Comprehensive immunoprofile analysis of prognostic markers in pancreaticobiliary tract cancers.

Pancreaticobiliary tract cancer has a poor prognosis with unmet needs in a new target treatment. Some studies have reported that an enhancement of T-cell immunity is associated with a good prognosis. The aim of this study is to investigate the immunoprofile as a prognostic marker of pancreaticobiliary tract cancers. Unresectable pancreatic ductal adenocarcinoma (PDAC, n = 80) and biliary tract cancer (BTC, n = 74) diagnosed between January 2012 and December 2018 in Samsung Medical Center were analyzed. Expression levels of CD8, FOXP3, PD-1, PD-L1, and CXCL13 in PDAC and BTC tissue samples were examined with immunohistochemical staining, which was evaluated with various clinical factors. In PDAC, higher degree of PD-L1 expression was significantly associated with shorter overall survival (OS) (p = 0.0095). On the other hand, higher infiltrations of PD-1+ immune cells (p = 0.0002) and CD8+ T cells (p = 0.0067) were associated with longer OS. In BTC, higher FOXP3+ (p = 0.0343) and CD8+ (p = 0.0028) cell infiltrations were associated with better survival. Low infiltration of CD8+ (p = 0.0148), FOXP3+ (p = 0.0208), PD-1+ (p = 0.0318) cells in PDAC, and FOXP3+ cells (p = 0.005) in BTC were considerably related to metastasis. In a combined evaluation of clinical factors and immunoprofiles, univariate analysis revealed that operation after chemotherapy (p < 0.0001), mass size (p = 0.0004), metastasis (p = 0.006), PD-L1 (p < 0.0001), PD-1 (p = 0.003) and CD8 (p = 0.0063) was significantly associated with OS in PDAC, and CD8 (p = 0.007) was statistically related to OS in BTC. In multivariate analysis, prognostic factors were operation after chemotherapy (p = 0.021) in PDAC and CD8 (p = 0.037) in BTC. Therefore, immunoprofile analysis of cells expressing CD8, FOXP3, PD-1, and PD-L1 might have prognostic values in patients with pancreaticobiliary tract cancers.

Role of Exosomes and Their Potential as Biomarkers in Epstein-Barr Virus-Associated Gastric Cancer.

Exosomes are a subtype of extracellular vesicles ranging from 30 to 150 nm and comprising many cellular components, including DNA, RNA, proteins, and metabolites, encapsulated in a lipid bilayer. Exosomes are secreted by many cell types and play important roles in intercellular communication in cancer. Viruses can hijack the exosomal pathway to regulate viral propagation, cellular immunity, and the microenvironment. Cells infected with Epstein-Barr virus (EBV), one of the most common oncogenic viruses, have also been found to actively secrete exosomes, and studies on their roles in EBV-related malignancies are ongoing. In this review, we focus on the role of exosomes in EBV-associated gastric cancer and their clinical applicability in diagnosis and treatment.

Indirect Clinical Validation of a Programmed Death-Ligand 1 Laboratory-Developed Test for Gastric/Gastroesophageal Junction Adenocarcinoma with 22C3 Antibody Concentrate.

BACKGROUND: The PD-L1 IHC 22C3 pharmDx used on the Dako Autostainer Link 48 (ASL48) staining platform is an established method for assessing programmed death-ligand 1 (PD-L1) expression in tumor tissue and determining patient eligibility for pembrolizumab treatment; however, the availability of this platform is limited in Europe and Asia. OBJECTIVES: The aims of this study were to develop and optimize protocols for the PD-L1 22C3 antibody concentrate with multiple immunohistochemistry staining platforms and to validate these protocols using PD-L1 combined positive score (CPS) with a cut-off of ≥ 1 in gastric or gastroesophageal junction adenocarcinoma. DESIGN: The 22C3 antibody concentrate was tested and optimized protocols were developed for use with three staining platforms: Dako ASL48, Ventana BenchMark ULTRA, and Leica BOND-MAX. Tumor specimens (N = 120) from patients with gastric or gastroesophageal junction adenocarcinoma were used for the validation study; these specimens were evaluated independently by three pathologists for PD-L1 CPS as a continuous variable and using a cut-off of ≥ 1. PD-L1 IHC 22C3 pharmDx used on the Dako ASL48 platform served as the reference or gold standard. RESULTS: The intraclass correlation coefficient of CPS as a continuous variable between the gold standard and each staining platform assessed was 0.910-0.989. When CPS was dichotomized based on a cut-off of ≥ 1, depending on the pathologist and the platform used, positive percentage agreement was 81-99% and negative percentage agreement was 90-100%. Interobserver agreement using the gold standard showed substantial agreement (κ = 0.779). CONCLUSION: The PD-L1 22C3 antibody concentrate can potentially be used with the laboratory-developed test on three commercially available immunohistochemistry staining platforms to determine PD-L1 expression in tumor samples from patients with gastric or gastroesophageal junction adenocarcinoma.

