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.

Co-occurrence CDK4/6 amplification serves as biomarkers of de novo EGFR TKI resistance in sensitizing EGFR mutation non-small cell lung cancer.

Despite the development of predictive biomarkers to shape treatment paradigms and outcomes, de novo EGFR TKI resistance advanced non-small cell lung cancer (NSCLC) remains an issue of concern. We explored clinical factors in 332 advanced NSCLC who received EGFR TKI and molecular characteristics through 65 whole exome sequencing of various EGFR TKI responses including; de novo (progression within 3 months), intermediate response (IRs) and long-term response (LTRs) (durability > 2 years). Uncommon EGFR mutation subtypes were significantly variable enriched in de novo resistance. The remaining sensitizing EGFR mutation subtypes (exon 19 del and L858R) accounted for 75% of de novo resistance. Genomic landscape analysis was conducted, focusing in 10 frequent oncogenic signaling pathways with functional contributions; cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGF-ß, p53 and ß-catenin/Wnt signaling. Cell cycle pathway was the only significant alteration pathway among groups with the FDR p-value of 6 × 10-4. We found only significant q-values of < 0.05 in 7 gene alterations; CDK6, CCNE1, CDK4, CCND3, MET, FGFR4 and HRAS which enrich in de novo resistance [range 36-73%] compared to IRs/LTRs [range 4-22%]. Amplification of CDK4/6 was significant in de novo resistance, contrary to IRs and LTRs (91%, 27.9% and 0%, respectively). The presence of co-occurrence CDK4/6 amplification correlated with poor disease outcome with HR of progression-free survival of 3.63 [95% CI 1.80-7.31, p-value < 0.001]. The presence of CDK4/6 amplification in pretreatment specimen serves as a predictive biomarker for de novo resistance in sensitizing EGFR mutation.

MicroRNA Expression Profiling in Hydatidiform Mole for the Prediction of Postmolar GTN : MicroRNA Profile in Postmolar GTN.

Objectives: The primary aim of the study was to identify miRNAs that were differentially expressed between complete hydatidiform moles (CHMs) that turned out to be gestational trophoblastic neoplasia (GTN) [GTN moles] and CHMs that regressed spontaneously after evacuation [remission moles]. The secondary aim was to study the profiles of miRNA expressions in CHMs. Methods: A case-control study was conducted on GTN moles and remission moles. We quantitatively assessed the expression of 800 human miRNAs from molar tissues using Nanostring nCounter. Results: From a pilot study, 21 miRNAs were significantly downregulated in GTN moles compared to the remission moles. Five of them (miR-566, miR-608, miR-1226-3p, miR-548ar-3p and miR-514a-3p) were downregulated for >4 folds. MiR-608 was selected as a candidate for further analysis on 18 CHMs (9 remission moles and 9 GTN moles) due to its striking association with malignant formation. MiR-608 expression was slightly lower in GTN moles compared to the remission moles, that is, 2.22 folds change [p = 0.063]. Conclusion: We identified 21 miRNAs that were differentially expressed between GTN moles and remission moles suggesting that miRNA profiles can distinguish between the two groups. Although not reaching statistically significant, miR-608 expression was slightly lower in GTN moles compared to remission moles.

Prevalence of Tissue BRCA Gene Mutation in Ovarian, Fallopian Tube, and Primary Peritoneal Cancers: A Multi-Institutional Study.

