Diagnostic Approaches to Investigate JAK2-Unmutated Erythrocytosis Based on a Single Tertiary Center Experience.

INTRODUCTION: Erythrocytosis is attributed to various clinical and molecular factors. Many cases of JAK2-unmutated erythrocytosis remain undiagnosed. We investigated the characteristics and causes of JAK2-unmutated erythrocytosis. METHODS: We assessed the clinical and laboratory results of patients with erythrocytosis without JAK2 mutations and performed targeted next-generation sequencing (NGS) panels for somatic and germline mutations. RESULTS: In total, 117 patients with JAK2-unmutated erythrocytosis were included. The median hemoglobin and hematocrit levels were 17.9 g/dL and 53.4%, respectively. Erythropoietin levels were not below the reference range. Thrombotic events were reported in 17 patients (14.5%). Among JAK2-unmutated patients, 44 had undergone targeted panel sequencing consisting of myeloid neoplasm-related genes, and 16 had one or more reportable variants in ASXL1 (5/44), TET2, CALR, FLT3, and SH2B3 (2/44). Additional testing for germline causes revealed eight variants in seven genes in eight patients, including NF1, BPGM, EPAS1, PIEZO1, RHAG, SH2B3, and VHL genes. One NF1 pathogenic, one BPGM likely pathogenic, and six variants of undetermined significance were detected. CONCLUSION: Somatic and germline mutations were identified in 36.4% and 33.3 % of the JAK2-unmutated group; most variants had unknown clinical significance. Not all genetic causes have been identified; comprehensive diagnostic approaches are crucial for identifying the cause of erythrocytosis.

Issues, challenges, and future perspectives of genetic counseling in Republic of Korea: Perspectives of laboratory physicians based on a 2022 survey.

The field of genetic counseling (GC) in the Republic of Korea has evolved from a single medical doctor's clinic to a multidisciplinary service with medical geneticists and non-medical professionals working as a team. Here, we assessed the current status of GC in the Republic of Korea based on professional surveys from the perspective of laboratory physicians. An electronic survey was designed and conducted, with the respondents being 50 certified laboratory physicians who were members of the Korean Society for Genetic Diagnostics. Among the 50 respondents, 12 (24%) operated GC clinics. The number of sessions and cases of GC have been on the rise over the last few years, and counseling for cancer genetics was the most common request. Most respondents considered a good understanding of the genetic test and the ability to interpret the test results as strengths of laboratory physicians as medical geneticists, while the lack of clinical experience was a weakness. Education programs regarding laboratory physicians' needs should be provided for high-quality counseling. Lastly, improving the efficiency of GC by strengthening the workforce through a multidisciplinary team is necessary.

A simplified two-marker immunohistochemistry strategy for Lynch syndrome screening in endometrial cancer patients.

OBJECTIVE: To examine the efficacy of MSH6 and PMS2 immunohistochemistry (IHC) as a screening method for Lynch syndrome in endometrial cancer patients. METHODS: Through multidisciplinary discussions, an institutional MSH6 and PMS2 IHC-initiated cascade test (MSH6, PMS2 IHC→microsatellite instability [MSI] assay→germline mismatch repair [MMR] gene sequencing) was developed to screen for Lynch syndrome in endometrial cancer patients. Testing was performed on a consecutive cohort of 218 newly diagnosed endometrial cancer patients who underwent surgery at a tertiary hospital in the Republic of Korea between August 2018 and December 2020. The number of MMR deficiencies (MSH6 or PMS2 loss in IHC) and. RESULTS: of subsequent tests (MSI assay and germline MMR gene sequencing) were examined. RESULTS: MMR deficiency was detected in 52 of the 218 patients (24.0%). Among these 52 patients, 34 (65.0%) underwent MSI testing, of which 31 (91.0%) exhibited high MSI. Of the 31 patients with MSI-high status, 15 (48.0%) underwent germline MMR gene sequencing. Subsequently, Lynch syndrome was diagnosed in five patients (33.0%). CONCLUSION: Lynch syndrome screening using MSH6 and PMS2 IHC-initiated cascade testing is a viable strategy in the management of endometrial cancer. A simplified strategy (MSH6 and PMS2 IHC→germline MMR gene sequencing) was proposed because most women with MMR deficiencies exhibited high MSI.

