Research Focus: Targeting Cancer Stem Cells

Research Focus: Targeting Cancer Stem CellsThis activity is supported by an educational grant from Boston Biomedical.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsIn this module, Max S. Wicha, MD, provides expert insight on cancer stem cells and emerging strategies to therapeutically target these cells. 1About These SlidesUsers are encouraged to use these slides in their own noncommercial presentations, but we ask that content and attribution not be changed. Users are asked to honor this intentThese slides may not be published or posted online without permission from Clinical Care Options (email permissions@clinicaloptions.com)DisclaimerThe materials published on the Clinical Care Options Web site reflect the views of the authors of the CCO material, not those of Clinical Care Options, LLC, the CME providers, or the companies providing educational grants. The materials may discuss uses and dosages for therapeutic products that have not been approved by the United States Food and Drug Administration. A qualified healthcare professional should be consulted before using any therapeutic product discussed. Readers should verify all information and data before treating patients or using any therapies described in these materials.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem Cells2FacultyMax S. Wicha, MDDistinguished Professor of OncologyDirector, University of Michigan Comprehensive Cancer CenterDepartment of Internal MedicineUniversity of MichiganAnn Arbor, Michigan

Max S. Wicha, MD, has disclosed that he has received consulting fees from MedImmune, Paganini, and Verastem; has received funds for research support from Dompe and MedImmune; and has ownership interest in OncoMed.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem Cells3Cancer Stem Cell HypothesisCancers are driven by cells with stem cell properties[1]Self-renewing and long livedImmune privileged, thus not eliminated by immune cellsMultipotent, thus able to contribute to tumor cell heterogeneityCancer stem cells arise from normal stem cells with acquired mutations that promote dysregulated self-renewalCancer stem cells contribute to tumor development, recurrence, and metastasesLess sensitive to chemotherapy and biologic agents1. Bruttel VS, et al. Front Immunol. 2014;5:360.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsThe idea that there was a relationship between stem cells and cancer is quite oldit goes back more than 100 years when it was a thought that there was a relationship between cells in the embryo and cancers. However, it is only in the last 15 years or so that the science of stem cells has advanced, leading to a blossoming of this field. How do we define exactly what a stem cell is? Stem cells have 2 primary characteristics: the ability to self-renew, meaning that they can form exact copies of themselves (in a sense, stem cells are immortal), and the capacity to differentiate (ie, to form other cell types within an organ). There are different types of stem cells, including embryonic stem cells and adult stem cells. Embryonic stem cells form in the embryo and have the capacity to form virtually any other cell in the body, which is why they are potentially useful for tissue regeneration and perhaps treating diseases such as Alzheimers disease. In addition, every organ has its own group of tissue-specific stem cells, which we call adult stem cells. These are much more limited in their capacity to differentiate. That is, a stem cell in the breast can only form other breast cells, and a stem cell in the colon can form only other colon cells.There is a third type of stem cell, the cancer stem cell. Cancer stem cells may arise from either tissue-specific stem cells or other self-renewing cells, but they have embryonic stem cell properties, and that is the key, we think, to how they drive cancer formation.[1]The cancer stem cell hypothesis comprises 2 important but independent components. The first component concerns the origin of tumors. The classical thinking is that cancers develop as a result of random mutations and that any cell can become a cancer cell. However, the cancer stem cell hypothesis posits that cancers can only form in cells that have a particular property, that of self-renewal. These cells can either be normal stem cells within each tissue or can be nonstem cells which, through mutation, have acquired the ability to self-renew. This concept has important implications for understanding where cancers come from and, more importantly, how we might treat and prevent them.The second part of the cancer stem cell hypothesis is that tumors are organized in a hierarchical fashion; not all the cells in a cancer are the same. At the apex of this hierarchy are the cancer stem cells. And the fact that cancers comprise cells with stem cell properties has important clinical implications.

Reference1. Bruttel VS, Wischhusen J. Cancer stem cell immunology: key to understanding tumorigenesis and tumor immune escape. Front Immunol. 2014;5:360.

4Tissue Specific Correlation Between Cancer Risk and Total Stem Cell Divisions2. Tomasetti C, et al. Science 2015;347:78-81.10-110-310-510510710910111013FAP colorectalBasal cellColorectalLynch colorectalSmall intestineAMLCLLHepatocellularPancreatic ductalMelanomaHCV hepatocellularHPV head & neckLung (smokers)FAP duodenumLung (non smokers)DuodenumMedulloblastomaHead osteosarcomaPelvis osteosarcomaGallbladderGlioblastomaOvarian germ cellThyroid medullaryThyroid follicularHead & neckTesticularEsophagealPancreatic isletArms osteosarcomaLegs osteosarcomaOsteosarcomaTotal Stem Cell DivisionsLifetime Riskclinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsAML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; FAP, familial adenomatous polyposis; HCV, hepatitis C virus; HPV, human papillomavirus.

The concept that cancers arise from self-renewing cells is a very interesting and important one, and I call your attention to a recent publication by Vogelstein and colleagues.[2] The goal of this study was to answer a very old question: Why do some organs have a much higher rate of cancer formation than others? They examined the relationship between the lifetime risk of cancer and the number of stem cell renewals in specific organs. What they found was a strong correlation between these 2 factors. For example, looking specifically at the colon, there is a high proportion of normal tissue stem cell divisions and a high risk of carcinogenesis. By contrast, the gall bladder has fewer stem cell divisions and a lower risk of carcinogenesis. These data reinforce the idea that self-renewing cells may be the source of cancers and, furthermore, that most cancers are caused by mutations within these self-renewing cell populations.

Reference2. Tomasetti C, Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science. 2015;347:78-81.

Stem Cells and Breast CarcinogenesisDIFFERENTIATIONEarly progenitorLate progenitorLuminal cellsMyoepithelial cellsAlveolar cellsStem cellQuiescent pool of stem cellsCancer stem cellMutation eventTumor formation3. Dontu G, et al. Cell Prolif. 2003;36(suppl 1):59-72.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsIn my laboratory, we focus on tumors of the breast and mammary gland, as the breast is a very interesting and important organ in which stem cells have been isolated and characterized. Of more importance, breast cancer is the leading cancer in women and is a large clinical problem, both in the United States and around the world. This slide illustrates how, within the normal breast, a breast stem cell can give rise to more copies of itself.[3] During pregnancy, the breast stem cell can self-renew and differentiate, producing cells that acquire receptors for hormones such as estrogen and that are able to make milk proteins. Breast cancers come from these self-renewing cell populations.

Reference3. Dontu G, Al-Hajj M, Abdallah WM, et al. Stem cells in normal breast development and breast cancer. cell Prolif. 2003;36(suppl 1):59-72.

6

20,000

CD44-CD24+cells200

CD44+CD24-cells Tumor Formation by Human Breast Cancer Stem Cells 5. Al-Hajj M, et al. Proc Natl Acad Sci U S A. 2003;100:3983-3988.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsThe first studies of stem cells in breast cancer were performed by our laboratory in collaboration with Michael Clarke and his colleagues. The experiments were modeled after research on leukemia formation by Lapidot and colleagues,[4] which showed that only a small fraction of cells in a human leukemia were able to transfer that leukemia into immunosuppressed mice. We wanted to know if the same was true in breast cancerwas there only a subset of cells capable of transferring the cancer into an immunosuppressed mouse? We identified breast cancer stem cells as CD44 positive and CD24 negative and found that injecting as few as 200 of these cells into the mammary gland of an immunosuppressed mouse consistently resulted in tumor formation.[5] However, 20,000 cells that did not express these protein markers were not tumorigenic. Most of cancer research has focused on those 20,000 cells, which form the bulk of the tumor, but we found that the cells with this stem cell biomarker pattern accounted for only 1% to 5% of the cells in human breast cancers.

References4. Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367:645-658.5. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100:3983-3988.

7ConventionalCancer therapyCSCs regenerate tumorCSC targetedtherapyTumor looses itsability to generatenew cellsTumor degenerates;patient is curedTumor regressesTumor recurs6. Reya T, et al. Nature. 2001;414:105-111.Treatment Implications of Cancer Stem CellsCSCCSCCSCclinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell.

Not only are cancer stem cells a rare population within tumors that can transfer the tumor into mice, but we and others have shown that these cells are the malignant seeds that cause metastasis.[6-8] Furthermore, both mouse models and human clinical trials have found that cancer stem cells are relatively more resistant to many of the therapies that we have developed, such as chemotherapy and radiation therapy. In other words, most therapies selectively kill the bulk cells in a tumor but not the cancer stem cells. Therefore, many of the therapies that have been developed can shrink a tumor but unfortunately are not curative when the cancer is metastatic. The cancer stem cells are comparable to resistant seeds in a weed, and the available treatments are only killing the leaves of the weed; the seeds and the roots grow back and the cancer recurs. If this is the case, then being able to cure cancers will require elimination of the cancer stem cells.There are numerous properties that make cancer cells resistant to chemotherapy and radiation.[9] First, they have different cell cycle kinetics. Many of the cancer stem cells are not cycling; they are actually quiescent cells. Numerous therapies target cells that are rapidly proliferating. Second, cancer stem cells have increased amounts of anti-apoptotic proteins, such as BCL-2 and BCL-X, that resist cell death. Third, cancer stem cells have more efficient DNA repair mechanisms than noncancer stem cells, and they have increased antioxidants that protect them against radiation therapy. In addition, they express transporter proteins that can pump chemotherapy agents out of the cells, such as the breast cancer resistance protein. Finally, cancer stem cells express high levels of ALDH (which we use as a marker for stem cells) that is able to metabolize some chemotherapeutic agents. Clearly, cancer stem cells have a wide arsenal of mechanisms that enable them to resist chemotherapy and radiation.

References6. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001.414:105-111. 7. Li F, Tiede B, Massagu J, et al. Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res. 2007;17:3-14.8. Liao WT, Ye YP, Deng YJ, et al. Metastatic cancer stem cells: from the concept to therapeutics. Am J Stem Cells. 2014;3:46-62.9. Colak S, Medema JP. Cancer stem cell: important players in tumor therapy resistance. FEBS J. 2014;281:4779-4791.

8Implications of CSC TherapeuticsTumor regression inadequate endpointPreclinical modelsPhase II-III clinical trialsExtrapolation to adjuvant settingsCSCs may be resistant to therapy (apoptosis)Effective therapies should target CSCs while sparing normal stem cells Circulating tumor cells may be useful in monitoring responseclinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell.

The cancer stem cell model has profound implications for how we develop new therapies to treat cancer. Many of our current therapies are based on the ability of treatments to cause shrinkage of tumors as assessed by Response Evaluation Criteria in Solid Tumors criteria. Unfortunately, tumor shrinkage does not correlate well with patient survival for many tumor types, including breast cancer. Therefore, the idea that we can use tumor shrinkage in either our preclinical models or our phase I and phase II clinical trials may have to be re-examined.Furthermore, our development of adjuvant therapies may have to be completely reanalyzed based on the cancer stem cell model. We think that regrowth of tumors from micrometastases in the adjuvant setting results from cancer stem cell regrowth, whereas tumor shrinkage measures a reduction in bulk tumor cells.Because cancer stem cells may be resistant to many of our current therapies, effective cancer stem celltargeting therapies need to be developed. However, this is a challenge because some of the pathways used by cancer stem cells may also be used by normal adult tissue stem cells, which are essential for maintaining organs such as the intestine and the bone marrow. So we need to develop therapies that spare the normal stem cells but target the cancer stem cells. We also have to develop novel methods to assess the efficacy of cancer stem celltargeting therapies. I will briefly discuss one such methodmonitoring circulating tumor cellsbelow.9InitialWk 3Wk 12302520151050Effect of Neoadjuvant Chemotherapy and Lapatinib on Breast CSCsPercent of CD44+ CD24- cells* in HER2-tumors before, during, and after neoadjuvant chemotherapyPercent of CD44+ CD24- cells* in HER2+ tumors before, during, and after lapatinib therapy*Cells isolated form biopsy samples.10. Li X, et al. J Natl Cancer Inst. 2008;100:672-679. N = 31CD44+/CD24-Observed PredictiveInitialWk 2Wk 4Wk 6302520151050N = 21Observed PredictiveCD44+/CD24-clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell.

