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Previous Article | Next Article 
Molecular and Cellular Biology, January 2007, p. 195-207, Vol. 27, No. 1
0270-7306/07/$08.00+0 doi:10.1128/MCB.01525-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Travis L. Leonard,1,
Jessica L. Biddle,1
Michael T. Debies,1 and
Edward J. Gunther1,2*
Jake Gittlen Cancer Research Foundation,1 Department of Medicine, Pennsylvania State College of Medicine, Hershey, Pennsylvania 170332
Received 16 August 2006/ Returned for modification 13 September 2006/ Accepted 9 October 2006
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alternatively, the tumor dormancy phenomenon might be explained by an emerging concept that places cancer stem cells at the root of solid tumors, such as breast cancers (27, 31). Somatic stem cells inhabit adult epithelial tissue compartments and maintain tissue homeostasis in the face of wear and injury. In order to perform these functions over the lifetime of the organism, somatic stem cells possess both a strong intrinsic cell survival program and a capability to enter into, and emerge from, protracted quiescence (6, 15). Robust survival and reversible quiescence are not only biological hallmarks of putative mammary stem cells; they are also clinical hallmarks of MRD as encountered in breast cancer patients. Therefore, mechanisms governing survival and quiescence in normal mammary stem cells may likewise govern survival and quiescence within dormant MRD. Viewed this way, the failure of adjuvant therapy to cure the majority of breast cancer patients at risk for relapse might be blamed on a population of malignant progenitor cells resident within MRD that persists despite treatment and ultimately reconstitutes the malignancy (2, 14).
At present, there is no convincing biological description of clinically important MRD lesions (i.e., the residual disease which gives rise to relapse) in breast cancer patients. Ethical barriers typically prevent routine procurement of the substantial amounts of tissue that might reveal MRD on histologic examination. Even when permissible, biopsy-driven studies provide only a static picture of residual disease. For example, cytokeratin-positive cells thought to represent disseminated breast cancer can be found in the bone marrow of patients at high risk for relapse (8). However, while these cells may serve as an important clinical marker of aggressive disease, whether these foci represent clinically important "culprit lesions" capable of generating overt recurrence in bone or elsewhere remains unknown. Since the MRD lesions that directly yield relapse in human breast cancer patients will remain uncharacterized and inaccessible for the foreseeable future, we have aimed to model latent breast cancer in the mouse. Toward this end, we previously described an inducible transgenic model that permits reversible, Wnt-initiated mammary tumorigenesis (17). Importantly, this model yields long-lived subclinical disease capable of engendering relapse, thereby modeling the clinical behavior of human breast cancer.
Here, residual disease lesions persisting long after reversal of Wnt-initiated mammary tumorigenesis were propagated in vivo to elucidate biological properties of dormant mammary cancer. We find that mammary ductal outgrowths derived from dormant malignancy are morphologically indistinguishable from normal ductal epithelium and are capable of following the developmental programs triggered by hormones of puberty and pregnancy. Nonetheless, these same outgrowths retain a latent malignant growth program that rapidly is reengaged upon restoration of oncogenic Wnt signaling. Our findings support the relevance of the cancer stem cell concept to a tractable preclinical model of dormant mammary cancer and implicate mechanisms governing mammary progenitor cell survival and quiescence in mammary cancer dormancy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of mammary neoplasia and tissue-recombinant hosts. Mammary neoplasia was generated in MTB/TWNT bitransgenic donor mice as previously described (17). Tumors were subjected to biopsy when they reached 1 to 1.5 cm in diameter, and hosts were subjected to Dox withdrawal 2 weeks after surgery to trigger tumor regression. After 4 weeks or more of subclinical disease, the mammary gland that previously harbored a clinically apparent tumor was harvested and the region suspected of harboring dormant MRD was identified and excised. This donor tissue immediately was placed in phosphate-buffered saline on ice and divided into fragments of approximately 1 mm in diameter which were then implanted into the inguinal mammary fat pads of 3-week-old wild-type FVB/N host females after surgical clearing of the endogenous mammary epithelium as described previously (37). In all cases, donor epithelium was permitted to engraft and mature within host mice in the absence of Dox treatment for a minimum of 12 weeks. Tumor explants were performed as described previously (17).
