Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Discovery of Novel Potential Prognostic Markers and Targeted Therapy to Overcome Chemotherapy Resistance in an Advanced-Stage Wilms Tumor.

Wilms tumor (WT), the most prevalent type of renal cancer in children, exhibits overall survival rates exceeding 90%. However, chemotherapy resistance, which occurs in approximately 10% of WT cases, is a major challenge for the treatment of WT, particularly for advanced-stage patients. In this study, we aimed to discover potential mutation markers and drug targets associated with chemotherapy resistance in advanced-stage WT. We performed exome sequencing to detect somatic mutations and molecular targets in 43 WT samples, comprising 26 advanced-stage WTs, of which 7 cases were chemotherapy-resistant. Our analysis revealed four genes (ALPK2, C16orf96, PRKDC, and SVIL) that correlated with chemotherapy resistance and reduced disease-free survival in advanced-stage WT. Additionally, we identified driver mutations in 55 genes within the chemotherapy-resistant group, including 14 druggable cancer driver genes. Based on the mutation profiles of the resistant WT samples, we propose potential therapeutic strategies involving platinum-based agents, PARP inhibitors, and antibiotic/antineoplastic agents. Our findings provide insights into the genetic landscape of WT and offer potential avenues for targeted treatment, particularly for patients with chemotherapy resistance.

SARS-CoV-2 variant with the spike protein mutation F306L in the southern border provinces of Thailand.

The southernmost part of Thailand is a unique and culturally diverse region that has been greatly affected by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak during the coronavirus disease-2019 pandemic. To gain insights into this situation, we analyzed 1942 whole-genome sequences of SARS-CoV-2 obtained from the five southernmost provinces of Thailand between April 2021 and March 2022, together with those publicly available in the Global Initiative on Sharing All Influenza Data database. Our analysis revealed evidence for transboundary transmissions of the virus in and out of the five southernmost provinces during the study period, from both domestic and international sources. The most prevalent viral variant in our sequence dataset was the Delta B.1.617.2.85 variant, also known as the Delta AY.85 variant, with many samples carrying a non-synonymous mutation F306L in their spike protein. Protein-protein docking and binding interface analyses suggested that the mutation may enhance the binding between the spike protein and host cell receptor protein angiotensin-converting enzyme 2, and we found that the mutation was significantly associated with an increased fatality rate. This mutation has also been observed in other SARS-CoV-2 variants, suggesting that it is of particular interest and should be monitored.

Exome Sequencing Reveals Novel Germline Variants in Breast Cancer Patients in the Southernmost Region of Thailand.

Germline carriers of pathogenic variants in cancer susceptibility genes are at an increased risk of breast cancer (BC). We characterized germline variants in a cohort of 151 patients diagnosed with epithelial BC in the southernmost region of Thailand, where the predominant ethnicity differs from that of the rest of the country. Whole exome sequencing was used to identify and subsequently filter variants present in 26 genes known to be associated with cancer predisposition. Of the 151 individuals assessed, 23, corresponding to 15.2% of the sample, exhibited the presence of one or more pathogenic or likely pathogenic variants associated with BC susceptibility. We identified novel germline truncating variants in BRIP1, CHEK2, MSH6, PALB2, and PTEN and annotated variants of uncertain significance (VUSs), both novel and previously documented. Therefore, it is advisable to use genetic testing as an additional risk screening method for BC in this area.

Population-based prevalence study of common congenital malformations of the alimentary tract and abdominal wall in Thailand: a study using data from the National Health Security Office.

