Oocyte collection and outcome following oncologic treatment: a retrospective multicentre study.

PURPOSE: This study assesses fertility treatment outcomes in female patients who had undergone successful oocyte retrieval following cancer therapy. METHODS: Between January 2020 and December 2022, we collected fertility treatment data from six participating centres in Spain and Germany. All patients associated with this data had undergone successful oocyte retrieval following cancer treatment. RESULTS: Women had most frequently been diagnosed with a haematological (41.9%), breast (22.6%) or gynaecological malignancy (12.9%); two thirds (67.7%) had previously received a chemotherapy, half a radiotherapy (53.3%) and 45.2% had undergone surgery. On average, 7 years (range 0-28) had passed between cancer treatment and first ovarian stimulation cycle. Forty-nine ovarian stimulation cycles had been conducted on these 31 women between 2004 and 2021 (mean age at first oocyte collection following treatment: 34.8 ± 5.7 years). On average, 7 oocytes were collected per cycle (range 0-26) and 11 were collected per patient (range 0-51). Out of the 190 oocytes collected for immediate use of artificial reproductive technique, 139 were fertilised at a rate of 73%. Live birth rate per fresh transfer was 45% (9/20); no births were reported following cryotransfer (0/10). Mean values of anti-Mullerian hormone (AMH) before stimulation declined with time since treatment; however, oocytes were successfully collected from four women with an AMH of <0.5 ng/ml, although no pregnancies were reported. Ten pregnancies were documented; 3 ended in miscarriage. Two twin and 5 single pregnancies resulted in nine live births. On average, children were carried to term. CONCLUSION: In this small cohort, oocytes were successfully collected after chemotherapy and radiotherapy, despite-in individual cases-low AMH values. Further studies are needed to enrich the database and ultimately provide appropriate counselling to female cancer patients regarding expectations and ART outcome following cancer therapy.

Updates in the Management of Central and Peripheral Nervous System Tumors among Patients with Neurofibromatosis Type 1 and Neurofibromatosis Type 2.

BACKGROUND: Neurofibromatosis type 1 and neurofibromatosis type 2 are unrelated, distinct genetic disorders characterized by the development of central and peripheral nervous system tumors. SUMMARY: Neurofibromatosis type 1 is the most common inherited tumor predisposition syndrome with a lifelong increased risk of benign and malignant tumor development, such as glioma and nerve sheath tumors. Neurofibromatosis type 2 classically presents with bilateral vestibular schwannoma, yet it is also associated with non-vestibular schwannoma, meningioma, and ependymoma. Historically, the number of effective therapies for neurofibromatosis-related neoplasms has been limited. KEY MESSAGE: In the past decade, there have been significant advances in the development of precision-based therapies for NF-associated tumors with an increased emphasis on functional outcomes in addition to tumor response. Continued scientific discovery and advancement of targeted therapies for NF-associated neoplasms are necessary to continue to improve outcomes for patients with NF.

R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells.