BLI. An isoflurane-suffused induction chamber was used to transiently anesthetize mice prior to administering 100 mg/kg ketamine and 10 mg/kg xylazine via intraperitoneal injection. Anesthetized mice then received a 135-mg/kg intraperitoneal injection of D-luciferin (Prolume, CA). Imaging was acquired and processed using an IVIS 50 imaging system and Living Image software (Xenogen, CA). The bioluminescence imaging (BLI) signals emanating from individual mammary glands and tumors were quantitated by measuring photon flux within software-generated regions of interest (ROIs). Settings were chosen such that ROIs were bounded by contour lines demarcated by a diminution in photon flux to 20% of the maximum flux detected within the ROI. In preliminary experiments, we determined that the BLI-based signal reproducibly reached a plateau level by 10 min after luciferin injection and varied little over the subsequent 10 min. Therefore, BLI data were acquired between 10 and 15 min after luciferin injection.
Tissue processing and immunohistochemistry. Tissue samples were fixed in 4% paraformaldehyde for 2 h on wet ice. Immunohistochemistry was performed on 4-µm paraffin-embedded tissue sections. Primary antibodies used were rat anti-keratin 8 (Troma I) at 1:100 from the Developmental Studies Hybridoma Bank at the University of Iowa; mouse anti-smooth muscle actin (anti-SMA) (M0851) at 1:250 from DakoCytomation, Denmark; and rabbit anti-keratin 6 (PRB-169P) at 1:7,500 from Covance, Berkeley, CA. Sections were incubated in primary antibody at 4°C overnight. Secondary antibodies and diaminobenzidine detection were performed using the following: a DakoCytomation Ark peroxidase kit (K395411) from DakoCytomation, Denmark (for SMA); a DakoCytomation Envision+ system-HRP (K4010 for keratin-6 and keratin-8; for keratin-8 the ant-mouse secondary antibody was replaced with anti-rat secondary antibody E0468). Sections were counterstained with hematoxylin. Mammary gland whole mounts were prepared as described previously (11). Images were captured using a Nikon DXM1200 digital camera on either a Nikon E600 microscope (sections) or a Nikon SMZ1000 stereoscope (whole mounts).
H-Ras mutation analysis. Genomic DNA was prepared from frozen tissue samples and used as the template in PCRs to amplify the relevant H-Ras coding region, using primers 5'-CGCTGTAGAAGCTATGACAG-3' (forward) and 5'-AAGCCCTCCCCTGTGCGCAT-3' (reverse). PCR products were identified and isolated by agarose gel electrophoresis and subjected to DNA sequencing using an ABI Prism model 377 or ABI model 3100 DNA sequencer.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether transgenic outgrowths progress toward Wnt-initiated adenocarcinoma, we generated a cohort of hosts harboring 18 tissue-recombinant MTB/TWNT mammary glands. Source tissue for 10 of the grafts was harvested from transgenic donor mice that were 8 weeks or more into continuous Dox treatment. Source tissue for the remaining 8 grafts was harvested from transgenic donors 2 to 4 weeks into a Dox withdrawal period that followed a 6- to 8-week "pulse" of Dox treatment. Hosts were maintained Dox naïve for a minimum of 12 weeks during the period of engraftment and graft maturation and then were monitored during chronic Dox treatment by serial BLI as well as weekly visual inspection and palpation.
Though a number of parameters might influence transgene expression in an individual cell, we anticipated that BLI signal would provide an approximate measure of the number of transgenic epithelial cells present in a tissue-recombinant mammary gland. The BLI signal emanating from grafts harboring hyperplasia-derived ductal outgrowths increased markedly over the first weeks of Dox treatment but increased only modestly, or not at all, thereafter when monitored for more than 6 months (17 of 18 grafts; a representative imaging series is depicted in Fig. 2b, and quantitative data are depicted in Fig. 3c). This suggested that hyperplasia-derived transgenic mammary epithelium underwent only a limited Dox-induced expansion in cell number. A single graft in this experiment (1 of 18) likewise generated a stable BLI signal out to 5.5 months but later generated an increased signal that lateralized to the right inguinal graft and heralded the development of a palpable right inguinal tumor with adenocarcinoma histology (Fig. 2b). The stochastic development of a single mammary tumor among 18 transgenic grafts monitored for 6 months is consistent with a requirement for cooperating event(s) en route to tumorigenesis in hyperplasia-derived outgrowths. The rate of tumorigenesis in this experiment, i.e., 1 tumor in 18 "at-risk" transgenic glands during 6 months of Dox treatment, is comparable to the 20-week mean tumor latency observed for Dox-treated MTB/TWNT mice, each of which harbors 10 "at-risk" transgenic glands (17).