BACKGROUND AND OBJECTIVE: Ovarian, fallopian tube, or primary peritoneal cancer patients with BRCA gene mutation have enhanced sensitivity to platinum-based regimens and PARP inhibitors. However, the knowledge regarding BRCA mutation in Thai patients is limited. This study aimed at identifying the prevalence and characteristics of somatic and germline BRCA 1 and 2 mutations in Thai patients with these cancers. MATERIALS AND METHODS: The paraffin blocks of tumors with histology of high grade serous, high grade endometrioid, or clear cell carcinoma obtained between June 2016 and December 2017 were analyzedto evaluate BRCA mutation using next-generation sequencing system. Blood or normal tissue paraffin blocks of positive patients were further tested for germline BRCA mutation. RESULTS: Tissue paraffin blocks of 178 patients were collected but only 139 were analyzed. Positive BRCA mutation was identified in 24 patients (17.3%): BRCA1 in 13 cases, BRCA2 in 10 cases, and BRCA1 and 2 in the rest one. Germline mutation study in blood or normal tissue in 23 positive patients revealed BRCA mutation in 14 cases, BRCA1 in 8 cases and BRCA 2  in 6 cases. Overall, the prevalence of somatic and germline mutation was 6.5% (9 out of 138 patients) and 8.7% (14 out of 138 patients), respectively. The most common histology associated with BRCA mutation was high grade serous cancer (27.3%). No significant difference was found between patients with or without BRCA mutation in terms of stage, outcome, platinum status, and survival outcome. CONCLUSION: BRCA mutation was demonstrated in less than 10% of Thai ovarian cancer patients. Higher rate of mutation was found in high grade serous cancer.
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Simplified Molecular Subtyping of Medulloblastoma for Reduced Cost and Improved Turnaround Time.

Molecular subtyping of medulloblastoma (MB) has become increasingly important for prognosis and management. Typically this involves detailed molecular genetic testing which may not be available in all centers. The purpose of the present study was to find a simplified approach to assign molecular subtypes of MB for routine use in centers with more limited resources. The molecular subtypes of MBs from 32 Thai patients, aged 0.5 to 35 years, were first determined by NanoString. These results were then compared with those obtained using a combination of limited immunohistochemistry (IHC) (ß-catenin, GAB-1, YAP-1, p75-NGFR, OTX2) and CTNNTB exon 3 mutation analysis. By NanoString assay, there were 6 MBs (19%) in the wingless (WNT) group, 8 (25%) in the sonic hedgehog (SHH) group, 7 (22%) in group 3, and 11 (34%) in group 4. Although ß-catenin immunostaining missed 4/6 WNT MBs, CTNNTB mutation analysis confirmed all WNT MB cases with amplifiable DNA. The IHC panel correctly assigned all the other molecular subtypes, except for 1 MB in group 4. Thus, our protocol was able to correctly categorized 31/32 cases or 97% of cases. Our study is the first to report molecular subtypes of MB in Southeast Asia. We found that molecular subgroups of MBs can be reliably assigned using a limited IHC panel of ß-catenin, GAB-1, YAP-1, p75-NGFR, OTX2, together with CTNNTB exon 3 mutation analysis. This simplified approach incurs lower cost and faster turnaround time compared with more elaborate molecular methodologies and should be beneficial to centers with reduced laboratory resources.

Genetic Characterization of Exotic Commercial Honey Bee (Hymenoptera: Apidae) Populations in Thailand Reveals High Genetic Diversity and Low Population Substructure.

Domestication of animal species is often associated with a reduction in genetic diversity. The honey bee, Apis mellifera Linnaeus, 1758, has been managed by beekeepers for millennia for both honey and wax production and for crop pollination. Here we use both microsatellite markers and sequence data from the mitochondrial COI gene to evaluate genetic variation of managed A. mellifera in Thailand, where the species is introduced. Microsatellite analysis revealed high average genetic diversity with expected heterozygosities ranging from 0.620 ± 0.184 to 0.734 ± 0.071 per locus per province. Observed heterozygosities were generally lower than those expected under Hardy-Weinberg equilibrium, both locally and across the population as a whole. Mitochondrial sequencing revealed that the frequency of two evolutionary linages (C-Eastern European and O-Middle Eastern) are similar to those observed in a previous survey 10 yr ago. Our results suggest that Thai beekeepers are managing their A. mellifera in ways that retain overall genetic diversity, but reduce genetic diversity between apiaries.

Germline mutations in Thai patients with nonmucinous epithelial ovarian cancer.

