Table ofContents

2

Introduction
02 Table of Contents
04 LabPMM Testing Services
06 LabPMM Locations
08 How to Order a Test
10 Patient Consent & Confidentiality
12 Partner with Us
14 An Ideal CDx Partner
16 Join the 60+ Pharma Companies Working with Us
18 CDx Commercialization Phasing

Companion Diagnostic Tests
20 Introduction

CDx - USA (FDA approved)
21 LeukoStrat® CDx FLT3 Mutation AssayCDx (CE-marked)
22 LeukoStrat® CDx FLT3 Mutation Assay

CDx - Japan (MHLW/PMDA approved)
23 LeukoStrat® CDx FLT3 Mutation Assay

Molecular Diagnostics Tests
24 Introduction
25 NPM1 Mutation Analysis

Clonality Tests
26 Introduction
27 B-cell Clonality
28 T-cell Clonality

Measurable Residual Disease NGS Tests
29 Introduction

AML
30 FLT3 ITD MRD Assay
31 NPM1 MRD Assay

Clonality
32 B-cell MRD Assay
33 T-cell MRD AssayNGS Cancer Panels
34 Introduction
35 MyAML®
36 MyMRD®
37 LabPMM Assay Guidelines

Multiparametric Flow Cytometry Panels
39 Introduction
40 Hematolymphoid Screening Panel - MFC
41 AML MRD Assay - MFC
42 CLL MRD Assay - MFC

3

LabPMM Testing
Services

LEARN MORE:
invivoscribe.com/clinical-lab-services

4

LabPMM Offers Efficient and Reliable
Standardized Tests that Meet the Highest
Quality and Service Standards.
» CRO Services» Global Stratification and enrollment of patients in clinical trials» Diagnostic testing, Research and Development» Worldwide experts in precision medicine and companion diagnostics (CDx) of hematological malignancies

The Laboratories for Personalized Molecular Medicine (LabPMM) is the Invivoscribe® global network of clinical reference labs located in the USA,
Europe and Asia, which provides international access to harmonized molecular testing.
LabPMM specializes in oncology with an emphasis on leukemias and lymphomas. We offer an ever-expanding menu of flow cytometry andmolecular assays to support medical diagnosis, research testing, stratification & enrollment of patients in clinical trials, optimization of treatments
and development of companion diagnostics (CDx).
The comprehensive test menu is focused on biomarkers that demonstrate clinical utility, providing data and information that is clinically actionable
and critical to making informed treatment decisions. Our CE-IVD assays and bioinformatics are further developed and manufactured consistently
under ISO 13485 in our FDA-registered facility, making them eligible to be submitted to worldwide regulatory authorities for registration.
LabPMM is one of the only international laboratory networks that offers both flow cytometry and NGS-based MRD testing for coherent comparisons
for a number of clinically significant biomarkers. Accordingly, test results generated in any of our laboratories in Europe, Asia, or the USA are
internationally concordant and reproducible. The harmonized testing provided by LabPMM assists healthcare providers in offering optimized andconsistent care for their patients, as the test results accurately and reproducibly stratify patients for international clinical trials, thus ensuring thatpatients receive optimal treatment and that drugs are approved quickly.

5

LabPMM Global
Locations

LEARN MORE:
invivoscribe.com/company/locations

6

LabPMM LLC
Located in San Diego, California, USA, it holds the following accreditations andcertifications: ISO 15189, CAP, and CLIA, and is licensed to provide diagnostic
laboratory services in the states of California, Florida, Maryland, New York,
Pennsylvania, and Rhode Island.

LabPMM GmbH
Based in Hallbergmoos (Munich), Germany. It is an ISO 15189 accredited
international reference laboratory offering diagnostic services.

LabPMM 合同会社

Located near Tokyo in Kawasaki (Kanagawa), Japan. It is an ISO 15189 and JAB
accredited international reference laboratory.

Invivoscribe Diagnostic
Technologies (Shanghai) Co., Ltd.
Located in Shangai, China. It is the newest CAP accredited international reference lab,supporting clinical trial work in China.