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Dox-dependent malignant behavior of RTD-derived epithelial outgrowths. Previously, we showed that solitary mammary tumors arise stochastically in MTB/TWNT mice during chronic Dox treatment, implying a requirement for spontaneous cooperating genome lesions (genetic and/or epigenetic) within somatic tissue. MTB/TWNT mammary tumors regressed upon Dox withdrawal, but a subset of these tumors relapsed and regrew in a Dox-independent manner, indicating that disease foci harboring latent malignant potential persist, as has been shown to occur in a variety of inducible transgenic solid-tumor models (7, 10, 17, 25, 34). We expected that mammary epithelium within these lesions would be profoundly impaired in its capacity to reconstitute a normal mammary ductal tree due to retention of cooperating genome lesions acquired during tumorigenesis. To test this hypothesis, macroscopic subclinical disease foci were identified in the mammary glands of MTB/TWNT mice whose tumors had regressed after Dox withdrawal, and fragments representing putative dormant MRD were propagated in the cleared fat pads of wild-type hosts in the absence of Dox. Since MRD represents a clinical term for the cells that beget relapse in human cancer patients, residual disease as it pertains to this mouse model is herein referred to as residual transgene-induced disease (RTD). A morphological and molecular description of in situ RTD lesions will be reported separately.
Tissue-recombinant mammary glands harvested from Dox-naïve hosts 12 weeks or more after implantation of RTD-derived epithelium consistently harbored mammary ductal outgrowths that extended centrifugally from the surgical implantation site and filled the majority of the host fat pad (Fig. 3a and data not shown). Thus, cooperating genome lesions presumed necessary for Wnt-initiated tumorigenesis did not prevent RTD-derived mammary epithelium from following the morphogenetic program of ductal elongation in response to hormones of puberty. Surprisingly, microscopic examination of whole mounts and tissue sections from these tissue-recombinant mammary glands typically revealed mammary ductal morphology that was indistinguishable from that of wild-type endogenous epithelium (Fig. 3a). Moreover, RTD-derived outgrowths rarely gave rise to spontaneous tumors in the absence of Dox. We observed only one spontaneous tumor in more than 50 RTD-derived outgrowths monitored in Dox-naïve hosts for a minimum of 12 weeks (Table 1). Remarkably, 10 RTD-derived outgrowths failed to yield spontaneous tumors in Dox-naïve hosts monitored for periods ranging from 6 months to 1 year despite harboring latent malignant potential (see below) (Table 1; see Fig. 8). Thus, RTD-derived epithelium in large part exhibits benign behavior when propagated in Dox-naïve host mice.
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Notably, in contrast to the relatively modest Dox-induced BLI signal emanating from glands harboring hyperplasia-derived epithelium, which typically leveled off after several months (Fig. 2b and 3c), the Dox-induced BLI signal emanating from glands harboring RTD-derived epithelium increased more rapidly (Fig. 3b and 3c) (for ease of comparison, BLI data in all figures are depicted using an identical photon flux color scale with identical minimum and maximum photon detection settings). Palpable mammary tumors consistently arose in tissue-recombinant glands within several weeks of starting Dox (Fig. 3d and Table 1), and these tumors were heralded by marked increases in BLI signal that were typically 10-fold or more in excess of the peak signal measured in hyperplasia-derived grafts (Fig. 3b and c). Tumors displayed histology characteristic of Wnt-initiated mammary adenocarcinoma and were invasive (see below). In all, 26 RTD-derived grafts yielded 23 clinically apparent tumors by 7 weeks of Dox treatment. In contrast, as shown above, tissue-recombinant glands generated from benign transgenic epithelium behaved differently, since none of 18 grafts yielded tumors over the first 24 weeks of Dox treatment (P < 10–7 by the log rank test). Thus, restoration of Wnt pathway activation rapidly restored a malignant phenotype to RTD-derived outgrowths, while outgrowths derived from benign transgenic epithelium yielded only hyperplasia under similar conditions. Moreover, tumors arose from RTD-derived outgrowths months before stochastic Wnt-initiated tumors would be expected.
