In this webinar, Medpace experts discuss key clinical, operational and laboratory considerations, lessons-learned, and best practices for accelerating the global development of safe and effective targeted therapeutics, using acute myeloid leukemia (AML) to highlight the complexities.

1. CHALLENGES AND CONSIDERATIONS IN
CLINICAL DEVELOPMENT OF TARGETED
AML THERAPIES

2. PROGNOSTIC
CLASSIFICATION OF
AML AND TARGETED
TREATMENTS
GREGORY A. HALE, M.D.

3. M A K I N G T H E C O M P L E X S E A M L E S S
ACUTE MYELOID LEUKEMIA
• Hematologic cancer arising from the clonal expansion of immature
malignant hematopoietic cells of the granulocytic or monocytic
lineage
‒ Cytopenias result in infection, hemorrhage, anemia
‒ Typically ill at diagnosis
• May be de novo, therapy-related, or arise from another marrow
disorder (myelodysplasia or myeloproliferative neoplasm)
• Most common leukemia in adults
‒ Median age onset 70 years
• 21,000 cases/year in US in 2019
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4. M A K I N G T H E C O M P L E X S E A M L E S S4

5. M A K I N G T H E C O M P L E X S E A M L E S S
Porwit and Saft, Journal Hematopathology 2011
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6. M A K I N G T H E C O M P L E X S E A M L E S S
ACUTE MYELOID LEUKEMIA
• Rapidly fatal without therapy
‒ Intensive chemotherapy and/or targeted therapies
‒ Death from disease or treatment-related complications
• Risk stratification based on cytogenetic and molecular alterations
‒ AML M3 is treated differently and has a better prognosis
‒ New agents available for AML subtypes
• 5-year overall survival rates vary by patient age
‒ 40% in young adults (18-60 years)
‒ 10% in older adults
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7. M A K I N G T H E C O M P L E X S E A M L E S S
AML THERAPY
• No change in therapy for 4 decades
‒ Cytarabine and anthracycline induction
• Considerations
‒ AML M3 (APL, promyelocytic) is treated differently
‒ AML risk group
‒ Patient age/condition: older versus younger patients
• Induction chemotherapy
‒ Cytarabine/daunorubicin: “7+3 regimen”
‒ Azacitidine or decitabine with venetoclax
• Post-remission therapy
‒ Intermediate dose cytarabine
‒ Allogeneic HSCT
• Supportive care
‒ Infection, bleeding, organ dysfunction
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8. M A K I N G T H E C O M P L E X S E A M L E S S
EVOLUTION OF AML THERAPY
• Improved characterization of genetic and molecular basis of AML
• Elucidated disease heterogeneity
‒ Numerous distinct disease subtypes recognize
• Define AML risk stratification
• Develop risk-adapted therapy
‒ Targeted against a specific abnormality
‒ Targeted against a specific pathway
‒ Targeted against a specific AML subset
• Within last 3 years, FDA has approved 8 new targeted agents for the
treatment of AML
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9. M A K I N G T H E C O M P L E X S E A M L E S S
TARGETED THERAPIES
• Cancer treatments that target the cancer’s specific genes, proteins, or
tissue environment
• Examples include monoclonal antibodies and small molecule drugs
‒ Breast (HER2), colon (VEGF, EGFR). Lung (EGFR, ALK, ROS), melanoma (BRAF)
• Requires a molecular knowledge of the cancer and the ability to match the
targeted therapy to the patient
‒ May be tumor agnostic or site agnostic: NTRK fusion
• On target, off tumor toxicities can be significant and different from
traditional cancer therapies
‒ Specificity for the target
‒ Normal tissue distribution
• Can be used alone or in combinations with chemotherapy and radiation
therapy
• May require development of companion diagnostic assay
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10. M A K I N G T H E C O M P L E X S E A M L E S S
IDENTIFICATION OF TARGETS
• Cytogenetics
• Flow cytometry
• Molecular analysis
• Next-generation sequencing (NGS)
‒ High-throughput method used to determine a portion of the nucleotide
sequence of an individual’s genome
‒ Utilizes gene sequencing technologies capable of processing multiple DNA
sequences in parallel
• Cellular abnormalities often occur in combination rather than in
isolation
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11. M A K I N G T H E C O M P L E X S E A M L E S S
NEW AGENTS FOR AML IN LAST 3 YEARS
• Midostaurin and gilteritinib
• Gemtuzumab ozogamicin
• Venetoclax
• Ivosidenib and enasidenib
• CPX-351
• Glasdegib
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12. M A K I N G T H E C O M P L E X S E A M L E S S12
Agent Mechanism FDA approval date Comments
Midostaurin FLT3 inhibitor April 2017 1st TKI approved for AML
1st approved for mutation-specific non-APL
Gemtuzumab
ozogamicin
Antibody-drug
conjugate targeting
CD33
July 2017 newly diagnosed or relapsed/refractory
CD33+ AML alone or with standard
chemotherapy
Enasidenib IDH2 inhibitor August 2017 Accelerated approval
CPX-351* Liposomal cytarabine
& daunorubicin
August 2017 Therapy-related and MDS-related AML
Ivosidenib IDH1 inhibitor July 2018 Single agent
Venetoclax BCL2 inhibitor November 2018 Accelerated approval in combination with
hypomethylating agent or low dose
cytarabine
Glasdegib Inhibits SMO,
hedgehog pathway
November 2018 in combination with low dose cytarabine for
elderly AML patients
Gilteritinib FLT3 inhibitor November 2018 relapsed or refractory AML with FLT3
mutation

