G-CSF and Neuroblastoma
In May 2015, the Baylor College of Medicine published a press release promoting the research conducted by Dr. Shohet and colleagues examining the impact of G-CSF on neuroblastoma cancer cells. This press release caused a significant amount of concern within the neuroblastoma community. Families started to question if it was safe to give G-CSF to their child and/or if G-CSF caused their child’s cancer to relapse. Not long after this press release, Solving Kids Cancer and the Coalition Against Childhood Cancer (CAC2) organized a webinar where Dr. Shohet presented the results of this research to parents and interested members of the community.[2,3,4]
The Agarwal et al (2015) paper published in the journal of Cancer Research reports that:
G-CSF promotes neuroblastoma cancer cells to grow and spread (metastasize) specifically by the presence of neuroblastoma cancer stem cells.
Cancer stem cells were first discovered in leukemia by Dr. John Dick in Toronto. Cancer Stem Cells (CSCs) are a subpopulation of cancer cells in tumors. They are able to make copies of themselves and all of the other sub-types of cells within a tumour. CSCs are also typically resistant to chemotherapy, are able to spread to different parts of the body, are the cells that are capable of forming tumours (tumorigenic) and are felt to play a central role in disease relapse. The rest of the tumor consists of cells that are typically sensitive to treatment and this is why there is an interest in targeting both regular cancer cells and CSCs with targeted drugs and therapies. There is strong consensus in the cancer research community that cancer stem cells do exist.
Blood, brain, and colon cancers are three that have identified cancer stem cell populations and this list of cancers with identified cancer stem cells has continued to expand over the past several years. Research continues to move forward in the area of cancer stem cells, including understanding the advantages and challenges of targeting cancer stem cells.[7,8] Not all cancers have been identified as having cancer stem cell components. As it stands within the neuroblastoma expert scientific community today, there are some who believe that neuroblastoma cancer stem cells exist and there are others who do not believe that this is the case. At this point in time, there is a mixed consensus that neuroblastoma cancer stem cells exist.
It is important to distinguish cancer stem cells from the normal stem cells that reside in most parts of our bodies. Normal stem cells are very important in maintaining and repairing our tissues, in learning and memory, and by making cells in our blood that respond to infections. G-CSF, the subject of this article, is used in neuroblastoma patients to mobilize blood stem cells to go into the circulation, helping the body produce more cells used to fight infections.
This article will therefore focus on the role of G-CSF in the spread and metastasis of neuroblastoma cells. It will not deal with the issue of neuroblastoma cancer stem cells. The identification of neuroblastoma cancer stem cells must be founded through dedicated research and be internationally recognized through expert consensus.
G-CSF and the G-CSF Receptor
Granulocyte-Colony Stimulating Factor, G-CSF, is a small protein that is naturally occurring in the body and is produced by cells to stimulate the bone marrow to produce granulocytes (a type of white blood cell) and stem cells, which are ultimately released into the blood stream. G-CSF that is naturally found in the body is called endogenous (found inside the body). G-CSF can be synthetically made and given to patients either through subcutaneous (under the skin) injections or by IV directly into the blood stream. Synthetic G-CSF, by trade name Filgrastim or Neupogen, is called exogenous (occurring outside of the body). G-CSF is described as a “growth factor” and/or a “cytokine”. [A helpful video illustrating white blood cells.]
G-CSF was first introduced into pediatric oncology in the 1990s and it is typically given in between cycles of chemotherapy to help patients produce white blood cells faster so that the immune system becomes stronger to help fight off possible infections. Chemotherapy often causes blood counts to drop, resulting in a decrease in white blood cells, which can negatively impact how the immune system is able to fight off infections. At the completion of a cycle of chemotherapy, patients can receive daily or weekly injections of G-CSF to help bolster their immune system since there is a significant risk when a patient contracts an infection when their immune system is weak (also called neutropenia).