Available services include: CDx FLT3, NGS Gene Panels, Clonality testing (B- & T-cell),
MRD assays, Custom assays, and Multiparametric Flow Cytometry
LabPMM® specializes in personalized flow cytometry and molecular testing services for oncology, including leukemia and lymphoma. We arecommitted to providing high-quality testing in support of Personalized Molecular Medicine®.
Our diagnostic and research portfolio includes a full range of oncology services, such as single gene assays for FLT3, NPM1, clonality testing of B- andT-cells, minimal or measurable residual disease (MRD) assessments and comprehensive next-generation sequencing (NGS) gene panels for AML andother hematologic malignancies.
Rapid turnaround times are vital to ensure that the physician can make timely informed treatment decisions. Our turnaround times for individual genetests are in the range of 1 to 3 days following sample receipt, while turnaround times for our NGS assays and gene panels are 7 to 21 days. When using
LabPMM, physicians receive results faster which expedites patient care and streamlines clinical trials. The reason is simple: all of our LabPMM sites
initiate tests the day of clinical sample receipt, avoiding delays caused by batching of samples for testing.
Customer support is an important aspect of our services. We provide responsive, timely support both via email and telephone. We are also boundby strict privacy laws and use only secure proven methods to communicate patient-related data and results.

7How to Ordera TestPlease contact your local LabPMM
site to receive the necessary forms
to initiate a services ordering account.
EMAIL:
inquiry@invivoscribe.com

8Ordering Details
Specimen Collection and Shipment
We advise our customers to send all specimens through an overnight delivery service. Please notify your local LabPMM site of urgent samples
so we know when to expect the specimens and can investigate any shipping issues if needed.

Specimens for DNA Assays
Collect blood and bone marrow specimens in sodium heparin, EDTA, or an ACD (acid citrate dextrose) tube. Specifically for FLT3 ITD MRD and NPM1
MRD, only samples collected in EDTA are accepted. Blood and bone marrow may be stored at 2-8 °C for up to 7 days.
Please ship blood and bone marrow at ambient temperature or with cool packs, do not freeze. Please ship previously isolated DNA at ambient
temperature, with cool packs, or on dry ice, as applicable. Previously isolated DNA may be stored indefinitely at -65°C to -85°C.

Specimens for Flow Cytometry Panels
Collect blood or bone marrow specimens in sodium heparin or EDTA tubes. Blood and bone marrow may be stored at 2-8 °C for up to 7 days.
Please ship blood and bone marrow at ambient temperature or with cool packs, do not freeze.

CDx FLT3 Mutation Assay
Peripheral blood or bone marrow aspirate samples collected in sodium heparin or EDTA tubes are accepted. Preserved specimens may be stored
at 2-8°C for up to 7 days. ACD specimen collection not accepted.

Accredited Menu
LabPMM, LLC (Americas)
CAP ISO 15189-accredited and CLIA-certified
CDx FLT3, FLT3 ITD MRD, NPM1, NPM1 MRD, NGS B-cell Clonality,
NGS T-cell Clonality, B-cell MRD, MyAML, MyMRD, MFC Hematolymphoid
Screening Panel, MFC AML MRD Assay, MFC CLL MRD Assay

New York State Licensed Tests
CDx FLT3, NPM1LabPMM GMBH (EU/Middle East/Africa)
DAkkS-accredited tests to ISO 15189
CDx FLT3, NPM1, NGS B-cell Clonality, NGS T-cell Clonality

LabPMM GK (Asia/Pacific)
Licensed Clinical Lab
CDx FLT3

Invivoscribe Diagnostic Technologies (Shanghai) Co., Ltd.
CAP-accredited
CDx FLT3, NGS B-cell Clonality

9Patient Consent
and Confidentiality

LEARN MORE:
invivoscribe.com/clinical-lab-services

10Patient Consent
» LabPMM will only process routine diagnostic samples submitted by medical institutions, whereby consent for diagnostic testing is obtainedby the submitting physician.
» N o data is forwarded to outside organizations without specific prior consent.
» Samples for the MyAML and MyMRD assays must have a completed patient consent form, signed by the patient and the submitting physician toconfirm that the patient has understood and given consent for the testing requested.

Patient Confidentiality» B
 y sending samples to LabPMM for routine diagnostic testing, patients will be protected by the strict data protection laws.
» Patient samples will only be reused for quality control of the assays requested on the original requisition form.
» At LabPMM GmbH, unless prior consent is given, primary samples or DNA from patient samples is retained for 12 months for quality control
purposes only. Thereafter patient samples are de-identified and destroyed.
» Ourdata servers are located in a facility in the USA. Data is encrypted prior to transfer and the transferred data is subject to the same safeguards
as data held in Germany.

