A newly identified form of cell death known as ferroptosis is characterized by the peroxidation of lipids in response to iron. Rapid progress in research on ferroptosis in glioma and neuroblastoma has promoted the exploitation of ferroptosis in related therapy. This manuscript provides a review of the findings on ferroptosis-related therapy in glioblastoma and neuroblastoma and outlines the mechanisms involved in ferroptosis in glioma and neuroblastoma. We summarize some recent data on traditional drugs, natural compounds and nanomedicines used as ferroptosis inducers in glioma and neuroblastoma, as well as some bioinformatic analyses of genes involved in ferroptosis. Moreover, we summarize some data on the associations of ferroptosis with the tumor immunotherapy and TMZ drug resistance. Finally, we discuss future directions for ferroptosis research in glioma and neuroblastoma and currently unresolved issues. Despite their small percentage (approximately 1%) among all invasive cancer cases, malignant central nervous system (CNS) tumors are representative tumor types in children and adolescents as well as the major cause of death related to cancer in males younger than 40 and females younger than 20. As a result, malignant CNS tumors are the third and fourth leading cause of cancer-related death among individuals in the age ranges of 0-14 and over 40 years old, respectively . NB is aFerroptosis is associated with iron and reactive oxygen species (ROS) and primarily results in cytological changes, including oxidative stress. As a result of strong membrane lipid peroxidation and oxidative stress, mitochondrial cristae are reduced or absent, the outer mitochondrial membrane is ruptured, and the mitochondrial membranes are condensed, resulting in weaker plasma membrane selective permeability and increased oxidative stress. At least three cytoprotective systems against ferroptosis with distinct subcellular localizations have been identified in recent studies: glutathione peroxidase 4 (GPX4) located in the cytoplasm and mitochondria; ferroptosis suppressor protein 1 (FSP1) located at the plasma membrane, which promotes ubiquinone regeneration; and dihydroorotate dehydrogenase (DHODH) located in the mitochondria. GPX4 can remarkably prevent ferroptosis by decreasing the levels of phospholipid hydroperoxides and thereby inhibiting lipid peroxidation mediated by lipoxygenase. FSP1, which promotes ubiquinone regeneration at the plasma membrane, uses NAD(P)H to catalyze the regeneration of nonmitochondrial coenzyme Q10 (CoQ10), which blocks ferroptosis by inhibiting lipid peroxide propagation. In parallel with mitochondrial GPX4, DHODH reduces ubiquinone (CoQ) to ubiquinol (CoQH2), an antioxidant capable of resisting ferroptotic activity, which inhibits ferroptosis within the inner mitochondrial membrane (independent of cytosolic GPX4 or FSP1) –7.The prognosis of glioma and neuroblastoma is not particularly satisfactory. Currently, it is necessary to develop effective therapeutic approaches for glioma and neuroblastoma. A valid way to circumvent therapeutic resistance in cancer cells is targeting the ferroptotic pathway because of the high level of iron accumulation and the accompanying increase in ROS production. However, ferroptosis-related therapy application in glioma and neuroblastoma is still challenging because several aspects of the mechanisms of ferroptosis are still unclear. In this article, we present the progress in ferroptosis research in glioma and neuroblastoma and relevant future perspectives.Ferroptosis is a form of nonapoptotic cell death that results from the accumulation of intracellular iron and increased toxic lipid peroxide reactive oxygen species. In the prevention of ferroptosis, antioxidant systems can help decrease oxidative stress. Inhibition of an antioxidant system can contribute to the induction of ferroptosis in tumor cells. As a result, antioxidant systems are capable of remarkably regulating ferroptosis in cells and are also one of the major areas of research on ferroptosis at present.The Xc system is also referred to as the cystine/glutamate reverse transporter protein. GPX4 essentially constitutes the selenoprotein family and mainly mediates the reduction of peroxides to the corresponding alcohol. This antioxidant system prevents ferroptosis by transporting cysteine through the Xc system for the synthesis of glutathione (GSH), which in turn helps GPX4 reduce peroxides. As a major antioxidant component, GSH participates in a wide range of redox reactions in the body to maintain physiological homeostasis. GPX4 can critically regulate ferroptosis and is known to determine cell fate. Upregulating or inhibiting these antioxidant systems to regulate ferroptosis can impact the development of various diseases. Moreover, studies have identified various drugs and molecules as inducers of ferroptosis that act by restricting Xc system activity , 9.There is increasing evidence that inhibiting GPX4 activity does not necessarily lead to ferroptosis in cells. FSP1 on the plasma membrane reduces ubiquinone with NADPH as a cofactor, thereby preventing the peroxidation of lipids. GSH is not required as a cofactor for this process, nor does this process depend on GPX4. As a result, in contrast to GPX4, FSP1 may be regarded as a ferroptosis inhibitor, and the expression of FSP1 confirms the sensitivity of cells to ferroptosis , 10, 11.th rate-limiting step in pyrimidine biosynthesis and is capable of catalyzing dihydroorotic acid (DHO) to be oxidized to orotate (OA) and CoQ (ubiquitin) for further reduction to CoQH2 (ubiquinone), which is associated with the respiratory complex and affects electron transfer in the oxidative respiratory chain. Further studies have shown that inhibition of DHODH results in ferroptosis in cells with low GPX4 expression and increases the sensitivity of cells with high GPX4 expression to ferroptosis. DHODH can act synergistically with GPX4 to inhibit mitochondria-related ferroptosis without dependence on FSP1  are the most common treatments for clinical glioma, but they have a poor prognosis in patients, particularly those who suffer from high-grade gliomas, including GBM. The exploration of new therapeutic methods and therapeutic targets for glioma remains a hot spot. Targeting the ferroptotic pathway can serve as an effective treatment for glioma Figure 1The Xc-GSH-GPX4 network serves as the primary antioxidant barrier against ferroptosis. As a direct target gene, recombinant solute carrier family 7, member 11 (SLC7A11) is repressed by p53. It is a key component of the cystine-glutamate antagonist system (xCT system), which mediates the uptake of extracellular cystine in exchange for glutamate within the cell. The direct interaction between ubiquitin hydrolase ovarian tumor domain protease domain, ubiquitin aldehyde binding protein 1 (OTUB1) and SLC7A11 stabilizes the SLC7A11 protein, and OTUB1 knockdown triggers SLC7A11 expression-dependent ferroptosis . MoreoveCurrent research indicates that glutathione peroxidase 4 plays a critical role in ferroptosis. It has been demonstrated that a number of molecules affect the expression of GPX4 in gliomas to regulate ferroptosis. RSL3 (a GPX4 inhibitor) inactivates GPX4 and induces glycolytic dysfunction in glioma cells with reduced ATP and pyruvate content as well as HKII, PFKP, and PKM2 protein levels, which in turn induces ferroptosis . KnockdoTP53 encodes p53 promoting cell cycle arrest, senescence, and apoptosis, which are three canonical functions of p53 involved in tumor suppression. This gene is the most frequently mutated tumor suppressor gene in all human cancers. The TP53 gene has been found to be activated under various conditions and to play an important role in the control of ferritin by regulating lipid, energy, and iron metabolism , 20. SLCIn recent years, ACSL4 was found to partially activate long-chain fatty acid metabolism and immune signal transduction, indicating that it might be a regulator of ferroptosis . ACSL4 oGPX7 is another member of the glutathione peroxidase family (GPX) and participates in oxidative stress and tumorigenesis. GPX7 silencing enhances oxidative stress associated with ferroptosis in glioma cells, while GXP7 deletion sensitizes gliomas to ferroptosis induced by erastin. In addition, miR-29b was found to repress GPX7 expression directly after transcription .Recent studies have confirmed that tetrahydrobiopterin (BH4), a significant cofactor for multiple enzymes, can remarkably inhibit ferroptosis. The GTP cyclohydrolase 1 (GCH1)-BH4 axis controls BH4 synthesis and reduces intracellular CoQ and ROS accumulation, thereby leading to ferroptosis inhibition. In addition, GCH1/BH4 exerts a selective inhibitory impact on nuclear receptor coactivator 4 (NCOA4)-mediated ferritin autophagy and affects iron metabolism , 35. ThiThe transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) controls the expression of genes associated with oxidative stress and can reliably maintain redox stability and resistance to oxidative stress. High levels of NRF2 lead to sensitivity in glioblastoma dependent on the expression of its proferroptotic target ATP binding cassette subfamily C member 1 (ABCC1), resulting in GSH depletion upon blockade of the Xc system by erastin .With ongoing research progress, the mechanisms regulating ferroptosis are becoming increasingly clear. Further research on the ferroptosis-related gene network will provide new ideas and broad opportunities for the treatment of glioma, not only primary high-grade gliomas such as GBM but also recurrent gliomas. However, there are a few issues that require further exploration. For example, we must determine how to more effectively and precisely induce ferroptosis in glioma cells and improve the efficacy and safety of this treatment.Chemotherapy is one of the basic therapeutic strategies for glioma. TMZ is currently one of the first-line chemotherapeutic drugs for glioma, especially high-grade glioma. However, with the widespread use of TMZ, the median survival time of GBM patients has improved by only approximately 2.6 months. Frustratingly, as GBM patients receive long-term TMZ therapy, resistance inevitably develops, resulting in treatment efficacy dropping significantly or even disappearing. New replacement drug regimens remain to be developed . In the From the The use of rationally designed nanomaterials for the treatment of cancer is an emerging field that has led to tremendous medical success. The administration of ferroptosis-inducing nanoformulations with accurately tuned physicochemical properties is as an extended and feasible therapeutic strategy for tumors. We compiled recent research advances related to the induction of ferroptosis in glioma cells by nanomaterials. Table 2 in situ in drug-resistant GBM tissues, which was combined with the effect of ferroptosis induction by Fe2+ in Fe3O4. Some studies have also been conducted on the combination of chemotherapeutic drugs with nanomaterials . This sonodynamic therapy (SDT) combines targeting of ferroptosis in glioma cells with SDT . Howeveraterials . Gallic aterials . A studyIn general, the different designs are interesting and well designed. In conclusion, to achieve ferroptosis-targeted therapy with nanomaterials, the following steps must be achieved: blood−brain barrier penetration, tumor targeting, and ferroptosis induction. Nanomaterials with properties that enable these events may be new agents for glioma therapy in the future. However, the design of different nanomaterials is relatively complicated, such as the camouflage achieved with different cell membranes and the encapsulation of different drugs, and further improvements and validation in industrial production and human experimental safety are still needed. However, we believe that with the continuous progress of medical-industrial crossover technology, an increasing number of nanoagents will start to capture attention and provide new insights for the treatment of glioma in combination with ferroptosis-inducing agents.TMZ is still a most effective drugs for glioma chemotherapy. Ferroptosis can considerably affect TMZ resistance in glioma, and ferroptosis resistance may serve as a mechanism of TMZ resistance in glioma. TMZ increases