MondoA drives malignancy in B-ALL through enhanced adaptation to metabolic stress.

Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X-like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.

The landscape of genomic alterations across childhood cancers.

Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.

Author Correction: The landscape of genomic alterations across childhood cancers.

In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.

DNA methylation-based classifier and gene expression signatures detect BRCAness in osteosarcoma.

Although osteosarcoma (OS) is a rare cancer, it is the most common primary malignant bone tumor in children and adolescents. BRCAness is a phenotypical trait in tumors with a defect in homologous recombination repair, resembling tumors with inactivation of BRCA1/2, rendering these tumors sensitive to poly (ADP)-ribose polymerase inhibitors (PARPi). Recently, OS was shown to exhibit molecular features of BRCAness. Our goal was to develop a method complementing existing genomic methods to aid clinical decision making on administering PARPi in OS patients. OS samples with DNA-methylation data were divided to BRCAness-positive and negative groups based on the degree of their genomic instability (n = 41). Methylation probes were ranked according to decreasing variance difference between two groups. The top 2000 probes were selected for training and cross-validation of the random forest algorithm. Two-thirds of available OS RNA-Seq samples (n = 17) from the top and bottom of the sample list ranked according to genome instability score were subjected to differential expression and, subsequently, to gene set enrichment analysis (GSEA). The combined accuracy of trained random forest was 85% and the average area under the ROC curve (AUC) was 0.95. There were 449 upregulated and 1,079 downregulated genes in the BRCAness-positive group (fdr < 0.05). GSEA of upregulated genes detected enrichment of DNA replication and mismatch repair and homologous recombination signatures (FWER < 0.05). Validation of the BRCAness classifier with an independent OS set (n = 20) collected later in the course of study showed AUC of 0.87 with an accuracy of 90%. GSEA signatures computed for this test set were matching the ones observed in the training set enrichment analysis. In conclusion, we developed a new classifier based on DNA-methylation patterns that detects BRCAness in OS samples with high accuracy. GSEA identified genome instability signatures. Machine-learning and gene expression approaches add new epigenomic and transcriptomic aspects to already established genomic methods for evaluation of BRCAness in osteosarcoma and can be extended to cancers characterized by genome instability.

Surgical treatment of a spontaneous spinal epidural hematoma under antithrombotic treatment of a multisystem inflammatory syndrome in children associated with SARS-COV-2.

Multisystem inflammatory syndrome in children (MIS-C) is a novel syndrome of multisystemic inflammation affecting children. This case report documents an exceptional and severe complication of an epidural hematoma in a 3-year-old boy under the treatment of MIS-C. During the course of the disease, the patient suffered from a hypocoagulable state and an extensive multisegmental epidural hematoma in the cervical spinal canal. This led to severe anterior spinal cord compression and tetraparesis. Extensive emergency surgery had to be carried out to reverse rapid clinical deterioration.

Prospective evaluation of multitarget treatment of pediatric patients with helical intensity-modulated radiotherapy.

BACKGROUND AND PURPOSE: Radiotherapy (RT) is persistently gaining significance in the treatment of pediatric tumors. However, individual features of a growing body and multifocal stages complicate this approach. Tomotherapy offers advantages in the treatment of anatomically complex tumors with low risks of side effects. Here we report on toxicity incidence and outcome of tomotherapy with a focus on multitarget RT (mtRT). MATERIALS AND METHODS: From 2008 to 2017, 38 children diagnosed with sarcoma were treated with tomotherapy. The median age was 15 years (6-19 years). Toxicity was graded according to the Common Terminology Criteria for Adverse Events v.4.03 and classified into symptoms during RT, acutely (0-6 months) and late (>6 months) after RT, and long-term sideeffects (>24 months). RESULTS: The main histologies were Ewing sarcoma (n = 23 [61%]) and alveolar rhabdomyosarcoma (n = 5 [13%]). RT was performed with a median total dose of 54 Gy (40.5-66.0 Gy) and a single dose of 2 Gy (1.80-2.27 Gy). Twenty patients (53%) received mtRT. Median follow-up was 29.7 months (95% confidence interval 15.3-48.2 months) with a 5-year survival of 55.2% (±9.5%). The 5­year survival rate of patients with mtRT (n = 20) was 37.1 ± 13.2%, while patients who received single-target RT (n = 18) had a 5-year survival rate of 75 ± 10.8%. Severe toxicities (grade 3 and 4) emerged in 14 patients (70%) with mtRT and 7 patients (39%) with single-target RT. Two non-hematological grade 4 toxicities occurred during RT: one mucositis and one radiodermatitis. After mtRT 5 patients had grade 3 toxicities acute and after single-target RT 4 patients. One patient had acute non-hematological grade 4 toxicities (gastritis, pericarditis, and pericardial effusion) after mtRT. Severe late effects of RT occurred in 2 patients after mtRT and in none of the single-target RT patients. No severe long-term side effects appeared. CONCLUSION: Our results showed acceptable levels of acute and late toxicities, considering the highly advanced diseases and multimodal treatment. Hence, tomotherapy is a feasible treatment method for young patients with anatomically complex tumors or multiple targets. Especially mtRT is a promising and innovative treatment approach for pediatric sarcomas, delivering unexpectedly high survival rates for patients with multifocal Ewing sarcomas in this study, whereby the limited number of patients should invariably be considered in the interpretation.

