Soo Kyung Nam

Clinical significance of intratumoral HER2 heterogeneity in gastric cancer

AIM To evaluate the clinical significance of intratumoral HER2 heterogeneity in gastric cancer (GC). METHODS A total of 322 GC tissues were evaluated by HER2 immunohistochemistry (IHC), of which 73 with IHC 2+ or 3+ were subjected to fluorescence in situ hybridisation (FISH). Also, 3-5 distinct spots in each case showing different HER2 staining intensities were evaluated individually by comparing IHC staining intensity with gene copy number (GCN). Minimum, average and maximum FISH scores were generated for each case. RESULTS Intratumoral heterogeneity of HER2 overexpression and gene amplification were 54 and 30 of 73 cases with IHC 2+ or 3+, respectively. These cases were characterised by diffuse or mixed Lauren type, HER2 IHC 2+, and low-level amplification. Kaplan-Meier survival analysis revealed that the heterogeneous overexpression was significantly associated with longer disease-free survival times than the homogeneous, and the high average GCN was most associated with poor outcome. Also, there was a strong correlation between the IHC and FISH results for each spot. Quantitative polymerase chain reaction (PCR) analysis of the cancer tissues and the cell-free plasma showed that HER2 gene copy by quantitative PCR on tissue correlated well with those by FISH, but plasma HER2 level was not. CONCLUSIONS Considering the high incidence of intratumoral HER2 heterogeneity in GC, accurate HER2 assessment would require larger tissues and more detailed guidelines. The guidelines should include the recommendation that FISH-scoring areas be selected with reference to a corresponding IHC slide. Also, the definition of HER2-positive tumours should be reassessed considering the intratumoral heterogeneity.

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), β-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for β-actin (97 bp) were performed. Results All formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and β-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 β-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.

Clinicopathologic implications of immune classification by PD-L1 expression and CD8-positive tumor-infiltrating lymphocytes in stage II and III gastric cancer patients

We co-assessed PD-L1 expression and CD8+ tumor-infiltrating lymphocytes in gastric cancer (GC), and categorized into 4 microenvironment immune types. Immunohistochemistry (PD-L1, CD8, Foxp3, E-cadherin, and p53), PD-L1 mRNA in situ hybridization (ISH), microsatellite instability (MSI), and EBV ISH were performed in 392 stage II/III GCs treated with curative surgery and fluoropyrimidine-based adjuvant chemotherapy, and two public genome databases were analyzed for validation. PD-L1+ was found in 98/392 GCs (25.0%). The proportions of immune types are as follows: PD-L1+/CD8High, 22.7%; PD-L1−/CD8Low, 22.7%; PD-L1+/CD8Low, 2.3%; PD-L1−/CD8High, 52.3%. PD-L1+/CD8High type accounted for majority of EBV+ and MSI-high (MSI-H) GCs (92.0% and 66.7%, respectively), and genome analysis from public datasets demonstrated similar pattern. PD-L1−/CD8High showed the best overall survival (OS) and PD-L1−/CD8Low the worst (P < 0.001). PD-L1 expression alone was not associated with OS, however, PD-L1−/CD8High type compared to PD-L1+/CD8High was independent favorable prognostic factor of OS by multivariate analysis (P = 0.042). Adaptation of recent molecular classification based on EBV, MSI, E-cadherin, and p53 showed no significant survival differences. These findings support the close relationship between PD-L1/CD8 status based immune types and EBV+, MSI-H GCs, and their prognostic significance in stage II/III GCs.

BRAF, PIK3CA, and HER2 Oncogenic Alterations According to KRAS Mutation Status in Advanced Colorectal Cancers with Distant Metastasis

