Genomic and Transcriptomic Characterization of Gastric Cancer with Bone Metastasis / Journal of the Korean Cancer Association, 대한암학회지

Purpose@#Bone metastasis (BM) adversely affects the prognosis of gastric cancer (GC). We investigated molecular features and immune microenvironment that characterize GC with BM compared to GC without BM. @*Materials and Methods@#Targeted DNA and whole transcriptome sequencing were performed using formalin-fixed paraffin-embedded primary tumor tissues (gastrectomy specimens) of 50 GC cases with distant metastases (14 with BM and 36 without BM). In addition, immunohistochemistry (IHC) for mucin-12 and multiplex IHC for immune cell markers were performed. @*Results@#Most GC cases with BM had a histologic type of poorly cohesive carcinoma and showed worse overall survival (OS) than GC without BM (p < 0.05). GC with BM tended to have higher mutation rates in TP53, KDR, APC, KDM5A, and RHOA than GC without BM. Chief cell-enriched genes (PGA3, PGC, and LIPF), MUC12, MFSD4A, TSPAN7, and TRIM50 were upregulated in GC with BM compared to GC without BM, which was correlated with poor OS (p < 0.05). However, the expression of SERPINA6, SLC30A2, PMAIP1, and ITIH2 were downregulated in GC with BM. GC with BM was associated with PIK3/AKT/mTOR pathway activation, whereas GC without BM showed the opposite effect. The densities of helper, cytotoxic, and regulatory T cells did not differ between the two groups, whereas the densities of macrophages were lower in GC with BM (p < 0.05). @*Conclusion@#GC with BM had different gene mutation and expression profiles than GC without BM, and had more genetic alterations associated with a poor prognosis.

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. Reevaluation 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 reevaluation. 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.

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.

MicroRNA-552 expression in colorectal cancer and its clinicopathological significance

Background@#MicroRNA-552 (miR-552) has been reported to correlate with the development and progression of various cancers, including colorectal cancer (CRC). This study aimed to investigate miR-552 expression in cancer tissue samples compared to normal mucosal tissue and its role as a diagnostic or prognostic marker in CRC patients. @*Methods@#Normal mucosal tissues and primary cancer tissues from 80 surgically resected CRC specimens were used. Quantitative real-time polymerase chain reaction was performed for miR-552 and U6 small nuclear RNA to analyze miR-552 expression and its clinicopathological significance. Immunohistochemistry for p53 and phosphatase and tension homolog (PTEN) was performed to evaluate their association with miR-552 expression. @*Results@#miR-552 expression was significantly higher in primary cancer tissues compared to normal mucosal tissues (p<.001). The expression level of miR552 was inversely correlated with that of PTEN (p=.068) and p53 (p=.004). Survival analysis showed that high miR-552 expression was associated with worse prognosis but this was not statistically significant (p=.255). However, patients with CRC having high miR-552 expression and loss of PTEN expression had significantly worse prognosis than others (p=.029). @*Conclusions@#Our results suggest that high miR-552 expression might be a potential diagnostic biomarker for CRC, and its combined analysis with PTEN expression can possibly be used as a prognostic marker.

Cerebral Oxygenation during Apnea in Preterm Infants: Effects of Accompanying Peripheral Oxygen Desaturation