We tested whether maintenance of RTD-derived tumors requires ongoing activation of oncogenic Wnt signaling. As shown previously for primary mammary tumors arising in the MTB/TWNT model (17), first-generation RTD-derived tumors rapidly extinguished TWNT transgene expression and regressed to yield subclinical disease upon Dox withdrawal (Fig. 3b). This reversibility enabled de novo propagation of subclinical RTD that persisted following regression of first-generation RTD-derived tumors in second-generation hosts according to the scheme depicted in Fig. 1. Remarkably, second-generation RTD-derived tumors likewise arose in a Dox-dependent manner and were reversible (Fig. 3b and data not shown).
"Mixed-lineage" histology of RTD-derived tumors. Wnt1 is frequently overexpressed due to insertional mutagenesis in MMTV-initiated tumors (26), and such mammary tumors are thought to arise from malignant transformation of a primitive mammary cell that serves as the progenitor for both the myoepithelial cell and luminal epithelial cell lineages (21). Furthermore, canonical Wnt signaling regulates stem cells in a variety of adult tissue compartments (30), suggesting that Wnt1 preferentially transforms a mammary stem cell. Experimental support for this hypothesis comes from immunohistochemical studies, which document that myoepithelial and luminal markers are coexpressed only in the subset of transgenic mouse mammary tumors initiated by Wnt pathway signaling components (22). Using these same markers to track the myoepithelial and luminal cell lineages, we investigated whether tumors arising from RTD-derived outgrowths retained the mixed-lineage character expected of antecedent primary tumors. RTD-derived tumors harbored large numbers of anti-SMA-positive cells indicative of the myoepithelial cell lineage as well as large numbers of keratin 8-positive cells indicative of the luminal epithelial cell lineage (Fig. 4). The patterns of immunoreactivity were highly conserved, such that neither lineage marker yielded an expression pattern that distinguished primary mammary tumors from either first- or second-generation tumors arising from RTD-derived outgrowths (Fig. 4).
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Synchronous transformation of RTD-derived outgrowths. We considered whether the capacity to generate mature, normal-appearing ductal outgrowths and the capacity to generate tumors might reside in distinct subpopulations of RTD-derived mammary epithelium. Specifically, if both benign and malignant subpopulations of transgenic mammary epithelium coengrafted, the benign epithelium might predominate at the leading edge of the outgrowth and account for the normal ductal morphology. Meanwhile, the malignant epithelium might reside at the engraftment site and remain capable of regenerating a Dox-dependent tumor but contribute little to ductal morphogenesis and elongation in the absence of Dox. To test this possibility, we examined the morphology of sibling RTD-derived grafts (i.e., grafts derived from the same source RTD lesion) in both Dox-naïve hosts and hosts given short-term Dox treatment.
As described above, RTD-derived epithelium from Dox-naïve hosts was morphologically indistinguishable from endogenous wild-type ductal epithelium. In contrast, by 4 days of Dox treatment, RTD-derived outgrowths showed expansion of ductal epithelium, manifesting as both luminal filling and an increase in ductal side branching (Fig. 5 and data not shown). These changes were particularly striking at the periphery of the graft, indicating that epithelial cells that traveled to the outskirts of the fat pad during ductal elongation were capable of participating in this morphological transition. By 2 weeks of Dox treatment a widespread, relatively homogeneous overgrowth of epithelium was apparent in RTD-derived grafts; this was morphologically distinct from, and far in excess of, the overgrowth characteristic of typical Wnt-initiated mammary hyperplasia (compare rightmost panels in Fig. 5 with rightmost panels in Fig. 2b). In addition, RTD-derived mammary epithelial cells showed marked nuclear enlargement and atypia at these early Dox treatment time points, and chronic Dox treatment of hosts harboring additional sibling grafts yielded palpable adenocarcinomas within 4 weeks (data not shown). Together, these findings are consistent with RTD outgrowths harboring a dynamic population of latent malignant cells that participate in ductal morphogenesis during puberty.