G-CSF is also given in larger quantities prior to the collection of stem cells (also called stem cell harvest). Stem cell collection is typically done after the first or second cycle of chemotherapy. Higher doses of G-CSF are given to the patient to produce large quantities of stem cells which the body pushes into the blood stream. A dialysis machine (apheresis) is used to extract the stem cells from the blood so that they can be given back to the patient at a future time in their treatment.
High-Risk Neuroblastoma Treatment and Timing of Cycles
The current Children’s Oncology Group (COG) and International Society of Pediatric Oncology- Europe Neuroblastoma (SIOPEN) protocols for high-risk neuroblastoma requires the cycles of chemotherapy to be carried out closely together. This intensive approach is carefully calculated to stop tumour cells from growing or returning in between cycles of chemotherapy. G-CSF makes it possible to provide the maximum dose of chemotherapy while keeping the time in between chemotherapy cycles as short as possible. It also works to reduce infections and gastrointestinal side-effects.
G-CSF and STAT3 in Neuroblastoma
G-CSF attaches to the G-CSF receptor (G-CSFR) on the surface of the neuroblastoma cell. The G-CSF receptor is also called CD114. When G-CSF attaches to the G-CSF receptor, multiple different enzymes (kinases) are activated which in turn activates a signalling molecule called STAT3 (through a process called phosphorylation). STAT3 goes into the nucleus and activates a number of downstream inflammatory signals which can trigger multiple genes to perform various tasks such as self-renewal (cell growth), cell death, drug/chemotherapy resistance and tumour growth.
Before the results of the Ladenstein (2010) paper were published, a 2008 article from researchers at Baylor College of Medicine sounded the initial alarm about the use of G-CSF in neuroblastoma patients. They identified that neuroblastoma cells do have G-CSF receptors causing increased cellular growth and decreased cellular death (apoptosis) when exposed to G-CSF. The results of this study[11,12] were limited by the cell lines used in the experiment; however the stimulation of neuroblastoma cells by G-CSF was enough to call for further examination.
A 2011 review article raised questions again about the use of G-CSF in neuroblastoma treatment and a subsequent 2013 paper elaborated on these concerns. The 2013 study identified of a subpopulation of neuroblastoma cells with a G-CSF receptor (CD114+) that were highly tumorigenic, or capable of cancer growth and proliferation, that resembled stem cells.
The investigators were able to separate the GCSF positive receptor cells and GCSF negative receptor cells. The neuroblastoma cells with the G-CSF receptors were implanted into mice and were found to be 5 to 10 times more potent in their ability to create malignant tumours. In other words G-CSF positive receptor cells (CD114+) are able to create more tumour cells than G-CSF negative receptor cells (CD114-). This subpopulation of cells is different from neuroblastoma tumor initiating cells[15,16] that in some studies have been defined by CD113 expression. It was found that the CD114+ neuroblastoma cells accounted for approximately 0.5-1.5% of the tumours at the time of diagnosis. This research did not examine the effect of G-CSF (exogenous) on neuroblastoma CD114+ cancer stem cells.
Summary of Dr. Agarwal et al’s 2015 Paper
As a follow-up to the 2013 paper, Dr. Agarwal and colleagues worked to understand the effects of G-CSF on neuroblastoma cells and how G-CSF may impact the growth and spread of neuroblastoma. Their theory is that G-CSF will act as a growth factor, a substance that will induce cellular growth, of cancer stem cells in high-risk neuroblastoma.