Systemic antitumor effect by regional hyperthermia combined with low-dose chemotherapy and immunologic correlates in an adolescent patient with rhabdomyosarcoma - a case report.

Introduction: An abscopal effect is a clinical observation whereby a local treatment is associated with regression of metastatic cancer at a site distant from the primary location of treatment. Here, we describe the clinical systemic effect induced by regional hyperthermia combined with low-dose chemotherapy and provide immunologic correlates.Case presentation: A 15-year-old patient had been diagnosed with alveolar rhabdomyosarcoma (ARMS). All previous treatment options failed in the patient including haploidentical stem cell transplantation and donor lymphocyte infusion. The patient presented with local and metastatic disease, and upon admission, underwent regional hyperthermia combined with low-dose chemotherapy. Immediately following therapy severe skin reactions were observed. Skin biopsies revealed an intraepithelial lymphocytic infiltration dominated by CD3+/CD8+ T cells with a regular network of dendritic cells. Clinical images compared before and during sequential treatment cycles showed complete metabolic response of the local tumor for more than 10 months of therapy. In addition, metastases completely regressed although they were not direct targets of regional hyperthermia. The systemic effect was associated with enhanced frequency of NK cells and T cells expressing the lectin-like natural-killer group 2 D activating receptor (NKG2D), an increase of the CD56bright subset of NK cells, as well as an increase of effector/memory and effector CD8+ and CD4+ T cells in the blood while the percentage of CD25+FOXP3+ regulatory T cells declined.Conclusions: Regional hyperthermia combined with low-dose chemotherapy had the potential to create a systemic effect which was associated with activation of NK cells and T cells.

Impact of Pathological Fractures on the Prognosis of Primary Malignant Bone Sarcoma in Children and Adults: A Single-Center Retrospective Study of 205 Patients.

BACKGROUND: The purpose of this study was to investigate whether pathological fractures (PF) influence the prognosis of patients with osteosarcoma (OS) or Ewing tumor (ET) regarding 5-year survival, occurrence of metastases, and local recurrence. METHODS: We retrospectively analyzed 205 patients with metastatic and nonmetastatic OS or ET. Survival analysis was performed for all patients and differentiated for patients with OS (n = 127) and ET (n = 78) as well as for adults (n = 101) and children (n = 104). RESULTS: Patients with PF showed survival rates of 64% compared to 83% for those without PF (p = 0.023). Local recurrence occurred in 7% of the patients without and in 24% of those with PF (p = 0.023). In patients with ET and in children, survival analysis showed no significant difference between patients with and without PF in survival and local recurrence rates. In patients with OS, survival rate decreased from 83 to 59% (p = 0.024) and local recurrence rate increased from 13 to 30% (p = 0.042). In adults, survival rate decreased from 78 to 51% (p = 0.004) and local recurrence rate increased from 13 to 42% (p < 0.001). In multivariate analysis, age and PF were associated with inferior survival. CONCLUSION: This study suggests that the occurrence of PF has a negative impact on survival and implicates an increased risk of local recurrence. In children and in patients with ET, PF did not have a prognostic impact.

