An artificial intelligence-assisted clinical framework to facilitate diagnostics and translational discovery in hematologic neoplasia.

BACKGROUND: The increasing volume and intricacy of sequencing data, along with other clinical and diagnostic data, like drug responses and measurable residual disease, creates challenges for efficient clinical comprehension and interpretation. Using paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) as a use case, we present an artificial intelligence (AI)-assisted clinical framework clinALL that integrates genomic and clinical data into a user-friendly interface to support routine diagnostics and reveal translational insights for hematologic neoplasia. METHODS: We performed targeted RNA sequencing in 1365 cases with haematological neoplasms, primarily paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) from the AIEOP-BFM ALL study. We carried out fluorescence in situ hybridization (FISH), karyotyping and arrayCGH as part of the routine diagnostics. The analysis results of these assays as well as additional clinical information were integrated into an interactive web interface using Bokeh, where the main graph is based on Uniform Manifold Approximation and Projection (UMAP) analysis of the gene expression data. At the backend of the clinALL, we built both shallow machine learning models and a deep neural network using Scikit-learn and PyTorch respectively. FINDINGS: By applying clinALL, 78% of undetermined patients under the current diagnostic protocol were stratified, and ambiguous cases were investigated. Translational insights were discovered, including IKZF1plus status dependent subpopulations of BCR::ABL1 positive patients, and a subpopulation within ETV6::RUNX1 positive patients that has a high relapse frequency. Our best machine learning models, LDA and PASNET-like neural network models, achieve F1 scores above 97% in predicting patients' subgroups. INTERPRETATION: An AI-assisted clinical framework that integrates both genomic and clinical data can take full advantage of the available data, improve point-of-care decision-making and reveal clinically relevant insights promptly. Such a lightweight and easily transferable framework works for both whole transcriptome data as well as the cost-effective targeted RNA-seq, enabling efficient and equitable delivery of personalized medicine in small clinics in developing countries. FUNDING: German Ministry of Education and Research (BMBF), German Research Foundation (DFG) and Foundation for Polish Science.

Functional characterization of novel or yet uncharacterized ATP7B missense variants detected in patients with clinical Wilson's disease.

Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P-type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic.

European standard clinical practice - Key issues for the medical care of individuals with familial leukemia.

Although hematologic malignancies (HM) are no longer considered exclusively sporadic, additional awareness of familial cases has yet to be created. Individuals carrying a (likely) pathogenic germline variant (e.g., in ETV6, GATA2, SAMD9, SAMD9L, or RUNX1) are at an increased risk for developing HM. Given the clinical and psychological impact associated with the diagnosis of a genetic predisposition to HM, it is of utmost importance to provide high-quality, standardized patient care. To address these issues and harmonize care across Europe, the Familial Leukemia Subnetwork within the ERN PaedCan has been assigned to draft an European Standard Clinical Practice (ESCP) document reflecting current best practices for pediatric patients and (healthy) relatives with (suspected) familial leukemia. The group was supported by members of the German network for rare diseases MyPred, of the Host Genome Working Group of SIOPE, and of the COST action LEGEND. The ESCP on familial leukemia is proposed by an interdisciplinary team of experts including hematologists, oncologists, and human geneticists. It is intended to provide general recommendations in areas where disease-specific recommendations do not yet exist. Here, we describe key issues for the medical care of familial leukemia that shall pave the way for a future consensus guideline: (i) identification of individuals with or suggestive of familial leukemia, (ii) genetic analysis and variant interpretation, (iii) genetic counseling and patient education, and (iv) surveillance and (psychological) support. To address the question on how to proceed with individuals suggestive of or at risk of familial leukemia, we developed an algorithm covering four different, partially linked clinical scenarios, and additionally a decision tree to guide clinicians in their considerations regarding familial leukemia in minors with HM. Our recommendations cover, not only patients but also relatives that both should have access to adequate medical care. We illustrate the importance of natural history studies and the need for respective registries for future evidence-based recommendations that shall be updated as new evidence-based standards are established.

Genetic susceptibility in children, adolescents, and young adults diagnosed with soft-tissue sarcomas.