11

Partner with Us

Your Ideal Partner for Laboratory
Services, Clinical Trial Testing, andCompanion Diagnostic Development.
EMAIL:
businessdevelopment@invivoscribe.com

12 Partner With Us

LabPMM (an Invivoscribe® company)
is Your Partner of Choice for Diagnostic,
Research, and Clinical Trial Services.
Our network of laboratories located in the USA, Europe and Asia specialize in internationally harmonized molecular testing, and flow cytometry,
and collectively have CLIA and ISO 15189 certifications via CAP and DAkkS. We also offer contract research organization (CRO) services, and are
a comprehensive companion diagnostics (CDx) and custom assay development partner, providing ISO 13485-compliant biomarker development,
cGMP manufacturing, regulatory capability, global laboratory services and commercialization.
We offer an ever expanding menu of molecular assays, including NGS gene and MRD panels, FLT3 and NPM1 mutation assays, and B- and T-cellclonality and MRD assessment. Recently, LabPMM expanded its testing capabilities to include multiparametric flow cytometry. Our comprehensive
test menu will now eliminate the need for partners to split primary specimens, dramatically decreasing turnaround times and allowing for coherent
comparison of flow-based and NGS-based MRD test results.

State of the Art
We thrive in international cooperation and in continuous investment in the advancement of precision medicine. We work with a full rangeof collaborators: key opinion leaders, leading healthcare institutions, and top-tier pharmaceutical companies. We work on the premise that all
those reliant on data and results from clinical testing (healthcare providers, pharmaceutical companies and most importantly, patients) will
benefit from better standardization and more consistent performance of molecular diagnostic tests.

Quality
Internationally-harmonized diagnostics through our global network of laboratories. We follow full QSR design control for assay and softwaredevelopment. Products are manufactured under cGMP and ISO 13485.

Partnership
We support partnerships worldwide to develop, validate, and commercialize custom biomarker assays and reagents. Our global distribution
network operates in more than 700 laboratories in 160 countries.

Expertise
With nearly 30 years of experience we are the foremost experts in providing molecular products and services for oncologic testing. We offer dedicatedsupport in design and development, manufacturing, software and bioinformatics, technical support, quality assurance, and global regulatory affairs.

Partner With Us 13

An Ideal CDx
Partner for DrugApprovals

EMAIL:
businessdevelopment@invivoscribe.com

14 Partner With Us

IVD Product Development» Nearly 30 years of assay development experience» Biomarker assays & software development under full ISO 13485 design controls» Comprehensive NGS gene panels that identify actionable biomarkers» Custom biomarker assay and CDx development

Global Regulatory, Quality andCommercial Expertise» Experienced staff & proven Quality Management System» Full adherence to FDA 21 CFR part 820 and ISO13485 standards» Registered Medical Device Establishment with the US FDA, KFDA, Saudi Arabia, and the PMDA
» Multiple CDx approvals supporting various drugs: by the FDA (US) and PMDA/MHLW (Japan)» CDx CE-marks in the EU» 50+ CE-marked IVDs available in the EU and select ROW markets; 60 tests registered with the ARTG in Australia» Marketing Authorization Holder (MAH) and National reimbursement for CDx in Japan» CDx submitted for reimbursement in the US» Supporting ongoing clinical drug trials in the US, EU, Japan, China and ROW

Clinical Testing
Global Clinical Reference Laboratory Network» A dozen years of clinical reference lab experience» Internationally standardized CDx and biomarker testing with labs serving the US, Europe, and Asia
» Standardized CAP/CLIA-certified Multiparametric Flow Cytometry panels for screening, trial enrollment and MRD monitoring» Comprehensive LymphoTrack® clonality/MRD assays and CAP and CLIA-certified NGS MyGene™ panels identify clinically actionable biomarkers» Complementary MRD assays for all biomarkers – potential for surrogate endpoints per agency inputs» Testing services have supported hundreds of enrollment sites worldwide

Manufacturing» FDA/CDRH-registered and ISO 13485-certified cGMP manufacturing facility based in San Diego» Comprehensive Dx and CDx Manufacturing:» CDx for USA (PMA), Japan, EU, and ROW markets» 50+ CE-IVDs (NGS assays + bioinformatics software) IUO & RUO assays, ASRs & GPRs
» DNA / RNA controls, MRD controls & proficiency panels

Partner With Us 15

Join the 75+
Pharma Companies
Worldwide Working
with UsEMAIL:
businessdevelopment@invivoscribe.com

16 Partner With Us

Integrated Approach to CDx Development
We provide efficient handling of all stages of CDx development, from the biomarker
discovery process to commercialization, including:» Supportive, collaborative relationship with our pharmaceutical partners» Dedicated CDx development team with extensive expertise in program management, feasibility studies, product development, quality control,
regulatory, and commercialization» Sense of urgency and commitment to partner's success