Effect of a single autologous cord blood infusion on beta-cell and immune function in children with new onset type 1 diabetes: a non-randomized, controlled trial.

BACKGROUND: The application of autologous cord blood in children with type 1 diabetes has been found to be safe, but not to preserve beta-cell function in a previous study, which, however, had not included a control group. OBJECTIVE: To compare the changes of metabolic and immune function over time between cord blood infused children and natural controls. SUBJECTS AND METHODS: Seven children with newly diagnosed type 1 diabetes underwent a single autologous cord blood infusion and 10 children were enrolled as natural controls in a non-randomized, controlled, open label intervention trial. Primary analyses were performed 1 year following cord blood infusion. Cases and controls were compared regarding metabolic [area under the curve (AUC) and peak C-peptide, insulin use, and HbA1c] and immune outcome (islet autoantibody titer and T-cell response), adjusted for age, gender, diabetes duration, and baseline levels. RESULTS: There were no significant adverse events related to the infusion. Metabolic and immune outcomes were not significantly different at 12 months follow-up between infused children and controls (e.g., adjusted p = 0.244 for AUC C-peptide, adjusted p = 0.820 for insulin use, adjusted p = 0.772 for peripheral regulatory T cells). Six-month change of AUC C-peptide correlated significantly with the number of infused CD34+ cells (r = 0.931, p = 0.002). CONCLUSIONS: An autologous cord blood infusion does not change the natural course of metabolic and immune parameters after disease onset. However, the content of CD34+ cells in the stored blood sample might offer potential for improvement of future cell therapies.

G-Protein coupled receptor 64 promotes invasiveness and metastasis in Ewing sarcomas through PGF and MMP1.

Metastatic spread in Ewing sarcomas (ES) is frequent and haematogenous. G-protein coupled receptor 64 (GPR64), an orphan receptor with normal expression restricted to human epididymis is specifically over-expressed in ES among sarcoma, but also up-regulated in a number of carcinomas derived from prostate, kidney or lung. Inhibition of GPR64 expression in ES by RNA interference impaired colony formation in vitro and suppressed local tumour growth and metastasis in Rag2(-/-) γC (-/-) mice. Microarray analysis after GPR64 knock down revealed a GPR64-mediated repression of genes involved in neuronal development like SLIT, drosophila, homolog of, 2 (SLIT2), and genes regulating transcription including pre-B cell leukemia homeobox 2 (PBX2). Concurrently, the suppression of GPR64 increased ES susceptibility to TRAIL induced apoptosis. Moreover, a GPR64-mediated induction of placental growth factor (PGF) in ES was observed. PGF suppression by RNA interference resulted in a reduction of metastatic growth similar to that observed after GPR64 knock down. Importantly, inhibition of GPR64 as well as PGF expression was associated with a reduced expression of matrix metalloproteinase (MMP) 1 and invasiveness in vitro. Furthermore, MMP1 knock down abrogated lung metastasis in Rag2(-/-) γC (-/-) mice. Thus, GPR64 expression in ES maintains an immature phenotype that is less sensitive to TRAIL-induced apoptosis and via its up-regulation of PGF and MMP1 orchestrates and promotes invasiveness and metastatic spread.

The Zyxin-related protein thyroid receptor interacting protein 6 (TRIP6) is overexpressed in Ewing's sarcoma and promotes migration, invasion and cell growth.

BACKGROUND INFORMATION: Ewing's sarcoma (ES) is the second most common bone-associated malignancy in children and is driven by the fusion oncogene EWS/FLI1 and characterised by rapid growth and early metastasis. Here, we explored the role of the Zyxin-related protein thyroid receptor interacting protein 6 (TRIP6) in ES. The Zyxin family comprises seven homologous proteins involved in migration and proliferation of many cell types of which Zyxin has been described as a tumour suppressor in ES. RESULTS: By interrogation of published microarray data (n = 1254), we observed that of all Zyxin proteins, only TRIP6 is highly overexpressed in primary ES compared with normal tissues. Re-analysis of published EWS/FLI1 gain- and loss-of-function microarray experiments as well as chromatin-immunoprecipitation assays revealed that TRIP6 overexpression is not mediated by EWS/FLI1. Microarray and subsequent gene-set enrichment analyses of ES cells with and without RNA interference-mediated TRIP6 knockdown demonstrated that TRIP6 expression confers a pro-proliferative and pro-invasive transcriptional signature to ES cells. While short-term proliferation was not considerably affected by TRIP6 knockdown, silencing of the protein significantly reduced migration, invasion, long-term proliferation and clonogenicity of ES cells in vitro as well as tumourigenicity in vivo. CONCLUSIONS: Taken together, our data indicate that TRIP6 acts, in contrast to Zyxin, as an oncogene that partially accounts for the autonomous migratory, invasive and proliferative properties of ES cells independent of EWS/FLI1.