LDH, MDA and iron levels and decreases GSH levels in glioma cells to induce ferroptosis. In addition, ROS levels and DMT1 expression are elevated, and GPX4 expression is decreased in cells treated with temozolomide; these events are under the regulation of the Nrf2/HO-1 pathway .In addition to ferroptosis inducers and xCT inhibitors, quinacrine (a compound capable of crossing the blood−brain barrier) has been found to impair autophagy but increase the sensitivity of glioblastoma stem cells (GSCs) to TMZ and trigger ferroptosis in GSCs . A long Further study of the ferroptosis mechanism in glioma TMZ resistance will contribute to new insights into the clinical reversal of glioma TMZ chemoresistance. However, details are still needed for clinical application.One of the most effective ways to treat cancer is to induce tumor cell death. Immunotherapy is considered a milestone in precision medicine. It elicits significant therapeutic responses in patients who have developed resistance to other conventional therapies . Howeverin vitro or increases the number of tumor-infiltrating CD8+ T cells in vivo, thereby enhancing tumor control , a soluble chaperone associated with the endoplasmic reticulum (ER), is one of the proteins that regulates the tumor microenvironment. As a result of ferroptosis, CRT is translocated onto tumor cells, where it can induce a robust immune response against the tumor . Neutrop control –81. In c control . Moreove control . These fin vitro and in vivo, triggering a vaccination-like effect in immunocompetent mice, in which ATP and high mobility group box 1 (HMGB1), the most typical injury-related molecular patterns associated with immunogenic cell death, can be passively released and act as immunogenic signals that affect the immunogenicity of early ferroptotic cancer cells  have demonstrated a broad clinical impact and early success. Some but not all cases of ICI response have been associated with the expression of immune checkpoint molecules, including PD-1 ligand (PD-L1) . Some paAlthough ICI immunotherapy has been shown to have significant positive effects in some cancer patients, there is still evidence of drug resistance in many tumors, including GBM, due to tumor heterogeneity, low tumor-infiltrating T-cell (TIL) levels, loss of target antigens and off-target toxicity , 90. ChiIn conclusion, with increasing research, immunotherapy is becoming more specific and individualized, which provides opportunities for therapy in glioma. The effects of ferroptosis and immunotherapy are bidirectional, i.e., ferroptosis can further influence the effect of immunotherapy by affecting the immune microenvironment, and the effect of immunotherapy can be further enhanced by enhancing ferroptosis. However, there are still some details and limits that need to be further researched for glioma therapy.With the development of sequencing technology and the creation of databases, bioinformatic analysis now plays an important role in identifying potential targets and drug effects and predicting prognosis. We compiled the recently published literature on biogenic analysis to provide potential new ideas for future research –105. As Although a large number of bioinformatic analysis studies currently provide us with ferroptosis-related targets in low-grade glioma (LGG) and GBM, they still have many limitations and points of controversy due to the lack of rigorous experimental support. Many of the studies relied on only computer technology. Bioinformatic analysis may be a future direction, but the validity and clinical significance of the molecules identified with this approach need to be further explored.Neuroblastoma is one of the most prevalent extracranial tumors in children, accounting for the majority of childhood cancer-related deaths, especially in high-risk cases. High-risk NB is characterized by the appearance of this disease after the age of 18 months, the amplification of MYCN , or the activation of mechanisms for telomere maintenance , 107. ThThe characteristics of NB are significantly different from those of glioma, and the focus is also different. Genomic amplification of the oncogene MYCN acts as an essential oncogenic event in high-risk NB, occurring in approximately 50% of high-risk cases, and MYCN amplification is strongly related to a poor NB prognosis (OS < 50%). MYCN-amplified NB shows a system-dependent increase in the level of the Xc-cystine/glutamate reverse transporter protein for ROS detoxification mediated by increased transcription of this receptor . As a reFerroportin (Fpn) is the only iron export protein that partially regulates the intracellular iron concentration. Fpn knockdown has been shown to increase the accumulation of iron-dependent lipid ROS to accelerate erastin-induced ferroptosis, and Fpn may be an appropriate target for NB treatment . MitochoThere are currently several drugs for NB treatment, including cisplatin, etoposide, vincristine, doxorubicin, and cyclophosphamide. These drugs are the most effective inducers of apoptosis. However, this type of drug therapy creates multidrug-resistant clones, which makes eradicating this type of tumor much harder and favors tumor recurrence . The indIn addition, withaferin A (WA), a natural ferroptosis inducer in NB, activates Kelch-like ECH-associated protein 1 to activate the nuclear factor-like 2 pathway and produces increased intracellular unstable Fe(II) levels after heme oxygenase-1 is excessively activated, inducing ferroptosis or inactivating GPX4 . ChloridDespite advances in multimodal treatment, midbody treatment and the prognosis of gliomas and neuroblastoma are discouraging. Ferroptosis is a newly identified form of programmed cell death (PCD) dependent on iron that differs from apoptosis, cell necrosis, and autophagy. It plays a very important role in GBM and NB. This article summarizes the mechanisms involved in the roles of ferroptosis in GBM and NB. To summarize, we report that (1) the GPX4 pathway remarkably affects GBM and NB and that direct or indirect inhibition of GPX4 disrupts lipid peroxidation. (2) MYCN amplification may be a potent target in NB. (3) Nanodrugs may be new therapeutic agents for treating glioma and neuroblastoma. (4) The complexity of the tumor immune microenvironment and regulatory mechanisms need to be further explored.Therefore, future research directions should include an in-depth study of ferroptosis, identification of key targets in the ferroptotic pathway and validation of their relationships in glioma and neuroblastoma, application of ferroptosis biomarkers in clinical prevention and monitoring, exploration of a new generation of ferroptosis-targeting systems, and finally, validation of the relationship between immunity and ferroptosis in glioma and neuroblastoma.HC and BL made equal contributions to the work. HX and GL took charge of project conception and design. HC and BL proposed the research and finalized the paper. QW, ZG and BF searched and summarized the literature. All authors contributed to the article and approved the submitted version.


Health professionals are often faced with the need to identify women at risk of manifesting poor psychological resilience following the diagnosis and treatment of breast cancer. Machine learning algorithms are increasingly used to support clinical decision support (CDS) tools in helping health professionals identify women who are at risk of adverse well-being outcomes and plan customized psychological interventions for women at risk. Clinical flexibility, cross-validated performance accuracy, and model explainability permitting person-specific identification of risk factors are highly desirable features of such tools.This study aimed to develop and cross-validate machine learning models designed to identify breast cancer survivors at risk of poor overall mental health and global quality of life and identify potential targets of personalized psychological interventions according to an extensive set of clinical recommendations.A set of 12 alternative models was developed to improve the clinical flexibility of the CDS tool. All models were validated using longitudinal data from a prospective, multicenter clinical pilot at 5 major oncology centers in 4 countries . A total of 706 patients with highly treatable breast cancer were enrolled shortly after diagnosis and before the onset of oncological treatments and were followed up for 18 months. An extensive set of demographic, lifestyle, clinical, psychological, and biological variables measured within 3 months after enrollment served as predictors. Rigorous feature selection isolated key psychological resilience outcomes that could be incorporated into future clinical practice.Balanced random forest classifiers were successful at predicting well-being outcomes, with accuracies ranging between 78% and 82% (for 12-month end points after diagnosis) and between 74% and 83% (for 18-month end points after diagnosis). Explainability and interpretability analyses built on the best-performing models were used to identify potentially modifiable psychological and lifestyle characteristics that, if addressed systematically in the context of personalized psychological interventions, would be most likely to promote resilience for a given patient.Our results highlight the clinical utility of the BOUNCE modeling approach by focusing on resilience predictors that can be readily available to practicing clinicians at major oncology centers. The BOUNCE CDS tool paves the way for personalized risk assessment methods to identify patients at high risk of adverse well-being outcomes and direct valuable resources toward those most in need of specialized psychological interventions. Adequate resilience is typically indicated by the ability to maintain good emotional functioning in the face of illness  or return to normal function shortly after adversity  and high [>9 years]), relationship status (single or with partner), children (yes or no), employment status (currently employed or not), type of employment , and monthly income . A total of 2 additional variables were aggregated over the first 3 months after enrollment (month 3): sick leave taken (in days) and considerable life stressors (other than BC) during the first 3 months after diagnosis (categorized as none or single event vs ≥2 events).The following lifestyle characteristics were recorded: current smoking, alcohol consumption , self-defined diet , and physical exercise .A number of clinical and biological variables were extracted from the patients’ medical records: Eastern Cooperative Oncology Group performance status; obesity; family history of BC; preexisting chronic physical illness (other than metabolic); psychotropic medications (including sleep medications); preexisting metabolic disease; preexisting anxiety or dysthymia; anemia; menopausal status ; serum levels of alanine aminotransferase, creatinine, and bilirubin; and blood cell count .BC-related variables included cancer stage (I vs II or III), tumor molecular profile , progesterone receptor positivity, estrogen receptor positivity, HER2 positivity, and Ki67 levels (≥25). Treatment-related variables were surgery at month 0, surgery at month 3, onset of chemotherapy at month 0, onset of chemotherapy at month 3, onset of radiotherapy at month 0, onset of radiotherapy at month 3, type of breast surgery (lumpectomy vs mastectomy), type of chemotherapy (adjuvant or neoadjuvant), type of endocrine therapy , anti-HER2 therapy, and systematic mental health support through month 3.Psychosocial characteristics were evaluated using standardized questionnaires that had been appropriately adapted and translated into 5 languages of the BOUNCE participants . The following domains were assessed: (1) several personality characteristics, (2) coping and the ability to cope, (3) perceived social support, (4) resilience as a trait, (5) illness perception and related behaviors, (6) global health status/QoL and anxiety and depression symptoms, and (7) patient affect at the time of measurement. A detailed list of available measures can be found in the study by Pettini et al  and in MCDS tool users could choose among three distinct endpoints: (1) overall mental health status at month 12 or month 18 (“good” or “poor”), (2) mental