BACKGROUND: Loss of the Ras GTPase-activating protein neurofibromin promotes nervous system tumor pathogenesis in patients with neurofibromatosis type 1 (NF1). Neurofibromin loss potentially hyperactivates classic Ras (H-Ras, N-Ras, K-Ras), M-Ras, and R-Ras (R-Ras, R-Ras2/TC21) subfamily proteins. We have shown that classic Ras proteins promote proliferation and survival, but not migration, in malignant peripheral nerve sheath tumor (MPNST) cells. However, it is unclear whether R-Ras, R-Ras2 and M-Ras are expressed and hyperactivated in MPNSTs and, if so, whether they contribute to MPNST pathogenesis. We assessed the expression and activation of these proteins in MPNST cells and inhibited them to determine the effect this had on proliferation, migration, invasion, survival and the phosphoproteome. METHODS: NF1-associated (ST88-14, 90-8, NMS2, NMS-PC, S462, T265-2c) and sporadic (STS-26T, YST-1) MPNST lines were used. Cells were transfected with doxycycline-inducible vectors expressing either a pan-inhibitor of the R-Ras subfamily [dominant negative (DN) R-Ras] or enhanced green fluorescent protein (eGFP). Methodologies used included immunoblotting, immunocytochemistry, PCR, Transwell migration, 3H-thymidine incorporation, calcein cleavage assays and shRNA knockdowns. Proteins in cells with or without DN R-Ras expression were differentially labeled with SILAC and mass spectrometry was used to identify phosphoproteins and determine their relative quantities in the presence and absence of DN R-Ras. Validation of R-Ras and R-Ras2 action and R-Ras regulated networks was performed using genetic and/or pharmacologic approaches. RESULTS: R-Ras2 was uniformly expressed in MPNST cells, with R-Ras present in a major subset. Both proteins were activated in neurofibromin-null MPNST cells. Consistent with classical Ras inhibition, DN R-Ras and R-Ras2 knockdown inhibited proliferation. However, DN R-Ras inhibition impaired migration and invasion but not survival. Mass spectrometry-based phosphoproteomics identified thirteen protein networks distinctly regulated by DN R-Ras, including multiple networks regulating cellular movement and morphology. ROCK1 was a prominent mediator in these networks. DN R-Ras expression and RRAS and RRAS2 knockdown inhibited migration and ROCK1 phosphorylation; ROCK1 inhibition similarly impaired migration and invasion, altered cellular morphology and triggered the accumulation of large intracellular vesicles. CONCLUSIONS: R-Ras proteins function distinctly from classic Ras proteins by regulating distinct signaling pathways that promote MPNST tumorigenesis by mediating migration and invasion. Mutations of the NF1 gene potentially results in the activation of multiple Ras proteins, which are key regulators of many biologic effects. The protein encoded by the NF1 gene, neurofibromin, acts as an inhibitor of both classic Ras and R-Ras proteins; loss of neurofibromin could cause these Ras proteins to become persistently active, leading to the development of cancer. We have previously shown that three related Ras proteins (the classic Ras proteins) are highly activated in malignant peripheral nerve sheath tumor (MPNST) cells with neurofibromin loss and that they drive cancer cell proliferation and survival by activating multiple cellular signaling pathways. Here, we examined the expression, activation and action of R-Ras proteins in MPNST cells that have lost neurofibromin. Both R-Ras and R-Ras2 are expressed in MPNST cells and activated. Inhibition of R-Ras action inhibited proliferation, migration and invasion but not survival. We examined the activation of cytoplasmic signaling pathways in the presence and absence of R-Ras signaling and found that R-Ras proteins regulated 13 signaling pathways distinct from those regulated by classic Ras proteins. Closer study of an R-Ras regulated pathway containing the signaling protein ROCK1 showed that inhibition of either R-Ras, R-Ras2 or ROCK1 similarly impaired cellular migration and invasion and altered cellular morphology. Inhibition of R-Ras/R-Ras2 and ROCK1 signaling also triggered the accumulation of abnormal intracellular vesicles, indicating that these signaling molecules regulate the movement of proteins and other molecules in the cellular interior. Video Abstract.

Health-related quality of life of children born to childhood cancer survivors in Germany.

OBJECTIVE: Rising childhood cancer survival rates have increased the importance of health-related quality of life (HRQL) assessment. While survivors show comparable HRQL to peers, concerns that cancer treatment could impact the health of prospective children were reported. No previous publications address HRQL of childhood cancer survivor offspring. METHODS: We assessed survivor offspring HRQL using the parental KINDL questionnaire. Matched-pair analysis was conducted with data from the general population (KiGGS study) using age, gender and education (1:1, n = 1206 cases). Multivariate analyses were conducted to detect the influence of parental diagnose and treatment on offspring HRQL. RESULTS: Overall, within KINDL dimensions, survivors reported comparable to higher HRQL for their children than the general population. Survivor parents reported significantly (p < 0.001) higher psychological (86.7% vs. 83.0%, Cohen's d = 0.3) and self-esteem (79.1% vs. 73.3%, Cohen's d = 0.5) well-being scores for younger children (3-6-year-olds). As time since diagnosis increased, parents reported higher well-being scores. Accordingly, recently diagnosed survivors reported significantly lower psychological well-being scores (p = 0.28; OR = 0.457; 95% CI = 0.228-0.918) for their children. With increasing age, average HRQL scores decreased in both cohorts; yet, this drop was less pronounced for survivor offspring. The biggest difference between age groups (7-10- vs. 14-17-year-olds) was found for school-specific well-being (6.2-point drop in survivor offspring vs. 18.2-point drop in KiGGS offspring). CONCLUSION: Comparable to higher parentally assessed HRQL was reported for survivor offspring compared to peers. These findings are reassuring and consistent with self-reported HRQL in childhood cancer survivors. Type of parental cancer diagnosis and treatment showed no negative impact on offspring HRQL.