Purpose@#Premature infants have immature respiratory control and cerebral autoregulation. We aimed to investigate changes in cerebral oxygenation during apnea with and without peripheral oxygen desaturation in premature infants. @*Methods@#This prospective observational study was conducted at Inha University Hospital. Near-infrared spectroscopy (NIRS)-monitored regional cerebral oxygen saturation (rScO2) and pulse oximeter-monitored peripheral oxygen saturation (SpO2) were assessed during the first week of life in 16 stable, spontaneously breathing preterm infants. Apneic episodes that lasted for ≥20 seconds or were accompanied by desaturation or bradycardia were included for analysis. The average rScO2 value during the 5-minute prior to apnea (baseline), the lowest rScO2 value following apnea (nadir), the time to recover to baseline (recovery time), the area under the curve (AUC), and the overshoot above the baseline after recovery were analyzed. @*Results@#The median gestational age and birth weight of the infants were 29.2 weeks (interquartile range [IQR], 28.5 to 30.5) and 1,130 g (IQR, 985 to 1,245), respectively. A total of 73 apneic episodes were recorded at a median postnatal age of 2 days (IQR, 1 to 4). The rScO2 decreased significantly following apneic episodes regardless accompanied desaturation. There were no differences in baseline, nadir, or overshoot rScO2 between the two groups. However, the rScO2 AUC for apnea with desaturation was significantly higher than that for apnea without desaturation. @*Conclusion@#Cerebral oxygenation can significantly decrease during apnea, especially when accompanied by reduced SpO2. These results add the evidence for the clinical utility of NIRS in monitoring premature infants.

Cerebral Oxygenation during Apnea in Preterm Infants: Effects of Accompanying Peripheral Oxygen Desaturation

Purpose@#Premature infants have immature respiratory control and cerebral autoregulation. We aimed to investigate changes in cerebral oxygenation during apnea with and without peripheral oxygen desaturation in premature infants. @*Methods@#This prospective observational study was conducted at Inha University Hospital. Near-infrared spectroscopy (NIRS)-monitored regional cerebral oxygen saturation (rScO2) and pulse oximeter-monitored peripheral oxygen saturation (SpO2) were assessed during the first week of life in 16 stable, spontaneously breathing preterm infants. Apneic episodes that lasted for ≥20 seconds or were accompanied by desaturation or bradycardia were included for analysis. The average rScO2 value during the 5-minute prior to apnea (baseline), the lowest rScO2 value following apnea (nadir), the time to recover to baseline (recovery time), the area under the curve (AUC), and the overshoot above the baseline after recovery were analyzed. @*Results@#The median gestational age and birth weight of the infants were 29.2 weeks (interquartile range [IQR], 28.5 to 30.5) and 1,130 g (IQR, 985 to 1,245), respectively. A total of 73 apneic episodes were recorded at a median postnatal age of 2 days (IQR, 1 to 4). The rScO2 decreased significantly following apneic episodes regardless accompanied desaturation. There were no differences in baseline, nadir, or overshoot rScO2 between the two groups. However, the rScO2 AUC for apnea with desaturation was significantly higher than that for apnea without desaturation. @*Conclusion@#Cerebral oxygenation can significantly decrease during apnea, especially when accompanied by reduced SpO2. These results add the evidence for the clinical utility of NIRS in monitoring premature infants.

Prediction of TP53 mutations by p53 immunohistochemistry and their prognostic significance in gastric cancer

Background@#Recently, molecular classifications of gastric cancer (GC) have been proposed that include TP53 mutations and their functional activity. We aimed to demonstrate the correlation between p53 immunohistochemistry (IHC) and TP53 mutations as well as their clinicopathological significance in GC. @*Methods@#Deep targeted sequencing was performed using surgical or biopsy specimens from 120 patients with GC. IHC for p53 was performed and interpreted as strong, weak, or negative expression. In 18 cases (15.0%) with discrepant TP53 mutation and p53 IHC results, p53 IHC was repeated. @*Results@#Strong expression of p53 was associated with TP53 missense mutations, negative expression with other types of mutations, and weak expression with wild-type TP53 (p<.001). The sensitivity for each category was 90.9%, 79.0%, and 80.9%, and the specificity was 95.4%, 88.1%, and 92.3%, respectively. The TNM stage at initial diagnosis exhibited a significant correlation with both TP53 mutation type (p=.004) and p53 expression status (p=.029). The Kaplan-Meier survival analysis for 109 stage II and III GC cases showed that patients with TP53 missense mutations had worse overall survival than those in the wild-type and other mutation groups (p=.028). Strong expression of p53 was also associated with worse overall survival in comparison to negative and weak expression (p=.035). @*Conclusions@#Results of IHC of the p53 protein may be used as a simple surrogate marker of TP53 mutations. However, negative expression of p53 and other types of mutations of TP53 should be carefully interpreted because of its lower sensitivity and different prognostic implications.

