Therapeutic implications of colon CSCs

Therapeutic implications of colon cancer stem cells by Eros Fabrizi and 3 co-authors, including Lucia Ricci-Vitiani, World J Gastroenterol 2010(Aug 21); 16(31): 3871-7. OA review. [FriendFeed entry][PubMed citation]. Abstract:

Colorectal cancer is the second most common cause of cancer-related death in many industrialized countries and is characterized by a heterogenic pool of cells with distinct differentiation patterns. Recently, the concept that cancer might arise from a rare population of cells with stem cell-like properties has received support with regard to several solid tumors, including colorectal cancer. According to the cancer stem cell hypothesis, cancer can be considered a disease in which mutations either convert normal stem cells into aberrant counterparts or cause a more differentiated cell to revert toward a stem cell-like behaviour; either way these cells are thought to be responsible for tumor generation and propagation. The statement that only a subset of cells drives tumor formation has major implications for the development of new targeted therapeutic strategies aimed at eradicating the tumor stem cell population. This review will focus on the biology of normal and malignant colonic stem cells, which might contribute to our understanding of the mechanisms responsible for tumor development and resistance to therapy.

Review about retroviral-induced leukemogenesis and the CSC hypothesis

Hematopoietic stem cells and retroviral infection by Prabal Banerjee, Lindsey Crawford, Elizabeth Samuelson and Gerold Feuer, Retrovirology 2010(Feb 4); 7(1): 8 [Epub ahead of print][Connotea bookmark][[FriendFeed entry][PubMed Citation][Full text (PDF)].

Includes sections on Leukemia Stem Cells/Cancer Stem Cells (LSC/CSC) and on The Cancer Stem Cell Hypothesis.

Analyzing tumors as ecosystems

Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype by So Yeon Park and 4 co-authors, including Kornelia Polyak, J Clin Invest 2010(Jan 25) [Epub ahead of print][FriendFeed entry][Connotea bookmark][Full text is publicly accessible (via Gratis OA)]. PubMed Abstract:

Intratumor genetic heterogeneity is a key mechanism underlying tumor progression and therapeutic resistance. The prevailing model for explaining intratumor diversity, the clonal evolution model, has recently been challenged by proponents of the cancer stem cell hypothesis. To investigate this issue, we performed combined analyses of markers associated with cellular differentiation states and genotypic alterations in human breast carcinomas and evaluated diversity with ecological and evolutionary methods. Our analyses showed a high degree of genetic heterogeneity both within and between distinct tumor cell populations that were defined based on markers of cellular phenotypes including stem cell-like characteristics. In several tumors, stem cell-like and more-differentiated cancer cell populations were genetically distinct, leading us to question the validity of a simple differentiation hierarchy-based cancer stem cell model. The degree of diversity correlated with clinically relevant breast tumor subtypes and in some tumors was markedly different between the in situ and invasive cell populations. We also found that diversity measures were associated with clinical variables. Our findings highlight the importance of genetic diversity in intratumor heterogeneity and the value of analyzing tumors as distinct populations of cancer cells to more effectively plan treatments.

The final sentence of the Discussion section of the full text:

In summary, in this study we have demonstrated the power of analyzing tumors as ecosystems and suggest that quantitative measures of intratumor diversity might be clinically useful biomarkers predicting prognosis and response to treatment.

Another recent article from the same group: Heterogeneity for Stem Cell–Related Markers According to Tumor Subtype and Histologic Stage in Breast Cancer by So Yeon Park and 5 co-authors, including Kornelia Polyak, Clin Cancer Res 2010; 16(3): 876–87 [Epub 2010(Jan 26)][FriendFeed entry][Connotea bookmark][PubMed Citation].

From the Conclusions section of the abstract:

Our findings suggest that in breast cancer, the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage.

CSC targeted to prevent relapse

Cancer stem cells targeted to prevent relapse by Vivek Sinanan, The Johns Hopkins News-Letter, October 26, 2009. In part, the news item appears to be based on this editorial: Controversies in cancer stem cells by Richard J Jones, J Mol Med 2009(Oct 23) [Epub ahead of print][PubMed Citation][Full text]. In turn, the editorial cites these articles, which will be published in J Mol Med 2009; 87(11):

• Cancer stem cells: controversies in multiple myeloma, Sarah K Brennan, William Matsui, J Mol Med 2009(Sep 17) [Epub ahead of print][PubMed Citation].