BACKGROUND: Genetic testing is widely recommended for all epithelial ovarian cancer (EOC) patients. However, an increased probability of identifying germline mutations has been reported in selected patients with risk factors such as a family history or personal history of cancer and high-grade serous carcinoma (HGSC) subtype. HGSC has been reported to be the most common subtype of EOC worldwide (approximately 70%). However, this subtype is less prevalent in Thai patients (reported as only 20%). The difference in the distribution of various subtypes of EOC may reflect the incidence of germline mutations in Thai EOC patients. AIM: To evaluate the frequencies of germline mutations in EOC patients and to compare the frequencies in those with and without clinical risk factors for hereditary ovarian cancer. METHODS: This cross-sectional study included 112 nonmucinous EOC patients who underwent primary surgery at our tertiary care hospital. Clinical risk factors for hereditary ovarian cancer were defined as follows: Age below 40 years, a significant family history of cancer, synchronous ovarian and endometrial cancer, and HGSC. Comprehensive germline mutations were detected by next-generation sequencing. RESULTS: Of a total of 112 patients, 82 (73.2%) patients had ≥ 1 risk factor and 30 (26.8%) patients had no risk factors. Germline mutations were detected in 26 patients: 20 (17.8%) patients had BRCA1/2 mutations, but 6 (5.4%) patients had mutations in other genes, including 1 in MLH1, 1 in MSH2, 1 in RAD51C, 2 in ATM and 1 in CDH1. Germline mutations were only detected in patients with risk factors (26 of 82, 31.7%), not in patients without risk factors (P < 0.001). A significant family history of cancer and HGSC were the only two significant risk factors associated with a higher proportion of germline mutations (56.3% vs 10% for those with and without a history of cancer, respectively, 40.8% vs 9.5% for those with and without HGSC). Germline BRCA mutations were detected in 38.8% of patients with HGSC but in only 1.6% of those with non-HGSC. An age below 40 years, personal history of breast cancer, and synchronous ovarian and endometrial cancer were not significant factors (14.3% vs 23.5%, 33.3% vs 21%, 22.2% vs 22.3%). CONCLUSION: Approximately one-third of EOC patients with risk factors had germline mutations. Almost all germline BRCA mutations were found in patients with the HGSC subtype. Selected patients with HGSC and a family history of cancer should be initially considered for genetic analysis in Thailand.

Immunohistochemistry for ATRX Can Miss ATRX Mutations: Lessons From Neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood with a median age of presentation of 17 months. A common theme in high-risk neuroblastoma is maintenance of telomeres, one mechanism for which involves alternate lengthening of telomeres (ALT) associated with ATRX gene mutations. Mutations are believed to result in loss of ATRX protein, and therefore immunohistochemistry is used to detect mutations. We screened 133 cases of neuroblastoma by ATRX immunohistochemistry, and found 9 cases with partial to total absence of ATRX. Sequencing for ATRX mutations detected a mutation in 1 of 9 cases, suggesting immunostaining was not reliable for detecting mutations. To correlate immunostaining with ALT, fluorescence in situ hybridization (FISH) for ALT was performed in 6 of these cases and 5 (from 4 patients) showed ALT, implying impaired ATRX protein function, despite the failure to identify a mutation. Two other cases with large deletions in the ATRX gene showed diffusely positive staining for ATRX protein but showed ALT by FISH. Four of the 6 patients with ALT-positive tumors were over 5 years old. Therefore, 29 additional patients 5 years old and above with ATRX-positive tumors were screened for ALT by FISH and 6 additional cases with ALT were detected, bringing the total to 29% (10/34) of children 5 years old and above, 70% of which showed positive ATRX immunohistochemistry. Patients with ATRX mutations in neuroblastoma tend to have a more chronic and progressive course of disease. Screening neuroblastoma tumors at diagnosis for ATRX mutations may help identify patients who might benefit from personalized therapy directed against ALT. However, relaying on negative immunohistochemistry for ATRX protein to identify ALT in neuroblastoma may miss a significant proportion of patients. The addition of FISH for ALT as part of the diagnostic workup, especially for older children (5 y old and above), would help ensure that patients are correctly identified for anti-ALT therapy.