Soft tissue sarcomas (STS) may arise as a consequence of germline variants in cancer predisposition genes (CPGs). We believe that elucidating germline sarcoma predisposition is critical for understanding disease biology and therapeutic requirements. Participation in surveillance programs may allow for early tumor detection, early initiation of therapy and, ultimately, better outcomes. Among children, adolescents, and adults diagnosed with soft-tissue sarcomas and examined as part of published germline sequencing studies, pathogenic/likely pathogenic (P/LP) variants in CPGs were reported in 7-33% of patients. P/LP germline variants were detected most frequently in TP53, NF1 and BRCA1/2. In this review, we describe reported associations between soft tissue sarcomas and germline variants in CPGs, with mentioning of locally aggressive and benign soft tissue tumors that have important associations with cancer predisposition syndromes. We also discuss recommendations for diagnostic germline genetic testing. Testing for sarcoma-predisposing germline variants should be considered as part of the routine clinical workup and care of any child, adolescent, or adult diagnosed with STS and take into account consequences for the whole family.

Rare and potentially fatal - Cytogenetically cryptic TNIP1::PDGFRB and PCM1::FGFR1 fusion leading to myeloid/lymphoid neoplasms with eosinophilia in children.

Myeloid/lymphoid neoplasms with eosinophilia (MLN-eos) are rare haematological neoplasms primarily affecting adults. The heterogeneous clinical picture and the rarity of the disease, especially in children, may delay an early diagnosis. MLN-eos are characterized by constitutive tyrosine kinase (TK) activity due to gene fusions. It is thus of importance to obtain a prompt genetic diagnosis to start a specific therapy. Here, we outline the clinical, genetic, and biochemical background of TK driven MLN-eos and report two extremely rare paediatric cases of MLN-eo, the used diagnostic methods, therapy and clinical outcomes. Our results demonstrate that, standard cytogenetic and molecular methods may not be sufficient to diagnose MLN-eo due to cytogenetically cryptic aberrations. We therefore recommend performing additional evaluation with fluorescence in-situ hybridization and molecular genetic methods (array-based comparative genomic hybridization and RNA sequencing) which will lead to the correct diagnosis. Following this diagnostic route we detected a TNIP1::PDGFRB and a PCM1::FGFR1 fusion in our patients. Thus, genetic diagnosis must be precise and quick in order to initiate adequate therapies with tyrosine kinase inhibitors or HSCT.

Epigenome-wide analysis of T-cell large granular lymphocytic leukemia identifies BCL11B as a potential biomarker.

BACKGROUND: The molecular pathogenesis of T-cell large granular lymphocytic leukemia (T-LGLL), a mature T-cell leukemia arising commonly from T-cell receptor αß-positive CD8+ memory cytotoxic T cells, is only partly understood. The role of deregulated methylation in T-LGLL is not well known. We analyzed the epigenetic profile of T-LGLL cells of 11 patients compared to their normal counterparts by array-based DNA methylation profiling. For identification of molecular events driving the pathogenesis of T-LGLL, we compared the differentially methylated loci between the T-LGLL cases and normal T cells with chromatin segmentation data of benign T cells from the BLUEPRINT project. Moreover, we analyzed gene expression data of T-LGLL and benign T cells and validated the results by pyrosequencing in an extended cohort of 17 patients, including five patients with sequential samples. RESULTS: We identified dysregulation of DNA methylation associated with altered gene expression in T-LGLL. Since T-LGLL is a rare disease, the samples size is low. But as confirmed for each sample, hypermethylation of T-LGLL cells at various CpG sites located at enhancer regions is a hallmark of this disease. The interaction of BLC11B and C14orf64 as suggested by in silico data analysis could provide a novel pathogenetic mechanism that needs further experimental investigation. CONCLUSIONS: DNA methylation is altered in T-LGLL cells compared to benign T cells. In particular, BCL11B is highly significant differentially methylated in T-LGLL cells. Although our results have to be validated in a larger patient cohort, BCL11B could be considered as a potential biomarker for this leukemia. In addition, altered gene expression and hypermethylation of enhancer regions could serve as potential mechanisms for treatment of this disease. Gene interactions of dysregulated genes, like BLC11B and C14orf64, may play an important role in pathogenic mechanisms and should be further analyzed.

