Biallelic Inactivation Research Articles

Biallelic inactivation of the NF1 tumour suppressor gene in juvenile myelomonocytic leukaemia: Genetic evidence of driver function and implications for diagnostic workup.

Juvenile myelomonocytic leukaemia (JMML) is characterized by gene variants that deregulate the RAS signalling pathway. Children with neurofibromatosis type 1 (NF-1) carry a defective NF1 allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild-type allele. Here we examined the two-hit concept in leukaemic cells of 25 patients with JMML and NF-1. Ten patients with JMML/NF-1 exhibited a NF1 loss-of-function variant in combination with uniparental disomy of the 17q arm. Five had NF1 microdeletions combined with a pathogenic NF1 variant and nine carried two compound-heterozygous NF1 variants. We also examined 16 patients without clinical signs of NF-1 and no variation in the JMML-associated driver genes PTPN11, KRAS, NRAS or CBL (JMML-5neg) and identified eight patients with NF1 variants. Three patients had microdeletions combined with hemizygous NF1 variants, three had compound-heterozygous NF1 variants and two had heterozygous NF1 variants. In addition, we found a high incidence of secondary ASXL1 and/or SETBP1 variants in both groups. We conclude that the clinical diagnosis of JMML/NF-1 reliably indicates a NF1-driven JMML subtype, and that careful NF1 analysis should be included in the genetic workup of JMML even in the absence of clinical evidence of NF-1.

Clinico-Genetic and Prognostic Analyses of 635 Patients with Myelodysplastic Neoplasms Based on the 2022 World Health Organization Classification

Myelodysplastic neoplasms (MDS) are a heterogeneous group of clonal myeloid neoplasms characterized by dysregulated hematopoiesis. In the past several years, major advances in molecular technologies and the development of next generation sequencing have deepened our understanding of MDS pathobiology. Revisions of the existing classification were warranted. Under the aegis of the International Agency for Research on Cancer, the 2022 World Health Organization (WHO) classification was released. The 2022 WHO proposed new categories including MDS with biallelic TP53 inactivation (MDS-bi TP53), MDS, hypoplastic (MDS-h) and MDS with fibrosis (MDS-f). In addition, the novel risk scoring system-Molecular International Prognostic Scoring System (IPSS-M) has been established. There are limited data that explore the application of the 2022 WHO classification of MDS in light of the IPSS-M. In this study, a cohort of 635 patients diagnosed as having primary MDS based on the 2022 WHO criteria was retrospectively reviewed. We aimed to elucidate the differences in genetic features and clinical outcomes among patients with different MDS subtypes, based on the 2022 WHO classification. The survival impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT) was also evaluated. TruSight myeloid sequencing panel and the HiSeq platform were used to analyze the alterations of 54 myeloid neoplasm-relevant genes. Comparison of the genetic profiles of patients with different subtypes of MDS showed that patients with MDS-low blasts (LB) and those with MDS with LB and ring sideroblasts (RS) exhibited comparable mutation profiles, while patients with MDS- SF3B1 had different mutation pattern from that of these two subtypes. Among patients with MDS-increased blasts-1 (IB1), MDS-IB2 and MDS-f, patients with MDS-IB2 had higher frequencies of NRAS (9% vs. 2%, P=0.019) and mono-allelic TP53 (7% vs. 1%, P=0.031) mutations, and a trend of more TET2 (19% vs. 10%, P=0.059) mutations than those with MDS-IB1. In addition, patients with MDS-f exhibited lower incidence of TET2 (0% vs. 19%, P=0.020), and STAG2 mutations (8% vs. 27%, P=0.050) as compared to those with MDS-IB. Furthermore, patients with MDS-bi TP53 had specific concurrent genetic alterations with few co-occurring mutations which differed from those found in patients with MDS-IB or MDS-f. We performed pairwise survival comparisons among each subtypes of MDS (Figure 1). In low-risk MDS, patients with MDS-h had significantly longer median leukemia-free survival (LFS) and overall survival (OS) (185.5 months for median LFS and OS) compared with those with MDS-LB and RS [38.3 months for median LFS ( P=0.013) and OS ( P=0.010)], while had a similar outcome compared to those with MDS-LB [170.2 months for median LFS ( P=0.152) and OS ( P=0.146)], and MDS- SF3B1 [114.6 months for median LFS ( P=0.549) and OS ( P=0.528)]. For patients with increased blasts or bi TP53 mutations, those with bi TP53 inactivation had the worst outcomes (median LFS: 3.9 months and OS: 4.5 months, all P<0.001) compared to those with MDS-f (median LFS: 16.6 months and OS: 17.7 months), MDS-IB2 (median LFS: 10.0 months and OS: 17.7 months), and MDS-IB1 (median LFS: 25.6 months and OS: 31.4 months). Patients with MDS-f had similar survival to those with MDS-IB2 and IB-1 in terms of LFS (16.6 vs. 25.6 months, P=0.271) and OS (17.7 vs. 31.4 months, P=0.194) . Multivariable analysis showed that IPSS-M ( P<0.001), old age (HR for LFS: 1.023; for OS: 1.032, P<0.001), and the 2022 WHO categorization ( P<0.001) could predict LFS and OS independently and that HSCT could improve LFS (HR, 0.492, P=0.001) (Table 1). Subgroup analysis of the impact of transplantation using time-dependent Cox regression revealed that allo-HSCT could prolong both LFS and OS in patients with MDS-IB1 (HR for LFS: 0.293, for OS: 0.350, P<0.001), LFS alone (HR: 0.534, P=0.049) in patients with MDS-IB2, and a trend of LFS (HR: 0.305, P=0.078) in patients with MDS-f. However, allo-HSCT failed to remedy the dismal outcomes for patients with MDS-bi TP53. In conclusion, based on the evidence gathered from this cohort analysis, the 2022 WHO classification promote efficient segregation of this heterogeneous disease, with focus on the differences in molecular features and prognoses across different disease subtypes, leading to accurate MDS diagnosis and effective risk-adapted treatment.

