The Growing Importance of Next-Generation Sequencing in Myeloid Molecular Testing

Table of Contents

Click on a chapter to get started.

CHAPTER 1: Introduction

See Table of Contents

CHAPTER 2: Myeloid Disorders

See Table of Contents

Major categories of myeloid malignancies

Myeloid malignancies arise from mutations in hematopoietic stem or progenitor cells.⁴ These clonal disorders often exhibit high degrees of heterogeneity, complex karyotypes, and multiple categories of somatic mutations.^1,2,4

The natural process of blood cell formation, hematopoietic stem cell differentiation, and generation of myeloid and lymphoid cell lineages.⁵ Hematopoietic disruptions in the myeloid lineage can lead to 3 major disease categories: acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), and myelodysplastic syndrome (MDS).⁴

The challenges of molecular testing in hemato-oncology

Due to their genetic complexity and high degree of heterogeneity, myeloid malignancies can be challenging for molecular analysis.1 Since some myeloid disorders can progress rapidly, the European LeukemiaNet (ELN) recommends that molecular testing for some biomarkers (eg, NPM1 and FLT3) be available on a rapid-turnaround basis, often within 48 to 72 hours.^1,15

Over the last decade, whole-genome and whole-exome sequencing studies have revealed a number of recurrent mutations associated with these disorders. As the list of biomarkers expands so do the molecular testing algorithms, making it impractical—if not implausible—for laboratories to keep pace using single-gene testing methods.^1,16 NGS helps overcome these challenges by empowering labs to perform a multi-gene assessment with a single test and enabling rapid results.^1,16

Ultimately, real progress depends on the ability of all molecular testing labs (institutional, hospital, and community) to keep pace and to deliver the answers that clinicians need at each stage of the care continuum. Realizing the promise of precision oncology and its potential for better outcomes in hemato-oncology and beyond entails a new molecular testing paradigm—one where in-house, streamlined biomarker profiling with rapid results is not only possible, but ubiquitous.

Next-generation sequencing
for molecular hematology testing overview.

Megan Lim, MD, PhD - Director of Hematopathology
University of Pennsylvania

AML testing complexities: Multiple biomarkers, modalities, and TATs

The complexities of hemato-oncology testing are evident in AML.^1,15 This traditional diagnostic scenario depicts a representative process of testing multiple biomarkers across several modalities with associated staggered turnaround times (TATs), all of which can contribute to deleterious delays in therapy selection and initiation.¹

CHAPTER 3: The Necessity for In-house NGS

See Table of Contents

Optimizing Molecular Testing with In-house NGS

In-house NGS testing overcomes these challenges by enabling labs to profile all relevant genetic aberrations simultaneously in a massively parallel fashion. NGS generally provides higher sensitivity, larger scale, and the ability to detect novel aberrations compared to traditional methods (Table 1).¹⁸

In fact, numerous commercially available panels feature target probes for all key mutations. From 1 test, a lab can identify all relevant point mutations, insertions, deletions, translocations, and copy number variations associated with a given disorder.16 There are panels for DNA targets and RNA targets, which are necessary to detect gene fusions.¹⁶ Some platforms even allow simultaneous assessment of DNA and RNA. The primary clinical benefit of in-house NGS testing is that labs can produce a complete mutational profile in as fast as 1 day with the latest technologies.

Dr. Bekim Sadokovic 
Western University 
Quote taken from:
Eur J Haematol. 2019 Sep;103(3):178-189. 

Expert Opinion

The classic approach to molecular hematology testing involves targeted single-analyte testing after initial pre-screening by other diagnostic modalities including flow cytometry, anatomic pathology, and clinical cytogenetics. This approach presents several limitations:

• Every patient specimen must be stabilized, DNA and RNA extracted and stored regardless of whether molecular testing will be performed. Subsequently, a specific targeted molecular assay may be activated for a subset of patient.
• Commonly, the assay targets (specific genes) chosen are a “best guess” based on preliminary findings from the initial pre-screening. 
• Targeted testing may exhaust available scarce tissue specimens such as bone marrow rendering additional targeted assays impossible.
• Turnaround times are negatively impacted due to delays in initiating targeted molecular testing.
• Furthermore, there are significant personnel requirements to manage the pre-screening tests and to determine which samples require targeted molecular assessment and by which specific molecular assays.

"Given that whole genome or exome sequencing has revealed a wide but limited-range of high-frequency mutations associated with hematologic malignancies such as myeloid neoplasms, including DNA sequence mutations and gene fusions, a DNA/RNA-based NGS panel provides an ideal alternative approach for comprehensive testing of patients as an initial, tier-1 screen in place of targeted triage, rather than as a secondary (tier-2) assay reflexive to other tests."

