6 The Role of extreme phenotype selection in cancer research

The Role of Extreme Phenotype Selection in Cancer Research Jose Luis Perez-Gracia, Maria Gloria Ruiz-Ilundain, Ignacio Garcia-Ribas, and Eva Carrasco

Introduction

The validity of such an approach is unquestionable, as reflected by some outstanding results that have been achieved with it. Some relevant examples are the correlation between the level of expression of thymidylate synthase and the efficacy of 5-fluorouracil in digestive tumors (Johnston et al., 1994) or the identification through microarrays of different gene-expression patterns in patients with diffuse large B-cell lymphoma (Alizadeh et al., 2000). This new methodology for classification of lymphoma offers important advantages over traditional clinical and pathologic classifications. However, it is clear that not all trials have achieved relevant results, and during the last few years we have seen an enormous proliferation of studies that reach conclusions of uncertain clinical significance, sometimes even contradicting previous results from similar studies. This fact has resulted in the need to design specific guidelines to validate the quality of such studies prior to publication (Editor, 1999). Several factors underlie this problem, and their detailed review is out of the scope of the present work, so we will focus on some of the most relevant ones. First, there is the possibility that, even if the genetic alterations studied are truly related to the outcome of the disease, they may not be the only or the main prognostic factor. In contrast with diseases caused by fully

The availability of modern techniques of molecular biology has opened a new era in the field of genetics. These techniques are used in a pure laboratory environment and in the setting of translational research, which represents the integration of basic science and clinical findings to improve our understanding of the biology underlying the clinics. In the field of cancer research, many of those studies have been focused on correlating the genetic characteristics of patients with cancer, either from normal or tumoral tissue, with their prognosis or with the efficacy of the treatments that are used. These studies follow the hypothesis that different individuals or tumors might harbor diverse genetic characteristics that may correlate with their prognosis. The selection of the genetic characteristics that are studied is usually based on theoretic hypotheses that correlate preclinical knowledge with tumor biology or with the mechanisms of action of antitumor agents. Nonetheless, with the incorporation of high-throughput techniques, such as microarrays or serial analysis of gene expression (SAGE), it has become common to screen the expression of large numbers of genes at the same time, with or without potential correlation with the end point studied. Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 2: Molecular Pathology, Colorectal Carcinoma, and Prostate Carcinoma

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Copyright 9 2005 by Elsevier (USA) All rights reserved.

66 penetrant genetic alterations, such as cystic fibrosis, in which the presence of the genetic alteration is inexorably linked with the development of the disease, cancer is usually a complex and multifactorial disease. Therefore, cancer prognosis depends on the interaction of many intrinsic and extrinsic factors. Consequently, the identification of genetic alterations that are truly associated with a significantly different prognosis becomes a difficult task because most of them have a reduced penetrance. Second, in most cases the patients selected for these studies do not have a truly characteristic phenotype. Instead, they belong to a general patient population that is classified in terms of their good or bad prognosis using conventional efficacy parameters such as survival or clinical response. This complicates the process of identifying genetic factors that correlate with a characteristic prognosis because in many instances none of the patients have a truly characteristic outcome. Moreover, a large part of the variability observed might be explained by clinical factors. Third, many studies are of retrospective nature and have a low potential to establish relationships of causality. Instead, they are just valid for generation of hypotheses. Moreover, the thresholds to define the relations between the factors studied and the prognosis are frequently based on retrospective statistical calculations, which may lead to additional biases. Last, technical and methodologic differences in the way that laboratory work is performed, and the intrinsic heterogeneity of any technique, contribute to more confusion in the interpretation of the results. The possibility to solve most of the previously mentioned issues is somewhat remote. We cannot change the nature of the disease, and the techniques that we have today, although constantly improving, have some limitations. Similarly, although some prospective studies are moving forward, cost and time will always limit their feasibility and perhaps their value. Yet, the inadequate selection of the phenotypes studied could provide a valid frame to improve the design of current trials. Indeed, a potentially more efficient strategy to isolate the genetic features associated with characteristic outcomes could be to focus research in few subjects with extreme, truly differentiated phenotypes. Such individuals may have a greater chance of carrying characteristic genotypes that are responsible for their distinctive prognosis than the general population. Such an approach is not new in medical research and has been used successfully before, including in oncology. The study of individuals either affected by multiple tumor syndromes, and/or with a markedly increased familiar risk of developing cancer, has led to the discovery of genetic alterations that explain such situations. Some well-known examples are the detection of p53

