Exploring Alternative Individualized Treatment Strategies in Colorectal Cancer

Exploring Alternative Individualized Treatment Strategies in Colorectal Cancer Peter M. Wilson,1 Robert D. Ladner,1 Heinz-Josef Lenz2 Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer in men and women in the United States, with a predicted 154,000 new cases this year. For > 40 years, 5-fluorouracil (5-FU) has remained the central agent in therapeutic regimens used in the treatment of CRC, with single-agent response rates (RRs) of 20%-25% in advancedstage disease. The past decade has witnessed the introduction of newer agents, such as the DNA-damaging agents oxaliplatin and irinotecan, which when used in combination with 5-FU, have dramatically increased RRs to 40%-50% in advanced disease and improved overall survival. The development of monoclonal antibodies targeting the epidermal growth factor receptor or vascular endothelial growth factor have now demonstrated additional clinical benefit for patients with metastatic disease, and the clinical development of these agents continues to progress. However, many patients will die, and a significant proportion will experience severe chemotherapy-induced toxicities, while deriving little or no benefit. Global efforts are currently under way to identify reliable and validated cassettes of markers with the ability to predict response and toxicity from a chemotherapeutic regimen. In addition, the ability to accurately predict patients with early-stage disease at high risk of recurrence will enable the appropriate administration of adjuvant therapy. The emerging cancer stem cell hypothesis continues to gain momentum with ongoing research, suggesting this might become one of the prime targets for future therapy. Together, these approaches are spearheading a paradigm shift toward individualized treatment strategies in CRC treatment. Clinical Colorectal Cancer, Vol. 7, Suppl. 1, S28-S36, 2007 Key words: Adjuvant therapy, Epidermal growth factor receptor, 5-Fluorouracil, Metastases, Monoclonal antibodies, Stem cells, Vascular endothelial growth factor

Introduction Colorectal cancer (CRC) remains the second leading cause of cancer-related death in men and women in the Western world, with an estimated 53,000 deaths in the United States in 2007.1 Approximately 70% of these malignancies will arise in the colon, with the remaining 30% arising in the rectum. Surgery remains the most effective treatment for carcinomas of the colon and, in some instances, is curative. Radiation therapy is an additional option, employed primarily in rectal cancer when appropriate. However, a significant percentage of patients present at diagnosis with late-stage or advanced disease, and systemic chemotherapy is essential to effectively manage the disease. 1Department of Pathology 2Division of Medical Oncology

University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, Los Angeles Submitted: Oct 10, 2007; Revised: Nov 20, 2007; Accepted: Nov 21, 2007 Address for correspondence: Heinz-Josef Lenz, MD, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, 1441 Eastlake Ave, Suite 3456, Los Angeles, CA 90033 Fax: 323-865-0061; e-mail: [email protected]

5-fluorouracil (5-FU) and the oral 5-FU prodrug capecitabine remain the most effective therapeutic agents in the treatment of CRC and are the preferred partners for the DNA-damaging agents irinotecan and oxaliplatin, achieving response rates (RRs) of 40%-50% in combination therapy and prolonging overall survival (OS).2-6 Novel biologic agents, such as the monoclonal antibodies (MoAbs) cetuximab, which targets the epidermal growth factor (EGF) receptor (EGFR), and bevacizumab, an inhibitor of vascular endothelial growth factor (VEGF), have recently demonstrated additional clinical response and survival for patients with metastatic CRC (mCRC) and have provided clinicians with more therapeutic options.7 However, for patients with stage II colon cancer, the role of adjuvant chemotherapy remains undefined, and there is clear evidence that a subset of patients are at high risk for disease recurrence. In the treatment of patients with rectal cancer, improved outcomes have been noted with the use of total mesorectal excision and preoperative concurrent chemoradiation therapy. Regardless of these recent advancements, selection of the most beneficial treatment strategy in treating CRC remains a challenge and is hindered by the lack of predictive and prognostic markers (Table 1). In

Dr Wilson has no relevant relationships to disclose. Dr Ladner has no relevant relationships to disclose. Dr Lenz has received research support from Roche, Bristol-Myers Squibb, Eli Lilly, sanofi-aventis, Novartis Oncology, Genentech BioOncology, ImClone, Pfizer, and AstraZeneca, has served as a paid consultant or been on an Advisory Board for Merck, Response Genetics, Genentech BioOncology, Amgen, Novartis Oncology, sanofi-aventis, Pfizer, Bristol-Myers Squibb, and ImClone, and has served on a Speaker's Bureau for Roche, Pfizer, Eli Lilly, sanofi-aventis, and Merck.

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addition, the high incidence of tumor drug resistance remains a major stumbling block to effective cancer treatment. In recent years, global research efforts have attempted to identify subsets of biochemical markers that can predict response to treatment (evaluated through clinical response, time to disease progression, and treatment-associated toxicities) and prognostic markers to assess the aggressiveness of the disease and the likelihood of recurrence after surgery. The science of pharmacogenomics is emerging as an increasingly useful molecular tool in the investigation of the disparity in drug efficacy by simultaneous analysis of variables in the patient and their disease, such as genetic polymorphisms in drug targets, metabolizing enzymes, and transporters and influential receptors.8 Accordingly, the identification of sensitive and validated predictive and prognostic markers combined with an increasing variety of therapeutic agents is now providing clinicians with the means to tailor a targeted and effective therapy to the molecular profile of the patient and their disease while minimizing and avoiding life-threatening toxicities.