Background Anti-EGFR antibody–based treatment is an important therapeutic strategy for advanced colorectal cancer (CRC); despite this, several mutations—including KRAS, BRAF, and PIK3CA mutations, and HER2 amplification—are associated with the mechanisms underlying the development of resistance to anti-EGFR therapy. The aim of our study was to investigate the frequencies and clinical implications of these genetic alterations in advanced CRC. Methods KRAS, BRAF, and PIK3CA mutations were determined by Cobas real-time polymerase chain reaction (PCR) in 191 advanced CRC patients with distant metastasis. Microsatellite instability (MSI) status was determined by a fragmentation assay and HER2 amplification was assessed by silver in situ hybridization. In addition, KRAS mutations were investigated by the Sanger sequencing method in 97 of 191 CRC cases. Results Mutations in KRAS, BRAF, and PIK3CA were found in 104 (54.5%), 6 (3.1%), and 25 (13.1%) cases of advanced CRC, respectively. MSI-high status and HER2 amplification were observed in 3 (1.6%) and 16 (8.4%) cases, respectively. PIK3CA mutations were more frequently found in KRAS mutant type (18.3%) than KRAS wild type (6.9%) (P = 0.020). In contrast, HER2 amplifications and BRAF mutations were associated with KRAS wild type with borderline significance (P = 0.052 and 0.094, respectively). In combined analyses with KRAS, BRAF and HER2 status, BRAF mutations or HER2 amplifications were associated with the worst prognosis in the wild type KRAS group (P = 0.004). When comparing the efficacy of detection methods, the results of real time PCR analysis revealed 56 of 97 (57.7%) CRC cases with KRAS mutations, whereas Sanger sequencing revealed 49 cases (50.5%). Conclusions KRAS mutations were found in 54.5% of advanced CRC patients. Our results support that subgrouping using PIK3CA and BRAF mutation or HER2 amplification status, in addition to KRAS mutation status, is helpful for managing advanced CRC patients.

Human papillomavirus in breast carcinomas of Korean women.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #3086 Introduction The association between human papillomavirus (HPV) and cervical cancer is well documented. However, it remains unclear whether there is also a correlation between HPV infection and human breast cancer. The aim of this study was to investigate the status of HPV-DNA in breast carcinoma of Korean women and to examine the possible association between HPV and breast cancer development. Patients and Methods We examined the HPV-DNA of 154 patients including 123 cases of breast carcinoma, 31 cases of intraductal papilloma, and 27 cases of adequate nipple from cancer patients using DNA-chip method. Results The HPV-DNA was detected in 8 breast carcinomas (6.5%), but in none of the intraductal papillomas. All detected HPV was high-risk groups; the HPV-18 and HPV-70 were found in each two cases and each one cases HPV-16, 31, 56/58 and 59. In one case of nipple, both low-risk (HPV-62) and high-risk (HPV-18) group HPV-DNA was obtained. The papillary carcinomas and the invasive ductal carcinomas with adjacent intraductal papillomas showed slightly increased incidence (11% vs. 3-4%), however, there was no significant difference. No correlation between the presence of HPV-DNA and specific prognostic predictors for the disease outcome was observed. Conclusions Our results suggest that the presence of high-risk group HPV in the breast might be related to breast carcinogenesis, especially papillary phenotype. Further studies are warranted. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3086.

The quantification of HER2 and MYC gene fragments in cell-free plasma as putative biomarkers for gastric cancer diagnosis.

Abstract Background: This study aimed to investigate the significance of circulating HER2 and MYC gene fragments quantification in the diagnosis of gastric cancer. Methods: Levels of HER2 and MYC genes were evaluated by fluorescence in situ hybridization and real-time PCR in 81 gastric cancer tissues, and by real-time PCR in 36 gastritis tissues. Real-time PCR for HER2 and MYC products was also performed on 184 plasma samples from 81 gastric cancers, eight gastric adenomas, 63 gastritis patients, and 32 healthy individuals. Results: HER2/HBB and MYC/HBB ratios in tissue and cell-free plasma from gastric cancer patients were significantly higher than those of gastritis tissue and cancer-free individuals. An optimized cut-off value of plasma target gene to HBB ratio, used to differentiate cancer patients from cancer-free individuals, was evaluated using receiver operating characteristic (ROC) curves. Values of 2.0 were calculated for HER2 [area under the ROC curve (AUC), 0.760] and 2.725 for MYC (AUC, 0.767). A combination model of HER2 and MYC provided a better differentiation condition than that for HER2 or MYC only (AUC, 0.850). HER2/HBB ratios in plasma from gastric cancer patients correlated with MYC/HBB ratios. Conclusions: Our findings suggest that the measurement of plasma HER2 and MYC gene levels could improve the screening of gastric cancer.

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.

Fibroblast Growth Factor Receptor 1 Gene Copy Number and mRNA Expression in Primary Colorectal Cancer and Its Clinicopathologic Correlation.

