Introduction

Personalized medicine, the treatment of patients based upon their individual genetic, epigenetic, and phenotypic makeup, is the ultimate goal of many researchers and clinicians trying to find less toxic and more effective therapies for cancer. While cancer as a whole is characterized by uncontrollably proliferating cells, the disease is being subdivided into increasingly smaller units, from organ-specific to gene-specific categories. An understanding of the molecular basis of some cancers and an increasing knowledge of how those cancers differ from those in other organs and in other individuals have led to the development of drugs enormously successful at treating those types of cancer. Yet many other candidate drugs based on the same kind of analysis fail in the clinic, leaving cytotoxic chemotherapy still the standard treatment for many patients.

From May 19–21 2010, academic and industry researchers, technology developers, and clinicians from around the world gathered in Barcelona, Spain, to discuss the progress and the challenges in the field of personalized cancer medicine. Presented by the New York Academy of Sciences, Taléncia, and Fundació la Caixa, the conference brought together over 300 attendees to learn about topics ranging from basic cell biology and how it goes awry in cancer cells to massive efforts to characterize cancer genomes. A special technology workshop highlighted the important role of technological advances in driving the efforts toward personalized medicine. Conference participants also discussed their concerns about the field, in particular the failure of the U.S. clinical trial system to keep up with the changes demanded by the new approach.

Researchers have made great progress in discovering the cellular processes that have become pathological in cancer. For example, it is clear that genetic and epigenetic changes that affect cell proliferation, DNA repair, programmed cell death, stem cell maturation or self-renewal, and other fundamental cellular and molecular activities can lead to cancer formation. New information is also coming to light about more systemic problems, such as the survival of cancer cells in different microenvironments, the cells' ability to stimulate angiogenesis, and the way they promote their own survival by interacting with noncancerous cells to acquire nutrients, suppress immune response, and more. Several speakers at the conference shared their research into these and other biological events that lead to cancer.

Post-diagnostic treatments are clearly important, but the best way to deal with any disease is to prevent it from ever occurring. To that end, scientists and clinicians are working together to find ways to determine whether a person is more or less likely to develop cancer and why. To associate genetic status with cancer susceptibility, researchers need to analyze large amounts of clinical and molecular data, sometimes collected over a lengthy clinical follow-up process. Japan has taken up this mantle with its BioBank Japan project, in which researchers and clinicians are collecting DNA, serum, and clinical information from 310,000 patients with common diseases, including some types of cancer. The information will be used to investigate genetic susceptibility to disease, molecular targets for drug discovery, genetic information for personalized medicine, and more.

The processes that lead to epigenetic changes, such as methylation, acetylation, and changes in chromatin structure, are less well understood than those that lead to DNA mutations, but are likely to be just as important in determining cancer susceptibility. Gene expression is highly dependent upon the physical state of the genes' promoter regions, which in turn is regulated by epigenetic mechanisms. Overexpression of genes that lead to cell proliferation or under expression of those that promote terminal cell differentiation, as well as other alterations in gene expression, can give rise to tumors. Speakers in the second session of the conference provided insight into the specific ways genetic and epigenetic changes can be linked to cancer development.

The third session of the conference focused on oncogenomics and tumor profiling. Researchers are using whole genome sequencing of tumors to find the underlying mutations. This genomic information can reveal the signaling pathways mutated in a particular tumor type as well as the variability among tumors within that type. In addition to chromosomes with point mutations and deletions, cancer cells are characterized by a large number of genomic rearrangements that can result in gene fusions and consequent oncogenic proteins. Special techniques have been developed to identify these rearranged chromosomes and to pinpoint the fusion genes that result. Some of the rearrangements affect microRNAs, which as a molecular class went unnoticed until 2001 when the human counterparts of the C. elegans versions were located. It may be possible to design therapies targeting microRNAs in oncogenic signaling pathways, as they can be downstream targets of processes that are often dysregulated in cancer.

While basic research is revealing new targets for cancer drugs, treatment currently consists of some combination of neoadjuvant therapy, surgery, and chemotherapy or targeted therapy. Because of the variety of treatment options, many of which are expensive and quite toxic, it is critical to determine which treatment is most likely to work for each patient. Breast cancer researchers are at the forefront of efforts to use gene expression signatures and other molecular data to guide treatment, predict risk, and understand epidemiological phenomena. There are several clinical trials underway to test some of these new tools, some of which were presented in the fourth session's talks about efforts to identify biomarkers for prognosis and treatment response.

The fifth and sixth sessions of the conference delved into a variety of approaches to developing targeted therapies for cancer. Some of these efforts take advantage of technologies that allow high-throughput analyses of genomic information and drug responses in cell culture. Others use preclinical mouse models to evaluate candidate target therapies or to counteract the drug resistance that often thwarts cancer treatment. Next-generation sequencing and microarray technology have also played an important part in the advance toward personalized cancer medicine, as a technology workshop highlighted.

Although the conference participants covered a broad range of topics in cancer medicine, three ideas kept coming to the fore in their talks: first, research is moving into a new era in which very large amounts of data are being generated and efforts must be made to share this information across research groups in academia and industry; second, the current clinical trial system in the United States is not keeping pace with changes in drug discovery; and third, as treatments become more personalized, the pharmaceutical industry, regulatory agencies, and patients will have to adjust to the fact that the number of people treated with any given drug will be much smaller than in the past.

The clinical trial system in the United States is regulated by the U.S. Food and Drug Administration. There are several features of the current system that need to be changed to enable personalized medicine to reach its full potential. For example, the current system prohibits the testing of combination therapies before each drug has been proven efficacious on its own. However, a growing understanding of the molecular mechanisms of drug action suggests that some drugs may only work in combination with others, and will fail in single drug clinical trials. In other instances, a drug's efficacy may depend on its administration early in the disease process, but clinical trials often test new drugs on late stage cancer patients. Finally, for targeted therapies, the patient population will need to be stratified, making it difficult to test a new compound in the number of patients currently required for a drug efficacy to be established. The clinical trial system will need to be modernized to account for these changes in drug development.

While cancer remains a challenging disease to treat, the advent of technologies and data processing tools that allow for the gathering and analysis of large amounts of information, the proven success of some personalized therapies, and the sheer breadth of cancer research efforts suggests that, from diagnosis to treatment, clinicians will have new and powerful ways to tackle the disease in the future.

Session 1: Biological Processes Leading to Cancer

Speakers:
Hans Clevers, Hubrecht Institute in Utrecht
William G. Kaelin, Jr., Harvard Medical School
Napoleone Ferrara, Genentech
Joan Massagué, Memorial Sloan-Kettering Cancer Center; Institute for Research in Biomedicine, Barcelona (Adjunct)

Highlights

• Crypt base columnar cells give rise to large tumors when mutated and appear to comprise the intestinal stem cell population.
• The dioxygenase family of enzymes may provide a new target for drug discovery.
• G-CSF, a myeloid growth factor secreted by tumor cells, upregulates expression of the angiogenesis-promoting the Bv8 gene in myeloid cells.
• Src-pathway activation is associated with late-onset bone metastasis of breast cancer.