13. M A K I N G T H E C O M P L E X S E A M L E S S
TYPES OF TARGETED AGENTS
• Mutation-targeted agents
‒ Act on oncogenic effectors of AML-associated mutations
‒ FLT3 and IDH inhibitors
• Nonmutation-targeted novel agents
‒ Disrupt metabolic or cell maintenance pathways without directly damaging
DNA or its repair
‒ Epigenetic modifiers and agents targeting apoptosis
• Targeted delivery of cytotoxic agents
‒ Antibody-drug conjugates
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14. M A K I N G T H E C O M P L E X S E A M L E S S
• FLT3: tyrosine kinase on hematopoietic cells
‒ Fms: fms-related tyrosine kinase 3 (FLT3)
‒ 25-30% of AML patients have FLT3 internal tandem (FLT3-ITD) mutation
‒ 5-10% of AML patients have FLT3 tyrosine kinase domain (FLT3-TKD)
mutation
• FLT3-ITD associated with higher WBC at diagnosis
• Higher allelic ratios associated with poor prognosis due to high
relapse rate
• Prognostic significance can be altered by the presence of other
mutations
‒ Concomitant NPM1 mutations have better prognosis than those without
MUTATION TARGETED AGENTS
FLT3 INHIBITORS
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15. M A K I N G T H E C O M P L E X S E A M L E S S
• Midostaurin: TKI that is active against several kinases
‒ VEGF, PDGF, KIT
‒ Randomized trial showed 23% reduction in risk of death
‒ Effect enhanced with chemotherapy
‒ Approved in combination with standard 7+3 chemotherapy
‒ Considered a component of post-remission therapy
• Gilteritinib
‒ Randomized trial showing improved CR rate (34% vs 15%)
• Newer generation FLT 3 inhibitors are being evaluated
‒ Crenolanib, quizartinib
MUTATION TARGETED AGENTS
FLT3 INHIBITORS
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16. M A K I N G T H E C O M P L E X S E A M L E S S
• IDH exists in 3 isoforms
‒ Catalyze oxidative decarboxylation of isocitrate to alfa-ketoglutarate
• Accumulation of 2-hydroxyglutarate, leading to epigenetic alterations and a block
in cell differentiation
‒ IDH1 mutations occur in 6-10% AML patients
‒ IDH2 mutations occur in 9-13% of AML patients
‒ Mutations are considered prognostically neutral
• Ivosidenib: targets IDH1
‒ Phase 1: objective RR 41.6% and cCR rate of 30.4%
• Enasidenib: targets IDH2
‒ Phase1/2: objective response rate 40.3% and CR rate of 19.3%
‒ New genetic alterations arise, contributing to relapse
• Both agents are well-tolerated
MUTATION TARGETED AGENTS
IDH INHIBITORS (ISOCITRATE DEHYDROGENASE)
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17. M A K I N G T H E C O M P L E X S E A M L E S S
• BCL2: antiapoptotic protein, oncogene
• Venetoclax: selective, potent BCL2 inhibitor
• Promising results in elderly AML patients (> 65 years)
‒ Combination with azacitidine or decitabine
‒ 67% cCR rate and median OS rate of 17.5 months
• Less promising results when combined with low dose cytarabine
• Adverse events tolerable
‒ Nausea, vomiting, mucositis, neutropenic fever
• No tumor lysis syndrome observed
NON-MUTATION TARGETED AGENTS
BCL2 ANTAGONISTS (B-CELL LEUKEMIA/LYMPHOMA 2)
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18. M A K I N G T H E C O M P L E X S E A M L E S S
• Dynamic, reversible post-translational modification of DNA and
histone proteins
‒ DNA methylation, lysine acetylation, lysine methylation, arginine methylation
• Regulate accessibility of DNA for the transcription machinery of the
cell
• Hypomethylating agents: decitabine and azacitidine
‒ Target DNA methyltransferases
‒ AML and MDS
• Used alone or in combination with chemotherapy
‒ Maintenance therapy
NON-MUTATION TARGETED AGENTS
EPIGENETIC MODIFIERS
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19. M A K I N G T H E C O M P L E X S E A M L E S S
• Hedgehog signaling pathway inhibitors
‒ Glasdegib inhibits protein smoothened (SMO) in this pathway
‒ Phase 3 trial of agent with cytarabine alone or in combination showed 49%
reduction in risk of death and improved CR rate (17% vs. 2%)
‒ Unknown how it compares with hypomethylating agents
‒ Approved in combination with low dose cytarabine for elderly or unfit patients
• BET inhibitors (bromodomain and extra-terminal motif)
• LSD1 inhibitors (lysine demethylase)
• DOT1L inhibitors (disruptor of telomeric silencing 1-like)
• Antibodies
• Cellular therapies
NON-MUTATION TARGETED AGENTS
MISCELLANEOUS
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20. M A K I N G T H E C O M P L E X S E A M L E S S
• Gemtuzumab ozogamicin (GO)
• Humanized anti-CD33 antibody conjugated to a derivative of cytotoxic
molecule calicheamicin
• Upon binding to the cell, the ADC is internalized, triggering release of the
cytotoxin
• Initial approval in 2000 for elderly AML patients in first relapse was
withdrawn 2010
‒ Black box warning for sinusoidal obstructive syndrome
‒ 2004 phase 3 randomized trial showed no survival benefit and demonstrated a
higher induction mortality rate in the GO arm (5% vs 1%).
• Pooled analysis of 5 randomized trials did demonstrate lower relapse and
high OS rates in favorable and intermediate AML patients
‒ Led to FDA approval in 2017 in combination with standard chemotherapy or alone
TARGETED DELIVERY
ANTIBODY-DRUG CONJUGATES
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21. M A K I N G T H E C O M P L E X S E A M L E S S
CHALLENGES
• Increase interest in developing less intense, targeted therapies for
AML patients
‒ Alone or in combination with chemotherapy
• None of these agents has yet achieved the results of ATRA or arsenic
trioxide in AML M3 or the TKIs in CML
• Objective, reproducible criteria need to be developed to avoid
selection bias
‒ Geriatric assessment for fitness
• The field is a moving target as the distinction between regimen
intensity changes with the development of new agents
• Primary problem in AML remains disease recurrence
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22. INTRODUCTION OF NGS
IN CLINICAL PRACTICE
IN AML
EL MUSTAPHA BAHASSI, PH.D