Clinicoradiological and histopathological characteristics and treatment outcomes of cerebral astroblastoma in children: a single-institution experience.

OBJECTIVE: Astroblastoma (AB) is a rare glial tumor. The optimal treatment and prognosis of this tumor remain unclear. The authors retrospectively analyzed the clinical characteristics, neuroimaging findings, histopathological results, and treatment outcomes of 7 patients with AB. METHODS: The study comprised 7 patients with pathologically proven AB who were surgically treated at Samsung Medical Center from November 1994 to January 2019. Clinicoradiological, histopathological, and surgical records were reviewed. RESULTS: The patients included 5 girls (71.4%) and 2 boys (28.6%), with a median age of 13 years. All patients showed contrast enhancement on preoperative MRI: 5 ABs (71.4%) showed a concomitant solid and cystic appearance, and 2 (28.6%) demonstrated a solid appearance. ABs in 6 patients (85.7%) showed a well-circumscribed, characteristic "bubbly" appearance on T2-weighted MRI. Gross-total resection (GTR) was achieved in all cases (100%). Six patients (85.7%) were diagnosed with high-grade AB and 1 (14.3%) with low-grade AB. Six (85.7%) of the 7 patients received adjuvant treatment after resection, including 5 (83.3%) with AB who received chemotherapy and radiotherapy and 1 (16.7%) who received proton therapy alone. The median clinical follow-up duration was 96 months (range 48-189 months). Two patients experienced recurrence, and all patients in this series were alive at the last follow-up. CONCLUSIONS: In this study, the clinicoradiological and histopathological features of AB were described. Based on the authors' limited experience with 7 cases, resection with the goal of GTR is currently the mainstream treatment for AB, and adjuvant radiation treatment should be considered after surgery.

Deep learning-based virtual cytokeratin staining of gastric carcinomas to measure tumor-stroma ratio.

The tumor-stroma ratio (TSR) determined by pathologists is subject to intra- and inter-observer variability. We aimed to develop a computational quantification method of TSR using deep learning-based virtual cytokeratin staining algorithms. Patients with 373 advanced (stage III [n = 171] and IV [n = 202]) gastric cancers were analyzed for TSR. Moderate agreement was observed, with a kappa value of 0.623, between deep learning metrics (dTSR) and visual measurement by pathologists (vTSR) and the area under the curve of receiver operating characteristic of 0.907. Moreover, dTSR was significantly associated with the overall survival of the patients (P = 0.0024). In conclusion, we developed a virtual cytokeratin staining and deep learning-based TSR measurement, which may aid in the diagnosis of TSR in gastric cancer.

Establishment and preclinical application of a patient-derived xenograft model for uterine cancer.

BACKGROUND: The patient-derived xenograft (PDX) model is a promising translational platform for duplicating the characteristics of primary tumors. Here, we established and characterized PDX models of uterine cancer to demonstrate their utility for preclinical drug testing. MATERIALS AND METHODS: We generated PDX tumors surgically derived from 58 cases of uterine cancer. Subrenal capsule xenografts and primary tumors were compared using microscopic examination, short tandem repeat analyses, and targeted sequencing analyses. A phosphatidylinositol 3-kinase (PI3K) inhibitor was administered to mice whose PDX tumors harbored a PTEN deletion or PIK3CA mutation. We also generated an orthotopic PDX model using uterine horn implantation. RESULTS: Thirty-three (56.9%) PDXs were successfully generated and passaged to maintain tumors. The histological features of the PDX tumors were stable over subsequent passages. By contrast, the proportions of epithelial and mesenchymal components of carcinosarcoma PDX models varied by generation. Targeted sequencing analyses revealed that all mutated cancer-related genes were stable during establishment and subgrafting. Treatment with a PI3K inhibitor cased a significant decrease in tumor weight in the clear cell carcinoma PDX harboring a frameshift PTEN deletion (p = 0.049) and in the serous carcinoma PDX harboring a missense PI3KCA mutation (p = 0.003) compared with matched controls. We also successfully established orthotopic PDX models (3/3; 100.0%). CONCLUSIONS: The histological and genetic features of PDXs were similar to those of primary tumors. This model is a promising translational platform for preclinical testing of new anticancer drugs and will enable the personalized development of therapeutic options for uterine cancer.

