Correction: The association between vitamin D receptor polymorphism and phases of chronic hepatitis B infection in HBV carriers in Thailand.

[This corrects the article DOI: 10.1371/journal.pone.0277907.].

Detection of EGFR T790M mutation using liquid biopsy for non-small cell lung cancer: Utility of droplet digital polymerase chain reaction vs. cobas real-time polymerase chain reaction.

BACKGROUND: Digital platforms for mutation detection yield higher sensitivity than non-digital platforms but lack universal positive cut-off values that correlate with the outcome of osimertinib treatment. This study determined compared droplet digital polymerase chain reaction (ddPCR) to the standard cobas assay for epithelial growth factor receptor (EGFR) T790M mutation detection in patients with non-small cell lung cancer. METHODS: Study patients had EGFR-mutant tumours with disease progression on first/second generation EGFR tyrosine kinase inhibitors, and osimertinib treatment after T790M mutation detection. T790M status was tested by cobas assay using liquid biopsy, and only by ddPCR if an EGFR mutation was identified but T790M was negative. Clinical efficacy of osimertinib was compared between patients with T790M detected by cobas vs. only by ddPCR. A positive cut-off value for ddPCR was determined by assessing efficacy with osimertinib. RESULTS: 61 patients had tumors with an acquired T790M mutation, 38 detected by cobas and an additional 23 only by ddPCR. The median progression-free survival (PFS) for the cobas- and ddPCR-positive groups was 9.5 and 7.8 months, respectively (p=0.43). For ddPCR, a fractional abundance (FA) of 0.1% was used as a cut-off value. The median PFS of patients with FA ≥0.1% and <0.1% was 8.3 and 4.6 months, respectively (p=0.08). FA ≥0.1% was independently associated with a longer PFS. CONCLUSION: Using ddPCR to follow up the cobas assay yielded more cases (38% of total) with a T790M mutation. A cut-off value of FA ≥0.1% identified patients who responded as well to osimertinib as those identified by cobas assay. This sequential approach should detect additional patients who might benefit from osimertinib treatment.

Tissue and Plasma-Based Highly Sensitive Blocker Displacement Amplicon Nanopore Sequencing for EGFR Mutations in Lung Cancer.

PURPOSE: The epidermal growth factor receptor (EGFR) mutation is a widely prevalent oncogene driver in non-small cell lung cancer (NSCLC) in East Asia. The detection of EGFR mutations is a standard biomarker test performed routinely in patients with NSCLC for the selection of targeted therapy. Here, our objective was to develop a portable new technique for detecting EGFR (19Del, T790M, and L858R) mutations based on Nanopore sequencing. MATERIALS AND METHODS: The assay employed a blocker displacement amplification (BDA)-based polymerase chain reaction (PCR) technique combined with Nanopore sequencing to detect EGFR mutations. Mutant and wild-type EGFR clones were generated from DNA from H1650 (19Del heterozygous) and H1975 (T790M and L858R heterozygous) lung cancer cell lines. Then, they were mixed to assess the performance of this technique for detecting low variant allele frequencies (VAFs). Subsequently, formalin-fixed, paraffin-embedded (FFPE) tissue and cell-free DNA (cfDNA) from patients with NSCLC were used for clinical validation. RESULTS: The assay can detect low VAF at 0.5% mutant mixed in wild-type EGFR. Using FFPE DNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and Cobas real-time PCR were 98.46%, 100%, and 100%, respectively. For cfDNA, the concordance rates of EGFR 19Del, T790M, and L858R mutations between our method and droplet digital PCR were 94.74%, 100%, and 100%, respectively. CONCLUSION: The BDA amplicon Nanopore sequencing is a highly accurate and sensitive method for the detection of EGFR mutations in clinical specimens.

Molecular-based classification of endometrial carcinoma in Northern Thailand: impact on prognosis and potential for implementation in resource-limited settings.

BACKGROUND: Endometrial carcinoma is molecularly categorized into four subgroups: polymerase-E exonuclease domain-mutant (POLE-mut), mismatch repair-deficient (MMR-d), p53-abnormal (p53-abn), and no specific molecular profile (NSMP). This classification scheme has been included into clinical recommendation for post-operative risk-based management, although there have been few Asian studies on this topic. The present study aimed to evaluate the prevalence and clinical outcomes of endometrial carcinoma using this classification in Northern Thailand and the feasibility of implementation in resource-limited settings. METHODS: Endometrial carcinomas from hysterectomy specimens were classified using immunohistochemistry for MMR proteins and p53, as well as POLE mutation testing. Clinicopathological variables and outcomes were analyzed. The costs of the molecular information-based approach were compared to those incurred by the conventional approach (without molecular classification). RESULTS: Of 138 patients, 52.9% in the NSMP subgroup, 28.2% were in the MMR-d, 13.8% in the p53-abn, and 5.1% in the POLE-mut. After adjusting for other variables, patients with POLE-mut showed the most favorable outcomes, while those with p53-abn had the poorest survival. When estimating the costs for post-operative management, the use of molecular classification resulted in a 10% increase over the conventional approach. However, the cost increased only by 1% if only POLE testing was used to identify patients for treatment omission. CONCLUSION: In Northern Thailand, endometrial carcinoma had comparable subgroup distribution and prognostic implications to previous reports, supporting the implementation of management guidelines that incorporate molecular information. In resource-limited settings, at least POLE mutation testing in early-stage patients should be considered.

