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.

Stromal expression of heat-shock protein 27 is associated with worse clinical outcome in patients with colorectal cancer lung metastases.

BACKGROUND: Pulmonary metastases are common in patients with primary colorectal cancer (CRC). Heat-shock protein 27 (Hsp27) is upregulated in activated fibroblasts during wound healing and systemically elevated in various diseases. Cancer-associated fibroblasts (CAFs) are also thought to play a role as prognostic and predictive markers in various malignancies including CRC. Surprisingly, the expression of Hsp27 has never been assessed in CAFs. Therefore we aimed to investigate the expression level of Hsp27 in CAFs and its clinical implications in patients with CRC lung metastases. METHODS: FFPE tissue samples from 51 pulmonary metastases (PMs) and 33 paired primary tumors were evaluated for alpha-SMA, CD31, Hsp27 and vimentin expression by immunohistochemistry and correlated with clinicopathological variables. 25 liver metastases served as control group. Moreover, serum samples (n=10) before and after pulmonary metastasectomy were assessed for circulating phospho-Hsp27 and total Hsp27 by ELISA. RESULTS: Stromal expression of Hsp27 was observed in all PM and showed strong correlation with alpha-SMA (P<0.001) and vimentin (P<0.001). Strong stromal Hsp27 was associated with higher microvessel density in primary CRC and PM. Moreover, high stromal Hsp27 and αSMA expression were associated with decreased recurrence-free survival after pulmonary metastasectomy (P=0.018 and P=0.008, respectively) and overall survival (P=0.031 and P=0.017, respectively). Serum levels of phospho- and total Hsp27 dropped after metastasectomy to levels comparable to healthy controls. CONCLUSIONS: Herein we describe for the first time that Hsp27 is highly expressed in tumor stroma of CRC. Stromal α-SMA and Hsp27 expressions correlate with the clinical outcome after pulmonary metastasectomy. Moreover, serum Hsp27 might pose a future marker for metastatic disease in CRC.

EGFR, BRAF and KRAS status in patients undergoing pulmonary metastasectomy from primary colorectal carcinoma: a prospective follow-up study.

BACKGROUND: Pulmonary metastasectomy is an integral part of the interdisciplinary treatment of patients with pulmonary metastases (PMs) from colorectal carcinoma (CRC). Although alterations in the epidermal growth factor receptor (EGFR) pathway are common in CRC, there is still insufficient data regarding PM. We hypothesized that EGFR expression and Kirsten rat sarcoma viral oncogene homolog (KRAS)/BRAF mutations (Mts) might be associated with clinicopathological variables and the outcome in patients undergoing pulmonary metastasectomy. METHODS: In this single-center study, 44 patients undergoing pulmonary metastasectomy from primary CRC were included and prospectively followed up. Tissue specimens of resected PMs were assessed. Restriction fragment length analysis was used for BRAF V600E and KRAS codons 12 and 13 Mt analyses. EGFR expression was evaluated by immunohistochemistry. Patients were followed up in 3-6-month intervals. RESULTS: EGFR expression was evident in 49 % of the PMs, whereas Mts in KRAS and BRAF were detected in 48 and 0 %, respectively. Time to lung-specific recurrence after metastasectomy was significantly decreased in patients with KRAS mutated PMs in univariate (p = 0.013) and multivariate analysis (p = 0.035), whereas EGFR expression had no impact on recurrence free survival. Moreover, KRAS Mts were associated with the number of PMs (p = 0.037) and with the lung as first site of recurrence after metastasectomy (p = 0.047). DISCUSSION: This is the first evaluation of EGFR pathway alterations in the setting of pulmonary metastasectomy. Our data suggest that patients with KRAS Mts are at high risk for early pulmonary recurrence and have a more diffuse pattern of metastasis. These findings may have impact on the therapeutic management of CRC patients with pulmonary spreading.

Carbonic anhydrase IX is associated with early pulmonary spreading of primary colorectal carcinoma and tobacco smoking.

OBJECTIVES: Pulmonary metastasectomy is performed routinely in selected patients with metastatic spreading to the lungs. According to current guidelines, the tumour biology should be taken into account when selecting patients for a resection. Carbonic anhydrase IX (CA9) expression has been shown to be a common feature in primary tumour growth and metastasis and it negatively affects the clinical outcome in various malignancies. Data on CA9 in pulmonary metastases are lacking. METHODS: Forty-four patients with primary colorectal cancer (CRC) who underwent curative pulmonary metastasectomy were included in this study. We determined the expression of CA9 in pulmonary metastases and corresponding primaries by immunohistochemistry. The expression level was correlated with clinical parameters and patients' smoking habits. Furthermore, the impact of nicotine treatment on phosphorylation of STAT3, HIF-1α and CA9 expression was assessed in HT29 cells. RESULTS: High expression of CA9 in resected pulmonary metastases and corresponding primary tumours correlated with early spreading to the lung (both P < 0.001). Moreover, CA9 expression was affected by the smoking habits of the patients. Treating HT29 cells with nicotine resulted in an induction of CA9 in vitro. This induction was associated with STAT3 phosphorylation and was independent of HIF-1α. CONCLUSIONS: This study provides first evidence of CA9 expression in pulmonary metastases of CRC and suggests a role of CA9 as a prognostic marker. Moreover, our in vitro and in vivo data indicate an association between tobacco smoking and CA9 expression. Immunohistochemical assessment of CA9 expression might serve as an additional tool in decision-making for selecting patients for pulmonary metastasectomy.