Masaharu Nomura

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (1) Stage-related protein candidates upon non-metastatic lung adenocarcinoma

We used formalin-fixed paraffin-embedded (FFPE) materials for biomarker discovery in cases of lung cancer using proteomic analysis. We conducted a retrospective global proteomic study in order to characterize protein expression reflecting clinical stages of individual patients with stage I lung adenocarcinoma without lymph node involvement (n=7). In addition, we studied more advanced stage IIIA with spread to lymph nodes (n=6), because the degree of lymph node involvement is the most important factor for staging. FFPE sections of cancerous lesions resected surgically from patients with well-characterized clinical history were subjected to laser microdissection (LMD) followed by Liquid Tissue solubilization and digestion trypsin. Spectral counting was used to measure the amounts of proteins identified by shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS). More than 500 proteins were identified from IA and IIIA cases, and non-parametric statistics showed that 81 proteins correlated significantly with stage IA or IIIA. A subset of those proteins were verified by multiple-reaction monitoring mass spectrometric quantitation (MRM assay), described in other paper in this issue. These results demonstrated the technical feasibility of a global proteomic study using clinically well documented FFPE sections, and its possible utility for detailed retrospective disease analyses in order to improve therapeutic strategy.

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma

A preceding paper suggested 81 candidates of stage-specifically expressed proteins for either stage IA or IIIA by global shotgun proteomics and spectral counting. Six proteins, a subset of these proteins, were chosen for a further verification study since they are potentially soluble and/or secretory, which nature is convenient for detecting them in blood in clinical practice. The multiple-reaction monitoring (MRM) quantitative analysis suggested that napsin-A and anterior gradient protein 2 homolog (hAG-2) out of the 6 candidates would be useful for determining stage IA or IIIA and are related to metastasis. In the study we noted that stage IIIA patients with better outcome showed napsin-A profiles similar to that of stage IA patients. We therefore examined 14 additional patients for analysis, which contained the IA-stage patients of poorer outcome and the IIIA-stage patients of better outcome. The MRM analysis of napsin-A for all patients suggests that napsin-A contents correlate with better outcome in stage IA. This and discovery studies demonstrate that direct isolation of tumor cells alone by laser microdissection (LMD) greatly reduces complexity on comprehensive analyses, and that MRM mass spectrometry using the endogenous internal standard is a feasible technology for quantitative verification of target proteins in formalin-fixed paraffin embedded (FFPE) tissues.

Pathological Vascular Invasion and Tumor Differentiation Predict Cancer Recurrence in Stage ia Non–Small-Cell Lung Cancer After Complete Surgical Resection

Introduction: The appropriate therapeutic strategy and postoperative management for patients with stage IA non–small-cell lung cancer (NSCLC) still remain a matter of debate because of the prognostic heterogeneity of this population, including the risk of cancer recurrence. The objective of the current study was to identify the clinicopathological factors that affect overall prognosis and cancer recurrence of stage IA NSCLC. Methods: We reviewed the data of 532 patients in whom complete resection of stage IA NSCLC had been performed. Overall survival and recurrence-free proportion (RFP) were estimated using the Kaplan–Meier method. RFP was estimated from the date of the primary tumor resection to the date of the first recurrence or last follow-up. We performed univariate and multivariate analyses to determine the independent prognostic factors. Results: On multivariate analyses, three variables were shown to be independently significant recurrence risk factors: histological differentiation (hazard ratio [HR] = 1.925), blood-vessel invasion (HR = 1.712), and lymph-vessel invasion (HR = 1.751). On subgroup analyses combining these risk factors, the 5-year RFP was 91.3% for patients with no risk factors, 79.5% for those with either poorly differentiated carcinoma or vascular invasion, (p < 0.001 for both), and 62.9% for those with both poorly differentiated carcinoma and vascular invasion (p = 0.068). Conclusion: These results indicated that vascular invasion and tumor differentiation have a significant impact on the prediction of cancer recurrence in patients with stage IA NSCLC. Patients with these predictive factors of recurrence may be good candidates for adjuvant chemotherapy.