Individualized Treatment: A Paradigm Shift in the Treatment of Colorectal Cancer In recent years, a paradigm shift has occurred in the management of CRC treatment. The “one-size-fits-all” approach to treatment and chemotherapy is no longer sufficient, and it is now widely accepted that a multitude of factors contribute to the success of a chemotherapeutic regimen. Individualized treatment strategies are becoming more prevalent as more clinicians recognize the heterogeneity in the patients and the diseases presented to them. Ultimately, the critical factor in designing an individualized treatment strategy is dependent on the disease presented and the goal of the therapy. Treatment strategies undergone with the intention of a curative outcome are frequently aggressive and involve combination therapies and/or biologic agents. Alternatively, palliative treatment can be oriented toward controlling disease, relieving symptoms, and minimizing toxicities. Therefore, simultaneous analysis of patient clinical and pathologic characteristics and molecular profiling of the patient and their specific disease, combined with an increasing arsenal of therapeutic agents, is spearheading the advancement of individualized CRC treatment. This review will not discuss at length individual markers for 5-FU, irinotecan, or oxaliplatin (for a comprehensive review of predictive molecules, see Longley et al9), but rather focuses more on several key areas where individualized treatment strategies are likely to have the greatest effect on the future of CRC treatment.

Adjuvant Therapy: To Treat or Not to Treat? Intervention with adjuvant therapy represents a niche in CRC treatment, which will greatly benefit from an individualized treatment strategy. Currently, pathologic stage represents the most important prognostic factor for patients with CRC. The TNM system, as defined by the American Joint Committee on Cancer (AJCC), is the most commonly used staging system and is based on depth of invasion of the bowel wall, extent of regional lymph node involvement, and presence of metastases.10

Table 1

Current Challenges and Goals in the Treatment of Colorectal Cancer

Challenges in the Treatment of Colorectal Cancer

The Goals of Individualized Therapy

Rigorous validation of molecular markers and their association with clinical outcome in large, randomized, multicenter, prospective trials

Identification of patients who will benefit from chemotherapy

Refining technological platforms and bioinformatics to accommodate the complexity of the multifaceted molecular map, which might determine outcome The implementation of these findings and methods to everyday clinical practice: benchside to bedside

Selection of new chemotherapeutic combinations based on diseasespecific molecular targets Inclusion of molecular marker screening in prospective trials Pharmacogenomics must be implemented early in drug development to assist in determining drug metabolism and avoid life-threatening toxicities

As the AJCC stage increases from stage I to stage IV, patients are faced with the reality of a decline in the 5-year survival rate from > 90% to < 10%.11,12 Since its introduction in 1957, the fluoropyrimidine 5-FU remains the mainstay of therapeutic regimens in the treatment of CRC. Its primary mechanism of action is inhibition of thymidylate synthase (TS), blocking the de novo synthesis of thymidine, an essential component of DNA synthesis, and initiating DNA damage. Additional mechanisms of toxicity are exerted through the incorporation of fluorinated nucleotides into DNA and RNA.13 Recently, the oral 5-FU prodrug capecitabine was introduced. Capecitabine is absorbed intact through the gastrointestinal mucosa and undergoes a 3-step enzymatic conversion to 5-FU. Previously, 5-FU modulated with leucovorin (LV) was the treatment of choice in the adjuvant setting, demonstrating a statistically significant increase in disease-free survival (DFS) and OS in patients with stage III disease. In the adjuvant treatment of 1987 patients with stage III colon cancer, capecitabine was also shown to be similarly effective when compared with the Mayo Clinic regimen of bolus 5-FU/LV.14 More recently, the DNA-damaging agent oxaliplatin has been combined with 5-FU/LV in the adjuvant setting. The addition of oxaliplatin to 5-FU–based adjuvant treatment resulted in a statistically superior DFS in stage III disease and is now considered the standard of care.15 Unexpectedly, although showing clear activity in metastatic disease, a recent study demonstrated that the topoisomerase I inhibitor irinotecan had no benefit in the stage III adjuvant setting; this is a reminder that large, randomized, prospective trials are a necessity to avoid jumping to conclusions before clinical assessment.16 However, implementation of adjuvant therapy for stage II disease remains in question, with conflicting reports. QUASAR-1 is the only single study that has demonstrated a statistically significant advantage to the administration of adjuvant therapy in stage II disease.17 Numerous previous trials that have included patients with stage II/III disease repeatedly did not demonstrate a statistically significant survival benefit for patients with stage II disease. A pooled analysis of Clinical Colorectal Cancer Supplement December 2007 • S29

Individualized Treatment Strategies in Colorectal Cancer Figure 1

Trial Schema for Eastern Cooperative Oncology Group 5202

Phase III Randomized Study of 5-FU, Oxaliplatin, and LV with or Without Bevacizumab in Stage II Colon Cancer

High Risk

R A 5-FU/LV/ N D Oxaliplatin O 5-FU/LV/ M I Oxaliplatin/ Z Bevacizumab E

Low Risk

Observation

Eligibility: Stage II (T3-T4, N0, M0) Tumor specimen available Age > 18 Years No distant sites of disease

This study is a large, randomized, prospective trial to determine the clinical usefulness of molecular markers. This simplified schematic demonstrates proposed stratification of patients with stage II disease based on genetic stability parameters. Several important clinical eligibility parameters are highlighted. Patients deemed to be at high risk from high MSI and loss of heterozygosity at 18q are randomized to 1 of 2 treatment arms, and those considered to be at low risk are registered for observation only. ECOG 5202 is in its early stages and has a target accrual of 3610 patients.30

7 studies demonstrated a 5-year OS of 81% in patients who received 5-FU–based adjuvant therapy and 80% in patients who underwent surgery alone.18 In specific cases, such as with stage II disease with a 10%20% tumor recurrence rate, it becomes imperative to decide which patients should receive therapy. In essence, these patients include those having a high likelihood of recurrence, and adjuvant therapy is suggested for patients with “high-risk” stage II and T4 tumor stage, bowel perforation, or clinical bowel obstruction.19 Identifying a reliable panel of prognostic and predictive markers for tumor recurrence is critical in selecting a tailored chemotherapy.

