Presenter : CR 周益聖 Supervisor: VS 曾主任 Cell 152, 714–726, February 14, 2013

Chronic lymphocytic leukemia CLL – Mature B lymphocyte ≧ 5000/uL in the peripheral blood Monoclonal B-lymphocytosis – Mature B lymphocyte < 5000/uL – No lymphadenopathy, organomegaly, cytopenia or symptoms Blood. 2008;111: ASH image bank

Staging NCCN Guideline V

Treatment indications Not indicated for Rai stage 0 or Binet stage A Absolute lymphocyte count alone is not an indication for treatment unless above x 10 /L or symptoms related leuastasis Binet stage C or Rai stage III or Rai stage IV progressive marrow failure / worsening of anemia and/or thrombocytopenia progressive or symptomatic splenomegaly progressive or symptomatic lymphadenopathy Progressive lymphocytosis – an increase of more than 50% over a 2-month period – lymphocyte doubling time (LDT) of less than 6 months Autoimmune anemia and/or thrombocytopenia that is poorly responsive to corticosteroids NCCN Guideline V Blood. 2008;111:

17p with poor prognosis N Engl J Med 343: , 2000

Unmutated IGHV as poor prgnositic factor of OS in CLL Blood Sep 15;94(6): Un vs mut: 117 vs 293 months, P=0.001

Whole exome sequencing Affordable, rapid, and comprehensive for detecting somatic coding mutations High sequencing depth (typically 100X–150X) Coding mutations likely encompass many of the important driver events that provide fitness advantage for specific clones Low cost of WES permits studies of large cohort to assess the impact of clonal heterogeneity on disease outcome

What are the Mutations In CLL? Fig 1

Will Age or IGHV status affect clonal or subclonal mutations? Fig 2

posterior probability distribution over CCF c : Consider a somatic mutation observed in a of N sequencing reads on a locus of absolute somatic copy number q in a sample of purity α. The expected allele-fraction f of amuta- tion present in one copy in a fraction c of cancer cells

Clonal mutation

Subclonal mutations

Comparison of mutational spectrum between subclonal and clonal sSNVs Mutation spectrum are similar in clonal and subclonal sSNVs

When did clonal or subclonal mutations occur? Fig 3

9/12(MYD88) and 12/14(Tri12) had at least one other mutations

Will clonal or subclonal evolution occur in chemotheray treated patient? Fig 4

C/T Untreated

C/T Treated

Genetic Evolution and Clonal Heterogeneity Result in Altered Clinical Outcome

Presence of Subclonal Drivers Mutations Adversely Impacts Clinical Outcome

Multivariate Cox regression models All patients 62 patients who had at least one driver

Proposed model of CLL clonal evolution Fig7

Discussion

Why CLL? Slow growing B cell malignancy – extended window for observing the process of clonal evolution Highly variable disease course Diverse combinations of somatic mutations  Presence, diversity, and evolutionary dynamics of subclonal mutations in CLL contribute to the variations observed in disease tempo and response to therapy

Different periods of clonal evolution First period prior to transformation – Passenger events accumulate in the cell that will eventually be the founder of the leukemia in proportion to the age of the patient Second period – the founding CLL mutation appears in a single cell and leads to transformation – Recurrent clonal mutations across patients del(13q), MYD88, and trisomy 12 Third period – subclonal mutations expand over time as a function of their fitness-integrating intrinsic factors (proliferation and apoptosis) and extrinsic pressures (interclonal competition and therapy) ATM, TP53,or RAS mutation

How does treatment affects evolution? Clonal equilibrium – Untreated patients – relative sizes of each subclone were maintained – some subclones emerge as dominant – more time is needed for a new fit clone to take over the population in the presence of existing dominant clones Emergence of fitter clones – treated patients – cytotoxic therapy typically removes the incumbent clones – shifts the evolutionary landscape in favor of one or more aggressive subclones – highly fit subclones likely benefit from treatment and exhibit rapid outgrowth

Therapeutic implications Presence of pretreatment subclonal driver mutations anticipated the dominant genetic composition of the relapsing tumor – therapies to prevent the expansion of highly fit subclones Potential hastening of the evolutionary process with treatment provides a mechanistic justification for the empirical practice of ‘‘watch and wait’’ as the CLL treatment paradigm Detection of driver mutations in subclones (a testimony toan active evolutionary process) may thus provide a prognostic approach in CLL

Conclusion WES to study tumor heterogeneity and clonal evolution – readily adopted for clinical applications Importance of evolutionary development as the engine driving cancer relapse Develop therapeutic paradigms that not only target specific drivers (i.e., ‘‘targeted therapy’’) but also the evolutionary landscape of these drivers