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Hepatoblastoma is characterized by driver mutations in CTNNB1 , making it an attractive biomarker for a liquid biopsy approach utilizing circulating tumor DNA (ctDNA). This prospective observational study sought to ascertain the feasibility of ctDNA detection in patients with hepatoblastoma and explore its associations with established clinical indicators and biomarkers, including serum Alpha-fetoprotein (AFP). We obtained 38 plasma samples and 17 tumor samples from 20 patients with hepatoblastoma. These samples were collected at various stages: 10 at initial diagnosis, 17 during neoadjuvant chemotherapy, 6 post-operatively, and 5 at disease recurrence. Utilizing a bespoke sequencing assay we developed called QUENCH, we identified single nucleotide variants and deletions in CTNNB1 ctDNA. Our study demonstrated the capability to quantitate ctDNA down to a variant allele frequency of 0.3%, achieving a sensitivity of 90% for patients at initial diagnosis, and a specificity of 100% at the patient level. Notably, ctDNA positivity correlated with tumor burden, and ctDNA levels exhibited associations with macroscopic residual disease and treatment response. Our findings provide evidence for the utility of quantitative ctDNA detection in hepatoblastoma management. Given the distinct detection targets, ctDNA and AFP-based stratification and monitoring approaches could synergize to enhance clinical decision-making. Further research is needed to elucidate the interplay between ctDNA and AFP and determine the optimal clinical applications for both methods in risk stratification and residual disease detection.

Genetic histone variants have been implicated in cancer development and progression. Mutations affecting the histone 3 (H3) family, H3.1 (encoded by HIST1H3B and HIST1H3C ) and H3.3 (encoded by H3F3A ), are mainly associated with pediatric brain cancers. While considered poor prognostic brain cancer biomarkers in children, more recent studies have reported H3 alterations in adult brain cancer as well. Here, we established reliable droplet digital PCR based assays to detect three histone mutations (H3.3-K27M, H3.3-G34R, and H3.1-K27M) primarily linked to childhood brain cancer. We demonstrate the utility of our assays for sensitively detecting these mutations in cell-free DNA released from cultured diffuse intrinsic pontine glioma (DIPG) cells and in the cerebral spinal fluid of a pediatric patient with DIPG. We further screened tumor tissue DNA from 89 adult patients with glioma and 1 with diffuse hemispheric glioma from Southwestern Sydney, Australia, an ethnically diverse region, for these three mutations. No histone mutations were detected in adult glioma tissue, while H3.3-G34R presence was confirmed in the diffuse hemispheric glioma patient.

Abnormal molecular processes occurring throughout the genome leave distinct somatic mutational patterns termed mutational signatures. Exploring the associations between mutational signatures and clinicopathological features can unravel potential mechanisms driving tumorigenic processes. We analyzed whole genome sequencing (WGS) data of tumor and peripheral blood samples from 37 primary breast cancer (BC) patients receiving neoadjuvant chemotherapy. Comprehensive clinico-pathologic features were correlated with genomic profiles and mutational signatures. Somatic mutational landscapes were highly concordant with known BC data sets. Remarkably, we observed a divergence of dominant mutational signatures in association with BC subtype. Signature 5 was overrepresented in hormone receptor positive (HR+) patients, whereas triple-negative tumors mostly lacked Signature 5, but expectedly overrepresented Signature 3. We validated these findings in a large WGS data set of BC, demonstrating dominance of Signature 5 in HR+ patients, mostly in luminal A subtype. We further investigated the association between Signature 5 and gene expression signatures, and identified potential networks, likely related to estrogen regulation. Our results suggest that the yet elusive Signature 5 represents an alternative mechanism for mutation accumulation in HR+ BC, independent of the homologous recombination repair machinery related to Signature 3. This study provides theoretical basis for further elucidating the processes promoting hormonal breast carcinogenesis., (© 2022 The Authors. Molecular Carcinogenesis published by Wiley Periodicals LLC.)