CHAPTER 4: NGS Methods

See Table of Contents

Simultaneous detection of multiple types of variants

Given the substantial phenotypic and genetic heterogeneity of myeloid disorders and the continually increasing number of alterations that need to be analyzed, many more laboratories are integrating NGS into their diagnostic algorithms.^1,3,17

By providing a complete analysis of genetic alterations, including single-nucleotide variants, translocations, and small insertions and deletions, NGS myeloid panels can enable enhanced differentiated disease classification, risk stratification, and improved therapeutic decisions.^1,3,17

Enlarge Image

Workflow Overview

Watch a brief workflow overview video below.

Click the slider arrows below to see workflow.

The Pathologists' Perspective

Workflow Automation

In the past, NGS adoption was limited mainly to academic institutions, specialty cancer centers, and large reference labs.^27,28 Due to the complexity of the technology, the high costs, and the need for specialized bioinformatics expertise, NGS testing was primarily outsourced when needs arose in the community hospital setting.^27,28

Significant advancements in NGS technology, workflow automation, integrated analysis, and reporting software—in some platforms—have leveled the playing field, making in-house NGS accessible for most community hospitals and labs.^22,27

Precision medicine and its promise of improved outcomes will be realized as more and more labs bring the latest highly automated NGS platforms in-house and genetic testing becomes routine in clinical practice.^1,27

Hear more from Dr. Morrison on Solving Common Hematopathology Testing Challenges with Next-Generation Sequencing.

Watch 2 minute video clip

Dr. Carl Morrison, MD
SVP, Scientific Development and Integrative Medicine
Roswell Park Comprehensive Cancer Center

Watch Full Presentation

Dr. Kojo Elenitoba-Johnson, MD
Peter C. Nowell, M.D. Professor of Pathology and Laboratory Medicine
Perelman School of Medicine at the University of Pennsylvania
Director, Center for Personalized Diagnostics
Director, Division of Precision and Computational Diagnostics

Read Entire Interview

Rapid Turnaround Time

Rapid assessment of genetic biomarkers in myeloid malignancies is vital for early diagnosis, first-line therapy choices, and ongoing clinical disease management.^3,29 This includes evaluation of therapy response and relapse/measurable residual disease (MRD) detection.^3,29 In particular, acute diseases like AML require rapid test results for key diagnostic and risk stratification markers (ie, PML-RARA gene fusion, FLT3 or IDH1/2, and NPM1).^3,15 The 2017 ELN guidelines recommend that results for FLT3 and NPM1 be available within 72 hours.¹⁵

"There are several protocols, including extensive amounts of genetics and immunophenotyping, which are required to establish the appropriate approach to treat patients. The sooner we can get results and deliver treatment, the better the outcomes."

Advances in AML risk-stratification guidelines and targeted therapies have made it increasingly important for clinicians to consider a wide range of genetic biomarkers to guide front-line patient-care decisions.^3,15,29 NGS helps to address this need by rapidly providing results for a broad number of genetic targets.³ The latest instruments enable a laboratory to sequence a patient sample in about 24 hours so clinicians can have the results in days.³   

NGS testing considerations in molecular oncology research

During a recent UAB study presentation, Craig Mackinnon, MD, PhD, discussed the key considerations for setting up a next-generation sequencing (NGS) panel in a molecular oncology research lab. Basic requirements included the capacity to target genes of interest and detect a wide range of variants, including single nucleotide variants (SNVs), small indels, and fusions, all while accommodating multiple specimen types of varying quality and input amounts.

Dr, Mackinnon concluded that automation, hands-on time, turnaround time, and the associated potential for cost savings should all be part of the evaluation.

Watch 1 minute video clip

Craig Mackinnon, PhD
Director, Genomic Diagnostics and Bioinformatics - Professor, Department of Pathology, University of Alabama at Birmingham

Watch Full Presentation

NGS testing considerations for molecular oncology³¹

CHAPTER 5: The Value of NGS Across the

Care Continuum

See Table of Contents

A giant step forward for disease management and
precision oncology

Advances in NGS have made simultaneous assessment of multiple target genes possible in daily laboratory analysis.^1,3 Targeted NGS panels designed explicitly for myeloid neoplasms can inform patient-management decisions across the entire continuum of care—from streamlining diagnosis, identifying disease subtypes, facilitating treatment decisions, assessing risk stratification, and providing prognostic insights to improving monitoring and measurable residual disease (MRD) detection.^1,3,17

As in-house NGS testing becomes ubiquitous at a community hospital-lab level, more patients will be able to benefit from these important advancements in precision oncology.