Fertility education for adolescent cancer patients: Gaps in current clinical practice in Europe.

OBJECTIVE: As adolescent cancer patients may suffer from infertility following treatment, fertility counselling is essential. Our aim was to explore the current situation in four European countries in terms of (I) education about the risk for infertility, (II) counselling on fertility preservation, (III) patients' knowledge on fertility, (IV) sufficiency of information and (V) uptake of cryopreservation. METHODS: In total, 113 patients (13-20 years) at 11 study centres completed a self-report questionnaire three and six months after cancer diagnosis. Multivariate logistic regression was used to estimate odds ratios (OR) with 95% confidence intervals (CI). RESULTS: As many as 80.2% of participants reported having received education about the risk for infertility prior to treatment, 73.2% recalled counselling on fertility preservation. Only 52.3% stated they felt sufficiently informed to make a decision. Inability to recall counselling on fertility preservation (OR = 0.03, CI: 0.00-0.47) and female gender (OR = 0.11, CI: 0.03-0.48) was associated with lower use of cryopreservation, whereas older age was associated with higher use. CONCLUSION: Fertility counselling was available to a relatively high proportion of patients, and it did influence the utilisation of cryopreservation. However, many patients did not feel sufficiently informed. Further improvement is needed to enable adolescent cancer patients to make an informed decision on fertility preservation.

ErbB4 promotes malignant peripheral nerve sheath tumor pathogenesis via Ras-independent mechanisms.

BACKGROUND: We have found that erbB receptor tyrosine kinases drive Ras hyperactivation and growth in NF1-null malignant peripheral nerve sheath tumors (MPNSTs). However, MPNSTs variably express multiple erbB receptors with distinct functional characteristics and it is not clear which of these receptors drive MPNST pathogenesis. Here, we test the hypothesis that altered erbB4 expression promotes MPNST pathogenesis by uniquely activating key cytoplasmic signaling cascades. METHODS: ErbB4 expression was assessed using immunohistochemistry, immunocytochemistry, immunoblotting and real-time PCR. To define erbB4 functions, we generated mice that develop MPNSTs with floxed Erbb4 alleles (P0-GGFß3;Trp53+/-;Erbb4flox/flox mice) and ablated Erbb4 in these tumors. MPNST cell proliferation and survival was assessed using 3H-thymidine incorporation, MTT assays, Real-Time Glo and cell count assays. Control and Erbb4-null MPNST cells were orthotopically xenografted in immunodeficient mice and the growth, proliferation (Ki67 labeling), apoptosis (TUNEL labeling) and angiogenesis of these grafts was analyzed. Antibody arrays querying cytoplasmic kinases were used to identify erbB4-responsive kinases. Pharmacologic or genetic inhibition was used to identify erbB4-responsive kinases that drive proliferation. RESULTS: Aberrant erbB4 expression was evident in 25/30 surgically resected human MPNSTs and in MPNSTs from genetically engineered mouse models (P0-GGFß3 and P0-GGFß3;Trp53+/- mice); multiple erbB4 splice variants that differ in their ability to activate PI3 kinase and nuclear signaling were present in MPNST-derived cell lines. Erbb4-null MPNST cells demonstrated decreased proliferation and survival and altered morphology relative to non-ablated controls. Orthotopic allografts of Erbb4-null cells were significantly smaller than controls, with reduced proliferation, survival and vascularization. ERBB4 knockdown in human MPNST cells similarly inhibited DNA synthesis and viability. Although we have previously shown that broad-spectrum erbB inhibitors inhibit Ras activation, Erbb4 ablation did not affect Ras activation, suggesting that erbB4 drives neoplasia via non-Ras dependent pathways. An analysis of 43 candidate kinases identified multiple NRG1ß-responsive and erbB4-dependent signaling cascades including the PI3K, WNK1, STAT3, STAT5 and phospholipase-Cγ pathways. Although WNK1 inhibition did not alter proliferation, inhibition of STAT3, STAT5 and phospholipase-Cγ markedly reduced proliferation. CONCLUSIONS: ErbB4 promotes MPNST growth by activating key non-Ras dependent signaling cascades including the STAT3, STAT5 and phospholipase-Cγ pathways. ErbB4 and its effector pathways are thus potentially useful therapeutic targets in MPNSTs.