PD-L1 expression in paired biopsies and surgical specimens in gastric adenocarcinoma: A digital image analysis study.

Programmed death-ligand 1 (PD-L1) expression in biopsies of gastric carcinoma may predict the results in corresponding surgical specimens. We compared PD-L1 immunohistochemistry (IHC) 22C3 pharmDx expression in paired biopsy and resection specimens. We also characterized the validity of a new PD-L1 assay using digital image analysis. PD-L1 IHC with 22C3 pharmDx and clone 73-10 was performed in 224 gastric cancer tissues (112 biopsies and paired surgical tissues) and the specimens were analyzed with the Leica Aperio Imagescope. For statistical analyses, the area under the receiver operating characteristic curve and R package were used. With 22C3 pharmDx, a PD-L1 combined positive score of ≥1 was found in 36 biopsied (32.14 %) and 53 surgical (47.32 %) samples. PD-L1 expression results were concordant in 71 cases (63.4 %) and discordant in 41 (36.6 %). The overall discordance rate was 36.61 % (95 % confidence interval 2.101-8.983) and the κ value was 0.254 with fair agreement. The sensitivity and specificity of biopsy PD-L1 to predict the results of the surgical specimen was 62 % and 73 %, respectively. The correlation of 22C3 pharmDx and clone 73-10 was high (correlation coefficient = 0.88). When only tumor cell staining was compared, this correlation was increased (correlation coefficient = 0.95). Our results indicated moderate association of PD-L1 expression between gastric biopsies and corresponding resected tumors. Results of PD-L1 assay with 73-10 are comparable to 22C3 pharmDx results.

Digital image analysis in pathologist-selected regions of interest predicts survival more accurately than whole-slide analysis: a direct comparison study in 153 gastric carcinomas.

Automatic quantification of biomarkers such as tumor-infiltrating lymphocytes and PD-L1 is one of the most studied topics in digital pathology image analysis (DIA). However, direct comparison between the DIA of a whole-slide image (WSI) and that of regions of interest (ROIs) chosen by pathologists has not been performed. In this study, we aimed to compare the prognostic value of tumor microenvironment markers CD8 and PD-L1, measured by DIA of WSIs and ROIs. We selected 153 primary gastric cancer tissues and stained them with CD8 and PD-L1. All IHC slides were scanned at ×200 magnification and ratios of CD8 and PD-L1 were measured in WSIs and ROIs from the invasive front, within the tumor, and the mucosa. Patients with high CD8 and PD-L1 ratios showed more favorable outcomes compared to those with low ratios. Pathologist-aided DIA predicted the survival of patients more accurately than WSI analysis (CD8, p = 0.025 versus p = 0.068; PD-L1, p = 0.008 versus p = 0.2). Although a high density of CD8+ T cells at the invasive front correlated best with patient survival, CD8 ratio in the mucosa could also predict patient outcome. In conclusion, CD8 and PD-L1 ratios measured by pathologist-aided DIA predicted survival more accurately than WSI analyses and ROIs at the invasive front correlated best with patient outcome.

Nivolumab for esophageal squamous cell carcinoma and the predictive role of PD-L1 or CD8 expression in its therapeutic effect.