Streamlined Diagnosis

Historically, most community-based labs have utilized traditional single-analyte molecular testing methods, outsourcing to reference labs as needed to augment their in-house capabilities.^27,28

This strategy presents a challenge when multiple biomarkers need to be tested, as is the case for myeloproliferative neoplasms (MPNs). Sequential iterative testing can contribute to substantial delays in diagnosis and treatment.^{1,3,16, 17} These delays can be time and cost-intensive for labs, frustrating for clinicians, and angst-provoking for patients.^{1,3,16, 17}

Today, multi-gene NGS panels enable labs to analyze all relevant genes using a single test that yields results within days.³ With NGS technology delivering expedited results, clinicians will have the information they need to optimize disease management sooner, and patients will have peace of mind.^1-3

Pathologist perspective: Utilizing NGS for MPN diagnosis and risk assessment

During a recent conference, Dr. Yi Ding of Geisinger Medical Laboratories described how her laboratory overcame the MPN testing challenge. Before they brought NGS in-house, MPN genetic testing took 4 to 6 weeks in their lab. Patient follow-ups were scheduled as far out as 3 months after the initial visit. By bringing a multi-gene NGS panel in-house, they dramatically reduced the time to diagnosis.

Takeaway: Multi-gene NGS panels that include genes and mutations recommended by National Comprehensive Cancer Network (NCCN) guidelines and deliver timely results offer unprecedented opportunities to streamline diagnosis and enhance disease management for improved outcomes and quality of life.^1-3,16,32

Watch 5 minute video clip

Yi Ding, MD, PHD
Systems & Core Laboratory Director of Molecular Diagnostics
Geisinger Medical Laboratories

Myeloproliferative neoplasms: Commonly mutated genes³³

PV, polycythemia vera; ET, essential thrombocythemia; PMF, primarymyelofibrosis

Disease Subclassification: AML

Many myeloid malignancies are classified by the presence of specific genetic mutations.^1,3 While an initial diagnosis can be achieved with histopathological and immunophenotyping tests, disease subtypes are usually determined through karyotyping and genetic analysis.1 Per the 2016 World Health Organization (WHO) classification, AML subtypes include recurrent genetic abnormalities and germline predisposition.^1,29 In fact, > 95% of AML cases harbor at least 1 somatic alteration.34 In addition, there are approximately a dozen genomic alterations and an average of 3 driver mutations identified per AML sample.³⁴

NGS offers critical advantages in AML disease subclassification as all relevant mutations can be identified in a single test.³⁴ Some of the latest NGS platforms deliver results within hours or days, so these mutational insights can be evaluated in conjunction with the histopathological or immunophenotyping results for a more complete picture of the disease pathology.³

Indeed, the inclusion of certain mutations in the key treatment guidelines (WHO classification, 2016, ELN, 2017, and the National Comprehensive Cancer Network (NCCN)) provides strong evidence for how mutational testing is influencing clinical disease management.^{18, 29,32,35} Genetic profiling and disease subclassification is expected to become increasingly important in daily clinical practice.³¹

2017 WHO Classification of Myeloid Malignancies

Risk Stratification: AML

Genetic abnormalities are powerful prognostic factors that can help inform therapy selection.^{15, 36} For patients with AML, genetic mutations can indicate the suitability for allogenic hematopoietic stem cell transplantation (allo HCT).^{15, 36} There are also multiple co-occurrent mutations that can impact risk stratification in AML.¹⁵ Results from conventional cytogenetics and NGS mutational screenings are routinely used in clinical practice.¹⁶

The 2017 ELN guideline groups genetic abnormalities into 3 levels of risk that range from favorable to adverse.¹⁵ One of the most common examples of genes with co-occurring mutations that impact risk stratification are NPM1 and FLT3-ITD.¹⁵ Risk level is dependent on the presence of both mutations and the allelic ratio of FLT3-ITD.¹⁵

2017 European LeukemiaNet (ELN) Guidelines¹³

Key Takeaway: Both the NCCN and ELN guidelines recommend that patients with suspected AML be screened for gene mutations.^15,32

NGS offers distinct advantages for risk stratification. Given the sheer number of genetic abnormalities that must be evaluated, a multi-gene NGS panel can consolidate many, if not all of the individual single-gene tests that would otherwise be required.^3,16 Additional NGS identification of SNVs, insertions, deletions, and rearrangements could provide added value.³⁷

Currently, NGS is the most cost-effective method of risk assessment for AML.³ By utilizing in-house NGS, labs could potentially accelerate time to results while simultaneously lowering costs and reducing the strain on its resources.^1,3,16