In Vitro Experiment A – Treating NB Cell Lines With or Without G-CSF in a Dish
For the in vitro experiments (with cell lines not in a mouse or other type of host), two different cell lines (NGP and SH-SY5Y cell lines) were used and the cells were sorted into two groups of G-CSF receptor positive cells (CD114+) and G-CSF receptor negative cells (CD114-). From each group, single cells were each put into a “well” of a plate/dish and treated with or without G-CSF for 28 days. Measurements were taken to quantify the amount of cellular growth (proliferation) that occurred with or without G-CSF. They found that the CD114+ neuroblastoma cells (G-CSF receptor positive cells) increased significantly in number; however, there was minimal to no change in the number of CD114- neuroblastoma cells (G-CSF receptor negative cells). For the SH-SY5Y cell line, the cellular growth increased as the dose of G-CSF increased. However, for the NGP cell line, cellular growth was high only for the lowest does of G-CSF and dropped off for the other two higher doses. An increased activation of STAT3 was observed in the CD114+ cells; however, no changes were observed in the activation of STAT3 in the CD114- cells.
In Vivo Experiment A – Treating NB Cell Lines With or Without G-CSF in Mice
In the first in vivo experiment (using mice), two different neuroblastoma cell lines (NGP and SH-SY5Y cell lines) were injected into mice into the renal (kidney) area. The day after the neuroblastoma cells were implanted into the mice, the experimental group was given G-CSF every day for 21 days and the control group was not given G-CSF. At the end of the 21 days, the mice were euthanized and the tumour and bone marrow was analyzed. Mice with the NGP cell line that were treated with G-CSF had a significant increase in tumour weight and an increase in the percentage of CD114+ cells (three times more than the control). Mice with the SH-SY5Y cell line that were treated with G-CSF had similar results. For both cell lines, a direct correlation between the size of the tumour mass and the percentage of CD114+ cells was observed. For those cell lines that were MYCN amplified and treated with G-CSF, the occurrence of bone marrow metastasis was significantly more than the control group who did not receive G-CSF. An increase in STAT3 was also seen in tumour cells that were treated with G-CSF in comparison to the control group that was not treated with G-CSF.
In Vivo Experiment B – Treating NB Cell Lines With or Without G-CSF in Immunocompromised Mice
The second in vivo experiment aimed to examine the impact of G-CSF that is produced naturally by the stromal cells that are found in the microenvironment of the tumour. “G-CSF knockout mice”, or mice that are unable to produce G-CSF, were used for this experiment. These mice were injected with a different neuroblastoma cell line that expressed MYCN and contained a small CD114+ population. This experiment found that G-CSF was not needed for the tumour to engraft in the mouse; however, tumour growth and spread were enhanced with G-CSF when it was injected into the mouse.
In Vitro Experiment B – Treating NB Cell Lines with a STAT3 Inhibitor
A subsequent in vitro experiment was conducted to observe the effect of blocking STAT3 in different neuroblastoma cell lines by treating the tumour cells with a small-molecule inhibitor called Stattic for 24 hours. Stattic is able to block STAT3 and all cell lines showed some degree of response to this inhibitor. CD114+ cells in particular were responsive to Stattic; however, CD114- cells were found to be resistant to the inhibitor.
In Vitro and In Vivo Experiment C – Treating NB Cell Lines with a STAT3 Inhibitor and Etoposide in a Dish and in Mice
In vitro, cell lines were treated with the STAT 3 Inhibitor (Stattic) and etoposide (VP-16 chemotherapy) and the neuroblastoma cells responded to the coordinated treatment. Next, the experiment was conducted in vivo using mice and two different neuroblastoma cell lines (NGP and SH-SY5Y). Mice were either not treated, treated with Stattic only, treated with etoposide only, or treated with Stattic and etoposide. It was found that etoposide did reduce the tumour burden; however, it was not able to significantly decrease disease metastasis or CD114+ cells. It is interesting to note that treatment with etoposide alone actually increases the percentage of CD114+ cells. Treatment with etoposide and Stattic further reduced the tumour burden and metastatic disease, and reduced the percentage of CD114+ cells. It is important to note that the mice were not given G-CSF in this experiment.