INTRODUCTION: Nivolumab, a programmed death 1 (PD-1) inhibitor, has recently demonstrated efficacy as second-line therapy for esophageal squamous cell carcinoma (ESCC) patients in a phase III trial. We report real-world clinical outcomes of nivolumab therapy for ESCC patients. METHODS: ESCC patients refractory/intolerant to at least one line of chemotherapy and who received nivolumab as a subsequent line of therapy were included. The efficacy and safety of nivolumab and the predictive role of PD-L1 and CD8 expression were analyzed. RESULTS: Fifty-eight patients were analyzed for safety and survival outcomes, while 57 were analyzed for objective response rates (ORR) excluding one with no measurable lesions. Eleven patients achieved a partial response, leading to an ORR of 19.3%. The median response duration was 6.5 months (range 4.1-22.4). The median progression-free survival (PFS) and overall survival were 2.1 (95% confidence interval [CI] 1.8-2.3) and 7.4 (95% CI 4.8-10.0) months, respectively. Among patients with adequate samples, 56.9% (29/51), 27.5% (14/51), and 17.6% (9/51) expressed a combined positive score (CPS) ≥ 1, ≥ 10, and ≥ 20, respectively, while 24.4% (11/45) and 57.5% (23/40) were positive for intratumoral and peritumoral CD8 + T cell infiltration, respectively. A significantly longer PFS was observed in patients with a CPS ≥ 20 (7.5 [95% CI 1.8-13.1] vs. 1.9 [1.4-2.3] months, P = 0.05), and a trend towards better survival was seen in those with CPS ≥ 10 or intratumoral CD8 + T cell infiltration. CONCLUSIONS: Nivolumab is a valuable option at subsequent treatment lines for patients with advanced ESCC.

DNA-protein biomarkers for immunotherapy in the era of precision oncology.

The use of biomarkers to guide patient and therapy selection has gained much attention to increase the scope and complexity of targeted therapy options and immunotherapy. Clinical trials provide a basis for discovery of biomarkers, which can then aid in development of new drugs. To that end, samples from cancer patients, including DNA, RNA, protein, and the metabolome isolated from cancer tissues and blood or urine, are analyzed in various ways to identify relevant biomarkers. In conjunction with nucleotide-based, high-throughput, next-generation sequencing techniques, therapy-guided biomarker assays relying on protein-based immunohistochemistry play a pivotal role in cancer care. In this review, we discuss the current knowledge regarding DNA and protein biomarkers for cancer immunotherapy.

Clinical outcomes of immune checkpoint inhibitors for patients with recurrent or metastatic head and neck cancer: real-world data in Korea.

BACKGROUND: Anti-PD1 inhibitors have been approved for the treatment of recurrent or metastatic head and neck cancer (HNC), as a result of Global Phase III trials. However, the clinical outcomes of immune checkpoint inhibitors in patients who are not eligible for clinical trials or have various medical conditions have not been fully elucidated. METHODS: We retrospectively reviewed 46 patients with recurrent or metastatic HNC who received pembrolizumab or nivolumab between June 2016 and June 2019. RESULTS: Thirty-five patients had head and neck squamous cell carcinoma (HNSCC) affecting the oropharynx, oral cavity, hypopharynx, larynx, nasal cavity, or paranasal sinuses, and eleven patients had nasopharyngeal cancer (NPC). The median progression-free survival (PFS) and overall survival (OS) were 3.7 months and 6.8 months, respectively, for patients with HNSCC, and 4.3 months and 11.8 months, respectively, for patients with NPC. The objective response rate (ORR) in all patients was 21%. Of 30 patients with HNSCC, 5 patients achieved complete response and 2 achieved partial response (ORR 23%); 1 of 8 NPC patients achieved partial response (13%). Patients who previously underwent radiotherapy had better OS than those who did not (median OS, 7.6 months vs. 2.3 months, p = 0.006). OS was longer in patients treated with pembrolizumab than in those treated with nivolumab (median OS, 11.8 months vs. 6.8 months, p = 0.017). CONCLUSION: Consistent with previous reports, immune checkpoint inhibitors showed promising efficacy in patients with previously treated recurrent or metastatic HNC in a real-world setting.

PTEN Protein Loss and Loss-of-Function Mutations in Gastric Cancers: The Relationship with Microsatellite Instability, EBV, HER2, and PD-L1 Expression.