Diagnosis and Prognosis: MDS

Detecting leukemic drivers with NGS informs disease management

Over the last decade, NGS has helped unravel the complex molecular underpinnings of myelodysplastic syndromes (MDS).³⁵ Dozens of recurrent mutations associated with the disease have been revealed.³⁵ Targeted sequencing will typically identify at least 1 mutation in the majority of cases.³⁵ While NGS does not replace conventional cytology, histology, and cytogenetics, it can provide meaningful insights from diagnosis and prognosis to treatment response in MDS patients.³⁵

MDS is a clonal disease arising from the proliferation of mutated hematopoietic stem cells.^12,35 The spectrum of clonal myeloid disorders ranges from clonal hematopoiesis of indeterminate potential (CHIP) to secondary acute myeloid leukemia (sAML).^35,38 MDS is characterized by peripheral blood cytopenia, dysplasia, and the absence of features that define acute leukemia.^12,38 In cases where clonal hematopoiesis is detected in the absence of a cytopenia or dysplasia, mutational data can help distinguish MDS from CHIP.³⁸ Typically, a larger number of mutations with a higher variant allelic frequency (VAF) are more supportive of an MDS diagnosis.³⁸

Notably, mutational data of clonal myeloid disorders can also help inform the level of risk for a particular disease to progress to another more serious disorder (eg, clonal cytopenia of undetermined significance [CCUS] evolving to MDS and MDS evolving to sAML).^12,38,39
Ongoing research in this area is likely to increase the clinical relevance of NGS for diagnosing and treating these disorders.^12,21,35

R/R = Relapse/Refractory
* Patients >75 years old or who have comorbidities that preclude the use of intensive induction chemotherapy

Recurrence Monitoring

Recurrence monitoring and measurable residual disease (MRD) detection are critical for hematological disorders.^1,3,17 While our improved understanding of the molecular landscape of AML has resulted in more informed and targeted first-line treatments, the threat of disease relapse is always looming.⁴⁰ Studies have shown that negative MRD (assessed by molecular techniques or immunophenotyping) may be a better predictor of survival as it’s associated with a lower risk of relapse.³

Chart: NGS identification of clonal and subclonal mutations in AML provides insights into prognosis, treatment, and response.²¹

This schematic depicts the subclonal genetic heterogeneity of AML development and progression and illustrates the critical role of NGS in the assessment of clonal and subclonal mutations throughout disease management.

Prognostic Value of MRD Detection for AML

By traditional standards, complete remission is achieved when a morphological assessment shows < 5% myeloblasts (immature myeloid cells) in the bone marrow, absence of circulating blasts, and hematologic recovery.²¹ Although most AML patients achieve complete remission after primary treatment, many eventually relapse, indicating that morphology is not the best predictor of survival.²¹

CHART: Studies demonstrate the prognostic value of NGS and multiparameter flow cytometry (MFC) MRD for AML relapse (~75% concordance)^{41,42  (Source)}

Enlarge Image

Molecular monitoring methods: MFC, qPCR, and NGS

Molecular diagnostic techniques, such as multiparameter flow cytometry (MFC) and quantitative real‐time PCR (qPCR) assays, are commonly used to measure and monitor disease burden following treatment of myeloid disorders.^41,42

MFC has been the gold standard for MRD detection, but it has drawbacks. For example, it is operator- and center-dependent and without a standardized method of enumerating flow-based MRD in AML.⁴² Most notably, MFC lacks sensitivity for detecting residual disease.¹

While qPCR assays are highly sensitive, most monitoring strategies for AML are limited to identifying the most common AML‐associated mutations and translocations.³⁸ Since only 15% to 25% of AML cases have these known molecular markers, and even fewer MDS cases have them, qPCR offers limited utility.⁴³

Another challenge for qPCR is tracking post-chemotherapy clonal evolution as non-founder clonal mutations may be lost or gained despite the persistence of the founding clones.38 With the considerable time, cost, and resources involved in designing patient-specific primer sets for recurrent somatic single nucleotide variants and small insertions, qPCR is impractical for AML monitoring.^1,3,16

Today, more labs rely on NGS‐based tumor-burden monitoring assays because they cover a broader range of genes and have the sensitivity to detect small clonal populations by identifying low‐frequency somatic mutations associated with the malignant clones.^1,21,37 Unlike qPCR‐based assays, targeted NGS panels can simultaneously detect many mutated genes and translocations found in myeloid malignancies—without the need to develop patient‐specific primer sets.^16,37

CHAPTER 6: Outlook & Key Considerations

See Table of Contents

Want to Learn More?

Speak with a representative about myeloid testing with next generation sequencing for your lab today.