• Brain cancer stem cells, Sara GM Piccirillo and 4 co-authors, including Khalid Shah, J Mol Med 2009(Sep 29) [Epub ahead of print][PubMed Citation].


• Colon cancer stem cells, Lucia Ricci-Vitiani and 4 co-authors, including Ruggero De Maria, J Mol Med 2009(Sep 2) [Epub ahead of print][PubMed Citation].


• Cancer stem cells-clinical relevance, Richard J Jones, J Mol Med 2009(Oct 10) [Epub ahead of print][PubMed Citation].

Targeting cancer stem cells

Griffin Securities Announces Investment Opinion on Targeting Cancer Stem Cells, Yahoo Finance, October 9, 2008. First paragraph:

Griffin Securities (“Griffin”), a research-driven investment banking firm, has released an industry report on the subject of targeting cancer stem cells (CSCs). The stem cell hypothesis offers a rational approach to preventing, diagnosing, and treating malignant growth. Covered in the report are five classes of drugs nearing the Phase II clinical trial that provides human proof-of concept data, a crucial stage of development: Notch signaling, PI3k/akt pathway, immunotherapeutics, molecular chaperons, and hedgehog signaling.

See also: Firms Seek to Prove Cancer Stem Cell Hypothesis by Keith A Markey (scientific director at Griffin Securities), Genetic Engineering & Biotechnology News, Oct 15, 2009 (Vol 29, No 18).

Comments: Information can be found online about some of the drugs mentioned in the article by Keith A Markey. Two examples:

1. Notch signaling: OMP-21M18
   

   A humanized monoclonal antibody directed against the N-terminal epitope of Notch ligand DLL4 (delta-like 4) with potential antineoplastic activity. Anti-DLL4 monoclonal antibody OMP-21M18 binds to the membrane-binding portion of DLL4 and prevents its interaction with Notch-1 and Notch-4 receptors, thereby inhibiting Notch-mediated signaling and gene transcription, which may impede tumor angiogenesis. Activation of Notch receptors by DLL4 stimulates proteolytic cleavage of the Notch intracellular domain (NICD); after cleavage, NICD is translocated into the nucleus and mediates the transcriptional regulation of a variety of genes involved in vascular development. The expression of DLL4 is highly restricted to the vascular endothelium.

   One Phase 1 trial of OMP-21M18 [ClinicalTrials.gov Identifier: NCT00744562].


2. Hedgehog signaling: IPI-926

   An orally bioavailable, cyclopamine-derived inhibitor of the Hedgehog (Hh) pathway with potential antineoplastic activity. Specifically, Hedgehog pathway inhibitor IPI-926 binds to and inhibits the cell membrane-spanning G-protein coupled receptor SMO, which may result in the suppression of Hh pathway signaling and a decrease in tumor cell proliferation and survival. SMO is activated upon binding of Hh ligand to the cell surface receptor Patched (PTCH); inappropriate activation of Hh signaling and uncontrolled cellular proliferation may be associated with SMO mutations. The Hh signaling pathway plays an important role in proliferation of neuronal precursor cells in the developing cerebellum and other tissues.

   One Phase 1 trial of IPI-926 [ClinicalTrials.gov Identifier: NCT00761696].

A search of the Canadian Cancer Trials database indicated no trial sites, for either of these two particular trials, that are located in Canada.

A contrarian view of 'stemness'

The 'stem cell' concept: is it holding us back? by Arthur D Lander, J Biol 2009(Sep 21); 8(8):70 [Epub ahead of print][Related FriendFeed entry][PubMed Citation][Full text is publicly accessible (via Libre OA)]. Abstract:

Developmental biology, regenerative medicine and cancer biology are increasingly occupied with the molecular characterization of stem cells. Yet recent work adds to a growing body of literature suggesting that 'stemness' cannot be reduced to the molecular features of cell types, and is instead an emergent property of cell lineages under feedback control.

Brief excerpt from the full text: "Like gene or phlogiston, the term 'stem cell' is a scientific concept. Stem cells are very much in the news, thanks to a dramatic upsurge in interest in their therapeutic potential".