Epidermal Growth Factor Receptor Tyrosine Kinase Defines Critical Prognostic Genes of Stage I Lung Adenocarcinoma

Purpose To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy. Patients and Methods Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF) in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK)-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM). “Gefitinib-sensitive” genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer. Results The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS) with a hazard ratio (HR) of 7.16 (P = 0.029) and 3.26 (P = 0.0072), respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC) histology in a Japanese cohort for OS and recurrence-free survival (RFS) with HRs of 8.79 (P = 0.001) and 3.72 (P = 0.0049), respectively. Conclusion The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis. Trial Registration The Gene Expression Omnibus (GEO) GSE31210

Prognostic impact of number of resected and involved lymph nodes at complete resection on survival in non-small cell lung cancer.

Background: Lymph node (LN) status is a major determinant of stage and survival in patients with lung cancer. In the 7th edition of the TNM Classification of Malignant Tumors, the number of involved LNs is included in the definition of pN factors in breast, stomach, esophageal, and colorectal cancer, and the pN status significantly correlates with prognosis. Methods: We retrospectively investigated the prognostic impact of the number of resected LNs (RLNs) and involved LNs in the context of other established clinical prognostic factors, in a series of 928 consecutive patients with non-small cell lung cancer (NSCLC) who underwent complete resection at our institution between 2000 and 2007. Results: The mean number of RLNs was 15. There was a significant difference in the total number of RLNs categorized between less than 10 and ≥10 (p = 0.0129). Although the incidence of LN involvement was statistically associated with poor prognosis, the largest statistically significant increase in overall survival was observed between 0 to 3 and ≥4 involved LNs (hazard ratio = 7.680; 95% confidence interval = 5.051–11.655, p < 0.0001). On multivariate analysis, we used the ratio between the number of involved LNs and RLNs. The number of RLNs was found to be a strong independent prognostic factor for NSCLC (hazard ratio = 6.803; 95% confidence interval = 4.137–11.186, p < 0.0001). Conclusion: Complete resection including 10 or more LNs influenced survival at complete NSCLC resection. Four involved LNs seemed to be a benchmark for NSCLC prognosis. The number of involved LNs is a strong independent prognostic factor in NSCLC, and the results of this study may provide new information for determining the N category in the next tumor, node, metastasis classification.

Klotho predicts good clinical outcome in patients with limited-disease small cell lung cancer who received surgery

BACKGROUND The important role of surgery in early-stage small cell lung cancer (SCLC) has been recognized, and curative surgical resection is recommended. However, the role of adjuvant chemotherapy for stage I SCLC has not yet been evaluated, and novel approaches focusing on the specific genomic characteristics of SCLC may be invaluable for customized therapy. In this study, we focused on the Klotho gene, which is an anti-aging gene known to be a potential tumor suppressor. We investigated whether the expression of Klotho, assessed by immunohistochemistry, can predict survival in patients with resected SCLC. METHODS The medical records of patients diagnosed as having limited-disease (LD) SCLC and treated by surgical resection (n=30) at Tokyo Medical University Hospital were retrospectively reviewed. The expression status of Klotho, and of the ATP-binding cassette (ABC) transporters MRP1, MDR and breast cancer resistant protein (BCRP), which can cause resistance to anticancer drugs, including irinotecan, was assessed by immunohistochemical analysis in resected surgical specimens of patients with early-stage SCLC. RESULTS Of the 30 patients, Klotho expression was seen in the specimens from 18 patients (60.0%), but not in those of the remaining 12 patients (40.0%). The immunostaining for Klotho was mostly localized in the cytoplasm. The expression of Klotho was significantly associated with the overall survival (OS) (ratio 0.088; 95% confidence interval 0.019-0.409; P=0.002). The administration of perioperative chemotherapy had no significant effect in improving the survival, as assessed by the Kaplan-Meier method. However, the patients showing Klotho expression in the resected specimens in p-stage I and II, may have benefited from perioperative chemotherapy. A multivariate analysis revealed no significant association between the expression status of MRP1, MDR or BCRP and the OS. CONCLUSION Expression of Klotho was predictive of a favorable outcome following resection in limited-disease SCLC patients, and the Klotho expression status may serve as a new biomarker for the need of additional therapies to be developed in the future.