Spatial Dissection of the Bone Marrow Microenvironment in Multiple Myeloma By High Dimensional Multiplex Tissue Imaging

Introduction Multiple myeloma (MM) is shaped by interactions between immune, stromal and tumor cells in the bone marrow (BM) microenvironment (BMME). Our understanding of these processes is based on high parametric flow cytometry and single cell transcriptomics, which lack spatial resolution. Thus, crucial aspects of the in situ tumor ecosystem, including cell-cell interactions, can only be predicted. Novel high dimensional, multiplex tissue imaging methods are able to capture similar granularity while also preserving spatial organization. By optimizing antibody selection, as well as staining, imaging and data processing protocols we have established a BMME-targeted CODEX workflow based on a 60 marker panel. Here, we conducted the first comprehensive multiplex protein imaging survey of the BMME across the spectrum of MM and precursor stages and leveraged paired FISH and whole genome sequencing (WGS) to elucidate the interplay of tumor intrinsic features and the BMME. Methods We imaged 489 BM trephine biopsies in tissue microarray format on the CODEX (Akoya Biosciences) platform. After quality control (≥750 cells per core, <25% unclassifiable cells) we retained data from 12 non-malignant controls, 13 MGUS, 20 smoldering MM, 349 uniformly treated newly diagnosed MM (NDMM) patients from a phase III clinical trial, as well as 22 relapsed MM samples with a median of 3 (range 1-6) tissue cores per sample, comprising a total of 3.1 million single cells. CODEX data was processed and analyzed using custom scripts. Cellular neighborhoods were computed by clustering the cell type composition of sliding windows (n=10 nearest neighboring cells) across the tissue. WGS (n=212) and FISH (n=309) were performed on CD138-purified BM mononuclear cells and analyzed using in-house pipelines. Results We identified 40 cell types, encompassing the relevant lineages (myeloid, lymphoid, stromal, endothelial, tumor), subtypes (e.g. T, B, NK cells) and functional states (e.g GRZB, Ki67, and PD1) of the BMME. The degree of plasma cell infiltration determined by CODEX correlated with the clinical pathologist's quantification based on classical immunohistology (r=0.77, p<0.0001), and the kappa/lambda status of MM cells matched the reported light chain restriction, supporting the validity of our data. At the compositional level, we observed shifts in relative cell type abundance from precursor to MM stages. Major changes in the BMME included enriched stromal, endothelial and CD8+ T cells as well as depleted polymorphonuclear (PMN), erythropoietic and CD4+ T cells in advanced stages. The highest proportion of monocytoid dendritic cells, monocytes, mast cells and cytotoxic CD8+ T cells was seen in relapsed MM. Leveraging the spatial resolution of the CODEX data, we identified multiple distinct cellular neighborhoods (CNs) defined by recurrent local cell type composition. CNs enriched for plasma cells in combination with different immune cell subsets including exhausted T cells increased towards the MM stage, while CNs dominated by PMNs, erythropoietic cells and CD206 negative macrophages decreased. To investigate links between MM subtypes and BMME patterns, we correlated CODEX data with MM-initiating and driver events. While patients with t(4;14) showed a significant increase in eosinophils, patients with gain1q had higher levels of endothelial and stromal subsets colocalizing in the same CN. Distinct types of stromal cells were also enriched in patients with double hits (≥2 FISH high risk features). RAS mutations and biallelic inactivation of tumor suppressor genes were associated with altered T cell subset composition. Further corroborating the importance of BMME architecture, a CN with colocalization of plasma and exhausted CD8 T cells was associated with unfavorable prognosis in NDMM patients. Conclusions High dimensional, multiplex tissue imaging of the BMME enables interrogation of cellular interactions and tissue architecture in situ in clinical samples at single cell resolution. To our knowledge this is the first highly multiplexed spatial study of the human BMME in a large clinical cohort and the first such study in multiple myeloma. We track characteristic shifts in tissue composition associated with disease progression, reveal genotype-phenotype associations in the BMME as a correlate of tumor-microenvironment co-evolution and detect prognostically relevant tissue architectural features.