Research into new cancer therapies still relies heavily on an understanding of the basic mechanisms of disease development and progression. The first session of the conference featured a variety of investigations ranging from the role of stem cells in colon cancer to the genetics and cell biology of metastasis, all of them exploring in some way the basis of oncogenesis and metastasis.

Covering cancer's genesis, the proliferation of cancer cells, their acquisition of nutrients, and cancer's deadly spread from one organ to another, speakers considered their research in the broader context of research into both fundamental cancer properties and the application of that knowledge in the search for applicable clinical solutions. Hans Clevers's talk elucidated the relationship between normal processes of regeneration and pathological regeneration: between intestinal cell renewal and intestinal cancer. Following the trail of cancer's impact on bodily functions, William Kaelin and Napoleone Ferrara both talked of tumors' seemingly intractable path to access the nutrients they need. Driven to seek those nutrients, tumors might also be driven by genetic and epigenetic markers to metastasize to specific organs, even years after their first appearance, as Joan Massagué explained.

The self-renewing intestine

Hans Clevers of the Hubrecht Institute in Utrecht described his group's efforts to link the physiology of intestinal self-renewal with the biology of colon cancer. The intestine is one of the most rapidly self-renewing tissues in the both adult humans and mice.

The source of the new cells is a small compartment called a crypt that lies between the villi of the small intestine. There are approximately 10 billion crypts in the intestine, each of which has from 6–10 stem cells that generate about 200 cells, called transit amplifying cells, every day. These daughter cells proliferate for 2–3 days, undergoing 5 cell cycles before differentiating and finally, dying at the tips of the villi 2–3 days later. Before Clevers's work scientists knew of 4–6 stem cells residing at the base of crypts, but they did not know precisely which crypt cells were these stem cells.

Proliferation of cells in self-renewing intestinal tissue is regulated by the Wnt signaling pathway. In colon cancer, the Wnt pathway is locked in the ON state. Stimulation of the Wnt signaling pathway in intestinal cells ultimately activates the TCF4 transcription factor, whose targets are all cancer genes. Many of these genes are expressed in the transit-amplifying cells, and others are expressed in the Paneth cells, a group of cells derived from the stem cells that are thought to sterilize the lumina of the crypts.

Lgr5 expression marks putative stem cells

To identify the intestinal stem cells, Clevers's team looked for genes expressed only in a few cells near the bottom of the crypts. They found that the Lgr5 gene was expressed in cycling crypt base columnar (CBC) cells, a small group of cells located between the Paneth cells that were first identified by Charles Philippe Leblond in 1974 and that subsequently fell into obscurity. Lgr5 encodes an orphan, G-protein coupled receptor related to the glycoprotein hormone receptors.

A number of experiments were performed to look for stem cell properties in Lgr5-expressing cells. A lineage-tracing experiment demonstrated that they produce the right number of daughter cells. Also, the marker for putative stem cells and their daughters persisted through the lifetime of the mouse, as expected. Multicolor lineage tracing showed that 10 individual stem cells were active at the same time in the crypt. Some crypts became clonal for a while then the competition started over. Based on these findings, the Lgr5-expressing CBC cells appear to comprise the intestinal stem cell population (Figure 2). Curiously, these cells are never quiescent, a property thought to be inherent in stem cells.

Figure 2. Lineage tracing of Lgr5-expressing crypt-base columnar (CBC) cells. The CBC cells give rise to all the requisite cell types of the epithelium.

Clevers's team next showed that single CBC cells cultured in matrigel culture with three growth factors can form crypt structures with all the requisite cell types. An unusual property of this system is that the Paneth cells appear to provide the niche that supports the stem cells although the Paneth cells are their daughter cells. Genetic manipulations that cause the gradual loss of Paneth cells result in the disappearance of the stem cells.

Relating his work on intestinal stem cells to cancer, Clevers described the results of deleting the APC (Adenomatous polyposis coli) gene, which plays an inhibitory role in the Wnt pathway and is often mutated in colon cancer, either in intestinal stem cells or in other intestinal cells. Transgenic mice with APC deleted from the stem cells rapidly developed large tumors. In contrast, mice with APC deleted from anywhere but the stem cells developed tiny adenomas that failed to grow any larger. Taken together these data support the hypothesis that the columnar base crypt cells are the stem cells in the intestine and that mutations that affect proliferation of these cells may play a role in the development of colon cancer.

New cancer targets from studies of the VHL tumor suppressor protein

The growth of tumors is limited by their inability to access nutrients and oxygen. Tumors overcome these limitations both by stimulating the growth of blood vessels and by activating a host of genes, via the hypoxia-inducible factor alpha (HIFα) transcription factors, that promote survival in hypoxic conditions. William Kaelin and his team at Harvard Medical School are teasing out the intricate pathway by which the HIFα transcription factors contribute to tumor survival. HIFα is targeted for degradation by the VHL tumor suppressor protein, whose mutation is a common early event in colon cancer. In the presence of oxygen, VHL serves as an ubiquitin ligase that targets the alpha subunit of HIFα. The oxygen sensitivity depends on the fact that HIFα has to undergo an oxygen-dependent posttranslational modification to be recognized by VHL. The main enzyme that modifies HIFα for this recognition is known as EglN1 (or PHD2), a prolyl hydroxylase.

Figure 3. The druggable pocket of EglN1 (PHD2). (Adapted from McDonough et al. 2006 Proc. Natl. Acad. Sci. 103(26): 9814-9819.)

Transcription factors are notoriously difficult to target with drugs because of their structure. By contrast, enzymes are often excellent targets, and EglN1 is no exception. EglN1 crystallizes as a homotrimer with a pocket that provides a possible inhibitor target site (Figure 3). Interestingly, EglN1 is a member of a larger enzyme family that includes EglN2, which is a protein induced in estrogenic stimulation of breast cancer. Knockdown of EglN2 leads to loss of cyclin D1, part of cell cycle progression, and EglN2 shRNA inhibits cancer cell proliferation. Taken together, these results provide evidence that this enzyme family may be a good target for new cancer drugs.

Myeloid cells and angiogenesis

Although tumors can turn on genes that help them survive in hypoxic conditions, ultimately they need a source of food and oxygen to survive. To get these resources, they secrete factors that promote angiogenesis. In 1989, Napoleone Ferrara and his team at Genentech identified and cloned the vascular endothelial cell growth factor gene (VEGF), which encodes a growth factor that plays a key role in promoting new blood vessel growth. The team later developed a monoclonal antibody, now available clinically, that targets VEGF. But as is the case in so many attempts to treat cancer, the patient eventually develops resistance to the treatment and succumbs to the disease. Ferrara's team is now trying to identify the distinguishing characteristics of tumors that are resistant to VEGF inhibition.