Impact of visceral pleural invasion on the survival of patients with non-small cell lung cancer.

BACKGROUND In this study, we investigated visceral pleural invasion (VPI) as a poor prognostic factor in patients with non-small cell lung cancer (NSCLC) according to the 7th edition of the TNM classification. METHODS Between January 2000 and December 2007, 886 consecutive patients with pathological T1a-T2b NSCLC underwent complete resection with systematic lymph node dissection in Tokyo Medical University. We statistically analyzed the association between VPI and clinicopathologic factors, or clinical outcomes. RESULTS The 5-year overall survival (OS) rates of the pl0, pl1, and pl2 patients were 80.8%, 63.7%, and 49.6%, respectively, with significant differences between pl0 and pl1 (p=0.002), pl1 and pl2 (p=0.03). Thus, the pl1 and pl2 patient groups were defined as patients with VPI. VPI was found to be a significant independent prognostic factor by multivariate survival analysis (p=0.0002). In patients with tumors ≤3 cm, especially with tumors ≤2 cm, VPI was significantly associated with an increased rate of lymph node metastasis, compared with non-VPI (p=0.0003 and p=0.015, respectively). Analysis of the OS of patients stratified by tumor size (≤3 cm, 3.1-5 cm, 5.1-7 cm) and VPI status showed that in any nodal status, patients with 3.1-5 cm/VPI tumors had significantly worse survival than patients with ≤3 cm/VPI tumors (p=0.019) and patients with 3.1-5 cm/non-VPI tumors (p=0.001). On the other hand, there was no significant difference in the OS between patients with 3.1-5 cm/VPI tumors and patients with 5.1-7 cm tumors regardless of lymph node metastasis (T2b tumors). Similar relationships were observed among these groups with N0 disease. CONCLUSION We identified the presence of VPI as an independent poor prognostic factor in patients with NSCLC of ≤7 cm. Tumors 3.1-5cm with VPI should be upstaged to T2b tumors in the future in the TNM classification of the Union of International Cancer Control staging system. In addition, the surgical strategy involving more extensive lymph node dissection for patients with ≤3 cm/VPI tumors, especially ≤2 cm/VPI, is warranted owing to more frequent lymph node metastasis.

Preferential expression of potential markers for cancer stem cells in large cell neuroendocrine carcinoma of the lung. An FFPE proteomic study

BackgroundLarge cell neuroendocrine carcinoma (LCNEC) of the lung, a subtype of large cell carcinoma (LCC), is characterized by neuroendocrine differentiation that small cell lung carcinoma (SCLC) shares. Pre-therapeutic histological distinction between LCNEC and SCLC has so far been problematic, leading to adverse clinical outcome. We started a project establishing protein targets characteristic of LCNEC with a proteomic method using formalin fixed paraffin-embedded (FFPE) tissues, which will help make diagnosis convincing.MethodsCancer cells were collected by laser microdissection from cancer foci in FFPE tissues of LCNEC (n = 4), SCLC (n = 5), and LCC (n = 5) with definite histological diagnosis. Proteins were extracted from the harvested sections, trypsin-digested, and subjected to HPLC/mass spectrometry. Proteins identified by database search were semi-quantified by spectral counting and statistically sorted by pair-wise G-statistics. The results were immunohistochemically verified using a total of 10 cases for each group to confirm proteomic results.ResultsA total of 1981 proteins identified from the three cancer groups were subjected to pair-wise G-test under p < 0.05 and specificity of a proteins expression to LCNEC was checked using a 3D plot with the coordinates comprising G-statistic values for every two group comparisons. We identified four protein candidates preferentially expressed in LCNEC compared with SCLC with convincingly low p-values: aldehyde dehydrogenase 1 family member A1 (AL1A1) (p = 6.1 × 10-4), aldo-keto reductase family 1 members C1 (AK1C1) (p = 9.6x10-10) and C3 (AK1C3) (p = 3.9x10-10) and CD44 antigen (p = 0.021). These p-values were confirmed by non-parametric exact inference tests. Interestingly, all these candidates would belong to cancer stem cell markers. Immunohistochmistry supported proteomic results.ConclusionsThese results suggest that candidate biomarkers of LCNEC were related to cancer stem cells and this proteomic approach via FFPE samples was effective to detect them.