Chromosomal Instability and Microsatellite Instability The 2 most characterized mechanisms of genomic instability are chromosomal instability (CIN) and microsatellite instability (MSI). The CIN phenotype is present in approximately 85% of sporadic colon cancers and is characterized by aneuploidy, chromosomal rearrangements, and mutations in mitotic checkpoint genes, microtubule spindle defects, and telomere dysfunction. The MSI phenotype is found in many different types of cancer and is characterized by a change in the length of DNA microsatellites as a result of the insertion or deletion of repeating units. This phenomenon is reputedly caused by defects in mismatch repair (MMR) genes such as MLH1, MSH2, or methylation of the MLH1 promoter. Microsatellite instability is frequently classified using the revised Bethesda-guidelines panel loci (Bat25, Bat26, Mfd15, D2S123, and D5S346) and the transforming growth factor (TGF)–β-RII locus. The National Cancer Institute–approved reference panel standardized the identifica-

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tion of patients having high MSI as those with the presence of instability in ≥ 30% of the markers tested.20 Definitive correlations between genetic alterations in tumors and their clinical behavior are infrequent. Several studies have investigated the prognostic significance of a number of markers which predict tumor recurrence, including the most promising, MSI status. A study in stage II/III CRC identified that patients with high MSI had improved survival and were more likely to exhibit better recurrence-free survival than those with microsatellite stable (MSS) phenotypes.21 Additional studies have analyzed the relationship between MSI and CRC prognosis and concluded that patients with CRC exhibiting MSI had a significantly better prognosis compared with patients having intact MMR, but did not benefit from the administration of 5-FU therapy in the adjuvant setting.22,23 The presence of a mutation in TGF-β-RII has been shown in a recent report to improve survival in patients who also possess MSI-high. The 5-year survival rate for patients with high MSI tumors and the TGF-β-RII mutation was 74% after adjuvant 5-FU–based therapy, compared with 46% in patients with high MSI tumors, which lacked the mutation in TGF-β-RII. Interestingly, 61% of stage III colon cancers in this study showed expression of the TGF-β-RII mutation, indicating that this high-frequency mutation might be useful in combination with MSI status as a prognostic marker for adjuvant therapy.24 Published data also strongly suggest that tumor MSI status is directly correlated with response to irinotecan, which might be of use to clinicians considering an irinotecan-containing therapy.25

Loss of Heterozygosity of 18q and 17p It is reported that allelic deletions involving chromosome 18q and 17p occur in > 70% of cases of CRC. Such deletions are believed to signal the existence of a tumor suppressor gene in the specific region. The tumor suppressor gene p53, often referred to as the “guardian of the genome” because of its critical role in genetic stability and detection of genotoxic stress, is located on 17p and is mutated in 40%-60% of CRCs.26 The status of p53 has been rigorously analyzed as a prognostic and predictive marker in CRC, with conflicting results. Retention of 18q alleles in MSS cancers points to a favorable outcome after adjuvant chemotherapy with 5-FU–based regimens for stage III colon cancer.24 The 18q chromosome contains DCC (deleted in colon cancer), a cell adhesion molecule whose elevated expression can lead to enhanced tumor growth and metastatic potential.27 Several studies have determined that patients with tumors displaying chromosome 18q loss appear to have a worse DFS and OS.28,29 Eastern Cooperative Oncology Group (ECOG) trial 5202 is a current prospective clinical trial and one of the first of its kind. It is randomizing patients with stage II disease based on their MSI and 18q status to observation versus chemotherapy with the intention of prospectively determining the prognostic value of molecular markers and the benefit of the addition of bevacizumab to the treatment arm (Figure 1).30 Insight into the genetic stability of a tumor might give clinicians the ability to tailor a targeted, more efficacious therapy. However, until the results of such prospective trials are pub-

Peter M. Wilson et al lished, the true significance of the genetic instability markers in predicting prognosis and recurrence will remain in question.

Figure 2

Simplified Diagram Illustrating Multiple Pathways and Downstream Effects Modulated by EGFR and VEGFR Signaling in Colorectal Cancer34 Bevacizumab