Beyond Pathogenic RUNX1 Germline Variants: The Spectrum of Somatic Alterations in RUNX1-Familial Platelet Disorder with Predisposition to Hematologic Malignancies.

Pathogenic loss-of-function RUNX1 germline variants cause autosomal dominantly-inherited familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD). RUNX1-FPD is characterized by incomplete penetrance and a broad spectrum of clinical phenotypes, even within affected families. Heterozygous RUNX1 germline variants set the basis for leukemogenesis, but, on their own, they are not transformation-sufficient. Somatically acquired secondary events targeting RUNX1 and/or other hematologic malignancy-associated genes finally lead to MDS, AML, and rarely other hematologic malignancies including lymphoid diseases. The acquisition of different somatic variants is a possible explanation for the variable penetrance and clinical heterogeneity seen in RUNX1-FPD. However, individual effects of secondary variants are not yet fully understood. Here, we review 91 cases of RUNX1-FPD patients who predominantly harbor somatic variants in genes such as RUNX1, TET2, ASXL1, BCOR, PHF6, SRSF2, NRAS, and DNMT3A. These cases illustrate the importance of secondary events in the development and progression of RUNX1-FPD-associated hematologic malignancies. The leukemia-driving interplay of predisposing germline variants and acquired variants remain to be elucidated to better understand clonal evolution and malignant transformation and finally allow risk-adapted surveillance and targeted therapeutic measures to prevent leukemia.

Clonal Evolution at First Sight: A Combined Visualization of Diverse Diagnostic Methods Improves Understanding of Leukemic Progression.

Patients with myeloid neoplasia are classified by the WHO classification systems. Besides clinical and hematological criteria, cytogenetic and molecular genetic alterations highly impact treatment stratification. In routine diagnostics, a combination of methods is used to decipher different types of genetic variants. Eight patients were comprehensively analyzed using karyotyping, fluorescence in situ hybridization, array-CGH and a custom NGS panel. Clonal evolution was reconstructed manually, integrating all mutational information on single nucleotide variants (SNVs), insertions and deletions (indels), structural variants and copy number variants (CNVs). To allow a correct integration, we differentiate between three scenarios: 1) CNV occurring prior to the SNV/indel, but in the same cells. 2) SNV/indel occurring prior to the CNV, but in the same cells. 3) SNV/indel and CNV existing in parallel, independent of each other. Applying this bioinformatics approach, we reconstructed clonal evolution for all patients. This generalizable approach offers the possibility to integrate various data to analyze identification of driver and passenger mutations as well as possible targets for personalized medicine approaches. Furthermore, this model can be used to identify markers to assess the minimal residual disease.

OnkoRiskNET: a multicenter, interdisciplinary, telemedicine-based model to improve care for patients with a genetic tumor risk syndrome.

BACKGROUND: Genetic tumor risk syndromes are responsible for at least five to ten percent of the 4 million cases of cancer diagnosed in Europe every year. Currently, the care of oncological patients suffers from a lack of specialists in medical genetics and also a lack of access to genetic care in rural areas and structured care pathways between oncologists and medical geneticists. As a result, genetic tumor risk syndromes are underdiagnosed with potentially fatal consequences for patients and their families. METHODS: The OnkoRiskNET study is supported by a grant from the Federal Joint Committee of the Federal Republic of Germany. The study will include 2,000 oncological index patients from oncology practices in Lower Saxony and Saxony after the start of the study in July 2021. Randomization is carried out by means of a stepped wedge design at the level of the practices. Patients either go through routine care or the new form of care with structured cooperation between medical geneticists and oncologists, case management and the use of telemedical genetic counseling. Using a mixed-methods approach, the following parameters will be evaluated in the control and intervention group: (1) Conducted genetic counseling sessions by patients with suspected tumor risk syndrome and their first degree relatives; (2) Patient satisfaction and psychological distress after genetic counseling and testing; (3) Factors influencing the acceptance and experience of telemedical genetic counseling; (4) Satisfaction of oncologists and medical genetics with the structured pathway; (5) Cost efficiency of the new form of care. DISCUSSION: OnkoRiskNET aims to close the gap in care through the formation of a cooperation network between practicing oncologists and specialists in medical genetics and the use of telemedical genetic counseling, thereby, increasing the diagnostic rate in genetic tumor risk syndromes and serving as a model for future genetic care in Germany. TRIAL REGISTRATION: Trial was registered on 01.12.2021 in the German Clinical Trial Register ( https://trialsearch.who.int/ ) with the DRKS-ID:  DRKS00026679 . TITLE: Cooperation network for the provision of local care for patients and families with a genetic tumour risk syndrome. Trial acronym: OnkoRiskNET. Protocol version 1.1.

