Tomoyuki Hishida

Podoplanin expression by cancer associated fibroblasts predicts poor prognosis of lung adenocarcinoma

Recent studies have reported increased podoplanin expression by cancer cells and stromal cells, but little is known about its expression and biological significance in adenocarcinoma of the lung. We examined podoplanin expression by both cancer cells and stromal cells in 177 consecutive lung adenocarcinoma cases and analyzed relations between podoplanin expression and both clinicopathological factors and outcome. Podoplanin expression was observed on the apical membrane of the cancer cells in only 9 of the 177 (5.1%) cases. By contrast, cancer‐associated fibroblasts (CAFs) were found to express podoplanin in 54 cases (30.5%). Podoplanin (+) CAFs were found only in invasive adenocarcinoma and none were found in noninvasive adenocarcinoma. Conventional prognostic factors were significantly correlated with podoplanin expression by CAFs. The univariate analyses and log‐rank test showed that podoplanin expression was significantly associated with shorter survival time (p < 0.001 and p < 0.001, respectively). We divided the cases into 3 groups according grade based on the proportion of CAFs expressing podoplanin [a grade 0 group (n = 123), a grade 1 group (n = 36) and a grade 2 group (n = 18)]. The result showed that conventional prognostic factors were significantly correlated with the grade of podoplanin expression by CAFs. Furthermore, the grade 2 group tended to have a shorter survival time than the grade 1 group (p = 0.092). The results of this study highlight the importance of podoplanin expression by CAFs and provide new insights into the biology of the cancer microenvironment in adenocarcinoma of the lung.

Stromal Macrophage Expressing CD204 is Associated with Tumor Aggressiveness in Lung Adenocarcinoma

Background: Tumor tissue is composed of variable numbers of cancer cells and stromal cells, and tumor-associated macrophages are recruited into cancer-induced stroma and produce a specific microenvironment. Alternatively, activated macrophages (M2 phenotype) are known to be related to tumor progression and outcome, and CD204 has been reported to be expressed in M2 macrophages in some tumors. Methods: To investigate whether CD204-positive macrophages reflect tumor aggressiveness in adenocarcinoma of the lung, we investigated the relationships between the numbers of CD204-positive stromal macrophages and both clinicopathological features and outcome in 170 consecutive resected cases. We also examined the relationships between the numbers of CD204-positive macrophages and the expression levels of cytokines involved in the migration and differentiation of M2 macrophages. Results: The numbers of CD204-positive macrophages were significantly correlated with several prognostic factors. The log-rank test showed a significant association between the numbers of CD204-positive macrophages and a poor outcome (p = 0.0073), whereas the numbers of macrophages expressing CD68, a pan-macrophage/monocyte marker, were of marginal prognostic significance (p = 0.0789). We evaluated associations between the levels of expression of the cytokines IL-6, IL-10, IL-12a, IL-12b, M-colony-stimulating factor, IFN-gamma-., and monocyte chemoattractant protein-1 in cancer tissue and the numbers of CD204-positive macrophages. The expression levels of IL-10 and monocyte chemoattractant protein-1, which are involved in differentiation, accumulation, and migration of M2 macrophages, were significantly correlated with the numbers of CD204-positive macrophages (p = 0.031 and p = 0.031, respectively). Conclusion: These findings demonstrated that CD204-positive macrophages clearly reflect the tumor-promoting phenotype of tumor-associated macrophages in lung adenocarcinoma.