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H-Ras mutations link RTD-derived outgrowths to antecedent malignancies. In our experiments tissue fragments, rather than defined cell lines, were implanted into host fat pads. Therefore, it was important to provide genetic evidence that cells within RTD-derived outgrowths are indeed clonally related to antecedent tumor cells. To demonstrate clonality, we took advantage of the observation that Wnt-initiated mammary tumors frequently acquire somatic mutations that activate endogenous H-Ras alleles during carcinogenesis. These mutations are not detectable in Wnt-initiated hyperplasia but are detectable in half of all adenocarcinomas (28; S. Gestl and E. Gunther, unpublished observations). These mutations, which correspond to activating ras mutations found in human cancers, can occur in any of three H-Ras codons (codons 12, 13, and 61) and provide a mutation signature with which to track clonally related neoplastic cells.
Endogenous H-Ras alleles from several sets of tissue samples comprised of antecedent tumors and their descendant RTD-derived outgrowths were amplified and analyzed by DNA sequencing. Somatic point mutations were identified in distinct H-Ras codons that clonally linked outgrowths to antecedent tumors in three distinct sets of tissue samples, each of which descended from an independent primary mammary tumor (Fig. 7, Table 1, and data not shown). Notably, a somatic H-Ras mutation signature indicating a clonal link with a malignant cell lineage was well represented within a Dox-naïve outgrowth that showed benign behavior and histologic evidence of lobuloalveolar differentiation triggered by hormones of pregnancy (Fig. 7).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our model, regenerative capacity and tumor reconstitution potential coexist within RTD-derived outgrowths, suggesting that RTD lesions harbor cancer stem cells. Since RTD-derived outgrowths generate functional mammary glands in the absence of Wnt pathway activation, our findings support a model in which oncogenic Wnt signaling acts to transform a mammary progenitor cell (as opposed to imparting progenitor cell properties), as suggested by others (21, 22). Definitive identification of a stem cell population requires prospective purification of that cell population to homogeneity prior to demonstrating both self-renewal and multipotency (41). Our experimental approach falls short of this exacting standard in that macroscopic tissue fragments rather than defined cell populations were implanted. However, since RTD-derived epithelium was propagated by immediate implantation, our protocol avoids one well-documented source of ambiguity in stem cell-related work by precluding the possibility that novel biological potentials were acquired during intervening cell culture (20). Even so, the ultimate description of a malignant stem cell population in our model likely will depend upon ongoing efforts to characterize and validate candidate mammary stem cell markers (35, 38). Inversely, RTD lesions, like those arising in our model, may prove useful in refining such markers, since RTD may embody a pool of cells highly enriched for multipotent (albeit malignant) progenitors.
In terms of clinical behavior, RTD persisting after reversal of Wnt-initiated mammary tumors resembles MRD in breast cancer patients, since both lesions are capable of engendering late relapse after a prolonged disease-free interval. However, the mechanisms regulating dormancy in RTD versus MRD may be distinct. Given that MRD lesions from breast cancer patients remain inaccessible, it is not yet possible to compare the properties of human MRD lesions with the properties of murine RTD lesions. In this regard, it is important to consider how Wnt-initiated mammary tumorigenesis as analyzed in a transgenic mouse model may differ from human breast carcinogenesis. For example, since malignant transformation appears to require fewer genetic events in murine than in human cells (29), MRD lesions in humans likely harbor a greater number of genetic aberrations than RTD lesions in mice. Whereas in our study RTD-derived ductal epithelium generated normal-appearing mammary outgrowths, it is possible that increased genetic complexity may render MRD from humans incapable of recapitulating normal mammary morphogenetic programs. Furthermore, comparative analyses of transgenic mammary cancer models suggest that distinct initiating oncogenes culminate in distinct tumor types that possess oncogene-specific histologic features and gene expression profiles (19, 33). Moreover, evidence suggests that distinct oncogenes may yield distinct mammary tumor types in part by targeting distinct mammary cell types (22). An important question is whether the properties of Wnt-initiated RTD described here are shared by RTD that arises when reversible mammary tumorigenesis is initiated by oncogenes believed to transform more committed mammary progenitors.