Glioblastoma with novel EGFR mutations (T790M and exon 20 insertion) yet unresponsive to osimertinib: A case report.

Glioblastoma (GBM) is a high-grade adult-type IDH-wildtype diffuse glioma, commonly harboring epidermal growth factor receptor (EGFR) amplification. Here, we describe a case of a 49-year-old man with a GBM harboring a TERT promoter mutation. Despite surgical and chemoradiation therapy, the tumor recurred. At that time, comprehensive genomic profiling by next-generation sequencing identified two rare mutations in EGFR: T790M and an exon 20 insertion. Based on these findings, the patient elected to undergo off-label therapy with osimertinib, a third-generation EGFR tyrosine kinase inhibitor that has shown promising results in non-small cell lung carcinoma, including metastatic to brain, with exactly the same EGFR mutations. Moreover, the drug has excellent central nervous system penetration. Even so, no clinical response was observed, and the patient succumbed to the disease. The lack of response may be related to the specific nature of the EGFR mutations, and/or other unfavorable tumor biology overriding any benefit from osimertinib.

The association between vitamin D receptor polymorphism and phases of chronic hepatitis B infection in HBV carriers in Thailand.

Vitamin D receptor (VDR) polymorphism partly regulates the immune system and is associated with hepatic flare in chronic Hepatitis B virus infection (HBV). Our study identified the association between two distinct phases, VDR polymorphisms and HBV inactive carrier (IC) and chronic hepatitis (CH). Chronic HBV patients were enrolled from February to August 2020. An HBV viral load (VL) < 2,000 IU/ml twice for 6 months apart, with no prior history of HBV treatment, defined the IC phase. Six common polymorphisms in the VDR gene, including CdX-2, GATA, FokI, Bsml, ApaI, and TaqI, were studied using real-time PCR. The different outcomes in allele, genotype, and haplotype frequencies in between groups and linkage disequilibrium (LD) mapping were analyzed. Among 324 enrolled patients, there were 163 patients in IC and 161 patients in CH phases. The mean vitamin D levels were not statistically different between groups. The proportion of allele frequencies of CdX-2 in IC and CH was 53.7% and 62.7% for G allele, and 46.3% and 37.3% for A allele (p 0.019). The proportion of GG genotype of CdX-2 was less frequently found in patients with IC compared to that in patients with CH (27% vs 41%, p 0.028). By multivariate analysis, CdX-2 G/A genotypes were independently associated with IC, with adjusted odd ratio (OR) 1.83 (1.10-3.04), p 0.019. The LD mapping of single nucleotide polymorphisms (SNP) revealed high LD scores in Bsml/ApaI/TaqI (BAT) haplotype in both groups while, CdX-2/GATA and GATA/FokI demonstrated high LD score only in CH group. CdX-2 G/A genotypes were independently associated with IC status in Thai patients with chronic HBV infection. The difference in LD of the CdX-2/GATA and GATA/FokI haplotypes in between groups may represent a non-random selection resulting in the variation of immune control.

Identification of Shared Neoantigens in BRCA1-Related Breast Cancer.

Personalized neoantigen-based cancer vaccines have been shown to be safe and immunogenic in cancer patients; however, the manufacturing process can be costly and bring about delays in treatment. Using off-the-shelf cancer vaccines targeting shared neoantigens may circumvent these problems. Unique mutational signatures and similar phenotypes found among BRCA1-mutated breast cancer make it an ideal candidate for discovering shared neoantigens within the group. We obtained genome sequencing data of breast cancer samples with or without somatic BRCA1 mutations (BRCA1-positive and BRCA1-negative, respectively) from the three public cancer databases; The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Catalogue of Somatic Mutations in Cancer (COSMIC); and from three studies with whole genome/exome sequencing data of samples with germline BRCA1 mutations. Data were analyzed separately within the same database/cohort. We found PIK3CA H1047R, E545K, E542K, and N345K recurrently in BRCA1-negative groups across all databases, whereas recurrent somatic mutations in BRCA1-positive groups were discordant among databases. For germline BRCA1-mutated breast cancer, TP53 R175H was unanimously the most frequent mutation among the three germline cohorts. Our study provides lists of potential shared neoantigens among BRCA1-related breast cancer, which may be used in developing off-the-shelf neoantigen-based vaccines.

KRAS Mutation in Pediatric Intracranial Germ Cell Tumors.

