According to the cancer stem cell hypothesis, only a small subset of the cells in a tumor are able to give rise to a tumor. The bulk of the cancer cells may cause symptomatic problems but not metastatic disease. Researchers have amassed plenty of laboratory and animal data that support the hypothesis, and it is consistent with some clinical observations, including the continued risk of relapse even after treatment has eliminated detectable disease. However, direct evidence that these putative cancer stem cells have an impact on patient outcomes has been lacking.
That pattern is starting to change, however. For example, data reported at the most recent San Antonio Breast Cancer Symposium showed that stem cell-like cells in breast tumors are resistant to neoadjuvant chemotherapy (OT, 3/25/08), and more recently, the results of several studies consistent with the hypothesis were reported at the American Association for Cancer Research and American Society for Clinical Oncology Annual Meetings.
Has to Be Relevant
“If we can't show clinically that cancer stem cells are relevant, then I don't know what we are doing; I don't know why we are doing this,” said William Matsui, MD, Assistant Professor of Oncology in the Division of Hematologic Malignancies at The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins School of Medicine, who studies both normal and cancer stem cells.
Demonstrating that cancer stem cells drive tumorigenesis or disease progression in patients, though, is not as easy as one would hope. The definitive experiment—which is entirely unethical and thus will not and should not be done—would be to transfer tumor samples from one person to another and show that disease arises in the recipient only if cancer stem cells are contained in the transferred sample.
In the absence of that experiment, Dr. Matsui sees two main approaches for demonstrating the clinical relevance of the hypothesis. Researchers can develop drugs that target the putative cancer stem cells and determine whether patients treated with these agents have longer survival than those treated with standard therapies. Alternately they can try to correlate patient outcomes with some kind of measure of stem cells in individual patient tumors.
Targeting Stem Cells in Myeloma
Dr. Matsui's group is using both approaches and reported early results from the clinical studies at the AACR meeting. In the first of the studies, Dr. Matsui and Carol Ann Huff, MD, an oncologist who specializes in multiple myeloma, hypothesized that targeting the stem cells could delay relapse in that disease.
The group had previously shown that the putative cancer stem cells in multiple myeloma resemble memory B lymphocytes and carry the CD20 cell surface protein. In contrast, the majority of the tumor cells are differentiated plasma cells that do not express CD20 in most patients.
With that in mind, the team reasoned that treating patients with cyclophosphamide (50 mg/kg a day for four days) plus rituximab, which binds CD20, might improve patient outcomes relative to standard therapy alone.
They enrolled patients with primary refractory disease, high-risk patients in their first remission, and patients who had relapsed disease that was responding to salvage therapy. The patients received two doses of rituximab prior to cyclophosphamide, then four weekly doses after their blood cell counts recovered from the chemotherapy, and up to four maintenance doses every three months for patients who did not show evidence of progression.
With 21 patients treated, one had a complete response, 16 had partial responses, and about four had progressive disease. The median duration of response was 194 days, with three patients remaining progression-free at 81 to 538 days. The median overall survival has not been reached with a median follow-up of 494 days.
To look for evidence of anti-stem cell activity, the researchers performed correlative studies on biopsy samples taken throughout the first year following cyclophosphamide treatment. The researchers saw an average 44% drop in the clonogenic growth of samples from the patients immediately after treatment, suggesting that the treatment did reduce the number of cancer stem cells present.
Patients whose biopsy samples showed a return of clonogenic growth to pretreatment levels within 12 months had a median progression-free survival of 185 days compared with 564 days for those patients whose clonogenic levels remained low at 12 months. The number of putative stem cells that give rise to clonogenic growth rose a median of 53 days prior to a patient relapsing.
Using flow cytometry, the researchers could see that the anti-CD20 antibody did bind to the putative stem cells, but did not kill them. “The cells are coated in Rituxan, but Rituxan isn't really doing anything,” Dr. Matsui said. “This has led us to think that maybe we have the right cellular target but the wrong agent to target them.”