Fertility knowledge and associated empowerment following an educational intervention for adolescent cancer patients.

INTRODUCTION/OBJECTIVES: Fertility preservation is a major concern for adolescent cancer patients; yet, educational gaps remain. Our intervention study examined whether specially designed educational materials regarding fertility preservation increase knowledge and empowerment of patients and parents. METHODS: Eleven paediatric-oncological centres in four European countries agreed to enrol all eligible patients and parents in a questionnaire survey at 3 and 6 months after diagnosis. Treating physicians were surveyed on their medical consultation regarding fertility. RESULTS: Educational intervention increased knowledge in both patients (n = 113 and n = 101 in the control and intervention groups, respectively) and parents (n = 111 and n = 99 in the control and intervention groups, respectively), but the difference did not achieve statistical significance (knowledge difference patients: 5.6% (t0)/13.1% (t1); parents: 6.4% (t0)/3.8% (t1)). Parents of older patients (OR = 1.3, 95%CI = 1.1-1.7) and higher educational groups (OR = 6.2, 95%CI = 2.1-18.3) in the intervention group (OR = 1.9, 95%CI = 1.03-3.7) achieved higher knowledge levels. Empowerment was significantly improved in both patients (p = 0.046, d = 0.27) and parents (p = 0.046, d = 0.48) in the intervention group. DISCUSSION/CONCLUSIONS: In our study, the use of specifically prepared flyers and brochures successfully raised the level of fertility preservation knowledge in parents of older patients as well as parents with higher educational levels. Overall, the intervention improved patient and parent empowerment. Subsequent projects will include simpler information and digital material to particularly reach out to younger and less educated individuals.

Suberanilohydroxamic acid (vorinostat) synergistically enhances the cytotoxicity of doxorubicin and cisplatin in osteosarcoma cell lines.

Osteosarcoma is the most common primary bone cancer in children and is a highly malignant disease, in which 25% of patients present with metastasis at diagnosis. Considerable advances in the treatment of localized disease have been achieved since the introduction of combined modality treatment, increasing the prognosis of overall survival to 70%. Yet, established therapies have only limited success in treating both metastatic disease and nonresponders to primary chemotherapy. Therefore, new therapeutic approaches are required, particularly for the control of osteosarcoma in these patient groups. Epigenetically modifying substances are a class of emerging drugs that have shown therapeutic potential in various hematological and solid cancers. We examined the cytotoxic effects of 5-azacitidine, 3-deazaneplanocin A, and suberanilohydroxamic acid (SAHA) on osteosarcoma cell lines HOS, MG-63, MNNG, and ZK-58. SAHA was the only chemical agent that exerted a strong, growth-limiting effect in all cell lines tested. The growth-limiting effect of SAHA was accompanied by features characteristic of apoptotic death. We found that cotreatment with SAHA and cisplatin showed strong synergism in all cell lines. The effect of cotreatment with SAHA and doxorubicin was cell line dependent. In the cell lines HOS, MG-63, and MNNG, the combined effect was synergistic, whereas in the cell line ZK-58, SAHA antagonized doxorubicin. The strong synergism of SAHA indicated that in combination with cisplatin, it might enable a promising add-on to current therapy regimens. However, considering the cell line-dependent effect that was found when SAHA was combined with doxorubicin, further experimentation is needed.

Management and Outcome of Ewing Sarcoma of the Head and Neck.

BACKGROUND: Ewing sarcoma (EWS) of the head and neck is rare. Multimodal treatment consists of chemotherapy and local treatment; however, local treatment for EWS of the head and neck is challenging. The first objective was to describe local treatment administered to the patients with localized EWS of the head and neck according to the EURO-E.W.I.N.G.99-trial, and to assess the impact on survival. The second objective was to systematically review the scientific literature available for this topic. PROCEDURE: Fifty-one patients were included. Local control consisted of surgery and/or radiotherapy (RT). Event-free survival (EFS) and overall survival (OS) were determined. Outcome was analyzed by comparing local treatment approaches. A Medline search was performed for EWS of the head and neck. RESULTS: Eighty-six percent of patients had localized disease. Most common primary sites included the skull (45%), maxilla (14%), and mandible (12%). Three-year EFS was 74% and 3-year OS was 87% for patients with localized disease. EFS was 40% for patients >15 years compared to 81% for patients <15 years. Local control consisted of surgery (S; 33%), RT (18%), or S + RT (45%). Related 3-year EFS was 81% (S), 80% (RT), and 72% (S + RT); 3-year OS was 80%, 76%, and 81%, respectively. CONCLUSIONS: In patients with EWS of the head and neck, age, and stage are important prognostic factors. Although not statistically significant, large tumor volume seems to be a negative prognostic factor. No difference in EFS and OS could be found when comparing patients treated with surgery, RT, or combined surgery and RT.