Clinical Characteristics of Infantile Colic

PURPOSE: To diagnose infantile colic from parent questionnaires, as well as investigating the risk factors and clinical course of infantile colic. METHODS: We retrospectively reviewed the medical records of 462 infants, with a corrected age of < 4 months at the time of visiting Inha University Hospital from January to December 2017. Parents responded to a 10-line questionnaire consisting of seven items relating to colic symptoms and three further items relating to underlying disease. The score was based on the number of days each symptom was evident during the preceding week. We defined infantile colic as the sum total being greater than seven points; if at least one of the three symptoms suggesting underlying disease was present, the infant was excluded from the diagnosis. RESULTS: One hundred and sixty-seven infants (36.1%) satisfied the criteria. The lower the gestational age, the more infantile colic they developed (P < 0.001). The prevalence of colic was higher in infants born with a birth weight < 2.5 kg (62.7% vs. 24.4%, P < 0.001) and in infants small for their gestational age, in the < 10th percentile (54.5% vs. 33.7%, P=0.003). The prevalence of colic was significantly different according to the type of feeding (P=0.001), being the lowest in breast-only feeding (29.8%), 32.8% in mixed feeding with breast milk and formula, and 49.7% in formula-only feeding. Colic symptoms improved by administering hydrolyzed formula (87.5%), low-lactose formula (47.1%), galactosidase (44.4%), and the probiotic Lactobacillus reuteri (34.5%). CONCLUSION: The prevalence of infantile colic was over 30%. Prematurity, lower birth weight, and small for gestational age were the risk factors of infantile colic. Clinical improvement was observed when active intervention was performed.

Clinical impact of admission hypothermia in very low birth weight infants: results from Korean Neonatal Network / 소아과

BACKGROUND: Preterm infants have difficulty maintaining body temperature after birth. However, clinical guidelines advocate that neonatal body temperature should be maintained at 36.5°C–37.5°C. PURPOSE: We aimed to investigate the incidence of admission hypothermia in very low birth weight (VLBW) infants and to determine the association of admission temperature with in-hospital mortality and morbidities. METHODS: A cohort study using prospectively collected data involving 70 neonatal intensive care units (NICUs) that participate in the Korean Neonatal Network. From registered infants born between January 2013 and December 2015, 5,343 VLBW infants born at less than 33 weeks of gestation were reviewed. RESULTS: The mean admission temperature was 36.1°C±0.6°C, with a range of 31.9°C to 38.4°C. Approximately 74.1% of infants had an admission hypothermia of <36.5°C. Lower birth weight, intubation in the delivery room and Apgar score <7 at 5 minutes were significantly related to admission hypothermia. The mortality was the lowest at 36.5°C–37.5°C and adjusted odd ratios for all deaths increased to 1.38 (95% confidence interval [CI], 1.04–1.83), 1.44 (95% CI, 1.05–1.97) and 1.86 (95% CI, 1.22–2.82) for infants with admission temperatures of 36.0°C–36.4°C, 35.0°C–35.9°C, and <35.0°C, respectively. Admission hypothermia was also associated with high likelihoods of bronchopulmonary dysplasia, pulmonary hypertension, proven sepsis, pulmonary hemorrhage, air-leak, seizure, grade 3 or higher intraventricular hemorrhage and advanced retinopathy of prematurity requiring laser therapy. CONCLUSION: A large portion of preterm infants in Korea had hypothermia at NICU admission, which was associated with high mortality and several important morbidities. More aggressive interventions aimed at reducing hypothermia are required in this high-risk population.