Combination of genomic instability score and TP53 status for prognosis prediction in lung adenocarcinoma

The genomic instability (GI) /homologous recombination deficiency (HRD) score, calculated as the sum of the events of loss of heterozygosity (LOH), large-scale state transition (LST) and telomere allele imbalance (TAI), is used to guide the choice of treatment in several cancers, but its relationship with genomic features, clinicopathological characteristics and prognosis in lung cancer is poorly understood, which could lead to population bias in prospective studies. We retrospectively analyzed 1011 lung cancer patients whose tumor samples were successfully profiled by high-throughput sequencing panel including GI/HRD score. Alterations of many cancer suppressor genes were associated with higher GI/HRD scores, biallelic inactivation of TP53 was correlated with a high GI/HRD score. A combination of two gene alterations exhibited a higher GI/HRD scores than single gene alterations. The GI/HRD score was associated with advanced stages in lung adenocarcinoma but not in lung squamous cell carcinoma. Furthermore, patients with higher GI/HRD scores had significantly shorter overall survival and progression-free survival than patients with lower GI/HRD scores. Finally, patients with a combination of a higher GI/HRD scores and TP53 alteration exhibited an extremely poor prognosis compared with patients with a lower GI/HRD scores and wild-type TP53 (overall survival, training cohort, hazard ratio (HR) = 8.56, P < 0.001; validation cohort, HR = 6.47, P < 0.001; progression-free survival, HR = 4.76, P < 0.001). Our study revealed the prognostic value of the GI/HRD score in lung adenocarcinoma, but not for all lung cancer. Moreover, the combination of the GI/HRD score and TP53 status could be a promising strategy to predict the prognosis of patients with lung adenocarcinoma.

Concurrent PTEN and PDGFRB Alterations Characterize Storiform Collagenoma.

Storiform collagenoma is a rare mesenchymal skin tumor that is composed of thickened collagen bundles arranged in a characteristic storiform pattern with a relatively hypocellular CD34-positive spindle cell component. Storiform collagenoma is most often sporadic, but multiple lesions can occur in Cowden syndrome, which is characterized by germline alterations in PTEN (phosphatase and tensin homolog) on chromosome 10. Here, we investigated the molecular pathogenesis of storiform collagenoma using a targeted next-generation DNA sequencing platform, including 5 sporadic cases and one case associated with Cowden syndrome. Recurrent PTEN alterations were identified in all cases, with biallelic PTEN inactivation observed in the case associated with Cowden syndrome and one sporadic case. Unexpectedly, we also identified recurrent activating mutations in the platelet-derived growth factor receptor beta (PDGFRB) gene. This included a missense substitution in the D5 Ig-like domain of PDGFRB in the Cowden syndrome-associated case. In addition, we report missense alterations in the juxtamembrane domain of PDGFRB in 4 of 5 (80%) sporadic cases, including mutations that have been previously described in sporadic myofibroma and myopericytoma. Therefore, we confirm the neoplastic nature of storiform collagenoma, we expand the spectrum of reported PDGFRB alterations in mesenchymal tumors and we suggest a possible collaborative role for PTEN and PDGFRB in the pathogenesis of storiform collagenoma.

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators.