MiR-129-5p exerts Wnt signaling-dependent tumor-suppressive functions in hepatocellular carcinoma by directly targeting hepatoma-derived growth factor HDGF.

BACKGROUND: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis. METHODS: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined. RESULTS: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs. CONCLUSIONS: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.

MicroRNA-449a Inhibits Triple Negative Breast Cancer by Disturbing DNA Repair and Chromatid Separation.

Chromosomal instability (CIN) can be a driver of tumorigenesis but is also a promising therapeutic target for cancer associated with poor prognosis such as triple negative breast cancer (TNBC). The treatment of TNBC cells with defects in DNA repair genes with poly(ADP-ribose) polymerase inhibitor (PARPi) massively increases CIN, resulting in apoptosis. Here, we identified a previously unknown role of microRNA-449a in CIN. The transfection of TNBC cell lines HCC38, HCC1937 and HCC1395 with microRNA-449a mimics led to induced apoptosis, reduced cell proliferation, and reduced expression of genes in homology directed repair (HDR) in microarray analyses. EME1 was identified as a new target gene by immunoprecipitation and luciferase assays. The reduced expression of EME1 led to an increased frequency of ultrafine bridges, 53BP1 foci, and micronuclei. The induced expression of microRNA-449a elevated CIN beyond tolerable levels and induced apoptosis in TNBC cell lines by two different mechanisms: (I) promoting chromatid mis-segregation by targeting endonuclease EME1 and (II) inhibiting HDR by downregulating key players of the HDR network such as E2F3, BIRC5, BRCA2 and RAD51. The ectopic expression of microRNA-449a enhanced the toxic effect of PARPi in cells with pathogenic germline BRCA1 variants. The newly identified role makes microRNA-449a an interesting therapeutic target for TNBC.

Evolution of severe (transfusion-dependent) anaemia in myelodysplastic syndromes with 5q deletion is characterized by a macrophage-associated failure of the eythropoietic niche.

Evolution of erythrocyte transfusion-dependent (RBC-TD) anaemia associated with haploinsufficiency of the ribosomal protein subunit S14 gene (RPS14) is a characteristic complication of myelodysplastic syndromes (MDS) with del(5q) [MDS.del(5q)]. Evaluating 39 patients with MDS.del(5q), <5% of anaemia progression was attributable to RPS14-dependent alterations of normoblasts, pro-erythroblasts, or CD34+ CD71+ precursors. Ninety-three percent of anaemia progression and 70% of the absolute decline in peripheral blood Hb value were attributable to disappearance of erythroblastic islands (Ery-Is). Ery-Is loss occurred independently of blast excess, TP53 mutation, additional chromosome aberrations and RPS14-dependent alterations of normoblasts and pro-erythroblasts. It was associated with RPS14-dependent intrinsic (S100A8+ ) and extrinsic [tumour necrosis factor α (TNF-α)-overproduction] alterations of (CD169+ ) marrow macrophages (p < 0.00005). In a mouse model of RPS14 haploinsufficiency, Ery-Is disappeared to a similar degree: approximately 70% of Ery-Is loss was related to RPS14-dependent S100A8 overexpression of marrow macrophages, less than 20% to that of CD71high Ter119- immature precursors, and less than 5% to S100A8/p53 overexpression of normoblasts or pro-erythroblasts. Marked Ery-Is loss predicted reduced efficacy (erythrocyte transfusion independence) of lenalidomide therapy (p = 0.0006). Thus, erythroid hypoplasia, a characteristic complication of MDS.del(5q), seems to result primarily from a macrophage-associated failure of the erythropoietic niche markedly reducing the productive capacity of erythropoiesis as the leading factor in anaemia progression and evolution of RBC-TD in MDS.del(5q).