health decline between baseline (month 0) and month 12 (or month 18), and (3) decline in global QoL between baseline (month 0) and month 12 (or month 18). Self-reported overall mental health status was indexed by the total score on the 14-item Hospital Anxiety and Depression Scale. Higher scores indicate more frequent psychological symptoms. The clinically validated cutoff score of 16/42 points in a wide range of languages was used to identify patients who reported potentially clinically significant symptoms ,34. GlobA total of 12 models were developed, cross-validated, and incorporated into the BOUNCE CDS tool varying on the type of end point examined by each and on the timing of measurement of predictor variables. Three sets of models were included in the CDS tool in terms of well-being end points at 12 or 18 months after enrollment : (1) typpoor mental health at the time of enrollment in the study, whereas type B and C models were designed for patients who reported good mental health  at that time.Type A models were more suitable for patients who reported The primary prediction models incorporated into the BOUNCE CDS tool assume that psychological and lifestyle predictor variables are recorded within a few weeks from the time of diagnosis and shortly after the onset of cancer treatments (up to approximately 3 months after diagnosis). Clinical and biological variables are typically available as part of the patient’s medical file at the time of diagnosis. However, it is often the case that a mental health professional is not called in until sometime later, and therefore, data on potential psychological and lifestyle predictors are not available until approximately 6 months after diagnosis. To accommodate the risk assessment needs for such cases, we designed distinct model subtypes according to the timing of the available predictor measurements (at month 0/month 3 and month 6). An ensemble learning supervised feature selection and classification scheme was developed based on the balanced random forest (BRF) classification model, a variation of the frequently used random forest  with intInitially, raw data were rescaled to 0 mean and unit variance, and ordinal variables were recoded into dummy binary variables. Cases and variables with >10% of missingness were excluded from the final data set. The remaining missing values were replaced with the global mean values.Feature selection was performed to select variables of the highest importance for each prediction. The selection was conducted inside a nested cross-validation pipeline using a BRF algorithm that assigns weights to the features and ranks them according to their relative importance. The top 20 features were retained according to cumulative feature importance and entered into the final classification models. Embedding the feature selection process into the nested validation loop ensured stable and representative results and a final robust and generalizable ML predictive model.To address the rather common problem of model overfitting or overly optimistic results in ML applications in clinical research, we adopted a nested cross-validation scheme -38. ModeF1-score, and area under the curve. All model hyperparameters for both the selection and classification BRF models were left to default values (instead of hyperparameter grid-type search) to enhance the generalizability of the results.The ML pipeline was implemented in Python (Python Software Foundation) using the scikit-learn ML library . Class idalex Python package [Model-agnostic analyses were applied to the final cross-validated models, which were trained using the most important 20 features . Global-level analyses explored the relative importance of the selected variables in distinguishing between the 2 classes and, thus, making accurate 12- or 18-month predictions based on the selected features. Analyses at the local (patient) level searched for key contributors to a given classification result after controlling for all other predictors in the model. This was made possible with the use of ceteris paribus profiles -44 as we package ,47 with  package -31).Given that the principal intended use of the CDS tool is to help health professionals plan customized psychological interventions for women at risk of poor mental health outcomes such as recovery from BC, an extensive set of personalized clinical recommendations was developed as follows: (1) potentially modifiable predictors of each of the key well-being outcomes that emerged from each of the 12 models included in the CDS tool were grouped into clinically meaningful intervention targets, (2) specific BOUNCE clinical recommendations were prepared for each set of intervention targets, and (3) health care professionals may choose to receive generic recommendations on how to address all substantial modifiable predictors that emerged from a given well-being prediction model or review predictor profile plots based on patient-level model-agnostic analyses and select specific intervention targets to receive appropriate treatment recommendations according to their expertise.The BOUNCE clinical recommendations are not meant as any type of systematic psychological therapy for the patients but rather as a set of ideas that the professional can use to stimulate patients’ involvement in their treatment, empower them, and help them self-manage their experience with illness and its psychological sequelae in a way that is tailored to the needs of each patient. The BOUNCE clinical recommendations focus on patients’ resources to cope with, adjust to, and deal with the cancer experience and aim to foster their resilience.Of the total cohort of 706 women enrolled, 537 (76.1%) were followed up for 12 months, and 495 (70.1%) were followed up for 18 months. The average age of the women followed up for 12 and 18 months, respectively, was 55.5 (SD 8.2) years and 55.8 (SD 8.2) years. Most of the women followed up for 12 and 18 months had completed at least a high school education , were married or in a relationship , and were diagnosed with stage-I or stage-II BC . The women followed up for 12 and 18 months were mostly treated with lumpectomy , endocrine treatment , radiotherapy , or chemotherapy .i relied on the clinical, biological, and psychological measurements available at month 0 and month 3, whereas model Aii relied on the clinical and biological measurements obtained at month 0 combined with the psychological measurements available at month 6. Correct prediction of poor outcomes was achieved for 78.9% (424/537) to 82% (440/537) of patients depending on the type and timing of the predictor measurements  reported minimal or mild symptoms of anxiety or depression. The remaining 19.9% (107/537) of women comprised the group displaying an overall “poor” mental health outcome. Model Aurements . MoreoveAs shown in Examples of breakdown profile plots for 2 randomly selected patients from each group are presented in reduced the probability of poor mental health  included relatively low depression symptoms, avoidant coping, and treatment side effects, accompanied by relatively high emotional functioning and optimism. However, certain patient scores emerged as risk factors that could increase, albeit modestly, the probability of an adverse mental health outcome, namely, a relatively high proportion of neutrophils, low levels of overall emotional functioning, and mindfulness accompanied by relatively high levels of anxiety at baseline. The ceteris paribus plots  the semantic tier, which includes all available data and serves as the basis for the analytical functionalities of the platform; (2) the AI tier, which consumes the available data and provides the main AI and ML modeling and execution capabilities of the platform; and (3) the application tier, which uses the services from the AI tier and presents the results of the models through the graphical user interface. The various layers communicate via well-defined representational state transfer application programming interfaces to implement the designed flow of information between them in a robust and solid manner.current mental health  category is shown , and the user can then choose to estimate the predicted patient status at 12 or 18 months down the line. The selected models are executed, and the risk assessment results are visualized along with the probability, credibility, and confidence of the prediction (step 5).The CDS tool workflow is shown in poor, the CDS tool displays ceteris paribus plots allowing the user to compare the patient’s actual scores on each predictor variable with the recorded scores of the patients in the BOUNCE prospective clinical study. This step allows the user to form a tentative personalized risk and vulnerability profile for a given patient, potentially comprising relatively high scores on certain “risk” variables (such as anxious preoccupation) or relatively low scores on specific “protective” variables (such as coping strategies). The shape of the ceteris paribus plots further allows the user to identify patient characteristics that, if modified, could lead to a substantial change in the probability of the predicted adverse well-being outcome.If the predicted status of a given patient is Finally, based on their clinical experience and specific clinical needs, users can select variables as targets for potential psychological interventions by clicking on their respective ceteris paribus plots. BOUNCE clinical recommendations are available in two forms: (1) an abbreviated version for clinicians who come in direct contact with the patient but are not trained in administering systematic psychological support and (2) an extended version for mental health professionals who have some training in psychological interventions. The BOUNCE clinical recommendations refer to a short list of basic cues and solutions that a health care professional can use to encourage the patient to more actively participate in their treatment and enhance their psychological health and QoL. The BOUNCE clinical recommendations are not meant as any type of systematic psychological therapy for the patients but rather as a set of ideas that the professional can use to stimulate patients’ involvement in their treatment, empower them, and help them self-manage their experience with illness and its psychological sequelae in a way that is tailored to the needs of each patient. The BOUNCE clinical recommendations focus on patients’ resources to cope with, adjust to, and deal with the cancer experience and aim to foster their resilience.The design of ML models included in the BOUNCE CDS tool was guided primarily by the potential future clinical utility of the forthcoming results. Thus, the supervised learning models included variables that can be readily available to practicing clinicians at major oncology centers in most countries, namely, medical, sociodemographic, and lifestyle variables integrated with a select set of psychosocial patient characteristics. In essence, the results of the BOUNCE predictive models presented in this paper are in full agreement with psychological theory and research on the factors involved in adaptation to severe illness, including BC ,49. Howe. Clinician input was also instrumental in limiting the set of features entered into the ML models according to conventional psychometric criteria. In addition, robust cross-validation procedures were adopted to ensure that only the most relevant features were retained in the final prediction models. The behavior of these variables at a global level (both within and between classes) was inspected and confirmed to be relevant to the clinical question/prediction at hand. This approach is in line with the notion of transparency, that is, making the prediction process of clinically relevant models much more apparent and interpretable to clinicians and other stakeholders. These steps are important to conform to the rising demand for good AI/ML practices in real-world settings and guard against the potential liability of medical experts using AI-/ML-based solutions [Importantly, we used a rigorous analytic approach to mitigate some of the commonly observed pitfalls of ML approaches, namely, overfitting and poor model generalizability. Furthermore, we demonstrated their