Samuel Aparicio, B.M., B.Ch., Ph.D., and Carlos Caldas, M.D. The Implications of Clonal Genome Evolution for Cancer Medicine N ENGL J MED 2013;368:842-51 (Feb 28, 2013) Samuel Aparicio, B.M., B.Ch., Ph.D., and Carlos Caldas, M.D. R3 변자민

INDEX Darwinian Evolution & Cancer The Decipherment of Clonal Evolution Implications for Clinical Oncology Genomic stratification Tumor monitoring The emergence of treatment resistance Mutational and clonal assessment in practice Implications for Drug Development and Clinical Testing of New agents It has long been hypothesized that screening for colorectal cancer can affect mortality from the disease in two ways: By detecting cancers at an early and curable stage By detecting and removing adenomas

DARWINIAN EVOLUTION & CANCER Darwin’s theory of evolution: “speciation” fundamental property of cancer “Tumor evolution” now a hot issue with the advent of comprehensive cancer genome sequencing technology It has long been hypothesized that screening for colorectal cancer can affect mortality from the disease in two ways: By detecting cancers at an early and curable stage By detecting and removing adenomas

‘Clonal structure and evolution in cancers’ by Peter Nowell in 1976 Clone: a group of cells related to each other by descent from a unitary origin Clonal relationship among cells arise with ‘selection’ operating on phenotypes Stable phenotype Consequence of fixed mutation (e.g. oncogenic mutations, drug-sensitivity mutations) Consequence of genetic drift (mutation over time) without selection Transient or unstable phenotype Not evident as lineage relationship in the genome

Figure 1. Clonal Evolution and Clonal Relationships

Human tumors are composed of evolving clones Hallmarks of Cancer Existence of clonal genotypes (i.e., not all mutations occur in the same cells) Expansion and decline of clonal population over time Existence of internal spatial variation in tumor composition Partial tumor responses to therapy and the emergence of drug-resistant malignant cells Seeding of metastatic cells from subclones Absence of an observable clonal structure based on genome aberrations in some cancers Existence of neutral clonal relationship without discernible phenotypic consequences

DECIPHERMENT of CLONAL EVOLUTION The next-generation sequencing device now provides a measure of allele prevalence for almost any aberration found in a genome ! It has long been hypothesized that screening for colorectal cancer can affect mortality from the disease in two ways: By detecting cancers at an early and curable stage By detecting and removing adenomas

Figure 2. Next-Generation Sequencing Measurements of Allele Prevalence and Clonal Analysis

3 key concepts Clonal-mutation prevalence is a compound measure of the population abundance of the mutation in question A clonal genotype refers to the set of common fixed mutations that define a clone Clonal lineage defines the relationship between clones as they evolve over time

→ Another important implication: Figure 3. Changes in Clonal Composition over Time, Estimated on the Basis of Changes in Clonal-Mutation Prevalence Determination of clones with genotypes that confer risk of progression or drug resistance → Another important implication: spatial and temporal variation in the composition of primary cancer (e.g. breast cancers, renal cancers) → important consequence:

IMPLICATIONS for CLINICAL ONCOLOGY I. Genomic Stratification Genomically distinct clones → variable tx responses Distinguish clinically relevant minor subclones Multiple sampling of primary tumors with or without assistance from functional imaging Stratification of targeted tx in metastatic disease Discordance of mutations between primary and corresponding metastatic disease Characterizing and measuring clonal heterogeneity is the key: important to rebiopsy tumors in metastatic disease II. Tumor Monitoring III. Emergence of Treatment Resistance IV. Mutational & Clonal Assessment in Practice

IMPLICATIONS for CLINICAL ONCOLOGY I. Genomic Stratification II. Tumor Monitoring III. Emergence of Treatment Resistance IV. Mutational & Clonal Assessment in Practice

Blood sample as liquid biopsy Bloodstream dissemination Blood sample as liquid biopsy Circulating tumor DNA sequencing: assess tumor dynamics (recurrence and tumor burden monitoring) Direct sequencing of plasma DNA: identify new mutations in circulating tumor (ctDNA) → early identification of resistance to targeted therapies death in situ Tumor DNA

post curative lobectomy Primary tumor c chromosomal rearrangement (e.g. ROS1 translocation) Post-mastectomy chemoTx Figure 4-1. Concept of Plasma DNA for Monitoring Circulating Tumor DNA and Clonal Evolution

KRAS wild type metastaic colorectal cancer Cetuximab treatment 3 months BRCA1 germline mutation ovarian cancer TP53 mutation in ctDNA Figure 4-2. Concept of Plasma DNA for Monitoring Circulating Tumor DNA and Clonal Evolution

IMPLICATIONS for CLINICAL ONCOLOGY I. Genomic Stratification Systemic genomic sampling of tumors a) before treatment, b) at the completion of treatment, and c) at the time of relapse Determining the mechanism of drug resistance Finding combinations of drugs to suppress clonal evolution II. Tumor Monitoring III. Emergence of Treatment Resistance IV. Mutational & Clonal Assessment in Practice Treatment of recurrent disease

IMPLICATIONS for CLINICAL ONCOLOGY I. Genomic Stratification II. Tumor Monitoring How might clonal analysis be used in practice in the near term? III. Emergence of Treatment Resistance IV. Mutational & Clonal Assessment in Practice Provision of targeted deep-sequence coverage for analysis of clonal-mutation prevalence by clinical genomics laboratories Example: KRAS mutations to stratify patients with colon cancer for EGFR mutations to predict benefits from erlotinib or gefitinib

IMPLICATIONS for DRUG DEVELOPMENT & CLINICAL TESTING of NEW AGENTS The genetic variability of cancer and its capacity to evolve mean that most single-target approaches are not sufficient The ability to follow which clonal genotypes are sensitive and which are resistant could be valuable in both the and late stages of drug development The mutational landscape of cancers suggests a vast array of possible potentially targetable pathways by prospectively developing sequenced polyclonal xenografted human tumors by mapping clonal genotypes of therapeutic resistance and sensitivity in patients

Mutation sequencing is gradually becoming adopted CONCLUSIONS Mutation sequencing is gradually becoming adopted →The clinical-trials community will need to retool to incorporate tissue biopsies and ongoing studies of tumor evolution as part of the correlative science of clinical trials