Neuregulin-1 overexpression and Trp53 haploinsufficiency cooperatively promote de novo malignant peripheral nerve sheath tumor pathogenesis.

Malignant peripheral nerve sheath tumors (MPNSTs) are Schwann cell-derived malignancies that arise from plexiform neurofibromas in patients with mutation of the neurofibromin 1 (NF1) gene. We have shown that the growth factor neuregulin-1 (NRG1) also contributes to human neurofibroma and MPNST pathogenesis and that outbred C57BL/6J × SJL/J transgenic mice overexpressing NRG1 in Schwann cells (P0-GGFß3 mice) recapitulate the process of neurofibroma-MPNST progression. However, it is unclear whether NRG1 acts predominantly within NF1-regulated signaling cascades or instead activates other essential cascades that cooperate with NF1 loss to promote tumorigenesis. We now report that tumorigenesis is suppressed in inbred P0-GGFß3 mice on a C57BL/6J background. To determine whether NRG1 overexpression interacts with reduced Nf1 or Trp53 gene dosage to "unmask" tumorigenesis in these animals, we followed cohorts of inbred P0-GGFß3;Nf1+/−, P0-GGFß3;Trp53+/− and control (P0-GGFß3, Nf1+/− and Trp53+/−) mice for 1 year. We found no reduction in survival or tumors in control and P0-GGFß3;Nf1+/− mice. In contrast, P0-GGFß3;Trp53+/− mice died on average at 226 days, with MPNSTs present in 95 % of these mice. MPNSTs in inbred P0-GGFß3;Trp53+/− mice arose de novo from micro-MPNSTs that uniformly develop intraganglionically. These micro-MPNSTs are of lower grade (WHO grade II-III) than the major MPNSTs (WHO grade III-IV); array comparative genomic hybridization showed that lower grade MPNSTs also had fewer genomic abnormalities. Thus, P0-GGFß3;Trp53+/− mice represent a novel model of low- to high-grade MPNST progression. We further conclude that NRG1 promotes peripheral nervous system neoplasia predominantly via its effects on the signaling cascades affected by Nf1 loss.

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system, a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Through in vitro pharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

Identification of therapeutic sensitivities in a spheroid drug combination screen of Neurofibromatosis Type I associated High Grade Gliomas.

Neurofibromatosis Type 1 (NF1) patients develop an array of benign and malignant tumors, of which Malignant Peripheral Nerve Sheath Tumors (MPNST) and High Grade Gliomas (HGG) have a dismal prognosis. About 15-20% of individuals with NF1 develop brain tumors and one third of these occur outside of the optic pathway. These non-optic pathway gliomas are more likely to progress to malignancy, especially in adults. Despite their low frequency, high grade gliomas have a disproportional effect on the morbidity of NF1 patients. In vitro drug combination screens have not been performed on NF1-associated HGG, hindering our ability to develop informed clinical trials. Here we present the first in vitro drug combination screen (21 compounds alone or in combination with MEK or PI3K inhibitors) on the only human NF1 patient derived HGG cell line available and on three mouse glioma cell lines derived from the NF1-P53 genetically engineered mouse model, which sporadically develop HGG. These mouse glioma cell lines were never exposed to serum, grow as spheres and express markers that are consistent with an Oligodendrocyte Precursor Cell (OPC) lineage origin. Importantly, even though the true cell of origin for HGG remains elusive, they are thought to arise from the OPC lineage. We evaluated drug sensitivities of the three murine glioma cell lines in a 3D spheroid growth assay, which more accurately reflects drug sensitivities in vivo. Excitingly, we identified six compounds targeting HDACs, BRD4, CHEK1, BMI-1, CDK1/2/5/9, and the proteasome that potently induced cell death in our NF1-associated HGG. Moreover, several of these inhibitors work synergistically with either MEK or PI3K inhibitors. This study forms the basis for further pre-clinical evaluation of promising targets, with an eventual hope to translate these to the clinic.