Reactivation of oncogenic Wnt signaling rapidly triggered malignant transformation of normal-appearing, RTD-derived mammary epithelium despite the presence of wild-type, host-derived stroma. Notably, Wnt1 acted as a master regulator of the malignant phenotype only in RTD-derived grafts and not in grafts derived from benign epithelium. Therefore, cooperating genome lesions acquired during tumorigenesis and retained within RTD encode a permissive state that renders RTD uniquely susceptible to Wnt-mediated transformation. Our experiments do not exclude the possibility that stromal cells contribute to this permissive state. Indeed, since interactions between the epithelium and stroma are essential for normal mammary gland morphogenesis (42), it seems likely that aberrant signaling from epithelium to stroma aids in assembling the malignant microenvironment. Nonetheless, our findings argue strongly that malignant transformation of RTD-derived outgrowths does not require cooperating somatic genome lesions in the stromal cell compartment. Were such lesions required, tumors would be expected to arise stochastically and focally upon Wnt pathway activation. Instead, restoration of oncogenic signaling triggers a widespread, homogeneous ductal morphology transition and rapidly yields an invasive adenocarcinoma.
Our experimental protocol provides a novel means of analyzing the biological potential of a mammary cancer genome. Recently, in a remarkable study, chimeric mice were generated using pluripotent cells harboring a melanoma genome that was reprogrammed via nuclear transfer (18). As part of this study, efforts to create similar mice using nuclei from mammary tumors were unsuccessful. Here, by propagating latent malignant epithelium, we studied a mammary cancer genome at the level of the organ rather than at the level of the organism. In doing so, we showed that activation of an endogenous H-Ras allele is compatible with the mammary epithelium completing mammary gland-specific developmental programs such as ductal elongation and lobuloalveolar differentiation. This finding contrasts with results obtained with transgenic mice that overexpress an exogenous mutant HRAS allele, as these transgenic mice have markedly abnormal ductal epithelium by puberty and fail to lactate (36). As such, our findings add to a growing body of data that point to distinct outcomes depending upon whether mutant Ras proteins are expressed at supraphysiologic levels versus levels governed by endogenous promoter elements. Further support for the notion that an activated ras allele expressed at physiologic levels can be broadly compatible with organogenesis comes from the recent identification of germ line-activating HRAS mutations in individuals with Costello syndrome (4).
Previously, reversal of Myc-initiated liver tumorigenesis in transgenic mice revealed dormant RTD with multilineage differentiation potential (34). Our work extends this finding in two important ways. First, we demonstrate experimental access to RTD arising in a model of breast cancer, a disease in which tumor dormancy plays a well-recognized and clinically important role. Second, we demonstrate propagation of RTD lesions in vivo, thereby defining a tractable experimental paradigm with which to investigate biological properties of dormant malignancy. It might be argued that our model was predisposed to reveal stem cell-like properties within RTD, since mammary tumorigenesis was driven by activation of the Wnt pathway, which has putative roles in regulating a variety of epithelial stem cells (30). However, recent studies implicate aberrant activation of embryonic signaling pathways in the pathogenesis of a sizable fraction of the most common and deadly human cancers. In addition to Wnt, these pathways include the Hedgehog and Notch pathways (6, 39). These same embryonic signaling pathways govern stem cells during adult tissue maintenance and repair in diverse tissue compartments. In this context, our results highlight the possibility that cellular programs governing normal stem cell homeostasis may be misappropriated to regulate dormancy in a wide variety of malignancies.
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ACKNOWLEDGMENTS |
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Animal housing was provided through a facility constructed with support from a Research Facilities Improvement Grant (C06 RR-15428-01) from the National Center for Research Resources, NIH. Image acquisition equipment was purchased through grant CA40145 to Gary Clawson of the Gittlen Foundation. This work was supported in part by grants from the National Cancer Institute (K08 CA79682 and R01 CA114001 to E.J.G.).
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FOOTNOTES |
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Published ahead of print on 23 October 2006.
These authors contributed equally to this work.
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