Jiro Okami

Quantitative detection of EGFR mutations in circulating tumor DNA derived from lung adenocarcinomas.

Purpose: Examination of somatic epidermal growth factor receptor (EGFR) mutations is now a diagnostic routine for treatment of cancer using EGFR tyrosine kinase inhibitors (EGFR-TKI). Circulating tumor DNA is a promising target for noninvasive diagnostics. We evaluated its utility by quantitatively detecting activating and resistant mutations, which were measured with BEAMing (beads, emulsion, amplification, and magnetics). Experimental Design: Twenty-three patients with lung cancer with progressive disease after EGFR-TKI treatment and 21 patients who had never been treated with EGFR-TKIs were studied. Their primary tumors were confirmed to have activating mutations. In the plasma DNA of each patient, the activating mutation found in the corresponding primary tumor and the T790M resistance mutation were quantified by BEAMing. Results: In 32 of 44 patients, activating mutations were detected in the plasma DNA [72.7%; 95% confidence interval (CI), 58.0%–83.6%]. The T790M mutation was detected in 10 of 23 patients in the first group (43.5%; 95% CI, 25.6%–53.4%). The ratio of T790M to activating mutations ranged from 13.3% to 94.0%. The peak of the distribution of the mutation allele fraction in the plasma DNA was in the 0.1% to 1% range. Conclusions: The major advantage of BEAMing is its ability to calculate the fraction of T790M-positive alleles from the alleles with activating mutations. This feature enables the detection of increases and decreases in the number of T790M mutations in cancer cells, regardless of normal cell DNA contamination, which may be useful for monitoring disease progression. Circulating tumor DNA could potentially be used as an alternative method for EGFR mutation detection. Clin Cancer Res; 17(24); 7808–15. ©2011 AACR.

Up-regulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus.

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies including squamous cell carcinomas (SCCs) of the esophagus, but little is known about COX-2 expression in premalignant esophageal squamous dysplasia. To elucidate the role of COX-2 in esophageal carcinogenesis, we examined the expression of this enzyme in normal squamous epithelium (n = 42), squamous dysplasia [high-grade dysplasia (HGD, n = 41; low-grade dysplasia (LGD, n = 33)]; carcinoma in situ (n = 16), mucosal invasive carcinoma (n = 18), and advanced SCC (n = 45). Immunohistochemistry showed a significantly high COX-2 expression in HGD compared with other lesions. The COX-2 score, an index determined by intensity and positivity of COX-2 staining (maximum 3.0), was 0.29 +/- 0.04 in normal esophagus, 1.75 +/- 0.11 in LGD, 2.89 +/- 0.05 in HGD, 2.17 +/-0.18 in CIS, 1.95 +/- 0.22 in mucosal invasive carcinoma, and 1.81 +/- 0.08 in advanced SCC. Results of reverse transcription-PCR assays confirmed those obtained by immunohistochemistry. COX-2 expression correlated with proliferation activity assessed by the proliferating cell nuclear antigen index in dysplastic lesions (P = 0.001) but not in SCCs. COX-2 expression in SCC did not correlate with various clinicopathological parameters including prognosis. Our results indicate that COX-2 is a sensitive marker for HGD and suggest that COX-2 may be involved in early stages of squamous carcinogenesis of the esophagus.

Intratumor heterogeneity of epidermal growth factor receptor mutations in lung cancer and its correlation to the response to gefitinib

Somatic mutations introduced into the epidermal growth factor receptor (EGFR) gene in non‐small‐cell lung cancer (NSCLC) are important factors to determine therapeutic responses to gefitinib. The current diagnostic test measures the overall EGFR mutation status of the cancer tissue, and may ignore the presence of non‐mutated, gefitinib‐unresponsive cancer cells. Twenty‐one NSCLC patients with EGFR mutations were recruited for the study. All patients were treated with gefitinib after surgical treatment. Fifty to sixty areas of NSCLC tumors were sampled from each tissue, and their EGFR mutation states were determined by a primer extension assay. This assay discriminates between EGFR mutation‐positive and ‐negative cancer cells within a single tumor tissue. Fifteen tissues consisted only of cells with EGFR mutations, but the remaining six tissues contained both mutated and non‐mutated cells. Time to disease progression and overall survival after gefitinib treatment were significantly shorter in those patients with EGFR heterogeneity (P = 0.009 and P = 0.003, respectively). A considerable proportion of NSCLC contains a heterogeneous population of both EGFR mutated and non‐mutated cancer cells, resulting in a reduced response to gefitinib. The intratumor genetic heterogeneity of a target molecule such as EGFR would be an important factor to consider when treating patients with molecular target agents. (Cancer Sci 2008; 99: 929–935)