Another excerpt from the full text:

For example, in the case of cancers that are stem cell driven, it is not clear that we actually have grounds to assume that the specific chemotherapeutic targeting of cancer stem cells will necessarily stop tumors in their tracks. Indeed, if feedback and lineage progression continue to take place in cancerous tissues, we might observe that, under different conditions - different stages of tumorigensis, different parts of a tumor, different amounts of tumor cells - that different cell types will assume the role of 'cancer stem cell'. The therapeutic implications of this possibility are clearly substantial.

An interview with Arthur Lander is at: Q&A: Is stem cell research misguided? by Bob Grant,TheScientist.com, September 29, 2009 [FriendFeed entry].

Comment: The major contrarian perspective in this opinion piece is the comparison of the stem cell concept with the phlogiston concept. This comparison is discussed further in the Q & A interview. The Comments section of Dr. Lander's article includes an answer to a question that I asked: What about purified stem cells? As part of a discussion on FriendFeed, I also asked: Is the debate about 'stemness' becoming another version of the nature-nurture debate? So far, no response to this question.

Diabetes drug metformin appears to target breast CSC

Diabetes Drug Metformin Shows Promise as a Breast Cancer Treatment, NCI Cancer Bulletin 2009(Sep 22); 6(18). First two paragraphs:

Low doses of the commonly used diabetes drug metformin may be an effective treatment for breast cancer, primarily because the drug appears to target breast cancer stem cells, Harvard Medical School researchers reported online September 14 in Cancer Research. Cancer stem cells, also called tumor-initiating cells, are thought to be relatively rare cells that can give rise to new tumors and are resistant to standard cancer treatments.

In the study, the combination of metformin and the chemotherapy agent doxorubicin was more effective than either drug alone at eliminating cancer cells and cancer stem cells in cultured cell lines of four genetically distinct breast cancer types, including HER2-positive and triple-negative breast cancers. In a breast cancer mouse model, the drug combination eliminated tumors and prevented regrowth, whereas doxorubicin alone only reduced tumor size and did not prevent regrowth, and metformin alone had little effect.

This bulletin is about the publication: Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth and Prolong Remission by Heather A Hirsch, Dimitrios Iliopoulos, Philip N Tsichlis and Kevin Struhl, Cancer Res 2009(Sep 14) [Epub ahead of print][PubMed Citation].

Bidirectional interconvertibility between CSCs and non-CSCs?

Cancer stem cells: mirage or reality? Piyush B Gupta, Christine L Chaffer and Robert A Weinberg, Nat Med 2009(Sep); 15(9): 1010-1012 [Epub 2009(Sep 4)]. [FriendFeed entry] PubMed Abstract:

The similarities and differences between normal tissue stem cells and cancer stem cells (CSCs) have been the source of much contention, with some recent studies calling into question the very existence of CSCs. An examination of the literature indicates, however, that the CSC model rests on firm experimental foundations and that differences in the observed frequencies of CSCs within tumors reflect the various cancer types and hosts used to assay these cells. Studies of stem cells and the differentiation program termed the epithelial-mesenchymal transition (EMT) point to the possible existence of plasticity between stem cells and their more differentiated derivatives. If present, such plasticity would have major implications for the CSC model and for future therapeutic approaches.

Excerpt from the full text:

Figure 1: Stem-differentiation hierarchy.

[Figure]

Increased plasticity may be present within cancer populations, enabling bidirectional interconvertibility between CSCs and non-CSCs.

Last two sentences of the full text:

However, if non-CSCs can indeed give rise to CSCs, this plasticity would frustrate attempts to cure tumors by eliminating CSCs alone, as therapeutic elimination of CSCs may be followed by their regeneration from residual non-CSCs, allowing tumor regrowth and clinical relapse. We, therefore, suspect that optimal therapeutic regimens will need to incorporate agents that target both CSCs and non-CSCs if truly curative therapies are ever to be achieved.

Comment: This article presents a novel model of the "stem-differentiation hierarchy" involving CSCs and non-CSCs. The model includes the possibility that "a dynamic equilibrium may exist between CSCs and non-CSCs within tumors" that "may be shifted in one direction or another by contextual signals within the tumor microenvironment that influence the probability of interconversion between the CSC and non-CSC compartments ...". It's unfortunate that the article isn't openly accessible. If it were OA (with an appropriate Creative Commons License), a copy of the model depicted in Figure 1 could have been included in this post.