Novel pathogenic PDX1 gene variant in a Korean family with maturity-onset diabetes of the young.

The diagnosis of maturity-onset diabetes of the young (MODY), a monogenic form of diabetes mellitus caused by a mutation in a single gene, is often uncertain until genetic testing is performed. We report a 13-yr-old Korean boy who was initially diagnosed with type 2 diabetes (T2DM). MODY was suspected because of his nonobese body habitus and family history of multiple affected members. Targeted panel sequencing of all MODY-related genes was performed using the NextSeq 550Dx platform (Illumina). Sanger sequencing was performed using blood samples from the parents, siblings, and other relatives. A frameshift variant in the 3' region of the last exon of PDX1 was detected in the patient and his family members with diabetes. PP1_Moderate criterion was applied and this variant was confirmed to be the genetic cause of diabetes in the family and classified as likely pathogenic. The study highlights the importance of genetic testing for nonobese, early-onset diabetic patients with multiple affected family members. Increased awareness and aggressive genetic testing for MODY are needed.

Comprehensive Study of Microsatellite Instability Testing and Its Comparison With Immunohistochemistry in Gastric Cancers.

PURPOSE: In this study, polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing was comprehensively analyzed and compared with immunohistochemistry (IHC) for mismatch repair (MMR) protein expression in patients with gastric cancer (GC). MATERIALS AND METHODS: In 5,676 GC cases, PCR-based MSI testing using five microsatellites (BAT-26, BAT-25, D5S346, D2S123, and D17S250) and IHC for MLH1 were performed. Re-evaluation of MSI testing/MLH1 IHC and additional IHC for MSH2, MSH6, and PMS2 were performed in discordant/indeterminate cases. RESULTS: Of the 5,676 cases, microsatellite stable (MSS)/MSI-low and intact MLH1 were observed in 5,082 cases (89.5%), whereas MSI-high (MSI-H) and loss of MLH1 expression were observed in 502 cases (8.8%). We re-evaluated the remaining 92 cases (1.6%) with a discordant/indeterminate status. Re-evaluation showed 1) 37 concordant cases (0.7%) (18 and 19 cases of MSI-H/MMR-deficient (dMMR) and MSS/MMR-proficient (pMMR), respectively), 2) 6 discordant cases (0.1%) (3 cases each of MSI-H/pMMR and MSS/dMMR), 3) 14 MSI indeterminate cases (0.2%) (1 case of dMMR and 13 cases of pMMR), and 4) 35 IHC indeterminate cases (0.6%) (22 and 13 cases of MSI-H and MSS, respectively). Finally, MSI-H or dMMR was observed in 549 cases (9.7%), of which 47 (0.8%) were additionally confirmed as MSI-H or dMMR by re-evaluation. Sensitivity was 99.3% for MSI testing and 95.4% for MMR IHC. CONCLUSIONS: Considering the low incidence of MSI-H or dMMR, discordant/indeterminate results were occasionally identified in GCs, in which case complementary testing is required. These findings could help improve the accuracy of MSI/MMR testing in daily practice.

Universal Screening for Lynch Syndrome Compared with Pedigree-Based Screening: 10-Year Experience in a Tertiary Hospital.