Dr. Russell and Dr. Shohet’s 2011 paper states that there is little evidence supporting that the use of G-CSF increases overall survival in the treatment of high-risk neuroblastoma. In the current Children’s Oncology Group (COG) protocol for high-risk neuroblastoma, G-CSF is utilized in between cycles of chemotherapy to limit low white blood cell counts (neutropenia) and hopefully reduce the possibility of infections. Along with this and the findings from their current research, Dr. Agarwal et al (2015) argue that the use of G-CSF during treatment for high-risk neuroblastoma should be reconsidered. “We give G-CSF after every cycle of chemotherapy, especially in these pediatric protocols for neuroblastoma, where we are using very intense doses of drugs, to help the white cells come back sooner,” he said. “We are giving a growth factor to help fight infection, but our data clearly show that we are also stimulating cancer stem cells at a time when we want them to be dying from the chemotherapy.” This conclusion is supported by the research conducted at Memorial Sloan Kettering Cancer Centre (MSKCC) that was reported in the journal Cancer in 2000. Neuroblastoma patients treated at MSKCC receive much less G-CSF than patients treated on other protocols.
Dr. Maris et al’s Letter Addressing Dr. Agarwal and Colleague’s Research
Dr. Agarwal et al’s 2015 paper garnered a great deal of attention in the neuroblastoma community by families and oncologists. A letter was written by Dr. Maris et al (2015) in response to the article. Dr. Maris et al’s (2015) letter recognized that CD114 is expressed on neuroblastoma cells and that this expression may be impacted by compounds and drugs such as chemotherapy. However, they do not agree with the paper’s conclusion to re-evaluate the use of G-CSF for the treatment of neuroblastoma.
The survival rates for high-risk neuroblastoma have increased over the last 25 years by approximately 20%, with G-CSF being a medication that was added to treatment regimens during this time. In addition, during the last 25 years the use of G-CSF has allowed for chemotherapy doses to be increased by almost four times to result in intensified treatment protocols. To support this argument, the authors point to the 2010 International Society of Paediatric Oncology Europe Neuroblastoma (SIOPEN) study that examined the use of G-CSF in a randomized clinical trial which illustrated that more intensive chemotherapy was safe, that it reduced chemotherapy induced complications and did not inhibit how the disease responded to treatment. Dr. Maris et al provided information in the letter illustrating that there is no difference in outcomes between those patients that received G-CSF and those who did not.
Dr. Maris et al also point out that the dose of G-CSF used in the in vitro studies may be too high (compared to those used in children) and result in findings that are not relevant. Some of the experiments done give G-CSF for 21 days; however, in clinical practice, G-CSF is only given to patients for about 8-10 days. They also point out that G-CSF is used in between chemotherapy cycles and that if there are negative effects from the G-CSF, these cells should be sensitive to the chemotherapies and possibly immunotherapies that the patient will receive in the future treatment cycles.
Dr. Kim and Dr. Shohet’s Response to the Letter from Dr. Maris et al
Dr. Kim et al’s response to the above letter explains the rationale for the dosing of G-CSF that was utilized in their experiments. They also question the belief that the effects of G-CSF would be addressed by subsequent chemotherapies and even immunotherapies. They feel that G-CSF may play a role in protecting neuroblastoma cancer cells from chemotherapy, making them resistant to damage. The researchers do feel that additional pre-clinical and clinical research is required and would like to see a discussion that weighs the benefits and risks of the use of G-CSF in the treatment for high-risk neuroblastoma. All parties agree that patient safety is the number one concern.
There are some additional questions which one can ask about the research conducted by Dr. Agarwal et al in their 2015 paper. There are many questions which arise from the work, and which will hopefully be addressed by future collaborative research. Some of these may be:
- In the neuroblastoma cell lines used, why were cells from actual patients not used in the experiments? The practice of using cells from patients, specifically those that have not spent much time in the tissue culture dish, is potentially important to validate their findings.