Investigations into the tumor environment have indicated that myeloid cells in the area also promote angiogenesis. To investigate whether recruitment of myeloid cells is a factor in resistance to VEGF inhibitors, Ferrara's team tested whether refractory tumors recruited more myeloid cells than sensitive, treatment responsive, cells. They transplanted GFP-labeled bone marrow cells from transgenic mice into lethally irradiated mice, then implanted either sensitive or resistant tumor cells into the chimeric mice. The group found that refractory cells did recruit more GFP-labeled cells and that the majority of cells associated with the refractory tumors consisted of CD11b⁺Gr1⁺ myeloid cells, which encompasses a broad population of immune cells. Ferrara noted that evidence suggests that these myeloid cells are both proangiogenic and immunosuppressive.

Analysis of gene expression in bone marrow from mice with resistant or sensitive tumors revealed a large number of differentially expressed genes, including growth factors and genes involved in mobilizing myeloid cells. Ferrara's team chose to focus on one such gene called Bv8, which has tissue-specific angiogenic properties.

Figure 4. In refractory cells, those insensitive to VEGF-A inhibition, Bv8 upregulation promotes angiogenesis when the VEGF pathway is blocked.

Ferrara's group found that tumors that are highly refractory to VEGF inhibitors produce G-CSF, a myeloid growth factor, which in turn dramatically upregulates expression of Bv8 in myeloid cells. Upregulation of Bv8 is a work-around for the blocked VEGF pathway (Figure 4). "G-CSF is highly likely to mediate communication between the tumor and bone marrow because it behaves like a classic hormone," said Ferrara. It is secreted into the blood, unlike VEGF, which binds tightly to heparin and is confined to the tumor microenvironment. Preliminary experiments show that early anti-Bv8 treatment inhibits the growth of human xenografts in mice though not as effectively as anti-VEGF treatment.

Resistance to a drug usually arises when the cells manage to restore the pathway that is blocked or to find an alternate route to bypass the drug's effects. The identification of such alternate routes gives drug developers a way to head off resistance and improve treatment regimens.

Understanding metastasis

Oncologists and their patients hope that the excision of a tumor before it has a chance to metastasize will be the end of the cancer. But even a clean excision is no guarantee that the cancer will not recur. A better understanding of the process of metastasis is crucial for the development of more effective cancer treatments for before and after the excision. Joan Massagué and his colleagues at Memorial Sloan-Kettering Cancer Center are trying to understand how the cells move from the circulation into a new tissue, survive in that microenvironment, and retain the ability to reinitiate tumors after they are established.

Different cancers have very different paths to recurrence. In breast adenocarcinoma, it may be decades from the time the initial tumor was excised to when the cancer returns. The metastatic cells that are derived from the initial tumors appear to be dormant over the years. When they do reappear, they generally have taken up residence in the lung, bone, or brain. In contrast, lung or pancreatic adenocarcinomas have very short latency periods and there is not much selectivity in which tissues they colonize.

One approach to studying metastasis is to examine the metastatic end-products. Massagué's group showed that metastatic cells from breast cancer patients tend to establish themselves in the same organ when transferred from one mouse to another. Furthermore they showed that cells with a predilection for one organ over another preexisted in the initial population.

The next step was to determine what was different about the type of metastatic cells that colonized one organ versus another. In a study of metastasis to lung tissue, they identified a set of 18 genes that predict relapse from lung infiltration and are found in the primary tumors of ER negative breast cancer patients. Among the genes, the group found the expected genes promoting tumor angiogenesis, vascular permeability, cell mobility, invadopodia, and collagenase production, but they also found genes that are expressed in response to signals in the tumor microenvironment. One such gene ANGPTL4 (angiopoietin-like 4) encodes a protein that disrupts the microvascular endothelial cell junctions, allowing the cells to move from blood vessels into the lung tissue. Interestingly, ANGPTL4 expression is driven by TGF-β released by the stroma in the primary tumor, paving the way for invasion when the cells arrive in the lung.

By looking at the expression of genes in metastatic tumors, Massagué's group is mapping out the steps required to establish tumors in specific organs. But this approach does not allow them to answer the question of what keeps the cells alive for such a long time before relapse. To investigate this issue, the team took another approach, correlating the expression levels in primary tumors of genes in the major growth and survival signaling pathways (TGF-β, Src, Ras, Wnt, etc.) with late-onset relapse. The Src pathway was the only one that was associated with metastasis to bone. Furthermore, they found that stromal cells in the bone secrete two factors that promote long-term survival of tumor cells with a high level of Src activity.

So, if highly Src-active tumor cells are associated with metastasis to bone and are supported for the long term by bone cells, can Src inhibitors prevent metastasis in high risk patients? Preliminary experiments in vitro (Figure 5) and in mouse models indicate that the approach may work in cancers that have a short latency period as long as treatment is administered early on in the latent state. The group's gene expression studies pointed to another potential target, in addition to Src, for inhibition. That target is an actin bundling protein called Fascin, which is important for invasive motility and has, in every gene expression study, been associated with aggressive metastatic cancer. Fascin is also implicated in attracting circulating tumor cells back to the primary tumor, a process that might be hindered by a recently developed Fascin inhibitor.

Figure 5. Src inhibition mainly suppresses cancer cell survival, not invasion or proliferation.

Metastasis is ultimately what kills cancer patients. But scientists, propelled by research like Massagué's into the precursors of metastatic tumors that arise early on in cancer progression, are ever closer to finding new treatments to prevent this deadly development. Already begun in Massagué's lab, successful identification of the genes expressed in these cellular forerunners to metastatic tumors will be crucial to attempts to prevent metastasis before it becomes terminal.

Session 3: Oncogenomics and Tumor Profiling

Speakers:
Todd R. Golub, The Broad Institute of Harvard and MIT
Arul Chinnaiyan, University of Michigan Medical School
Joe W. Gray, Lawrence Berkeley Laboratory (talk presented by Laura Vant Veer)
René Bernards, The Netherlands Cancer Institute, Amsterdam
Carlo M. Croce, The Ohio State University Medical Center

Highlights

• Whole cancer genome sequencing can reveal previously unidentified oncogenes.
• Translocations that fuse androgen-driven regulatory sequences to ETS family transcription factors can be found in 60%–70% of prostate cancers.
• Putative cancer drugs can be tested in cell lines classified by breast cancer subtypes.
• The Ras signaling pathway confers resistance to retinoic acid therapy for neuroblastoma by blocking retinoic acid-induced transcription.
• MicroRNAs are downstream targets of pathways that are commonly dysregulated in cancer.

Finding mutations is itself a cumbersome task, but sorting out the mutations relevant to cancer is even more of a difficult process. Scientists, however, have successfully employed a variety of new methods to do just that. Todd Golub and his team categorized cancers by their genetic signature, which required them first to map mutations and then to determine their statistical significance. His group's work yielded some very interesting results about the development of cirrhosis into hepatocellular carcinoma. The need to sort through great quantities of genetic data for meaningful mutations inspired Arul Chinnaiyan and his group to develop and apply an algorithm specifically designed to analyze tumor microarray data to find cancer-associated chimeric DNA sequences and the genes that regulate them.