Background: MYCN amplification (MNA), segmental chromosomal aberrations (SCA) and ALK activating mutations are biomarkers for risk-group stratification and for targeted therapeutics for neuroblastoma, both of which are currently assessed on tissue biopsy. Increase in demand for tumor genetic testing for neuroblastoma diagnosis is posing a challenge to current practice, as the small size of the core needle biopsies obtained are required for multiple molecular tests. We evaluated the utility of detecting these biomarkers in the circulation., Methods: Various pre-analytical conditions tested to optimize circulating-tumor DNA (ctDNA) copy number changes evaluations. Plasma samples from 10 patients diagnosed with neuroblastoma assessed for SCA and MNA using single nucleotide polymorphism (SNP) array approach currently used for neuroblastoma diagnosis, with MNA status assessed independently using digital-droplet PCR (ddPCR). Three patients (one in common with the previous 10) tested for ALK activating mutations p.F1174L and p.F1245I using ddPCR., Results: Copy number detection is highly affected by physical perturbations of the blood sample (mimicking suboptimal sample shipment), which could be overcome using specialized preservative collection tubes. Pre-analytical DNA repair procedures on ctDNA before SNP chromosome microarray processing improved the lower limit of detection for SCA and MNA, defined as 20% and 10%, respectively. We detected SCA in 10/10 (100%) patients using SNP array, 7 of which also presented MNA. Circulating-free DNA (cfDNA) and matched tumor DNA profiles were generally identical. MNA was detected using ddPCR in 7/7 (100%) of MNA and 0/12 (0%) non-MNA cases. MNA and ALK mutation dynamic change was assessed in longitudinal samples from 4 and 3 patients (one patient with both), respectively, accurately reflected response to treatment in 6/6 (100%) and disease recurrence in 5/6 (83%) of cases. Samples taken prior to targeted treatment with the ALK inhibitor Lorlatinib and 6-8 weeks on treatment showed reduction/increase in ALK variants according to response to treatment., Conclusions: These results demonstrate the feasibility of ctDNA profiling for molecular risk-stratification, and treatment monitoring in a clinically relevant time frame and the potential to reduce fresh tissue requirements currently embedded in the management of neuroblastoma.

Liquid biopsy is rapidly gaining traction for potentially revolutionizing cancer diagnosis and treatment through blood-based utilization of shed biomolecules. This approach can provide a global picture of the cancer in real time, at multiple time points, and with minimal invasiveness. In this review, we familiarize cancer biobanks with the principles used for liquid biopsy work and highlight unique aspects of applying liquid biopsy approaches to pediatric cancers to enable high-quality and efficient translational research.

Driver mutations in the CTNNB1 gene (encoding β-catenin) are a hallmark of sporadic hepatoblastoma (HBL). Our results show that CTNNB1 circulating tumour DNA (ctDNA) is readily detected in patients diagnosed with localised HBL, with serial sampling along the course of therapy and follow up providing a sensitive mechanism to monitor tumour dynamics and response to treatment. This exciting potential for CTNNB1 ctDNA to serve as a biomarker for treatment response in HBL holds clinical value, and requires assessment in a larger cohort of mixed tumour stages and recurrent disease., (© 2020 Wiley Periodicals LLC.)

In view of the relatively limited efficacy of immunotherapies targeting the PD-1-PD-L1 axis in triple-negative breast cancer (TNBC) and of published reports on tumor-promoting roles of TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs), we determined the incidence of TNFR2+ TILs in TNBC patient tumors, their association with disease outcome and relations with PD-1+ TILs. Using a cohort of treatment-naïve TNBC patients with long follow-up (n = 70), we determined the presence of TNFR2+ TILs and PD-1+ TILs by immunohistochemistry. TILs (≥ 1% of cellular mass) and TNFR2+ TILs (≥ 1% of total TILs) were detected in 96% and 74% of tumors, respectively. The presence of TILs at > 5% of tumor cell mass ("Positive TILs"), as well as of positive TNFR2+ TILs (> 5%), was independently associated with good prognosis, and combination of both parameters demonstrated superior outcome relative to their lower levels. PD1+ TILs (> 5/hot spot) were detected in 63% of patients. High levels of PD-1+ TILs (> 20/hot spot) showed an unfavorable disease outcome, and in their presence, the favorable outcome of positive TNFR2+ TILs was ablated. Thus, TNFR2+ TILs are strongly connected to improved prognosis in TNBC; these findings suggest that TNFR2+ TILs have favorable effects in TNBC patients, unlike the tumor-promoting roles attributed to them in other cancer systems. Overall, our observations propose that the TNFR2+ TIL subset should not be targeted in the course of TNBC therapy; rather, its beneficial impacts may become into power when anti-PD-1 regimens-that may potentiate immune activities-are administered to TNBC patients.