A continuing clinical education course to maintain clinical competencies and foster new clinical knowledge during the graduate school years of MD-PhD training.

Introduction: Most students in MD-PhD programs take a leave of absence from medical school to complete PhD training, which promotes a natural loss of clinical skills and knowledge and could negatively impact a student's long-term clinical knowledge. To address this concern, clinical refresher courses in the final year of PhD training have traditionally been used; however, effectiveness of such courses versus a longitudinal clinical course spanning all PhD training years is unclear. Methods: The University of Alabama at Birmingham MD-PhD Program implemented a comprehensive continuing clinical education (CCE) course spanning PhD training years that features three course components: (1) clinical skills; (2) clinical knowledge; and (3) specialty exposure activities. To evaluate course effectiveness, data from an anonymous student survey completed at the end of each semester were analyzed. Results: Five hundred and ninety-seven surveys were completed by MD-PhD students from fall 2014 to 2022. Survey responses indicated that the majority of students found the course helpful to: maintain clinical skills and knowledge (544/597, 91% and 559/597, 94%; respectively), gain exposure to clinical specialties (568/597, 95%), and prepare them for responsibilities during clinical clerkships. During semesters following lockdowns from the COVID-19 pandemic, there were significant drops in students' perceived preparedness. Conclusions: Positive student survey feedback and improved preparedness to return to clinic after development of the course suggests the CCE course is a useful approach to maintain clinical knowledge during research training.

Parenthood for childhood cancer survivors: unfounded fear of cancer development in offspring and related health behaviors.

Current literature reveals no increased risk for adverse non-hereditary health outcomes in the offspring of childhood cancer survivors (CCS), yet survivors reported concerns regarding their offspring's health. To investigate how the fear of cancer development in offspring influences parental behavior related to health and prevention, survey reports from 256 European adult CCS and 256 age- and sex-matched siblings who participated in a multicenter study on offspring health were analyzed in the present study. Analyses of covariance and chi-square tests were conducted to test for differences between CCS and siblings in outcome variables (all related to healthy parenting behavior). CCS reported higher fear levels (p = 0.044, Partial η2 = 0.01) and less alcohol consumption (p = 0.011, Phi = 0.12) and smoking (p = 0.022, Phi = 0.11) during pregnancy than siblings. In survivor families, children were breastfed less often (p < 0.001, Phi = 0.18). Partial correlation analyses showed that CCS' fear levels decreased with increasing age (r = -0.16, p = 0.014), time since oncological therapy (r = -0.19, p = 0.003), and number of children (r = -0.21, p = 0.001). Overall, due to their own experiences with cancer, many CCS harbor misperceptions regarding the health outcomes of their offspring. Although the fear decreases with increasing distance from the active disease, any fear should be taken seriously, even if unfounded, and combated through targeted educational measures.

Clinical Reasoning: A 12-Month-Old Male Child With Staring Episodes, Ataxia, and Right-sided Weakness.

Baylisascaris procyonis, or raccoon roundworm, is a rare cause of eosinophilic meningoencephalitis with historically poor clinical outcomes. Symptoms of neural larval migrans begin approximately 2-4 weeks after ingestion with fatigue, nausea, fever, and lethargy and then rapidly progress to weakness, incoordination, ataxia, seizures, altered mental status, and finally coma. Only 31 other cases of CNS Baylisascaris neural larval migrans have been reported, with more than 25% being lethal. Of the remaining cases, all but 3 were neurologically devastated largely because of delays in diagnosis and treatment. We present a case of an infant with Baylisascaris neural larval migrans manifested as right hemiparesis, ataxia, and cortical blindness. Eosinophilia was missed at an outside hospital due to misidentification of eosinophils as monocytes on automated cell differential. Repeated testing of serum and CSF revealed marked eosinophilia consistent with eosinophilic meningoencephalitis, and serum antibody testing through the Centers of Disease Control confirmed Baylisascaris infection. Notably, this child had a remarkably positive outcome with near complete recovery of neurologic function after treatment with albendazole and steroids. Although eosinophilic meningoencephalitis is rare, accounting for less than 3% of all lumbar punctures with pleocytosis, this case illustrates (1) the importance of early disease recognition and treatment to improve patient outcomes and (2) the fact that automated cell differentials may misidentify eosinophils as monocytes.