PURPOSE: Universal screening for Lynch syndrome (LS) refers to routine tumor testing for microsatellite instability (MSI) among all patients with colorectal cancer (CRC). Despite its widespread adoption, real-world data on the yield is lacking in Korean population. We studied the yield of adopting universal screening for LS in comparison with pedigree-based screening in a tertiary center. MATERIALS AND METHODS: CRC patients from 2007-2018 were reviewed. Family histories were obtained and were evaluated for hereditary nonpolyposis colorectal cancer (HNPCC) using Amsterdam II criteria. Tumor testing for MSI began in 2007 and genetic testing was offered using all available clinicopathologic data. Yield of genetic testing for LS was compared for each approach and step. RESULTS: Of the 5,520 patients, tumor testing was performed in 4,701 patients (85.2%) and family histories were obtained from 4,241 patients (76.8%). Hereditary CRC (LS or HNPCC) was present in 69 patients (1.3%). MSI-high was present in 6.9%, and 25 patients had confirmed LS. Genetic testing was performed in 41.2% (47/114) of MSI-high patients, out of which 40.4% (19/47) were diagnosed with LS. There were six additional LS patients found outside of tumor testing. For pedigree-based screening, Amsterdam II criteria diagnosed 55 patients with HNPCC. Fifteen of these patients underwent genetic testing, and 11 (73.3%) were diagnosed with LS. Two patients without prior family history were diagnosed with LS and relied solely on tumor testing results. CONCLUSION: Despite widespread adoption of routine tumor testing for MSI, this is not a fail-safe approach to screen all LS patients. Obtaining a thorough family history in combination with universal screening provides a more comprehensive 'universal' screening method for LS.

Concordance of circulating tumor DNA and matched formalin-fixed paraffin-embedded tumor tissue in gastric cancer as a predictor of recurrence.

PURPOSE: Combined analysis of the variant composition of circulating tumor DNA (ctDNA) from cell-free plasma and DNA from tumor tissue could provide insight into the implications of the genetic alterations responsible for the intratumoral and intertumoral heterogeneity of gastric cancer. We aimed to evaluate the usefulness of this approach in these patients. METHODS: Cell-free plasma and formalin-fixed paraffin-embedded tumor tissue samples from 46 patients with gastric cancer were examined. Targeted deep sequencing was performed using a commercially available kit. RESULTS: The cell-free DNA (cfDNA) concentration was higher in stage II-IV versus stage I patients and in larger versus smaller tumors. Only 12 of the 36 (33.3%) alterations in the tumor tissue samples were in concordance with those in the ctDNA samples. Two variants were in concordance in stage I samples and 10 in stage II-IV samples. Actionable variants that were detected in concordance were in the stage II-IV samples. Preoperative ctDNA positivity of actionable variants was significantly associated with cfDNA concentration, lymphatic invasion, N stage, and TNM stage. Cancer recurrence was significantly associated with tumor size, lymphatic/vascular invasion, TNM stage, and ctDNA-tumor tissue variant concordance. CONCLUSION: Preoperative ctDNA genetic analysis using a multigene panel offers substantial clinical benefits when performed in conjunction with targeted deep sequencing of tumor tissue. Concordance between preoperative ctDNA and tumor tissue mutations may serve as a prognostic indicator in patients with gastric cancer.

Clinical staging and genetic profiling of Korean patients with primary lymphedema using targeted gene sequencing.

Lymphedema is a progressive disease caused by lymphatic flow blockage in the lymphatic pathway. Primary (hereditary) lymphedema is caused by genetic mutations without secondary causes. We performed clinical profiling on Korean primary lymphedema patients based on their phenotypes using lymphoscintigraphy and made genetic diagnoses using a next-generation sequencing panel consisting of 60 genes known to be related to primary lymphedema and vascular anomalies. Of 27 patients included in this study, 14.8% of the patients had lymphedema of the upper extremities, 77.8% had lymphedema of the lower extremities and 7.4% had 4-limbs lymphedema. Based on the International Society of Lymphology staging, 14, 10, and 3 patients had stage 3, 2, and 1 lymphedema, respectively. Only one family was genetically confirmed to harbor likely pathogenic variants in CELSR1. The proband was carrying two likely pathogenic variants in CELSR1, while her symptomatic mother was confirmed to carry only one of the variants. Furthermore, two other variants of uncertain significance in CELSR1 were detected in other patients, making CELSR1 the most commonly altered gene in our study. The clinical and genetic profile of hereditary lymphedema reported here is the first such data series reported for South Korea.

Immunohistochemical Staining to Identify Concomitant Systemic Mastocytosis in Acute Myeloid Leukemia with RUNX1::RUNX1T1.

