Reanalysis of Cancer Drugs

Most cancer chemotherapy protocols in current use have been established empirically, and combination therapies have generally been designed based on single-agent activity and nonoverlapping toxicities. However, advances in our understanding of the molecular pathophysiology of human cancers indicate that certain molecular features of neoplastic cells can be associated with their sensitivity to chemotherapeutic agents and therefore can be used as the basis for rational drug selection and therapy design. In addition, the ability to discover markers that predict responsiveness to therapy is possible today as never before. Specimens (fixed tissues, immortalized cells, frozen specimens, body fluids, and so forth) obtained 5–10 years before the clinical outcome is known represent a major reservoir of information. Exploiting this resource can shorten the time necessary for retrospective trials and can then be used empirically when prospective trials are mounted. Furthermore, prognostic and predictive measures can be determined with microarrays (1).Identifying potential responders is critical to successful drug treatment and is potentially a major use of biomarkers. Such biomarker-based strategies can be key to the development and approval of new therapies. For instance, Koo et al. (2, 3) have observed a striking correlation between activating ras mutations in human tumor cells and enhanced sensitivity to deoxycytidine analogs as well as to topoisomerase (topo) II inhibitors. Compared with tumor cells with wild-type ras alleles, human tumor cell lines harboring or transformed with activated ras oncogenes display significantly enhanced cytotoxic sensitivity to deoxycytidine analogs, including 1-β-d-arabinofuranosylcytosine (cytarabine) and gemcitabine. Activated ras oncogenes similarly sensitize human tumor cells to topo II inhibitors and more prominently sensitize human tumor cells to epipodophyllotoxin-derived inhibitors such as etoposide and teniposide. Furthermore, these findings are supported by retrospective studies in patients with acute myeloid leukemia (AML) showing that the presence of ras oncogenes in leukemic cells is associated with increased complete remission rate, longer complete remission duration, and improved overall survival of the patients in response to treatment with cytarabine or the combination of cytarabine and a topo II inhibitor (4, 5). These results indicate that the ras oncogene plays a determinant role in rendering tumor cells sensitive to deoxycytidine analogs and topo II inhibitors and may provide a rationale for the design of potentially synergistic combination therapy (deoxycytidine analog + topo II inhibitor) for other cancers bearing frequent ras mutations. In Wilms’ tumor, the most common pediatric cancer of the urinary tract, previous studies have identified topo IIα overexpression; the highly effective chemotherapeutic regimens are mainly constituted with topo II inhibitors (6, 7, 8). These results may provide a molecular basis for the success of topo II inhibitor-based treatment of Wilms’ tumors and indicate that topo II overexpression is a biomarker for the choice of topo II inhibitor treatment.Strategies for selecting those most likely to respond to therapy have already proven beneficial to patients. For example, only patients with breast cancer expressing Her-2/neu respond to trastuzumab (Herceptin). Without identification of the susceptible subpopulation, Herceptin would not have been made available for treatment of this poor-prognosis breast cancer (9). The findings of Staudt (10) that gene expression profiles can be used prospectively to define lymphomas that respond to standard chemotherapy (Fig. 1) reaffirm that the thesis proposed herein has promise and that other examples of this utility will derive from the proposed analyses.This article proposes to explore the possibilities of analyzing (reanalysis) chemotherapeutic drugs and protocols in practice through conventional and newer molecular and/or imaging technologies to discover surrogate end point or other biomarkers predicting and measuring effectiveness of the therapies. These analyses should first be performed through clinical trials using approved drugs and established protocols. The studies can be prospectively designed or developed from retrospective studies, where appropriate patient material is available. The purpose of these studies will be severalfold: (a) first, to identify potential surrogate end point and other biomarkers markers that will be informative of patient outcome and response; and (b) second, to identify when a specific protocol should be used (e.g., as indicated by biomarkers and/or gene expression profiles). As candidate biomarker(s) are identified, they should be verified or validated against currently used standards or biomarkers. Efficacy in diagnosis, prognosis, or molecular classification for treatment must be demonstrated.Whereas the thrust of this paper is on evaluation of responses to approved cytotoxic therapies, other types of drugs and protocols should also be evaluated. It would be particularly beneficial to validate biomarkers of response to the newer molecular therapies that do not produce dramatic tumor shrinkage. Interestingly, modulation of expression of the molecular target might not always be a good predictor of response. For example, both epidermal growth factor receptor (EGFR)- and Her-2-overexpressing tumors are sensitive to the EGFR-selective tyrosine kinase inhibitor gefitinib (Iressa), and sensitivity to this agent is correlated with its ability to down-regulate Akt. However, EGFR-overexpressing MDA-468 cells, which lack PTEN function, are resistant to Iressa, and Iressa is unable to down-regulate Akt activity in these cells. This resistance to Iressa is attributable to EGFR-independent constitutive Akt activation caused by PTEN loss. Reconstitution of PTEN function through tetracycline-inducible expression restores Iressa sensitivity and reestablishes EGFR-stimulated Akt signaling (11). Gene or protein expression patterns such as these could be identified in other settings and with other drugs. Just as for the analysis of cytotoxic drugs, patients might be evaluated after a short treatment