Background: The study aimed to estimate the prevalence of major congenital anomalies of the alimentary system and the abdominal wall in Thailand using a nationwide hospital discharge database from the National Health Security Office (2017-2020). Methods: The study extracted data from records with International Classification of Diseases-10 (ICD-10) codes related to esophageal malformation (ESO), congenital duodenal obstruction (CDO), jejunoileal atresia (INTES), Hirschsprung's disease (HSCR), anorectal malformation (ARM), abdominal wall defects (omphalocele (OMP) and gastroschisis (GAS)), and diaphragmatic hernia from the database with patient age selection set to less than 1 year. Results: A total of 2539 matched ICD-10 records were found in 2376 individuals over the 4-year study period. Concerning foregut anomalies, the prevalence of ESO was 0.88/10 000 births, while that of CDO was 0.54/10 000 births. The prevalence figures of INTES, HSCR, and ARM were 0.44, 4.69, and 2.57 cases per 10 000 births, respectively. For abdominal wall defects, the prevalences of OMP and GAS were 0.25 and 0.61 cases/10 000 births, respectively. The mortality in our cases was 7.1%, and survival analysis found that associated cardiac defects had a statistically significant influence on survival in most anomalies studied. In HSCR, both Down syndrome (DS) (hazard ratio (HR)=7.57, 95% confidence interval (CI)=4.12 to 13.91, p<0.001) and cardiac defects (HR=5.82, 95% CI=2.85 to 11.92, p<0.001) were significantly associated with poorer survival outcomes. However, only DS (adjusted HR=5.55, 95% CI=2.63 to 11.75, p<0.001) independently predicted worse outcomes by multivariable analysis. Conclusions: Our analysis of the hospital discharge database found that the prevalence of gastrointestinal anomalies in Thailand was lower than that reported in other countries, except for HSCR and anorectal malformations. Associated Down syndrome and cardiac defects influence the survival outcomes of these anomalies.

Prevalence of Pathogenic Germline Mutations in 13 Hereditary Cancer-Related Genes in Breast Cancer Patients in Narathiwat Province, Thailand.

BACKGROUND: BRCA1 and BRCA2 genes are known to increase breast cancer's lifetime risk. Early identification of women with this inherited risk can potentially reduce the risk of breast and/or ovarian cancer and, together with early screening, decrease the mortality rate. OBJECTIVE: This study explored the frequency and distribution of genetic variants in consecutive cases of breast cancer in Narathiwat province, one of the three provinces in the southernmost Thai border. MATERIAL & METHOD: A series of 64 consecutive breast cancer patients who underwent treatment in two general hospitals in the province during the period from the year 2021 to 2022. Genotyping studies were performed using a whole exome sequencing platform. Moderate to high penetrance variants recommended by the National Comprehensive Cancer Network (NCCN) guidelines 2022 (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, NF1, PALB2, PTEN, RAD51C, RAD51D, STK11, TP53) were annotated and filtered for pathogenic, likely pathogenic, or high-impact variants. RESULTS: Pathogenic germline variants were found in 8/64 cases (12.5%), namely BRCA1 in 3 (4.7%), BRCA2 in 4 (6.3%), ATM in 1 (1.6%), and PALB2 in 1 (1.6%). One patient had two concomitant germline mutations in BRCA2 and ATM. CONCLUSION: This is the first study on the frequency of germline mutations in BRCA1/2 and other breast cancer-predisposing genes in the southernmost provinces of Thailand. At least one pathogenic germline mutation was identified in 12.5% of the study patients, which suggests that genetic testing in this population has a high potential to provide benefits.

Correlation between image-defined risk factors and surgical complications in patients with neuroblastoma: a retrospective study.

BACKGROUND: Image-defined risk factor (IDRF) is a common tool used for neuroblastoma risk group classification. We speculated that anatomical evaluation by IDRF might be correlated with surgical complications and the oncologic outcome. Here, we investigated correlation between IDRF with outcomes of surgery of neuroblastoma patients. METHODS: Medical records and computed tomography images of neuroblastoma patients who underwent a surgery at Songklanagarind Hospital between 2002 and 2019 were retrospectively reviewed. IDRFs were analyzed for correlation with surgical complications, overall survival, progression-free survival and local recurrence within 2 years. RESULTS: Forty-five patients were enrolled in the study. Sixteen (35%) patients had low IDRF score at diagnosis (score ≤ 5). Other 29 (64%) patients had high IDRF score (score ≥ 6). High IDRF group significantly had higher incidence of organ injury and more intraoperative blood loss. At post-chemotherapy, high IDRF was not only associated with higher operative complications, but also associated with 2-year overall survival and progress-free survival. CONCLUSIONS: Neuroblastoma patients whose IDRF score, either at diagnosis or after neoadjuvant therapy, was 6 or higher had increased risk of surgical complication. This evidence prompts pediatric surgeons to prepare more for safe surgery in this group of patients.

A novel pathogenesis concept of biliary atresia approached by combined molecular strategies.