Systemic mastocytosis with associated hematological neoplasm (SM-AHN) poses diagnostic challenges because of the coexistence of atypical mast cell proliferation and hematological neoplasms. We assessed the presence of SM-AHN in patients with acute myeloid leukemia (AML) with RUNX1::RUNX1T1 from 2014 to 2020. Bone marrow (BM) samples were evaluated for mast cell aggregates using CD117 and CD25 immunohistochemical (IHC) staining. The KIT D816V variant burden at diagnosis and post induction was assessed using droplet digital PCR. Among 23 patients diagnosed as having AML with RUNX1::RUNX1T1, four (17.4%) were also diagnosed as having SM-AHN. No significant differences in clinical characteristics or overall survival (P=0.565) were observed between patients with or without SM-AHN, except for the presence of KIT variants (P=0.040). After induction therapy, IHC staining revealed the presence of mast cell aggregates in the BM, and the KIT D816V variant burden decreased with decreasing blast count and was similar in BM aspirates, smear slides, and sections. Concomitant SM-AHN was not infrequent in AML patients with RUNX1::RUNX1T1. This study showed the importance of CD117 and CD25 IHC staining after induction chemotherapy for SM-AHN screening, especially in patients with KIT variants.

Tissue miR-200c-3p and circulating miR-1290 as potential prognostic biomarkers for colorectal cancer.

Epithelial-mesenchymal transition (EMT)-related cancers generally elicit low immune responses. EMT is regulated by several microRNAs (miRNAs) in cancers. Thus, this study aimed to evaluate the prognostic potential of EMT-related miRNAs as biomarkers in colorectal cancer (CRC). Formalin-fixed paraffin-embedded tumor and normal tissue and plasma samples were obtained from 65 patients with pathologically confirmed CRC. In addition, plasma samples were obtained from 30 healthy volunteers. Immunohistochemical staining for E-cadherin, ZEB1, PD-1, PD-L1, CD3, CD4, CD8, Foxp3, and CD68 was conducted on tissue samples. Droplet digital polymerase chain reaction (ddPCR) analysis was performed to evaluate miR-21-5p, 34a-5p, 138-5p, 200a-3p, 200b-5p, 200c-3p, 630, 1246, and 1290 expression in tissue samples and miR-630, 1246, and 1290 expression in plasma samples. miR-21-5p, 34a-5p, 630, 1246, and 1290 expression was higher in tumor tissues than in normal tissues (P < 0.05). EMT was significantly associated with reduced tumor-infiltrating T cells. Moreover, miR-21-5p, miR-34a-5p, miR-200a-3p, and miR-200c-3p expression was negatively correlated with T cell density (P < 0.05). High tissue levels of miR-200c-3p were associated with poor overall survival (OS) (P < 0.001). CRC patients with the EMT phenotype had poor OS; however, PD-L1 positivity and abundant PD-1 positive immune cells were correlated with better OS (P < 0.05). miR-1246 and miR-1290 levels were significantly higher in the plasma of patients with CRC than in the plasma of healthy controls (P < 0.05). High plasma levels of miR-1290 were correlated with advanced stage and poor OS (P < 0.05). The tissue expression of miR-200c-3p and plasma levels of miR-1290 measured by ddPCR indicate their potential as prognostic biomarkers for CRC.

Current Issues, Challenges, and Future Perspectives of Genetic Counseling in Korea.

Genetic testing has become increasingly integrated into all areas of healthcare, and complex genetic testing usage continues to grow; thus, the demand for genetic counseling (GC) is likely to increase. However, it is unclear whether the current clinical GC capacity is sufficient for meeting the existing demand. This review describes the current issues, challenges, and future perspectives of GC in Korea based on a professional survey conducted among laboratory physicians. In view of the growing GC demand in the clinical setting, participants expressed a concern about the lack of support from the national healthcare insurance policy and legal requirements, such as certification, for GC practice. The implementation of genetic testing in the overall healthcare system in Korea is in an early phase. Proper implementation can be achieved through education and training of specialists, collaboration among healthcare personnel, proper regulatory oversight, genomic policies, and public awareness. Understanding the current GC capacity, issues, and challenges is a prerequisite for effective strategic planning by healthcare systems considering the expected growth in the demand for clinical genetic services over the next few decades.