period with molecularly targeted agents; those who did not show the expression pattern could be spared further, possibly futile treatment.In the following sections, techniques and material sources for assessing and developing new biomarkers are delineated. Sample clinical settings for the conduct of research to identify and validate prognostic and predictive (drug effect and clinical outcome) markers are discussed. A separate section on AML provides data from completed and ongoing clinical trials as a prototype. The article concludes with specific recommendations for fostering research in this area. These include development of a collaborative initiative to facilitate access to both novel technologies as they become available (e.g., genomic, proteomic, functional imaging technologies, and so forth) and clinical trial samples (e.g., serum, biopsy tissue, DNA, imaging data, and so forth). The initiative could leverage existing National Cancer Institute (NCI) technology, clinical networks, and translational research programs. For example, the NCI’s Specialized Programs of Research Excellence program has fostered multidisciplinary teams to address biomarker identification and validation.11 A bioinformatics system to aid in the collection and sharing of key data elements (e.g., outcome, demographic, and so forth) could be implemented in accord with the NCI’s Cancer Bioinformatics Informatics Grid project.12 Patient and tissue access might best be achieved via established collaborative physician networks (e.g., Oncology Cooperative Groups). Finally, the initiative could also draw from other successful local, national, and international collaborations that support translational research and biomarker development, such as MdBIO,13 the NIH roadmap,14 the European Organization for the Treatment of Cancer,15 and the United Kingdom’s National Cancer Research Institute.16 Together, these efforts will promote the discovery of surrogate end point or other biomarkers to predict and measure effectiveness of therapies.Technologies that could be used to develop biological markers to predict drug response or clinical outcomes for a cancer chemotherapeutic agent include the following: immunologically based assays for a given protein, peptide, or glycoprotein; PCR assays for quantitation of the expression of a specific gene product; genomic analysis; gene expression profiling to characterize the transcription level of multiple genes; proteomics; DNA methylation patterns; detection of circulating cancer cells; and functional imaging. Each of these is described below. Table 1 lists examples of data resources on potential biological markers that may be identified with these technologies.Conventional methods for detection of tumor markers in body fluids involving isolation of the marker (usually a protein or glycoprotein), developing a specific antibody to the marker, and developing an immunologically based assay using the antibody, such as an ELISA, will continue to be useful in evaluating biomarkers. ELISA is the standard assay for detection of well-known serum markers such as carcinoembryonic antigen, human chorionic gonadotrophin, α-fetoprotein, CA-125, and prostate-specific antigen (12).More recently, the development of the PCR technique has allowed determination of transcription levels of the above serum markers as well as of additional markers such as oncogenes in circulating cancer cells, serum, urine, and fecal samples (12, 13, 14).Comparative genomic hybridization and spectral karyotyping are two techniques used to measure tumor-associated chromosomal alterations. In comparative genomic hybridization, tumor DNA and normal reference DNA with different fluorescent labels are hybridized simultaneously to normal chromosome spreads. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorophores along the target chromosomes (15). Spectral karyotyping allows visualization of all 23 pairs of human chromosomes by labeling each pair with a different fluorescent color (16).The Human Genome Project has revealed detailed information the of human This has allowed the development of DNA microarrays that the detection of of human on a as as a different of target DNA are (a) from a for a specific gene and (b) highly specific of the target in this can be used to for or may be to prognosis, or drug response. an of can be used to tumor tumor and/or tumor tumor a of can be used to For example, a a of in can be used to characterize types of lymphomas that in and predict patient survival a gene expression has been identified that survival of patients with breast cancer can be as the of the protein expression of a tissue or specific cell to define a of The in to provide an to the of the is used to define the complete of that is by a tissue, or In the of the is used to a protein expression that the of a cell that from normal cells of the tissue in which the tumor techniques are available to protein expression These include the technique of techniques to based on and so and analysis (e.g., and These techniques data that detailed bioinformatics or more may be in a all of the that can in the protein of human of the that a cancer to be an the determination of protein expression patterns based on pattern of serum has been used to the specific in each of the may not be known It is that based on of protein patterns for other cancers can also be This could provide a to use for cancer diagnosis, prognosis, and drug methylation of DNA in a cell provides an of The of DNA methylation generally on sequences, the are in of of all and methylation of these is associated with gene methylation of in cancer tissue has been associated with of tumor DNA can be in body fluids such as serum, or breast from patients with or breast cells are not in and their detection the presence of a cells can be from against a of cells, they specific cell not on cell in circulating cells have been to with and drug response in patients with or breast their use as biomarkers for drug response and and circulating cell levels in may as a biomarker for tumor and their detection and quantitation may the of therapy cell levels have been to significantly in the of and breast cancer to levels in patients who complete remission after chemotherapy to