Expression of podoplanin, CD44, and p63 in squamous cell carcinoma of the lung

Recent molecular biological studies have identified podoplanin as a candidate cancer stem cell (CSC) marker in squamous cell carcinoma (SqCC). The purpose of this study was to examine the expression pattern of podoplanin, and the other stem cell markers CD44 and p63, and their relationship to clinico‐pathological features including survival in pulmonary SqCC. We examined histologically the expression of podoplanin, CD44, and p63 in 162 consecutive SqCC by immunostaining. Podoplanin expression was observed in 107 (66%) tumors, CD44 in 145 (89.5%), and p63 in 151 (93.2%), respectively. In 95.3% of the podoplanin‐positive tumors, tumor cells showing strong expression were localized in the periphery of the tumor nests. However, this peripheral localization was observed in only 55.9% of the CD44‐positive and 43% of p63‐positive tumors. In 88.8% of the podoplanin‐positive tumors, positive cells were localized more peripherally in the tumor nests than CD44‐ or p63‐positive cells and when CD44 and p63 expressions were compared in these podoplanin‐positive tumors, p63‐positive layers in the periphery of the tumor nests were broader compared to CD44‐positive layers. These findings suggest tumor cells are aligned in the “hierarchical distribution pattern” according to the expression of these three markers. Patients who had podoplanin‐positive tumors with the “hierarchical pattern” resulted in significantly better overall survival than those who had podoplanin‐negative tumors (P = 0.043). These results suggest that podoplanin expression would reflect the most immature status in the differentiation process of SqCC, and SqCC with hierarchical expression would be a well‐organized tumor group with lower biological aggressiveness based on the CSC concept. (Cancer Sci 2009)

Long-Term Outcomes of 50 Cases of Limited-Resection Trial for Pulmonary Ground-Glass Opacity Nodules

Introduction: From 1998 to 2002, we performed a trial of prospective limited resection for pulmonary ground-glass opacity lesions 2 cm or smaller. This is the second report on the long-term outcome. Methods: The enrollment criteria of the trial were as follows: pulmonary peripheral nodule less than 2 cm, diagnosis or suspected diagnosis of clinical T1N0M0 carcinoma with ground-glass opacity and lack of evident pleural indentations or vascular convergence on high-resolution computed tomography. Limited-resection (wedge or segment) specimens were intraoperatively examined by frozen section. If the nodule was confirmed as Noguchi type A or B with a resection margin of greater than 1 cm, the incision was sutured and the patient followed up. The median surveillance period was 10 years. Results: In a total of 50 enrolled participants, there were two Noguchi type A, 23 type B and 15 type C adenocarcinomas; five atypical adenomatous hyperplasias, four fibroses, and one granuloma. Although there were no patients with recurrence within the first 5 years, in four patients who underwent limited-resection pulmonary adenocarcinoma developed more than 5 years after the initial resection, of either cut-end recurrence or metachronous primary disease. Conclusions: Of 26 patients who underwent limited resection, adenocarcinoma developed in four after more than 5 years. These were possibly cut-end recurrences. We concluded that 5 years is not a sufficient period for follow-up, and that limited resection should still be done only in a trial setting, even for small ground-glass opacity lesions.

Late Recurrence of Non-Small Cell Lung Cancer More Than 5 Years After Complete Resection: Incidence and Clinical Implications in Patient Follow-up

BACKGROUND The purpose of this study was to evaluate the risk of late recurrence in patients who had undergone complete resection for non-small cell lung cancer (NSCLC) and remained recurrence-free for > or = 5 years. METHODS Between 1993 and 2002, 1,358 patients with NSCLC underwent complete primary tumor resection and systematic lymph node dissection. Of these, 819 patients remained recurrence-free for 5 years. Recurrence-free probability was estimated from the benchmark of 5 years after primary tumor resection to the date of first recurrence or last follow-up, using the Kaplan-Meier method. Multivariate Cox regression was used to test the relationship of recurrence-free probability to various clinicopathologic factors. RESULTS Of the 819 patients who were free of recurrence at 5 years, 87 (11%) developed a subsequent recurrence. The recurrence-free probabilities at 3 years and 5 years from the point of 5 years after primary tumor resection were 92% and 87%, respectively. The 5-year recurrence-free probabilities from the point of 5 years after primary tumor resection were 81% for patients with intratumoral vascular invasion (P < .001), and 89%, 84%, and 65% for patients with N0, N1, and N2 cancers, respectively (P < .001). Multivariate Cox analysis demonstrated that intratumoral vascular invasion and nodal involvement significantly influenced recurrence 5 years after complete resection (P = .030, P = .022, respectively). CONCLUSIONS Patients with NSCLC with selected tumor characteristics have a significant risk of late recurrence. Therefore, 5 years might not be a sufficient amount of time to declare that NSCLC has been cured.