Drug Development Process
Phase I

Preclinical

Phase II

Assay
Assay
Development
Development

Phase III

Start
Clinical Trial

Regulatory
Submission

Rx/Dx
Co-submission

Rx Commercialization

Rx/Dx
Co-approval

Biomarker
Development
Process
Biomarker ID/
Content IP

Assay Design

Assay
Feasibility

Testing andDevelopment

Biomarker Development Process
CDx Development

Prospective
Testingduring Phase III
Rx Study

Regulatory
Submission

CDx
Commercialization

Partner With Us 17

CDx
Commercialization
Phasing

EMAIL:
businessdevelopment@invivoscribe.com

18 Partner With Us

Planning» Develop plan in collaboration with partner» Share market insights and collective knowledge» Align Rx / Dx commercial objectives» Define commercialization strategy and country level prioritization/timing» Determine activities requiring partner support

Pre-Launch» Identify and nurture key opinion leaders (KOLs)» Align CDx development studies with commercialization studies» Voice of customer to drive product requirements and commercialization considerations» Prepare marketing message for PMA submission

Launch» Establish lab network» Create physician awareness» Activate KOL network to start commercialization studies» Complete HEOR studies, prepare HTAs, request reimbursement coverage» Initiate PR and marketing campaigns

Post-Launch» Expand lab network and physician awareness after reimburement established and guidelines updated» Continue to nurture KOLs
» Monitor and adjust activities to achieve market adoption» Transition to maintenance campaigns

Examples of FLT3 CDx Partnerships
Novartis

Astellas» FLT3 CDx for RYDAPT® (midostaurin)» Breakthrough designation in the USA» FDA, Swissmedic and EU approval in 2017

» FLT3 CDx for XOSPATA® (gilteritinib fumarate)» More than 140 international collection sites» PMDA approval in Japan in 2018
» Reimbursement approved in Japan» FDA approval in USA in 2018

Daiichi-Sankyo» FLT3 CDx for VANFLYTA® (quizartinibhydrochloride)» Bridging study for multiple labs in the USA,
Europe and Asia
» PMDA approval in Japan in 2019

» EU approval in 2019

Partner With Us 19

Companion
Diagnostic Testing
According to the U.S. FDA definition, a companion diagnostic is a medical
device, often an in vitro device (IVD), which provides information that isessential for the safe and effective use of a corresponding drug or biologicalproduct. The use of a companion diagnostic will therefore help cliniciansand healthcare providers determine whether a patient is likely to benefit fromthe drug in question and monitor the response. In the European Union under
the new IVDR, the definition of a companion diagnostic expands to requirepatient screening before and/or during treatment for those likely
to experience benefit and/or increased risk as a result of treatment withthe corresponding medicinal products.
The use of assays that have not been specifically validated for the safety andeffectiveness of a drug or biological product may deliver inaccurate resultsthat could harm the patient. For instance, a false positive result could leadto treatment with a drug without the proven benefits, exposing the patient
to potential toxic side effects. Likewise, a false negative test result could
withhold or delay a potentially beneficial treatment, putting the patient
at risk.
Companion diagnostics help demonstrate drug efficacy and accelerate
approval. They have become an important tool for improving individual
patient treatment.
LabPMM embraces international harmonization and partnering. We workwith key opinion leaders to standardize molecular diagnostic testing and
we are also partnered with pharmaceutical companies to develop
companion diagnostic tests. Our proud history of partnerships have led
to outstanding work towards internationally standardized testing,exemplified by FDA and PMDA approval of the first companion diagnostic
for acute myeloid leukemia.