Pharmacogenetic Signatures to Predict Recurrence

VEGF

Cetuximab EGF

VEGF

At the 43rd Annual Meeting of the American Society of Clinical Oncology (ASCO), held June 1-5, 2007 in Chicago, IL, data were presented that demonstrated P P P P P P P P with high statistical significance VEGFR VEGFR EGFR EGFR the importance of a number of Nucleus molecular markers in predicting recurrence in stage II/III disease. Activation of Signaling Cascades The results of this study identified polymorphisms in several angioSTAT Ras PI3K MAPK Cell Cycle Survival Progression genic genes, vascular endothelial DNA repair and inhibition of apoptosis growth factor (VEGF) receptor (VEGFR)–2 AC repeat and EGFR AC repeat, adrenomedullin, and Proliferation Metastasis Angiogenesis Differentiation interleukin (IL)–1β that were sigMigration and Blood vessel invasion recruitment nificantly associated with risk of Growth factors recurrence in patients with stage II colon cancer. These data represent the foundation of a pharmacogenomic signature, implicating genes involved in the tumor angiogenesis Activation of the VEGR and/or the EGFR axis results in activation of downstream signaling cascades resulting in enhanced tumor processes. pathway as critical factors in idenAdapted with permission from Willson et al. Gastrointest Cancer Res 2007. In press. tifying patients with a high risk of recurrence.31 An additional group used microarray analysis in an burden incurred in a therapeutic strategy that incorporates a biologic agent and will represent a major advancement in indiattempt to identify a signature of recurrence for stage II disease. vidualized treatment of CRC. The authors identified a 23-gene signature profile in a small number of patients with stage II disease linked with a 13-fold higher Epidermal Growth Factor Receptor risk of recurrence; this was the first study incorporating a global The EGFR (ErbB-1; HER1) is a transmembrane receptor with genomic approach for predicting the prognosis of stage II colon an intracellular tyrosine kinase (TK) domain. Major activating 32 cancer. Identification of additional markers of recurrence in ligands include EGF and transforming growth factor (TGF)–α. combination with large, prospective, validation trials will assist cliThe EGFR pathway is activated after homodimerization of 2 nicians in identifying the small subset of patients who are likely to EGFRs or heterodimerization with additional members of the have disease recurrence and administer an effective individualized ErbB family. Activation via phosphorylation of the TK initiates therapy. Equally as important, this process will identify patients several intracellular signaling pathways, including phosphatiwho are at low risk and require no postoperative chemotherapy. dylinositol 3-kinase (PI3K)/AKT, mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcripThe Biologic Agents: Defining tion 3 (STAT3), which promote downstream influence on cell Their Role cycle regulation, proliferation, migration, angiogenesis, and inhiThe introduction of the biologic agents that target recepbition of apoptosis (Figure 2).33,34 Dysregulation of the EGFR tor-mediated tumor processes have been proven to provide meaningful clinical benefit in CRC treatment. However, our pathway promotes growth and progression of many cancers, knowledge of the precise mechanisms of action, resistance, and including CRC, and is associated with aggressive disease and poor optimal scheduling and administration of these agents is still prognosis.35 It is reported that 50%-70% of CRCs exhibit EGFR in its infancy. A greater understanding of these issues will assist expression, but as yet there is no conclusive evidence regarding in identifying the population of patients who will benefit from the role of EGFR as a prognostic marker.36 A recent retrospecthese agents. This will not only ensure efficacy and increased tive analysis investigated EGFR as a prognostic indicator in stage response rates (RRs), but will justify the additional financial II CRC and concluded that it was an independent predictor of Clinical Colorectal Cancer Supplement December 2007 • S31

Individualized Treatment Strategies in Colorectal Cancer recurrence (P = .05) and that elevated expression was associated with poor survival (P = .01).37 Clearly, more research is needed to define the precise prognostic role of EGFR in CRC.

Targeting Epidermal Growth Factor Receptor Monoclonal antibodies (MoAbs) represent an important clinical option in the inhibition of EGFR by binding to the extracellular domain and inhibiting activation of intracellular signaling cascades. The most promising MoAb targeting EGFR is cetuximab. Recently, the FDA approved another anti-EGFR MoAb, panitumumab, for the treatment of mCRC. Clinical trials are already reporting promising results for panitumumab in the treatment of advanced CRC.38 The antitumor activity of cetuximab has been attributed to several distinct mechanisms, including direct inhibition of TK activity and blockade of the EGFR signaling pathways, which results in pro-apoptotic, antiangiogenic, and anti-invasive effects. Additional mechanisms of action include recruitment of the host immune system through antibody-dependent cellular cytotoxicity.39 A series of phase II/III clinical trials evaluated the clinical efficacy of cetuximab and demonstrated that it has activity when used in combination with FOLFOX (5-FU/LV/oxaliplatin), FOLFIRI (5-FU/LV/irinotecan), and also as monotherapy in advanced CRC. Cetuximab has been extensively introduced into clinical regimens and its clinical benefit is well documented; however, the patients who will derive clinical benefit from this agent need to be identified in order to direct this therapy appropriately. Within the past few years, several studies have identified a number of markers that can discriminate between patients who are likely to respond to cetuximab and those who will not. A recent study identified mutations in K-ras as a significant predictor of response to cetuximab therapy (P = .0003) and OS (P = .016) in a group of 30 patients with mCRC.40 K-ras is a tumor suppressor gene that is mutated in many different types of cancers and in approximately 50% of cases of CRC. It plays a key role in the transduction of signals from membrane-bound receptors via multiple downstream effector pathways. K-ras mutation results in constituitive activation of these pathways and, consequently, unregulated proliferation and impaired differentiation. More recently, further data was present indicating that K-ras mutations preclude tumor shrinkage in CRC treated with cetuximab.41 At the 43rd Annual Meeting of the ASCO, data were presented indicating that EGFR gene status, K-ras mutation, and loss of phosphatase and tensin homologue on chromosome 10 (PTEN) were all predictive of resistance to cetuximab.42 Further compelling evidence was recently published demonstrating with high statistical significance that patients with tumors that express high levels of the EGFR ligands epiregulin and amphiregulin are more likely to have disease control with cetuximab.43 Additionally, patients whose tumors do not possess K-ras mutations have a significantly higher disease control rate than patients with K-ras mutations. The efficacy of panitumumab alone in CRC was recently shown to be confined to patients with tumors lacking K-ras mutations; interestingly, 43% of patients displayed such mutations in this study.44 Ongoing research will determine whether the high asso-

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ciation of K-ras status and response extends to panitumumab in combination with additional agents. The accumulating research strongly suggests that the K-ras mutation, which can render the EGFR pathway constituitively active irrespective of blocking EGFR with an antibody, should be considered as a selection marker in patients with CRC who are candidates for EGFR-targeted therapies including the MoAbs panitumumab and cetuximab.

