Safety and tolerability of topically administered autologous, apoptotic PBMC secretome (APOSEC) in dermal wounds: a randomized Phase 1 trial (MARSYAS I).

Developing effective therapies against chronic wound healing deficiencies is a global priority. Thus we evaluated the safety of two different doses of topically administered autologous APOSEC, the secretome of apoptotic peripheral blood mononuclear cells (PBMCs), in healthy male volunteers with artificial dermal wounds. Ten healthy men were enrolled in a single-center, randomized, double-blinded, placebo-controlled phase 1 trial. Two artificial wounds at the upper arm were generated using a 4-mm punch biopsy. Each participant was treated with both topically applied APOSEC and placebo in NuGel for 7 consecutive days. The volunteers were randomized into two groups: a low-dose group (A) receiving the supernatant of 12.5 × 106 PBMCs and a high-dose group (B) receiving an equivalent of 25 × 106 PBMCs resuspended in NuGel Hydrogel. Irradiated medium served as placebo. The primary outcome was the tolerability of the topical application of APOSEC. All adverse events were recorded until 17 days after the biopsy. Local tolerability assessment was measured on a 4-point scale. Secondary outcomes were wound closure and epithelization at day 7. No therapy-related serious adverse events occurred in any of the participants, and both low- and high-dose treatments were well tolerated. Wound closure was not affected by APOSEC therapy.

Tumor-infiltrating lymphocyte subsets and tertiary lymphoid structures in pulmonary metastases from colorectal cancer.

The presence of tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) reflects an active inflammatory tumor microenvironment. High density of TILs as well as presence of TLS is associated with improved survival in various solid cancer types. We aimed to describe the density and distribution of TILs and TLS in pulmonary metastases (PMs) from primary colorectal cancer (CRC) and its correlation with clinicopathological variables. Fifty-seven CRC pulmonary metastasectomy specimen (PM) and 31 matched primary CRC specimen were included. Cluster of differentiation (CD)3+, CD8+, CD45RO+ and FoxP3+ TILs were evaluated by immunohistochemistry and density was scored semiquantitatively. TLS were evaluated based on morphological criteria. Survival time was defined from pulmonary metastasectomy to death or last follow up. A marked infiltration with CD3+, CD8+, CD45RO+ and FoxP3+ TILs was evident in CRC PM and matched primary CRC. Further assessment of the immune infiltrate in PM showed that a high density of FOXP3+ TILs at the invasive margin [HR 2.40 (1.11-6.96); P = 0.031] and low density of CD8+ cells in TLS [HR 0.30 (0.14-0.79); P = 0.016] were associated with a worse prognosis in univariate analysis. Moreover, a low CD8/FoxP3-ratio of TILs at the invasive margin (P = 0.042) and in TLS (P = 0.027) conferred an impaired prognosis after pulmonary metastasectomy. Our findings suggest that CRC PM harbor an immune active microenvironment. The balance of CD8+ and FoxP3+ T-cells at the tumor border and in TLS provides prognostic information in patients with CRC PM.

Clinical impact of c-MET expression and mutational status in patients with colorectal cancer lung metastases.

OBJECTIVES: The c-MET tyrosine kinase is known to play a key role in tumour promotion in a variety of cancers. The prognostic significance of c-MET pathway alterations has previously been described in primary colorectal cancer (CRC). However, data on the expression and genetic mutational status of c-MET in CRC pulmonary metastases (PM) are lacking. We aimed to assess the clinical implications of alterations in the c-MET pathway in patients undergoing pulmonary metastasectomy. METHODS: From April 2009 to November 2013, all patients with complete CRC lung metastasectomy were included in this study and prospectively followed up. Tissue samples of 51 PM and 33 paired primary CRCs were stained immunohistochemically for c-MET and phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Genetic alterations of MET were detected using an exome panel on a next generation sequencing (NGS) platform. Serum hepatocyte growth factor (HGF) levels were measured in a patient subset (n = 10) before and after metastasectomy. RESULTS: c-MET expression was significantly higher at the invasive front of metastases compared with central tumour areas (P = 0.020) and was associated with nuclear pSTAT3 expression (P = 0.042). pSTAT3 but not c-MET overexpression in PM was associated with time to tumour recurrence after metastasectomy (P = 0.036). Expression levels of neither c-MET nor pSTAT3 had an impact on time to lung-specific recurrence. However, patients with c-MET or pSTAT3 overexpression in PM had a significantly worse overall survival after metastasectomy (P = 0.023 and 0.008, respectively). Mutations in the MET gene were identified in 20 patients of our cohort by NGS, which failed to be of prognostic relevance. Serum HGF did not significantly differ between patients with PM and healthy controls. CONCLUSIONS: To the best of our knowledge, this is the first structured evaluation of the c-MET axis in the context of pulmonary metastasectomy for CRC. Our results suggest that overexpression of c-MET/pSTAT3 is associated with an impaired prognosis following complete resection. Moreover, this work suggests that the value of c-MET tyrosine kinase inhibitors in the treatment of patients with CRC lung metastases should be assessed in clinical trials.