Interested in the efficacy of cancer drugs, Joe Gray and René Bernards investigated, respectively, particular drugs' ability to inhibit breast cancer cell growth and the signaling pathways that affect retinoic acid sensitivity in neuroblastoma. Carlo Croce wrapped up the session by discussing his use of microRNA signatures to characterize tumor behavior. MicroRNAs, he said, make great therapeutic targets because they are the post-transcriptional regulators for genes that affect everything from epigenetic dysfunction to DNA repair.

Statistically significant mutations in multiple myeloma

Todd Golub of the Broad Institute of Harvard and MIT and his group are using genome sequencing and expression profiling to find gene signatures that define different cancer types. In one approach, the team is using whole genome sequencing to find a gene signature for multiple myeloma, a relatively rare but incurable hematological disorder. They began by sequencing tumor/normal pairs using a combination of whole genome and whole exome shotgun sequencing on an Illumina platform. They identified the nonsilent mutations in each tumor and confirmed the existence of the mutations by genotyping. Determining which mutations are statistically significant was challenging, and required correcting for gene size and base composition. Nevertheless they have identified several statistically significant genes, some of which are new to multiple myeloma and to cancer in general.

In an effort to understand the role of some of the genes whose function in cancer is unclear, Golub's group correlated their expression patterns with that of all other genes in the transcriptome in a collection of multiple myeloma samples. The group was able to link these genes with certain cellular processes, expanding our understanding of their role in oncogenesis.

Golub's team was also interested in determining whether rare mutations, which individually weren't statistically significant, in aggregate identified pathways that were hitherto unlinked to the disease. They identified several such pathways and work is now underway to determine how these pathways are contributing to multiple myeloma progression.

Development of a gene signature

Whereas large scale sequencing efforts can be used to find new oncogenes, large scale gene expression studies can be used to classify patients' cancers and perhaps determine prognosis. To identify a gene signature for hepatocellular carcinoma, Golub's postdoc Yujin Hoshida began by looking at gene expression in tumors that had been excised from liver tissue.

Once he had identified a signature, he asked if it was possible to predict risk of hepatocellular carcinoma in patients with cirrhosis, the condition that often precedes development of hepatocellular carcinoma. Initial results of that investigation are indeed promising. Golub's lab is also encouraged by preliminary testing of a way to use gene expression profiles to predict patients with high risk and treat them specifically rather than treating a large, heterogeneous group of patients who may have very different outcomes and very different treatment needs.

Translocations in prostate cancer

Chromosomal translocations can, much like point mutations, create new gene products, contribute to the overall dysregulation of genes, genetic instability, and ultimately to cancer formation. Translocation can result in, among many other possibilities, the fusion of a promoter sequence from one gene to the coding sequence of another or the fusion of two gene sequences together, which can lead to dysfunctional or fusion proteins, some of which are immune to typical post-translational regulation. The range of outcomes to these translocation events is vast, and counted among them are a few mutations known specifically for their association with oncogenesis.

One of the most successful drugs developed for cancer therapy is Gleevec, a drug used to target the Bcr-Abl fusion oncoprotein that causes chronic myelogenous leukemia. Bcr-Abl is produced by a translocation between chromosomes 9 and 22, and the resulting chimeric chromosome is known as the Philadelphia chromosome. Encouraged by the successful targeting of an oncogenic fusion product, Arul Chinnaiyan and his colleagues at the University of Michigan Medical School are taking a bioinformatics approach to look for gene fusions in common solid tumors, such as those found in prostate cancer. They developed an algorithm called COPA that identifies genes that are expressed at very high levels, as might occur if, for example, a foreign promoter had been driving expression of a gene it normally doesn't regulate. Application of the algorithm to a compendium of tumor microarray profiling data identified the ETS transcription factor as highly upregulated in prostate cancer. Further analysis revealed that the regulatory region of an androgen-driven gene had been fused to the ETS gene.

Chinnaiyan's team went on to identify a host of other gene fusions in prostate cancer that can be used to classify the disease into subtypes. Similar to their first find, these fusion genes largely contain androgen-driven regulatory sequences fused to the genes of ETS family transcription factors. Chinnaiyan's group and others have found that 60%–70% of prostate cancers from prostatectomy cohorts bear these ETS gene fusions. They occur early in prostate cancer and can be used as a diagnostic marker of the disease. Several different kinds of genetic experiments in model systems have been used to verify that the fusion genes are oncogenic.

Figure 8. Diagram showing the many steps in the pathway from TMPRSS2-ERG gene fusion to the transcription and translation of the dysfunctional ETS protein and then to its blocking of prostate cell differentiation, some of which processes present possible targets for drug therapies.

After investigating the translocations' importance to cancer development, the team was eager to understand how they arose. In androgen-responsive cultured prostate cells, the androgen-sensitive regulatory gene region (TMPRSS2) and the ETS gene (ERG) were brought in close physical proximity by the addition of androgen, and irradiating cells induced the translocation required to have TMPRSS2 and ERG on the same chromosome. The induced translocations are cell type specific; for example the Bcr-Abl fusion can't be induced in prostate cells.

To understand the role of the fusion genes in prostate cancer the group developed a compendium of ChIP (chromatin immunoprecipitation) sequencing analyses in prostate cancer cell lines to map the genomic landscape of the master regulators of prostate cancer. The results suggested that the androgen receptor signaling that ordinarily drives differentiation of prostate cells is blocked in several ways by the actions of the fusion gene (TMPRSS2-ERG), leading to epigenetic silencing and oncogenesis.

Scientists are developing diagnostic tests to identify the fusion gene in the blood or urine of prostate cancer patients. At the same time, a number of therapeutic approaches can be explored for when the gene is present, such as blocking androgen receptor signaling, degrading the fusion gene transcript with siRNA, using small molecule inhibitors that directly engage the ETS gene, or finding synthetic lethal interactions to kill cells bearing the fusion mutation.

Chinnaiyan's group is now using next-generation sequencing to analyze the transcriptome of prostate cancer cell lines and identify other chimeric genes, which might also play a role in the cancer's behavior. The approach has been validated and a robust pipeline has been developed. In recent work, for instance, the team has identified fusions of B-RAF and C-RAF in the group of prostate cancers that do not bear the ETS fusions, as well as in other types of cancer. Patients bearing these RAF fusions may respond to treatment with RAF and MEK inhibitors, treatment avenues made available only through investigation of chimeric genes and their effects on oncogenesis.

Subsets, specific therapies in breast cancer

Joe Gray of the University of California, San Francisco and his group are surveying the range of breast cancers by looking at a large number of cell lines established from breast cancer tumors. Gray was unable to attend the event but Laura van't Veer presented his work in his stead. Gray's team began by characterizing 50 breast cancer cell lines to ensure that they were representative of human tumors. Using gene, protein, and phosphoprotein expression profiling, they found that the cell lines could be assigned to the different breast cancer subtypes defined by Charles Perou and others (Figure 9).