Cancer cell heterogeneity: an essay

Heterogeneity in Cancer: Cancer Stem Cells versus Clonal Evolution by Mark Shackleton, Elsa Quintana, Eric R Fearon and Sean J Morrison, Cell 2009(Sep 4); 138(5): 822-29. [FriendFeed entry][Full text]. Summary:

The identification and characterization of cancer stem cells might lead to more effective treatments for some cancers by focusing therapy on the most malignant cells. To achieve this goal it will be necessary to determine which cancers follow a cancer stem cell model and which do not, to address technical issues related to tumorigenesis assays, and to test the extent to which cancer cell heterogeneity arises from genetic versus epigenetic differences.

Globe & Mail article on CSCs

Cancer stem cells spur hope, skepticism by Jill Colvin, The Globe and Mail, August 27, 2009. [WebCite cache][Twitter entry][FriendFeed entry]. Excerpt:

Ontario Cancer Institute researcher John Dick, arguably the world's leading authority in the field, first identified stem cells in leukemia in the 1990s. Today, few doubt they play a key role in blood cancers.

In addition to this comment about the crucial contributions of John Dick, research on brain-tumour stem cells by Samuel Weiss is noted. There's also mention of the MIT and Harvard joint venture, Identification of Selective Inhibitors of Cancer Stem Cells by High-Throughput Screening, published in Cell 2009(Aug 13). [See also this post in CSC News, August 14, 2009].

The criticisms by Scott Kern (such as: The fuzzy math of solid tumor stem cells: a perspective) of application the CSC hypothesis to colon, breast, or lung cancer are outlined. There's also a comment attributed to Richard Hill:

The hypothesis also hinges on the assumption that these cells are rare. Otherwise, traditional cancer therapies that shrink tumours would be killing them, too, and there would be no need to develop specialized treatments.

The "game-changing paper" by a team led by Sean Morrison, Efficient tumour formation by single human melanoma cells is also discussed. [See another post in CSC News, December 3, 2008, about this paper]. This research supports the view that "... stem cells may be key for some kinds of cancers and not for others".[This paper has attracted much attention. See, for example, Cancer Stem Cells May Not Be the Supervillains We Thought by Alexis Madrigal, Wired Science, December 3, 2008].

The article in The Globe & Mail ends with a paragraph about the hope that "new drug combinations that target all cell types" [associated with tumors] will be found, and a quote from William Hahn: "Whether the hypothesis is correct or incorrect in its full-blown beauty is really not important in the end" . [What really matters, from a clinical perspective, is whether or not efforts to target CSCs, in addition to other kinds of cells associated with tumors, will lead to improved outcomes for patients].

The article has already attracted some comments from readers.

Disclosure: I'm a co-author of some early papers about the CSC hypothesis, such as: J Natl Cancer Inst 1983(Jan); 70(1): 9-16.

CD133 and poorer prognosis in locally advanced colon cancer

Higher percentage of CD133+ cells is associated with poor prognosis in colon carcinoma patients with stage IIIB by Chun-Yan Li and 11 co-authors, including Xiao-Shi Zhang, J Transl Med 2009(Jul 7); 7: 56. [Full text via Libre OA][PMC version of the full text][PubMed Abstract] Final paragraph of the Abstract:

CONCLUSION: The fact that a higher percentage CD133+ cells were strongly associated with a poorer prognosis in patients with locally advanced colon cancer implicated that CD133+ cancer cells contribute to the tumor progression, and the overpopulation hypothesis of cancer stem cell seems reasonable.