Vascular Endothelial Growth Factor Vascular endothelial growth factor–A is a secreted ligand with specific receptors that are primarily expressed by angioblasts and endothelial cells.45 The VEGF pathway plays a major role in tumor growth and angiogenesis. The generation of new life-bringing blood vessels carrying oxygen, nutrients, growth factors, and hormones is an important factor essential for proliferation of solid tumors. Activation of the VEGF/VEGF receptor (VEGFR) axis triggers multiple intracellular signaling pathways, such as PI3K, MAPK, and the focal adhesion pathway. Activation of this signaling cascade results in increased vascular endothelial cell proliferation, enhanced permeability, invasion, migration, and survival (Figure 2). Overexpression of VEGF and increased circulating VEGF have been associated with tumor progression and poor prognosis in several gastrointestinal (GI) tumor types, including CRC.46-48 Many solid tumors are also reported to secrete elevated levels of VEGF to stimulate vascularization and initiate metastasis.49 Vascular endothelial growth factor–positive tumors were also associated with a 50% chance of disease recurrence compared with 11% in VEGF-negative tumors.50 Vascular endothelial growth factor–positive tumors also exhibit a 4.5-fold higher rate of recurrence in resected stage III CRC than VEGF-negative tumors.51 As mentioned previously, genes involved in tumor angiogenesis were also reported to predict recurrence in stage II/III disease.

Targeting Vascular Endothelial Growth Factor Because of its critical role in promoting tumor growth, angiogenesis, and metastasis, VEGF has become a target for therapeutic intervention. Bevacizumab is a recombinant humanized MoAb targeted against VEGF and was the first antiangiogenic drug approved by the FDA for treatment in mCRC in combination with 5-FU–based therapy, providing a statistically significant improvement in OS and progression-free survival when compared with 5-FU–based therapy alone in mCRC.52,53 Although the efficacy of antiangiogenic therapy is now well documented, the identification of biomarkers to determine patients who will benefit from these agents is of great interest. Preclinical data suggest that dysregulation and mutation in the Ras/Raf/MEK/ERK54 and p53 pathways55 might modulate the efficacy of anti-VEGF therapies, including bevacizumab. A retrospective study by Ince et al analyzed the expression and mutation status of K-ras, B-Raf, and p53 to predict which patients were more likely to respond to bevacizumab.56 The authors reported no statistical significance between mutations of K-ras, B-Raf, or p53 (mutation and overexpression) and the increase in median survival associated with the

Peter M. Wilson et al addition of bevacizumab to IFL Figure 3 Targeting Stem Cells in Cancer Treatment (irinotecan/5-FU/LV) therapy, but did note that patients with no mutations in K-ras or B-Raf Tumorigenic stem cell had statistically significant better OS irrespective of treatment received. An additional study analyzed host factors involved in Solid tumor or the negative regulation of angiometastatic site genesis as potential predictors of Chemotherapy-induced response to antiangiogenic theraapoptosis of high-proliferating pies, including microvessel denbulk of tumor cells sity (MVD), the antiangiogenic Drug-induced stem cell thrombospondin (THBS) family differentiation into highly proliferative intermediate stage member THBS-2, and VEGF.57 The results of the study indicated that patients with mCRC will benefit from the addition of bevacizumab regardless of VEGF, THBS-2 expression, and MVD. With this distinct lack of predicStem cell–fueled tumor recurrence tive molecules for bevacizumab, Standardized chemotherapies effectively current and future studies are eliminate actively proliferating differentiated cells analyzing biomarkers involved in the activation of VEGF signaling (Src kinases), downstream A simplified schematic demonstrating proposed evasion of tumor stem cells from standard chemotherapies used in the treatment of molecules such as carcinoembryCRC. Surviving stem cells possess the ability to regenerate and refuel tumor growth even after successful chemotherapy and can remain after surgical resection, leading to future disease recurrence. Studies suggest that these stem cells can be induced to onic antigen–related cell adhedifferentiate into more actively proliferating cells by treatment with existing and novel therapeutic “prodifferentiation” agents such as sion molecule and neuropilin-1 histone deacetylase inhibitors. These more actively dividing cells will be highly susceptible to standard chemotherapies and DNAdamaging agents. (a novel VEGFR). While VEGF is the best characterized of the breast, prostate, brain, pancreatic, and head and neck cancer. angiogenic-promoting molecules, clear evidence exists that In January 2007, O’Brien et al presented compelling evidence VEGF-independent angiogenesis is mediated through addifor the existence of colon cancer stem cells and the hierarchial tional pathways that include IL-8, IL-1β, and neuropilin-1.58 organization of human colon cancer.60 These stem-like cells Characterizing the VEGF-mediated angiogenesis and determinwere identified using the CD133 marker and were able to maining the influence of additional angiogenic pathways will be tain themselves as well as differentiate and re-establish tumor imperative in the clinical development and pursuance of the heterogeneity upon serial transplantation in xenograft models. antiangiogenic therapies. Ricci-Vitiani et al also presented data demonstrating that CRC The Invisible Enemy: The Cancer is potentially created and propagated by a small number of Stem Cell Hypothesis undifferentiated tumorigenic CD133+ cells.61 Future efforts The rejuvenation of the cancer stem cell hypothesis is must be directed toward the development of functional assays to another exciting area of research that must be pursued. There identify possible stem cell populations in solid tumors, including is a growing body of evidence suggesting that cancers develop CRC. Analysis of these cells could be the most important progfrom a small subpopulation of cells with self-renewal propernostic marker in the critical decision of whether or not to initiate ties analogous to organ stem cells that acquire epigenetic and chemotherapy and choosing the appropriate treatment strategy. genetic changes required for tumorigenicity or might represent Furthermore, our knowledge of the pathways involved in proliferative progenitors that acquire self-renewal capabilities.59 the regulation of self-renewal and cell fate in these systems is If these cells retain the hallmarks of tissue stem cells in their steadily improving. Molecular pathways such as Wnt, Notch, infrequent replication, they might represent a subpopulation and Hedgehog are well known to regulate self-renewal of of cells intrinsically resistant to conventional therapies. Future normal stem cells; however, tumor suppressor genes, such as strategies would therefore require objective targeting of the PTEN and p53, have also been implicated in the regulation of cancer stem cell self-renewal. In cancer stem-like cells, these minority stem cell population, which fuels tumor growth and pathways are believed to be dysregulated, leading to the loss of regeneration in combination with targeting the bulk of highly tightly-controlled self-renewal, which results in the generation proliferating tumor cells (Figure 3). These cancer stem-like cells of tumors that are inherently resistant to conventional theraare no longer the invisible enemy, as they have been identified in Clinical Colorectal Cancer Supplement December 2007 • S33