In fact, Andrzej J. Jakubowiak, MD, PhD, Professor of Internal Medicine at the University of Michigan Comprehensive Cancer Center, recognized the potential of an anti-CD20 antibody to kill myeloma stem cells but wanted more killing power than rituximab has.
“When I was thinking of developing a clinical trial, I thought that targeting these cells with a cold antibody would have some chance of success, but very low,” he said in an interview. “These cells have theoretically a very great ability to resist many other therapies and presumably they are in very small numbers, so if you eliminate 50% or 60% or 80% of these cells, you may still end up with enough self-renewing cells that the impact of the treatment may not be clinically seen.”
Therefore, he initiated a trial in myeloma patients with tositumomab (Bexxar), an anti-CD20 antibody that is conjugated with radioactive iodine. Tositumomab is used as consolidation treatment for patients who had reached a plateau in their response either to initial therapy—as a sandwich treatment between initial standard therapy and transplant for those eligible—or after a first or second salvage therapy.
Dr. Jakubowiak declined to discuss many specifics because he has not yet presented or published extensive data from the trial, but he did say that he has enrolled eight patients thus far, four patients who are heading for transplants and four who have previously undergone transplantation. Of the four patients beyond transplant, only one showed a decline in monoclonal protein three months after tositumomab therapy. One patient had progressive disease and three others had stable disease at three months.
Whether the antibody therapy has improved those outcomes over what would have occurred with standard therapy is unclear. “I do believe we have some encouraging information that would give us some sense that this intervention is working, but it is too early to say,” he said.
All four of the patients in the pre-transplant group have had a good or complete response and none had relapsed with a “reasonably good period of observation,” he said. In this setting, with the post-tositumomab transplant, it is even harder to judge the impact of the antibody therapy on clinical outcomes because the transplant follows the antibody therapy.
As in Dr. Matsui's trial, Dr. Jakubowiak has planned extensive correlative science studies, which may help answer the question about the role of the putative stem cells in disease. He said he expects to present more complete data in December at the American Society of Hematology Annual Meeting.
Pancreatic Cancer Correlations
In a distinct set of experiments, Dr. Matsui's group has been using correlative outcome data to decipher the role of putative cancer stem cells in pancreatic cancer. “One way to maybe show clinical relevance is to show that in some tumors there are more stem cells, and if you have more stem cells when you are newly diagnosed, you live a shorter amount of time,” he said.
With that model in mind, the researchers examined tumor specimens from nearly 300 patients with pancreatic cancer. When they assayed the samples for cells that express aldehyde dehydrogenase (ALDH), a marker for the putative cancer stem cells in pancreatic disease, 90 samples were ALDH positive and 179 were ALDH negative. The patients with ALDH-positive cells survived for a median of 14 months compared with 18 months for the patients whose tumors lacked ALDH-staining cells.
This observation is consistent with data that Max Wicha, MD, Professor of Internal Medicine and Director of the University of Michigan Comprehensive Cancer Center, and colleagues saw when looking at tumor samples from breast cancer patients (Cell Stem Cell 2007;1:555-567). Both studies indicate that the presence of cancer stem cells correlates with shorter survival, although they do not demonstrate a causal relationship between the two observations.
To learn whether the presence of putative cancer stem cells increased the likelihood of metastases, Dr. Matsui's group took one more step: They took advantage of a rapid autopsy program available for pancreatic cancer patients at Johns Hopkins Medical Institute and looked for the presence of ALDH cells in metastatic lesions.
In eight cases, the researchers had tumor samples from the primary tumor isolated previously and from metastatic disease at the time of death. Two of the original tumors were ALDH-positive. Of the six tumors initially ALDH-negative, four had converted to ALDH-positive disease in the metastatic setting.
“I think that it does show some clinical relevance,” Dr. Matsui said. “It shows that if you have these cells around, it predicts your survival, and the way they do that is by mediating the metastatic spread of disease, and that is what ultimately kills you.”
