A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features.

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study.

PURPOSE: Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS: We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS: Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION: Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

Clinical significance of circulating tumor cells and cell-free DNA in pediatric rhabdomyosarcoma.

Liquid biopsy analysis represents a powerful and noninvasive tool to uncover biomarkers for disseminated disease assessment and longitudinal monitoring of patients. Herein, we explored the value of circulating and disseminated tumor cells (CTC and DTC, respectively) and cell-free DNA (cfDNA) in pediatric rhabdomyosarcoma (RMS). Peripheral blood and bone marrow samples were analyzed to detect and enumerate CTC and DTC, respectively. We used the epithelial cellular adhesion molecule (EpCAM)-based CellSearch platform coupled with an automatic device to collect both EpCAM-positive and EpCAM-low/negative CTCs. The standard assay was implemented, including the mesenchymal marker desmin. For selected cases, we molecularly profiled primary tumors and liquid biopsy biomarkers using whole-exome sequencing and droplet digital PCR, respectively. RMS patients with metastatic disease had a significantly higher number of CTCs compared to those with localized disease, whereas DTCs were detected independently of disease presentation. The use of the desmin marker remarkably increased the identification of CTCs and DTCs in RMS samples. Of note, CTC clusters were detected in RMS patients with disseminated disease. Further, cfDNA and CTC molecular features closely reflected the molecular makeup of primary tumors and informed of disease course.

Case Report: Circulating Tumor Cells as a Response Biomarker in ALK-Positive Metastatic Inflammatory Myofibroblastic Tumor.

Inflammatory myofibroblastic tumors (IMTs) are locally aggressive malignancies occurring at various sites. Surgery is the mainstay of treatment and prognosis is generally good. For children with unresectable or metastatic tumors, however, outcome is particularly severe, limited also by the lack of predictive biomarkers of therapy efficacy and disease progression. Blood represents a minimally invasive source of cancer biomarkers for real-time assessment of tumor growth, particularly when it involves the analysis of circulating tumor cells (CTC). As CTCs potentially represent disseminated disease, their detection in the blood correlates with the presence of metastatic lesions and may reflect tumor response to treatment. Herein, we present a case report of a 19-year-old boy with an ALK-positive IMT of the bladder, proximal osteolytic and multiple bilateral lung lesions, who received ALK inhibitor entrectinib postoperatively and underwent longitudinal CTC analysis during treatment. Antitumor activity of entrectinib was demonstrated and was accompanied by regression of lung lesions, elimination of CTCs from the blood and no development of relapses afterwards. Therapy continued without any clinical sign of progression and 24 months since the initiation of treatment the patient remains symptom-free and disease-free.

Circulating miR-26a as Potential Prognostic Biomarkers in Pediatric Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) arises from myogenic precursors that fail to complete muscle differentiation and represents the most frequent soft tissue sarcoma in children. Two major histological subtypes are recognized: alveolar RMS, characterized by a more aggressive behavior and a greater proneness to metastasis, and embryonal RMS which accounts for the 80% of cases and carries a better prognosis. Despite the survival of patients with localized tumors has progressively improved, RMS remains a challenging disease especially for metastatic patients and in case of progressive or recurrent disease after front-line therapy. MicroRNAs, a class of small non-coding RNA, have emerged as crucial players in cancer development and progression, and their detection in plasma (circulating miRNAs) represents a promising minimally invasive approach that deserve to be exploited in clinical practice. We evaluated the utility of circulating miRNAs as diagnostic and prognostic biomarkers in children with RMS profiling miRNAs from plasma of a small cohort of RMS patients and healthy donors (HD) using a qPCR Cancer Panel. An assessment of hemolysis status of plasma using miR-451/miR-23a ratio was performed as pre-analytical analysis. Statistical analysis revealed that miRNAs expression pattern clearly distinguished RMS patients from HD (p < 0.05). Interestingly, plasma levels of muscle-specific miR-206 were found to be significantly increased in RMS patients compared to HD, whereas levels of three potential tumor-suppressor miRNAs, miR-26a and miR-30b/30c, were found lower. Reduced levels of circulating miR-26a and miR-30b/c were further measured in an independent larger cohort of patients (validation set) by digital droplet PCR. In particular, we evidenced that miR-26a absolute plasma levels were associated with fusion status and adverse outcome (p < 0.05). Taken together, these findings demonstrate the potential of circulating miRNA as diagnostic and prognostic biomarker in children affected by this malignancy and enforced the key role of miR-26a in pediatric rhabdomyosarcoma.

