Seiichi Kakegawa

Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma

Epidermal growth factor receptor (EGFR) gene mutations have been reported to be clinically significant in non-small cell lung cancer (NSCLC). However, because most previous studies focused only on adenocarcinomas, EGFR mutations in other histotypes are poorly investigated. We evaluated the frequency of EGFR gene mutations in squamous cell carcinoma (SCC) and its clinicopathological features. In total, 89 frozen tumor specimens that had been first diagnosed as SCCs, were examined for EGFR mutations in exons 19 and 21 using direct sequencing, PNA-enriched sequencing and SmartAmp2. Additionally, pathological investigation, including immunostaining for p63 and TTF-1, alcian blue staining and EGFR mutation-specific immunohistochemistry in mutation-positive samples was also performed. The frequency of EGFR mutations was 5.6% (5/89); all mutations were deletions in EGFR exon 19. Immunohistological investigation of these samples revealed that two of five were positive for p63 and TTF-1 staining, and showed production of mucin, as evidenced by alcian blue staining. Consequently, three of the samples were considered to be true SCC at final pathological diagnosis, while the remaining two samples were revised to adenosquamous carcinoma and adenocarcinoma. The final frequency of the EGFR mutations in true SCC was 3.4% (3/87). In conclusion, EGFR mutations were found in a small, but significant, number of SCC tumor samples and thus EGFR mutational analysis was useful in the accurate diagnosis of SCC. Our data demonstrate that EGFR mutational analysis should be performed not only in adenocarcinoma, but also in SCC to allow accurate diagnosis and treatment.

Clinicopathological features of lung adenocarcinoma with KRAS mutations.

KRAS and epidermal growth factor receptor (EGFR) mutations are thought to play an important role in the carcinogenesis of lung adenocarcinoma. However, clinicopathological findings of KRAS mutated adenocarcinoma cases have not yet been fully clarified. The authors analyzed the relationship between the KRAS mutation and corresponding clinicopathological findings, focusing on nonmucinous and mucinous bronchioloalveolar elements.

Oncogenic KRAS-induced interleukin-8 overexpression promotes cell growth and migration and contributes to aggressive phenotypes of non-small cell lung cancer.

The CXC chemokine interleukin‐8 (IL‐8) is an angiogenic growth factor that is overexpressed in various cancers, including non‐small cell lung cancer (NSCLC). Previously, IL‐8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS‐driven NSCLC. Using microarray analysis, we identified IL‐8 as the most downregulated gene by shRNA‐mediated KRAS knockdown in NCI‐H1792 NSCLC cells where IL‐8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL‐8, while IL‐8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL‐8 expression was downregulated by shRNA‐mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL‐8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL‐8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL‐8 expression, while the p38 inhibitor increased IL‐8 expression. Attenuation of IL‐8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL‐8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL‐8 expression in NSCLC; IL‐8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS‐mutated adenocarcinomas; and IL‐8 plays a role in cell growth and migration in oncogenic KRAS‐driven NSCLC.

Mutation detection of epidermal growth factor receptor and KRAS genes using the smart amplification process version 2 from formalin-fixed, paraffin-embedded lung cancer tissue.

Recent evidence indicates that the presence of epidermal growth factor receptor (EGFR) or KRAS mutations in non-small cell lung cancer (NSCLC) can predict the response of the tumor to gefinitib. However, it is difficult to detect these mutations using formalin-fixed, paraffin-embedded (FFPE) tissues because the fixation process and aging can damage the DNA. In this study, we describe our work in adapting the Smart Amplification Process version 2 (SmartAmp2) to detect EGFR or KRAS mutations in DNA extracted from FFPE tissues. We were able to detect these mutations in 37 (97%) of 38 FFPE lung cancer tissue samples within 60 minutes with the SmartAmp2 assay and to confirm the correlation between EGFR mutations in FFPE tissues and gefitinib responsiveness. All mutations had previously been confirmed in the 38 samples using DNA extracted from frozen tissues. Electrophoresis results indicated that PCR analysis was not reliable for DNA extracted from FFPE tissue when primers with a long amplicon (>300 bp) were used. This study confirms that the SmartAmp2 assay is suitable for use with DNA extracted from FFPE as well as frozen tissues.

