The role of perioperative blood transfusion in augmenting pancreatic cancer progression remains unclear due to clinical variability amongst patients and individual tumor biology, although large clinical series of patients with pancreatic cancer suggest a negative impact of transfusion on survival.17,18
Similarly, the specific constituent(s) of allogeneic blood that mediate TRIM as it relates to cancer progression are unclear. Plasma, platelets, and leukocytes, which inherently accompany NLR pRBCs, and proteins (cytokines or chemokines) that accumulate during PRBC storage have been implicated in the pathogenesis of TRIM.34–37
Clinical studies in non-cancer patients support the link between TRIM and the length of storage of banked blood.24,38–40
Furthermore, in vitro
data have demonstrated that tumor cell growth is enhanced by blood that has been stored for increasing time periods.41
The release of bioactive substances by leukocytes and platelets and the accumulation of these substances during storage is generally accepted, 21,42,43
and animal studies suggest that early removal of leukocytes from banked blood ameliorates the tumor growth promoting effects of blood transfusion.36,37,44
However, five randomized controlled trials designed to evaluate the effect of allogeneic white blood cell transfusion on cancer recurrence failed to yield convincing results with regard to an enhanced TRIM effect from stored versus fresh pRBCs.11
This study has demonstrated that the acellular plasma fraction from stored pRBCs promotes cellular proliferation and Pan02 migration (surrogates for cancer growth and metastasis) and promotes in vivo
pancreatic cancer growth. Although previous clinical studies have shown similar conclusions10,17,18,45
these studies are difficult to interpret given the large number of variables inherent to any cancer patient population including tumor biology, tumor stage, and untoward events during surgery as well as innumerable host factors. Use of this well established immunocompetent model of pancreatic cancer29
has allowed us to control for many factors that cause clinical trials to be dismissed. First, a clonal pancreatic cancer cell tumor was surgically introduced into the pancreas itself, which allowed for consistent tumor biology within all test animals. Second the use of an identical tumor inoculum (cell number) in all mice as well as ultrasound examination to confirm tumor presence allows control for “tumor stage” prior to administration of plasma from pRBCs. Third, the use of syngeneic animals controls for “patient variability”, and fourth, the same surgical stress is applied to all animals.
Interestingly, when examining the affect of the plasma from pRBCs on cellular proliferation in vitro, it appears that presence of leukocytes is the predominant factor that promotes proliferation although increasing storage time trended toward increasing proliferation in the Pan02 group as well. Moreover, as opposed to proliferation, storage time appeared to be more important in Pan02 migration even though the presence of leukocytes was clearly important in the stored D.42 blood.
The significant difference in tumor growth and metastasis observed in the animals that received plasma from pRBCs suggests that infusion of bioactive substances within the plasma of pRBCs promotes tumor progression, irrespective of storage time. In addition, acellular plasma harvested from stored D.42 pRBCs did not differentially affect tumor size and metastatic burden when infused into tumor-bearing animals. However, there was a trend toward more severe disease in the D.42 animals, with these animals demonstrating a higher rate of jaundice and ascites formation. Current transfusion literature supports these findings somewhat, suggesting that transfusion of blood with increased storage time augments the detrimental effects of TRIM. 40,41,46,43
Although levels of biologically active cytokines increase during storage of pRBCs, pre-storage leukoreduction decreases the cancer promoting effects of such transfusions;21,48,49
moreover, contrary to the presented data, these studies showed reduction of white blood cells before storage reduced the metastasis formation to control levels. However, until recently, little was known about the effects of pre-storage leukoreduction on platelet-derived growth factors, e.g. VEGF, PDGF or EGF all of which may have a role in tumor growth and may be eliminated from pRBCs by the current generation of filters. Interestingly, the infusion of the plasma fraction from day 1 and day 42 of storage from non-leukoreduced packed pRBCs were not different. These findings suggest that substances with sufficient bioactivity to promote tumor growth are present in the plasma after only one day of storage.
An unexpected finding was the observed rapid decline in the health of the mice harboring pancreatic cancer and receiving plasma versus tumor-bearing control animals. After sham surgery and plasma transfusion, mice with tumors appeared ill within days and failed to gain appropriate weight while tumor bearing control animals gained weight in parallel with non-tumor bearing controls until late in the study. The causes for this “clinical” picture appears to have been identified at necropsy where predominantly animals receiving plasma from D.1 and D.42 NLR pRBCs suffering biliary, gastrointestinal and urinary tract obstruction and a large portion of the mice treated with the plasma from D.42 NLR pRBCs plasma developing ascites within the study period. This difference may be more profound than is initially apparent as human clinical studies have demonstrated that presence of malignant cells in the peritoneum is a strong negative prognostic marker in pancreatic cancer, independent of the size of the primary tumor.50
Thus, there may be a differential effect of stored pRBCs versus fresher erythrocytes. The rapid decline in health in these animals closely parallels cancer-related morbidity witnessed in human patients and suggests that substances within plasma are promoting a virulent cancer progression that is not due to surgical trauma or the presence of tumor alone, but rather a combination of plasma transfusion and surgery in a tumor-bearing host. Although the observed increased morbidity may be related to storage age of the blood, it will require further study for elucidation; furthermore, these findings suggest that transfusion of pRBCs may “tip the balance” of the tumor-host interaction in favor of the tumor.
There are several limitations of this study that warrant discussion: 1) plasma contains many substances that could affect the host response, and the effect of individual agents is difficult to determine. 2) Human plasma was used in this model, and the mouse immune response to these foreign proteins is difficult to predict. Using blood from a syngeneic source would be ideal, but would be logistically difficult considering the need for similar harvesting, processing and storage techniques to make the model clinically relevant. Multiple studies by Silliman, et al 23,51
have demonstrated acute lung injury after transfusion of plasma from stored but not fresh pRBCs that is reproducible using multiple blood donors with no idiosyncratic effects noted in those studies.23,51,52
3) Many of the parameters used to determine disease severity, such as identification of cancerous lesions (lymph node, liver, peritoneal metastases) as well as jaundice and visceral obstruction are subjective. Two blinded observers were employed for confirmation of findings at necropsy to avert bias. Finally, statistical comparisons were necessarily made at high multiplicity comparing tumor growth, which should be viewed with some caution. Despite these issues, this model has clinical relevance and warrants further confirmation with more sophisticated studies in the future.
Blood products are given intravenously to cancer patients in the perioperative period everyday and the effects on cancer-related morbidity and death have yet to be proven. This study has attempted to control for many of the variables present in the clinical setting. In summary, this data suggests that bioactive substances capable of affecting the tumor biology within an organism are present within 24 hours of storage of erythrocytes. The substances and mechanism of action remain to be elucidated; however, these studies offer an avenue for investigation and identification of strategies for intervention.