The first evidence that cancer stem cells in patients with breast cancer were resistant to chemotherapy was reported by Li and colleagues[10] in 2008. The authors assessed the proportion of CD24+ CD44- cancer stem cells in biopsies obtained from patients before, during, and after neoadjuvant chemotherapy (for HER2-negative tumors) or lapatinib therapy (for HER2-positive tumors).In patients with HER2-negative cancer who were given neoadjuvant chemotherapy, the percentage of stem cells increased even as the tumor shrank. In patients with HER2-positive cancer who received lapatinib therapy, the frequency of cancer stem cells did not increase. In fact, it decreased slightly by Week 6 along with the tumor shrinking. We think that the remarkable clinical efficacy of HER2-blocking agents is due to their ability to target breast cancer stem cells.

Reference10. Li X, Lewis MT, Huang J, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008;100:672-679.10CXCR1Cell deathIL-8CSCIL-8Cell death ChemotherapyBulk tumor cellsSelf-renewalBystander effectsChemotherapy Induces Self-Renewal in CSCs via IL-8 and CXCR1Reparixin12. Ginestier C, et al. J Clin Invest. 2010;120:485-497.Tclinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell.

In the previous slide, we saw that when patients are treated with cytotoxic chemotherapy, the percentage of cancer stem cells increases. This occurs because cancer stem cells are relatively resistant to chemotherapy. Moreover, using mouse models, we found that the absolute number of stem cells also increased following chemotherapy or radiation therapy. We concluded that chemotherapy and radiation therapy may have an unwanted consequence of actually increasing the number of cancer stem cells. How does that occur? Chemotherapy induces cell death and dying cancer cells secrete cytokines such as interleukin (IL)-8. We discovered that cancer stem cells have a receptor for IL-8 called CXCR1. When IL-8 binds to CXCR1, it causes the stem cell to both block its own cell death pathways and trigger self-renewal processes.[11,12] This pathway may have developed to preserve normal tissue stem cells within injured tissues; distress signals from dying cells initiate the normal stem cells to proliferate in order to repair the tissue. However, when this happens in the tumor environment, the end result is increased numbers of cancer-forming stem cells. Now that we have identified this pathway we can develop specific agents to block it. Indeed, a small molecule inhibitor of CXCR1 has been developedreparixin (formerly repertaxin).

References:11. Charafe-Jauffret E, Ginestier C, Iovino F, et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res. 2009;69:1302-1313.

12. Ginestier C, Liu S, Diebel ME, et al. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J Clin Invest. 2010;120:485-497.

11Chemotherapy Plus CXCR1 Blockade Targets Breast CSCs in Mouse Model-40 -30 -20 -10 0 10 20 30Days After InjectionTumor Size (mm)ControlReparixinDocetaxelReparixin/docetaxel13. Ginestier C, et al. J Clin Invest. 2010;120:485-497.CSC Population (%)ControlReparixinDocetaxelReparixin/Docetaxel2016128409876543210clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell.

We tested the ability of reparixin to block the increase in cancer stem cells following cytotoxic chemotherapy in a mouse model of breast cancer.[13] This slide illustrates both tumor size and the frequency of cancer stem cells in mice with breast tumors that were treated with reparixin and docetaxel. Cancer stem cells were identified using an agent that acts as a substrate for ALDH and becomes fluorescent after interacting with the enzyme. When reparixin was administered along with chemotherapy, the increase in cancer stem cells caused by docetaxel was completely blocked.

Reference:13. Ginestier C, Liu S, Diebel ME, et al. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J Clin Invest. 2010;120:485-497.12Reparixin Plus Paclitaxel in Pts With HER2-Negative MBCPhase Ib pilot studyN = 33 pts with HER2-negative MBC, 3 prior lines of CT (not including neoadjuvant CT), RECIST 1.1, ECOG PS 0-1, no brain metastases3-day run-in with reparixin (400 mg, 800 mg, or 1200 mg 3 x daily) followed by paclitaxel 80 mg/m2/wk + reparixin for 21 daysTreatment continued until disease progression, toxicity, withdrawalSafety assessed after 1 cycle15 serious AEs reported; none related to reparixinGrade 3/4 AEs in 30% (10/33) 5 hematologic 1 discontinuation due to reversible, reparixin-induced GI reaction2 CR, 6 PR among 18 pts eligible for tumor assessment DOR: 2-20 mos 15. Schott AF, et al. SABCS 2014. Abstract P6-03-01.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsAE, adverse event; CR, complete response; CT, chemotherapy; DOR, duration of response; ECOG, Eastern Cooperative Oncology Group; GI, gastrointestinal; MBC, metastatic breast cancer; PR, partial response; PS, performance status

Based on these preclinical data, reparixin is currently under evaluation in a phase Ib clinical trial in combination with paclitaxel in 33 patients with HER2-negative breast cancer.[14] The first analysis of this trial showed that the combination therapy had very few adverse events; most were due to the chemotherapy.[15] Among evaluable patients, there were 2 complete responses, 6 partial responses, and 4 patients with stable disease for 16 weeks. On the basis of these data, a randomized phase II study is planned to evaluate reparixin plus chemotherapy and paclitaxel vs chemotherapy and paclitaxel.

References:14. ClinicalTrials.gov. Pilot study to evaluate reparixin with weekly paclitaxel in patients with HER2 negative metastatic breast cancer (MBC). Available at: https://clinicaltrials.gov/ct2/show/NCT02001974. Accessed February 17, 2015. 15. Schott AF, Wicha MS, Perez RP, et al. A phase Ib study of the CXCR1/2 inhibitor reparixin in combination with weekly paclitaxel in HER2 negative breast cancer: first analysis. Program and abstracts of the 2014 San Antonio Breast Cancer Symposium; December 9-13, 2014; San Antonio, Texas. Abstract P6-03-01.

13HER2 overexpression increases the frequency of cancer stem cells as determined by ALDH activity staining, tumor formation, and expression of stem cell related genesTrastuzumab reduces the frequency of cancer stem cells in HER2+ cell lines

403020100% ALDH Activity-Positive Cells% ALDH Activity-Positive CellsCells:MCF7Sum149Sum159HCC-1954HER2:--+--+--++--Effect of Trastuzumab on CSC PhenotypePercent of CSCs Among Multiple Cell Lines With and Without HER2 ExpressionPercent of CSCs Among Cell Lines Exposed to Trastuzumab17. Korkaya H, et al. Oncogene. 2008;27:6120-6130.Cells:Sum159HER2:--+403020100Trastuzumab-Trastuzumab+clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem Cells1414ALDH, aldehyde dehydrogenase; CSC, cancer stem cell.

Arguably, the greatest advance in breast cancer treatment during the last decade is the development of HER2-targeted therapies. As many as 60% of patients with HER2-positive breast cancers experience a complete pathologic response, which is an important endpoint in clinical trials of breast cancer treatments.[16] We think that the remarkable clinical efficacy of HER2-blocking agents is due to the ability of these agents to target breast cancer stem cells.We performed a study in which the HER2 gene was spliced into multiple breast cancer cell lines.[17] The result was an increase in the frequency of cancer stem cells, as determined by ALDH activity staining and tumor formation in mice. Furthermore, when these cells were treated with trastuzumab, the cancer stem cell population declined. Of course, HER2-targeting agents were developed before we knew about breast cancer stem cells. It is only in retrospect that we think their efficacy is due to the role of HER2 in driving the formation of cancer stem cells.

References:16. Valachis A, Mauri D, Polyzos NP, et al. Trastuzumab combined to neoadjuvant chemotherapy in patients with HER2-positive breast cancer: a systematic review and meta-analysis. Breast. 2011;20:485-490.

17. Korkaya H, Paulson A, Iovino F, Wicha MS. HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene. 2008;27:6120-6130.

Adjuvant Trastuzumab Beneficial for Both HER2- and HER2+ PtsEndpoint for Central HER2 Assay*ACT,n/NACTH, n/NRelative Risk (95% CI)P ValueP Value for InteractionDisease progressionHER2 positive163/87585/8040.47 (0.37-0.62)< .001.47HER2 negative20/927/820.34 (0.14-0.80).014DeathHER2 positive55/87538/8040.66 (0.43-0.99).047.08HER2 negative10/921/820.08 (0.01-0.64).017*HER2 assay results considered negative if both fluorescence in situ hybridization and immunohistochemistry assays negative.19. Paik S, et al. N Engl J Med. 2008;358:1409-1411. clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsACT, doxorubicin/cyclophosphamide/paclitaxel; ACTH, doxorubicin/cyclophosphamide/paclitaxel + trastuzumab.

The relationship between HER2 and breast cancer stem cells was further elucidated by a study that initially was perplexing. In the past, the treatment of breast cancer with HER2-targeted therapies was limited to patients with HER2-positive tumors, as they were thought to be the only group that would benefit from these therapies. Paik and colleagues[18] reanalyzed biopsies from the National Surgical Adjuvant Breast and Bowel Project, a phase III trial of trastuzumab plus adjuvant chemotherapy for HER2-positive breast cancer, and found that 18% of patients initially classified as HER2 positive were actually HER2 negative. Then, they assessed the efficacy of the trastuzumab-containing regimen according to the level of HER2 expression, with the assumption that the greatest efficacy would occur among patients with the highest HER2 expression. However, they found no significant correlation between trastuzumab benefit and HER2 expression.[19] In fact, patients who were HER2 negative benefited as much from the treatment as HER2-positive patients. The relative risk of disease progression was 0.47 among patients with HER2-positive cancer, which translates to a 53% reduction in recurrence. However, in the HER2-negative group, the benefit was even greater, with a 0.34 relative risk of disease progression, or a 66% reduction in recurrence with trastuzumab plus adjuvant chemotherapy. There was an even greater difference in the relative risk of death between HER2-positive and HER2-negative patients (0.66 vs 0.08, respectively). This was quite shocking, and it was unclear how HER2-negative patients could benefit from a HER2-blocking agent.Work in our laboratory suggests that this counterintuitive outcome may be due to the fact that HER2 is selectively expressed in the cancer stem cell population. We performed a comparative assessment of HER2 in nonstem cells vs cancer stem cells in luminal breast cancer cell lines and found that the stem cells show greatly increased HER2 expression.[20] Therefore, we think that even in tumors classified as HER2 negative, HER2 may be selectively expressed in the cancer stem cell population. This may explain why trastuzumab, which is able to reduce the HER2-expressing stem cell population, may have clinical benefit even in HER2-negative patients. These data suggest that we may have developed adjuvant therapies using the wrong paradigm. In general, adjuvant therapies were developed by using agents capable of reducing tumor mass and administering them in the adjuvant setting. However, in the case of trastuzumab, in the advanced setting tumor shrinkage is only seen in patients with HER2 amplification because HER2 is expressed in all of the tumor cells. By contrast, in the adjuvant setting, HER2 is selectively expressed in cancer stem cells, which are in the bone marrow, and therefore, the patients may benefit from adjuvant trastuzumab. What this is telling us is that we may have to reconsider how adjuvant therapies are actually targeted. Notably, it is now clear that other cancers also express HER2, including bladder and lung cancers.[21] I think we will see more trials of HER2-targeted therapies in other tumor types. I would just caution that assessing tumor shrinkage as an endpoint in adjuvant therapy trials could be misleading; it will be important to examine the cancer stem cell population as well. Moreover, based on our work, we think that HER2 gene amplification is not the only clinically relevant measure. HER2 expression is capable of driving stem cells even in cells without gene amplification.