Mutation of the PRDM16 gene causes human dilated and non-compaction cardiomyopathy. The PRDM16 protein is a transcriptional regulator that affects cardiac development via Tbx5 and Hand1, thus regulating myocardial structure. The biallelic inactivation of Prdm16 induces severe cardiac dysfunction with post-natal lethality and hypertrophy in mice. The early pathological events that occur upon Prdm16 inactivation have not been explored. This study performed in-depth pathophysiological and molecular analyses of male and female Prdm16csp1/wt mice that carry systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes through transcriptomics, proteomics, and metabolomics. Kinetic modelling of cardiac metabolism was performed in silico with CARDIOKIN. Prdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt cardiac tissue of both sexes showed reductions in metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and the tricarboxylic acid cycle. Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. An accumulation of triacylglycerides exclusively in male Prdm16csp1/wt hearts suggests a sex-specific metabolic adaptation. Metabolic modelling using CARDIOKIN identified a reduction in fatty acid utilization in males as well as lower glucose utilization in female Prdm16csp1/wt cardiac tissue. On the level of transcripts and protein expression, Prdm16csp1/wt hearts demonstrate an up-regulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre-B-cell leukaemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional up-regulation was detected for Prdm16 itself, probably through an autoregulatory mechanism. Monoallelic, global Prdm16 mutation diminishes cardiac performance in Prdm16csp1/wt mice. Metabolic alterations and transcriptional dysregulation in Prdm16csp1/wt affect cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype, indicating sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in the PRDM16 associated cardiac pathology.

Germline and somatic drivers in inherited hematologic malignancies.

Inherited hematologic malignancies are linked to a heterogenous group of genes, knowledge of which is rapidly expanding using panel-based next-generation sequencing (NGS) or whole-exome/whole-genome sequencing. Importantly, the penetrance for these syndromes is incomplete, and disease development, progression or transformation has critical clinical implications. With the earlier detection of healthy carriers and sequential monitoring of these patients, clonal hematopoiesis and somatic driver variants become significant factors in determining disease transformation/progression and timing of (preemptive) hematopoietic stem cell transplant in these patients. In this review, we shed light on the detection of probable germline predisposition alleles based on diagnostic/prognostic 'somatic' NGS panels. A multi-tier approach including variant allele frequency, bi-allelic inactivation, persistence of a variant upon clinical remission and mutational burden can indicate variants with high pre-test probability. We also discuss the shared underlying biology and frequency of germline and somatic variants affecting the same gene, specifically focusing on variants in DDX41, ETV6, GATA2 and RUNX1. Germline variants in these genes are associated with a (specific) pattern or over-/underrepresentation of somatic molecular or cytogenetic alterations that may help identify the underlying germline syndrome and predict the course of disease in these individuals. This review is based on the current knowledge about somatic drivers in these four syndromes by integrating data from all published patients, thereby providing clinicians with valuable and concise information.

THU504 Is Carney Complex A Predisposing Syndrome For Breast Cancer? Prospective Study Of 50 Women

Abstract Disclosure: P. Vaduva: None. F. Violon: None. A. Jouinot: None. L. Bouys: None. S. Espiard: None. F. Bonnet-Serrano: None. N. Marie Odile: None. C. Cardot-bauters: None. G. Raverot: None. S. Hieronimus: None. H. Lefebvre: None. M.L. Nunes: None. A. Tabarin: None. L. Groussin Rouiller: None. G. Assié: None. M. Sibony: None. M. Vantyghem: None. P. Eric: None. J. Bertherat: None. Objective: Carney Complex (CNC) is a rare hereditary genetic syndrome, mostly due to inactivating pathogenic variants of the tumor suppressor gene PRKAR1A. It has a wide spectrum of manifestations with frequent pigmented skin lesions, cardiac myxomas, primary pigmented nodular adrenocortical dysplasia (PPNAD) causing Cushing syndrome, acromegaly and thyroid cancers. Breast benign tumors (fibroadenomas, ductal adenomas and myxoid lesions) have been associated with CNC, but so far, association with malignancy has not been investigated. Methods: The present study was designed to describe the characteristics of breast tumors diagnosed in CNC patients and their association with other manifestations of CNC and PRKAR1A genotype. Since breast cancer is the most frequent cancer in women and the leading cause of death from cancer worldwide, malignant breast lesions were carefully analyzed. This cohort comes from a 3 years’ follow-up multicenter French prospective study of CNC patients (Espiard, JCEM 2020). The 50 included women were investigated for CNC manifestations and particularly breast tumors, with breast imaging (echography+/-mammography), genetic and hormonal investigations, in order to characterize them and assess the frequency and average age of breast cancer. Results: Among the 38 women with breast imaging, 14 (28%) had breast tumors, half of them being bilateral. Ten women (20%) presented with benign tumors. Six women presented (12%) with breast carcinomas: five had invasive cancer under 50 years of age (10%) with one contralateral recurrence (4 ductal adenocarcinomas and one solid intracystic papillary carcinoma) and one had ductal carcinoma in situ. The occurrence of breast cancer was more frequent in the CNC women with PRKAR1A pathogenic variants than in the general population (OR=6.2[1.6-17.3]; p=0.006). The mean age at breast cancer diagnosis was 44.7 years old, 17 years younger than in the general population. Cumulative risk of breast cancer for women under 40 years of age was much higher in CNC women, compared to women worldwide (OR=592.1[79.7-3817.4]; p=9.58). Breast cancer had good prognosis factors: 5 out of 6 tumors were T1N0M0, so stage I. One woman had metastatic breast cancer, still alive after 15 years follow up. All breast cancers were negative for HER2, with 100% positivity for estrogen receptor and 50% for progesterone receptor. They all occurred in individuals with familial CNC and PRKAR1A pathogenic variants. Loss of heterozygosity at the PRKAR1A locus observed in 2 investigated breast carcinomas tumor tissue suggests that PRKAR1A bi-allelic inactivation could promote breast cancer development. Conclusions: Breast carcinoma occurs frequently and at an early age in women with CNC, suggesting that CNC predisposes to breast carcinoma. This suggests that adequate screening strategy (starting around 40 years old) and follow up should be discussed in CNC women. Presentation: Thursday, June 15, 2023