Characteristics of Moyamoya Syndrome in Pediatric Patients With Neurofibromatosis Type 1.

BACKGROUND: Moyamoya syndrome (MMS) is a progressive cerebral arteriopathy with increased incidence in children with neurofibromatosis type 1 (NF1). Despite the potential for significant neurological morbidity including stroke, little is known about the natural history, and no guidelines exist for screening and management of NF1-associated MMS. METHODS: We identified 152 literature cases of children aged ≤18 years with NF1-associated MMS. A meta-analysis was performed evaluating clinical and neuroimaging findings and patient outcomes. Data from 19 patients with NF1-associated MMS from our center treated from January 1995 to July 2020 were abstracted via chart review and similarly analyzed for clinical and neuroimaging features. RESULTS: Meta-analysis of literature cases showed a median age of MMS diagnosis of 6 years (interquartile range 3 to 10.8 years). Optic pathway gliomas were more common in patients with MMS (42%) compared with historical prevalence. Stroke or transient ischemic attack (TIA) was present at diagnosis in 46%. TIA and stroke were more common in patients with bilateral versus unilateral MMS (62% vs 34%, P = 0.001) and in children aged <4 years versus those aged ≥4 years (61% vs 40%, P = 0.02). Compared with the literature cases, our cohort was more frequently asymptomatic (42% vs 25%) and less likely to present with TIA or stroke (32% vs 46%) at diagnosis. CONCLUSIONS: These data suggest there is an aggressive form of MMS in children with NF1 <4 years of age. Therefore, early screening should be considered to facilitate early detection and treatment of cerebral arteriopathy.

The Use of Assisted Reproductive Technology by European Childhood Cancer Survivors.

CCS often wish to have biological children yet harbour concerns about fertility impairment, pregnancy risks and the general health risks of prospective offspring. To clarify these concerns, health outcomes in survivor offspring born following ART (n = 74, 4.5%) or after spontaneous conception (n = 1585) were assessed in our European offspring study by descriptive and bivariate analysis. Outcomes were compared to a sibling offspring cohort (n = 387) in a 4:1 matched-pair analysis (n = 1681). (i) Survivors were more likely to employ ART than their siblings (4.5% vs. 3.7%, p = 0.501). Successful pregnancies were achieved after a median of one cycle with, most commonly, intracytoplasmic sperm injection (ICSI) using non-cryopreserved oocytes/sperm. (ii) Multiple-sibling births (p < 0.001, 29.7% vs. 2.5%), low birth weight (p < 0.001; OR = 3.035, 95%-CI = 1.615−5.706), and preterm birth (p < 0.001; OR = 2.499, 95%-CI = 1.401−4.459) occurred significantly more often in survivor offspring following ART utilisation than in spontaneously conceived children. ART did not increase the prevalence of childhood cancer, congenital malformations or heart defects. (iii) These outcomes had similar prevalences in the sibling population. In our explorative study, we could not detect an influence on health outcomes when known confounders, such as multiple births, were taken into account.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed. This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Establishment and genomic characterization of a sporadic malignant peripheral nerve sheath tumor cell line.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived neoplasms that occur sporadically or in patients with neurofibromatosis type 1 (NF1). Preclinical research on sporadic MPNSTs has been limited as few cell lines exist. We generated and characterized a new sporadic MPNST cell line, 2XSB, which shares the molecular and genomic features of the parent tumor. These cells have a highly complex karyotype with extensive chromothripsis. 2XSB cells show robust invasive 3-dimensional and clonogenic culture capability and form solid tumors when xenografted into immunodeficient mice. High-density single nucleotide polymorphism array and whole exome sequencing analyses indicate that, unlike NF1-associated MPNSTs, 2XSB cells have intact, functional NF1 alleles with no evidence of mutations in genes encoding components of Polycomb Repressor Complex 2. However, mutations in other genes implicated in MPNST pathogenesis were identified in 2XSB cells including homozygous deletion of CDKN2A and mutations in TP53 and PTEN. We also identified mutations in genes not previously associated with MPNSTs but associated with the pathogenesis of other human cancers. These include DNMT1, NUMA1, NTRK1, PDE11A, CSMD3, LRP5 and ACTL9. This sporadic MPNST-derived cell line provides a useful tool for investigating the biology and potential treatment regimens for sporadic MPNSTs.