Dynamic molecular changes associated with epithelial-mesenchymal transition and subsequent mesenchymal-epithelial transition in the early phase of metastatic tumor formation

Metastatic tumor formation via vessel route begins with cancer cell extravasation from vessel lumen, migration into the connective tissue surrounding vessels, and invasion into target organ parenchyma. Epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) have been recognized to play an important role in metastatic process, however, how and where these biological changes take place in the early phase of metastatic tumor development has never been clarified. We morphologically evaluated 34 small intrapulmonary metastases formed after cancer cell extravasation from lymphatics (lymphogenic metastasis) and 40 formed in the absence of extravasation (aerogenous metastasis) in human specimens and found that isolated or small clusters of invasive cancer cells (tumor budding) were frequently observed in lymphogenic metastasis (24/34; 71%), but were never observed within aerogenous metastasis. We immunostained 34 lymphogenic metastases for 13 molecular markers of EMT and MET and scored the immunostaining intensity of cancer cells floating in lymphatic vessels (LVs), migrating into the connective tissue surrounding vessels [bronchovascular bundle (BVB)], and growing in lung parenchyma (LP). Cancer cells within BVBs stained more weakly for E‐cadherin (p < 0.001), β‐catenin (p < 0.001), and Geminin (p < 0.001) and more strongly for MMP‐7 (p = 0.046) and Laminin‐5 γ2 (p = 0.037) than tumor cells in LVs. However, cancer cells in LP exhibited resurgent E‐cadherin (p = 0.011), β‐catenin (p < 0.001), and Geminin (p = 0.037) expression and reduced MMP‐7 (p = 0.038) and Laminin‐5 γ2 (p = 0.001) expression in comparison with cancer cells in BVBs. Our results suggested that in the early phase of metastatic tumor formation cancer cells undergo dynamic phenotypic change associated with EMT and subsequent MET.

Immunohistochemical differential diagnosis between thymic carcinoma and type B3 thymoma: diagnostic utility of hypoxic marker, GLUT-1, in thymic epithelial neoplasms

There are only a few immunohistochemical markers that are useful for differentiating thymic carcinomas from type B3 thymomas. The purpose of this study is to examine the additional markers that would be useful for differentiating between thymic carcinoma and thymoma type B3. We performed a tissue microarray analysis of surgically resected thymic tumor specimens from12 cases of thymic carcinoma, 7 cases of type B3 thymoma, and 68 cases of other types of thymoma. Immunostaining using 49 antibodies was scored based on staining intensity and the percentage of cells that stained positive. Seven proteins that were selected by the staining scores, namely, GLUT-1 (167 vs 4), CA-IX (110 vs 15), c-kit (162 vs 44), CD5 (33 vs 0), MUC-1 (54 vs 0), CEA (42 vs 0), and CK18 (110 vs 42), were significantly higher in the thymic carcinomas than in the type B3 thymomas. The staining sensitivity and specificity of the antibodies for thymic carcinoma were GLUT-1, sensitivity 72% and specificity 100%; CA-IX, 58 and 71%; c-kit, 72 and 85%; CD5, 33 and 100%; CK18, 58 and 71%; MUC-1, 25 and 100%; and CEA, 33 and 100%. Glucose transporter 1 (GLUT-1) is the best marker for thymic carcinoma because it had the highest sensitivity and specificity. Positive immunostaining for a combination of three markers, namely, GLUT-1, CD5, and CEA, enabled differentiation of thymic carcinoma with 91.6% sensitivity and 100% specificity. In conclusion, we identified GLUT-1 as an additional marker that will be useful for differentiating thymic carcinoma from type B3 thymoma, especially in biopsy specimens that have been crushed or are otherwise difficult to examine morphologically in thymic tumors.