Vascular endothelial cell-specific disruption of the profilin1 gene leads to severe multiorgan pathology and inflammation causing mortality.

Actin-binding protein Profilin1 is an important regulator of actin cytoskeletal dynamics in cells and critical for embryonic development in higher eukaryotes. The objective of the present study was to examine the consequence of loss-of-function of Pfn1 in vascular endothelial cells (ECs) in vivo. We utilized a mouse model engineered for tamoxifen-inducible biallelic inactivation of the Pfn1 gene selectively in EC (Pfn1EC-KO). Widespread deletion of EC Pfn1 in adult mice leads to severe health complications presenting overt pathologies (endothelial cell death, infarct, and fibrosis) in major organ systems and evidence for inflammatory infiltrates, ultimately compromising the survival of animals within 3 weeks of gene ablation. Mice deficient in endothelial Pfn1 exhibit selective bias toward the proinflammatory myeloid-derived population of immune cells, a finding further supported by systemic elevation of proinflammatory cytokines. We further show that triggering Pfn1 depletion not only directly upregulates proinflammatory cytokine/chemokine gene expression in EC but also potentiates the paracrine effect of EC on proinflammatory gene expression in macrophages. Consistent with these findings, we provide further evidence for increased activation of Interferon Regulatory Factor 7 (IRF7) and STAT1 in EC when depleted of Pfn1. Collectively, these findings for the first time demonstrate a prominent immunological consequence of loss of endothelial Pfn1 and an indispensable role of endothelial Pfn1 in mammalian survival unlike tolerable phenotypes of Pfn1 loss in other differentiated cell types.

Identifying novel mechanisms of biallelic TP53 loss refines poor outcome for patients with multiple myeloma

Biallelic TP53 inactivation is the most important high-risk factor associated with poor survival in multiple myeloma. Classical biallelic TP53 inactivation has been defined as simultaneous mutation and copy number loss in most studies; however, numerous studies have demonstrated that other factors could lead to the inactivation of TP53. Here, we hypothesized that novel biallelic TP53 inactivated samples existed in the multiple myeloma population. A random forest regression model that exploited an expression signature of 16 differentially expressed genes between classical biallelic TP53 and TP53 wild-type samples was subsequently established and used to identify novel biallelic TP53 samples from monoallelic TP53 groups. The model reflected high accuracy and robust performance in newly diagnosed relapsed and refractory populations. Patient survival of classical and novel biallelic TP53 samples was consistently much worse than those with mono-allelic or wild-type TP53 status. We also demonstrated that some predicted biallelic TP53 samples simultaneously had copy number loss and aberrant splicing, resulting in overexpression of high-risk transcript variants, leading to biallelic inactivation. We discovered that splice site mutation and overexpression of the splicing factor MED18 were reasons for aberrant splicing. Taken together, our study unveiled the complex transcriptome of TP53, some of which might benefit future studies targeting abnormal TP53.