Hospital Visits from Respiratory Diseases of Early and Late Preterm Infants

PURPOSE: We aimed to evaluate the respiratory illness-related hospital visits (out-patient clinics, emergency room, and re-admission) of preterm infants, and compare them according to corrected age and prematurity. METHODS: We reviewed the medical records of preterm infants born at < 37 weeks of gestation admitted to the neonatal intensive care unit (NICU) at Inha University Hospital between January 2012 and June 2015. Infant follow-up appointments in both neonatology and pulmonology out-patient clinics occurred for at least 2 years after NICU discharge. RESULTS: The proportion of infants who visited the hospital due to any respiratory illness was as high as 50% until 12 months of corrected age, and subsequently decreased over time. Hospital admission was significantly higher in early preterm infants ( < 34 weeks of gestation) compared to late preterm infants (≥34 and < 37 weeks of gestation). The proportion of infants who were re-admitted due to lower respiratory tract illness was significantly higher until 6 months of corrected age compared to the later, and did not differ between early and late preterm infants. CONCLUSION: The proportion of hospital visits of preterm infants due to respiratory disease was high until 12 months of corrected age. Most notably, the re-admission proportion from lower respiratory tract illness was high under 6 months in both early and late preterm infants. Preterm infants within this age that are visiting the hospital with respiratory symptoms should be carefully observed and followed up.

Ligand-Independent Epidermal Growth Factor Receptor Overexpression Correlates with Poor Prognosis in Colorectal Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Molecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients’ survival with CRC. MATERIALS AND METHODS: The expression of EGFR ligands, including heparin binding epidermal growth factor-like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH. RESULTS: Unlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands–/EGFR+ group showed a significant association with the worst disease-free survival (DFS; p=0.018) and overall survival (OS; p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis. CONCLUSION: Ligand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.

Stromal Expression of MicroRNA-21 in Advanced Colorectal Cancer Patients with Distant Metastases

BACKGROUND: The aim of this study was to determine the regional heterogeneity and clinicopathological significance of microRNA-21 (miR-21) in advanced colorectal cancer (CRC) patients with distant metastasis. METHODS: miR-21 expression was investigated by using locked nucleic acid- fluorescence in situ hybridization in the center and periphery of the primary cancer and in distant metastasis from 170 patients with advanced CRC. In addition, α-smooth muscle actin and desmin were evaluated to identify cancer-associated fibroblasts (CAFs) by using immunohistochemistry. RESULTS: The miR-21 signal was observed in the cancer stroma. The expression of miR-21 (a score of 1-4) in the center and periphery of the primary cancer and in distant metastasis was observed in specimens from 133 (78.2%), 105 (61.8%), and 91 (53.5%) patients, respectively. miR-21 expression was heterogeneous in advanced CRC. Discordance between miR-21 expression in the center of the primary cancer and either the periphery of the primary cancer or distant metastasis was 31.7% or 44.7%, respectively. miR-21 stromal expression in the periphery of the primary cancer was significantly associated with a better prognosis (p=.004). miR-21 expression was significantly associated with CAFs in the center of the primary cancer (p=.001) and distant metastases (p=.041). CONCLUSIONS: miR-21 expression is observed in cancer stroma related to the CAF quantity and frequently presents regional heterogeneity in CRC. Our findings indicate that the role of miR-21 in predicting prognosis may be controversial but provide a new perspective of miR-21 level measurement in cancer specimens.

Effects of Fixation and Storage of Human Tissue Samples on Nucleic Acid Preservation

BACKGROUND: Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. METHODS: To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), beta-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for beta-actin (97 bp) were performed. RESULTS: All formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and beta-actin by PCR. hGH was successfully detected in all MFPE samples, but in only 46.7% of the FFPE samples. Prolonged formalin fixation resulted in fewer GAPDH and beta-actin PCR products, and amplification of hGH was not successful. The PCR and reverse transcription-PCR results were significantly affected by the duration of decalcification. The yield, purity, and integrity of mRNA progressively decreased with increased storage periods of paraffin blocks. CONCLUSIONS: Fixation and storage should therefore be standardized in order to improve the quality of molecular pathologic diagnosis.