Sustained response to bevacizumab in a patient with mosaic neurofibromatosis type 2 carrying the NF2:c.784C>T p.(Arg262*) variant.

Neurofibromatosis type 2 (NF2) is a tumor predisposition syndrome characterized by the growth of schwannomas, especially bilateral vestibular schwannomas (VS), meningiomas, and ependymomas. The anti-VEGF antibody bevacizumab has shown efficacy for VS in some NF2 patients. However, there is limited data on the effect of bevacizumab on non-vestibular tumors, and on the correlation between therapy response and genotype. Here, we report on a 33-year-old patient with bilateral VS, 14 additional intracranial or spinal schwannomas, and a meningioma treated with bevacizumab, off-label in the European Union, for 2 years. The genotype of the patient was determined by mutational analysis of NF2, SMARCB1, and LZTR1 on DNA of multiple tissues. Additionally, we performed volumetric measurements of quantifiable non-vestibular tumors (n = 8) on MRI scans from 5 pre-therapeutic and 2 therapeutic years, and pure-tone audiometry of the non-deaf ear. A heterozygous NM_000268.3(NF2):c.784C>T p.(Arg262*) variant was identified in DNA from 3 schwannomas, but not in leukocyte or oral mucosa DNA, and no rare SMARCB1/LZTR1 variants were detected, establishing the diagnosis of definite NF2 mosaicism. While schwannomas had progressed with a mean annual growth rate of 38% pre-therapeutically, volume stabilization or reduction of all schwannomas along with improvement of pain and neurological deficits, including hearing impairment, were observed under 24 months of bevacizumab. In summary, this is the first report of a sustained response to bevacizumab in a patient shown to carry the frequent mosaic NF2:c.784C>T p.(Arg262*) variant. Our results may be of particular relevance to guide treatment decisions in mosaic NF2 patients harboring this variant.

Guiding the global evolution of cytogenetic testing for hematologic malignancies.

Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

Ago-RIP Sequencing Identifies New MicroRNA-449a-5p Target Genes Increasing Sorafenib Efficacy in Hepatocellular Carcinoma.

BACKGROUND: Patients with hepatocellular carcinoma (HCC) have very limited treatment options. For the last fourteen years, the multi-tyrosine kinase inhibitor sorafenib has been used as standard-of-care therapeutic agent in advanced HCC. Unfortunately, drug resistance develops in many cases. Therefore, we aimed to find a way to mitigate drug resistance and to improve the sorafenib efficacy in HCC cells. MicroRNAs play a significant role in targeting genes involved in tumor control suggesting microRNA/sorafenib combination therapy as a promising treatment option in advanced HCC. METHODS: MiR-449a-5p target genes were identified by Ago-RIP sequencing and validated by luciferase reporter assays and expression analyses. Target gene expression and survival data were analyzed in public HCC datasets. Tumor-relevant functional effects of miR-449a-5p and its target genes as well as their impact on the effects of sorafenib were analyzed using in vitro assays. An indirect transwell co-culture system was used to survey anti-angiogenic effects of miR-449a-5p. RESULTS: PEA15, PPP1CA and TUFT1 were identified as direct target genes of miR-449a-5p. Overexpression of these genes correlated with a poor outcome of HCC patients. Transfection with miR-449a-5p and repression of miR-449a-5p target genes inhibited cell proliferation and angiogenesis, induced apoptosis and reduced AKT and ERK signaling in HLE and Huh7 cells. Importantly, miR-449a-5p potentiated the efficacy of sorafenib in HCC cells via downregulation of PEA15, PPP1CA and TUFT1. CONCLUSIONS: This study provides detailed insights into the targetome and regulatory network of miR-449a-5p. Our results demonstrate for the first time that targeting PEA15, PPP1CA and TUFT1 via miR-449a overexpression could have significant implications in counteracting sorafenib resistance suggesting miR-449a-5p as a promising candidate for a microRNA/sorafenib combination therapy.