Infantile Sinonasal Myxoma Is Clinically and Genetically Distinct From Other Myxomas of the Craniofacial Bones and From Desmoid Fibromatosis.

Sinonasal myxomas are rare benign tumors of the maxillary bone and sinus. There is published evidence that sinonasal myxomas occurring in children up to 3 years of age ("infantile sinonasal myxomas") are clinically distinctive and harbor Wnt signaling pathway alterations. Here, we characterized 16 infantile sinonasal myxomas and compared them to 19 maxillary myxomas and 11 mandibular myxomas in older patients. Clinical follow-up was available for 21 patients (46%) overall (median: 2.6y; range: 4mo to 21y), including 10 of 16 infantile sinonasal myxomas (62%). None of the 8 resected infantile sinonasal myxomas recurred, despite positive margins in 6 of them. One incompletely resected infantile sinonasal myxoma underwent partial regression without additional treatment. In contrast, 4 of the 11 other myxomas with follow-up recurred (36%), including one that recurred twice. Imaging studies demonstrated all infantile sinonasal myxomas to be expansile lesions arising from the anterior maxillary bone adjacent to the nasal aperture, with peripheral reactive bone formation. Histologically, infantile sinonasal myxomas showed short, intersecting fascicles of bland fibroblastic cells with prominent stromal vessels. Examples with collagenous stroma showed some morphologic overlap with desmoid fibromatosis, although none showed infiltrative growth into adjacent soft tissue. Immunohistochemistry demonstrated nuclear β-catenin expression in 14 of 15 infantile sinonasal myxomas (93%), in contrast to 4 of 26 other myxomas of craniofacial bones (15%). Smooth muscle actin was expressed in only 1 of 11 infantile sinonasal myxomas (9%). Next-generation sequencing was successfully performed on 10 infantile sinonasal myxomas and 7 other myxomas. Infantile sinonasal myxomas harbored CTNNB1 point mutations in 4 cases (D32Y, G34E, G34R, and I35S), and none harbored alterations to the phosphorylation sites T41 and S45 that are altered in 99% of CTNNB1 -mutant desmoid fibromatoses. Three tumors showed alterations consistent with biallelic APC inactivation. Three infantile sinonasal myxomas that showed strong nuclear β-catenin expression were negative for CTNNB1 and APC alterations. Sequencing was negative for CTNNB1 or APC alterations in all 7 myxomas of craniofacial bones in older patients. Four of these myxomas in older patients (57%) showed copy number alterations, and all lacked known driving alterations. These findings support the notion that infantile sinonasal myxomas are clinically and genetically distinctive, and we propose the use of the diagnostic term "infantile sinonasal myxoma" to distinguish this tumor type from other myxomas of the craniofacial bones. Infantile sinonasal myxoma should be distinguished from desmoid fibromatosis because of its unique clinical presentation, more indolent clinical behavior, different morphology, different immunohistochemical profile, and different genetics. Given its indolent behavior even when marginally excised, infantile sinonasal myxoma can be managed with conservative surgery.

A novel marker integrating multiple genetic alterations better predicts platinum sensitivity in ovarian cancer than HRD score.

Introduction: Platinum-based chemotherapy is the first-line treatment strategy for ovarian cancer patients. The dismal prognosis of ovarian cancer was shown to be stringently associated with the heterogeneity of tumor cells in response to this therapy, therefore understanding platinum sensitivity in ovarian cancer would be helpful for improving patients' quality of life and clinical outcomes. HRDetect, utilized to characterize patients' homologous recombination repair deficiency, was used to predict patients' response to platinum-based chemotherapy. However, whether each of the single features contributing to HRD score is associated with platinum sensitivity remains elusive. Methods: We analyzed the whole-exome sequencing data of 196 patients who received platinum-based chemotherapy from the TCGA database. Genetic features were determined individually to see if they could indicate patients' response to platinum-based chemotherapy and prognosis, then integrated into a Pt-score employing LASSO regression model to assess its predictive performance. Results and discussion: Multiple genetic features, including bi-allelic inactivation of BRCA1/2 genes and genes involved in HR pathway, multiple somatic mutations in genes involved in DNA damage repair (DDR), and previously reported HRD-related features, were found to be stringently associated with platinum sensitivity and improved prognosis. Higher contributions of mutational signature SBS39 or ID6 predicted improved overall survival. Besides, arm-level loss of heterozygosity (LOH) of either chr4p or chr5q predicted significantly better disease-free survival. Notably, some of these features were found independent of HRD. And SBS3, an HRD-related feature, was found irrelevant to platinum sensitivity. Integrated all candidate markers using the LASSO model to yield a Pt-score, which showed better predictive ability compared to HRDetect in determining platinum sensitivity and predicting patients' prognosis, and this performance was validated in an independent cohort. The outcomes of our study will be instrumental in devising effective strategies for treating ovarian cancer with platinum-based chemotherapy.