Relationship among genetic polymorphism of SLCO1B1, rifampicin exposure and clinical outcomes in patients with active pulmonary tuberculosis.

AIMS: Rifampicin is a key drug for the treatment of tuberculosis (TB). Little is known for the relationship between the rifampicin pharmacokinetics and genetic polymorphisms in the Asian population. We aimed to investigate relationship between genetic polymorphism of SLCO1B1 and rifampicin exposure and its impact on clinical outcomes in Korean patients with active pulmonary TB. METHODS: From February 2016 to December 2019, patients with active pulmonary TB who were taking rifampicin for >1 week were prospectively enrolled. Serial or 1-time blood sampling was conducted to determine rifampicin concentrations. The genotype of 4 single nucleotide polymorphisms of SLCO1B1 was determined. To estimate the drug clearance and exposure, population pharmacokinetics analysis was conducted. Clinical outcomes such as time to acid-fast bacteria culture conversion, chest radiograph score changes from baseline, and all-cause mortality were also evaluated. The exposure among different SLCO1B1 genotype was compared and relationship between drug exposure and clinical outcomes were explored. RESULTS: A total of 105 patients (70 males and 35 females) were included in the final analysis. The mean age of patients was 55.4 years. The mean drug clearance and exposure were 13.6 L/h and 57.9 mg h/L, respectively. The genetic polymorphisms of SLCO1B1 were not related to rifampicin clearance or exposure. As the rifampicin exposure increased, the chest radiographs improved significantly, but the duration of acid-fast bacteria culture conversion was not related to the drug exposure. CONCLUSION: SLCO1B1 gene polymorphisms did not influence rifampicin concentrations and clinical outcomes in Korean patients with active pulmonary TB.

Improving self-sufficiency in organ transplantation in Korea.

After attaining a qualified medical capacity in organ transplantation, Korea has been struggling to increase the number of deceased organ transplants to reach self-sufficiency. As one of these efforts, Korea revised the organ transplantation law in 2010 by adding three articles the mandatory reporting based on the recommendation of the 3rd Global Consultation on Organ Transplantation of Madrid in 2010. Along with the new constitution, considerable efforts have been initiated to upgrade the deceased organ transplantation system while maintaining the virtues of fairness, justice, and transparency. The Korean Society of Transplantation played a critical role in revising the law as well as in establishing organizations such as the Korean Organ Donation Agency (KODA; 2009), the Vitallink (2009), and the Korean Organ Transplantation Registry (KOTRY; 2010). By the activities of these professional organizations, Korea could implement fundamental programs such as mandatory reporting and could develop various education programs for organ donation for students and the general population. As a result, the number of deceased donors increased from 1.08 p.m.p. (2000) to 9.23 p.m.p.(2020). Further efforts are needed to increase the number of organ donor cardholders and family consent rates by well-designed, target-specific education programs to overcome traditional negative cultural barriers toward organ donation. The community atmosphere of honoring and thanking donors and their families should be nurtured by national and regional activities of life-sharing weeks linked with organ donor memorial parks.

Author Correction: Comparative analysis of HER2 copy number between plasma and tissue samples in gastric cancer using droplet digital PCR.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparative analysis of HER2 copy number between plasma and tissue samples in gastric cancer using droplet digital PCR.

In this study, we measured the human epidermal growth factor receptor 2 (HER2) copy number in both tissue and plasma samples of gastric cancer patients by using droplet digital polymerase chain reaction (ddPCR) method. Eighty gastric cancer patients were enrolled and both formalin-fixed and paraffin-embedded tissue and preoperative plasma samples were collected. HER2 status was determined by HER2 immunohistochemistry (IHC)/silver in situ hybridization (SISH) in tissue samples and ddPCR of the target gene HER2 and the reference gene eukaryotic translation initiation factor 2C, 1 in both tissue and plasma. The concordance rate of tissue HER2 status determined by IHC/SISH and HER2 ddPCR was 90.0% (72/80), and the sensitivity and specificity of tissue ddPCR were 85.0% and 95.0%, respectively. The concordance rate of plasma ddPCR and IHC/SISH was 63.8% (51/80). The sensitivity, specificity, positive predictive value, and negative predictive value of plasma HER2 ddPCR were 37.5%, 90.0%, 79.0%, and 59.0%, respectively. As HER2 measurement by tissue ddPCR showed a high concordance rate with HER2 status by IHC/SISH, it could replace tissue IHC/SISH testing in gastric cancer. These findings may contribute to the development of tissue and plasma HER2 testing that would be useful in daily practice.