Figure 9. Cell lines model luminal, basal, and claudin low transcriptional subtypes. Expression profiling allowed the assignment of cell lines to breast cancer subtypes.

Looking for growth inhibition, the group tested 77 compounds on each of 50 cell lines and graded the cells from sensitive to resistant according to their response. Gray's team found that 18 compounds were most effective on the slower-growing cell lines, 17 compounds were associated with copy number abnormalities, and 31 were specific for cells belonging one or another breast cancer subtype defined by their transcription profile.

The study also shed light on the mechanism of cells' response to compounds that target the mitotic apparatus. Work done in collaboration with Carlos Caldas showed that mitotic apparatus protein inhibitors tend to work on the basal subtype of breast cancer. To put this information to practical use, they developed a mitotic network activity index (MNAI) that is the sum of the expression levels of genes in the network. MNAI was associated with subtype and outcome in the 1000 primary breast tumors sampled. The signature overlaps with a chromosome instability signature developed by Carter and others. Interestingly, this network is only found in tumors and has yielded therapeutic targets that can be validated in these tumors.

Finding mechanisms and biomarkers of drug resistance in cancer

Many cancer patients do not respond to current targeted therapeutic approaches but those who do, respond very well, stipulated René Bernards of the Netherlands Cancer Institute. Thus, it is crucial to develop better ways to identify patients who will respond to the drug and to stop overtreating patients who will not respond. But identifying likely responsive patients requires moving beyond known biomarkers and known therapeutic targets. Bernards framed his talk with the question: "How can biomarkers of a response be found when there are no likely suspects [such as EGFR or Bcr-Abl] to consider?"

Such is the case with neuroblastoma, a frequent childhood tumor with heterogeneous clinical behavior. While N-MYC amplification is found in approximately 20% of neuroblastoma cases and loss-of-heterozygosity of 1p36 is found in approximately 30% of cases, not much else is known about the genetics of the disease, which limits what can be used to narrow down potential drug treatment populations. Treatment relies on induction chemotherapy followed by autologous bone marrow transplantation or continued chemotherapy. Retinoic acid, which differentiates neuronal cells, is used for maintenance therapy. Unfortunately, retinoic acid treatment has limited therapeutic benefit.

Classical genetic approaches, which are unbiased, are well suited for a search for causal genes. For example, a gain-of-function screen can be used to look for cases where overexpression of a gene results in altered response to a drug. The opposite approach is also useful—a loss-of-function screen can identify cases where the suppression of a gene results in a drug response.

Bernards described one such genetic screen: a loss-of-function screen looking for responsiveness to drugs in neuroblastoma. While the genetic technique may be classical, its implementation is decidedly modern. Bernards' team used a large library of short-hairpin RNA (shRNA) vectors that target mRNA for destruction to test the effect of individually suppressing thousands of genes. Because each shRNA contains a sequence unique to its target, it serves as a molecular bar code that can be used to identify target genes.

Figure 10. A schematic of the process of shRNA screening to determine the shRNAs that confer resistance to cancer drugs.

In the shRNA bar code screen Bernards described, neuroblastoma cells that were very responsive to retinoic acid were infected with the shRNA library. These were divided into two replicate cultures, a control and a dish treated with retinoic acid. Cells that are responsive to the drug will stop dividing, whereas those that have acquired an shRNA that confers drug resistance will continue to grow, increasing the abundance of that shRNA in the culture. After a few weeks, a comparison of the levels of shRNA by PCR amplification and hybridization to a microarray identifies both shRNAs that confer resistance and those that contribute to cell death, thereby illuminating mechanisms of drug resistance and susceptibility.

The analysis identified two independent shRNAs that knocked down the expression of the NF-1 neurofibromatosis tumor suppressor gene (Figure 11), conferring drug resistance upon the cell culture. NF-1 encodes a GTPase-activating protein that inhibits Ras signaling but that had not previously been implicated in retinoic acid signaling. Further experiments showed that loss of NF-1 conferred retinoic acid resistance in a neuroblastoma cell line. An activated Ras protein that cannot be rendered inactive by NF-1 also confers resistance to retinoic acid on the cells. Furthermore, Bernard's team determined that the Ras signaling pathway was making cells resistant to retinoic acid by blocking retinoic acid-induced transcription.

Figure 11. Red boxed plates show restoration of sensitivity to Retinoic acid (RA) with MEK inhibition. Retinoic-acid induced transcription can then continue, resulting in apoptosis.

Previously, the group had discovered that such transcription in neuronal cells requires a co-activator called ZNF423, which binds to the retinoic acid receptor complex at promoters. Microarray analysis of NF-1 knockdown cells showed that ZNF423 transcription was repressed and that expression of the gene in these cells restores retinoic acid sensitivity. These findings have clinical implications as well; patients with low levels of NF-1 or ZNF423 have a poor prognosis compared to those with high levels. Approximately 6% of neuroblastoma patients show a genetic aberration in the NF-1 gene.

Genetic experiments yielded an important, clinically relevant finding: Of the three pathways that mediate Ras signaling, only the MEK pathway affects the retinoic acid sensitivity of neuroblastoma cells. Small molecule inhibitors of MEK restore retinoic acid sensitivity to resistant cells. As his work on retinoic acid drug resistance demonstrates, and as Bernards stressed, it is critically important to understand not just potential perturbations in signaling pathways, but also the crosstalk between those pathways, the many ways they affect one another.

MicroRNAs and cancer

Carlo Croce of the Ohio State University Medical Center introduced another genetic contributor to cancer formation: microRNA genes. He and his team made use chromosomal alterations such as deletions and translocations to identify genes involved in cancer as did many of the conference presenters. But they encountered difficulty with a deletion found in some cases of chronic lymphocytic leukemia (CLL), which prompted them to try to unravel the complex genetic mechanisms involved in the disease's pathogenesis. Believing initially that CLL-inducing mutations would be in genes that code for proteins, they sequenced 2MB of DNA in the region of the deletion and characterized every protein-coding gene. Disappointingly, they failed to find a protein-coding gene that was specifically altered in CLL. However, the presence of a very rare translocation in the same region allowed them to map the chromosomal breakpoint to the nucleotide level and a small deletion in another patient gave them the evidence they needed to locate the relevant gene precisely on the chromosome.

Figure 12. Histology of spleen cells (a, b, & c) and bone marrow (d, e, & f) in mice with and without the dysregulation of mIR155. Image c shows the disruption over time (from a to b to c) of cell structure caused by leukemic cells in a transgenic mouse with mIR155 dysregulated. Image f shows the change to bone marrow histology (compared to the wild type in image d) induced by mIR155 dysregulation. Dysregulation of a single microRNA can cause cancer, but it takes time.