generalizability and interoperability by reducing their algorithmic bias while facilitating the interpretation of results according to the key challenges of XAI . The guiolutions . In thistechnological advances. Chief among these is the development of ML classification models that provide input to a CDS system for predicting the psychosocial status of women with BC throughout the cancer continuum. This is built using predictive modeling technologies through a multidisciplinary approach involving social sciences and humanities, medicine, and computer science. To facilitate clinical utility, the CDS tool is supported by a dynamic electronic platform to allow for the input of targeted psychosocial, QoL, and lifestyle data by the patient and of medical data by the clinician. It should be noted that the analysis pipeline built into the CDS tool is comprehensive, considering a wide range of patient characteristics, and adaptable to the data that may actually be available to support a clinical decision for a given patient. Importantly, the tool can enhance clinician decision-making in terms of transparency and trust in XAI/ML models. Finally, the personalized risk assessment output is accompanied by 2 layers of clinical recommendations to accommodate both the specific needs of a given patient and the level or expertise and preference of the clinician.The BOUNCE approach encapsulates a number of The BOUNCE solution presented in this paper would not be possible without the extensive database of multimodal, multi-scale measurements obtained in a multicenter clinical study at 5 oncology centers with extensive experience in the comprehensive treatment of large numbers of patients with BC. The prospective study featured several measurement time points to consider multiple trajectories to clinical recovery and patient well-being . Crucially, the study followed participants over a period of 18 months after the initial diagnostic procedures for BC. During this time, women are faced with several life-changing stressors . This period is crucial for the assessment of potential vulnerabilities that could predispose women to poor psychological well-being, as well as for providing expert support to promote behavioral and cognitive emotional adaptation strategies. In addition, the study measured multiple indexes of psychological resilience to ensure that all theoretically relevant aspects of this complex construct are considered and, ultimately, validated against objectively measurable clinical and psychosocial end points. The highest-ranking predictors of adverse mental health and QoL outcomes across the 12 models included in the CDS tool involved several common variables that can be classified into the following “clusters”: (1) negative affect, (2) coping with cancer responses and self-efficacy to cope with cancer, (3) a sense of control/positive expectations , (4) social and family support, (5) certain lifestyle factors , and (6) certain treatment-related symptoms . These findings are consistent with the notion that adaptation to a severe health crisis is a complex process that is determined by (1) a variety of personal and interpersonal resources, such as expectations, lifestyle, or social support, which may buffer the negative impact of the situation and facilitate adaptation; (2) cognitive emotional processes, such as affect, emotion regulation, and self-efficacy to cope with cancer, which guide behaviors such as preoccupation and helplessness; and (3) contextual and specific stressor-related factors that may affect adaptation directly or indirectly, such as physical symptoms ,53.Data-related limitations should be considered in evaluating these results. The availability of a larger data set, possibly by oversampling patients in the smaller, “poor” outcome classes, may have enhanced the consistency of the results and possibly improved the overall prediction accuracy. We attempted to address this limitation by controlling for the ratio of predictors to cases. Moreover, the subsets of patients entered into each of the 12 alternative models were highly overlapping, ensuring the comparability of the model results. Importantly, the BOUNCE approach and model construction is flexible and adaptable to different cultural and clinical settings. Above all, the most direct method to validate these results and support the clinical utility of the BOUNCE CDS tool is through a prospective randomized controlled trial comparing personalized interventions informed by the tool’s clinical recommendations with a conventional intervention that does not consider individualized risk profiles.The effort to build appropriate and valid clinical recommendations has 2 additional limitations. The first refers to the development of the BOUNCE clinical recommendations, and the second refers to their implementation. Specifically, the development of the BOUNCE clinical recommendations was based on the results of the prospective study that was conducted in 4 countries. Thus, the critical predictors to which the BOUNCE clinical recommendations correspond may vary for patients in different cultural settings. Carrying out similar research efforts in these countries as well may be an appropriate response to this limitation. In addition, the limited resources at many oncology centers may prevent the implementation of these clinical recommendations. However, it is noteworthy that the implementation of the BOUNCE clinical recommendations is not very demanding.Our results highlight the importance of early psychological responses to cancer diagnosis and related treatments and stress the necessity for timely intervention strategies to prevent substantial deterioration of patient well-being. The end product of the BOUNCE project paves the way for personalized risk assessment methods to identify patients at high risk of adverse well-being outcomes. This is a crucial step toward directing valuable resources to those patients with a more acute need for specialized psychological interventions. Finally, the BOUNCE approach highlights the usefulness of ML methods combined with carefully timed longitudinal measurements of a wide range of potential predictor variables. Such thoroughly cross-validated models can substantially reduce the length (and patient burden) of assessment batteries targeting complex psychological processes such as resilience and increase their feasibility in future research and clinical applications.


Diffuse intrinsic pontine gliomas (DIPGs) are high-grade gliomas (HGGs) that occur primarily in children, and represent a leading cause of death in pediatric patients with brain tumors with a median overall survival of only 8–11 months.While these lesions were previously thought to behave similarly to adult HGG, emerging data have demonstrated that DIPG is a biologically distinct entity from adult HGG frequently driven by mutations in the histone genes H3.3 and H3.1 not found in adult glioma. While biopsy of DIPG was historically felt to confer unacceptable risk of morbidity and mortality, multiple studies have demonstrated that stereotactic biopsy of DIPG is safe, allowing not only for improved understanding of DIPG but also forming the basis for protocols for personalized medicine in DIPG. However, current options for personalized medicine in DIPG are limited by the lack of efficacious targeted therapies for the mutations commonly found in DIPG. Multiple treatment modalities including targeted therapies, immunotherapy, convection-enhanced delivery, and focused ultrasound are in various stages of investigation.Increasing frequency of biopsy for DIPG has identified distinct driving mutations that may serve as therapeutic targets. Novel treatment modalities are under investigation. Diffuse intrinsic pontine gliomas (DIPGs) are high-grade gliomas (HGGs) which occur primarily in children. They belong to the category of diffuse midline glioma (DMG), as defined in the current WHO classification, which occur in midline locations and share similar genetic changes and clinical behavior. DIPG/DMG represents 10–15% of all pediatric brain tumors and is a leading cause of death in children with brain tumors . The medAttempts at understanding the tumor biology of DIPG were previously limited by the lack of available tissue. Due to its location in the pons, biopsy of these tumors was thought to be associated with unacceptable morbidity , 7. In tTissue obtained from stereotactic biopsy Fig. , in combEarly data from biopsy studies have further demonstrated that DIPG represents a biologically distinct entity from GBM and other adult glioma. Well-characterized adult glioma mutations including IDH1, BRAF, FGFR1, MYB, and TERT are atypical for DIPG , 13, 14.These mutations have been also associated with distinct clinical features: K27M H3.1 tumors frequently resemble anaplastic astrocytoma on histopathology and tend to occur in younger patients, K27M H3.3 tumors are associated with TP53 and ATRX mutations and more frequently resemble glioblastoma , 14, andFollowing the novel identification of multiple mutations in DIPG and an increased understanding of the heterogenous nature of these lesions, trials emerged paired stereotactic biopsy with molecularly targeted therapies. The DIPG Biology and Treatment Study both evaluated the feasibility of stereotactic biopsy at diagnosis for genetic analysis and designation of treatment groups and subsequently treated enrolled patients with FDA-approved therapies based on identified mutations : all patAnother early feasibility study, PNOC003, incorporated whole exome sequencing and RNA sequencing of paired tumor and normal tissues in DIPG. Following sequencing, medical treatment utilizing of up to 4 FDA-approved drugs was utilized based on the tumor's genetic profile . PNOC003Given the high prevalence of histone mutations in DIPG, HDAC inhibitors and other agents that target histone methylation have been an area of intense research and clinical interest. The HDAC inhibitor panobinostat has been shown to be effective in preclinical studies, producing an increase in H3K27 trimethylation, a partial rescue of the global hypomethylation phenotype found in DIPG , and ultmTOR inhibitors have also been investigated in an effort to target the increased activation of the PI3K/AKT/mTOR pathway, with dual mTOR inhibitors showing promise in some in vitro and xenograft models . HoweverThe ACVR1 mutation found in 20% of patient has also been an area of preclinical investigation, with ALK2 inhibitors demonstrating modest preclinical efficacy and an ability to cross the blood-brain barrier , 26, andDIPG is associated with a minimal immune infiltrate , and DIP3. CED was notable for producing negligible systemic exposure; in a study by Souweidane et al. utilizing radioimmunotherapy agent, the ratio of lesion-to-whole body radiation absorbed was higher than 1,200. However, concomitant biopsy may not be feasible with CED as it may negatively affect drug distribution, and the logistics of analyzing tissue and establishing a molecularly targeted regimen would therefore potentially require multiple invasive procedures in order to provide molecularly targeted agents via CED.Penetration of the blood-brain barrier of agents administered systemically requires molecular weight of <500 Da and high lipophilicity ; only 5%Early preclinical data for the use of focused ultrasound have shown promise in HGG. In a rat model, focused ultrasound in combination with the photosensitizing agent 5-aminolevulinic acid under MR thermometry surveillance produced an increase in survival and decreased tumor growth; 5-aminolevulinic acid and focused ultrasound did not improve survival separately . FocusedA number of trials are ongoing and in development for the treatment of DIPG using a variety of novel approaches and combinations Table . ONC201 The authors have no conflicts of interest to declare.No authors received funding for this study.Cecilia Dalle Ore performed the initial review and wrote the initial draft of the manuscript. Christina Coleman compiled additional information and contributed to the writing of the manuscript. Nalin Gupta supervised writing of the manuscript and edited the finalized manuscript. Sabine Mueller supervised the design and writing of the finalized manuscript.