23. M A K I N G T H E C O M P L E X S E A M L E S S
• Many of the important AML biomarkers were discovered from NGS
(whole genome, whole exome, panel sequencing) studies
• In NGS, millions of small fragments of DNA are immobilized on a
solid surface, amplified, and sequenced in parallel
• During sequencing a signal (light, pH change) is detected when a
base is incorporated
• Short contiguous reads are generated
• Reads are aligned to a reference sequence and analyzed
• Analysis is computationally intense
• Discovered variants are curated to determine their pathogenicity
NGS
ALSO KNOW AS MASSIVELY PARALLEL SEQUENCING
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24. M A K I N G T H E C O M P L E X S E A M L E S S
COMPARISON OF NGS APPLICATIONS
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25. M A K I N G T H E C O M P L E X S E A M L E S S
NGS VS NON-NGS TESTS
• NGS allows testing in a single run for all types of variants, including
single-nucleotide variations, insertions, deletions, exon duplications,
gene copy number changes, and known translocations
• Current non-NGS molecular diagnostic tests fail to detect known
genomic alterations in patients who, in actuality, possessed these
mutations.
• NGS identified actionable genomic alterations in 65% of lung cancers
tumors deemed without targetable genomic alterations by earlier
extensive non-NGS testing.
• Furthermore, a large percent of these patients' genomic alterations
had a targeted agent accessible through a clinical trial
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Drilon A. et al. 2015 Clinical Cancer Research