Possible Delayed Cut-End Recurrence After Limited Resection for Ground-Glass Opacity Adenocarcinoma, Intraoperatively Diagnosed as Noguchi Type B, in Three Patients

Introduction: In our limited resection trial of pulmonary peripheral ground-glass opacity (GGO) lesions from 1998 to 2002, limited resection of Noguchi type A and B carcinomas seemed to have a positive outcome. However, recently three of the 24 patients, with mixed GGO lesions intraoperatively diagnosed as type B, developed a solid lesion at the cut-end scar. Methods: Medical records and radiology and pathology findings of the three patients were reviewed. We also analyzed epidermal growth factor receptor gene mutations when possible. Results: Radiologically, these three second tumors were clearly cut-end scar area recurrences. However, other pathologic and mutation findings suggest metachronous primary cancers developed in Case 1, cut-end recurrence in Case 2, and needle biopsy implantation in Case 3. It is difficult to definitively conclude whether the second tumors were recurrences or metachronous primaries. Conclusions: These second tumors have convinced us that our initial caution in concluding GGO lesions can be cured by limited resection was very appropriate. The recurrences definitely indicate that continuing follow-up attention for more than 5 years is needed after limited resection even for GGO bronchioloalveolar carcinomas.

Risk Factors for Tumor Recurrence in Patients With Early-Stage (Stage I and II) Non-small Cell Lung Cancer: Patient Selection Criteria for Adjuvant Chemotherapy According to the Seventh Edition TNM Classification

OBJECTIVES The purpose of this study was to evaluate risk factors for tumor recurrence in patients with completely resected early-stage non-small cell lung cancer (NSCLC). METHODS Between July 1992 and December 2007, 1,967 consecutive patients with stage I and II NSCLC with diagnoses based on the seventh edition TNM classification underwent complete resection. All patients were divided into three groups according to the stage and presence of lymph node metastasis: stage I, patients with stage I, T1-T2aN0M0 disease; stage IIN0, patients with stage II, T2b-T3N0M0, node-negative disease; and stage IIN1, patients with stage II, T1-2N1M0, node-positive disease. Freedom from recurrence rate was estimated using the Kaplan-Meier method, and recurrence risk factors were identified by univariate and multivariate analyses. RESULTS The 5-year freedom from recurrence rates for stage I, stage IIN0, and stage IIN1 patients were 84%, 61%, and 54%, respectively. By multivariate analyses, three variables (histologic differentiation, vessel invasion, and visceral pleural invasion) in stage I and two variables (adenocarcinoma histology and visceral pleural invasion) in stage IIN0 and stage IIN1 were shown to be independently significant risk factors for recurrence. According to subgroup analyses that combined these risk factors in each group, the 5-year freedom from recurrence rate was 63% for stage I with three risk factors, whereas those for stage IIN0 and stage IIN1 without risk factors were 83% and 78%, respectively. CONCLUSION In patients with stage I and II NSCLC, we identified risk factors for recurrence. When these factors are combined, high- and low-risk subgroups can be identified within each group.

Risk factors of postoperative respiratory infections in lung cancer surgery.

Background: Postoperative infections have been a major issue in lung cancer surgery. We changed our perioperative prophylactic antibiotic policy to a single dose of cefazolin before and after surgery in July 2002. Objective: To identify the risk factors of postoperative pneumonia and empyema in lung cancer patients undergoing surgical resection. Methods: From July 1992 through September 2003, 2105 patients underwent primary lung cancer resection at our division. We reviewed 1855 eligible patients for possible risk factors of pneumonia and empyema. Results: Postoperative respiratory infections developed in 69 (3.7%) patients. There were 58 (3.1%) pneumonia cases and 18 (1.0%) cases of empyema. The mortality rate was 0.8% (15 patients). Nine (0.5%) patients died from postoperative respiratory infections. Multivariate analysis showed age 75 years or older, forced expiratory volume in 1 second as a percentage of forced vital capacity (FEV1%) less than 70%, advanced pathologic stage, and induction therapy to be independent risk factors of pneumonia. For postoperative empyema, advanced age was the significant factor. Twelve of 18 patients (67%) with empyema were complicated with bronchopleural fistula. The infection incidence rate did not change significantly after we modified our prophylactic antibiotic policy to a single dose of cefazolin before and after surgery. Conclusions: Lung cancer patients with advanced age, low FEV1%, advanced pathologic stage, or induction therapy had a risk for pneumonia after lung cancer surgery. Postoperative empyema was associated with advanced age.