After the publication of the original article [1], we were notified the upper panel of the Fig. 1, where the patients' codes are listed, was cropped by mistake so the patients 1-8 are repeated.

Background: Emerging mutations in the ESR1 gene that encodes for the estrogen receptor (ER) are associated with resistance to endocrine therapy. ESR1 mutations rarely exist in primary tumors (~ 1%) but are relatively common (10-50%) in metastatic, endocrine therapy-resistant cancers and are associated with a shorter progression-free survival. Little is known about the incidence and clinical implication of these mutations in early recurrence events, such as local recurrences or newly diagnosed metastatic disease., Methods: We collected 130 archival tumor samples from 103 breast cancer patients treated with endocrine therapy prior to their local/metastatic recurrence. The cohort consisted of 41 patients having at least 1 sample from local/loco-regional recurrence and 62 patients with metastatic disease (of whom 41 newly diagnosed and 28 with advanced disease). The 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) were analyzed either by targeted sequencing or by droplet digital PCR. Progression-free survival (PFS), disease-free survival (DFS), and distant recurrence-free survival (DRFS) were statistically tested by Kaplan-Meier analysis., Results: The prevalence of ESR1 mutations was 5/41 (12%) in newly diagnosed metastatic patients and 5/28 (18%) for advanced metastases, detected at allele frequency > 1%. All mutations in advanced metastases were detected in patients previously treated with both tamoxifen (TAM) and aromatase inhibitors (AI). However, in newly diagnosed metastatic patients, 4/5 mutations occurred in patients treated with TAM alone. PFS on AI treatment in metastatic patients was significantly shorter for ESR1 mutation carriers (p = 0.017). In the local recurrence cohort, ESR1 mutations were identified in 15/41 (36%) patients but only 4/41 (10%) were detected at allele frequency > 1%. Again, most mutations (3/4) were detected under TAM monotherapy. Notably, 1 patient developed ESR1 mutation while on neoadjuvant endocrine therapy. DFS and DRFS were significantly shorter (p = 0.04 and p = 0.017, respectively) in patients that had ESR1 mutations (> 1%) in their loco-regional recurrence tumor., Conclusions: Clinically relevant ESR1 mutations are prevalent in newly diagnosed metastatic and local recurrence of endocrine-treated breast cancer. Since local recurrences are amenable to curative therapy, these mutations may inform the selection of subsequent endocrine therapies.

Purpose: Molecular evolution of tumors during progression, therapy, and metastasis is a major clinical challenge and the main reason for resistance to therapy. We hypothesized that microRNAs (miRNAs) that exhibit similar variation of expression through the course of disease in several patients have a significant function in the tumorigenic process., Experimental Design: Exploration of evolving disease by profiling 800 miRNA expression from serial samples of individual breast cancer patients at several time points: pretreatment, posttreatment, lymph nodes, and recurrence sites when available (58 unique samples from 19 patients). Using a dynamic approach for analysis, we identified expression modulation patterns and classified varying miRNAs into one of the eight possible temporal expression patterns., Results: The various patterns were found to be associated with different tumorigenic pathways. The dominant pattern identified an miRNA set that significantly differentiated between disease stages, and its pattern in each patient was also associated with response to therapy. These miRNAs were related to tumor proliferation and to the cell-cycle pathway, and their mRNA targets showed anticorrelated expression. Interestingly, the level of these miRNAs was lowest in matched recurrent samples from distant metastasis, indicating a gradual increase in proliferative potential through the course of disease. Finally, the average expression level of these miRNAs in the pretreatment biopsy was significantly different comparing patients experiencing recurrence to recurrence-free patients., Conclusions: Serial tumor sampling combined with analysis of temporal expression patterns enabled to pinpoint significant signatures characterizing breast cancer progression, associated with response to therapy and with risk of recurrence. Clin Cancer Res; 22(14); 3651-62. ©2016 AACR., (©2016 American Association for Cancer Research.)

The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UAS)ru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i) The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA) pathway, targeting the transcription factors (TFs), Com2 and Sko1; (ii) high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK) pathway and its corresponding TF Sko1; (iii) elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv) the nitrogen source repressed UASru activity through Sum1; and (v) the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis.