The altered immune microenvironment of human liver in metastatic disease

Abstract

Globally, metastatic colon cancer is a significant disease with poor outcomes. Liver metastases are the leading cause of death and, despite maximal medical management, recurrent metastases are seen in up to 70% of patients with colon cancer. Surgical resection remains the only curative treatment option for these patients, despite major advances in immunotherapies for other malignant conditions. The complex immunological micro-environment of the liver and its manipulation during the development and growth of metastases presents a unique challenge to the development of effective immunotherapy for this cohort and this is the focus of this thesis.

The Department of Hepatobiliary and Liver Transplant Surgery at St. Vincent s University Hospital is the National Centre for Surgical Liver Disease. In this regard, patients are referred to St. Vincent s from the other 32 hospitals across Ireland for consideration of liver surgery. Between 1st January 2005 and 31st December 2014, 504 consecutive patients underwent hepatic metastatectomy at the Centre, 378 of whom returned to their referring hospitals for adjuvant therapy following surgery. Each patient underwent preoperative staging, comprising computed tomography of the chest, abdomen, and pelvis and primovist-enhanced magnetic resonance imaging of the liver. All patients were discussed at a weekly multidisciplinary team conference attended by oncologists, radiologists, pathologists and six hepatobiliary surgeons, where a consensus decision on resectability and sequence of treatment was made. On admission, prior to surgical resection, all patients had a routine full blood count taken where total blood leukocyte levels, including neutrophils and lymphocytes were analysed. Survival data was sought from each patients primary care physician. Elevated neutrophil counts (>8) and neutrophil lymphocyte ratio (NLR) > 5 in patients blood preoperatively was negatively associated with overall survival (p=0.018, p=0.027 respectively). However, in those who had received neoadjuvant chemotherapy, no association between elevated blood NLR and overall survival was demonstrated (p=0.93). Similarly, in those undergoing repeat resection, neither serum neutrophil count >8 (p=0.42) nor neutrophil lymphocyte ratio >5 (p=0.81) correlated to postoperative survival.

The microenvironment generated by any tumour is a dynamic interface between tumour cells and host stromal cells, fibroblasts, endothelial cells immune populations and their secretomes. Cytokines and chemokines within this microenvironment play key roles in the regulation of immune cell infiltration of the tumour and their activity. Immune cells recruited to malignant sites actively secrete cytokines and chemokines which attract and dictate the activity of tumour cells and other immune cells within the metastatic niche. Healthy human liver is replete with immune cells poised to provide effective anti-tumour surveillance. We hypothesise that for sustained tumour growth within the liver, its inherent immune surveillance mechanism is compromised. We sought to characterise the altered immune microenvironment of metastatic liver with a view to identifying biomarkers that may be associated with patient outcome, and potential targets for the development of immunotherapy.

Liver tissue was obtained from 25 patients from the above cohort undergoing hepatectomy for CRLM at SVUH between November 2014 and March 2015. On follow up at 18 months, 13 of these 25 went on to develop intrahepatic recurrence. Normal liver tissue was obtained from 12 donor livers at the time of organ retrieval prior to liver transplantation. Histological analysis revealed that periportal polymorphonuclear leukocytes were depleted and lymphocytes were increased in metastatic liver when compared to donor liver tissue. Interestingly, liver NLRs were lower in liver tissue distal to the metastasis when compared to donor. Patients with the fewest PMNs in liver tissue distal to their tumour had a shorter time to intrahepatic recurrence (P<0.001). This is the inverse of what we found on analysis of our cohort s peripheral blood NLR, where elevated neutrophils were negatively associated with patient survival, emphasising the unique immunological environment of the liver .

Conditioned media from three areas of metastatic liver were analysed by protein array and compared to donor liver samples. A number of cytokines, chemokines and growth factors were found in high concentrations in all samples of liver tissue emphasising the immunological complexity of the hepatic microenvironment. Twelve cytokines were found to be differentially expressed in metastatic liver compared to donor liver tissue. Multiplex analysis of each of these differentially expressed cytokines (IL-6, CXCL1, CXCL5, G-CSF, GM-CSF, VEGF, LIF, and CCL3) confirmed higher expression in CRLM-bearing liver compared to donor liver tissue. Even in liver distal from the tumour (often described as normal in other studies), levels of GM-CSF, LIF and IP10 were significantly elevated when compared to donor liver tissue indicating that the entire organ responds to the presence of malignancy. In vitro chemotactic assays revealed that cancer cells migrated towards conditioned medium from all regions of metastatic liver but not towards donor liver CM, supporting the view that the whole organ is effected by cancer growth.

This study adds to increasing evidence that the host immune system is altered in the presence of malignant disease. In particular, the immune microenvironment of metastatic liver is manifest by a changing cytokine profile and changing immune cell concentrations in tissue at a distance from the tumour. As a result of these alterations, metastatic liver is chemo attractive to CRC tumour cells. Following curative liver resection, recurrent metastatic disease is common and correlates with decreased neutrophil densities in metastatic hepatic parenchyma. Contrary to our findings in metastatic liver, increased neutrophil levels and high NLR in peripheral blood were associated with poorer overall outcomes, however the mechanism by which high NLR contributes to malignant progression is still poorly understood. A better understanding of these changes will be required in order to successfully manipulate the hepatic immune response following the establishment of metastasis.