Shinya Neri

Surgical Treatment of Local Recurrence After Stereotactic Body Radiotherapy for Primary and Metastatic Lung Cancers

Introduction: Stereotactic body radiotherapy (SBRT) has been proposed as an alternative to surgery for the treatment of lung cancer. The treatment of local recurrence that occurs after SBRT has not been reported. We present surgical outcomes for local recurrence following SBRT for primary and metastatic lung cancers. Methods: Seven of the patients who received SBRT between 2002 and 2008 underwent salvage surgery for local recurrence. These seven patients (two with stage I non-small cell lung cancer and five with metastatic tumors) were operable, although they refused surgery and chose SBRT for the first treatment as a less invasive procedure. Results: Six of the seven patients underwent lobectomy, and the other patient underwent segmentectomy. None of the seven patients had direct pleural adhesion resulting from SBRT, although, in general, radiation fibrosis occurs after radiotherapy and induces pleural adhesion that makes surgery difficult. Conclusions: Our study suggests that SBRT may not be the cause of difficulties encountered during the surgical process, and surgery is a good alternative treatment for local recurrence after SBRT. During follow-up after SBRT, the appearance of tumor regrowth on lung images resembled that of inflammatory changes such as radiation pneumonitis. We suggest that close follow-ups should be mandatory after SBRT.

Podoplanin-expressing cancer-associated fibroblasts lead and enhance the local invasion of cancer cells in lung adenocarcinoma

Cancer‐associated fibroblasts (CAFs) communicate with cancer cells and play important roles in cancer invasion. We previously reported that local invasion of cancer cells was frequently observed in lung adenocarcinoma patients with podoplanin (PDPN)‐expressing CAFs. However, the underlying mechanisms of this phenomenon have remained unclear. In this study, we established a novel collagen invasion assay model in which cancer cells and CAFs were cocultured; we analyzed the mechanisms governing how cancer cell invasion was promoted by PDPN(+)CAFs. By observing the dynamic movement of both CAFs and cancer cells in the collagen matrix, we found that PDPN(+)CAFs invaded the matrix to a greater extent, with more cancer cells invading within the “tracks” created by the CAFs, compared with control CAFs. The knockdown of PDPN in CAFs decreased the invasion of both the CAFs and the cancer cells. PDPN(+)CAFs displayed a higher RhoA activity and treatment with a ROCK inhibitor cancelled the increased invasion ability of PDPN(+)CAFs and subsequently decreased the number of invaded cancer cells. After intravenous injection in the mouse tail vein, PDPN(+)CAFs invaded and promoted cancer cell invasion into the lung parenchyma, compared with control CAFs. Among the patients with lung adenocarcinoma, we observed some cases with PDPN(+)CAFs at the invasive front of the tumor. These cases predominantly exhibited pleural invasion of cancer cells, known as pathological invasiveness. Our results indicated that PDPN(+)CAFs were tumor‐promoting CAFs that lead and enhance the local invasion of cancer cells, suggesting that the invasion activity of CAFs themselves could be rate‐determining for cancer cell invasion.

Podoplanin-Positive Cancer-Associated Fibroblasts in the Tumor Microenvironment Induce Primary Resistance to EGFR-TKIs in Lung Adenocarcinoma with EGFR Mutation

Purpose: The biologic characteristics of microenvironmental constituents, especially cancer-associated fibroblasts (CAF), can be key regulators of the cellular sensitivity to molecular-targeted therapy. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) have marked therapeutic effects against non–small cell lung cancer (NSCLC) with EGFR mutations, but some patients have exhibited primary resistance to EGFR-TKIs. We recently reported that podoplanin-positive fibroblasts are associated with a tumor-promoting phenotype of CAFs in lung adenocarcinoma. The aim of this study was to evaluate whether the susceptibility of NSCLC to EGFR-TKIs could be affected by podoplanin-expressing CAFs. Experimental Design: We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell lines cocultured with podoplanin-expressing CAFs. We also examined the association between the expression of podoplanin in CAFs in surgical specimens and EGFR-TKI response of postoperative recurrent patients with EGFR mutations (N = 106). Results: Lung adenocarcinoma cell lines became more resistant to EGFR-TKI when cocultured with podoplanin-expressing CAFs, compared with control CAFs in vitro. The knockdown of podoplanin expression on CAFs cancelled the resistance to EGFR-TKIs in cancer cells. Compared with control CAFs, the cancer cells that were cocultured with podoplanin-positive CAFs continued to exhibit significantly higher p-ERK levels after treatment with gefitinib. Furthermore, postoperative recurrent patients with podoplanin-positive CAFs had a significantly lower overall response rate to EGFR-TKIs compared with those with podoplanin-negative CAFs (53% vs. 83%; P < 0.01). Conclusions: Podoplanin-positive CAFs play an important role in primary resistance to EGFR-TKIs and may be an ideal therapeutic target for use in combination therapy with EGFR-TKIs. Clin Cancer Res; 21(3); 642–51. ©2014 AACR.