References:18. Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testingNational Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst. 2002;94:852-854.

19. Paik S, Kim C, Wolmark N. HER2 status and benefit from adjuvant trastuzumab in breast cancer. N Engl J Med. 2008;358:1409-1411.

20. Ithimakin S, Day KC, Malik F, et al. HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab. Cancer Res. 2013;73:1635-1646.

21. Iqbal N, Iqbal N. Human epidermal growth factor receptor 2 (HER2) in cancers: overexpression and therapeutic implications. Mol Biol Int. 2014;2014:852748.

15Tumor Expression of CSC Marker Predicts Metastasis and Survival in BCRetrospective analysis of 109 pts with inflammatory breast cancer identified expression of the CSC marker ALDH1 as an independent predictor of early metastasis and poor survival

Mos After DiagnosisMos After DiagnosisProbability of Specific SurvivalProbability of Metastasis-Free Survival23. Charafe-Jauffret E, et al. Clin Cancer Res. 2010;16:45-55. 1.00.80.60.40.20020406080100P = .0337ALDH1 positiveALDH1 negative1.00.80.60.40.20020406080100ALDH1 positiveALDH1 negativeP = .0152clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsALDH, aldehyde dehydrogenase; BC, breast cancer; CSC, cancer stem cell.

As I mentioned previously, cancer stem cells are also important because they act as the seeds of metastasis. We demonstrated that injecting fluorescently labeled breast cancer stem cells into the tails of mice led to metastatic tumors in the bone and other organs.[22] On the other hand, the same experiment performed with nonstem cells did not result in metastasis. Only cancer stem cells have enough self-renewal potential to form a clinically significant metastasis. Moreover, we performed a retrospective analysis of 109 patients with inflammatory breast cancer and found that expression of the cancer stem cell marker ALDH was associated with significantly lower rates of survival and significantly increased rates of metastasis.[23]

Reference:22. Charafe-Jauffret E, Ginestier C, Iovino F, et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res. 2009;69:45-55.23. Charafe-Jauffret E, Ginestier C, Iovino F, et al. Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin Cancer Res. 2010;16:45-55.16CSC Implications for Metastasis

TDC onlyCSC with FULL malignant potentialCSC with PARTIAL malignant potential

Subsequently to other sites

Metastases in mos to few yrs

No metastasesDormancy followed by metastases after many yrs:

Secondary Oncogenic Hits and/or Changes in MicroenvironmentNoCSC1TumorCSCclinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell; TDC, terminally differentiated cell.

In addition to seeding metastasis, cancer stem cells may also contribute to tumor dormancy. As clinicians, we have all seen patients who we think are cured of breast cancer experience a recurrence many years later. Evidence now suggests that there may be dormant stem cells in the bone marrow that can reactivate and cause recurrence many years later.[24,25] Thus, it will be important to develop therapies capable of eliminating this cancer stem cell population.

References:24. Ono M, Kosaka N, Tominaga N, et al. Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells. Sci Signal. 2014;7:ra63.

25. Quints-Cardama A, Grgurevic S, Rozovski U, et al. Detection of dormant chronic myeloid leukemia clones in the bone marrow of patients in complete molecular remission. Clin Lymphoma Myeloma Leuk. 2013;13:681-685.

17Tumor-Specific CSC Markers Tumor TypeCSC Surface MarkersSolid tumorsBrain[26]CD133+ CD49f+ CD90+Breast[27]ALDH+ ESA+ CD44+ CD24-/lowColon[28]CD133+ CD44+ CD166+ EpCAM+ CD24+Lung[29]CD133+ ABCG2++Melanoma[30]CD20+Pancreatic[31]CD133+ CD44+ EpCAM+ CD24+Prostate[32]CD133+ CD44+ CD24-HematologicAML[33]CD34+ CD38+Leukemia[34]CD34+ CD38- HLA-DR- CD71- CD90- CD117- CD123+26. Singh SK, et al. Nature. 2004;432:396-401. 27. Al-Hajj M, et al. Proc Natl Acad Sci U S A. 2003;100:3983-3988. 28. OBrien CA, et al. Nature. 2007;445:106-110. 29. Eramo A, et al. Cell Death Differ. 2008;15:504-14. 30. Fang D, et al. Cancer Res. 2005;65:9328-9337. 31. Li C, et al. Cancer Res. 2007;67:1030-1037. 32. Du L, et al. Clin Cancer Res. 2008;14:6751-6760. 33. Lapidot T, et al. Nature. 1994;367:645-648. 34. Guzman ML, et al. Cancer Control. 2004;11:97-104.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsABCG2, ATP-binding cassette subfamily G member 2; ALDH, aldehyde dehydrogenase; AML, acute myeloid leukemia; CSC, cancer stem cell; EpCAM, epithelial cell adhesion molecule; ESA, epithelial-specific antigen.