Inactivation of phosphatase and tensin homolog (PTEN) is caused by multiple mechanisms, and loss of PTEN activity is related to the progression of various cancers. In gastric cancer (GC), the relationship between the loss of PTEN protein expression and various genetic alterations remains unclear. The effects of microsatellite instability (MSI), Epstein-Barr virus (EBV), HER2 overexpression, and PD-L1 expression on PTEN mutation have not been fully explored. We performed comprehensive cancer panel tests with a cohort of 322 tumor samples from patients with advanced GC. Immunohistochemistry for PTEN protein was performed in all cases, and the loss of protein expression was defined as a complete absence of nuclear staining. In total, 34 cases (10.6%) had pathogenic PTEN mutations, of which 19 (55.9%) showed PTEN protein loss. The most common PTEN variants associated with protein loss were p.R130 (n = 4) followed by p.R335, p.L265fs, and deletions (n = 2). All the ten nonsense mutations identified in the samples resulted in PTEN inactivation. In the remaining 288 GC cases with wild-type PTEN, protein loss was found in 35 cases (12.2%). Thus, PTEN mutations were significantly associated with PTEN protein loss (p = 5.232 × 10-10), high MSI (p = 3.936 × 10-8), and EBV-positivity (p = 0.0071). In conclusion, our results demonstrate that loss-of-function mutations in PTEN are a frequent genetic mechanism of PTEN inactivation in GC.

Dysregulated miRNA in a cancer-prone environment: A study of gastric non-neoplastic mucosa.

Understanding cancer-prone environments is important to efficiently detect and prevent cancers. The associations between miRNA and cancer-prone environments are still largely unknown in gastric cancer (GC). Six miRNAs that are differentially expressed during gastric carcinogenesis were selected, and quantitative real-time PCR was performed in an independent training set (fresh non-tumor and tumor samples from 18 GC patients) and validation sets (set 1 with formalin-fixed paraffin-embedded non-tumor and tumor samples from 19 solitary GC and set 2 with 37 multiple GC patients). The results were compared with those of 37 gastric mucosa from 20 healthy volunteers. The expression levels of miR-26a, miR-375, and miR-1260 in gastric mucosa from healthy volunteers were statistically higher than that of non-tumorous gastric mucosa located 3 cm apart from the GC in the training set (miR-26a, P < 0.0001; miR-375, P = 0.0049; miR-1260, P = 0.0172), validation set 1 (miR-26a and miR-375, P < 0.0001; miR-1260, P = 0.0008), and validation set 2 (miR-26a, miR-375, and miR-1260, P < 0.0001). And a combination of miR-26a and miR-1260 showed the highest area under the curve value of 0.89. miRNAs are differentially expressed in non-neoplastic gastric mucosa and can be used as a biomarker to predict cancer-prone environments.

Tumor Mutational Burden Determined by Panel Sequencing Predicts Survival After Immunotherapy in Patients With Advanced Gastric Cancer.

Objective: Panel-based sequencing is widely used to measure tumor mutational burden (TMB) in clinical trials and is ready to enter routine diagnostics. However, cut-off points to distinguish "TMB-high" from "TMB-low" tumors are not consistent and the clinical implications of TMB in predicting responses to immune checkpoint blockade (ICB) in gastric cancer are not clearly defined. We aimed to assess whether TMB is associated with the response to immunotherapy and to examine its relation with other biomarkers of immunotherapy response in advanced gastric cancer. Design: In total, 63 patients with advanced gastric cancer treated with ICB were included in the study. Panel-based TMB in gastric tumor samples, treatment responses to ICB, clinicopathological data, and time to progression were retrospectively analyzed. Microsatellite instability (MSI) status, Epstein-Barr virus (EBV) positivity, and programmed death-ligand 1 (PD-L1) combined positive score (CPS) were also analyzed. Results: TMB ranged from 0 to 446 mutations/megabase (mt/mb) and was significantly associated with MSI (P < 0.001), PD-L1 CPS (P = 0.022), response to ICB (P = 0.04), chemotherapy (P = 0.02) and older patient age (≥65 years; P = 0.0014). The cut-off point of 14.31 mt/mb determined by log-rank statistics for progression-free survival divided the tumors into eight (12.7%) TMB-high and 55 (87.3%) TMB-low tumors. The median TMB of the chemo-refractory group was significantly higher (8.43 mt/mb) compared to that of chemo-naïve group (3.42 mt/mb) (P = 0.02). Patients with TMB-high tumors showed prolonged progression-free survival in univariate [HR, 0.32; 95% confidence interval (CI), 0.12-0.90] and multivariate (HR, 0.21; 95% CI, 0.07-0.69) analyses. In area under the receiver operating curve (AUC) analysis of TMB, PD-L1, EBV, MSI, and their combination, the AUC value was the highest for EBV (0.97), followed by MSI (0.96), PD-L1 (0.81), the combination (0.78), and TMB (0.56). Conclusion: In addition to EBV, MSI, and PD-L1 CPS, TMB could be used as a predictive biomarker in patients with advanced gastric cancer treated with ICB and may aid clinical decision making.