Tumor promoting effect of podoplanin-positive fibroblasts is mediated by enhanced RhoA activity

There is growing evidence that stromal fibroblasts can promote tumor progression via several mechanisms. We previously reported that podoplanin (PDPN) expressed on stromal fibroblasts is functionally protein responsible for the promotion of tumor formation in mouse subcutaneous tissue. The purpose of the present study was to reveal the molecular mechanism by which PDPN on stromal fibroblasts promotes tumor formation. The subcutaneous co-injection of the human lung adenocarcinoma cell line A549 and human fibroblasts (hFbs) overexpressing wild-type podoplanin (WT-PDPN) promoted subcutaneous tumor formation, compared with the co-injection of A549 and control hFbs (64% vs 21%). On the other hand, hFbs expressing PDPN mutant in which the cytoplasmic domain of PDPN was deleted (PDPN-Del.IC), resulted in a relatively lower level of tumor formation (33%). Since PDPN reportedly regulates RhoA activity through its cytoplasmic domain, we measured the activation state of RhoA in hFbs expressing WT-PDPN. RhoA activity was 2.7-fold higher in WT-PDPN expressing hFbs than in control hFbs. Furthermore, the subcutaneous co-injection of hFbs expressing constitutive active RhoA (G14VRhoA) and A549 cells enhanced tumor formation compared with the co-injection of the same cell line and control hFbs. These results indicate that enhanced RhoA activity in hFbs expressing PDPN may be one of the mechanisms resulting in the promotion of tumor formation, suggesting that biomechanical remodeling of the microenvironment by stromal fibroblasts may play important roles in tumor progression.

Prognostic impact of microscopic vessel invasion and visceral pleural invasion in non-small cell lung cancer: a retrospective analysis of 2657 patients.

Objective:We aimed to assess the prognostic significance of microscopic vessel invasion (MVI) and visceral pleural invasion (VPI) in non–small cell lung cancer (NSCLC). Background:VPI is included in the current tumor-node-metastasis (TNM) classification in NSCLC; however, MVI is not incorporated in TNM classification. Methods:From August 1992 to December 2009, 2657 consecutive patients with pathological T1-4N0-2M0 NSCLC underwent complete resection. In addition to conventional staging factors, we evaluated MVI histologically and analyzed its significance in NSCLC recurrence prognosis. The recurrence-free period in several NSCLC subgroups was analyzed using the Kaplan-Meier method and Cox regression analysis. Results:The proportion of patients with a 5-year recurrence-free period was 52.6% and 87.5%, respectively, in those with and without MVI (P < 0.001). Multivariate analysis showed that MVI, similarly to VPI, was found to be an independently significant predictor of recurrence [hazard ratio (HR): 2.78]. In particular, MVI and VPI were the 2 strongest significant independent predictors of recurrence in 1601 patients with pathological stage I disease treated without adjuvant chemotherapy (HR: 2.74 and 1.84, respectively). In each T subgroup analysis, evident and significant separation of the recurrence-free proportion curves were observed among the 3 groups (VPI and MVI absent, VPI or MVI present, and VPI and MVI present). Conclusions:This study demonstrated that MVI was a significant independent risk factor for recurrence in patients with a resected T1-4N0-2M0 NSCLC. Further data on MVI prognostic impact should be collected for the next revision of the TNM staging system.

Immunophenotypic features of metastatic lymph node tumors to predict recurrence in N2 lung squamous cell carcinoma.

Patients with mediastinal lymph node metastasis (N2) in squamous cell carcinoma (SqCC) of the lung have poor prognosis after surgical resection of the primary tumor. The aim of this study was to clarify predictive factors of the recurrence of pathological lung SqCC with N2 focusing on the biological characteristics of both cancer cells and cancer‐associated fibroblasts (CAFs) in primary and metastatic lymph node tumors. We selected 64 patients with pathological primary lung N2 SqCC who underwent surgical complete resection and investigated the expressions of four epithelial–mesenchymal transition‐related markers (caveolin, clusterin, E‐cadherin, ZEB2), three cancer stem cell‐related markers (ALDH‐1, CD44 variant6, podoplanin) of cancer cells, and four markers of CAFs (caveolin, CD90, clusterin, podoplanin) in both primary and matched metastatic lymph node tumors in the N2 area. In the primary tumors, the expressions of all the examined molecules were not related to recurrence. However, in the metastatic lymph node tumors, high clusterin and ZEB2 expressions in the cancer cells and high podoplanin expression in the CAFs were significantly correlated with recurrence (P = 0.03, 0.04, and 0.007, respectively). In a multivariate analysis, only podoplanin expression in the CAFs in metastatic lymph node tumors was identified as a significantly independent predictive factor of recurrence (P = 0.03). Our study indicated that the immunophenotypes of both cancer cells and CAFs in metastatic lymph node tumors, but not primary tumors, provide useful information for predicting the recurrence of pathological N2 lung SqCC.