I Molecular Pathology germ-line mutations in patients with the Li-Fraumeni syndrome (Malkin et al., 1990), described through the identification of an excess in the risk of death by rhabdomyosarcoma in siblings (Li et al., 1969; Miller, 1968), or the finding that patients with hereditary retinoblastoma present an inactivation of both copies of the retinoblastoma tumor-suppressor gene (Cavenee et al., 1983), as was wisely predicted by Knudson (1971). Nonetheless, the selection of phenotypes with increased risk to develop cancer is not the only approach that has led to successful results. The identification of complete deficiency of dihydropyirimidine dehydrogenase activity in peripheral blood mononuclear cells of a patient who developed severe toxicity after administration of 5-fluorouracil is an illustrative example (Diasio et al., 1988). Although in the initial report the molecular explanation for such deficiency was not identified, subsequent studies have demonstrated that genetic alterations are associated with this deficiency of enzymatic activity (Van Kuilenburg et al., 1999). The common factor for all the previous examples is that the key step that led to the final discovery was the identification and the study of a characteristic phenotype. As seen, the yield of this strategy is very high because only a few patients~or even just one--need to be studied to identify which factors determine the phenotype. This is logical because the only hypothesis tested is whether the observed phenotype (which is so unique that chance does not play a part in it) is related with a determined cause. In contrast, the hypothesis that a specific alteration confers a determined prognosis, either improved or worsened, is more uncertain because it actually implies two hypotheses: that the patients studied really have a characteristic prognosis and that the factor being studied explains such difference. Therefore, it is more difficult to achieve reliable conclusions. Quite surprisingly, the success achieved by these and other similar studies has not led to the development of a research methodology to obtain the maximum benefit of this strategy. Rather than from a systematic and solid scientific approach, the identification of characteristic phenotypes has mostly been based on isolated observations from bright clinicians. Another interesting consideration is that the selection of characteristic phenotypes has usually been limited to subjects with negative phenotypes, mainly those presenting an increased risk to develop one or multiple tumors. This is logical because such individuals or families are relatively easy to identify because their high incidence of cancer is unusual. Nonetheless, the possibility that subjects with positive phenotypes exist should also be considered as an alternative to optimize the strategy of studying extreme phenotypes.

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6 The Role of Phenotype Selection in Cancer Research

As we have seen in the previous examples, the strategy of correlating very characteristic phenotypes with their respective genotypes has been very successful. Therefore, it is clear that a consistent methodology should be developed to standardize and to obtain the maximum benefit from this strategy. This methodology should not only be directed to the identification of negative phenotypes but should also contemplate the possibility of identifying individuals with markedly positive phenotypes, either because of unusually positive outcomes from diseases of bad prognosis or because of a low risk to develop those diseases. In the following sections we will develop these possibilities.

Phenotype Selection of Patients with Cancer with Long-Term Survival Nowadays, a majority of advanced solid tumors are considered to be incurable with current treatment alternatives, and their median survival is very low. However, even in the tumor types with lowest survivals, there are some rare exceptions. Some case reports of unexplained long-term survivors of diseases, such as gastric cancer (Miyaji et al., 1996), colon cancer (Mukai et al., 2000), pancreatic cancer (Silberstein et al., 2000), or myeloma (Dutcher et al., 1984), in apparently incurable situations can be found in the medical literature. Therefore, these individuals could represent examples of extreme phenotypes because their prolonged survival is highly unusual, considering their disease, and it is unlikely that this is a consequence of the treatment that they received. If the existence of patients with cancer presenting an unusually prolonged survival would be confirmed, their study perhaps would explain the causes underlying that fact. These causes could be related to tumoral factors (such as alterations in mechanisms of drug resistance or cell-cycle regulation) or host factors (such as differences in drug metabolism, immunologic response, or deoxyribonucleic acid [DNA] repair mechanisms) in addition to other potential external factors. Although these and other potential explanations represent many hypotheses to study, their limitation to one or few subjects would represent a great advantage to the efficiency of this research. Evidently, the finding of more than one individual within the same family with unexpected long-term survival following a confirmed diagnosis of a tumor with a very poor prognosis would strongly argue in favor of the hypothesis that such phenotype is characteristic and that it is secondary to genetic factors. Obviously, the histologic diagnosis and the disease staging of these individuals should be based on solid

evidence because it is possible that some of them may have been erroneously staged or even diagnosed of cancer, that being the underlying explanation for their characteristic prognosis.