First identification of Ewing's sarcoma-derived extracellular vesicles and exploration of their biological and potential diagnostic implications.

BACKGROUND INFORMATION: Exosomes are small RNA- and protein-containing extracellular vesicles (EVs) that are thought to mediate hetero- and homotypic intercellular communication between normal and malignant cells.Tumour-derived exosomes are believed to promote re-programming of the tumour-associated stroma to favour tumour growth and metastasis. Currently, exosomes have been intensively studied in carcinomas. However, little is known about their existence and possible role in sarcomas. RESULTS: Here, we report on the identification of vesicles with exosomal features derived from Ewing's sarcoma(ES), the second most common soft-tissue or bone cancer in children and adolescents. ES cell line-derived EV shave been isolated by ultracentrifugation and analysed by flow-cytometric assessment of the exosome-associated proteins CD63 and CD81 as well as by electron microscopy. They proved to contain ES-specific transcripts including EWS-FLI1, which were suitable for the sensitive detection of ES cell line-derived exosomes by qRT-PCRin a pre-clinical model for patient plasma. Microarray analysis of ES cell line-derived exosomes revealed that they share a common transcriptional signature potentially involved in G-protein-coupled signalling, neurotransmitter signalling and stemness. CONCLUSIONS: In summary, our results imply that ES-derived exosomes could eventually serve as biomarkers for minimal residual disease diagnostics in peripheral blood and prompt further investigation of their potential biological role in modification of the ES-associated microenvironment

[Associations with ADHD and parental distress with in play in early childhood]. / ADHS und das Ausmaß der elterlichen Stressbelastung bei mangelnder Spielfähigkeit im Säuglings- und Kleinkindalter.

OBJECTIVE: According to Papousek (2004), "disinterests in play" in early childhood is a possible early indicator of AD(H)D at school age. The phenomenon of disinterest in play is associated with psychological distress in parents and children as well as with disturbances in the parent-child-relationships Papousek, 2003. We investigated the association between disinterest in play in early childhood and AD(H)D and their associations with maternal distress. METHOD: Mothers of 35 children with AD(H)D [age: M (SD)= 9.0 (1.1) years; males: 30 (85.7 %)] and mothers of 42 children without AD(H)D [age: M (SD) = 8.8 (1.0) years; males: 35 (83.3 %)] gave their written informed consent to participate in the study. Using a questionnaire on the ability to play during the first 4 years of age, subjects were grouped into 39 children with lower interest in play and 38 children with higher interest in play. Maternal stress was assessed using a German adaptation of the Parenting Stress Index Abidin, 1995. RESULTS: The rate of AD(H)D and maternal stress was significantly higher in the group of children with lower interest in play. Mothers of children showing both AD(H)D and lower interest in play scored highest, mothers of children presenting neither behavior disorder scored lowest. CONCLUSIONS: The results agree with the assumption that disinterest in play in early childhood is associated with the development of AD(H)D and an additional stressor for mothers.

TCR-transgenic T cells and YB-1-based oncolytic virotherapy improve survival in a preclinical Ewing sarcoma xenograft mouse model.