- Since Dr. Ladenstein confirmed that there was no difference in outcome between the 238 participants who were involved in the randomized study to look at the impact with and without G-CSF, how does one prove that G-CSF hurts survival? However, all patients in this study were given G-CSF for transplant-based consolidation. Because of that, is it possible to use this study to conclude anything about the impact of G-CSF on outcome?
- It is understood that the research done involved proof of principle experiments. Is it possible that additional research could be done using the same chemotherapeutic and immunotherapy combinations that are currently used to treat high-risk neuroblastoma instead of just one chemotherapy drug?
There are additional questions which could be asked about possible future research in this area:
- Is there any difference in terms of the effect of G-CSF on neuroblastoma cells for tumours with specific genetic markers such as the ALK gene mutations?
- What is the impact of immunotherapy (e.g., dinutuximab, 3F8, and others) on neuroblastoma cells with G-CSF positive receptors?
- Plerixafor (a CXCR4 antagonist) has been used to mobilize stem cells from the bone marrow and into the blood stream. It is used in conjunction with G-CSF and has been able to allow for the collection of more stem cells than just G-CSF alone.[30,31] Is it possible that the use of something like Plerixafor may reduce the need for G-CSF (however, use of plerixafor may not completely remove need for G-CSF)? To try and reduce the side-effects from these drugs, researchers are looking into other drugs which may help to mobilize stem cells to the blood stream (one drug is called Me6TREN and is in pre-clinical studies).
- Is there any difference in terms of the impact of G-CSF on neuroblastoma cells when different forms of G-CSF are used such as the pegylated version of the drug?
- Is there any concern about the role G-CSF may play in the infiltration of disease across the blood-brain barrier and into the central nervous system (CNS)?
- In the 2015 paper, Stattic was the only STAT inhibitor tested. Is it possible that other STAT inhibitors could be tested? For example, work was done on a STAT inhibitor called AZD1480 (however, this drug was discontinued in Phase II trials in 2012.)
- It is important to note that there are other cancers which have tumours that express both G-CSF and its receptor. Some of these cancers have found similar laboratory results showing that G-CSF has an impact on the growth and spread of cancer. What can we learn from these other cancers from the laboratory results and from trials in which patients with these types of cancers received G-CSF?
- A 2007 research study found that Ewing Sarcoma (ES) is one of these cancers and research showed that the administration of G-CSF caused tumour growth (vascular expansion, angiogenesis) and cancer spread in animal models. It called for the future use of G-CSF to be investigated.
- There are articles that go as far back as 1990s discussing the proliferation and spread effect of G-CSF on gliomas[36,37] and skin carcinoma cells.[38,39]
There is no doubt that the research into G-CSF and neuroblastoma cells conducted by Dr. Agarwal and colleagues (2015) has resulted in a significant amount of concern from neuroblastoma patients and families. Families are questioning whether they should continue to give their child G-CSF injections as part of their treatment protocol. In these cases, it is most important to speak to your Oncologist and/or Care Team to discuss any concerns that you may have.
“These experiments are ongoing. We can tell you that G-CSF absolutely increases the extent of metastatic disease seen in the mice treated with chemo”, says Dr. Shohet. “In addition, we will be opening a clinical trial in the very near future that will use standard induction chemotherapy (first six cycles before transplant) without any G-CSF administration. This will be a safety and efficacy pilot study. We no longer use G-CSF in the intermediate risk treatment protocols at TCH [Texas Children’s Hospital]. We agree that additional work should be done and is ongoing in our lab. However, we strongly believe that our published peer reviewed data is valid and additional unpublished data from our NIH [National Institute of Health] funded project continues to support our findings. G-CSF clearly alters the growth and spread of NB in mouse models.”
There are many open questions and much research that could be conducted on the impact of G-CSF on neuroblastoma cancer stem cells. It would be exceptionally valuable to see a cross-institutional and multi-national research team convened to look into the effect of G-CSF on high-risk neuroblastoma. In addition, it would be helpful to study the effects of GCSF in mice who were treated with regimens that resemble the current COG or SIOPEN treatment protocols and deliver GCSF at doses and frequencies similar to those received by patients. The questions are significant and should be addressed with experimental rigour and mutual respect.