Acquired resistance to a GPRC5D-directed T-cell engager in multiple myeloma is mediated by genetic or epigenetic target inactivation.

Bispecific antibodies targeting GPRC5D demonstrated promising efficacy in multiple myeloma, but acquired resistance usually occurs within a few months. Using a single-nucleus multi-omic strategy in three patients from the MYRACLE cohort (ClinicalTrials.gov registration: NCT03807128 ), we identified two resistance mechanisms, by bi-allelic genetic inactivation of GPRC5D or by long-range epigenetic silencing of its promoter and enhancer regions. Molecular profiling of target genes may help to guide the choice of immunotherapy and early detection of resistance in multiple myeloma.

Molecular-based targeted therapies in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma refractory to atezolizumab/bevacizumab

The "French Medicine Genomic program 2025" has been designed to give patients with cancers that are refractory to systemic treatments access to off-label therapies adapted to their specific genomic profile. Herein, we reported the results of this program in patients with advanced hepatocellular carcinoma (HCC) and hepato-cholangiocarcinoma (H-CCK). In one center, all patients with HCC or H-CCK who progressed under atezolizumab/bevacizumab with available tumor frozen samples benefited from whole-genome/-exome and RNA sequencing. Targeted therapies were matched to genomic alterations following the recommendations of a molecular tumor board and radiological response and overall survival were assessed. Among 135 patients with HCC and H-CCK treated by atezolizumab/bevacizumab, 20 patients benefited from genomic analysis after progression (16 HCC; 4H-CCK). Nineteen patients had analyzable data, 70% were male, median age was 57 years, 65% had metastatic disease and 45% had vascular invasion. Among these 19 patients, 14 patients (76%) harbored at least one actionable genomic alteration and 9/14 received an adapted targeted therapy (45%). One patient with H-CCK showing CDK4 amplification was treated with palbociclib and achieved a partial radiological response for 16 months. Another patient with H-CCK, high HER2 overexpression and a high homologous recombination score was treated with trastuzumab/olaparib and had stable disease. One patient with an HCC and bi-allelic inactivation of TSC2 achieved a complete radiological response under everolimus. The remaining six treated patients (all HCC) had progressive disease, including three patients treated with trametinib, two with everolimus and one with olaparib. Molecular-based guided therapy is feasible in patients with HCC/H-CCK progressing under atezolizumab/bevacizumab and may be useful in a small subset of patients. The use of whole-genome/-exome and RNA sequencing in clinical practice has not been reported in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma. Herein, we performed a pilot study which suggested that whole-genome/-exome and RNA sequencing is feasible on tumor biopsies from patients refractory to atezolizumab/bevacizumab, with a small subset of patients exhibiting at least one actionable genomic alteration and receiving an adapted targeted therapy. This proof-of-concept study suggests that this clinical strategy could benefit a small subset of patients. Finally, validation of this approach will be required in a larger cohort of patients.

Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Human 'knockouts' of CSF3 display severe congenital neutropenia.

Colony-stimulating factor 3 (CSF3) is a key factor in neutrophil production and function, and recombinant forms have been used clinically for decades to treat congenital and acquired neutropenia. Although biallelic inactivation of its receptor CSF3R is a well-established cause of severe congenital neutropenia (SCN), no corresponding Mendelian disease has been ascribed to date to CSF3. Here, we describe three patients from two families each segregating a different biallelic inactivating variant in CSF3 with SCN. Complete deficiency of CSF3 as a result of nonsense-mediated decay (NMD) could be demonstrated on RT-PCR using skin fibroblasts-derived RNA. The phenotype observed in this cohort mirrors that documented in mouse and zebrafish models of CSF3 deficiency. Our results suggest that CSF3 deficiency in humans causes a novel autosomal recessive form of SCN.

Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour

PurposeRetinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken.MethodsMYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells.ResultsThe expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis.ConclusionIn conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour.