Individualized Treatment Strategies in Colorectal Cancer pies. This therefore poses an immediate question: are there any therapies currently used in the global treatment of cancer that are targeting the stem-like cell populations? The mechanisms in place governing stem cell fate are under tight regulatory control, but the potential to alter these regulatory pathways is a real possibility. Recent studies have shown that several currently used drugs, such as colony-stimulating factors, statins, angiotensin-II receptor antagonists/angiontensin-converting enzymes (ACE)inhibitors, erythropoietin, nitric oxide donors, and glitazones, have modulatory activity on stem cell functions. Furthermore, a series of new pharmacologic agents, such as the chemokine receptor antagonist AMD3100,62 glycogen synthase kinase-3 inhibitors,63 and histone deacetylase inhibitors (HDACIs), that modulate the growth, differentiation, and mobilization of stem cells have recently been discovered and are currently under evaluation in in vivo experimental models and preliminary clinical trials. A recent study demonstrated that antiangiogenic therapies combined with cytotoxic agents are effective at reducing the stem-like cell fraction in a population of glioma cells, giving rise to hopes that currently approved agents might be successful in targeting the stem-like cell population.64 A recent workshop convened by the American Association for Cancer Research and attended by a wide variety of cancer biology experts highlighted the importance of pursuing this promising field, and a task force is currently being formed to expedite progress in the cancer stem cell field. Progress in this field offers a real possibility of identifying novel targets that could allow therapies to overcome drug resistance, improve therapeutic efficacy, and potentially make cancer treatment curative while minimizing adverse toxicities.65

The Future of Predictive Medicine in Colorectal Cancer Until recently, the analysis of single genes in predicting response to therapy and disease prognosis has, on occasion, proven insightful but ultimately lacked resolution and sensitivity. Regardless of recent advances in pharmacogenomic technologies, it is becoming increasingly apparent that response to therapy and disease progression are largely driven by complex, multifactorial pathways, and that analysis of a single marker is unlikely to accurately predict response or prognosis with sufficient resolution and consistency to become an integral part of clinical practice. The 2006 ASCO update of recommendations for the use of tumor markers in GI cancer recently reviewed the current literature investigating the use of tumor markers in screening, treatment, and surveillance of CRC.66 The conclusions of this report clearly stated that there is insufficient evidence to recommend the routine use of p53, TS, dihydropyrimidine dehydrogenase, thymidylate phosphorylase, K-ras, 18q loss of heterozygosity, or MSI status in CRC treatment, primarily because of conflicting data in the literature, inconsistencies in methodology for detection/measurement of genes and enzyme activity, and variation in data analysis, including statistical analyses used as well as their interpretation. In fact, with much conflict in the published literature, the distinct lack of validated markers for CRC is startling. In an effort to address this, the

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emergence and increasing progress in gene expression profiling, or microarray analysis, has already had a significant impact in the classification and prognosis of multiple forms of cancer. Expression profiling has already been used in colon cancer, and the use of defined cassettes of genes can now discriminate with reasonably high resolution among tumors of different stage and prognosis.67 Furthermore, efforts are now under way to identify panels of markers that can accurately predict response to chemotherapy, introducing the possibility of delivering the correct therapy with maximal antitumor efficacy in the first-line setting and enabling more patients with advanced and metastatic disease to undergo complete surgical resection with curative intent.68 In addition, efforts to build “prognosis predictors” with the ability to accurately classify patients with intermediatestage disease are holding promise, allowing for more appropriate administration of adjuvant therapy.69 The reality remains that the use of molecular profiling in CRC to predict clinical response, progression, and toxicity is still in its infancy. To move forward, there must be more coordinated effort in the identification and validation of these markers before analysis of genetic information will become a routine part of clinical CRC treatment. Retrospective analyses have clearly demonstrated the proof of principle in such approaches. However, the design of new prospective trials must encompass a more comprehensive and disciplined unilateral approach that clearly defines protocols, primary endpoints, and statistical analyses. The importance of the laboratory workbench is imperative in determining the functional significance of the many mutations and polymorphic variants that exist within the patient population. Such functional analyses will assist in unraveling the complex and multifactorial mechanisms of drug metabolism and action. The continuing evolution of high-throughput technologies, such as microarray gene profiling, proteomic profiling, and the newly developing field of metabolomics, will improve the resolution and sensitivity with which we can detect such markers and will be invaluable in the mechanistic analysis of the promising new classes of drugs emerging in CRC, which include the insulin-like growth factor inhibitors, the mammalian target of rapamycin inhibitors (mTOR), and the HDACIs. Together, this systems biology approach will give clinicians the ability to tailor specific therapies to the molecular profile of patients and their diseases and will revolutionize the treatment of CRC with improved RRs and quality of life.