Cholestatic jaundice is one of the most common neonatal conditions. BA, a correctable cholangiopathy, presents with cholestatic jaundice within the first weeks of life. The inflammation of bile ducts leads to progressive fibrosclerosis involving biliary trees, followed by cirrhosis and liver failure. With the use of modern molecular studies, this research aimed to define a novel pathogenesis by exploring variations. We performed genetic discovery by using supervised and unsupervised approaches. Ultimately, a combination of genetic variations and survival data was analyzed to strengthen the novel concept in this study. In this study, coding regions were explored to identify rare deleterious variants within genes from the first analysis together with gene sets reported in PFIC, and diseases with hyperbilirubinemia. Our unsupervised prioritization was primarily designed to identify novel causal genes from nonsynonymous variants derived by three biostatistical algorithms: enrichment analysis, burden test, and trio study. Survival analysis was integratively evaluated with a combination of identified causal genes. The individuals with identified variants from the supervised approach were frequently related to the severity of cirrhosis and poor postoperative outcome. In the unsupervised approach, nonsynonymous variants were enriched. Cilium and muscle related pathways had a significant correlation. CCDC8 was statistically significant gene in which six cases carried mutations identified through burden analysis. Individuals who carried variants in corresponding genes and significant pathways had significantly lower native-liver survival than individuals in whom none of these variants were identified (log-rank p value 0.016). This study explored genetic variations by multiple strategies. Different pathways of cholestatic diseases have been found to be associated with BA. Therefore, BA may be characterized as a shared sequela of many cholestatic disorders. Susceptibility in those pathways suggested an association with BA and strengthened this proposed novel hypothesis. The results emphasized the consequences of many disruptive pathophysiologies.

An evaluation of the association between radiological parameters and survival outcomes in pediatric patients with hepatoblastoma.

BACKGROUND: We evaluated the survival outcomes following hepatic resection as a treatment modality in pediatric patients with hepatoblastoma at a single institution, and to identify radiological parameters associated with poorer survival outcomes. METHODS: This was a retrospective cohort study. Medical records were reviewed, pertaining to pediatric patients diagnosed with hepatoblastoma who underwent surgical resection at a university hospital in Thailand between 2004 and 2021. Radiological parameters, clinical factors, and pathological data were also collected. Survival analysis was performed, and prognostic factors were identified using logistic regression analysis. RESULTS: Forty-two suitable patients were identified. Three cases with incomplete data were excluded, resulting in 39 cases being analyzed. Except for two, all patients received preoperative chemotherapy following the Thai Pediatric Oncology Group regimen. The two- and five-year overall survival rates were 78.0% and 70.9%, respectively. Upon analysis, the radiological parameters associated with poorer survival were poor response to neoadjuvant chemotherapy, presence of metastasis, post-chemotherapy tumor diameter, Post treatment extent of disease (POSTTEXT) Stage IV disease, presence of portal vein involvement, and presence of residual disease; poor neoadjuvant-response, portal vein involvement, and metastasis were independently associated with worse outcomes. In patients with non-metastatic hepatoblastoma who had at least a 25% reduction in size following neoadjuvant chemotherapy, the 5-year survival rate was 90.9% (95% CI 50.8-98.6%). CONCLUSIONS: Although preoperative evaluation of the tumor extent staging did not significantly affect survival, portal vein involvement as per POSTTEXT staging, stable or increasing tumor size, and metastasis following neoadjuvant chemotherapy were associated with poor overall survival. LEVEL OF EVIDENCE: IIB.

Impact of EFEMP1 on the survival outcome of biliary atresia in Thai infants.

Genome-wide association studies (GWASs) have identified a genetic associated between EFEMP1 and biliary atresia (BA). To examine the susceptibility of single nucleotide polymorphisms (SNPs) in EFEMP1 in Thai BA patients, we performed an analysis of the genetic associations and biological interactions with previously reported key SNPs in ADD3, a key gene associated with BA. The study also used high-throughput sequencing to detect novel variants in both genes. In addition, the clinical impact of EFEMP1 SNPs in terms of survival association was also evaluated. The genotypes of 60 BA patients and 179 controls were evaluated using a TaqMan genotyping assay for rs2501577 and rs17095355 in ADD3 and rs6761893 and rs727878 in EFEMP1. The genotype frequencies were analyzed together with the SNP-SNP interactions. Fine mapping by whole-exome sequencing was performed to identify deleterious variants within both genes, and the survival analysis results were analyzed with the EFEMP1 SNPs. The recessive genotypes of rs2501577, rs17095355 and rs6761893 showed significantly higher frequencies in the BA patients than the controls, and a logistic regression showed that minor alleles of those SNPs increased the BA risk by ORs of 1.86, 1.67, and 1.84, respectively. Moreover, the SNP-SNP interference suggested that a combination of recessive alleles from the 2 genes resulted in an additive risk to BA. In addition, rare missense variants in the gene coding sequences were identified in 7 cases. Immunohistochemical studies revealed a pattern of ADD3 downregulation and EFEMP1 overexpression in the bile ducts of BA patients. Patients with the AA genotype of rs6761893 had significantly lower 5-year native liver survival (34.0%) than those with AT/TT (75.0%), with a log-rank p value of 0.041. Variants in EFEMP1 are associated with the occurrence of BA in Thai patients. In addition, these variants have an additive influence on BA risk when combined with ADD3 variants. Moreover, rs6761893 in EFEMP1 was indicative of survival in Thai BA patients.