PD-L1 expression in gastric cancer determined by digital image analyses: pitfalls and correlation with pathologist interpretation.

Programmed death ligand-1 (PD-L1) immunohistochemistry is used to predict response to anti-PD-L1 therapy. However, intra- and inter-observer variability influence the prediction of PD-L1 expression. Unlike evaluations of non-small cell lung cancer according to PD-L1 expression determined by tumor proportion score, gastric cancer is evaluated using combined positive scores. We aimed to compare the results of digital image analysis and pathologists' interpretations for predicting response to pembrolizumab in gastric cancer patients. Gastric cancer tissues from patients enrolled in a clinical trial of pembrolizumab were reviewed, and 39 cases were analyzed. PD-L1 22C3 PharmDx (Dako) immunohistochemistry slides were interpreted by digital image analysis and by the consensus of two pathologists, and the results of interpretations were compared with the eventual clinical responses to pembrolizumab. In direct comparisons of digital image analyses and pathologists' interpretations, 33 (84.6%) out of 39 cases showed concordant results. In 6 (15.4%) cases, weak staining of PD-L1, anthracotic pigment deposits, or decalcification artifacts caused discordant results, and all the cases were challenging. In statistical analyses, PD-L1 expression as interpreted by pathologists and digital image analysis did not differ significantly for predicting responses to pembrolizumab (P = 0.1856). Digital image analyses and pathologists' interpretations showed concordant results for PD-L1 combined positive scores in most cases, and the prediction performances of each were not significantly different.

Outcomes of endoscopic submucosal dissection for intestinal-type adenocarcinoma with anastomosing glands of the stomach.

BACKGROUND AND AIM: Gastric intestinal-type adenocarcinoma with anastomosing glands (IAAG) is characterized by architectural abnormality with frequent anastomosing glands and low-grade cytologic atypia. Clinicopathologic features and long-term outcomes of endoscopic submucosal dissection (ESD) for IAAG remain unclear. METHODS: This study included 2828 patients who underwent ESD for early gastric cancers (EGCs) (78 IAAGs [2.6%] and 2893 well-differentiated [WD] or moderately differentiated [MD] EGCs [97.4%]). Clinicopathologic features and short-term and long-term outcomes of ESD for IAAG were reviewed and compared with those for WD or MD EGCs. RESULTS: Gastric IAAGs were larger and more likely to be confined to the lamina propria than WD or MD EGCs. Histological heterogeneity, flat or depressed lesion and lateral resection margin (LRM) involvement were observed with significantly higher frequencies in IAAGs than in WD or MD EGCs. En bloc with R0 resection and curative resection rates of IAAGs were 79.5% and 73.1%, respectively, and both were significantly lower than those of WD or MD EGCs (93.8% and 82.9%). LRM involvement accounted for 57.1% of the non-curative resection cases in gastric IAAGs. Half of IAAGs with LRM involvement had a crawling pattern at tumor periphery. Among patients undergoing curative ESD for IAAG, no recurrences occurred during a median 52 months of follow-up. No lymph node metastasis was found in any of IAAG patients undergoing additional surgery after ESD. CONCLUSIONS: Gastric IAAGs have distinct clinicopathologic features from WD or MD EGCs. Given the favorable long-term outcomes after curative resection, ESD can be indicated for early gastric IAAGs.

Cancer Panel Assay for Precision Oncology Clinic: Results from a 1-Year Study.