One important question is: Are cancer stem cells from different tumors the same? We and others have identified many different cancer stem cell markers across many malignancies.[26-34] Notably, many of the markers are remarkably conserved between different cancer stem cells because these represent more primitive cells that share common pathways. We were also interested in determining if cancer stem cells within a single tumor are a homogeneous or heterogeneous population. By examining the expression of various stem cell markers in breast tumors, we found that the cells located on the invasive edge of a tumor were distinct from those in the interior.[35] Specifically, CD44+ CD24- cells were located on the invasive edge at the periphery whereas cells expressing ALDH were found in the interior of the tumors. Based on their morphology, we call these distinct stem cell subsets mesenchymal-like (CD44+ CD24-) or epithelial-like (ALDH+). The former is relatively quiescent whereas the latter has a high proliferative potential. We think that the mesenchymal-like cells on the invasive edge are responsible for metastasis, and the epithelial-like cells harbor the proliferative capacity to establish a new tumor at the metastatic site. Notably, cancer stem cells are able to transition between these 2 states, thereby explaining how a dormant tumor could reactivate after many years.

References:26. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature. 2004;432:396-401.

27. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100:3983-3988.

28. OBrien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445:106-110.

29. Eramo A, Lotti F, Sette G, et al. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 2008;15:504-514.

30. Fang D, Nguyen TK, Leishear K, et al. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res. 2005;65:9328-9337.

31. Li C, Heidt DG, Dalerba P, et al. Identification of pancreatic cancer stem cells. Cancer Res. 2007;67:1030-1037.

32. Du L, Wang H, He L, et al. CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res. 2008;14:6751-6760.

33. Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367:645-648.

34. Guzman ML, Jordan CT. Considerations for targeting malignant stem cells in leukemia. Cancer Control. 2004;11:97-104.

35. Liu S, Cong Y, Wang D, et al. Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts. Stem Cell Reports. 2013;2:78-91.

18Therapeutic Agents Targeting CSC Survival and Self-Renewal36. Liu S, et al. J Clin Oncol. 2010;28:4006-4012. 37. Prudhomme GJ. Curr Pharm Des. 2012;18:2838-2849. -CATIL-8CXCR1Self-renewal drug resistance metastasisTARGET DNACancer stem cellNOTCHDLL/JAGWNTLPR/FZDTarextumab(OMP-59R5)Demcizumab(anti-DLL4)Vantictumab(anti-FZD)Ipafricept(OMP-54F28)ReparixinFAKDefactinib-CAT, STAT3, NanogBBI608clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsBased on much research during the last decade, the signaling pathways that regulate cancer stem cells are being elucidated. Signaling pathways such as Notch, Wnt, and CXCR1 along with downstream effectors including the transcription factors -catenin, Nanog, and STAT3 play key roles in stem cell self-renewal and maintenance. These pathways, and other intrinsic and extrinsic pathways, are the targets of multiple therapeutic agents as shown in this figure.[36,37]

References:36. Liu S, Wicha MS. Targeting breast cancer stem cells. J Clin Oncol. 2010;28:4006-4012.

37. Prud'homme GJ. Cancer stem cells and novel targets for antitumor strategies. Curr Pharm Des. 2012;18:2838-2849.

19CSC-Targeted Therapies: Preliminary Results from Phase I TrialsTreatmentTumorKey OutcomesTarextumab + etoposide/platinum[38]SCLCMTD not reached; DLT: 1 nausea; tumor reduction in 9/10 ptsDemcizumab + carbo/pem[39]NSCLCDLT: 1 reversible pulmonary HTN/heart failure; ORR: 13/28; SD: 11/28Ipafricept[40]MultipleNo grade 3 AEs; SD: 9/26BBI608 + paclitaxel[41]Multiple MTD not reached; DCR: 10/15BBI608[42]Multiple DCR: 4/6BBI503[43]Multiple MTD not reached; SD: 11/2038. Spigel DR, et al. ASCO 2014. Abstract 7601. 39. McKeage MJ, et al. ASCO 2014. Abstract 2544. 40. Jimeno A, et al. ASCO 2014. Abstract 2505. 41. Hitron M, et al. ASCO 2014. Abstract 2530. 42. Jonker DJ. ASCO 2014. Abstract 2546. 43. Laurie SA, et al. ASCO 2014. Abstract 2527. clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsAE, adverse event; CR, complete response; CSC, cancer stem cell; DLT, dose-limiting toxicity; HTN, hypertension; MTD, maximum tolerated dose; NSCLC, non-small-cell lung cancer; PFS, progression-free survival; PR, partial response; SCLC, small-cell lung cancer; SD, stable disease; TTP, time to progression.

Numerous clinical trials have been developed to assess the safety and efficacy of various therapies targeting cancer stem cells, both as single agents and in combination with chemotherapy. Preliminary results have been reported from early-stage clinical trials with some of these therapies as shown here.[38-43] In general, these agents were associated with manageable toxicity profiles with the most common adverse events including grade 1/2 fatigue and gastrointestinal events. Notably, prolonged demcizumab treatment was associated with cardiopulmonary toxicity so the length of exposure to this agent is being limited going forward.[39]

References:38. Spigel DR, Spira AI, Jotte RM, et al. Phase 1b of anticancer stem cell antibody OMP-59R5 (anti-Notch2/3) in combination with etoposide and cisplatin (EP) in patients (pts) with untreated extensive-stage small-cell lung cancer (ED-SCLC). Program and abstracts from the 2014 American Society of Clinical Oncology Annual Meeting; May 30 - June 4, 2014; Chicago, Illinois. Abstract 7601.

39. McKeage MJ, Kotasek D, Markman B, et al. A phase 1b study of the anticancer stem cell agent demcizumab (DEM), pemetrexed (PEM), and carboplatin (CARBO) in pts with first-line nonsquamous NSCLC. Program and abstracts from the 2014 American Society of Clinical Oncology Annual Meeting; May 30 - June 4, 2014; Chicago, Illinois. Abstract 2544.

40. Jimeno AJ, Gordon MS, Chugh R, et al. A first-in-human phase 1 study of anticancer stem cell agent OMP-54F28 (FZD8-Fc), decoy receptor for WNT ligands, in patients with advanced solid tumors. Program and abstracts from the 2014 American Society of Clinical Oncology Annual Meeting; May 30 - June 4, 2014; Chicago, Illinois. Abstract 2505.