Phenotype Selection of Individuals Potentially Protected from Developing Cancer It is well known that the risk of developing cancer is not uniform. Today we know that several genetic or environmental factors may increase the risk of individuals to develop certain types of cancer. Moreover, we also know that such increase in the risk is not uniform, but gradual. Whereas some of the factors such as passive or active smoking lead to mild or moderate increases, respectively, in the risk of cancer, other factors such as inactivation of both retinoblastoma tumor-suppressor genes markedly increase the risk of developing cancer. Therefore, assuming that the risk of cancer is not uniform, one further step would be to hypothesize that just as some individuals present an increased risk, other subjects may have lower risk than would be expected because of their environment and habits and that this decrease may also be gradual. These subjects would represent the left tail of a gaussian distribution showing the risk to develop cancer. If these individuals exist, their identification and the study of the causes of such protection would increase our knowledge about cancer and perhaps could also yield potentially useful treatments against it. This decrease in the risk to develop cancer could be secondary, for example, to factors related to improved mechanisms of DNA repair, cell-cycle regulation, metabolism of carcinogens, or immunologic response. Theoretically, the potential existence of such individuals could be supported by the intrinsic nature of the evolution process. Indeed, because cancer is a frequent and ancient disease (probably, inherent to life) organisms have developed mechanisms to protect themselves against neoplastic disorders, and probably the most efficient mechanisms have been selected during the course of evolution. However, the question remains whether individuals with genetic characteristics that confer them a significant protection against certain neoplastic disorders really exist. It is interesting to note that, if we look to diseases different than cancer, we will see that this is the case and that such an approach is again not new in medicine. Some intriguing examples can be used to illustrate that individuals with genetic alterations that confer protection against some diseases may exist, and, moreover, the use of an adequate methodology of phenotype selection may

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lead to identifying such alterations. Probably one of the most outstanding examples has been the identification of genetic alterations that confer to individuals that carry them a complete protection against infection by certain strains of the human immunodeficiency virus (HIV). The chemokine coreceptor CCR-5 allows the entry of HIV into its target cells, and it is well known that some alterations in the gene encoding CCR-5 confer to the individuals bearing them complete protection against the infection by certain strains of HIV. This phenotype has been observed in individuals with different genotypes: homozygotic deletions in the gene encoding CCR-5 (Liu et al., 1996) and heterozygotic mutations in CCR-5, when associated with the mentioned deletion in the other allele (Quillent et al., 1998). Quite interestingly, the discovery of these genotypes was based on the identification of the characteristic phenotypes of the individuals bearing them. Because the CCR-5 mutations had not been associated with any abnormalities, an astute observation that some individuals highly exposed to HIV never developed the infection gave the impetus to identify them (Rowland-Jones et al., 1995). Secondary to this observation, CCR-5 and other coreceptors for HIV have become a relevant target in the investigation of HIV infection. Another interesting example in which a characteristic phenotype has been successfully used to identify the underlying genotype is the relation between certain factor VII genotypes and the risk of myocardial infarction (Girelli et al., 2000). Elevated plasmatic levels of coagulation factor VII have been suggested to correlate with the risk of death as a result of coronary artery disease, and polymorphisms in the factor VII gene are associated with variations in levels of factor VII. Therefore, these polymorphisms were studied in 311 individuals with severe, angiographically documented coronary atherosclerosis, of whom 175 had a history of previous myocardial infarction and the rest did not. Among patients with no history of previous myocardial infarction, there was a significantly higher number of patients with determined genotypes than among patients with myocardial infarction. Therefore, this finding suggests that those genotypes confer a protection against developing myocardial infarction. This type of study must always be interpreted with because it is subject to a potentially high risk of bias, as reviewed by Gambaro et al. (2000). However, this example is of particular interest because the individuals that were found to be protected against ischemic disease had not developed myocardial infarction despite having a strong risk factor to develop it--documented coronary atherosclerosis~rather than being just normal healthy subjects (although the study did include a control