Next-generation sequencing (NGS)-based cancer panel tests are actively being applied in the clinic for precision oncology. Given the importance of NGS panel tests in the palliative clinical setting, it is critical to understand success rates, factors responsible for test failures, and the incidence of clinically meaningful genetic alterations. We performed NGS cancer panel test with tumors from the stomach (n = 234), colorectum (n = 196), and rare tumors (n = 105) from 535 recurrent or metastatic cancer patients for 1 year. Sequencing was successful in 483 (95.3%) archival tumor samples to find single nucleotide variant (SNV), copy number alteration (CNA), and fusion. NGS testing was unsuccessful in 52 (9.7%) specimens due to inadequate tissue (n = 28), low tumor volume (n = 19), and poor quality of nucleic acid (n = 5). According to the Tier system, variants were classified as Tier IA, 0.8%; IIC, 10.3%; IID, 2.0%; III, 66.7% for gastric: Tier IA, 3.6%; IIC, 11.6% for colorectal: Tier IA, 1.6%; IIC, 13.5%; IID, 0.5%; III, 70.8% for melanoma, and Tier IA, 9.1%; IIC, 1.8%; IID, 1.0%; III, 66.4% for GIST. In total, 30.8% of 483 sequenced cases harbored clinically meaningful variants. In Tier IA, KRAS and ERBB2 were the most commonly altered genes. Interestingly, we identified CD274 (PD-L1) amplification, PTPN11 (SHP2) SNV, TPM3-NTRK1 fusion, and FGFR3-TACC3 fusion as a rare (<2%) alteration having therapeutic targets. In conclusion, although small biopsy samples constitute half of cases, informative NGS results were successfully reported in >90% of archival tissue samples, and 30.8% of them harbored clinically meaningful variants.

CCNE1 amplification is associated with liver metastasis in gastric carcinoma.

With targeted therapies becoming the new standard of care in oncology, next generation sequencing (NGS) is emerging as a valuable method for analyzing the molecular underpinnings of individual tumors. Cyclin E1, encoded by CCNE1 causes activation of E2F mediated transcription and drives cells from G1 into S phase with cyclin-dependent kinase 2 (CDK2). CCNE1 amplification has been found in 11-12% of gastric cancers, but the clinical significance of this amplification remains controversial, and its association with liver metastasis has not been studied. This study included 226 patients diagnosed with advanced gastric adenocarcinoma. We performed multi-gene panel tests containing 143 genes using DNA and RNA obtained from primary (n = 197; 120 endoscopic biopsies and 77 resections) or metastatic cancer tissues (n = 29; 26 biopsies, 2 excisions, and 1 fin. needle aspiration). Among the 226 cases, 28 cases (12.4%) had CCNE1 amplification, almost half of which (n = 13, 46.4%) showed liver metastasis. In patients with CCNE1 amplification (n = 28), TP53 mutations (n = 23, 82.1%) and ERBB2 amplification (n = 8, 28.6%) were the most frequent concurrent genetic alterations. In contrast, 42 (21.2%) of 198 patients without CCNE1 amplification showed liver metastasis. CCNE1 amplification was significantly associated with liver metastasis (p = 0.004; odds ratio, 3.219). Our results show that CCNE1 amplification is significantly associated with liver metastasis in a TP53-mutated gastric cancer subtype. Given the frequent association of CCNE1 amplification with liver metastasis, close follow up for liver metastasis and further clinical trials targeting CDK2 inhibitors are warranted.

Tumor Heterogeneity Index to Detect Human Epidermal Growth Factor Receptor 2 Amplification by Next-Generation Sequencing: A Direct Comparison Study with Immunohistochemistry.

Intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS)-based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. GC tissues were obtained via biopsy (N = 103) and gastrectomy (N = 65). HER2 3+ by IHC was observed in 27 cases (16%), and 14 of 27 HER2 IHC 3+ cases (52%) showed intratumoral heterogeneity (<90% of tumor cells are HER2 3+). Of 27 HER2 3+ cases, 19 (70%) were detected by NGS. All eight cases with discrepant IHC and NGS results harbored intratumoral heterogeneity. The receiver operating characteristic curve analysis showed that the optimal value of THI to be detected by NGS was a score of 72 for biopsy specimens and 120 for resection specimens. In conclusion, intratumoral heterogeneity of HER2 expression is observed in 52% of metastatic GC cases. NGS laboratories should be aware that accuracy of ERBB2 copy number alteration detection by NGS is influenced by the THI.