Involvement of ribonucleotide reductase M1 subunit overexpression in gemcitabine resistance of human pancreatic cancer.

Pancreatic cancer is the most lethal of all solid tumors partially because of its chemoresistance. Although gemcitabine is widely used as a first selected agent for the treatment of this disease despite low response rate, molecular mechanisms of gemcitabine resistance in pancreatic cancer still remain obscure. The aim of this study is to elucidate the mechanisms of gemcitabine resistance. The 81‐fold gemcitabine resistant variant MiaPaCa2‐RG was selected from pancreatic cancer cell line MiaPaCa2. By microarray analysis between MiaPaCa2 and MiaPaCa2‐RG, 43 genes (0.04%) were altered expression of more than 2‐fold. The most upregulated gene in MiaPaCa2‐RG was ribonucleotide reductase M1 subunit (RRM1) with 4.5‐fold up‐regulation. Transfection with RRM1‐specific RNAi suppressed more than 90% of RRM1 mRNA and protein expression. After RRM1‐specific RNAi transfection, gemcitabine chemoresistance of MiaPaCa2‐RG was reduced to the same level of MiaPaCa2. The 18 recurrent pancreatic cancer patients treated by gemcitabine were divided into 2 groups by RRM1 levels. There was a significant association between gemcitabine response and RRM1 expression (p = 0.018). Patients with high RRM1 levels had poor survival after gemcitabine treatment than those with low RRM1 levels (p = 0.016). RRM1 should be a key molecule in gemcitabine resistance in human pancreatic cancer through both in vitro and clinical models. RRM1 may have the potential as predictor and modulator of gemcitabine treatment.

Pulmonary Resection in Patients Aged 80 Years or Over with Clinical Stage I Non-small Cell Lung Cancer: Prognostic Factors for Overall Survival and Risk Factors for Postoperative Complications

Introduction: This retrospective study was designed to identify the predictors of long-term survival and the risk factors for complications after surgery in patients aged 80 years or older with clinical (c)-stage I non-small cell lung cancer. Methods: The Japanese Joint Committee of Lung Cancer Registry collated the clinicopathological profiles and outcomes of 13,344 patients who underwent pulmonary resection for primary lung cancer in 1999. The data of 367 patients aged 80 years or older with c-stage I non-small cell lung cancer were analyzed for prognostic factors and risk factors for postoperative complications. Results: The median age was 82 years (range, 80-90 years). Of the total patient number, 102 (27.8%) had some form of comorbidity diagnosed preoperatively. Thirty-one (8.4%) patients presented with postoperative complications, and the operative mortality was 1.4%. The 5-year survival rates were 55.7% for c-stage I patients, 62.0% for c-stage IA, and 47.2% for c-stage IB. Advanced pathologic stage and comorbidity were significant independent predictors of shortened survival (p < 0.0001 and p = 0.032, respectively). Comorbidity and mediastinal lymph node dissection were identified as factors that increased the risk of postoperative complications (p < 0.0001 and p = 0.036, respectively). Survival rates were independent of the extent of pulmonary resection (lobectomy or limited resection). Conclusions: Octogenarian patients with c-stage I lung cancer in this study had a satisfactory long-term outcome and low-mortality rate. Comorbidity is a factor associated with both prognosis and operative risks. A selection of the patients who would be curable without mediastinal lymph node dissection after an accurate preoperative staging is beneficial to decrease the postoperative complications because this procedure is a risk factor.