Though there was no protein-coding gene there, the discovery of human microRNA genes led them to ask whether such genes might be found at that spot. In fact, two microRNA genes, called mIR15 and mIR16 were affected by the CLL-related translocation. Further analysis revealed that approximately 70% of CLL cases have disruptions in mIR15 and mIR16, and most if not all cases of the indolent form of the disease have these aberrations. In mouse models loss of the mIR15 and mIR16 resulted in the appearance of the indolent form of CLL, providing further evidence for the microRNA's contribution to oncogenesis. Croce's research shows that a small gene signature of 13 microRNAs can be used to diagnosis whether CLL is the indolent or aggressive form of the disease.

After determining that microRNA genes played a role in oncogenesis, the team still needed to uncover the nature of that role and, more specifically, the target of microRNA genes' regulation. Target prediction for microRNAs is difficult but Croce's group was able to find that one of the top targets for mIR15 and mIR16 was the oncogene the group had cloned in 1984, Bcl2. Bcl2 levels were shown to be increased in leukemia cells lacking mIR15 and mIR16. Adding back mIR15 and mIR16 reduced Bcl2 levels, as expected if it were a target of the microRNA genes, and eventually caused the cells to die. For this reason, Croce proposed that replacing mIR15 and mIR16 in cancer cells lacking these genes is a promising therapeutic option since the end result is apoptosis of the cancer cells.

Two different microRNAs were found to be critical in the regulation of another oncogene, TCL1, and the researchers were able to prove that TCL1 was the oncogene that caused the aggressive form of CLL. Deregulated expression of TCL1 caused T-cell leukemia in mice, but it also, somewhat surprisingly gave rise, with 100% penetrance, to B-cell leukemia in mice. In each of these studies and in the group's work on microRNA genes in lung tissue adenocarcinoma, the transformation of mutated cells to malignancy took time, sometimes up to 15 months, indicating to Croce that some additional genetic or epigenetic changes might be required for pathogenesis (Figure 12).

Croce also noted that the same microRNA can act as a tumor suppressor or an oncogene depending on the cell type in which they are functioning, and this tissue-specificity has allowed Croce's team to establish microRNA gene signatures for several other solid tumors, all of which depend on the tumors' location. Since microRNAs are downstream targets of pathways that are commonly dysregulated in cancer, they are excellent candidates for therapeutics, he said. They are targetable, if they're lost you can replace them, and if they're overexpressed you can knock them down. And, because they regulate processes such as epigenetic dysfunction and DNA repair, microRNAs can tackle mutations that are very difficult to target in the DNA itself.

Session 5: Targeted Therapies I

Speakers:
Alan Ashworth, The Institute of Cancer Research, London
D. Gary Gilliland, Merck and Co. Inc.
José Baselga, Massachusetts General Hospital (formerly at Vall d'Hebron Institute of Oncology (VHIO) and Vall d'Hebron University Hospital, Barcelona)

Highlights

• Genome-scale siRNA screen reveals that the CHK1 gene might enhance cytotoxicity of gemcitabine.
• PARP inhibitors provide second, and lethal hit, to cells with BRCA2 mutations.
• mTor inhibitors in combination with IGF1 receptor antibodies show promising results against cancers with PI3K mutations.

There are a number of ways that cancer therapy can fail. Some oncogenic proteins are not inhibited by currently available drug compounds. In other cases, resistance arises to drugs that were once effective. Speakers in the fifth session discussed some of the ways their research tackles drug inefficacy or resistance. Alan Ashworth spoke of using the synthetic lethal approach to kill cells with mutations in the homologous recombination repair system. Gary Gilliland considered Merck's efforts to find possible combination therapies by using siRNA in both synthetic lethality and enhancer screens. José Baselga presented his work on drug resistance, highlighting the importance of understanding how signaling pathways function and interact with each other in devising more effective treatments.

Synthetic lethality and DNA repair defects

Alan Ashworth of the Institute of Cancer Research discussed synthetic lethal approaches that exploit defects in DNA repair. A network of repair pathways works to repair an average of 10,000 DNA lesions in every cell every day. The rationale for targeting DNA repair defects in tumors is threefold: germline mutations in DNA repair genes, such as in BRCA1 and BRCA2, predispose individuals to cancer; many tumors carry signs of genomic/genetic instability as a result of unchecked mutations; and most chemotherapeutic agents work by damaging DNA, thus, defects in the repair pathways confer sensitivity to these drugs.

Carriers of BRCA1 or BRCA2 mutations have one wild-type copy of the gene, and the loss of this copy over time predisposes carriers to cancer. In homozygous mutant BRCA1 or BRCA2 cells, the normally efficient homologous recombination repair pathway is inactivated so an alternative pathway that haphazardly puts together broken DNA ends takes over. The result is a propensity to develop cancer. Ashworth's team looked for ways to target only the cells that had lost both copies of BRCA1 or BRCA2, not the heterozygous but phenotypically healthy cells.

Historically, researchers have been unable to develop drugs that work on cells that are dysfunctional due to the lack a critical protein, such as a DNA repair protein. But a classical genetic approach, called synthetic lethality, can take advantage of the cell's altered state, even in the case of a missing component, and thus is a promising answer to this problem. Synthetic lethality, originally described by Theodor Dobzhansky in 1946, is a situation where two mutations are not lethal when each occurs singly in a cell, but are lethal when combined. In the modern interpretation of this phenomenon, a drug stands in for the second mutation required to kill the cell.

Ashworth's group used PARP (Poly ADP ribose polymerase) inhibitors to inhibit base-excision repair in BRCA2 mutant cells, hoping to create a situation where inhibition of two different DNA repair pathways leads to cell death. In cells lacking only 1 copy of BRCA2, the homologous recombination pathway should still be functional so those cells should remain viable. Indeed they found that BRCA2-deficient mouse embryonic stem cells were extremely sensitive to PARP inhibition, whereas heterozygous BRCA2 cells behave exactly like normal cells (Figure 16).

Figure 16. Genome instability is clearly visible even on the chromosome level for BRCA2 mutant cells with inhibited PARP.

But, both for clinical implications and to show that the proposed mechanism of action was correct, Ashworth's team wanted to reveal the limits of this inhibition by finding cells that developed resistance to the synthetic lethal PARP treatment. BRCA2 mutant cells were treated with PARP inhibitor for several months until resistance developed. Analysis of the BRCA2 genes in the resistant cells showed that a functional BRCA2 gene had been reconstituted in these cells. The clinical implication for this work is that the best results to treatments are likely to be achieved early in the course of the disease when there are fewer cells to kill before resistance can arise.

The finding that PARP inhibitors show synthetic lethality in BRCA2 mutant cells allowed the researchers to redirect the route through the clinical trial system so that after finding the highest nontoxic dose in an unselected patient population, they could test the drug in cancer patients with the BRCA2 mutation. The results of the trial were very encouraging with a total clinical benefit rate of 59% in a very heavily pretreated patient population. The phase 2 results are also encouraging.

But what about all the cancers that aren't caused by BRCA mutations? The same approach can be used in cases where a second hit is required for lethality. Ashworth's team turned to the PTEN (phosphatase and tensin homolog) gene, mutations or deletions in which are common across a broad spectrum of cancers. Studies have shown that PTEN plays a role in regulating Rad51, a key component of the homologous recombination pathway. After showing that homologous recombination efficiency was reduced in PTEN mutant cells, Ashworth's group treated the cells with PARP inhibitors. While the sensitivity of these cells was not as great as in the BRCA mutant cells, the result was still very good and they are trying to start clinical trials in patients with PTEN mutations.