Rare Diseases (RD) do not have an exact definition since local authorities define the criteria in different ways, from fewer than 5 people in 10,000, according to the European Union, to the standard world average of 40 cases per 100,000 people . DespiteThe rapidly changing landscape of RD requires us, periodically, to update diagnostic omics and bioinformatics workflows of known and newly discovered clinical entities as well as the roadmaps for tailored patient treatment. These challenging issues paved the way to RDMMTS Special Issue IV, which includes 12 contributions—9 research and 3 review articles—focusing on different RD. In a few rare diseases, such as juvenile angiofibromas (JA), diagnostics and treatment are at an advanced stage, but etiopathogenesis is controversial. The study by Schick et al.  fills thThe diagnostic and therapeutic aspects are at the root of the paper by Magri and co-workers, which describes a patient affected by limb-girdle muscular dystrophy (LGMD) caused by molecular defects in the SGCB gene . They reThe generation of iPSC-neurons from patients with neurodevelopmental diseases has provided an in vitro platform to test known drugs and small molecules able to rescue or attenuate disease biomarkers  or to scThe article by Grosse et al.  adds theNUP98-mutated and RTS patients, indicated by converging dysregulated gene networks, supports this first-described NUP98 nucleoporopathy, adding to the list of inherited NUPs disorders and expanding the well-known role of NUP98 in cancer.The study by Colombo et al.  attests Fas, FasL and Casp10 genes and detected, only in patient 2, a Fas variant, resulting in an in-frame premature truncation at a highly conserved protein domain, previously reported in an APLS patient [The issue of the scarcity of reliable biomarkers acting as a bridge towards RD diagnosis and the search of therapies is adequately addressed by Delgadillo and coworkers , who rep patient . To lookde novo discovery [Drug discovery is a significant issue for rare diseases, especially considering financial investments required for de novo discovery. Funding is limited due to the lack of attention by pharmaceutical industries and public investments ,21. In tiscovery ,23. The The paper by Monticelli and co-workers relies on a described cell model, which had previously allowed the authors to identify the role of acetylsalicylic acid in potentiating the approved pharmacological chaperone therapy for Fabry Disease (FD) . In thisThe perspective of combining drugs to optimize therapies underlies the paper by Iacobucci and co-workers, which focuses on enzyme replacement therapy (ERT) for FD . Their pDrugs can act by different, unpredictable mechanisms, and the cellular context usually has a primary role in the response. This consideration represented the core of the work described in the paper by Pantoom and co-workers, who focused their attention on the mechanism of ambroxol in increasing β-glucosidase, the enzyme mutated in Gaucher Disease (GD) . AmbroxoIDUA gene and deficit of the lysosomal alpha-L-iduronidase enzyme, accumulation of glycosaminoglycans (GAGs) in the lysosomes, lysosomes dysfunction and multisystemic disease. The timeline clinical manifestations of severe MPS1 are described with special focus on the constellation of skeletal abnormalities (“Dysostosis Multiplex”) which do not benefit even from the therapeutic option of donor hematopoietic stem cell transplantation (HSCT) due to poor enzyme diffusion in this anatomical compartment. Enzyme replacement therapy (ERT) of the human recombinant IDUA, approved by the FDA in 2003 and considered the standard of care, proved of benefit to precociously treated patients, but only when combined to HSCT did it improve the outcomes of skeletal manifestations. Gene therapy and gene editing approaches are promising options, mainly investigated on mouse models, but few clinical applications have been tested in MPSI patients. Alternative approaches—including (i) blocking GAG production instead than promoting their catabolism; (ii) suppressing non sense mediate mRNA decay in case of nonsense mutations to bypass the termination codon; and (iii) adopting the chaperone option in case of mutations that impair protein folding, trafficking and accumulation—are explored in mouse models, but the applicability to patients is limited. Given that the skeletal issues, often refractory to the current therapeutic strategies, are crucial in determining the disease severity and the quality of life of MPSI patients, the authors conclude that the pathomechanism underlying impaired bone development and ossification should be better dissected to design tailored therapies to be administered as early as possible to MPSI children. Three review articles complete RDPMTSIV. The review by De Ponti et al.  comprehePACS1 missense mutation (c.607C > T) (p.R203W), hypothesized to act by gain-of-function (GOF) or a dominant negative mechanism. Different therapies that prevent accumulation of the mutant protein have been developed—first of all antisense oligonucleotides (ASO), targeting the mutant mRNAs for degradation, though ASO are incapable of crossing the blood–brain barrier and require direct central nervous system administration. The role of PACS1 in regulating membrane trafficking through the interaction with a number of client proteins, whose function is altered in the patients, fueled a patent application in which fibroblasts from patients with a stronger binding of mutated PACS1 to Histone Deacetylase 6 (HDAC6) revert to the normal cellular phenotype upon treatment with wide-spectrum or specific HDAC6 inhibitors [The review article on Schuurs–Hoeijmakers Syndrome or PACS1 (phosphofurin acidic cluster sorting protein 1) neurodevelopmental disorder (PACS1-NDD) (OMIM 615009  underlinhibitors . Other aThe review by Yang et al.  focuses


Drosophila flies, thus establishing the species as a model system for studies in mitochondrial evolutionary biology. In this review, we comprehensively review these studies, from those focusing on genetic processes shaping evolution within the mitochondrial genome, to those examining the evolutionary implications of interactions between genes spanning mitochondrial and nuclear genomes, and to those investigating the dynamics of mitochondrial heteroplasmy. We synthesize the contribution of these studies to shaping our understanding of the evolutionary and ecological implications of mitochondrial genetic variation.Mitochondria are key to energy conversion in virtually all eukaryotes. Intriguingly, despite billions of years of evolution inside the eukaryote, mitochondria have retained their own small set of genes involved in the regulation of oxidative phosphorylation (OXPHOS) and protein translation. Although there was a long-standing assumption that the genetic variation found within the mitochondria would be selectively neutral, research over the past 3 decades has challenged this assumption. This research has provided novel insight into the genetic and evolutionary forces that shape mitochondrial evolution and broader implications for evolutionary ecological processes. Many of the seminal studies in this field, from the inception of the research field to current studies, have been conducted using  Since their formal discovery in the 1890s, mitochondria were for the most part studied by biochemists and physiologists seeking to understand the biochemical and metabolic pathways regulating mitochondrial respiration . During Drosophila melanogaster, was first proposed as a genetic model by Charles W. Woodworth at the start of the 20th century and has since become one of the most widely used and genetically best described eukaryotic organisms  in the late 1980s, accelerated sequencing efforts in ensuing years, but it was not until the inception of next-generation technologies that data acquisition was truly revolutionized  was that of humans, a feat that required substantial efforts because technology at the time was reliant on the cloning of restriction fragments into appropriate vectors prior to sequencing and deciphering of fragments using large polyacrylamide gels , shaped largely by drift and reaching fixation by chance . Such a Drosophila.In the sections that follow in this review, we will overview evidence from subdisciplines within the field of mitochondrial evolutionary biology, which together have tested the neutrality assumption for mtDNA variation, and that suggest capacity for a complex interplay of positive (Darwinian), balancing and negative (purifying) selection in shaping the accumulation and maintenance of the genetic variation within the mitochondrial genomes of A prediction to arise from neutrality theory is that the ratio of nonsynonymous substitutions  to synonymous substitutions  found between species should equal the ratio of nonsynonymous to synonymous polymorphisms found within species . A neutrDrosophila have generally found that the strict neutrality prediction does not hold true for most protein-coding genes. Rather, the pattern for most mitochondrial genes is indicative of a general excess of polymorphisms within species, which ultimately do not reach fixation , which generates ATP via oxidative phosphorylation (OXPHOS), depends on coordination between genes distributed over both nuclear and mitochondrial genomes . By the Drosophila; petite and poky mutants of yeast and Neurospora, respectively—were among the first studies to link phenotypic changes directly to the mitochondria and later to the effects of mtDNA mutations themselves  to explore possible conditions under which cytonuclear fitness interactions could maintain joint polymorphisms at the interacting cytoplasmic  and nuclear loci. The results of these models suggested only very limited capacity for the maintenance of such polymorphisms, primarily restricted to cases of strong differential selection on these loci within (across life stages) and between the sexes , 1998. AD. melanogaster  and kept as a population. As such, any phenotypic differences detected between strains could be shaped by differences in cytonuclear genotype or by differences in any other sources of variance that might typically shape interindividual phenotypic variation across populations—by either environmental (e.g. shared environments of flies of each strain) or residual nuclear variation that may build up across the strains that share the same target nuclear genotype. This replication (or lack of genotype replication) issue continues to be relevant to numerous studies covered in this review and among contemporary studies of mitonuclear interactions; it represents an important methodological design consideration for future research in this field. Furthermore, a lack of replication at the level of the focal genotype (mito × nuclear interaction) may not only impede capacity to partition dedicated epistatic effects of mitochondrial and nuclear genetic variation from other sources of confounding variance but may also lead to nonoptimal specification of the error variances in the associated statistical models. In the absence of this replication, then instead of the error variance (reflected by the denominator degrees of freedom) representing the total number of mitonuclear strains used in an experiment, it may be represented either as the number of “shared environments” in which focal individuals are maintained prior to or during the experiments  or as the total number of focal individuals assayed . Error variances at these levels may lead to pseudo-replication, inflation of test statistics, anticonservative P-values, and higher rates of Type 1 error than intended   were placed alongside 3 nuclear backgrounds, with each mitonuclear combination created in 10 replicates. They then measured flies of each strain for mass-specific metabolic rate, as gauged by CO2 production, across 3 cohorts of flies of young adult age, for each sex and replicate. While they failed to detect effects of nuclear genetic background, or mitonuclear interactions, on metabolic rate, they did detect effects of mtDNA haplotype (with differences in metabolic rate of up to 20% across the 3 haplotypes), which were partially contingent on the sex of the flies. Specifically, flies harboring the D haplotype exhibited lower metabolic rate than those bearing I or II haplotypes. Flies carrying the I haplotype differed to those with the II haplotype, but this