41. Hitron M, Stephenson J, Chi KN, et al. A phase 1b study of the cancer stem cell inhibitor BBI608 administered with paclitaxel in patients with advanced malignancies. Program and abstracts from the 2014 American Society of Clinical Oncology Annual Meeting; May 30 - June 4, 2014; Chicago, Illinois. Abstract 2530.

42. Jonker DJ, Stephenson J Edenfield WJ, et al. A phase I extension study of BBI608, a first-in-class cancer stem cell (CSC) inhibitor, in patients with advanced solid tumors. Program and abstracts from the 2014 American Society of Clinical Oncology Annual Meeting; May 30 - June 4, 2014; Chicago, Illinois. Abstract 2546.

43. Laurie SA, Jonker DJ, Edenfield WJ, et al. A phase 1 dose-escalation study of BBI503, a first-in-class cancer stemness kinase inhibitor in adult patients with advanced solid tumors. Program and abstracts from the 2014 American Society of Clinical Oncology Annual Meeting; May 30 - June 4, 2014; Chicago, Illinois. Abstract 2527.

20CSC-Targeted Therapies: Ongoing Randomized TrialsTrialPhaseDiseaseTreatmentCO23[44]IIImCRCBBI608 vs BSCBRIGHTER[45]IIIGastric/GEJBBI608 + Pac vs Pac + Placebo COMMAND[47]IIPleural mesotheliomaDefactinib vs placebo DENALI[48]IINSCLCDemcizumab + Carbo/Pem vsCarbo/Pem + placebo YOSEMITE[49]IIPancreaticDemcizumab + Gem/nab-P vs Gem/nab-P + placebo ALPINE[50]Ib/IIPancreaticTarextumab + Gem/nab-P vsGem/nab-P + placebo PINNACLE[51]Ib/IISCLCTarextumab + E/Plt vs E/Plt + placebo44. ClinicalTrials.gov. NCT01830621. 45. ClinicalTrials.gov. NCT02178956. 47. ClinicalTrials.gov. NCT01870609. 48. ClinicalTrials.gov. NCT02259582. 49. ClinicalTrials.gov. NCT02289898. 50. ClinicalTrials.gov. NCT01647828. 51. ClinicalTrials.gov. NCT01859741. clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsBSC, best supportive care; carbo, carboplatin; CSC, cancer stem cell; E, etoposide; GEJ, gastroesophageal junction; mCRC, metastatic colorectal cancer; nab-P, albumin-bound paclitaxel; NSCLC, non-small-cell lung cancer; Pac, paclitaxel; Pem, pemetrexed; Plt, platinum agent, SCLC, small-cell lung cancer.

Some cancer stem celltargeted therapies are now being evaluated in randomized clinical trials. Two phase III trials are testing BBI608 either as a single agent in patients with metastatic colorectal cancer and disease progression through all available standard treatment options (CO23) or in combination with weekly paclitaxel for patients with advanced gastric or gastroesophageal junction cancer who progressed following initial therapy with chemotherapy (BRIGHTER).[44,45] The CO23 trial was closed to accrual following the first interim analysis.[46] Defactinib is being compared with placebo in patients with malignant pleural mesothelioma who have achieved at least stable disease following 4 cycles of pemetrexed/platinum doublet regimen.[47] In addition, 2 Notch pathway inhibitors, demcizumab and tarextumab, are being evaluated in combination with standard chemotherapy regimens in randomized phase II trials for patients with lung cancer or pancreatic cancer.[48-51]

References:44. ClinicalTrials.gov. BBI608 and best supportive care vs placebo and best supportive care in patients with pretreated advanced colorectal carcinoma. Available at: https://clinicaltrials.gov/ct2/show/NCT01830621. Accessed February 17, 2015.

45. ClinicalTrials.gov. A study of BBI608 plus weekly paclitaxel to treat gastric and gastro-esophageal junction cancer (BRIGHTER). Available at: https://clinicaltrials.gov/ct2/show/NCT02178956. Accessed February 17, 2015.

46. Jonker DJ, Stephenson J Edenfield WJ, et al. A phase I extension study of BBI608, a first-in-class cancer stem cell (CSC) inhibitor, in patients with advanced solid tumors. Program and abstracts from the 2014 American Society of Clinical Oncology Annual Meeting; May 30 - June 4, 2014; Chicago, Illinois. Abstract 2546.

47. ClinicalTrials.gov. Placebo controlled study of VS-6063 in subjects with malignant pleural mesothelioma (COMMAND). Available at: https://clinicaltrials.gov/ct2/show/NCT01870609. Accessed February 17, 2015.

48. ClinicalTrials.gov. A study of carboplatin, pemetrexed plus placebo vs carboplatin, pemetrexed plus 1 or 2 truncated courses of demcizumab in subjects with non-squamous non-small cell lung cancer (DENALI). Available at: https://clinicaltrials.gov/ct2/show/NCT02259582. Accessed February 17, 2015.

49. ClinicalTrials.gov. Study of gemcitabine, Abraxane plus placebo versus gemcitabine, Abraxane plus 1 or 2 truncated courses of demcizumab in subjects with 1st-line metastatic pancreatic ductal adenocarcinoma (YOSEMITE). Available at: https://clinicaltrials.gov/ct2/show/NCT02289898. Accessed February 17, 2015.

50. ClinicalTrials.gov. A phase 1b/2 study of OMP-59R5 in combination with nab-paclitaxel and gemcitabine in subjects with previously untreated stage IV pancreatic cancer (ALPINE). Available at: https://clinicaltrials.gov/ct2/show/NCT01647828. Accessed February 17, 2015.

51. ClinicalTrials.gov. A phase 1b/2 study of OMP-59R5 in combination with etoposide and platinum therapy in subjects with untreated extensive stage small cell lung cancer (PINNACLE). Available at: https://clinicaltrials.gov/ct2/show/NCT01859741. Accessed February 17, 2015.

