The Genom of Homo sapiens.pdf

26 WILSON ET AL.Prostate CancerProstate cancer is responsible for 3% of all deaths inmen over 55 years of age and is second only to lung canceras a cause of cancer death in the western world(Chakravarti and Zhai 2003). Here, prostate cancer is themost commonly diagnosed cancer, predominatelythrough the use of the serum marker prostate-specificantigen (PSA) assays. Numerous epidemiological andmolecular biological studies have accumulated evidencethat favors a significant but heterogeneous hereditarycomponent in prostate cancer susceptibility (Stanford andOstrander 2001). There have been seven susceptibilityloci mapped to date, with three loci having been characterizedfor specific genes and their constituent mutations.The role of these loci in hereditary and sporadic disease isstill debatable, however, and it could be very modest(Verhage and Kiemeney 2003). However, besides ageand race, family history is the only well-established riskfactor, where epidemiological studies show that first-degreerelatives of prostate cancer patients have a two- tothreefold increased risk of prostate cancer (Steinberg etal. 1990). The mode of heritability is still subject to debateand, indeed, may be different for early-onset versuslate-onset disease.Recently, multiple genes have been identified with putativerelevance to prostate carcinogenesis, with a modelfor prostate cancer progression that includes the potentialcontribution of inflammation to the development of preneoplasticor neoplastic lesions. The different stages ofprostate carcinogenesis are characterized more by the accumulationof multiple somatic genome alterations thanby any one genetic lesion, although specific changes mayincrease the likelihood of further neoplastic transformation.Overall, an understanding of the contributions of somaticgene defects to prostate carcinogenesis may facilitatethe development of novel targeted therapeuticapproaches for clinical management or more specific diagnosis(Gonzalgo and Isaacs 2003). To this end, we haveelected to profile the mutational spectrum of two broadgroups of the human “kinome” (Manning et al. 2002);namely, the genes that encode tyrosine kinase and tyrosine-kinase-likeproteins. The role of kinases as biologicalcontrol points, the causal role of protein kinase mutationsand dysregulation in human disease, and theirtractability as drug targets make these genes logicalchoices for our study of prostate carcinogenesis.Our approach to this analysis has focused on mRNAtranscripts purified from tumor tissue and reverse-transcribedinto cDNA templates. This strategy was chosen inorder to minimize the number of primers required for amplificationand sequencing, thereby maximizing the numberof patient samples that can be evaluated.Acute Myeloid LeukemiaAcute myeloid leukemia (AML) is the most commonform of leukemia and the most common cause of deathfrom leukemia. Although conventional chemotherapycan cure 25–45% of patients, most either die of relapse orfrom complications associated with treatment (Stirewaltet al. 2003). AML is a heterogeneous disease, characterizedby a myriad of genetic defects that have been foundto include translocations involving oncogenes and transcriptionfactors, activation of signal transduction pathways,and alterations to growth factor receptors. It appearsthat these multiple affected pathways interact in theonset of leukemogenesis, such that multiple genetic abnormalitiesare necessary for the development of overtleukemia (Kelly et al. 2002). Consequently, it is thoughtthat a single target of therapy may be widely useful intreating the spectrum of AML subtypes.Mutations in several receptor tyrosine kinases (RTKs)have been frequently described in AML, including FMS,KIT, and FLT3. FLT3 is the most frequently mutatedRTK in AML, found in ~15–40% of AML patients(Nakao et al. 1996; Kiyoi et al. 1997, 1988; Yokota et al.1997; Iwai et al. 1999; Xu et al. 1999; Abu-Duhier et al.2000, 2001; Meshinchi et al. 2001; Stirewalt et al. 2001;Yamamoto et al. 2001; Schnittger et al. 2002; Thiede etal. 2002). Two common mutations have been found inFLT3, both of which constitutively activate the receptor.One is an internal tandem duplication (ITD) within exons11 and 12 whose length varies from 3 to >400 bp(Schnittger et al. 2002). The prevalence of FLT3 ITDsvaries with age, from 10–15% in pediatrics to 25–35% inolder adults. The other is a point mutation in exon 17, typicallyin codon 835, which is found in 5–10% of AML patients(Abu-Duhier et al. 2001; Yamamoto et al. 2001).In addition to the FLT3 mutations, mutations in theRAS gene superfamily occur in ~15–25% of AML cases(Bos et al. 1987; Farr et al. 1988; Bartram et al. 1989;Radich et al. 1990; Vogelstein et al. 1990; Neubauer et al.1994). These are predominantly point mutations incodons 12, 13, and 61 and act to prevent the conversionof RAS from an active to an inactive form, thus constitutivelyactivating downstream pathways (Byrne and Marshall1998). In general, our hypothesis is that AML cellscontain a variety of acquired mutations, such as those describedabove, but that only some of them are relevant fordisease pathogenesis. To characterize these mutationsand to begin associating their presence with disease initiationand progression, we are producing mutational profilingdata for ~450 candidate genes, initially using samplesderived from a cohort of 47 AML patients for whichbanked tumor (bone marrow) and germ-line (skin) samplesare in hand. The resulting mutational profiles will becorrelated back to the AML subtypes and clinical outcomesfor these patients, as a means to discover and characterizethe mutations (and other polymorphisms) thatcan be putatively associated with AML pathogenesis.This set of activities constitutes a “discovery phase” ofthe AML project, and will culminate in the selection of asmaller number of genes to be the subjects of further mutationalprofiling work on a second set of AML patientswith matched tumor and germ-line samples. Other typesof AML studies, funded by this project, also will be consideredin the selection of genes to be examined in thissecond, or “validation phase” of the project. These studiesinclude functional genomics-based approaches usingseveral mouse models of AML, as well as microarraybasedcomparative genome hybridization studies of