Replication-Based Rearrangements Are a Common Mechanism for SNCA Duplication in Parkinson's Disease.

BACKGROUND: SNCA multiplication is a genomic cause of familial PD, showing dosage-dependent toxicity. Until now, nonallelic homologous recombination was suggested as the mechanism of SNCA duplication, based on various types of repetitive elements found in the spanning region of the breakpoints. However, the sequence at the breakpoint was analyzed only for 1 case. OBJECTIVES: We have analyzed the breakpoint sequences of 6 patients with PD who had duplicated SNCA using whole-genome sequencing data to elucidate the mechanism of SNCA duplication. METHODS: Six patient samples with SNCA duplication underwent whole-genome sequencing. The duplicated regions were defined with nucleotide-resolution breakpoints, which were confirmed by junction polymerase chain reaction and Sanger sequencing. The search for potential non-B DNA-forming sequences and stem-loop structure predictions was conducted. RESULTS: Duplicated regions ranged from the smallest region of 718.3 kb to the largest one of 4,162 kb. Repetitive elements were found at 8 of the 12 breakpoint sequences on each side of the junction, but none of the pairs shared overt homologies. Five of these six junctions had microhomologies (2-4 bp) at the breakpoint, and a short stretch of sequences was inserted in 3 cases. All except one junction were located within or next to stem-loop structures. CONCLUSION: Our study has determined that homologous recombination mechanisms involving repetitive elements are not the main cause of the duplication of SNCA. The presence of microhomology at the junctions and their position within stem-loop structures suggest that replication-based rearrangements may be a common mechanism for SNCA amplification. © 2020 International Parkinson and Movement Disorder Society.

Whole Exome Sequencing Identifies Novel Genetic Alterations in Patients with Pheochromocytoma/Paraganglioma.

BACKGROUND: Pheochromocytoma and paragangliomas (PPGL) are known as tumors with the highest level of heritability, approximately 30% of all cases. Clinical practice guidelines of PPGL recommend genetic testing for germline variants in all patients. In this study, we used whole exome sequencing to identify novel causative variants associated with PPGL to improve the detection of rare genetic variants in our cohort. METHODS: Thirty-six tested negative for pathogenic variants in previous Sanger sequencing or targeted gene panel testing for PPGL underwent whole exome sequencing. Whole exome sequencing was performed using DNA samples enriched using TruSeq Custom Enrichment Kit and sequenced with MiSeq (Illumina Inc.). Sequencing alignment and variant calling were performed using SAMtools. RESULTS: Among previously mutation undetected 36 patients, two likely pathogenic variants and 13 variants of uncertain significance (VUS) were detected in 32 pheochromocytoma-related genes. SDHA c.778G>A (p.Gly260Arg) was detected in a patient with head and neck paraganglioma, and KIF1B c.2787-2A>C in a patient with a bladder paraganglioma. Additionally, a likely pathogenic variant in BRCA2, VUS in TP53, and VUS in NFU1 were detected. CONCLUSION: Exome sequencing further identified genetic alterations by 5.6% in previously mutation undetected patients in PPGL. Implementation of targeted gene sequencing consisted of extended genes of PPGL in routine clinical screening can support the level of comprehensive patient assessment.

A novel strategy for interpreting the T-SPOT.TB test results read by an ELISPOT plate imager.