RAS Mutation in Mucinous Carcinoma of the Ovary

Objective: This study was designed to identify genetic mutation in mucinous carcinoma of the ovary of the patients in King Chulalongkorn Memorial hospital, Bangkok, Thailand and study the relationship between genetic mutation and patients' prognosis. Methods: Fifty cases of primary mucinous carcinoma of the ovary were selected. DNA was analyzed for genetic mutation using ColoCarta Panel v1.0 and MassArray® System. Demographic data and clinical information of the participants were reviewed from electronic medical records and government data services. Results: Median of disease-free survival is 171.33 +/- 9.04 months and the median overall survival is 171.37 +/- 9.03 months. Twelve percent of the participants had recurrence and all of recurrent cases died from disease or its complication. We found three mutations which were KRAS (27 cases, 54%), PIK3CA (4 cases, 8%) and BRAF (1 case, 2%). Among the KRAS-mutated patients, the majority of the cases (25 cases, 92.6%) were in stage I. Recurrence and disease related mortality were not observed in the KRAS mutated patients. Conclusion: The genetic mutation analysis found three mutations which were KRAS 27 cases (54%), PIK3CA 4 cases (8%) and BRAF 1 case (2%) The ovarian mucinous carcinoma patients with KRAS mutation in our study showed excellent prognosis.

Cyclin D1 expression as a potential prognostic factor in advanced KRAS-mutant non-small cell lung cancer.

BACKGROUND: East Asian, including Thailand, lung cancer population may have a relatively lower prevalence of KRAS mutations than Caucasians. We investigated the prevalence and clinical characteristics of KRAS-driven non-small cell lung cancer (NSCLC) patients and the expression of cyclin D1, one of the KRAS downstream targets. METHODS: Lung cancer patients who received treatment at the King Chulalongkorn Memorial Hospital between January 2015 and July 2017 were enrolled. We identified KRAS mutations using allele specific PCR KRAS mutation testing. Cyclin D1 expression was determined using immunohistochemistry. RESULTS: After excluding 376 EGFR mutations and inadequate samples, we enrolled 95 patients eligible for KRAS mutation testing. KRAS mutations were identified in 28 out of 95 patients. There were 26 patients with KRAS codon 12/13 and 2 patients with KRAS codon 61 mutations. The prevalence of KRAS mutations among informative samples was 28 out of 357 (7.8%) which was relatively lower than that reported in Caucasian population. Smoking and male were significantly associated with KRAS mutations. The prognosis of KRAS-mutant NSCLC patients in particular codon 61 mutations was worse than that found in KRAS- and EGFR-wild-type (KRAS WT/EGFR WT) NSCLC patients (P=0.048). The levels of cyclin D1 expression in KRAS-mutant NSCLC were significantly higher than those in KRAS WT/EGFR WT NSCLC (P=0.02). A better prognosis of KRAS-mutant NSCLC patients with low cyclin D1 expression was observed when compared with those with high cyclin D1 expression (median overall survival 41.7 vs. 3.5 months, P=0.037). CONCLUSIONS: We found a moderate prevalence of KRAS mutations in lung cancer in Thailand. Clinical characteristics were similar to those of Caucasian population. Most KRAS-mutant NSCLC had high cyclin D1 expression. Cyclin D1 expression may serve as a useful prognostic biomarker in KRAS-mutant lung cancer. Validation of this finding in larger cohort is required.

Tubulovillous Adenoma of Vagina With Both KRAS and APC Mutations: Case Report.

Adenomatous polyps of the vulva and vagina are extremely rare. We report a case of a 74-yr-old women with a tubulovillous adenoma occurring in the vagina, and a second one occurring later in the vulva. Tumor cells in both lesions were CK7, CK20, CDX-2, and showed intact mismatch-repair proteins. A G13D (c.38G>A, p.Gly13Asp) mutation in the KRAS gene was identified in both masses. As well, a novel frameshift truncating mutation (c.4320delA, p.Pro1441fsTer32) in the APC gene was detected only in the vaginal mass, ruling out the possibility that the vulvar mass was a local recurrence of the vaginal mass. This is the first identification of KRAS and APC gene mutations in adenomatous polyps involving the female lower genital tract.