References 1. American Cancer Society [Web site]. Estimated new cancer cases and deaths by sex for all sites, US, 2007. Available at: http://www.cancer. org/downloads/STT/CAFF2007leadingsites.pdf. Accessed: November 20, 2006. 2. Douillard JY, Cunningham D, Roth AD, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet 2000; 355:1041-7. 3. Giacchetti S, Perpoint B, Zidani R, et al. Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracilleucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol 2000; 18:136-47. 4. Tournigand C, André T, Achille E, et al. FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: a

Peter M. Wilson et al randomized GERCOR study. J Clin Oncol 2004; 22:229-37. 5. Hoff PM, Ansari R, Batist G, et al. Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study. J Clin Oncol 2001; 19:2282-92. 6. Van Cutsem E, Twelves C, Cassidy J, et al. Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study. J Clin Oncol 2001; 19:4097-106. 7. Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004; 351:337-45. 8. McLeod HL, Yu J. Cancer pharmacogenomics: SNPs, chips, and the individual patient. Cancer Invest 2003; 21:630-40. 9. Longley DB, Allen WL, Johnston PG. Drug resistance, predictive markers and pharmacogenomics in colorectal cancer. Biochim Biophys Acta 2006; 1766:184-96. 10. Greene FL, Stewart AK, Norton HJ. A new TNM staging strategy for node-positive (stage III) colon cancer: an analysis of 50,042 patients. Ann Surg 2002; 236:416-21; discussion 421. 11. Meyerhardt JA, Mayer RJ. Systemic therapy for colorectal cancer. N Engl J Med 2005; 352:476-87. 12. O’Connell JB, Maggard MA, Ko CY. Colon cancer survival rates with the new American Joint Committee on Cancer sixth edition staging. J Natl Cancer Inst 2004; 96:1420-5. 13. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003; 3:330-8. 14. Twelves C, Wong A, Nowacki MP, et al. Capecitabine as adjuvant treatment for stage III colon cancer. N Engl J Med 2005; 352:2696-704. 15. André T, Boni C, Mounedji-Boudiaf L, et al. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 2004; 350:2343-51. 16. Saltz LB, Niedzwiecki D, Hollis D, et al. Irinotecan fluorouracil plus leucovorin is not superior to fluorouracil plus leucovorin alone as adjuvant treatment for stage III colon cancer: results of CALGB 89803. J Clin Oncol 2007; 25:3456-61. 17. Gray RG, Barnwell J, Hills R. QUASAR: a randomized study of adjuvant chemotherapy vs. observation in 3238 colorectal cancer patients. Proc Am Soc Clin Oncol 2004; 22(14 suppl):246 (Abstract 3501). 18. Gill S, Loprinzi CL, Sargent DJ, et al. Pooled analysis of fluorouracilbased adjuvant therapy for stage II and III colon cancer: who benefits and by how much? J Clin Oncol 2004; 22:1797-806. 19. Benson AB III, Schrag D, Somerfield MR, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol 2004; 22:3408-19. 20. Piñol V, Castells A, Andreu M, et al. Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer. JAMA 2005; 293:1986-94. 21. Gryfe R, Gallinger S. Microsatellite instability, mismatch repair deficiency, and colorectal cancer. Surgery 2001; 130:17-20. 22. Lim SB, Jeong SY, Lee MR, et al. Prognostic significance of microsatellite instability in sporadic colorectal cancer. Int J Colorectal Dis 2004; 19:533-7. 23. Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol 2005; 23:60918. 24. Watanabe T, Wu TT, Catalano PJ, et al. Molecular predictors of survival after adjuvant chemotherapy for colon cancer. N Engl J Med 2001; 344:1196-206. 25. Fallik D, Borrini F, Boige V, et al. Microsatellite instability is a predictive factor of the tumor response to irinotecan in patients with advanced colorectal cancer. Cancer Res 2003; 63:5738-44. 26. Kern SE, Fearon ER, Tersmette KW, et al. Clinical and pathological associations with allelic loss in colorectal carcinoma [corrected]. JAMA 1989; 261:3099-103. 27. Khine K, Smith DR, Goh HS. High frequency of allelic deletion on chromosome 17p in advanced colorectal cancer. Cancer 1994; 73:28-35. 28. Lanza G, Matteuzzi M, Gafá R, et al. Chromosome 18q allelic loss and prognosis in stage II and III colon cancer. Int J Cancer 1998; 79:390-5. 29. Popat S, Houlston RS. A systematic review and meta-analysis of the relationship between chromosome 18q genotype, DCC status and colorectal cancer prognosis. Eur J Cancer 2005; 41:2060-70. 30. ClinicalTrials.gov [Web site]. Oxaliplatin, leucovorin, and fluorouracil