Liquid Biopsy in Pediatric Renal Cancer: Stage I and Stage IV Cases Compared.

Pediatric renal cancer is rare, and robust evidence for treatment recommendations is lacking. In the perspective of personalized medicine, clinicians need new biomarkers to improve risk stratification and patients' follow-up. Herein, we analyzed some liquid biopsy tools, which have been never tested in pediatric renal cancer: namely, circulating tumor cells (CTCs); the expression of M30, an apoptosis marker, to test CTC metastatic potential; and c-MET expression in CTCs, because of its role in renal cancer progression and drug-resistance. Furthermore, we evaluated the Circulating Endothelial Cells (CECs), whose utility we previously demonstrated in adult metastatic renal cancer treated with anti-angiogenic therapy. We compared two renal cell carcinomas of clear-cell type, stage I and IV, which underwent surgery and surgery plus Sunitinib, respectively. Baseline CTC level and its changes during follow-up were consistent with patients' outcome. In case 2, stage IV, the analysis of CECs performed during Sunitinib revealed a late response to treatment consistent with poor outcome, as the finding of M30-negative, viable cells. Noteworthily, few CTCs were MET-positive in both cases. Our study highlights the feasibility for a change in the prognostic approach and follow-up of childhood renal cancer, with a view to guide a better treatment design.

Pediatric sarcomas display a variable EpCAM expression in a histology-dependent manner.

EpCAM is a transmembrane glycoprotein typically overexpressed in cancer of epithelial origin and mainly involved in the epithelial-to-mesenchymal transition (EMT) of tumor cells that spread and disseminate. Strategies for the targeting and capture of EpCAM-expressing tumor cells are showing promise in cancers prone to metastatize, both as diagnostic tools and potential therapies. Sarcomas are among the most aggressive tumors in children, with a common mesenchymal origin that comprises both soft tissue sarcomas (STS) and bone sarcomas. The aim of this study was to assess EpCAM expression in pediatric sarcomas and correlate its expression with disease progression. To do so, we analyzed a set of cell lines and primary tumor tissues from rhabdomyosarcoma (RMS), Ewing sarcoma (ES), synovial sarcoma (SS) and desmoplastic small round cell tumor (DSRCT) STS, or osteosarcoma (OS) bone cancer. We demonstrated that EpCAM was variably expressed in pediatric sarcomas, with DSRCT, a rare, aggressive and almost fatal tumor type, characterized by the highest EpCAM expression levels. Interestingly, although EpCAM expression was lower in RMS tumors, high levels at diagnosis correlated with reduced patients' overall survival (p < 0.05). Indeed, membrane-bound EpCAM was detected in circulating sarcoma tumor cells, revealing its potential to be used as dissemination biomarker in this type of childhood cancers. This reinforces the concept that pediatric sarcomas do express both epithelial and mesenchymal markers and reside in an intermediate condition that most likely contributes to their aggressive phenotype and low survival rate.

Secondary Osteosarcoma After Bone Marrow Transplant: An Aggressive Disease.

We describe three children who developed an osteosarcoma after receiving treatment for acute lymphoblastic leukemia, which included an allogeneic bone marrow transplant (BMT). We discuss the therapeutic options. None of the patients responded to conventional chemotherapy, but one patient given regorafenib showed a temporary response. We conclude that osteosarcoma after BMT has an aggressive course and it is worth further investigating multikinase inhibitors in this setting.

Prognostic Value of Circulating IGFBP2 and Related Autoantibodies in Children with Metastatic Rhabdomyosarcomas.

Insulin-like growth factor-binding protein 2 (IGFBP2) is a tumor-associated protein measurable in patients' biopsies and blood samples. Increased IGFBP2 expression correlates with tumor severity in rhabdomyosarcoma (RMS). Thus, we examined the plasmatic IGFBP2 levels in 114 RMS patients and 15 healthy controls by ELISA assay in order to evaluate its value as a plasma biomarker for RMS. Additionally, we looked for the presence of a humoral response against IGBFP2 protein measurable by the production of anti-IGFBP2 autoantibodies. We demonstrated that both circulating IGFBP2 protein and autoantibodies were significantly higher in RMS patients with respect to controls and their combination showed a better discriminative capacity. IGFBP2 protein identified metastatic patients with worse event-free survival, whereas both IGFBP2 and anti-IGFBP2 antibodies negatively correlated with overall survival. Our study suggests that IGFBP2 and anti-IGFBP2 antibodies are useful for diagnostic and prognostic purposes, mainly as independent negative prognostic markers in metastatic patients. This is the first study that reports a specific humoral response in RMS plasma samples and proves the value of blood-based biomarkers in improving risk assessment and outcome of metastatic RMS patients.