Sublobar Resection Provides an Equivalent Survival After Lobectomy in Elderly Patients With Early Lung Cancer

BACKGROUND Sublobar resection is indicated for early-stage non-small cell lung cancer in patients with a perioperative risk associated with impaired medical conditions. This study was conducted to investigate the clinical impact of this procedure in the elderly. METHODS The patients who underwent complete resection for stage IA non-small cell lung cancer from 1990 and 2007 were enrolled (n = 764). Two age groups were defined as elderly (≥75 years) and younger (<75 years) patients. The 5-year survival, recurrence, and postoperative complications after sublobar resection were compared with those after standard lobectomy according to age group. RESULTS There were 133 elderly patients (79 standard lobectomies and 54 sublobar resections) and 631 younger patients (539 standard lobectomies and 92 sublobar resections). While the 5-year survival after sublobar resection was significantly inferior to that after standard lobectomy in the younger group (64.0% and 90.9%, respectively, p < 0.0001), however, no substantial difference was observed in the elderly (67.6% and 74.3%, p = 0.92). Locoregional recurrence rates were higher in patients after sublobar resection than those after standard lobectomy in both the elderly (11.1% vs 1.3%) and the younger (12.0% vs 1.5%) groups. No significant difference in postoperative complications was observed between the types of surgery in the elderly. CONCLUSIONS Sublobar resection for stage IA is considered to be an appropriate treatment in the elderly patients as this procedure provides an equivalent long-term outcome in comparison with lobectomy. A larger scale study with matching patients is necessary to confirm the noninferiority of sublobar resection in comparison with standard lobectomy in this population.

Occult mediastinal lymph node metastasis in NSCLC patients diagnosed as clinical N0-1 by preoperative integrated FDG-PET/CT and CT: Risk factors, pattern, and histopathological study

BACKGROUND Integrated F18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) is widely used for mediastinal lymph node (MLN) staging in patients with non-small cell lung cancer (NSCLC). However, FDG-PET/CT has certain limitations. Prediction of occult MLN metastasis could allow selection of candidates for preoperative cervical mediastinoscopy or endobronchial ultrasound-guided transbronchial needle aspiration. This study defined risk factors for occult MLN metastasis in patients with NSCLC patients who were diagnosed as clinical N0-1 by preoperative integrated FDG-PET/CT and CT. METHODS Consecutive patients with NSCLC who underwent staging using integrated FDG-PET/CT as an adjunct to CT prior to lung resection from October 2006 to September 2009 were evaluated retrospectively. The prevalence of MLN metastasis in patients diagnosed as clinical N0-1 was analyzed according to clinicopathological factors such as tumor location, tumor size, histology, and FDG uptake by the primary tumor. Risk factors for occult MLN metastasis were defined by multivariate analysis. Patterns of occult MLN metastasis were also analyzed and the involved MLNs were further examined histopathologically. RESULTS The incidence of MLN metastasis was 11% (24 patients of 224). Multivariate analysis identified adenocarcinoma (P=0.04), tumors located in upper or middle lobe (P=0.02), tumor size >3 cm (P=0.01), and SUV(max) of primary tumor >4.0 g/ml (P=0.04) as significant risk factors for MLN metastasis. The pattern of occult MLN metastasis was typical for NSCLC cases. The size of metastatic foci were small, with 68% of foci smaller than 4.0mm. CONCLUSIONS The present study demonstrated that adenocarcinoma, tumors located in the upper or middle lobe, tumor size >3 cm, and SUV(max) of primary tumor >4.0 g/ml are risk factors for occult MLN metastasis in patients with NSCLC who were diagnosed as clinical N0-1 by preoperative integrated FDG-PET/CT and CT. Patients with tumors located in the right upper or middle lobe are considered candidates for cervical mediastinoscopy because the involved metastatic mediastinal lymph nodes are easily accessible by these modalities.

