant MPNSTs that can be targeted by enhancer blockade.

Metabolic compensation activates pro-survival mTORC1 signaling upon 3-phosphoglycerate dehydrogenase inhibition in osteosarcoma.

Osteosarcoma is the most common pediatric and adult primary malignant bone cancer. Curative regimens target the folate pathway, downstream of serine metabolism, with high-dose methotrexate. Here, the rate-limiting enzyme in the biosynthesis of serine from glucose, 3-phosphoglycerate dehydrogenase (PHGDH), is examined, and an inverse correlation between PHGDH expression and relapse-free and overall survival in osteosarcoma patients is found. PHGDH inhibition in osteosarcoma cell lines attenuated cellular proliferation without causing cell death, prompting a robust metabolic analysis to characterize pro-survival compensation. Using metabolomic and lipidomic profiling, cellular response to PHGDH inhibition is identified as accumulation of unsaturated lipids, branched chain amino acids, and methionine cycle intermediates, leading to activation of pro-survival mammalian target of rapamycin complex 1 (mTORC1) signaling. Increased mTORC1 activation sensitizes cells to mTORC1 pathway inhibition, resulting in significant, synergistic cell death in vitro and in vivo. Identifying a therapeutic combination for PHGDH-high cancers offers preclinical justification for a dual metabolism-based combination therapy for osteosarcoma.

Germline DNMT3A mutation in familial acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by recurrent clonal events, including mutations in epigenetically relevant genes such as DNMT3A, ASXL1, IDH1/2, and TET2. Next-generation sequencing analysis of a mother and son pair who both developed adult-onset diploid AML identified a novel germline missense mutation DNMT3A p.P709S. The p.P709S protein-altering variant resides in the highly conserved catalytic DNMT3A methyltransferase domain. Functional studies demonstrate that the p.P709S variant confers dominant negative effects when interacting with wildtype DNMT3A. LINE-1 pyrosequencing and reduced representation bisulphite sequencing (RBBS) analysis demonstrated global DNA hypomethylation in germline samples, not present in the leukaemic samples. Somatic acquisition of IDH2 p.R172K mutations, in concert with additional acquired clonal DNMT3A events in both patients at the time of AML diagnosis, confirms the important pathogenic interaction of epigenetically active genes, and implies a strong selection and regulation of methylation in leukaemogenesis. Improved characterization of germline mutations may enable us to better predict malignant clonal evolution, improving our ability to provide customized treatment or future preventative strategies.

Immuno-genomic landscape of osteosarcoma.

Limited clinical activity has been seen in osteosarcoma (OS) patients treated with immune checkpoint inhibitors (ICI). To gain insights into the immunogenic potential of these tumors, we conducted whole genome, RNA, and T-cell receptor sequencing, immunohistochemistry and reverse phase protein array profiling (RPPA) on OS specimens from 48 pediatric and adult patients with primary, relapsed, and metastatic OS. Median immune infiltrate level was lower than in other tumor types where ICI are effective, with concomitant low T-cell receptor clonalities. Neoantigen expression in OS was lacking and significantly associated with high levels of nonsense-mediated decay (NMD). Samples with low immune infiltrate had higher number of deleted genes while those with high immune infiltrate expressed higher levels of adaptive resistance pathways. PARP2 expression levels were significantly negatively associated with the immune infiltrate. Together, these data reveal multiple immunosuppressive features of OS and suggest immunotherapeutic opportunities in OS patients.

Features of non-activation dendritic state and immune deficiency in blastic plasmacytoid dendritic cell neoplasm (BPDCN).

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, male-predominant hematologic malignancy with poor outcomes and with just one recently approved agent (tagraxofusp). It is characterized by the abnormal proliferation of precursor plasmacytoid dendritic cells (pDCs) with morphologic and molecular similarities to acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) in its presentation within the bone marrow and peripheral blood. To identify disease-specific molecular features of BPDCN, we profiled the bone marrow, peripheral blood, and serum samples from primary patient samples using an in-house hematologic malignancy panel ("T300" panel), transcriptome microarray, and serum multiplex immunoassays. TET2 mutations (5/8, 63%) were the most prevalent in our cohort. Using the transcriptome microarray, genes specific to pDCs (LAMP5, CCDC50) were more highly expressed in BPDCN than in AML specimens. Finally, the serum cytokine profile analysis showed significantly elevated levels of eosinophil chemoattractants eotaxin and RANTES in BPDCN as compared with AML. Along with the high levels of PTPRS and dendritic nature of the tumor cells, these findings suggest a possible pre-inflammatory context of this disease, in which BPDCN features nonactivated pDCs.

FusionPathway: Prediction of pathways and therapeutic targets associated with gene fusions in cancer.

Numerous gene fusions have been uncovered across multiple cancer types. Although the ability to target several of these fusions has led to the development of some successful anti-cancer drugs, most of them are not druggable. Understanding the molecular pathways of a fusion is important in determining its function in oncogenesis and in developing therapeutic strategies for patients harboring the fusion. However, the molecular pathways have been elucidated for only a few fusions, in part because of the labor-intensive nature of the required functional assays. Therefore, we developed a domain-based network approach to infer the pathways of a fusion. Molecular interactions of a fusion are first predicted by using its protein domain composition, and its associated pathways are then inferred from these molecular interactions. We demonstrated the capabilities of this approach by primarily applying it to the well-studied BCR-ABL1 fusion. The approach was also applied to two undruggable fusions in sarcoma, EWS-FL1 and FUS-DDIT3. We successfully identified known genes and pathways associated with these fusions and satisfactorily validated these predictions using several benchmark sets. The predictions of EWS-FL1 and FUS-DDIT3 also correlate with results of high-throughput drug screening. To our best knowledge, this is the first approach for inferring pathways of fusions.

KMT2D/MLL2 inactivation is associated with recurrence in adult-type granulosa cell tumors of the ovary.

Adult-type granulosa cell tumors of the ovary (aGCTs) are rare gynecologic malignancies that exhibit a high frequency of somatic FOXL2 c.C402G (p.Cys134Trp) mutation. Treatment of relapsed aGCT remains a significant clinical challenge. Here we show, using whole-exome and cancer gene panel sequencing of 79 aGCTs from two independent cohorts, that truncating mutation of the histone lysine methyltransferase gene KMT2D (also known as MLL2) is a recurrent somatic event in aGCT. Mono-allelic KMT2D-truncating mutations are more frequent in recurrent (10/44, 23%) compared with primary (1/35, 3%) aGCTs (p = 0.02, two-sided Fisher's exact test). IHC detects additional non-KMT2D-mutated aGCTs with loss of nuclear KMT2D expression, suggesting that non-genetic KMT2D inactivation may occur in this tumor type. These findings identify KMT2D inactivation as a novel driver event in aGCTs and suggest that mutation of this gene may increase the risk of disease recurrence.