BACKGROUND: The T-SPOT.TB can be read by an ELISPOT plate imager as an alternative to a labor-intensive and time-consuming manual reading, but its accuracy has not been sufficiently discussed to date. METHODS: 1,423 test results obtained from manual reading using a microscope and an ELISPOT plate imager were compared. The agreement of qualitative test results was assessed using Cohen's kappa coefficient. The relationship of spot counts was studied using Bland-Altman analysis. RESULTS: The overall percent agreement of the qualitative test results was 95.43% with a kappa coefficient of 0.91. Positive test results with the maximum net spot count of 8 and borderline test results showed relatively high discordance. The agreement of spot counts in panel A, panel B, and nil control was good, and variability did not increase with higher spot counts. On the basis of study findings, a novel strategy for interpreting the test results by an ELISPOT plate imager was proposed. CONCLUSIONS: To increase diagnostic accuracy, positive test results with the maximum net spot count of 8 and borderline test results should be manually confirmed. Our strategy could be a practical guide for laboratories to build their own strategies for interpreting the test results by an ELISPOT plate imager.

Digital polymerase chain reaction for detecting c-MYC copy number gain in tissue and cell-free plasma samples of colorectal cancer patients.

We focused on the utility of the droplet digital polymerase chain reaction (ddPCR) for detecting c-MYC gene copy number (GCN) gain in cell-free plasma and tumor tissue of colorectal cancer (CRC) patients. c-MYC GCN status was determined using dual-color silver in situ hybridization (SISH) and ddPCR in retrospective cohort 1 (192 CRC patients) and prospective cohort 2 (64 CRC patients). In cohort 1, c-MYC GCN gain was observed in 34 (17.5%) patients by SISH, and in 7 (3.6%) patients by ddPCR. c-MYC GCN by SISH significantly correlated with ddPCR results (ρ = 0.532, P < 0.001). Although 40 cases (20.7%) showed intratumoral genetic heterogeneity, it did not cause discordance in results obtained by the two methods. c-MYC GCN gain, by both SISH and ddPCR was independently correlated with worst prognosis (P = 0.002). In cohort 2, c-MYC GCN estimation in tissue by ddPCR was also significantly associated with results obtained by SISH (ρ = 0.349, P = 0.005), but correlated with plasma ddPCR with borderline significance (ρ = 0.246, P = 0.050). Additionally, detecting c-MYC GCN gain in plasma with ddPCR might have relatively low sensitivity but high specificity. Our study suggests that ddPCR can be a useful tool for detecting c-MYC GCN gain as a potential prognostic biomarker in CRC tissue samples; however, this will need further verification in plasma samples.

Spectrum of MNX1 Pathogenic Variants and Associated Clinical Features in Korean Patients with Currarino Syndrome.

BACKGROUND: The major genetic cause of Currarino syndrome (CS), a congenital malformation syndrome typically characterized by sacral agenesis, anorectal malformation, and presence of a pre-sacral mass, is known to be pathogenic variants in motor neuron and pancreas homeobox 1 (MNX1), which exist in almost all familial cases and 30% of sporadic cases. Less commonly, a large deletion or a complex rearrangement involving the 7q36 region is associated with CS. We investigated the spectrum of MNX1 pathogenic variants and associated clinical features in the Korean patients with CS. METHODS: We enrolled 25 patients with CS, including 24 sporadic cases and one familial case. Direct sequencing of MNX1 and multiplex ligation-dependent probe amplification were performed. We also analyzed clinical phenotypes and evaluated genotype-phenotype correlations. RESULTS: We identified six novel variants amongst a total of six null variants, one missense variant, and one large deletion. The null variants included four frameshift variants (p.Gly98Alafs* 124, p.Gly145Alafs*77, p.Gly151Leufs*67, and p.Ala216Profs*5) and two nonsense variants (p.Tyr186* and p.Gln212*). The missense variant, p.Lys295Gln, was located in the highly-conserved homeobox domain and was predicted to be deleterious. A large deletion involving the 7q36 region was detected in one patient. Pathogenic variants in MNX1 were detected in 28% of all CS cases and 25% of sporadic cases. The clinical phenotype was variable in patients with and without pathogenic variants; no significant genotype-phenotype correlation was observed. CONCLUSIONS: This study revealed the spectrum and phenotypic variability of MNX1 pathogenic variants in the Korean population.