with or without bevacizumab in treating patients who have undergone surgery for stage II colon cancer. Available at: http://www.clinicaltrials. gov/ct/gui/show/NCT00217737. Accessed: November 20, 2006. 31. Lurje G, Schultheis AM, Hendifar AE, et al. VEGF and VEGF receptor-2 (VEGFR2) gene polymorphisms predict tumor recurrence in stage II and III colon cancer. J Clin Oncol 2007; 25(18 suppl):164s (Abstract 4004). 32. Wang Y, Jatkoe T, Zhang Y, et al. Gene expression profiles and molecular markers to predict recurrence of Dukes’ B colon cancer. J Clin Oncol 2004; 22:1564-71. 33. Herbst RS, Langer CJ. Epidermal growth factor receptors as a target for cancer treatment: the emerging role of IMC-C225 in the treatment of lung and head and neck cancers. Semin Oncol 2002; 29(1 suppl 4):27-36. 34. Wilson PM, Ladner RJ, Lenz HJ. Predictive and prognostic makers in colorectal cancer. Gastrointest Cancer Res 2007. In press. 35. Ciardiello F. Epidermal growth factor receptor tyrosine kinase inhibitors as anticancer agents. Drugs 2000; 60(suppl 1):25-32; discussion 41-2. 36. McKay JA, Murray LJ, Curran S, et al. Evaluation of the epidermal growth factor receptor (EGFR) in colorectal tumours and lymph node metastases. Eur J Cancer 2002; 38:2258-64. 37. Resnick MB, Routhier J, Konkin T, et al. Epidermal growth factor receptor, c-MET, beta-catenin, and p53 expression as prognostic indicators in stage II colon cancer: a tissue microarray study. Clin Cancer Res 2004; 10:3069-75. 38. Gibson TB, Ranganathan A, Grothey A. Randomized phase III trial results of panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, in metastatic colorectal cancer. Clin Colorectal Cancer 2006; 6:29-31. 39. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000; 19:6550-65. 40. Lièvre A, Bachet JB, Le Corre D, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 2006; 66:3992-5. 41. De Roock W, De Schutter J, De Hertogh G, et al. KRAS mutations preclude tumor shrinkage of colorectal cancers treated with cetuximab. J Clin Oncol 2007; 25(18 suppl):196s (Abstract 4132). 42. Romagnani E, Martin V, Ghisletta M, et al. EGFR gene status, K-Ras mutation and PTEN expression predict cetuximab response in metastatic colorectal cancer (mCRC). Presented at: the 2007 Gastrointestinal Cancers Symposium; January 19-21, 2007; Orlando, FL. Abstract 427. 43. Khambata-Ford S, Garrett CR, Meropol NJ, et al. Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol 2007; 25:3230-7. 44. Amado RG, Wolf M, Freeman D, et al. KRAS Mutation predicts lack of response to EGFR inhibitors. Presented at: 14th European Cancer Conference; September 27, 2007; Barcelona, Spain. Abstracts 7LB and 3014. 45. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997; 18:4-25. 46. Cascinu S, Graziano F, Catalano V, et al. An analysis of p53, BAX and vascular endothelial growth factor expression in node-positive rectal cancer. Relationships with tumour recurrence and event-free survival of patients treated with adjuvant chemoradiation. Br J Cancer 2002; 86:744-9. 47. Lee JC, Chow NH, Wang ST, et al. Prognostic value of vascular endothelial growth factor expression in colorectal cancer patients. Eur J Cancer 2000; 36:748-53. 48. Nakayama Y, Sako T, Shibao K, et al. Prognostic value of plasma vascular endothelial growth factor in patients with colorectal cancer. Anticancer Res 2002; 22:2437-42. 49. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993; 362:841-4. 50. Cascinu S, Staccioli MP, Gasparini G, et al. Expression of vascular endothelial growth factor can predict event-free survival in stage II colon cancer. Clin Cancer Res 2000; 6:2803-7. 51. Ishigami SI, Arii S, Furutani M, et al. Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer. Br J Cancer 1998; 78:1379-84. 52. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350:2335-42. 53. Mass RD, Fyfe G, Hambleton J, et al. Bevacizumab in combination

Clinical Colorectal Cancer Supplement December 2007 • S35

Individualized Treatment Strategies in Colorectal Cancer

54.

55. 56.

57.

58.

59. 60.

with 5-FU/leucovorin improves survival in patients with metastatic colorectal cancer: a combined analysis. Proc Am Soc Clin Oncol 2004; 23:274 (Abstract 3616). Watnick RS, Cheng YN, Rangarajan A, et al. Ras modulates Myc activity to repress thrombospondin-1 expression and increase tumor angiogenesis. Cancer Cell 2003; 3:219-31. Yu JL, Rak JW, Coomber BL, et al. Effect of p53 status on tumor response to antiangiogenic therapy. Science 2002; 295:1526-8. Ince WL, Jubb AM, Holden SN, et al. Association of k-ras, b-raf, and p53 status with the treatment effect of bevacizumab. J Natl Cancer Inst 2005; 97:981-9. Jubb AM, Hurwitz HI, Bai W, et al. Impact of vascular endothelial growth factor-A expression, thrombospondin-2 expression, and microvessel density on the treatment effect of bevacizumab in metastatic colorectal cancer. J Clin Oncol 2006; 24:217-27. Huang S, Mills L, Mian B, et al. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma. Am J Pathol 2002; 161:125-34. Clarke MF, Becker MW. Stem cells: the real culprits in cancer? Sci Am 2006; 295:52-9. O’Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007; 445:106-10.

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61. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature 2007; 445:111-5. 62. Cashen AF, Nervi B, DiPersio J. AMD3100: CXCR4 antagonist and rapid stem cell-mobilizing agent. Future Oncol 2007; 3:19-27. 63. Meijer L, Flajolet M, Greengard P. Pharmacological inhibitors of glycogen synthase kinase 3. Trends Pharmacol Sci 2004; 25:471-80. 64. Bao S, Wu Q, Sathornsumetee S, et al. Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res 2006; 66:7843-8. 65. Clarke MF, Dick JE, Dirks PB, et al. Cancer stem cells–perspectives on current status and future directions: AACR Workshop on Cancer Stem Cells. Cancer Res 2006; 66:9339-44. 66. Locker GY, Hamilton S, Harris J, et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J Clin Oncol 2006; 24:5313-27. 67. Barrier A, Boelle PY, Roser F, et al. Stage II colon cancer prognosis prediction by tumor gene expression profiling. J Clin Oncol 2006; 24:4685-91. 68. Boyer J, Allen WL, McLean EG, et al. Pharmacogenomic identification of novel determinants of response to chemotherapy in colon cancer. Cancer Res 2006; 66:2765-77. 69. Eschrich S, Yang I, Bloom G, et al. Molecular staging for survival prediction of colorectal cancer patients. J Clin Oncol 2005; 23:3526-35.