effect was contingent on sex—males with haplotype I exhibited reduced metabolic rate than those with haplotype II, but the pattern was reversed in females, with those with haplotype I exhibiting increased metabolic rate , all of which derive from the 1 collection site in North Carolina, USA . Many of these studies have been conducted in nogaster . The resD. subobscura bearing haplotypes I or II (D. subobscura haplotypes (I–XIII) taken from a solitary wild population into an identical common coevolved nuclear background. The authors also included 1 haplotype (VIII) sourced from a geographically isolated island, which was also introgressed into the common nuclear background, as well as its own coevolved nuclear background. The authors then examined haplotype-specific differences in larvae–adult development time, fertility, and longevity across these strains. The experiments revealed significant differences across haplotypes in all measured traits, although in contrast to previous findings  or alongside a nuclear background of the congeneric Drosophila mauritiana (interspecific combination). Disruption of the cytonuclear lineage via introgression of haplotypes siI and siII into the nuclear background of D. mauritiana revealed a decrease in COX activity in crude fly homogenates . Furthermore, analyses based on in situ analyses of mitochondrial function from permeabilized fibers of flight muscle have tended to show results contrary in direction to those that used isolate mitochondria, with increased catalytic capacity and COX activity in flies harboring the siII relative to siIII haplotype , typically expressed alongside 1 or several distinct nuclear genetic backgrounds sourced from D. melanogaster , this approach provides a means to test whether the magnitude of effect size associated with the mitonuclear interaction is increased when the interacting mitochondrial and nuclear genotypes are sourced from divergent species (interspecific interactions) rather than from the same species (intraspecific interactions). Second, under the prediction that strong selection on the mitonuclear genotype will lead to tight intergenomic coadaptation, this approach provides an opportunity to test the prediction that disruption of species-specific mitonuclear complexes will lead to phenotypic disruption , and offspring of reciprocal crosses in which 1 member of the cross contained an mtDNA haplotype derived from D. simulans in a D. melanogaster nuclear background, and the other member contained a D. melanogaster mtDNA haplotype alongside D. melanogaster nuclear background. This design enabled the authors to test 2 predictions: first, that the magnitude of effect associated with the mitonuclear interaction will increase with the degree of molecular divergence separating the haplotypes used in each reciprocal cross; and second, that reciprocal crosses resulting in interspecific combinations of mitonuclear genotype would result in reductions in longevity relative to intraspecific combinations. The results supported the first prediction that the magnitude of mitonuclear effects increased with the degree of genetic divergence. Contrary to the second prediction, however, offspring inheriting the D. simulans haplotype was not consistently short-lived as might be predicted if interspecific mitonuclear combinations had resulted in disruption of coadapted mitonuclear lineages. This result is notable given that >500 nucleotide substitutions separate the mtDNA of D. simulans and D. melanogaster, implying that this level of divergence between haplotypes is insufficient to result in consistent intergenomic incompatibilities. Instead, the interspecific fly strains exhibited a large range of variation in longevity falling within that observed for the intraspecific mitonuclear combinations  haplotype expressed against the Oregon (OreR) nuclear background, which was associated with both low productivity and low female relative juvenile fitness. This specific mitonuclear interaction has since been the focus on intense study (reviewed in An interspecific mitonuclear incompatibility section). Investigation of the mito-X chromosome combinations indicated that mito-X interactions affected male, but not female, juvenile relative fitness, but that the interspecific mito-X combinations did not result in greater effects on fitness than the intraspecific combinations (A subsequent study by D. simulans mtDNA haplotype (simw501) and the D. melanogaster OreR nuclear background first reported in D. simulans haplotypes and 1 D. melanogaster haplotype expressed against 2 different D. melanogaster haplotypes). They confirmed that the interaction between simw501 mtDNA and the OreR nuclear background led to delays in development from egg to adulthood of ∼2 days , decreased fecundity of females of ∼50%, and shortening of the thoracic mechanosensory bristles of adult flies. Remarkably these phenotypes were not observed when the same haplotype was placed alongside a second D. melanogaster nuclear background (AutW132) and were not observed for a second simulans haplotype in the D. melanogaster background. Thus, the interspecific incompatibility is mediated by polymorphisms contained to specific mtDNA and nuclear variants of each of the species of this particular cross. Sequencing of D. simulans mtDNA revealed a SNP specific to the Tyrmt:tRNA in the simw501 haplotype, located at the base of the anticodon stem changing a G:C  to a G:U. The authors used meiotic mapping to locate the nuclear polymorphism interacting with the mitochondrial tRNA to a gene encoding the aminoacyl-tRNA synthetase for tyrosine in the mitochondria, showing a solitary nonsynonymous polymorphism, which changes a conserved alanine to a valine at 1 point of the amino acid sequence of the gene, separating the 2 nuclear backgrounds. They then created transgenic strains that differed at this nonsynonymous polymorphism. Experiments on these transgenic strains confirmed that this polymorphism was mediating the mitonuclear interaction. The authors hypothesized that the incompatibility was mediated by a reduced pool of tyrosine charged tRNAs, compromising protein translation in the mitochondria. In support of this hypothesized model, assays of mtETS enzyme complexes I, III, and IV , using mitochondria isolated from homogenized adult flies of both sexes revealed decreased enzyme activities, while complex II  was unaffected, as was the abundance of mitochondria as indicated by levels of citrate synthase . Subsequent studies have leveraged the same set of strains to further characterize this incompatibility. Providencia rettgeri, reporting lower survival and reproductive success of female flies harboring this incompatibility relative to those of other genotypes, with these effects manifesting specifically following infection. This result suggests that this mitonuclear incompatibility may result in an energy deficiency and resulting allocation trade-off between investments into immune defense versus other components of life history  in flies harboring the incompatibility . Clancy first assayed flies of each strain for survival, revealing differences across haplotypes in their effects on survival. Clancy then investigated whether survival was affected by mitonuclear interactions, by assaying longevity of male F1 offspring generated by crosses between females of 3 of these mitochondrial strains and males of 8 distinct isofemale lines that provided target nuclear backgrounds. F1 flies thus possessed 1 haploid copy of the w1118 nuclear background and a haploid copy of a target nuclear background. All strains had been cured of Wolbachia, enabling Clancy to home in on mitochondrial effects on longevity, and whether these effects were contingent on nuclear background. Clancy found clear mitonuclear interactions for male longevity. In particular, longevity differences between the 3 haplotypes tested were much larger on some nuclear backgrounds than others  as had been used by Subsequent studies have leveraged an expanded set of strains representing 13 haplotypes, originally collated by Sex specificity of mitochondrial haplotype effects and Mitochondrial gene by environment interactions sections below.Several other studies have leveraged the same panel as used by D. melanogaster in Australia, testing 2 predictions. The first prediction was that frequencies of mtDNA haplotypes would exhibit latitudinal clines, with some haplotypes predominating at higher frequency in low-latitudinal, subtropical localities, and others predominating in high-latitudinal, temperate localities. The second prediction was that spatial distributions of haplotypes would correspond to the direct effects of these haplotypes on the thermal tolerance phenotypes of flies, with haplotypes that are at higher population frequencies at low latitudes conferring higher tolerance to heat stress and those at higher population frequencies at high latitudes higher tolerance to cold stress. The authors identified 2 main groups of haplotypes segregating along the Australian eastern seaboard, each characterized by 1 major haplotype, which were delineated by 15 polymorphisms. The haplotypes exhibited a latitudinal cline, with the A1 haplotype predominating in lower latitude populations and the B1 haplotype at higher latitudes. The authors then created genetic strains in which each haplotype was placed alongside a standardized nuclear background and then characterized each strain for its capacity to confer tolerance to both extreme heat and extreme cold stress. Consistent with predictions, the A1 haplotype conferred higher tolerance to heat stress but lower tolerance to cold stress than B1 haplotypes. Thus, the thermal tolerances of the haplotypes corresponded to their spatial distributions in natural populations, consistent with the hypothesis that climatic selection had shaped the distributions of these haplotypes in the Australian fruit fly population  and cytochrome c oxidase II gene , which can perturb sperm development and function leading to male sterility or subfertility while leaving female fertility intact  on bioenergetic function have been shown to be sex specific; males at 15 and 25 days of adult age suffer reduced OXPHOS respiratory capacity relative to other haplotypes and generally low mass-specific metabolic rate, while bioenergetic and metabolic function in females carrying this haplotype is normal. Furthermore, males that carry this haplotype are characterized by lower levels of citrate synthase  present in permeabilized wing fibers than males harboring other haplotypes, while females with this haplotype exhibit normal mitochondrial quantities  in which resilience to heat stress was much reduced in males but not females, relative to other B1 sub-haplotypes  reported a lack of general male bias in effects , reporting no statistically significant mtDNA- or mitonuclear-mediated effects on sex differences in development time or sex ratio of egg clutches, albeit not formally testing for statistical interactions between mtDNA and sex or mDNA, nuclear genotype, and sex on development time or sex ratio.Notably, while numerous studies have provided support for the weak and strong forms of Mother's Curse in D. melanogaster and 1 from D. simulans, on genome-wide patterns of gene expression when placed alongside 2 different nuclear backgrounds from D. melanogaster. The authors reported generally larger effects of mtDNA substitution on patterns of differential expression in females than in males. Notably, interspecies substitution of mtDNA (D. simulans mtDNA in D. melanogaster nuclear backgrounds) tended to result in downregulation of mtDNA-encoded OXPHOS genes, with the exception of mt:ND2, which was upregulated (D. simulans and 2 from D. melanogaster), placed alongside 2 different D. melanogaster nuclear backgrounds, reporting effects of mtDNA haplotype on the expression of numerous genes, with no apparent sex bias. The authors further partitioned the analyses into effects of mtDNA haplotypes in general  and effects at the level of the 2 species (effects of D. simulans haplotypes vs. effects of D. melanogaster). Notably, and consistent with the results of the study of In interspecific studies testing the role of mitochondrial and nuclear variation in the regulation of genome-wide patterns of transcript expression, results have been mixed. In 1 study, The general discrepancy in the direction and strength of