Circulating CD14+CD204+ Cells Predict Postoperative Recurrence in Non–Small-Cell Lung Cancer Patients

Background: The expression of CD204 on macrophages in the stroma of the primary tumor is reportedly correlated with an unfavorable prognosis for lung cancer. The purpose of this study is to investigate the correlation among the number of CD204+ tumor–associated macrophages infiltrating the stroma of the primary tumor, the number of circulating CD14+CD204+ cells from the pulmonary vein (PV), and recurrence-free probability in non–small-cell lung cancer patients. Methods: Human mononuclear cells were isolated from the PV of resected lungs. We examined the expressions of CD14 and CD204 on these cells by flow cytometry. Immunohistochemical staining for CD204 was performed in the resected specimens. Results: The number of CD14+CD204+ cells from the PV was found to be correlated with the number of CD204+ tumor–associated macrophages identified in the stroma of the tumor. Significantly more cases with high levels of CD14+CD204+ cells from the PV were found to have developed early recurrences. CD14+CD204+ cells, which were polarized to the tumor-promoting phenotype cultured in lung cancer cell line–conditioned medium, facilitated the lung metastasis of cancer cells more effectively than CD14+CD204− cells in our in vivo mouse model. In multivariate analysis, only the high number of CD14+CD204+ cells from the PV was found to be a statistically significant independent risk factor for early recurrence. Conclusion: Our results showed the possibility that circulating CD14+CD204+ cells contribute to the metastasis of cancer cells. The blockage of circulating CD14+CD204+ cells activity may prevent postoperative recurrence in resected non–small-cell lung cancer patients.

CD200-positive cancer associated fibroblasts augment the sensitivity of Epidermal Growth Factor Receptor mutation-positive lung adenocarcinomas to EGFR Tyrosine kinase inhibitors

Cancer associated fibroblasts (CAFs) play important roles in the chemotherapeutic process, especially through influencing the resistance of tumor cells to molecular targeted therapy. Here we report the existence of a special subpopulation of patient-specific-CAFs that augment the sensitivity of EGFR gene mutation-positive lung cancer to the EGFR-tyrosine kinase inhibitor (EGFR-TKI), gefitinib. When cocultured with EGFR mutation positive lung cancer cells, these CAFs increased the apoptic effect of gefitinib on cancer cells, whereas, in the absence of gefitinib, they did not affect cancer cell viability. The assay using different single cell-derived clones demonstrated that the aforementioned sensitizing ability is clone-specific. Microarray analysis revealed that CD200 was expressed at much higher levels in this CAFs. Knocking down of CD200 expression deprived CAFs of their sensitizing potential, suggesting that CD200 is the functional molecule responsible for the effect. Immunohistochemical analysis of samples from patients receiving postoperative gefitinib treatment revealed that the individuals whose resected lung adenocarcinomas contained CD200-positive CAFs tended to have longer progression free survival of gefitinib when they recurred after surgery. These results suggest that CD200-positive CAFs can augment the sensitivity to EGFR-TKIs and may possess far reaching applications in the therapeutic use of EGFR-TKIs.

Tracheal stent placement via a tracheostomy for tracheal stenosis after inhalation injury

Tracheal stenosis is one of the late complications of inhalation injury. While tracheal stenosis is usually mild and asymptomatic, it sometimes appears as a severe fixed or dynamic airway obstruction [1]. Although tracheal stenosis in burns can be critical, the treatment remains undefined. We present a case of severe tracheal stenosis complicated by subglottic stenosis after inhalation injury, which we treated successfully with silicone stent placement through a tracheostomy port.

Clonal heterogeneity in osteogenic potential of lung cancer-associated fibroblasts: promotional effect of osteogenic progenitor cells on cancer cell migration

BackgroundCancer-associated fibroblasts (CAFs) consist of heterogeneous cell population in terms of their differentiation potential. The functional differences in tumor progression between CAFs with mesenchymal stem/progenitor cells (MSCs/MPCs) characteristics and CAFs without MSCs/MPCs characteristics are not clarified.MethodsCAFs and vascular adventitial fibroblasts (VAFs, which contain MSCs/MPCs) were isolated from nine primary lung cancers and were cultured in osteogenic or adipogenic medium to assess their multi-lineage differentiation. Next, we established nine single-cell-derived clones from the primary culture of CAFs and examined their differentiation potential. The effects of each single-cell-derived clone on the proliferation and migration of lung adenocarcinoma cell line, A549, were analyzed.ResultsThe nine samples of VAFs and CAFs showed various degrees of osteogenic differentiation. Although the VAFs displayed the ability to undergo adipogenic differentiation, all cases of the CAFs did not. CAFs clones presented varying degrees of osteogenic differentiation. Four clones displayed comparable levels of osteogenic potential with that of the VAFs, and two clones were completely negative. As compared to the CAFs clones that possessed lower osteogenic potential, CAFs clones with higher osteogenic potential did not confer proliferative activity in A549 cells. On the contrary, these clones significantly promoted the migration of A549 cells as compared to the clones with lower osteogenic potential.ConclusionOur studies clearly indicate that CAFs derived from lung cancer are heterogeneous population that consists of cells with varying osteogenic potentials and that CAFs with higher osteogenic potential have a greater tumor-promoting function through the enhancement of cancer cell migration.