26. M A K I N G T H E C O M P L E X S E A M L E S S
NGS VS NON-NGS TESTS
• Most NGS assays require as little as 10 ng of DNA, whereas non-
NGS tests, like FISH and IHC tests, require several histologic
sections of the formalin-fixed, paraffin-embedded (FFPE) specimen
block for a single run.
• Swift turnaround time for comprehensive DNA sequencing to provide
actionable data to clinical physicians
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27. M A K I N G T H E C O M P L E X S E A M L E S S
• Analyzed the genomes of 200 clinically
annotated adult cases of de novo AML
• Used either WGS (50 cases) or WES (150
cases), along with RNA and microRNA
sequencing and DNA-methylation analysis.
• A total of 23 genes were significantly
mutated, and another 237 were mutated in
two or more samples.
• Nearly all samples had at least 1
nonsynonymous mutation in one category
of genes that are almost certainly relevant
for pathogenesis
GENOMIC AND EPIGENOMIC LANDSCAPES OF AML
THE CANCER GENOME ATLAS NETWORK
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28. M A K I N G T H E C O M P L E X S E A M L E S S
FUNCTIONAL CATEGORIZATION OF MUTATED GENES
IN MYELOID MALIGNANCIES
THE ORGANIZATION OF MUTATED GENES INTO FUNCTIONALLY RELATED CATEGORIES
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Matynia et al., 2015. Archives of Pathology and Laboratory Medicine

29. M A K I N G T H E C O M P L E X S E A M L E S S
FUSIONS DETECTED IN THE AML COHORT IN THE
CANCER GENOME ATLAS (TCGA)
GENE FUSIONS
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In-frame (green) and out-of-frame (orange)