Objectives: Fibroblast growth factor receptor 1 (FGFR1) has been reported to be overexpressed in colorectal cancer (CRC) and suggested to be a therapeutic target. In this study, we investigated FGFR1 expression and amplification in CRC and its correlation with clinicopathologic parameters. Methods:FGFR1 dual-color fluorescence in situ hybridization and mRNA in situ hybridization were performed on tissue array blocks composed of 291 consecutive primary CRCs. Results: Of the 291 CRC cases, FGFR1 gene amplification was found in 11 (3.8%) cases, high FGFR1 polysomy in 4 (1.4%) cases, and FGFR1 gene copy number (GCN) gain (GCN >2) in 77 (26.5%) cases. FGFR1 GCN gain was significantly associated with left-sided location, lymph node metastasis, distant metastasis, and higher TNM stage (p < 0.05). FGFR1 GCN gain also correlated with poor patient survival (p = 0.015). FGFR1 mRNA overexpression (score 3-4) was present in 11.7% (34/291) of the patients and was significantly associated with FGFR1 GCN alteration (Pearson correlation coefficient, r = 0.463; p < 0.001). Conclusion:FGFR1 GCN gain was more frequently found (26.5%) than gene amplification (3.8%) and correlated with aggressive clinical behavior in consecutive CRC patients. FGFR1 GCN alteration was associated with a high FGFR1 mRNA level.

Correlation between microsatellite instability-high phenotype and occult lymph node metastasis in gastric carcinoma.

The aim of this study is to investigate the association of microsatellite instability (MSI) status with nodal status in gastric carcinoma (GC). MSI status was investigated in 623 consecutively resected GCs. To detect occult lymph node (LN) metastasis, immunohistochemistry (IHC) using antibodies against pan‐cytokeratin was performed in 391 node‐negative cases by initial histologic examination. MSI‐high (MSI‐H) phenotype was found in 68 GC cases (10.9%) and was significantly associated with increased patient age, antral location, intestinal type, absence of venous/perineural invasion, and expanding growth type (p < 0.05). When the nodal status was evaluated, the number of metastatic LNs of MSI‐H tumors tended to be lower than that of microsatellite stable/MSI‐low (MSS/L) tumors (1.49 ± 3.15 vs 4.37 ± 9.81; p = 0.052), but the MSI‐H phenotype was associated with the presence of lymphatic invasion (p = 0.036) and IHC‐positive occult LN metastasis (p = 0.007). By multivariate analysis, MSI‐H phenotype was significantly associated with IHC‐positive occult LN metastasis (Odds ratio, 2.654; p = 0.044). MSI status and occult LN metastasis were not prognostic factors by survival analysis. Our findings suggest that the relationship between MSI status and regional LN metastasis may have some clinical and biologic implications to be elucidated.

Somatic mutational profiles of stage II and III gastric cancer according to tumor microenvironment immune type

We aimed to determine somatic mutational profiles of stage II/III gastric cancers (GCs) according to their tumor microenvironment immune types (TMITs), which classify cancer based on co‐assessment of PD‐L1 expression and CD8+ tumor infiltrating lymphocytes. Eighty patients with stage II/III GC were classified as follows: TMIT I (PD‐L1+/CD8High), TMIT II (PD‐L1−/CD8Low), TMIT III (PD‐L1+/CD8Low), and TMIT IV (PD‐L1−/CD8High). Deep targeted sequencing using a panel of 170 cancer‐related genes was performed on an Illumina HiSeq‐2500 system. Most frequently mutated genes included GNAQ (41.3%), TP53 (38.8%), CREBBP (35.0%), and MAP3K1 (35.0%). PIK3CA mutations were observed more frequently in TMIT I (45.8%) and III (66.7%), than in II (12.0%) and IV (8.0%). Other genes with enriched mutations within TMIT I included ATM (33.3%), BRCA2 (33.3%), MAP3K4 (29.2%), and FLT4 (25.0%). FGFR3, MAP3K1, and RUNX1 mutations were more frequently found in TMIT II. TMIT III had a unique somatic mutation profile harboring enriched mutations of histone modifiers including CREBBP and KMT2A, and we found FGFR2 amplification exclusively within TMIT IV. Fuzzy clustering analysis based on somatic mutation frequencies identified a hypermutated group (cluster 1) and a hypomutated group (cluster 2). Cluster 1 had significant associations with TMIT I, EBV+ GCs, and MSI‐H GCs (P = .023, .014, and .004), and had better overall survival (P = .057) than Cluster 2. TMIT I, EBV+, and MSI‐H GCs were estimated to have greater tumor mutational burden (P = .023, .003, and .015). By analyzing somatic mutation profiles according to TMIT classification, we identified TMIT‐specific genetic alterations that provide clues for biological linkage between GC genetics and microenvironment.