Neuroblastoma Canada is extremely thankful for the support, feedback and expertise of Dr. David Kaplan and Dr. Meredith Irwin in the writing of this article. Neuroblastoma Canada would also like to thank Dr. Jason Shohet for providing clarification and feedback on this article.
[Cover image from Wikipedia.]
1 May 12, 2015. Study Links Tumor Recurrence to Growth Factor Commonly Used in High-Risk Neuroblastoma Treatment. Accessed online at: https://www.bcm.edu/news/cancer-pediatric/tumor-recurrence-growth-factor-neuroblastoma
2 Dr. Jason Shohet. May 20, 2015. G-CSF, Cancer Stem Cells and Relapsed Neuroblastoma: A New Study Opens Up Questions and Opportunities. CAC2 and Solving Kids’ Cancer Presentation. Accessed online at: https://www.youtube.com/watch?v=Uq_s8YpnCDM&feature=youtu.be
3 Solving Kids’ Cancer. G-CSF and Neuroblastoma Stem Cells. Accessed online at: http://www.ncca-uk.org/g-csf-and-neuroblastoma-stem-cells/
4 There was also a presentation made by Dr. Shohet at the 2014 Children’s Neuroblastoma Cancer Foundation Parent Neuroblastoma conference. This can be accessed online at: https://www.youtube.com/watch?v=TtURfe7Py-0
5 Agarwal, S., Lakoma, A., Chen, Z., Hicks, J., Metelitsa, L.S., Kim, E.S., and Shohet, J.M. (2015). G-CSF Promotes Neuroblastoma Tumorigenicity and Metastasis Via STAT3-Dependent Cancer Stem Cell Activation. Cancer Research, Vol. 75, No. 12, pps. 2566-2579.
6 Bonnet, D. And Dick, J.E. (1997). Human Acute Myeloid Leukemia is Organized as a Hierarchy that Originates from a Primitieve Hematopoietic Cell. Nature Medicine, Vol. 3, No. 7, pps. 730-737.
7 Youzhi, L., Rogoff, H.A., Keates, S., Gao, Y., Murikipudi, S., Mikule, K., et al. (2015). Suppression of Cancer Relapse and Metastasis by Inhibiting Cancer Stemness. PNAS, Vol. 112, No. 6, pps. 1839-1844.
8 Plaks, V., Zong, N., and Werb, Z. (2015). The Cancer Stem Cell Niche: How Essential is the Niche in Regulating Stemness of Tumor Cells? Cell Stem Cell, Vol. 16, No. 3, pps. 225-238.
9 Ladenstein, R., Valteau-Couanet, D., Brock, P., et al. (2010). Randomized Trial of Prophylactic Granulocyte Colony-Stimulating Factor During Rapid COJEC Induction in Pediatric Patients with High-Risk Neuroblastoma: The European HR-NBL1/SIOPEN Study. Journal of Clinical Oncology, Vol. 28, No. 21, pps. 3516-3524.
10 Gay, A.N., Chang, S., Rutland, L., Yu, L., Byeseda, S., et al. (2008). Granulocyte Colony Stimulating Factor (GCSF) Alters the Phenotype of Neuroblastoma Cells: Implications for Disease Free Survival of High-Risk Patients. Journal of Pediatric Surgery, Vol. 43, No. 5, pps. 837-842.
11 Botting, R.A., and McLachlan, C.S. (2009). Does Cellular Heterogeneity Influence Neuroblastoma Cell Line Proliferation and Invasiveness with Granulocyte Colony-Stimulating Factor? Journal of Pediatric Surgery, Vol. 44, No. 12, pps. 2436-2437.