30. M A K I N G T H E C O M P L E X S E A M L E S S
• European Leukemia Network (ELN) recommends genetic testing for all
patients with newly diagnosed AML. This includes:
1. conventional cytogenetics
2. screening for (a minimum of) 6 gene mutations including NPM1, CEBPA, RUNX1,
FLT3, TP53, ASXL1
3. screening for gene rearrangements including PML-RARA, CBFB-MYH11, RUNX1-
RUNX1T1 and BCR-ABL1
• National Comprehensive Cancer Network (NCCN) guidelines recommend:
1. conventional cytogenetics
2. testing for (a minimum of) 9 gene mutations including NPM1, CEBPA, RUNX1,
FLT3, TP53, ASXL1, IDH1, IDH2 and c-KIT
3. testing for gene rearrangements identically to the ELN recommendations.
• According to the NCCN and ELN, multiplex gene panels and NGS analysis
may be used to obtain further information regarding prognosis, treatment
decisions and eligibility for clinical trial participation
CURRENT GUIDELINES REGARDING NGS ANALYSES
FOR THE DIAGNOSIS OF AML
ELN AND NCCN
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31. M A K I N G T H E C O M P L E X S E A M L E S S
MYLEOID NGS GENE PANELS
• Currently, commercially available myleoid NGS gene panels include:
‒ SureSeq myPanel™ NGS Custom AML (Oxford Gene Technology,Oxfordshire, UK)
‒ Leuko-Vantage Myeloid Neoplasm Mutation Panel (Quest Diagnostics, Madison,
NJ, USA)
‒ AmpliSeq® Myeloid Sequencing Panel (Illumina)
‒ Human Myeloid Neoplasms Panel (Qiagen, Venlo, The Netherlands)
‒ Lab-developed panels
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32. M A K I N G T H E C O M P L E X S E A M L E S S
CHALLENGES TO HARNESSING WGS IN CLINICAL
MEDICINE & PUBLIC HEALTH
• Accuracy
‒ 99.99% accuracy x 3 billion nucleotides
‒ = 300,000 errors per patient
• Time consuming/expensive
• Interpretation of the identified variants
• Data storage and access in the medical record
• Education of providers, patients and public
• Issues of consent and reporting
• Incidental findings:
‒ Some are trivial or indeed beneficial but some are problematic and we will
occasionally discover lethal, untreatable late onset conditions
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33. M A K I N G T H E C O M P L E X S E A M L E S S
CHALLENGES IN IMPLEMENTING NGS IN CLINICAL
ROUTINE DIAGNOSTICS IN HEMATO-ONCOLOGY
Challenge Background Current and future approach
Discrimination of leukemia-
related mutations from
polymorphisms or passenger
mutations
• Driver mutations expected to
occur at higher allele
frequency in patient samples
than passenger mutations;
• Driver mutations more likely to
have an impact on protein
function than polymorphisms
or passenger mutations
• Optimization of cancer-specific
databases including reporting
of rare physiological gene
variants
• Implementation of novel
bioinformatic algorithms based
on prediction of functional
impact
• Quantitative and dynamic VAF
monitoring (separately and
together with other mutations)
at follow-up
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VAF: variant allele frequency

34. M A K I N G T H E C O M P L E X S E A M L E S S
CHALLENGES IN IMPLEMENTING NGS IN CLINICAL
ROUTINE DIAGNOSTICS IN HEMATO-ONCOLOGY
Challenge Background Current and future approach
Discrimination of leukemia-
related somatic mutations from
pathogenic germline alterations
Challenge to differentiate
acquired somatic mutations from
germline pathogenic variants at
diagnosis
• Mutation detection in germline
control samples (e.g., skin
fibroblasts, saliva) in genes
such as RUNX1, CEBPA
• Thorough medical family
history followed by molecular
genetic tests in relatives if
necessary
• High and stable VAF (e.g., 40–
50%) at follow-up despite
clinical response to treatment
may be indicative for germline
alteration
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35. M A K I N G T H E C O M P L E X S E A M L E S S
CHALLENGES IN IMPLEMENTING NGS IN CLINICAL
ROUTINE DIAGNOSTICS IN HEMATO-ONCOLOGY
Challenge Background Current and future approach
Discrimination of true genetic
alterations from PCR,
sequencing and post-sequencing
artifacts
Many artefacts are known to
arise during NGS library
preparation, sequencing and data
analysis
• Error correction using
molecular identifiers that
individually label original input
DNA molecules
• Refinement of error-correction
computational methods in
post-sequencing NGS data
analysis
• Confirmation using Sanger
sequencing
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36. M A K I N G T H E C O M P L E X S E A M L E S S
CHALLENGES IN IMPLEMENTING NGS IN CLINICAL
ROUTINE DIAGNOSTICS IN HEMATO-ONCOLOGY
Challenge Background Current and future approach
Limited sensitivity of NGS for
minimal residual disease (MRD)
assessment
Mutations detected at diagnosis
may be re-identified at best to a
VAF of 1–2%
• Error-corrected sequencing
using molecular identifiers
• Complementation of NGS by
established MRD tools like
real-time PCR and flow
cytometry
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37. M A K I N G T H E C O M P L E X S E A M L E S S
• NGS allows a real insight into the biology of the cancer
• NGS is important in predicting prognosis which can help make
treatment decisions
• The data from NGS can help build clinical and genomic databases
that can be used for retrospective single institution or multiple
institution studies
CONCLUSIONS
ADVANTAGES AND PITFALLS
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38. M A K I N G T H E C O M P L E X S E A M L E S S
• Always a trade-off between time, cost, coverage, depth etc.
• Expert annotation and analysis: Bioinformatics and expertise in
mutation annotation
• Certain mutations are not assessed well with NGS (FLT3 and CALR
duplications)
• The NGS results are not the whole picture for prognostication or
prediction in AML. Other factors such as age, CBC counts, large
indels can be as important or even more important than the
information provided by NGS
CONCLUSIONS
ADVANTAGES AND PITFALLS
38