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group formed by healthy subjects). Therefore, there was a greater chance of finding true protective factors, rather than just absence of disease, as could be expected in a group formed by individuals with a normal population risk. The bottom line of both examples is that the study of subjects with a lower than normal risk of developing a disease may unveil protective host factors for that disease, just as the study of subjects with an elevated risk of developing cancer can lead to the discovery of cancerrelated genetic alterations, as we have seen in some of the examples presented before. In the field of cancer, preclinical evidence is available that does provide a proof for the potential existence of cancer-protective genetic alterations. One outstanding example is the development of a "super p53" mice model, which carries supernumerary copies of the p53 gene in the form of large genomic transgenes (Garcia-Cao et al., 2002). These mice show a decreased risk to develop rumors in comparison with wild-type mice, as was shown in several tumor-induction models using chemical carcinogens. These mice present completely normal phenotypes, including a normal lifespan and aging process, in contrast with other mice models that also overexpress p53 and show premature aging. This is probably because the additional p53 copies inserted are under normal regulatory control, as opposed to the other mice models. Although the insertion of additional copies of p53 in the genotype of these mice was artificially induced, their normal phenotype raises the question of whether this same phenomenon could have taken place through spontaneous mechanisms. In that case, the individuals affected would be partially protected against the development of certain tumors. In the clinical setting, several studies have targeted the identification of genetic profiles that might be associated with a decrease in the risk to develop cancer, although, as summarized later in this chapter, the results of most of them have not been encouraging. Some polymorphisms in the methylenetetrahydrofolate reductase gene have been associated with a decreased risk of developing acute lymphocytic leukemia (Skibola et al., 1999) or colorectal cancer (Chen et al., 1996) in certain population subsets. Polymorphisms of enzymes such as microsomal epoxide hydrolase (London et al., 2000), myeloperoxidase (London et al., 1997), or NAD(P)H:quinone reductase (Chen et al., 1999), which are involved in the metabolism of determined carcinogens, have also been related to some protection against lung cancer or colorectal cancer (Harth et al., 2000) in some population subsets and/ or ethnic groups. However, other studies, such as that of microsomal epoxide hydrolase, have shown no

6 The Role of Phenotype Selection in Cancer Research

association (Smith et al., 1997) or even an inverse relationship (Benhamou et al., 1998). Determined genotypes of some cytochrome P450 enzymes, such as CYP1A1 or CYP2D6, have also been correlated with a decreased risk of lung cancer. Nonetheless, metaanalyses have failed to confirm this observation (Christensen et al., 1997; Houlston et al., 2000) or have just described a small protective effect with a nonappreciable relationship to individual susceptibility (Rostami-Hodjegan et al., 1998). Two common polymorphisms of the p 2 1 W A F 1 / C i p l gene have been correlated with a potential protective role against ovarian cancer (Milner et al., 1999). Certain genotypes of the glutathione S-transferase M1 have also been related with the risk of lung cancer and other aerodigestive tract cancers, but again a meta-analysis has failed to confirm such results (Houlston et al., 1999). Some human leukocyte antigen (HLA) alleles have been linked in cases and control studies with a decreased susceptibility to renal cell carcinoma (Ozdemir et al., 1997), melanoma (Ichimiya et al., 1996), or lung cancer (Tokumoto et al., 1998). Finally, even women who are homozygotic for determined polymorphic alleles of the B R C A - 1 gene have been associated with a decreased risk of breast cancer (Dunning et al., 1997). The ambiguous and clinically not very relevant results of some of these studies may be explained by methodologic flaws, as detailed elsewhere (Gambaro et al., 2000), but may also be related in part with an inadequate selection of the populations studied. In contrast with the former studies in which subjects were selected by a very characteristic phenotype~a definite protection against developing HIV or myocardial infarction was observed despite high risk factors. The later studies compared the risk of patients who have developed cancer with a control group formed by normal subjects. In these subjects, the risk of developing cancer was probably neither increased nor decreased, perhaps the only exception being that some groups were formed by smokers, and it was therefore unlikely that clinically relevant information would be discovered. A potentially more efficient approach would be to study individuals with a truly characteristic phenotype that indicates that they have a markedly reduced familiar or individual risk to develop cancer. Families with a very low or ideally null incidence of cancer over several generations (perhaps despite crossing with high-risk families), could be considered to have a reduced familiar risk. Subjects that do not develop cancer despite important exposure to widely recognized intrinsic factors or extrinsic ones could be considered to have a reduced individual risk. An example of individuals protected against cancer despite high intrinsic risk factors could