12 Olutoye, O.O., Kim, E.S., and Shohet, J.M. (2009). Reply. Journal of Pediatric Surgery, Vol. 44, No. 12, pp. 2437.
13 Russell, H. and Shohet, J.M. (2011). G-CSF Counteracts Chemotherapy Toxicity in Neuroblastoma. Nature Reviews Clinical Oncology, 8, pps. 6-8.
14 Hsu, D.M., Agarwal, S., Behnam, A., Coarfa, C., et al. (2013). G-CSF Receptor Positive Neuroblastoma Subpopulations are Enriched in Chemotherapy – Resistant or Relapsed Tumors and are Highly Tumorigenic. Cancer Research, Vol. 73, No. 13, pps. 4134-4146.
15 Hansford, L.M., McKee, A.E., Zhang, L., George, R.E., Gerstle, J.T., et al. (2007). Neuroblastoma cells isolated from bone marrow metastases contain a naturally enriched tumor-initiating cell. Cancer Research, 67, pps. 11234–11243.
16 Coulon, A., Flahaut, M., Muhlethaler-Mottet, A., Meier, R., Liberman, J., et al. (2011). Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model. Neoplasia, 13, pps. 991–1004.
17 Agarwal, S., Lakoma, A., Chen, Z., Hicks, J., Metelitsa, L.S., Kim, E.S., and Shohet, J.M. (2015). G-CSF Promotes Neuroblastoma Tumorigenicity and Metastasis Via STAT3-Dependent Cancer Stem Cell Activation. Cancer Research, Vol. 75, No. 12, pps. 2566-2579.
18 Russell, H. and Shohet, J.M. (2011). Pediatric Oncology: G-CSF Counteracts Chemotherapy Toxicity in Neuroblastoma. Nature Reviews Clinical Oncology, 8, pps. 6-8. http://www.nature.com/nrclinonc/journal/v8/n1/full/nrclinonc.2010.195.html
19 Lowry, F. (2015). Is it Safe to Use G-CSF in Neuroblastoma Patients? Medscape, May 26, 2015. Accessed online May 26, 2015 from: http://www.medscape.com/viewarticle/845310
20 Kushner, B.H., Heller, G., Kramer, K., and Cheung, N-K. (2000). Granulocyte-Colony Stimulating Factor and Multiple Cycles of Strongly Myelosuppressive Alkylator-Based Combination Chemotherapy in Children with Neuroblastoma. Cancer, Vol. 89, No. 10, pps. 2122-2130.
21 Lowry, F. (2015). Is it Safe to Use G-CSF in Neuroblastoma Patients? Medscape, May 26, 2015. Accessed online May 26, 2015 from: http://www.medscape.com/viewarticle/845310
22 Maris, J.M., Healy, J., Park, J., Ladenstein, R., and Potschger, U. (2015). G-CSF is a Cancer Stem Cell-Specific Growth Factor – Letter. Cancer Research, Vol. 75, No. 18, pps. 3991.
23 Irwin, M.S. and Park, J.R. (2015). Neuroblastoma: Paradigm for Precision Medicine. Pediatric Clinics of North America, 62, pps. 225-256. http://www.ncbi.nlm.nih.gov/pubmed/25435121
24 Ladenstein, R., Valteau-Couanet, D., Brock, P., et al. (2010). Randomized Trial of Prophylactic Granulocyte Colony-Stimulating Factor During Rapid COJEC Induction in Pediatric Patients with High-Risk Neuroblastoma: The European HR-NBL1/SIOPEN Study. Journal of Clinical Oncology, Vol. 28, No. 21, pps. 3516-3524. http://jco.ascopubs.org/content/28/21/3516.full
25 From Dr. Shohet in Personal Communication: “As we noted in our reply to the letter to the editor in Cancer Research, all the patients received G-CSF as part of the transplant regimen. The authors were not looking at the effect of G-CSF on any biologic endpoints, only side effects of treatment. We would expect the use of G-CSF to lead to increased treatment failures during induction (promoting drug resistance) and increased late relapses (driven by metastatic stem cells that evading initial therapy). As noted, dissecting whether G-CSF is detrimental to overall survival will require detailed controlled clinical trials. We believe that there is minimal evidence in the literature mandating the use of this potent cytokine and will be studying this further both in models and in clinical trials at TCH [Texas Children’s Hospital]”.