Stuttering priapism in a pediatric patient with pheochromocytoma-induced thrombocytosis.

Priapism is an erection of more than 4 h without sexual stimulation. Ischemic priapism may lead to irreversible erectile dysfunction after a long-lasting period. Stuttering priapism is characterized by a pattern of recurrence that may progress to an unrelenting ischemic crisis, which is a urological emergency. Few reports have revealed that priapism is associated with essential thrombocythemia. The reactive thrombocytosis is uncommonly manifested by pheochromocytoma and rarely causes thrombotic events even if the platelet count is extremely high. We presented priapism related to reactive severe thrombocytosis in a 12-year-old male with pheochromocytoma. The cornerstone of care was prompt medical and surgical intervention by a multidisciplinary team approach to save life and preserve erectile function.

Suspected Cat-to-Human Transmission of SARS-CoV-2, Thailand, July-September 2021.

A veterinarian in Thailand was diagnosed with COVID-19 after being sneezed on by an infected cat owned by an infected patient. Genetic study supported the hypothesis of SARS-CoV-2 transmission from the owner to the cat, and then from the cat to the veterinarian.

Juvenile Hyaline Fibromatosis: Report of a Case with a Novel ANTXR2 Gene Mutation.

BACKGROUND Juvenile hyaline fibromatosis is a rare autosomal recessive disorder with unknown prevalence characterized by abnormal development of hyalinized fibrous tissue usually in the skin, mucosa, bone, and often the internal organs. Here, we report the case of a 7-year-old girl from a family with ANTXR2 mutation confirming JHF. CASE REPORT The girl presented with multiple painless soft-tissue swellings affecting the ears, forehead, and scalp. Excisional biopsies of the masses reported positive immunohistochemical staining for collagen type VI in the extracellular matrix area, which indicated collagen VI accumulation. Genetic analysis was performed using whole-exome sequencing. The variants were further validated using Sanger sequencing in trio-based approach. We identified a novel mutation, c.1273_1293delinsTCTTGTGGGTTTGGCT in exon 15 of ANTXR2 gene, leading to a frameshift of the amino acid from codon 425 to all the rest of the amino acid chain (p.Pro425Serfs). The change of an encoded protein interrupted lysosome-mediated degradation of collagen VI. This finding was compatible with her parents whose genetic tests were both positive for the same heterogenous deletion/insertion mutation. The patient was treated with surgical excision of the tumor masses, which had to be repeated several times due to recurrences. CONCLUSIONS This novel mutation in exon 15 of the ANTXR2 gene may help improve understanding of genotype-phenotype correlations for this syndrome and provide the basis for diagnostic testing. A multidisciplinary team approach including genetic molecular testing is required for an accurate diagnosis and management of JHF for conducting genetic counseling for affected families as a part of holistic management.

Fecal microbiome alterations in pediatric patients with short bowel syndrome receiving a rotating cycle of gastrointestinal prophylactic antibiotics.