Translation of knowledge about CSC into clinical use

Radiation Therapy Oncology Group Translational Research Program Stem Cell Symposium: Incorporating Stem Cell Hypotheses into Clinical Trials. Authors: Wendy A Woodward, Robert G Bristow, Michael F Clarke, Robert P Coppes, Massimo Cristofanilli, Dan G Duda, John R Fike, Dolores Hambardzumyan, Richard P Hill, Craig T Jordan, Luka Milas, Frank Pajonk, Walter J Curran, Adam P Dicker, Yuhchyau Chen. Int J Radiat Oncol Biol Phys 2009(Aug 1); 74(5): 1580-91 [Epub 2009(Jun 17)]. PubMed Abstract:

At a meeting of the Translation Research Program of the Radiation Therapy Oncology Group held in early 2008, attendees focused on updating the current state of knowledge in cancer stem cell research and discussing ways in which this knowledge can be translated into clinical use across all disease sites. This report summarizes the major topics discussed and the future directions that research should take. Major conclusions of the symposium were that the flow cytometry of multiple markers in fresh tissue would remain the standard technique of evaluating cancer-initiating cells and that surrogates need to be developed for both experimental and clinical use.

Lecture on stem cells and cancer

The 2009 Summer Symposium of the David H. Koch Institute for Integrative Cancer Research of MIT was on Understanding Metastasis. The Symposium was held on June 19, 2009. One of the lectures in the morning session was on "Stem cells and cancer", by Sean Morrison (University of Michigan) [FriendFeed entry]. A video (35 min) of the lecture can be accessed via the program. One example of a noteworthy slide is the one that's shown for 10 seconds, from 28:38 to 28:48 (entitled: "Nobody has yet tested which cells actually contribute to disease in patients"), in which it's pointed out that the "Fate of cells within patients is unclear ...".

Another lecture, at the same Symposium, addresses this issue (within the context of the theme of the Symposium, "Understanding Metastasis"). It's "Monitoring the fate of cancer cells during metastasis", by Ann Chambers (Regional Cancer Center, London, Ontario). A video (25 min) of her lecture can also be accessed via the program.

A review of the CSC paradigm

Controversial Cancer Stem Cells Offer New Direction For Treatment, ScienceDaily, June 25, 2009. [FriendFeed entry]. First paragraph:

In a review in Science, a University of Rochester Medical Center researcher sorts out the controversy and promise around a dangerous subtype of cancer cells, known as cancer stem cells, which seem capable of resisting many modern treatments.

Based on this review: The Increasing Complexity of the Cancer Stem Cell Paradigm by Jeffrey M Rosen and Craig T Jordan, Science 2009(Jun 26); 324(5935): 1670-3. First paragraph:

The investigation and study of cancer stem cells (CSCs) have received enormous attention over the past 5 to 10 years but remain topics of considerable controversy. Opinions about the validity of the CSC hypothesis, the biological properties of CSCs, and the relevance of CSCs to cancer therapy differ widely. In the following commentary, we discuss the nature of the debate, the parameters by which CSCs can or cannot be defined, and the identification of new potential therapeutic targets elucidated by considering cancer as a problem in stem cell biology.

Blog post about the complexity of CSC

Complexity of cancer stem cells by Alexey Bersenev, Hematopoiesis, May 6, 2009. [Twitter link to blog post] Final paragraph:

But not everything about CSC is so pessimistic. We know many good examples of successful targeting and eradication in tumors where the CSC model could be applied. We still have a lot of ways, other than surface molecules and signaling pathways, to target them. We are still developing a models to validate the concept. We can see clinical relevance and significance of CSC. Field is developing tremendously right now and a model is in the making.

… debating the existence of CSCs or their frequency is not a particularly useful exercise, and the scientific community would be well served to move beyond these issues. Rather, the more pertinent question is whether studying and targeting CSC is important for developing better forms of therapy. The answer to that query seems somewhat less clear.
Craig Jordan (U of Rochester)

Questioning the CSC hypothesis

Cancer's culprit: Breast cancer's genetic profile calls the cancer stem cell hypothesis into question by Elie Dolgin, The Scientist 2009(Apr); 23(4): 59. Publicly accessible (free registration required). Excerpts:

In 2007, the CSC hypothesis was thrown for a loop, however, after a team led by Kornelia Polyak, of the Harvard Medical School and Dana-Farber Cancer Institute in Boston, probed a bit deeper into the Michigan team's findings. In this month's Hot Paper, [reference 2] Polyak's team compared the genetic profiles of the putative breast CSCs with other more differentiated cells, and found several genetic differences between the two cell populations. This "raises doubts about whether they are direct descendents of one another," says Polyak.

.....