Modeling human brain rhabdoid tumor by inactivating tumor suppressor genes in induced pluripotent stem cells

Atypical teratoid/rhabdoid tumor (ATRT) is a rare childhood malignancy that originates in the central nervous system. Over ninety-five percent of ATRT patients have biallelic inactivation of the tumor suppressor gene SMARCB1. ATRT has no standard treatment, and a major limiting factor in therapeutic development is the lack of reliable ATRT models. We employed CRISPR/Cas9 gene-editing technology to knock out SMARCB1 and TP53 genes in human episomal induced pluripotent stem cells (Epi-iPSCs), followed by brief neural induction, to generate an ATRT-like model. The dual knockout Epi-iPSCs retained their stemness with the capacity to differentiate into three germ layers. High expression of OCT4 and NANOG in neurally induced knockout spheroids was comparable to that in two ATRT cell lines. Beta-catenin protein expression was higher in SMARCB1-deficient cells and spheroids than in normal Epi-iPSC-derived spheroids. Nucleophosmin, Osteopontin, and Ki-67 proteins were also expressed by the SMARCB1-deficient spheroids. In summary, the tumor model resembled embryonal features of ATRT and expressed ATRT biomarkers at mRNA and protein levels. Ribociclib, PTC-209, and the combination of clofilium tosylate and pazopanib decreased the viability of the ATRT-like cells. This disease modeling scheme may enable the establishment of individualized tumor models with patient-specific mutations and facilitate high-throughput drug testing.

The Contribution of Oxidative Stress to NF1-Altered Tumors.

The neurofibromatosis-1 gene (NF1) was initially characterized because its germline mutation is responsible for an inherited syndromic disease predisposing tumor development, in particular neurofibromas but also various malignancies. Recently, large-scale tumor sequencing efforts have demonstrated NF1 as one of the most frequently mutated genes in human cancer, being mutated in approximately 5-10% of all tumors, especially in malignant peripheral nerve sheath tumors and different skin tumors. NF1 acts as a tumor suppressor gene that encodes neurofibromin, a large protein that controls neoplastic transformation through several molecular mechanisms. On the other hand, neurofibromin loss due to NF1 biallelic inactivation induces tumorigenic hyperactivation of Ras and mTOR signaling pathways. Moreover, neurofibromin controls actin cytoskeleton structure and the metaphase-anaphase transition. Consequently, neurofibromin deficiency favors cell mobility and proliferation as well as chromosomal instability and aneuploidy, respectively. Growing evidence supports the role of oxidative stress in NF1-related tumorigenesis. Neurofibromin loss induces oxidative stress both directly and through Ras and mTOR signaling activation. Notably, innovative therapeutic approaches explore drug combinations that further increase reactive oxygen species to boost the oxidative unbalance of NF1-altered cancer cells. In our paper, we review NF1-related tumors and their pathogenesis, highlighting the twofold contribution of oxidative stress, both tumorigenic and therapeutic.

Novel scheme for defining the clinical implications of TP53 mutations in myeloid neoplasia

BackgroundTP53 mutations (TP53MT) occur in diverse genomic configurations. Particularly, biallelic inactivation is associated with poor overall survival in cancer. Lesions affecting only one allele might not be directly leukemogenic, questioning the presence of cryptic biallelic subclones in cases with dismal prognosis.MethodsWe have collected clinical and molecular data of 7400 patients with myeloid neoplasms and applied a novel model by identifying an optimal VAF cutoff using a statistically robust strategy of sampling-based regression on survival data to accurately classify the TP53 allelic configuration and assess prognosis more precisely.ResultsOverall, TP53MT were found in 1010 patients. Following the traditional criteria, 36% of the cases were classified as single hits, while 64% exhibited double hits genomic configuration. Using a newly developed molecular algorithm, we found that 579 (57%) patients had unequivocally biallelic, 239 (24%) likely contained biallelic, and 192 (19%) had most likely monoallelic TP53MT. Interestingly, our method was able to upstage 192 out of 352 (54.5%) traditionally single hit lesions into a probable biallelic category. Such classification was further substantiated by a survival-based model built after re-categorization. Among cases traditionally considered monoallelic, the overall survival of those with probable monoallelic mutations was similar to the one of wild-type patients and was better than that of patients with a biallelic configuration. As a result, patients with certain biallelic hits, regardless of the disease subtype (AML or MDS), had a similar prognosis. Similar results were observed when the model was applied to an external cohort. In addition, single-cell DNA studies unveiled the biallelic nature of previously considered monoallelic cases.ConclusionOur novel approach more accurately resolves TP53 genomic configuration and uncovers genetic mosaicism for the use in the clinical setting to improve prognostic evaluation.