sex specificity between results of studies conducted at the intraspecific scale, and those at the interspecific scale, is intriguing. D. simulans mtDNA haplotypes expressed against D. mauritiana nuclear backgrounds. However, other studies to test the performance of interspecific combinations of D. simulans and D. melanogaster mtDNA haplotypes, expressed alongside D. melanogaster nuclear backgrounds, have failed to provide consistent evidence that the interspecific mitonuclear combination performs more poorly than the intraspecific combination in either sex  into 3 nuclear backgrounds of D. melanogaster, one of which was daughterless GAL4 driver, which could be crossed to a UAS-SIR2 nuclear background or control, to achieve overexpression of SIR2, which is a family of signaling proteins involved in metabolic regulation. They then screened these genotypes across 5 different diets that differed in their yeast and sugar concentrations, reporting extensive mitochondrial haplotype-by-diet interactions and mitochondrial-by-nuclear-by-diet interactions for life span and providing evidence that the effect of SIR2 on longevity changes across mtDNA haplotypes , when assayed on 4 diets differing in protein-to-carbohydrate ratios. They reported that outcomes of mitonuclear interactions on development time were contingent on the diets in which the flies were assayed  was larger in males than females  and the D. melanogaster OreR nuclear background, highlighted in An interspecific mitonuclear incompatibility section, is temperature sensitive in its effects. The negative effects of the incompatibility are reduced when juveniles develop at 16°C and increased when juveniles develop at warmer temperatures. Moreover, larval metabolic rate is normal at 16°C but elevated relative to other mitonuclear genotypes at 25°C, while metabolic plasticity of larvae is reduced at cooler temperatures  to establish large fly populations with specific proportions of each haplotype  alongside 3 different nuclear backgrounds . A series of experiments was initiated—firstly with population cages established with 500 pairs of flies for each mtDNA haplotype at a 1:1 sex ratio and a discrete generation cycle, with 1 replicate cage for each nuclear background, and then a series of smaller culture bottles with smaller number of flies, with the mtDNA haplotypes always competing alongside the same nuclear background. By monitoring haplotype frequencies after each of 5 generations, the authors observed linear shifts in haplotype frequencies, with haplotype I exhibiting consistent increases in frequency, particularly when the experiments were conducted under larger effective population sizes (in the population cages rather than culture bottles and hence less subject to frequency changes being dictated by drift). There was however 1 exception—haplotype I decreased in frequency when competing in the nuclear background matched to haplotype VIII, suggesting that haplotypes may perform better when placed alongside their own putatively coevolved nuclear backgrounds, implicating a role for mitonuclear interactions in moderating outcomes of selection on the mtDNA haplotypes  against 1 another across 4 population cages in D. subobscura. Results showed that when placed in competition alongside its natural heterogeneous nuclear background, haplotype II consistently reached fixation or near fixation after 13–16 generations  that had been reported to co-segregate in linkage disequilibrium with haplotypes I and II, to probe for nuclear factors that may impact haplotype frequencies  when expressed alongside 3 distinct nuclear backgrounds (2 homozygous backgrounds that were associated to the 2 mtDNA haplotypes under study and 1 hybrid nuclear background) across replicate populations. Haplotype frequencies did not change on either of the 2 homozygous backgrounds, while the frequency of the Argentina haplotype increased only in the first 2 generations on the hybrid nuclear background but not in subsequent generations  or when it was introduced into the cages in its coevolved nuclear background. The results of these experiments were contingent on the nuclear context used. In cages where the haplotypes had been introduced via isofemale lines in which the original mitonuclear associations were preserved, the siII haplotype outcompeted the siIII haplotype, but this pattern was reversed when the haplotypes were competing alongside the nuclear background originally associated with the siIII , siII consistently increased in frequency across all replicate cages, whereas siI decreased and siIII maintained its starting frequency. Following 15 generations of the experiment, the authors perturbed the haplotype frequency of each cage and then tracked ensuing haplotype frequencies across a further 28 generations of culture. They observed the same signature of positive selection on the siII haplotype, whereas siI was purged and siIII maintained at lower population frequencies.Similarly, using haplotypes from D. melanogaster in Texas, USA, which differed in their length only . Rand competed these haplotypes across 6 population cages—half initiated with high frequencies of the long variant, and the other half initiated with intermediate frequencies—but found no difference in the competitive abilities of the length variants across generations at the population level  and a control haplotype, Dahomey. Prior to the experiment, each of these haplotypes had been backcrossed independently into replicate mass-bred populations sourced from Dahomey; thus, the 2 haplotypes could be admixed at various starting frequencies and always competed against a heterogeneous nuclear background sourced from Dahomey. Rather than being set up in large cages, each population was maintained inside a 40-ml vial. This meant that population sizes were much smaller  than in a typical population cage , but the benefit was that replication of experimental populations was high (84 populations per experiment). The Brownsville haplotype was seeded into populations at 4 different starting frequencies—0, 25, 50, and 75%, The authors observed that populations that were initially seeded with at least 50% of the Brownsville haplotype exhibited numerical suppression in population size over the course of 10 generations of monitoring, as a result of the negative effects of the Brownsville haplotype on male fertility. Notably, despite this population suppression effect, the frequency of the Brownsville haplotype increased across generations in these populations, exhibiting signatures of an adaptive response to positive selection  or abiotic (e.g. temperature or dietary variation) environment . These studies, reconciled by the results of population cage studies, have inspired researchers to test whether trajectories of mitochondrial haplotype evolution, within and across replicate populations, may respond adaptively to laboratory-imposed regimes of natural selection. This approach, known as experimental evolution, extends on classic population cage studies, typically through inclusion of a treatment of divergent natural selection  or a treatment of divergent starting frequencies of competing haplotypes (when testing for effects of negative frequency-dependent selection). Although the framework of experimental evolution provides one of the most powerful mechanisms to observe adaptive evolution in action and delineate adaptive from nonadaptive responses to natural selection (Intraspecific studies section), we outlined a study of D. melanogaster exhibit a latitudinal cline across the eastern seaboard of Australia, with fly strains harboring the A1 haplotype exhibiting heightened resilience to extreme heat stress but reduced tolerance to cold stress, relative to those with the B1 haplotype. These thermal tolerant attributes of the haplotypes aligned with their spatial distribution along the latitudinal cline; the A1 haplotype predominates in the subtropical north of Australia, B1 haplotypes in the temperate south. Based on these findings, it is predicted that that the A1 haplotype would have a selective advantage over B1 under warmer conditions and potentially respond adaptively by increasing in population frequency under such environments. To test this, Wolbachia, which is known to infect populations of flies in east coast Australia) and a group that remained untreated and that therefore carried Wolbachia infection. Consistent with previous findings of Wolbachia , or mild cytoplasmic incompatibility driven by varying levels of infection in the admixed populations, or complex interactions between mtDNA haplotype, Wolbachia and temperature, will have interfered with the capacity of the mtDNA to respond to selection in the presence of a Wolbachia infection.Above , but with this effect waning as ratios decreased. Indeed, at ratios of 1:16, Alstonville was outcompeted by a haplotype sourced from Dahomey. Further experiments conducted on these 2 haplotypes confirmed that switches in diets provided to population cages across the course of a 28 generation long experiment (from protein-to-carbohydrate ratios of 1:2 to 1:16 and then back to 1:2) resulted in corresponding changes in the population frequencies of the Alstonville and Dahomey haplotypes across population cages, with Alstonville always increasing under high protein and decreasing under low protein. The authors reported these frequency changes were mediated by differences between haplotypes in larval development across the 2 diets   .D. suboscura  or via biparental inheritance of mitochondria . Heteroplasmy due to mutation accumulation is probably pervasive and can for instance explain the penetrance of many mitochondrial diseases in humans, in cases where mtDNA molecules carrying pathogenic mutations increase in frequency over time to the point in which they outnumber their wild-type counterparts within a given cell or tissue lineage, crossing a biochemical threshold at which disease is expressed . HeteropDrosophila, through for example observation of biparental inheritance resulting from crosses between isofemale lines in D. simulans, in which a small proportion of mating pairs consistently produced heteroplasmic offspring  estimated at 6%  into fertilized eggs . D. simulans that carried 2 divergent mitochondrial haplotypes (siII and siIII) differing at about 1.5% of nucleotide sites. Assessing changes in heteroplasmy levels across 10 generations revealed a rapid decrease of siIII haplotype frequency within the first 3 generations  and 2 strains of D. mauritiana (carrying the maI or maII mtDNA type) even exhibited a clear hierarchy in competitive haplotype advantage, with siII bearing the greatest advantage, followed by siIII, maI, siI, and finally maII . When the temperature-sensitive mtDNA haplotype was heteroplasmic alongside other wild-type haplotypes, high temperature selected for a decrease in the abundance of the mutant haplotype across generations, despite genetic complementation , tam. The researchers showed that reducing expression of tam enhanced the effect of purifying selection via elimination of the mutant mtDNA recombinant haplotype. This effect of enhanced purifying selection upon downregulation of tam extended to other heteroplasmic combinations, resulting in consistent increases of the haplotype associated with higher mETS functionality , which can lead to expression of severe disease phenotypes, including hearing loss, exercise intolerance, and lactic acidosis. Since then, hundreds of mtDNA mutations have been implicated in a wide range of multisystem disorders, including aging  gene could be rescued by expression by a GAL4/UAS-enabled alternative oxidase (25t) in the nuclear gene-encoding mitoribosomal protein S12 of D. melanogaster were shown to be sensitive to the mtDNA haplotype background alongside which the tko25t was expressed. Of 4 mtDNA backgrounds studied, 1 particular mtDNA background accentuated the negative effects of tko25t, leading to high numbers of melanotic modules and hemolymph abnormalities, indicators of defective innate immunity, and developmental lethality  and interspecific tests section). One challenge to further development of Drosophila as a model for the study of mitochondrial disease will hinge on the ability to unambiguously identify and isolate single disease-causing mutations in the mtDNA sequence. The most effective avenue to achieve this will be through development of mitochondrial genome editing to disassociate single mutations within haplotypes linked to disease.Despite proven potential, the number of  limited . This isDrosophila model  and extrinsic environments (e.g. climate and diet). Whether or not these epistatic interactions can mediate evolutionary responses to natural selection within populations or patterns of reproductive isolation between incipient species remains open to question and would benefit from attention of further studies that embrace approaches such as experimental evolution, which enable evolutionary trajectories to be tracked in real time. The evolutionary and ecological implications of sex-specific mitochondrial genetic variation also require further attention: firstly, to clarify the generality by which such sex specificity is male biased and consistent with predictions of the Mother's Curse hypothesis; and secondly, to determine whether such sex specificity may contribute to dynamics of sexually antagonistic coevolution or outcomes of sexual selection generally.Drosophila model is ideally positioned to address the outstanding questions of the future and further advance understanding of the ecological and evolutionary significance of mitochondrial genetic variation.Moreover, the underpinning loci involved in mitonuclear interactions remain largely elusive, and identifying these loci should be a priority for future research. Researchers are increasingly embracing next-generation sequencing technologies to dissect the functional basis of mitonuclear interactions at the genome-wide scale, revealing new insights into the broader influence of mitochondrial sequence variation in the regulation of the nuclear gene expression and the mechanisms linking mitochondrial genotype to phenotype (