Usefulness of Peptide Nucleic Acid (PNA)-Clamp Smart Amplification Process Version 2 (SmartAmp2) for Clinical Diagnosis of KRAS Codon12 Mutations in Lung Adenocarcinoma Comparison of PNA-Clamp SmartAmp2 and PCR-Related Methods

KRAS is an oncogene that can be activated by mutations. Patients with non-small cell lung cancer who have KRAS mutations do not respond to tyrosine kinase inhibitors; therefore, accurate detection of KRAS mutations is important for deciding therapeutic strategies. Although sequencing-related techniques have been frequently used, they are usually too complex, have low sensitivity, and are time-consuming for routine screening in clinical situations. We evaluated peptide nucleic acid (PNA)-clamp smart amplification process version 2 (SmartAmp2) as a detection method for KRAS codon 12 mutations in patient specimens compared with traditional sequencing and polymerase chain reaction-related methods. Among 172 lung adenocarcinoma samples, direct sequencing, enzyme-enriched sequencing, and PNA-enriched sequencing showed that 16 (9.3%), 26 (15.7%), and 28 (16.3%) tumors, respectively, contained KRAS mutations in codon 12. Using PNA-clamp SmartAmp2, we could identify 31 (18.0%) tumors that had KRAS mutations in codon 12 within 60 minutes, three of which were undetected by polymerase chain reaction-related methods. On the other hand, we examined 30 nonmalignant peripheral lung tissue specimens and found no mutations in any of the samples using PNA-clamp SmartAmp2. In this study, we confirmed that PNA-clamp SmartAmp2 has high sensitivity and accuracy and is suitable for the clinical diagnosis of KRAS codon 12 mutations.

Clinicopathological and therapeutic significance of CXCL12 expression in lung cancer.

Interactions between CXCL12 and its receptors CXCR4 or CXCR7 are involved in tumor growth and metastasis in various types of human cancer. However, CXCL12 expression and its role in lung cancer are not fully elucidated. Here we examined the expression of CXCL12 in 54 lung cancer cell lines consisting of 23 small cell lung cancers (SCLCs) and 31 non-small cell lung cancers (NSCLCs). CXCL12 was overexpressed in lung cancer cell lines compared to non-malignant human bronchial epithelial cell lines (N = 6). CXCL12 expression was positively but weakly correlated with the expression of CXCR4 or CXCR7. We also examined CXCL12 expression in 89 NSCLC specimens and found that CXCL12 expression was significantly higher in tumor specimens from female patients, non-smokers and adenocarcinoma patients. Small interfering RNAs targeting CXCL12 inhibited cellular proliferation, colony formation and migration of CXCL12-overexpressing lung cancer cells; however, this inhibition did not occur in lung cancer cells that lacked CXCL12. Furthermore, the anti-CXCL12 neutralizing antibody mediated inhibitory effects in three lung cancer cell lines that overexpressed CXCL12, but not in two CXCL12 non-expressing lung cancer cell lines nor two non-malignant bronchial epithelial cell lines. The present study demonstrates that: CXCL12 is concomitantly overexpressed with CXCR4 or CXCR7 in lung cancers; CXCL12 is highly expressed in NSCLCs from females, non-smokers and adenocarcinoma patients; and disruption of CXCL12 inhibits the growth and migration of lung cancer cells. Our findings indicate that CXCL12 is required for tumor growth and provide a rationale for the anti-CXCL12 treatment strategy in lung cancer.

Video-assisted thoracic lobectomy with bronchoplasty for lung cancer, with special reference to methodology.