Notably, Baylor oncologist Jenny Chang, together with her colleague Jeffrey Rosen, compared breast tumors in patients before and after chemotherapy, and found a three-fold enrichment in cells resembling CSCs following treatment, signifying that tumorigenic cells are, indeed, more resilient. [reference 5] "There are different subpopulations of [cancer] cells that we need to target separately," says Chang. These results lend credibility to the CSC hypothesis where it really counts, adds Wicha. "It's a valid model because it actually predicts behavior in the clinic," he says.

Reference 2: M. Shipitsin et al., "Molecular definition of breast tumor heterogeneity," Cancer Cell, 11:259-73, 2007. [PubMed Citation].

Reference 5: X. Li et al., "Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy," J Natl Cancer Inst, 100:672-9, 2008. [PubMed Citation].

John Dick interviewed by Monya Baker

John Dick: careful assays for cancer stem cells by Monya Baker, Nature Reports Stem Cells 2009(Mar 26) [Full text is publicly accessible]. Excerpt:

What does the field need to move forward now?

It needs controversy.

The field of cancer stem cells needs controversy?

That's a little tongue in cheek. But it does. Controversy sparks better and better science. What it does is it actually eliminates sloppy thinking. There's been a real rush onto the cancer stem cell bandwagon in the last couple of years. People are talking about cancer stem cells here, there and everywhere, and in any old cell line. There was a huge slippage in the kind of criteria and rigor. People were using this terminology without any thought or any rigor based on some cell-surface marker or something like that. .....

[Thanks to Alexey Bersenev, via Twitter/cells_nnm].

Focal points for discussion about CSC

Cancer Stem Cells: Controversial or Just Misunderstood? Craig T Jordan, Cell Stem Cell 2009(Mar 6); 4(3): 203-5. Summary:

While a broad range of expertise has recently come to bear on the intriguing topic of cancer stem cells, the overall relevance of stem cells as they relate to cancer remains in dispute. In this commentary, underlying points of contention are described with the aim of defining focal points for discussion and future consideration.

Thanks to Alexey Bersenev: twitter.com/cells_nnm/status/1286475878

Elimination of CSC in a mouse model of CML

Targeting a chronic problem: elimination of cancer stem cells in CML by Claus Nerlov, EMBO J 2009(Feb 4); 28(3): 167-8 [PubMed Citation][Openly accessible full text][Version in PMC]. First paragraph:

The eradication of tumours by targeting malignant stem cell populations, or cancer stem cells (CSCs), is a promising new strategy for cancer treatment. In a paper published in a recent issue of The EMBO Journal, pharmacogenetic evidence has been obtained that this is indeed feasible in a mouse model of human chronic myeloid leukaemia. These and further similar experiments will be essential to determine the validity and practical usefulness of the CSC hypothesis.

This article is a commentary about: Cancer induction by restriction of oncogene expression to the stem cell compartment by María Pérez-Caro and 12 co-authors, including Isidro Sánchez-García, EMBO J 2009(Jan 7); 28(1): 8-20 [Epub 2008(Nov 27)][PubMed Citation][Openly accessible full text][Version in PMC]. Excerpt from the last paragraph of the Discussion section of the full text:

The data presented here further show for the first time the in vivo physiological relevance of the CSC suppression using a model system representing in vivo biology of the human CML disease. .....

Pointers for future research on CSC

Looking ahead in cancer stem cell research by John E Dick, Nat Biotechnol 2009(Jan); 27(1): 44-6 [PubMed Citation]. Leading sentence:

The history of the stem cell field offers pointers for future research on cancer stem cells.

The full text is publicly accessible. Excerpts:

Since the beginning of the era inaugurated by the great experimental pathologists, microscopic examination has revealed that many tumors, be they liquid or solid, exhibit morphologic heterogeneity. Tumors are also heterogeneous functionally, as demonstrated most dramatically by studies in which murine or human tumors were re-transplanted into syngeneic or xenogeneic immune-compromised recipients. Remarkably, such experiments have also included human autotransplants. The collective conclusion from these studies was that tumor re-initiation is variable and often rare, requiring 10³ to 10⁷ cells.

.....

Ultimately, the relevance of the CSC model will be determined by clinical data.

See also:

The CSC hypothesis: recalling some history (December 30, 2008).

Tumorigenic cells not rare in human melanoma (December 3, 2008).