39. M A K I N G T H E C O M P L E X S E A M L E S S
SUMMARY
• In the last 5-10 years, NGS has been introduced in most specialized
Hematologic laboratories with various myeloid NGS panels
• NGS will pave the way towards a more detailed clinical evaluation
and optimized therapeutic decisions as NGS costs are rapidly
decreasing and technology is continuously improving
• However, challenges remain before NGS becomes routine:
‒ Cost and reimbursement
‒ Data analysis and storage
‒ Variant interpretation
‒ Limited sensitivity (ie. for MRD)
‒ Other technical aspects of testing (ie. PCR, Dup.) can affect the results
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40. OPERATIONAL
CONSIDERATIONS FOR
CONDUCTING CLINICAL
TRIALS IN PATIENTS
WITH AML
JEFF VASSALLO, PH.D.

41. M A K I N G T H E C O M P L E X S E A M L E S S
• Recent FDA approval of 8 targeted agents for the treatment of
patients with AML
• There are 541 actively recruiting clinical trials for the treatment of
patients with AML
• Investigators and patients have many choices
‒ Large academic centers have the most patients and clinical trials
‒ Number of eligible patients is limited by
• Rapidly increasing number of conventional therapies
• Number of clinical trials
• Increasingly smaller group of patients defined by molecular abnormalities
OPERATIONAL CONSIDERATIONS
LESSONS LEARNED – COMPETITIVE LANDSCAPE
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42. M A K I N G T H E C O M P L E X S E A M L E S S
• Recruitment challenges
‒ Time needed to fully characterize the patient’s AML
• Identification of potential therapeutic targets
‒ Complex and burdensome procedures
‒ Pre- / post-transplant patients
‒ Older patient population
‒ Frequent exclusion of hydoxyurea and azoles
• Investigators lack enthusiasm for protocols without published data
OPERATIONAL CONSIDERATIONS
LESSONS LEARNED - PROTOCOL
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43. M A K I N G T H E C O M P L E X S E A M L E S S
• Identify a physician champion who is a KOL
• Conduct site feasibility to understand patients and competing trials
‒ Select sites with consideration for KOLs, startup timeline, patients and
competing trials
‒ Identify sites which routinely characterize abnormalities for the intended
therapeutic agent
• Leverage master protocols which enable molecular characterization
of driver mutations to match patient’s AML abnormality with a
potential therapeutic targeted agent
‒ M2Gen / ORIEN
‒ BEAT AML (LLS)
OPERATIONAL CONSIDERATIONS
BEST PRACTICES - LANDSCAPE OF OPPORTUNITIES
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44. M A K I N G T H E C O M P L E X S E A M L E S S
• Investigators and patients prefer simple protocols
‒ Ensure all assessments support objectives
‒ Remove complex and burdensome procedures
• Limit number of bone marrow biopsy/aspirate
‒ Include patients treated with hydroxyurea or azoles
• Consider advisory board to facilitate protocol development
‒ Patients and parents on advisory board
• Publish scientific data supporting molecular targets, safety and
efficacy
‒ “Trust us” is not a good strategy
OPERATIONAL CONSIDERATIONS
BEST PRACTICES - PROTOCOLS WITH A COMPETITIVE ADVANTAGE
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45. M A K I N G T H E C O M P L E X S E A M L E S S
• Successful trial execution is predicated on relationships and expertise
‒ Key stakeholders
• Regulatory authorities / Scientific Review Committees / IRB / EC
• Sponsor
• CRO
‒ Medical Monitor
‒ Nurse Practitioner
‒ Clinical Trial Manager / Clinical Research Associates
• Investigators
• Patients
• Comprehensive understanding of patients, protocols and therapies
• Ensure best and most efficient operational practices are implemented
OPERATIONAL CONSIDERATIONS
BEST PRACTICES - TEAM OF AML EXPERTS
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46. QUESTIONS
THANK YOU