Ashworth's group is also developing assays for pharmacodynamic analysis of the consequence of PARP inhibitor treatment and for a Rad51 homologous recombination biomarker to aid in optimization of this and related treatments.

High throughput screening & combination therapies

A major problem with translational research is how to take all the information being generated and translate it into clinical benefit for patients. All too often in drug development, treatment results may show statistical significance but have very little effect on the patient's condition when put to the test clinically, commented Gary Gilliland of Merck, & Co. Not only is this a problem for the patient, it is a problem for the pharmaceutical industry because companies are required to test new drugs against the latest standard of care in the control population. This standard may be an expensive combination therapy that is barely more effective than the drug that had preceded it. Gilliland suggested that we need new therapeutic paradigms that will match the right drug to the right patient, permit the development of cocktails of targeted agents, and improve the value to discrete patient populations, without requiring pharmaceutical companies to demonstrate drug efficacy across a large population exhibiting a broad range of treatment needs.

As mounting evidence suggests that combination therapies hold the most promise for getting better treatment outcomes and avoiding resistance, Merck is using RNAi screens to inform combination chemotherapy regimens. Short interfering RNAs (siRNAs) that block the expression of their targets can be used in synthetic lethality screens and in enhancer screens to identify targets that may work in such combinations, by finding pathways that are upregulated or downregulated in tumors. Merck is taking advantage of the power of automation with ultra-highthroughput screens and multiparametric assays to identify biologically relevant siRNA hits.

Gilliland described the results of one such screen, a genome-scale siRNA screen for enhancers of gemcitabine (a common chemotherapy drug) cytotoxicity that yielded the CHK1 gene as a hit. Since gemcitabine is a drug that will be used as the standard-of-care arm of clinical trials, CHK1 inhibitors can be tested to see if they improve gemcitabine's ability to kill cancer cells in those trials.

The length and expense of clinical trials is no guarantee that drugs will make it through, and indeed, many do not. Gilliland suggested a number of changes that could be made during each trial phase to improve the odds of success. In phase 1, researchers need to show that the drug affects cell function. In addition, they must develop a test to select patients and validate the test in the clinic. In phase 2, researchers must at least see tumor shrinkage in a defined subpopulation. They must follow CLIA (Clinical Laboratory Improvement Amendments) regulations of clinical lab research performed on samples from humans. In phase 3, a new drug must be tested in all patients and shown to work only in subpopulations. Alternatively, an in vitro diagnostic can be developed to select patients who fall into the targeted population, so that the drugs never have to be tested on people who are unlikely to respond significantly.

To increase scientists' ability to find the most appropriate test populations, Merck has set up a very large scale effort to collect tumors on every patient that comes into participating hospitals. These tumors will be analyzed to identify signatures of tumors and treatments will be selected based upon the specific tumor phenotypes. The profiles of these patients will be stored at the hospitals, and the plan is to collect 25,000 clinically annotated tumor specimens over five years, of which 75% will be primary tumors and 25% will be metastatic tumors. A small subset of those collected will be biopsy samples. Gilliland noted that in 2008, the hospitals, to increase the samples' utility, began collecting large enough samples to split for DNA and RNA analysis (Figure 17).

Figure 17. Moffitt/M2GEN collaboration — pre-selective, biomarker-driven enrollment. Schematic of biomarker-driven, branched, subpopulation-based trials.

As a consensus emerges that companies and academia need to be much more open with their data, Merck has decided to share the mRNA profiles with the participating hospital system and ultimately with the public. In the recognition of the new paradigm, Lilly, Pfizer, and Merck have teamed up to form the Asian Research Cancer group. The power of new techniques such as next-generation sequencing and other high-throughput technologies, and a new openness to sharing data are beginning to change the landscape of cancer drug development.

Combating nonresponsiveness: countering PI3K mutations

Even after seemingly successful drugs have been developed to treat cancer, they may show less success in certain subsets of the treatment population. Expanding the treatable population for existing drugs is one of the reasons José Baselga of the Vall d'Hebron Institute of Oncology (now at Massachusetts General Hospital), investigates mutations in the PI3K pathway. Activating mutations or deletions in the PI3K/Akt/TOR pathway are extremely common in cancer.

As others have noted during this conference, breast cancers can be divided into subgroups based on their expression of receptors for hormones that stimulate cell proliferation. About 33% of hormone-responsive breast cancers and about 22% of HER2⁺ (human epidermal growth factor receptor) cancers have PI3K mutations. The incidence of the mutation is lower in triple negative tumors. Researchers have found that PI3K mutations interfere with response to trastuzumab, which is a monoclonal antibody that targets HER2 receptors and therefore HER2⁺ tumors.

The PI3K pathway can be targeted at many points by agents that inhibit pathway components. Baselga's group hypothesized that mTOR (mammalian target of rapamycin) inhibitors may be useful with anti-estrogen therapy against estrogen receptor positive (ER⁺) breast cancers. In a phase 2 neoadjuvant study of Letrozole (an anti-estrogen agent) with or without Everolimus (an mTor inhibitor), the group saw statistically significant results with the combination therapy, that is with Letrozole and Everolimus together. The results held up when another determinant of efficacy, a cell proliferation marker called Ki67, was employed. Based on these findings, Novartis has started a large phase 3 trial of this combination therapy on patients with advanced disease.

New PI3K inhibitors, as well as a dual inhibitor of PI3K and mTor, have been developed since Everolimus and some of them are entering clinical trials as well. Pleased with the direction of this research, Baselga generalized the trials' results: "When we achieve a good dose of these inhibitors, we do block the pathway in patients."

Applying more broadly the lessons learned in the search for PI3K and mTor inhibitors, Baselga outlined the questions that must be answered when trying to develop drugs for these pathways: What is the best target in the pathway? Will toxicities differ? Will the drugs' efficacy depend on where in the pathway a patient has a mutation, as is the case with the B-RAF inhibitor? Will specificity to patients' phenotypes make the drugs safer overall? Which combinations will be best? And what alternative pathways will cells turn to when one is successfully blocked?

Figure 18. Combination therapies can be an effective strategy to resuscitate treatment efficacy by knocking out compensatory ("work around") pathways in addition to the primary pathways.

Combination therapy is thought to be the best way to combat the development of resistance to treatment, but challenges arise even with this approach. Baselga's group found that in a study of Everolimus, phospho-Akt levels rose, indicating that a compensatory pathway (Figure 18) had been activated, and that treatment efficacy was likely reduced. This can be averted by using a combination of an antibody against the IGF1 receptor, a part of the compensatory pathway, and an mTOR inhibitor. Further analysis showed that activated Erk levels were also raised with mTOR inhibitor treatment. This result is context specific so the pathway driving Erk activation must first be determined before that compensatory pathway can also be blocked.