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be potential subjects with familiar adenomatous polyposis developing cancer significantly later than would be expected or not developing it at all. Also, an example of an individual protected against cancer despite high extrinsic risk factors could be a subject that does not develop a tumor despite heavy exposure to radiation. Different combinations of these and other strategies or different ones could also be pursued, always realizing that if such families or individuals exist, it would be naive to attribute their characteristic phenotype to chance, at least until other causes have been ruled out. Evidently, the chance of yielding positive results always will be directly related to the discrepancy between the risk of developing cancer and the actual phenotype. This methodology raises a number of issues. The main one is obviously how to select the individuals to study. In some cases, such as the selection of patients with cancer with long-term survival, it is clear that the best way would be to do it through the physicians that see those patients, and, therefore, appropriate training and awareness should be created among them. However, in the case of individuals potentially protected from cancer, patient selection becomes more complicated because they are in fact healthy subjects. Therefore, quite complex epidemiologic studies would be required. A second issue is what should be studied in these subjects, if they are identified. As we have hypothesized, in the case of long-term survivors, their phenotype may be the result of either intrinsic factors (related to the tumor or to the host) or to external ones. Therefore, all of them should be analyzed, and ideally samples from the tumor, the host, and environmental factors should be studied. In the case of individuals potentially protected from cancer, only host and environmental factors would need to be studied. One important limitation is the kind of samples that can be collected. Evidently, the availability of fresh tissue would be preferred because it makes it possible to perform a greater variety of studies, including highthroughput techniques, which would be useful for this kind of research, given their high potential to screen the expression of numerous genes. Nonetheless, it is clear that these samples or even others, such as paraffinembedded tissue, will not always be available. Therefore, access to adequate material might be an important limitation to perform these type of studies. Lastly, ethical issues are another potential point of concern because the study and the storage of genetic materials are subject to strict regulations to protect the privacy and the rights of the individuals. This becomes even more problematic if we consider the possibility

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that some of the target individuals might have been lost to follow-up, or they may not even be alive. Moreover, one additional ethical problem would be to approach individuals and try to estimate their risk to develop cancer as being high or low without being certain about it and without knowing the consequences that this may imply for those people. In summary, the study of individuals with very characteristic phenotypes has been useful to describe the mechanisms underlying them. This has been confirmed in subjects with high risk to develop determined diseases and in others who seem to be protected against certain diseases. Therefore, it seems logical to pursue this strategy as a valid methodology for the study of other diseases, including cancer. We propose to create databases compiling clinical and environmental information and appropriate samples from individuals who are either long-term survivors of theoretically incurable tumors or who seem to be protected against certain neoplastic disorders. The study of such data could perhaps help to provide a useful interpretation of the information that the sequencing of the human genome is yielding and could help to increase our current knowledge of cancer and to discover new therapeutic strategies against this disease. Even in the age of computer-aided molecular biology, observation should remain a better way to generate valid hypothesis than speculation.

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6 The Role of Phenotype Selection in Cancer Research Miller, R.W. 1968. Deaths from childhood cancer in sibs. N. Engl. J. Med. 279:122-126.

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Rowland-Jones, S., Sutton, J., Ariyoshi, K., Dong, T., Gotch, E, McAdam, S., Whitby, D., Sabally, S., Gallimore, A., and Corrah, T. 1995. HIV-specific cytotoxic T-cells in HIV-exposed but uninfected Gambian women. Nat. Med. 1:59-64. Silberstein, E., Walfisch, S., Lupu, L., and Sztarkier, I. 2000. Twelveyear survival after the diagnosis of locally advanced carcinoma of the pancreas: A case report. J. Surg. Oncol. 75:142-145. Skibola, C.E, Smith, M.T., Kane, E., Roman, E., Rollinson, S., Cartwright, R.A., and Morgan, G. 1999. Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. Proc. Natl. Acad. Sci. U.S.A. 96:12810-12815. Smith, C.A., and Harrison, D.J. 1997. Association between polymorphism in gene for microsomal epoxide hydrolase and susceptibility to emphysema. Lancet 350:630-633. Tokumoto, H. 1998. Analysis of HLA-DRBl-related alleles in Japanese patients with lung cancer-relationship to genetic susceptibility and resistance to lung cancer. J. Cancer Res. Clin. Oncol. 124:511-516.

Van Kuilenburg, A.B., Vreken, P., Abeling, N.G., Bakker, H.D., Meinsma, R., Van Lenthe, H., De Abreu, R.A., Smeitink, J.A., Kayserili, H., Apak, M.Y., Christensen, E., Holopainen, I., Pulkki, K., Riva, D., Botteon, G., Holme, E., Tulinius, M., Kleijer, W.J., Beemer, EA., Duran, M., Niezen-Koning, K.E., Smit, G.P., Jakobs, C., Smit, L.M., Moog, U., Spaapen, L.J., and Van Gennip, A.H. 1999. Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency. Hum. Genet. 104:1-9.