Few studies have described video-assisted thoracic surgery (VATS) to bronchoplasty with pulmonary resection. Here, we report the successful implementation of VATS bronchoplasty, as determined retrospectively. Between 2005 and 2010, 362 patients underwent elective lung resection for malignant or benign lung tumors. Of these patients, VATS lobectomy with bronchoplasty was performed in seven patients (four men, three women; median age, 72.9 years). The medical records were retrospectively reviewed. Of the seven patients, six had primary lung cancer (PLC), and one had metastatic cancer of the lung. The surgical procedures were lobectomy with wedge bronchoplasty. The patients with PLC also underwent mediastinal or hilar lymph node dissection. The median total operating time was 230 min, and the median blood loss was 152 ml. The median postoperative hospital stay was seven days, without major postoperative complications. The most important feature of the described method is that the surgeon mainly observes the operative field directly, through a working wound; the surgical team observes via a monitor. An advantage for the surgeon is the ability to use the same instruments in VATS as are used in conventional thoracotomy, as well as the same suturing techniques in vascular reconstruction, especially involving the pulmonary artery.

Establishment of a human lung cancer cell line with high metastatic potential to multiple organs: gene expression associated with metastatic potential in human lung cancer

Convenient and reliable multiple organ metastasis model systems might contribute to understanding the mechanism(s) of metastasis of lung cancer, which may lead to overcoming metastasis and improvement in the treatment outcome of lung cancer. We isolated a highly metastatic subline, PC14HM, from the human pulmonary adenocarcinoma cell line, PC14, using an in vivo selection method. The expression of 34,580 genes was compared between PC14HM and parental PC14 by cDNA microarray analysis. Among the differentially expressed genes, expression of four genes in human lung cancer tissues and adjacent normal lung tissues were compared using real-time reverse transcription polymerase chain reaction. Although BALB/c nude mice inoculated with parental PC14 cells had few metastases, almost all mice inoculated with PC14HM cells developed metastases in multiple organs, including the lung, bone and adrenal gland, the same progression seen in human lung cancer. cDNA microarray analysis revealed that 981 genes were differentially (more than 3-fold) expressed between the two cell lines. Functional classification revealed that many of those genes were associated with cell growth, cell communication, development and transcription. Expression of three upregulated genes (HRB-2, HS3ST3A1 and RAB7) was higher in human cancer tissue compared to normal lung tissue, while expression of EDG1, which was downregulated, was lower in the cancer tissue compared to the normal lung. These results suggest that the newly established PC14HM cell line may provide a mouse model of widespread metastasis of lung cancer. This model system may provide insights into the key genetic determinants of widespread metastasis of lung cancer.

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma.

The presence of EGFR mutations is correlated with a positive therapeutic response to tyrosine kinase inhibitors; therefore, the accurate detection of EGFR mutations is crucial when deciding appropriate therapeutic strategies. Recently, the rapid and sensitive assay smart amplification process version 2 (SmartAmp2) was developed. However, this method can only detect one type of mutation in EGFR exon 19; therefore, we applied the PNA technology to the SmartAmp2 assay to develop PNA-clamp SmartAmp2 for the detection of many types of deletions in EGFR exon 19, in a single reaction. This new assay was evaluated using 172 clinical samples. Thirty-nine (22.7%) samples were found to have deletions by PNA-clamp SmartAmp2; whereas 30 (17.4%) and 38 (22.1%) tumors were found to have deletions by direct sequencing and PNA-enriched sequencing, respectively. Three cases, in which we detected mutations with PNA-clamp SmartAmp2, but not with direct sequencing, were treated with gefitinib, and all cases showed a partial therapeutic response. Using clinical samples, we demonstrated that PNA-clamp SmartAmp2 can detect various types of mutations in EGFR exon 19 in a relatively short time and with high sensitivity. This method detected small amounts of mutant DNA and identified patients for whom clinical information was previously unavailable from other tests. This test may contribute to the administration of efficient therapeutic strategies.

Primary Thymic Mucosa-Associated Lymphoid Tissue Lymphoma: Diagnostic Tips

Mucosa-associated lymphoid tissue (MALT) lymphoma arising in the thymus is extremely rare and little is known regarding its clinicopathological features. This study examined the clinicopathological features of nine cases of thymic MALT lymphoma. Most patients had autoimmune disease or hyperglobulinemia, and they also had cysts in the tumors. Both increased serum autoantibody levels and polyclonal serum immunoglobulin levels remained essentially unchanged after total thymectomy in all patients. Thymic MALT lymphoma needs to be included in the differential diagnosis in Asian patients with a cystic thymic mass accompanied by autoimmune disease or hyperglobulinemia.