21Percent CD44+/CD24- Cells in Serial BiopsiesBreast CSC Frequency Following Docetaxel Plus NOTCH Inhibitor Phase Ib study of MK0752 + docetaxel in pts with locally advanced or metastatic breast cancer (N = 30)Dose-limiting toxicities: pneumonitis, hand-foot syndrome, LFT elevation, diarrhea (n = 1 per each); 1 death following grade 5 toxicityPR in 11/24, SD in 9/24, PD in 3/24CSCs assessed in serial biopsies by CD44+/CD24- phenotype, ALDH+ phenotype, sphere formation10 pts consented to biopsies; 6/10 evaluable repeat biopsiesDecreases in CSCs vs baseline observed in 3/5; initial increases in CSCs consistent with mechanism of action52. Schott AF, et al. Clin Cancer Res. 2013;19:1512-1524. 4035302520151050CD44+/CD24- (%)BaselinePost Cycle 1Post Cycle 3SurgeryPt9Pt23Pt21Pt26Pt28Pt29clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsALDH, aldehyde dehydrogenase; CSC, cancer stem cell; LFT, liver function test; PD, progressive disease; PR, partial response; SD, stable disease.

It has been difficult to determine how best to assess the efficacy of these novel cancer stem cell therapies. Of course, the ultimate endpoint is patient survival; we want to ensure that patients are living longer. However, survival endpoints take a considerably long time to mature. Therefore, developing intermediate endpoints is important to verify that these agents are targeting cancer stem cells. We cannot use classical endpoints such as tumor regression, since as noted above, cancer stem cells account for only a small percentage of cells in a tumor. One method to assess the efficacy of cancer stem celltargeting agents is by quantifying cancer stem cells in serial biopsies of patients receiving treatment. We performed such an analysis as part of a phase Ib study of docetaxel plus MK0752, an inhibitor of the Notch pathway.[52] We identified stem cells based on the markers CD44/CD24, ALDH, or their ability to form spheres in tissue culture, which is a measure of their proliferative ability. Among 6 patients with available serial biopsies, approximately one half experienced a decrease in stem cells compared with baseline. It is difficult to obtain serial biopsies and it is accompanied by some risk of complications. Therefore, much effort is focused on developing other methods to monitor cancer stem cells.

References:52. Schott AF, Landis MD, Dontu G, et al. Preclinical and clinical studies of gamma secretase inhibitors with docetaxel on human breast tumors. Clin Cancer Res. 2013;19:1512-1524.22Isolation of Circulating CSCsOne FDA-approved method[53]Approved for prognostic evaluation and therapeutic response in circulating tumor cells of breast, colon, and prostate cancerAutomated system to isolate EpCAM+ cytokeratin+ CD45- cells from whole bloodAdditional methods includeDensity-based gradient separation of CTCs from whole blood[54]Microfluidic chip that positively selects EpCAM+ cells from whole blood[55]53. Riethdorf S, et al. Clin Cancer Res. 2007;14:920-928. 54. Gertler R, et al. Recent Results Cancer Res. 2003;162:149-155. 55. Nagrath S, et al. Nature. 2007;450:1235-1239.clinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell; CTC, circulating tumor cells; FDA, US Food and Drug Administration.

One potential method for monitoring cancer stem cells is sampling circulating tumor cells, which are found in patients with many tumor types. Currently, there is one method for isolating these cells that has been certified by the US Food and Drug Administration.[53] However, we have found that it is not able to isolate the mesenchymal-like form of stem cells. Therefore, our group and others are developing additional protocols for isolating circulating cancer stem cells, some examples of which include density-based gradient separation and microfluidic chips.[54,55]

References:53. Riethdorf S, Fritsche H, Muller V, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch System. Clin Cancer Res. 2007;14:920-928.

54. Gertler R, Rosenberg R, Fuehrer K, et al. Detection of circulating tumor cells in blood using an optimized density gradient centrifugation. Recent Results Cancer Res. 2003;162:149-155.

55. Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumor cells in cancer patients by microchip technology. Nature. 2007;450:1235-1239.

23CSCs Contribute to Tumor HeterogeneityClassical model of tumor heterogeneity56. Meacham CE, et al. Nature. 2013;501:328-337.Mutations in CSCsCSC model of tumor heterogeneityclinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem CellsCSC, cancer stem cell.

In summary, one of the largest challenges in developing effective cancer treatments is the fact that cancers are heterogeneous. There are 2 forms of this heterogeneity.[56] One is the classical model of heterogeneity, which develops from mutation and selection of cells within a tumor. A second degree of heterogeneity arises from cancer stem cells that are able to differentiate and form the bulk cells in a tumor. Moreover, a cancer stem cell may evolve and mutate during tumorigenesis so that multiple distinct stem cells within a given tumor emerge. The challenge in the future is to develop therapies that can eliminate both the stem cells and the bulk cells in tumors. Our hope is that this kind of approach will cure many more patients with cancer. Finally, I want to add a note regarding the prevention of cancer. The nutrient sulforaphane, found in broccoli, has been shown to reduce the proportion of cancer stem cells in a mouse model of breast cancer.[57] Perhaps dietary and exercise interventions may serve in cancer prevention by changing stem cell populations.

References:56. Meacham CE, Morrison SJ. Tumor heterogeneity and cancer cell plasticity. Nature. 2013;501:328-337.

57. Li Y, Zhang T, Korkaya H, et al. Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells. Clin Cancer Res. 2010;16:2580-2590.

24Go Online for More CCO Coverage of Cancer Stem Cells!Additional CME-certified program on cancer stem cells with expert faculty commentary on all the key studies clinicaloptions.com/oncologyclinicaloptions.com/oncologyResearch Focus: Targeting Cancer Stem Cells25