The classification of myelodysplastic neoplasms (MDSs) in patients with a previous primary tumor is controversial, as an efficient consensus risk factor-based classification of secondary MDSs has not yet been established. The current classifications consider separate etiologies based on exposure to cytotoxic therapy and genetic predisposition, despite recent studies on the germline landscape and clonal hematopoiesis of indeterminate potential (CHIP) supporting the contention that these different risk factors are overlapping. This review wants to summarize the current status of knowledge of secondary MDS etiologies and foresees future classifications that assess all risk factors and their interactions for achieving a differential diagnosis of patients at risk to aid in routine clinical decision-making related to most adequate clinical management.There is a great deal of controversy in the hematologic community regarding the classification of secondary myelodysplastic neoplasms (MDSs). Current classifications are based on the presence of genetic predisposition and MDS post-cytotoxic therapy (MDS-pCT) etiologies. However, since these risk factors are not exclusive for secondary MDSs and there are multiple overlapping scenarios, a comprehensive and definitive classification is yet to come. In addition, a sporadic MDS might arise after a primary tumor fulfills the diagnostic criteria of MDS-pCT without a causative cytotoxicity. In this review, we describe the triggering pieces of a secondary MDS jigsaw: previous cytotoxic therapy, germline predisposition and clonal hematopoiesis. Epidemiological and translational efforts are needed to put these pieces together and ascertain the real weight of each of these pieces in each MDS patient. Future classifications must contribute to understanding the role of secondary MDS jigsaw pieces in different concomitant or independent clinical scenarios associated with the primary tumor. Myelodysplastic syndromes or, as renamed in the latest World Health Organization (WHO) classification , myelodyThe development of a secondary myelodysplasia is benign and often reversible if the offending trigger is withdrawn . In VienMDS post-cytotoxic therapy (MDS-pCT) cases are aggressive hematologic malignant neoplasms. The incidence of these neoplasms is rare , but the mortality rates are higher compared to primary MDS, with a 5-year survival rate of 10% vs. 31% in primary MDS, and a median survival of around 8–10 months . The diaIt has been estimated that 10% of patients with non-Hodgkin lymphoma , 8.2% ofMLL translocation at 11q23 or RUNX1/AML1 at 21q22 and a low frequency of complex karyotypes [On the other hand, the latency period for the appearance of MDS-pCT after treatment substantially varies depending on the type of primary cancer and the treatment regimen . This isryotypes ,27 -mutant murine hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, and that the effect was specific to Trp53-mutant HSPCs and was not observed in HSPCs with other clonal hematopoiesis mutations [On the other hand, immunomodulatory drugs were introduced for treating MM in the late 90s, leading to significantly improved overall survival. During the following decade, lenalidomide replaced thalidomide as the most used immunomodulatory drug for MM due to its higher efficacy and lower toxicity. Several clinical trials have found significantly higher rates of secondary myeloid neoplasms in lenalidomide-treated arms than in those without lenalidomide in relapsed/refractory, transplant-eligible and transplant ineligible MM patients . A definutations . A recenutations .TP53 mutations were found to be enriched in MDS-pCT patients, while spliceosome mutations are more frequent in de novo MDS, which might partially explain the complex karyotype and unfavourable clinical outcomes of MDS-pCT patients [PPM1D gene, a negative regulator of the DNA damage response pathway, are also frequent in MDS-pCT patients, as they confer a competitive advantage under the selective pressure of chemotherapy [Scarce information has been published regarding prognostic models or associations between MDS-pCT and concrete clinical outcomes and progression . In addipatients . Likewisotherapy .Over the last decade, large-scale genomic studies have described the landscape of genomic variants in many of the most relevant types of cancer with the initial and fundamental objective of providing prognostic, diagnostic and pathogenic information based on the acquired alterations detected. However, the co-assessment of germ tissue in these series has transformed the understanding of how inherited variants influence cancer development . Within SAMD9, SAMD9L, SRP72, TERC and TERT genes, together with other genes typically mutated in sporadic MDS, such as TP53, GATA2, DDX41, ANKRD26, ETV6, CEBPA, ASXL1 and RUNX1, have been associated with the germline development of the disease [The identification of clinical features and molecular biomarkers linked to this entity is important since its clinical management differs from sporadic MDS . To this disease . Differe disease .GATA2 haploinsufficiency, among others [Furthermore, several hereditary syndromes have also been associated with MDS development . These Mg others   maintains the therapy-related myeloid neoplasm (TRMN) category as an entity . NeverthBoth classifications are based on fitting, including two delimited etiologies for secondary MDS, based on therapy toxicity and genetic predisposition A. HoweveBRCA1, BRCA2, PALB2, CHEK2 and TP53 [Thus, genetic predisposition might increase the susceptibility to cytotoxic agents. The treatment of a primary tumor may affect bone marrow cells differently depending on the presence of mutations affecting the DNA damage repair system. In this sense, it has been described that between 16 and 21% of cancer survivors who developed MDS-pCT had a germline mutation associated with inherited cancer susceptibility genes . Severaland TP53 ,63 and Fand TP53 .TP53 (31%), DNMT3A (19%), IDH1/2 (13%), NRAS (13%), TET2 (12%), NPM1 (10%), PPM1D (9%) and PTPN11 (9%) [Similarly, recent studies showed that cancer therapy shapes the fitness landscape of clonal hematopoiesis by promoting the onset or the increment of cytopenia and clonal dysplasia, such as idiopathic cytopenia of undetermined significance (ICUS), clonal cytopenia of undetermined significance (CCUS), idiopathic dysplasia of unknown significance (IDUS) or CHIP . CHIP waN11 (9%) . Since CN11 (9%) .In addition to these overlapping scenarios, both primary and secondary tumors might not be associated with any risk factor and do not occur concomitantly. Current therapeutic strategies have reduced the mortality among cancer patients, with an increase in survival rates entailing an increased frequency of age-dependent secondary pathologies with no relationship with the primary tumor. Thus, both tumors might arise independently or from different risk factors. The MDSs of these patients would have no relationship with the cytotoxic therapy of the primary tumor or a common genetic origin (syndrome), but they mimic the clinical definition of a secondary MDS.With a few exceptions, the cytogenetic abnormalities and molecular similarities between secondary MDSs of different etiologies hamper adequate classification of differential diagnoses ,21,36 T. FurtherWe foresee future classifications of secondary MDSs assessing all risk factors and their interactions for a better assessment of the etiology of the cancer. Therefore, overlapping risk factors (CHIP and germline predisposition), together with the cytotoxic therapy effect, would result in the clinical definition of different concomitant scenarios, including a single or variety of responsible risk factors. Finally, clinical scenarios in which both tumors are independent or do not share risk factors must be considered as they mimic the clinical manifestation of secondary MDS but must be identified to prevent background noise.Therefore, we suggest the following scenarios .(a) Concomitant tumors are related to secondary MDS when it involves the etiology of the primary tumor:solely contribute to MDS without other known risk factors;trigger CHIP or drive an increase in CHIP;contribute depending on germline predisposition or susceptibility;contribute together with CHIP and the germline landscape.(a.1) MDS-pCT disease; the hematologic disease arises through exposure to a cytotoxic therapy when treating a primary tumor. Treatment might do the following:(a.2) Syndrome; secondary MDS arises from a common genetic origin together with the first tumor.(b) Independent tumors mimic the clinical appearance of the previous scenarios but do not share risk factors:Sporadic, correlative tumors with no shared risk factors;MDS-pCT-like, in which cytotoxic therapy does not participate in the development of a secondary myeloid tumor.Secondary MDS occurs with germline predisposition, in which a primary tumor might also have a genetic predisposition but is not common with that of the MDS.In summary, the role of risk factors in the different etiologies of MDS scenarios is still unclear. Furthermore, the classification of the etiologies of MDS becomes even more challenging when it occurs in a patient with a previous primary tumor. The first step to understand the molecular pathophysiology and the role of risk factors in different secondary MDSs is to have an adequate classification even with no prognostic biomarkers or molecular descriptions. Current classifications are based on etiologies related to well-delimited risk factors and cytotoxic therapy. However, the current knowledge regarding the biology of hematological malignancies supports the integration of genetic predisposition and CHIP into future MDS-pCT classifications to reflect the biological diversity and etiologies of MDS-pCT and their impact on outcomes. Thus, future classifications must consider the concomitance of single and overlapping risk factors, as well as independence regarding the relationship between primary and secondary tumors.Deciphering the contribution of risk factors in combination with cytotoxic treatments will allow for the differential diagnoses of patients at risk to aid in routine clinical decision-making at the translational level to provide adequate clinical management. In addition, understanding the pathologic mechanisms underlying different etiologies will improve the development of prognostic biomarkers and therapy-oriented guidance of primary tumors for patients at risk of developing secondary MDS and will also help in genetic counseling related to the suitability of hematopoietic progenitor cell transplants (HPTs) from a related donor.