Prognostic Factors and the Significance of Treatment After Recurrence in Completely Resected Stage I Non-small Cell Lung Cancer

OBJECTIVE The objective of this study was to identify the clinicopathologic factors influencing postrecurrence survival (PRS) in and the effect of postrecurrence therapy (PRT) on patients with completely resected stage I non-small cell lung cancer (NSCLC). METHODS We reviewed the data of 919 patients in whom complete resection of stage I NSCLC had been performed. RESULTS Of the 919 patients, 170 (18.5%) had recurrent disease. Initial PRT was performed in 118 patients (69.1%) (surgery in eight, chemotherapy in 79, radiotherapy in 10, and chemoradiotherapy in 21). On multivariate analyses, PRT (hazard ratio [HR], 0.542; 95% CI, 0.344-0.853; P = .008), female sex (HR, 0.487; 95% CI, 0.297-0.801; P = .005), and differentiation (HR, 1.810; 95% CI, 1.194-2.743; P = .005) demonstrated a statistically significant association with favorable PRS. Bone metastasis (HR, 3.288; 95% CI, 1.783-6.062; P < .001), liver metastasis (HR, 4.518; 95% CI, 1.793-11.379; P = .001), chemotherapy (HR, 0.478; 95% CI, 0.236-0.975; P = .040), epidermal growth factor receptor-tyrosine kinase inhibitors treatment (EGFR-TKIs) (HR, 0.460; 95% CI, 0.245-0.862; P = .015), and nonadenocarcinoma (HR, 2.136; 95% CI, 1.273-3.585; P = .004) were independently and significantly associated with PRS in the 118 patients who underwent any PRT. Subgroup analysis with a combination of these five PRS factors in the patients who underwent any PRT revealed median PRS times of 42.4 months for 20 patients lacking all five risk factors and 18.8 months for 98 patients with at least one of these risk factors (P = .001). CONCLUSIONS PRT, sex, and differentiation were independently associated with PRS. In the patients who underwent any PRT, PRS was related to EGFR-TKIs, chemotherapy, histology, and initial recurrence sites. One challenge for the future will be to create systematic treatment strategies for recurrent NSCLC according to the risk factor status of individual patients.

A proposal for combination of total number and anatomical location of involved lymph nodes for nodal classification in non-small cell lung cancer.

BACKGROUND We previously reported the prognostic impact of the number of involved lymph nodes (LNs) on survival in non-small cell lung cancer (NSCLC). However, it remains unknown whether the total number or anatomic location of involved LNs is a superior prognostic factor. METHODS A total of 689 patients with NSCLC who underwent complete resection involving dissection of the hilar and mediastinal LNs with curative intent of ≥ 10 LNs were enrolled. The association between the total number of LNs (nN) involved and survival was assessed by comparison with the anatomic location of LN involvement (pathologic lymph node [pN]), the present nodal category. RESULTS We classified the patients into five categories according to the combined pN and nN status as follows: pN0-nN0, pN1-nN1-3, pN1-nN4-, pN2-nN1-3, and pN2-nN4. Although there was no statistically significant difference between the pN1-nN4- and pN2-nN1-3 categories, pN2-nN1-3 had better prognoses than pN1-nN4-. On multivariate analysis, the nN category was an independent prognostic factor for overall survival and disease-free survival (vs nN4-; the hazard ratios of nN0 and nN1-3 for overall survival were 0.223 and 0.369, respectively, P < .0001 for all), similar to the pN category. We propose a new classification based on a combination of the pN and nN categories: namely, N0 becomes pN0-nN0, the N1 category becomes pN1-nN1-3, the N2a category becomes pN2-nN1-3 + pN1-nN4-, and the N2b category becomes pN2-nN4. Each survival curve was proportional and was well distributed among the curves. CONCLUSIONS A combined anatomically based pN stage classification and numerically based nN stage classification is a more accurate prognostic determinant in patients with NSCLC, especially in the prognostically heterogeneous pN1 and pN2 cases. Further large-scale international cohort validation analyses are warranted.