Increased lymphangiogenesis in lung metastases from colorectal cancer is associated with early lymph node recurrence and decreased overall survival.

Pulmonary metastasectomy (PM) is an accepted treatment modality in colorectal cancer (CRC) patients with pulmonary tumor spread. Positive intrathoracic lymph nodes at the time of PM are associated with a poor prognosis and 5-year survival rates of <20 %. Increased lymphangiogenesis in pulmonary metastases might represent an initial step for a subsequent lymphangiogenic spreading. We aimed to evaluate the presence of lymphangiogenesis in clinically lymph node negative patients undergoing PM and its impact on outcome parameters. 71 patients who underwent PM for CRC metastases were included in this dual-center study. Tissue specimens of pulmonary metastases and available corresponding primary tumors were assessed by immunohistochemistry for lymphatic microvessel density (LMVD) and lymphovascular invasion (LVI). Results were correlated with clinical outcome parameters. LMVD was 13.9 ± 8.1 and 13.3 ± 8.5 microvessels/field (mean ± SD) in metastases and corresponding primary CRC; LVI was evident in 46.5 and 58.6 % of metastases and corresponding primary CRC, respectively. Samples with high LMVD had a higher likelihood of LVI. LVI was associated with early tumor recurrence in intrathoracic lymph nodes and a decreased overall survival (p < 0.001 and p = 0.029). Herein, we present first evidence in a well-defined patient collective that increased lymphangiogenesis is already present in a subtype of pulmonary metastases of patients staged as N0 at the time of PM. This lymphangiogenic phenotype has a strong impact on patients' prognosis. Our findings may have impact on the post-surgical therapeutic management of CRC patients with pulmonary spreading.

Elevated inflammatory parameters and inflammation scores are associated with poor prognosis in patients undergoing pulmonary metastasectomy for colorectal cancer.

OBJECTIVES: Pulmonary metastasectomy (PM) has evolved to become a standard treatment for colorectal cancer lung metastases. However, biomarkers to estimate the prognosis after PM are currently missing. We therefore investigated the prognostic impact of inflammatory-related biomarkers and scores in patients undergoing curative PM for colorectal cancer. METHODS: We analysed prospectively collected datasets of 52 patients treated in our institution between April 2009 and June 2014. Fibrinogen (cut-off 325 mg/dl), C-reactive protein (CRP, cut-off 0.5 mg/dl), the modified Glasgow prognostic score (mGPS) and the neutrophil-to-lymphocyte ratio (NLR) at the time of PM were tested for their prognostic power, and correlated to time to recurrence (TTR), time to lung-specific recurrence (TTLR) and overall survival (OS). RESULTS: Median OS after PM of all patients (n = 52, 21 females, 31 males, mean age ± standard deviation: 62.65 ± 11.41 years) was 36 months [95% confidence interval (CI) 24.7-47.3 months, number of events: n = 20/38.5%]. In univariable survival analyses, high fibrinogen [hazard ratio (HR) 5.51, 95% CI 1.21-25.17], elevated CRP (HR 2.81, 95% CI 1.08-7.28), mGPS >0 (HR 2.81, 95% CI 1.08-7.28) and an NLR of 4 or higher (HR 3.05, 95% CI 1.02-9.13) was associated with poor OS. Median TTR was 15 months for all patients (number of events: n = 35/67.3%). Fibrinogen (HR 3.79, 95% CI 1.32-10.94) and NLR (HR 2.99, 95% CI 1.20-7.46) but not CRP (P = 0.102) and mGPS (P = 0.102) were found to indicate TTR. With regard to TTLR (number of events: n = 26/50%), only NLR predicted early lung recurrence (HR 3.02, 95% CI 1.06-8.564). After multivariable analyses, fibrinogen was the only significant OS predictor. However, all investigated inflammatory biomarkers and scores were prognostic for TTR in multivariable analyses. Finally, we divided the study population into an inflammatory phenotype (one or more inflammatory marker/score-elevated) and a non-inflammatory phenotype group. The inflammatory phenotype was prognostic in uni- and multivariable analyses for all three outcome parameters (OS, TTR and TTLR). CONCLUSIONS: Inflammatory markers provided promising prognostic information in this cohort of curative PM patients after colorectal cancer. Further validation is needed to verify the prognostic role of these markers and establish them in clinical routine.