cells, circulating cells from also potential as a surrogate marker for tumor prognostic and has not been in both tumor and cancer patients that is associated with increased of circulating technologies use to most used in clinical settings include and and In the in imaging methods revealed or treatment as or conventional detection and the use of imaging agents have on a and the evaluation of in tissue For drug development and the of imaging other biomarkers as those tissue and are the visualization of and of tumor the ability to changes and the of the technologies development using and may particularly useful in drug recently, imaging of specific drug and drug target has as a of functional imaging. imaging can be used to the of an drug (e.g., the and target of a or to the target (e.g., detection of receptor expression and modulation of particularly are used for such For example, drugs can be with the or and tissue of these drugs can be by imaging. technique is evaluation of target of a by molecular imaging techniques are particularly for drug and when used for this may be to responders and to a given and can also a key role in measuring response to The functional of key biological of neoplastic is for example, and responsiveness imaging and (e.g., cell and (e.g., and and resistance to chemotherapy in imaging (e.g., or have promise for the utility of imaging in measuring the response to examples of imaging to cancer drug development are and already is well established for use in the and of and as well as cancers of the and and The imaging has promise in predicting tumor response to in a techniques described above have specific the material material sources and their are described in the following is the of material for all analyses described in this article and is for certain types of particularly gene expression the of is to cell types and separate normal from tumor to functional genomic and/or cell can also be established for assays and surrogate biomarker are used for to biomarker can be developed for biomarker between patients or between and where appropriate samples are available. DNA and protein may be obtained from tissues, this material is not proven to be for material sources can be used in analyses of DNA, and cancer cells from a of from or example, leukemic cells can be from body to circulating cells from are also available for a of tumors of circulating cancer and cells is described of a candidate biomarker and provides the best for evaluation of the biomarker and for correlation with clinical clinical where such are possible is cancers in which serum biomarkers or are used to patient response to A is where tissue samples or are available and after when of the cancer A is in which and or imaging is available. for evaluation of biomarkers in these settings are in the following serum biomarkers are an of diagnosis, prognosis, and or in tumors (e.g., in For example, in α-fetoprotein, human chorionic gonadotrophin, and may tumors to be seen on and are for 1 after treatment These standard with can be used to the presence or expression of specific new biomarkers is predictive or correlated clinical response to the standard therapy as well as therapy is successful in a in cancer is to identify those of patients likely to respond to therapy and/or to In support of the a of additional biomarkers and used in combination with standard serum biomarkers could identify those with survival cancer patients samples are available both and patients treatment provide a in which to the expression of specific biomarkers is predictive of or correlated with responses to For instance, breast cancer a to the the use of chemotherapy has been chemotherapy may the of and may breast therapy in patients of the of their tumors However, the success of this is evaluation a II that treatment associated with a clinical response in breast cancer patients is that chemotherapy given in the after overall and has not been proven that chemotherapy A potential trial design would include and correlation of biomarker expression and response in and/or before and after treatment with as well as correlation with clinical response by clinical of and This is by of the gene associated with in gene expression profiling of biopsy samples before identified a pattern of expression of a of that correlated with response The gene expression a and and predictive cancer is the of and the basis of chemotherapeutic in cancer is is a in the of standard therapy for cancer to the presence or expression of specific biomarkers in biopsy and/or is predictive of or correlated with clinical response. overall a of patients with cell cancer respond to therapy with increased of the short of the and the of and imaging data with and cell cancer provides a for trials understanding patient response to standard The trial would biomarker presence and expression before and after therapy and would biomarker presence and response with clinical responses by clinical and A can be used in cell which to standard therapy of cancer It is already known that the presence of the mutations prognostic the prognostic potential of other markers as carcinoembryonic antigen, and is also For example, a used carcinoembryonic antigen and to identify a of in chemotherapy may be appropriate this the of prognostic and treatment effect biomarkers in AML is and strategies for research are discussed. A potential trial design is in A in which sensitivity to standard chemotherapy is highly AML is well to the discovery and of biomarkers in all of cancer drug development and therapy selection and prognosis, as well as predicting Such is to significantly patient with to of patients and (e.g., to patient for ongoing has to characterize prognostic and predictive in from to specific gene mutations. In the development and of new therapies have been by these The promise of this has already been in other the a in of myeloid leukemia and in acute leukemia is of and prognostic of this to discovery of the protein as the critical in myeloid which in the development of molecular for this biomarker that can when standard The development of a inhibitor of the tyrosine has transformed treatment in patients with myeloid leukemia is a that is of different acute acute acute acute