BACKGROUND: Pediatric patients with short bowel syndrome (SBS) are at risk of developing small intestinal bacterial overgrowth (SIBO). Prevention of SIBO using cyclic enteric antibiotics has been implemented to control the balance in microbial ecosystems, although its effectiveness has not been well studied. PURPOSE: This study aimed to explore the change in the gut microbial composition in SBS patients during cyclic antibiotic phases and antibiotic-free period, and to compare the microbiota composition between healthy controls and SBS patients. METHOD: SBS patients taking oral metronidazole alternating with trimethoprim-sulfamethoxazole (TMP-SMT) and antibiotic-free conditions as a '10-day cyclic protocol' were involved in fecal microbiome study using Illumina 16S sequencing. RESULTS: When healthy control possessed the majority of Bacteroidetes spp. (54%) and Firmicutes spp. (33%), the microbial composition in SBS patients especially Firmicutes spp. and Proteobacteria spp. was prominently changed in each phase of treatment. In antibiotic-free period, SBS patients displayed 49% Firmicutes and 36% Proteobacteria. However, higher Proteobacteria than Firmicutes were detected at the commencement of metronidazole (58% versus 33%). Similarly, 56% Proteobacteria and 27% Firmicutes were found during TMP-SMT. Escherichia coli increased prominently during the antibiotic periods. CONCLUSION: Prophylactic antibiotics change the gut microbiota composition in an unfavorable direction, especially when repeatedly used for a long period. This practice should be reconsidered. LEVEL OF EVIDENCE: III.

Variants Associated with Infantile Cholestatic Syndromes Detected in Extrahepatic Biliary Atresia by Whole Exome Studies: A 20-Case Series from Thailand.

Biliary atresia (BA) is the most severe form of obstructive cholangiopathy occurring in infants. Definitive diagnosis of BA usually relies on operative findings together with supporting pathological patterns found in the extrahepatic bile duct. In infancy, overlapping clinical patterns of cholestasis can be found in other diseases including biliary hypoplasia and progressive familial intrahepatic cholestasis. In addition, BA has been reported as a phenotype in some rare genetic syndromes. Unlike BA, other cholangiopathic phenotypes have their own established genetic markers. In this study, we used these markers to look for other cholestasis entities in cases diagnosed with BA. DNA from 20 cases of BA, diagnosed by operative findings and histopathology, were subjected to a study of 19 genes associated with infantile cholestasis syndromes, using whole exome sequencing. Variant selection focused on those with allele frequencies in dbSNP150 of less than 0.01. All selected variants were verified by polymerase chain reaction-direct sequencing. Of the 20 cases studied, 13 rare variants were detected in 9 genes: 4 in JAG1 (Alagille syndrome), 2 in MYO5B (progressive familial intrahepatic cholestasis [PFIC] type 6), and one each in ABCC2 (Dubin-Johnson syndrome), ABCB11 (PFIC type 2), UG1A1 (Crigler-Najjar syndrome), MLL2 (Kabuki syndrome), RFX6 (Mitchell-Riley syndrome), ERCC4 (Fanconi anemia), and KCNH1 (Zimmermann-Laband syndrome). Genetic lesions associated with various cholestatic syndromes detected in cases diagnosed with BA raised the hypothesis that severe inflammatory cholangiopathy in BA may not be a distinct disease entity, but a shared pathology among several infantile cholestatic syndromes.

Single nucleotide polymorphisms within Adducin 3 and Adducin 3 antisense RNA1 genes are associated with biliary atresia in Thai infants.

BACKGROUND: A genome-wide association study in East Asians suggested a genetic association between biliary atresia (BA) and a cluster of variants within the Adducin 3 (ADD3) and ADD3 antisense RNA1 (ADD3-AS1) genes. Another study in Thai neonates reported an association between BA and rs17095355. To validate those findings, this study aimed to analyze the BA association with single nucleotide polymorphisms (SNPs) and the additive influence of ADD3 and ADD3-AS1 in Thai neonates. METHODS: DNAs from 56 BA cases and 166 controls were genotyped for rs2501577, rs11194981, rs12268910 (ADD3) and rs17095355 (ADD3-AS1), using TaqMan PCR. Genotype distributions were compared between the groups, and SNP-SNP interactions were analyzed by combination of allelotypes. RESULTS: The risk allele frequencies of rs2501577, rs11194981, and rs17095355 in the BA group were significantly higher than in the controls. Univariate analysis showed that recessive variants in the three SNPs were associated with BA risk at ORs of 1.81 (95% CI 1.32-2.50), 1.58 (95% CI 1.14-2.20) and 1.92 (95% CI 1.39-2.66), respectively. SNP-SNP interaction analysis showed that the SNP combination of the two genes rs17095355 and rs2501577 provided an additive increase in BA risk. CONCLUSION: ADD3 and ADD3-AS1 variants increased susceptibility to BA, suggesting that these genes may play an additive role in the pathogenesis of the disease. In addition, these interactions may give a clue to the overexpression of the ADD3 protein in the liver of BA patients.