Rhabdomyosarcoma Cells Produce Their Own Extracellular Matrix With Minimal Involvement of Cancer-Associated Fibroblasts: A Preliminary Study.

BACKGROUND: The interplay between neoplastic cells and surrounding extracellular matrix (ECM) is one of the determinant elements for cancer growth. The remodeling of the ECM by cancer-associated fibroblasts (CAFs) shapes tumor microenvironment by depositing and digesting ECM proteins, hence promoting tumor growth and invasion. While for epithelial tumors CAFs are well characterized, little is known about the stroma composition of mesenchymal cancers, such as in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma during childhood and adolescence. The aim of this work is to identify the importance of CAFs in specifying RMS microenvironment and the role of these stromal cells in RMS growth. METHODS: We assessed in two dimensional (2D) and three dimensional (3D) systems the attraction between RMS cells and fibroblasts using epithelial colon cancer cell line as control. CAFs were studied in a xenogeneic mouse model of both tumor types and characterized in terms of fibroblast activation protein (FAP), mouse PDGFR expression, metalloproteases activation, and ECM gene and protein expression profiling. RESULTS: In 2D model, the rate of interaction between stromal and malignant cells was significantly lower in RMS with respect to colon cancer. Particularly, in 3D system, RMS spheroids tended to dismantle the compact aggregate when grown on the layer of stromal cells. In vivo, despite the well-formed tumor mass, murine CAFs were found in low percentage in RMS xenogeneic samples. CONCLUSIONS: Our findings support the evidence that, differently from epithelial cancers, RMS cells are directly involved in their own ECM remodeling, and less dependent on CAFs support for cancer cell growth.

Cell-Free DNA in Pediatric Rhabdomyosarcoma: Potential and Challenges.

Rhabdomyosarcoma is an aggressive solid tumor that may disseminate hematogenously giving metastasis which represents the most important prognostic factor. Chances of an effective cure in childhood cancer rely on the capacity to make an early and accurate diagnosis, detect metastatic disease or relapse, and predict the response to treatment.Liquid biopsy is a very promising blood test for cancer detection and noninvasive disease monitoring. This method has a great advantage to use blood and plasma, a more accessible biological material, quick and easy to obtain with minimal pain and risk for patients. In particular, circulating free DNA (cfDNA) represents a tumor biomarker detected in plasma that gives information on biology and genetic background of tumor.Moreover, cfDNA mutation detection could be a reliable method to monitor the efficacy of treatment and to discover novel targets for a personalized treatment in pediatric solid tumor. Here, we describe an optimized protocol to cfDNA isolation from small amount of plasma, as well as a method to assess the quantity and quality of cfDNA. Finally, we propose ddPCR as a reliable method to detect mutations at low frequency in cfDNA obtained from pediatric rhabdomyosarcoma samples.

NELL1, whose high expression correlates with negative outcomes, has different methylation patterns in alveolar and embryonal rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), which represents the most frequent soft tissue sarcoma in pediatric populations, is classified into two major subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS subtype, which shows greater aggressiveness and proneness to metastasis with respect to ERMS, are characterized, in about 75% of cases, by specific chromosomal translocations that involve PAX and FOXO1 genes. Many findings have demonstrated that PAX/FOXO1-positive ARMS have a worse prognosis than PAX/FOXO1-negative ones and that distinct molecular features characterize RMS with different gene fusion statuses. DNA methylation, which presently represents a challenging research area, is involved in the modulation of gene expression.We performed a genome-wide DNA methylation analysis using reduced-representation bisulfite sequencing (RRBS) in RMS samples and we found that fusion-positive alveolar and embryonal subgroups have different DNA methylation signatures and that ARMS fusion-positive subtypes are characterized by overall hypomethylation levels. While NELL1 was found to be hypomethylated and transcriptionally enhanced in RMS alveolar subtypes, high NELL1 expression levels, which proved to be correlated with negative RMS prognostic factors such as fusion status and histology (P < 0.0001), were found to discriminate between RMS patients with different outcomes (P < 0.05).In conclusion, our results demonstrated that different DNA methylation patterns distinguish between different RMS subgroups and they suggest that epigenetic signatures could be useful for risk stratification of patients.

MicroRNA-27a Contributes to Rhabdomyosarcoma Cell Proliferation by Suppressing RARA and RXRA.

BACKGROUND: Rhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: The expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA). CONCLUSIONS: Study results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.

High IGFBP2 expression correlates with tumor severity in pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor-binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR-negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS.

New miRNA labeling method for bead-based quantification.

BACKGROUND: microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies. RESULTS: Here we have applied with an innovative approach, the Luminex xMAP technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies. CONCLUSIONS: We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex xMAP technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.

Defining the gene expression signature of rhabdomyosarcoma by meta-analysis.

BACKGROUND: Rhabdomyosarcoma is a highly malignant soft tissue sarcoma in childhood and arises as a consequence of regulatory disruption of the growth and differentiation pathways of myogenic precursor cells. The pathogenic pathways involved in this tumor are mostly unknown and therefore a better characterization of RMS gene expression profile would represent a considerable advance. The availability of publicly available gene expression datasets have opened up new challenges especially for the integration of data generated by different research groups and different array platforms with the purpose of obtaining new insights on the biological process investigated. RESULTS: In this work we performed a meta-analysis on four microarray and two SAGE datasets of gene expression data on RMS in order to evaluate the degree of agreement of the biological results obtained by these different studies and to identify common regulatory pathways that could be responsible of tumor growth. Regulatory pathways and biological processes significantly enriched has been investigated and a list of differentially meta-profiles have been identified as possible candidate of aggressiveness of RMS. CONCLUSION: Our results point to a general down regulation of the energy production pathways, suggesting a hypoxic physiology for RMS cells. This result agrees with the high malignancy of RMS and with its resistance to most of the therapeutic treatments. In this context, different isoforms of the ANT gene have been consistently identified for the first time as differentially expressed in RMS. This gene is involved in anti-apoptotic processes when cells grow in low oxygen conditions. These new insights in the biological processes responsible of RMS growth and development demonstrate the effective advantage of the use of integrated analysis of gene expression studies.

Gene expression profiling identifies potential relevant genes in alveolar rhabdomyosarcoma pathogenesis and discriminates PAX3-FKHR positive and negative tumors.

We analyzed the expression signatures of 14 tumor biopsies from children affected by alveolar rhabdomyosarcoma (ARMS) to identify genes correlating to biological features of this tumor. Seven of these patients were positive for the PAX3-FKHR fusion gene and 7 were negative. We used a cDNA platform containing a large majority of probes derived from muscle tissues. The comparison of transcription profiles of tumor samples with fetal skeletal muscle identified 171 differentially expressed genes common to all ARMS patients. The functional classification analysis of altered genes led to the identification of a group of transcripts (LGALS1, BIN1) that may be relevant for the tumorigenic processes. The muscle-specific microarray platform was able to distinguish PAX3-FKHR positive and negative ARMS through the expression pattern of a limited number of genes (RAC1, CFL1, CCND1, IGFBP2) that might be biologically relevant for the different clinical behavior and aggressiveness of the 2 ARMS subtypes. Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR.

A leukemia-enriched cDNA microarray platform identifies new transcripts with relevance to the biology of pediatric acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: Microarray gene expression profiling has been widely applied to characterize hematologic malignancies, has attributed a molecular signature to leukemia subclasses and has allowed new subclasses to be distinguished. We set out to use microarray technology to identify novel genes relevant for leukemogenesis. To this end we used a unique leukemia-enriched cDNA microarray platform. DESIGN AND METHODS: The systematic sequencing of cDNA libraries of normal and leukemic bone marrow allowed us to increase the number of genes to yield a new release of a previously generated cDNA microarray. Using this platform we analyzed the expression profiles of 4,670 genes in bone marrow samples from 18 pediatric patients with acute lymphoblastic leukemia (ALL). RESULTS: Expression profiling consistently distinguished the leukemia patients into three groups, those with T-ALL, B-ALL and B-ALL with MLL/AF4 rearrangement, in agreement with the clinical classification. Our platform identified 30 genes that best discriminate these three subtypes. Using mini-array technology these 30 genes were validated in another cohort of 17 patients. In particular we identified two novel genes not previously reported: endomucin (EMCN) and ubiquitin specific protease 33 (USP33) that appear to be over-expressed in B-ALL relative to their expression in T-ALL. INTERPRETATION AND CONCLUSIONS: Microarray technology not only allows the distinction between disease subclasses but also offers a chance to identify new genes involved in leukemogenesis. Our approach of using a unique platform has proven to be fruitful in identifying new genes and we suggest exploration of other malignancies using this approach.