Stem Cells Express Mesenchymal Markers
Part of the reason his group looked for the ALDH cells in metastatic disease is that they and others have found that putative cancer stem cells express mesenchymal cell markers, even though the bulk of the tumor cells express epithelial markers.
For example, Robert Weinberg, PhD, of the Whitehead Institute and Professor of Biology at Massachusetts Institute of Technology, and colleagues reported that putative cancer stem cells isolated from mammary tumors express mesenchymal markers, such as vimentin, and have upregulated the activity of several developmental pathways that are involved in the epithelial mesenchymal transition (EMT).
These observations are significant because EMT dramatically shifts the phenotype of cells from an immotile polarized epithelial to a motile mesenchymal cell. Thus, cancer stem cells that behave like mesenchymal cells are likely to promote invasion and metastasis.
In the study reported at the most recent San Antonio Breast Cancer Symposium, Jenny Chang, MD, Medical Director of the Lester and Sue Smith Breast Center and Professor of Medicine at Baylor College of Medicine, found that the percentage of putative breast cancer stem cells increased in patient tumors following neoadjuvant treatment with standard chemotherapy. Building on that work, Dr. Chang reasoned that if EMT is important in cancer stem cell biology, then mesenchymal gene expression should also increase following standard neoadjuvant chemotherapy.
To find out, she characterized the gene-expression patterns in cancer stem cells isolated from breast tumors. Regardless of whether the cancer stem cells were isolated from estrogen receptor positive or negative tumors, the cancer stem cells had “strikingly homogeneous” gene expression signatures, she reported at this year's ASCO Annual Meeting. The stem cell signature included interesting pathways such as Notch and AKT/PI3K that are implicated in EMT and stem cell phenotypes.
She then compared the cancer stem cell signature with the breast cancer tumor types previously defined by gene-expression patterns, including luminal, basal, ERBB2-overexpressing, normal-like, and the recently described Claudin-low.
The cancer stem cell signature closely correlated with the Claudin-low signature. Interestingly, the Claudin-low tumors appear to have lost tight-junctions and cell adhesions, which are common traits of epithelial cells, and show an upregulation of EMT genes, such as snail, slug, and vimentin, and a loss of e-cadherin, which is expressed by epithelial cells.
If the cancer stem cell hypothesis is correct, and standard chemotherapy removes the bulk of the tumor but leaves behind the cancer stem cells, then the residual tumor in breast cancer patients after chemotherapy and endocrine therapy ought to contain a signature that is enriched in the cancer stem cell signature.
Looking at paired tumor samples from 18 patients taken before and after letrozole therapy, the researchers found that “overwhelmingly after chemotherapy, our stem cell signature was increased,” Dr. Chang said. The same thing was seen in samples from 12 women treated with docetaxel.
“Therefore, post conventional chemotherapy and post conventional endocrine therapy, the residual tumors are enriched for the cancer stem cell signature as well as the Claudin-low signature,” she said. “And these two signatures show considerable overlap.”
Finally, when the expression of two markers of EMT in paired tumor samples taken before and after letrozole therapy were compared, an increase was found following therapy. Specifically, vimentin increased after therapy in 14 of 23 women tested. Also MMP2 increased in 47 of 60 patients tested. The changes in expression of the two genes were statistically significant when compared across the population.
“Specific inhibitors to pathways active in our cancer stem cell signature may enhance the efficacy of our therapies,” Dr. Chang concluded.
Despite the increase in clinically based experiments, no one expects the transition of the theory to the clinic to occur quickly. Instead, progress will likely require moving back and forth between laboratory science and clinical experiments, which will take time. However, if the theory is right, it could ultimately have profound effects on patient outcomes, those interviewed for this article said.
“The accumulating data suggest that a lot of our current therapies may not be effective because they are targeting the wrong cell populations,” said cancer stem cell research pioneer Max Wicha, MD. “Our therapies need to be directed at this population, either through their elimination or potentially pushing their differentiation so that they lose their self-renewal potential.”