Baselga highlighted the newest direction his research is taking: incorporating molecular/dynamic imaging in early clinical trials, developing quantitative tissue-based biomarkers (fluorescent immunohistochemistry and protein arrays), and understanding mechanisms of primary and acquired resistance. Garnering this new knowledge will be enormously helpful in developing new, more effective, and more personalized cancer treatments.

Technology Workshop

Presenters:
Brian Pollok, Life Technologies
Klaus Pantel, University Medical Center, Hamburg-Eppendorf
Quyen Nguyen, University of California, San Diego
John McPherson, Ontario Institute for Cancer Research, Toronto (presented by Brian Pollok)
Michèl Schummer, The Fred Hutchinson Cancer Research Center

Highlights

• Circulating tumor cells and disseminated tumor cells can be detected using a novel cell sorting process.
• Fluorescent labeling of tumors and nerves may help surgeons remove tumors completely without damaging nearby nerves.
• Next-generation sequencing techniques enable researchers to perform many analyses that were cost prohibitive or unfeasible in the past.
• Publicly available data can be mined for information about possible cancer biomarkers.

The development of personalized medicine requires technologies that can process samples and analyze large amounts of data rapidly and inexpensively. Basic research, drug discovery, and clinical care all rely heavily on such technologies. Brian Pollok of Life Technologies emphasized the need to develop technologies that work together, from the determination of sequences to gaining an understanding of the function of biological molecules and systems, covering the entire workflow.

Detection of circulating tumor cells

Useful technologies have to advance with, and sometimes ahead of, the biological understanding of the system under investigation. For instance, scientists know that early tumor cell dissemination is a key step in tumor progression, so it is clearly important to be able to detect cancer cells as they spread. However, detection of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) requires the ability to pick out one tumor cell in the background of 10⁵–10⁷ normal cells. The enrichment methods that are currently available for positive selection of CTCs/DTCs are all flawed in some way because tumor cells are heterogeneous. Klaus Pantel of University Medical Center Hamburg-Eppendorf and his team decided to approach the problem from a different angle, by weeding out the leukocytes using an antibody to the CD45⁺ marker expressed on those cells (negative selection) instead of by searching for the few CTCs and DTCs among the leukocytes. For tumor cell identification cells enriched by positive or negative selection are stained for cytokeratin, which is expressed on epithelial cells and serves as a marker for CTCs and DTCs. By using an automated microscope system to look for the stained cells, they found that 36% of primary tumor breast cancer patients and 44.6% of prostate cancer patients with a primary tumor had these keratin-stained DTCs in bone marrow, as the most prominent site of full blown metastasis, whereas only 1% of people with nonmalignant disease had positive staining.

Since it is easier to screen blood than bone marrow, and blood would therefore be the desired way to test patients clinically, Pantel's group validated an automated system called CellSearch™ based on the technique described above, that has been FDA approved for detection of CTCs in the blood. The group was able to show that the number of CTCs in the blood correlated with prognosis in patients with advanced stages of cancer. The ability to detect CTCs can also be very useful in assessing the efficacy of treatment, as was the case in recent clinical trial called Geparquattro in Germany.

In addition to counting the number of CTCs in the blood, it may be useful to characterize the cells according to their expression profiles. Doing just that, Pantel's colleague Sabine Riethdorf found that while cells taken from the primary tumor of a breast cancer patient did not express HER2, CTCs from that patient were HER2⁺. It is not clear whether there was selection for this trait or whether a minor clone in the primary tumor was HER2⁺. The ability to detect HER2⁺ cells after Herceptin treatment will allow the researchers to examine resistance to the treatment.

In collaboration with the company Eppendorf, Pantel's group devised a method of performing molecular characterization of a single CTC with PCR analysis or whole genome amplification. Comparative genome hybridization and array hybridization can identify amplifications and deletions in the sample.

To perform functional characterization of CTCs, Pantel's group, in collaboration with Alix-Panabières' team, developed the EPISPOT assay, where cells are cultured in a dish with antibodies to secreted tumor proteins, the cells are washed off, then a set of fluorescent-conjugated antibodies is used to detect the bound antibodies. This assay can be used in several perturbations for functional studies. Cells lines of DTCs have also been established for functional analyses.

The advances in detecting and characterizing circulating/disseminated tumor cells will help researchers estimate risk for metastatic relapse or progression, stratify patients and monitor therapy, identify therapeutic targets and resistance mechanisms, and gain greater insights into the biology of metastatic development, Pantel noted.

Novel techniques to enhance surgery

Figure 23. Fluorescent nerve labeling could help surgeons avoid dangerous incisions during tumor excision operations. Lower panel shows the efficacy of combining fluorescent labeling techniques for highlighting nerves.

Although drug discovery gets most of the attention in discussions of cancer treatment, Quyen Nguyen of the University of California, San Diego, reminded the audience that surgery is still the primary way to treat the disease when it is first detected. Nguyen and her colleagues are developing new technologies to aid surgeons in their efforts to remove tumors completely without damaging nearby nerves. To do this, they have constructed activatable cell penetrating peptides, fluorescently labeled polycationic peptides coupled via a cleavable linker to a neutralized polyanionic peptide. Proteases expressed by diseased tissues cleave the linker and the activated peptide binds to the diseased tissue. This enables surgeons to visualize the tumor more effectively and ensure that they are not leaving behind cancerous tissue. Using a related technique, they have been able to fluorescently label nerves (Figure 23), which will help surgeons avoid severing them during tumor removal.

"Next generation sequencing"

Technology that facilitates inexpensive sequencing of individual genomes will be required to realize the goal of personalized medicine fully. Brian Pollok, speaking for John McPherson of the Ontario Institute for Cancer Research in Toronto, reviewed the three main platforms for "next generation sequencing" and presented examples of the kinds of information about genetic alterations these techniques can produce. Innovations in genomic sequencing techniques have allowed researchers to identify chromosomal rearrangements, compare the genome of tumor cells with normal cells from the same patient, and find personalized tumor biomarkers, such as chromosomal rearrangements, to study the progression of disease and the efficacy of therapy. Going forward, there is intense interest in single molecule sequencing that requires no pre-amplification and that would allow researchers to establish haplotypes (a set of alleles that are located on the same chromosome).

Looking for biomarkers in public databases

There is a lot of gene expression information available in public databases that can be mined for breast cancer research. Michèl Schummer of the Fred Hutchinson Cancer Research Center described his work looking for early markers of poor-prognosis breast cancer, comparing tumor and adjacent tissue samples from poor-outcome breast cancer patients and tissue from healthy women who had undergone breast reduction surgery. Using the public information to guide their choice of potential targets, he used PCR to screen for differential expression among the three groups. He found three genes that showed elevated expression in cancers with poor outcome measures and low expression in all other tissues. These might be useful as blood markers for early detection of breast cancers with poor prognoses.

All of the techniques presented in the technology workshop highlight the important contribution of technological advances in moving research forward.