26 Kim, E.S., Agarwal, S., and Shohet, J.M. (2015). G-CSF is a Cancer Stem Cell-Specific Growth Factor – Response. Cancer Research, Vol. 75, No. 18, pps. 3992.
27 Maris, J.M., Healy, J., Park, J., Ladenstein, R., and Potschger, U. (2015). G-CSF is a Cancer Stem Cell-Specific Growth Factor – Letter. Cancer Research, Vol. 75, No. 18, pps. 3991.
28 In communication from Dr. Shohet: “This will be an important line of research. We are currently working on determining how the immune microenvironment interacts with the CSCs.”
29 Broxmeyer HE, Orschell CM, Clapp DW, et al. (2005). Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. Journal of Experimental Medicine, No. 201, Vol. 8, pps. 1307-1318.
30 DiPersio, J.F., Micallef, I.N., Stiff, P.J., et al. (2009). Phase III prospective randomized doubleblind placebo-controlled trial of plerixafor plus granulocyte colony-stimulating factor compared with placebo plus granulocyte colony-stimulating factor for autologous stem cell mobilization and transplantation for patients with non-Hodgkin’s lymphoma. Journal of Clinical Oncology, Vol. 27, No. 28, pps. 4767-4773.
31 DiPersio, J.F., Stadtmauer, E.A., Nademanee, A. (2009). Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma. Blood, Vol. 113, No. 23, pps. 5720-5726.
32 Zhang, J., Ren, X., Shi, W., et al. (2014). Small molecule Me6TREN mobilizes hematopoietic stem/progenitor cells by activating MMP-9 expression and disrupting SDF-1/CXCR4 axis. Blood, Vol. 123, No. 3, pps. 428-441.
33 Yan, S., Li, Z., and Thiele, C.J. (2013). Inhibition of STAT3 with Orally Active JAK Inhibitor, AZD1480, Decreases Tumor Growth in Neuroblastoma and Pediatric Sarcomas In Vitro and In Vivo. Oncotarget, Vol. 4, No. 3, pps. 433-445.
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35 Morales-Arias, J., Meyers, P.A., Bolontrade, M.F. et al. (2007). Expression of Granulocyte-Colony-Stimulating Factor and its Receptor in Human Ewing Sarcoma Cells and Patient Tumor Specimens: Potential Consequences of Granulocyte-Colony-Stimulating Factor Administration. Cancer, Vol. 110, No. 7, pps. 1568-1577.
36 Muelle, M.M., Herold-Mende, C.C., Riede, D., Lange, M., Steiner, H.H., Fusenig, N.E. (1999). Autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor in human gliomas with tumor progression. American Journal of Pathology, 155, pps. 1557-1567.
37 Wang, J., Yao, L., Zhao, S., Zhang, X., et al. (2012). Granulocyte-Colony Stimulating Factor Promotes Proliferation, Migration and Invasion in Glioma Cells. Cancer Biology and Therapy, Vol. 13, No. 6, pps. 386-400.
38 Mueller, M.M., Peter, W., Mappes, M., et al. (2001). Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis and autocrine growth regulation by granulocyte colony-stimulating factor and granulocytemacrophage colony-stimulating factor. American Journal of Pathology, 159, pps. 1567-79.
39 Mueller, M.M., Fusenig, N.E. (1999). Constitutive expression of G-CSF and GM-CSF in human skin carcinoma cells with functional consequence for tumor progression. International Journal of Cancer, 83, pps. 780-789.