Background: Ewing sarcoma (EwS) is an aggressive and highly metastatic bone and soft tissue tumor in pediatric patients and young adults. Cure rates are low when patients present with metastatic or relapsed disease. Therefore, innovative therapy approaches are urgently needed. Cellular- and oncolytic virus-based immunotherapies are on the rise for solid cancers. Methods: Here, we assess the combination of EwS tumor-associated antigen CHM1319-specific TCR-transgenic CD8+ T cells and the YB-1-driven (i.e. E1A13S-deleted) oncolytic adenovirus XVir-N-31 in vitro and in a xenograft mouse model for antitumor activity and immunostimulatory properties. Results: In vitro both approaches specifically kill EwS cell lines in a synergistic manner over controls. This effect was confirmed in vivo, with increased survival using the combination therapy. Further in vitro analyses of immunogenic cell death and antigen presentation confirmed immunostimulatory properties of virus-infected EwS tumor cells. As dendritic cell maturation was also increased by XVir-N-31, we observed superior proliferation of CHM1319-specific TCR-transgenic CD8+ T cells only in virus-tested conditions, emphasizing the superior immune-activating potential of XVir-N-31. Conclusion: Our data prove synergistic antitumor effects in vitro and superior tumor control in a preclinical xenograft setting. Combination strategies of EwS-redirected T cells and YB-1-driven virotherapy are a highly promising immunotherapeutic approach for EwS and warrant further evaluation in a clinical setting.

Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Correct mRNA processing at a mutant TT splice donor in FANCC ameliorates the clinical phenotype in patients and is enhanced by delivery of suppressor U1 snRNAs.

The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5'ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5'ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations.

Preemptive immunotherapy in childhood acute myeloid leukemia for patients showing evidence of mixed chimerism after allogeneic stem cell transplantation.

Previous studies have shown that children with acute myeloid leukemia (AML) who developed mixed chimerism (MC) were at high risk for relapse after allogeneic stem-cell transplantation (allo-SCT). We investigated the feasibility of intensified preemptive immunotherapy in children receiving allo-SCT for AML. Eighty-four children were registered in our trial from May 2005 to April 2009; of these, 71 fulfilled the inclusion criteria and were treated according to the study protocol. Serial and semiquantitative analyses of posttransplantation chimerism were performed. Defined immunotherapy approaches were considered in MC patients. Continuous complete chimerism (CC) was observed in 51 of 71 patients. MC was detected in 20 patients and was followed by immunotherapy in 13. Six of 13 MC patients returned to CC without toxicity and remained in long-term remission. Overall, the probability of event-free survival (pEFS) was 66% (95% confidence interval [95% CI] = 53%-76%) for all patients and 46% (95% CI = 19%-70%) in MC patients with intervention; however, this number increased to 71% (95% CI = 26%-92%) in 7 of 13 MC patients on immunotherapy who were in remission at the time of transplantation. All MC patients without intervention relapsed. These results suggest that MC is a prognostic factor for impending relapse in childhood AML, and that preemptive immunotherapy may improve the outcome in defined high-risk patients after transplantation.

A costimulatory chimeric antigen receptor targeting TROP2 enhances the cytotoxicity of NK cells expressing a T cell receptor reactive to human papillomavirus type 16 E7.

Immune cells modified to express a tumor-reactive T cell receptor (TCR) have shown limited efficacy as stand-alone therapy against solid tumors. Genital and oropharyngeal carcinomas induced by human papillomavirus (HPV) type 16 express constitutively its E6 and E7 oncoproteins, which makes them convenient targets for adoptive cell immunotherapy. However, viral antigen presentation by tumor cells is low and limits the anti-tumor efficacy of CD8+ T cells. To enhance the functionality of immune effector cells, we have devised a strategy combining a costimulatory chimeric antigen receptor (CAR) with a TCR. We used a clinically tested TCR specific to E7 (E7-TCR) of HPV16 and a newly constructed CAR targeting the trophoblast cell surface antigen 2 (TROP2), which carried the intracellular costimulatory domains CD28 and 4-1BB, but was devoid of the CD3ζ domain. Flow cytometry analyses showed a notable upregulation of activation markers and of cytolytic molecule release by NK-92 cells genetically engineered to express CD3, CD8 and both E7-TCR and TROP2-CAR, after co-incubation with HPV16+ cervical cancer cells. Furthermore, the E7-TCR/TROP2-CAR NK-92 cells demonstrated enhanced antigen-specific activation and augmented cytotoxicity against tumor cells compared with NK-92 cells expressing the E7-TCR alone. A costimulatory TROP2-CAR can synergistically cooperate with the E7-TCR in NK cells thereby enhancing their signaling strength and antigen-specific cytotoxicity. This approach might improve the outcome of adoptive cell immunotherapies for HPV16+ cancer patients that are currently under investigation.