and acute is also in the that to of other including are of the most of outcome and can treatment strategies For example, chromosome to gene highly The and or of chromosome which are in are associated with of the factor transcription These define the AML which is associated with the most including response to therapy A Cancer and trial overall survival with cytarabine therapy in complete response and overall survival years in these patients These patients also have a response and outcome with of cytarabine therapy However, of to AML patients to their detection of the has been with to and to predict in AML patients have as they are in a of patients years of with is in molecular markers that can be used to AML patients by of the presence or of chromosome It is that of AML patients have a specific in a molecular marker that can be using PCR In to those that specific chromosomal (e.g., and expression of other can be and has potential in and Wilms’ tumor gene and tumor factor as well as novel such as and acute and that to constitutive activation of the which a tyrosine kinase growth factor are in of AML patients from both and A the prognostic of in AML trials by the United Research of significantly associated with and overall survival In a retrospective prognostic in patients with normal A separate retrospective trial that the associated with could be by of targeted chemotherapy with a topo II a possible of therapy in patients in the is identified II clinical trials assessing the potential of inhibitors of and other (e.g., and have in AML Interestingly, new sensitivity (e.g., and novel mutations the activation to the inhibitors and In addition, overexpression in the of to be associated with survival Together, these findings that of the of is an in the design and conduct of clinical trials the of inhibitors in trials may characterize molecular markers and/or from leukemic cells, these with clinical features of the and correlation with clinical responses in both therapy and therapy The findings of Koo et al. (2, 3) on activated ras above are an of this use of biomarkers in a standard II inhibitor protocol should be used for the therapy a design may be for the this the biomarker could be as a trial to or studies for a novel In support of this an ongoing will specific chromosomal with clinical molecular treatment response response duration, and in patients with acute or who are to be with and regimens trials in AML are well to the of new genomic, proteomic, and gene expression analyses to identify prognostic and predictive ongoing efforts are to identify the gene expression patterns and associated with in AML patients in For example, the expression pattern of in AML using analysis could from responders to chemotherapy research in this is to facilitate the of drug response in AML and is A is to identify potential molecular in analyses to identify a novel that in of AML on cancers to those in AML have been to to between tumors and also in to frequent access and of the The access in tumors to and the of tissue specimens, along with the certain of imaging and serum provide to develop biomarkers in the treatment settings in this article a clinical research to and the basis for and and the of responses to approved drugs. genomic, functional genomic, proteomic, and imaging technologies will be standard of and treatment protocols to this These new may also facilitate the development and evaluation of new drugs, including new and molecularly targeted drugs. of this research will of approved drugs. for this are as is of and so forth) both and based on clinical evaluation provides data to treatment therapies, and so The best for this clinical research will be settings where tissue will be available both and tissue biopsy is not and for multiple and longer both molecular and clinical will be particularly on available tissue with and data, retrospective studies will be However, for the most prospective studies will be and are to be these studies will cytotoxic standard of therapies where outcomes are and of new technologies with clinical responses can be obtained most This research would be by evaluation of therapy drug and with newer molecular targeted and other of A collaborative initiative the other and is to these The of this will be to facilitate access to both novel technologies as they become available (e.g., genomic, proteomic, functional imaging technologies, and so forth) and clinical trial samples (e.g., serum, biopsy tissue, DNA imaging data, and so forth). It is that the will an role in fostering this collaborative technology, translational and clinical networks could be for the proposed For example, the NCI’s Cancer Bioinformatics Informatics Grid is a to facilitate access to data, data and It is that the proposed bioinformatics system to and outcome, demographic, and other clinical data would be implemented in accord with the Cancer Bioinformatics Informatics Grid The initiative could also draw from the Specialized Programs of Research Excellence fostering multidisciplinary teams to address biomarker identification and validation.11 to patients might be best achieved via the NCI’s established collaborative physician networks (e.g., Oncology Cooperative efforts to facilitate translational research and biomarker development may also the proposed include a the growth of The NIH is a of to the of research from the to the efforts are supported by the European Organization for the Treatment of The United Kingdom’s National Cancer Research is a the and cancer research in the United Research Cancer Research United Research of The National Cancer Research Institute has the National Cancer Research which provides support for clinical cancer The National Cancer Research the National Cancer Research the of novel and from the clinical with the of the proposed of these include the following: (a) to cancer research networks the United to a of clinical trials has also been (b) to facilitate collection and analysis of biological samples associated with clinical data via the National Cancer to develop an to access and analysis of data all National Cancer Research clinical and so initiative is in with NCI’s Cancer Bioinformatics Informatics Grid to to address in the and to with for Research