BACKGROUND: Intracranial germ cell tumors (IGCTs) are rare, highly curable neoplasms. KRAS is a gene in the KIT/RAS signaling pathway, and KRAS mutations have been reported in patients diagnosed with IGCTs. OBJECTIVES: To describe the clinicopathologic and molecular features of KRAS mutation and the treatment outcome of children diagnosed with IGCTs. METHODS: Patients diagnosed with IGCTs at the Department of Pediatrics, King Chulalongkorn Memorial Hospital from 2007 to 2016 were retrospectively reviewed. DNA was extracted from formalin-fixed, paraffin-embedded tissue and used for molecular study. Mutations in codons 12, 13, and 61 of the KRAS gene were detected using the cobas® KRAS mutation test and pyrosequencing. RESULTS: Eighteen patients were diagnosed with IGCTs (11 males and 7 females): nine with germinomas and nine with non-germinomatous GCTs (NGGCTs). The age range of the patients was 5-14 years (median 10.5 years). Elevated markers were revealed in approximately 25% of the patients. Four patients (two with germinomas and two with NGGCTs) had leptomeningeal involvement. All patients underwent tumor biopsy and received neoadjuvant chemotherapy. Radiotherapy was administered in 16 patients, and craniospinal radiation was administered only in patients with leptomeningeal metastasis. With a median follow-up of 26 months, overall survival was 88.9% in the patients with germinomas and 37% in the patients with NGGCTs. Mutation of the KRAS gene was detected using pyrosequencing in one patient. The mutation located at codon 61, with frequency 38.3% units, nucleotide substitution CAA > CTA, and amino acid substitution, was Q61L. The patient carrying the mutant gene was diagnosed with germinoma with cerebrospinal fluid metastasis and eventually died from treatment-related toxicity. CONCLUSION: Our study revealed the treatment outcomes of IGCTs in Thai children. The metastatic germinoma patient with KRAS codon 61 mutation had a poor outcome, supporting that Q61L has a clinical correlation with IGCTs.

Prognostic factors in completely resected lymph-node-negative pulmonary adenocarcinoma.

Background: Lymph node involvement is one of the important prognostic factors for early-stage lung cancer. However, in lymph node-negative (N0) lung cancer the recurrent rate may be as high as 30%. We aimed to study potential prognostic factors including clinicopathological factors and epidermal growth factor receptor (EGFR) mutation status in this lung cancer population. Methods: We retrospectively reviewed the medical records and pathological examinations of patients with completely resected N0 pulmonary adenocarcinoma treated in our institute between 2009 and 2016. We used Cobas® test to determine EGFR mutation status. Recurrence-free survival (RFS) was analyzed by univariable and multivariable Cox regression analyses. Results: We recruited 220 patients with median duration of follow up 5 years. Majority of these patients were in stage I (80%) and did not receive adjuvant therapy (86%). There were 53% with EGFR mutations which comprised of exon 19 deletion 51% and L858R 43%. Recurrence occurred in 64 out of 220 patients (29%). The median time to recurrence was 2.1 years. Statistically significant prognostic factors in both univariate and multivariate analyses included tumor size ≥4 centimeter (cm) (HR: 1.94; 95% CI: 1.03-3.67), visceral pleural invasion (HR: 2.53; 95% CI: 1.34-4.79), tumor necrosis (HR: 2.45; 95% CI: 1.13-5.31) and bronchial resection margin <2 cm (HR: 1.96; 95% CI: 1.10-3.51). However, presence of sensitizing EGFR mutation was not found to be a significant prognostic factor (HR: 1.20; 95% CI: 0.66-2.18; P=0.56). Conclusions: In N0 surgically resected lung adenocarcinoma, there were significant pathological prognostic factors including tumor 4 cm or more, visceral pleural invasion, tumor necrosis and bronchial resection margin less than 2 cm. Mutation of EGFR is not a significant prognostic factor to determine the risk of recurrence in this population and their risks shall be determined by the other poor prognostic factors.

Pediatric fibromyxoid tumor with PLAG1 fusion: An emerging entity with a novel intracranial location.

Translocations involving PLAG1 occur in several tumors, most commonly pleomorphic adenoma and lipoblastoma. Recently, a distinctive soft tissue tumor with a PLAG1 fusion has been reported in the pediatric age group. These are low grade tumors with a fibroblastic or mixed fibroblastic and myxoid morphology but no other lines of differentiation. They are typically immunopositive for desmin and CD34. The partner genes for these tumors have included YWHAZ, EEF1A1, ZFHX4l, CHCHD7, and PCMTD1. We report another case of this fibromyxoid tumor with a PLAG1 fusion, this time with COL3A1 as the partner gene. The fusion placed expression of a full-length PLAG1 protein under the control of the constitutively active COL3A1 promoter. Overexpression of PLAG1 was confirmed by diffusely positive immunostaining for PLAG1. The most novel aspect of this tumor is the intracranial location. Opinion has been divided over whether these tumors are a specific entity, or related to lipoblastoma, since that tumor also typically occurs in soft tissue in the pediatric age group and shows many of the same gene fusions. However, lipoblastoma has never been reported in an intracranial location and, thus, our case provides compelling evidence that this fibromyxoid tumor is indeed a distinct entity.