Treatment strategy for patients with small peripheral lung lesion(s): intermediate-term results of prospective study

BACKGROUND This prospective study was undertaken to establish a novel management algorithm using new indicators to decide the type of lung resection for small peripheral lung lesions. METHODS Inclusion criteria were: (1) <or=20mm peripheral lung lesion(s) and (2) absence of significant lymph node swelling on preoperative CT. Along with the conventional criteria, the percentage of ground-glass opacity (GGO) (>or=50% as GGO type and <50% as solid type) on high-resolution CT scan was employed. In accordance with such indicators, a wide wedge resection (WWR), segmentectomy or lobectomy was planned for individual patients. The primary endpoint was to estimate the effectiveness of limited resection in patients with lung cancer by analyzing their locally disease-free survival rates at 5 and 10 years. RESULTS Of 179 patients enrolled between 1997 and 2002, 90 were male and 89 female. They were divided into 77 GGO types and 102 solid types. During surgery, conversions from limited resections to standard operations were performed on six patients to avoid the risk of local-regional recurrence. Finally, WWR was performed on 73 patients, segmentectomy on 26 and lobectomy on 80, respectively. There were 138 lung cancers and 41 non-cancers. Of 138 cancer patients, 114 patients are alive and 24 died. There were no local-regional recurrences among the 58 cancer patients who underwent limited resection. CONCLUSIONS This intermediate-term outcome suggests that the selection of the type for lung resection using this management algorithm for small peripheral lung lesions was effective for preventing both local-regional recurrences and the excessive resection of normal lung tissue.

Aberrant Expression of Connexin 26 Is Associated with Lung Metastasis of Colorectal Cancer

Purpose: Connexin 26 (Cx26) is one of the gap junction–forming family members classically considered to be tumor suppressors. However, recent studies show association of elevated expression of Cx26 with poor prognosis in several human malignancies. Furthermore, Cx26 has been observed to be indispensable to spontaneous metastasis of melanoma cells. Here, we assessed Cx26 expression in primary colorectal cancer (CRC) and the metastatic lesions to elucidate its role in metastasis. Experimental Design: Cx26 expression was assessed in 25 adenomas, 167 CRCs, and normal mucosa, together with the metastatic lesions. Results: Normal mucosa and adenomatous tissue expressed Cx26 mainly in the plasma membrane, whereas cancer cells mostly contained Cx26 in the cytoplasm. The incidence of aberrant Cx26 expression varied widely in CRC (mean, 49.5 ± 35.5%), and the expression levels were confirmed by Western blot and quantitative reverse transcription–PCR. Clinicopathologic survey revealed association of high expression with less differentiated histology and venous invasion (P = 0.0053 and P = 0.0084, respectively). Notably, high Cx26 expression was associated with shorter disease-free survival and shorter lung metastasis–free survival in 154 curatively resected CRC sets (P = 0.041 and P = 0.028, respectively). Survey of metastatic lesions revealed that lung metastasis, but not liver and lymph nodes metastases, expressed higher Cx26 than the CRC series or corresponding primary CRCs (P < 0.0001 and P = 0.0001, respectively). Conclusions: These findings suggest that aberrant expression of Cx26 plays an essential role in lung metastasis. Thus, Cx26 is a promising therapeutic target, particularly for CRC patients who develop lung metastasis.

Spheroid Culture of Primary Lung Cancer Cells with Neuregulin 1/HER3 Pathway Activation

Introduction: Primary culture of cancer cells is expected to be useful for investigating the biology of cancer and predicting chemosensitivity for individual patients, yet has been hampered by technical difficulties. We recently developed the cancer tissue–originated spheroid (CTOS) method for the primary culture of colorectal cancer cells. In the present study, we applied this system to the primary culture of non–small-cell lung cancer. Methods: We used 125 surgical specimens and 18 pleural effusions for CTOS preparation. Partially digested tumor fragments were cultured in a medium for embryonic stem cells. CTOSs were subjected to sensitivity assay and signal transduction assay for the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib. We also investigated the effects of growth factors in culturing lung cancer CTOS. Results: The success rate of CTOS preparation from surgical specimens was 80.0%. The CTOS method was also suitable for culturing tumor spheroids from pleural effusions. CTOSs from lung cancer consisted mostly of pure cancer cells. CTOSs and CTOS-derived xenografts retained the characteristics of the original tumors. In vitro assay results showed that EGFR mutation status and expression levels corresponded with erlotinib sensitivity, confirming previous clinical findings. Furthermore, we found that neuregulin 1, a ligand of HER3, potently induced CTOS growth. Conclusions: The CTOS method enables us to obtain primary lung tumor cells of high viability and purity. CTOS could be a new platform for studying lung cancer biology.