Companion
Diagnostic Testing20

United States - FDA Approved
Intended Use
The LeukoStrat CDx FLT3 Mutation Assay is a PCR-based in vitro
diagnostic test designed to detect internal tandem duplication(ITD) and tyrosine kinase domain (TKD) mutations D835 and I836
in the FLT3 gene in genomic DNA extracted from mononuclear cells
obtained from peripheral blood or bone marrow aspirates of patientsdiagnosed with acute myelogenous leukemia (AML).
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in theassessment of patients with AML for whom RYDAPT® (midostaurin)
treatment is being considered.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in theassessment of patients with AML for whom XOSPATA® (gilteritinib)
treatment is being considered.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in theassessment of patients with FLT3-ITD+ AML for whom VANFLYTA®
(quizartinib) treatment is being considered.
The test is for use on the 3500xL Dx Genetic Analyzer.
» Mutations in the FLT3 gene are the most common mutations
found in AML» Presence of a FLT3 activation mutation in patients with AML may
be prognostic and clinically actionable.
» The LeukoStrat CDx FLT3 Mutation Assay is used as aid in theselection of AML patients for whom midostaurin, gilteritinib, or
quizartinib are being considered» NCCN, ELN and CAP Guidelines recommend FLT3 testing to informpatient treatment decisions.
» Gilteritinib was approved by the FDA for the treatmentof adult patients with relapsed/ refractory FLT3mut+ AML» Midostaurin was approved by the FDA for the treatment of adultpatients with newly diagnosed AML who are FLT3 mutation positive» Quizartinib was approved by the FDA for the treatment of adult 		
patients with newly diagnosed FLT3-ITD+AML» First-to-market, this CDx is FDA approved (PMA#P160040)
as a predictive test for the efficacy of midostaurin therapy in all AML
patients, regardless of cytogenetics.

Test Name
LeukoStrat® CDx FLT3 Mutation Assay

Assay Type
Capillary Electrophoresis

Method Description

Predictive test for the efficacy
of RYDAPT® (midostaurin), XOSPATA®
(gilteritinib), or VANFLYTA® (quizartinib)
Primers targeting both in and around the juxtamembrane region for
ITD testing and kinase domain of the FLT3 gene are used to amplify
DNA extracted from a patient sample.
The TKD PCR product is further digested with a restriction enzyme.
The ITD PCR products and the digested TKD PCR products areanalyzed on a capillary electrophoresis instrument.
FLT3 ITDs are detected by a change in the expected size of a wild
type fragment. An amplicon larger than the wild type fragment
indicates the presence of FLT3 ITD. The TKD digestion pattern
identifies loss of the normal gene sequences and ensures thatdigestion occurred.

Indications for Testing» At initial diagnosis or relapse of AML» As an aid in the assessment of patients with AML for whom
RYDAPT® (midostaurin) treatment is being considered.
» As a tool to identify AML patients eligible for treatment withXOSPATA® (gilteritinib).
» As an aid in the assessment of patients with FLT3-ITD+ AML for
whom VANFLYTA® (quizartinib) treatment is being considered.

Interpretation
An interpretive report will be issued, indicating whether the patientis eligible for midostaurin, gilteritinib, or quizartinib treatment

Turnaround Time
2 to 3 business days
>95% patient samples are reported within 48 hours of receipt

Specimen Requirements
Recommended Specimen Volume (Preservative)
2 mL of peripheral blood in Sodium Heparin or EDTA
0.5mL of bone marrow in Sodium Heparin or EDTA

Minimum Specimen Volume (Preservative)

1mL of peripheral blood in Sodium Heparin or EDT
0.25 mL of bone marrow in Sodium Heparin or EDTA

Shipping Conditions
2°C to 8°C up to 72 hours; do not freeze.

Specimen Stability
2°C to 8°C up to 7 days

The LeukoStrat® CDx FLT3 Mutation Assay is designed to detect ITD
and TKD mutations in the FLT3 gene. The assay is performed on DNA
isolated from mononuclear cells obtained from peripheral blood orbone marrow aspirates of patients diagnosed with AML.

Companion Diagnostic Testing 21

CE-marked

Intended Use
The LeukoStrat® CDx FLT3 Mutation Assay is a PCR-based in vitro
diagnostic test designed to detect internal tandem duplications(ITD) and tyrosine kinase domain (TKD) mutations D835 and I836
in the FLT3 gene in genomic DNA extracted from mononuclear cells
obtained from peripheral blood or bone marrow aspirates of patientsdiagnosed with acute myelogenous leukemia (AML).
In regions where midostaurin is available, the LeukoStrat® CDx FLT3
Mutation Assay is used as an aid in the assessment of patients with
AML for whom RYDAPT® (midostaurin) treatment is being considered.
In regions where gilteritinib fumarate is available, the LeukoStrat®
CDx FLT3 Mutation Assay is used as an aid in the assessmentof patients with AML for whom XOSPATA® (gilteritinib fumarate)
treatment is being considered.

» Mutations in the FLT3 gene are the most common mutations foundin AML» Presence of a FLT3 activation mutation in patients with AML may
be prognostic and clinically actionable.
» NCCN, ELN and CAP Guidelines recommend FLT3 testing to informpatient treatment decisions.
» The LeukoStrat® CDx FLT3 Mutation Assay is used as an aid in theassessment of AML patients for whom midostaurin and/or
gilteritinib fumarate treatment is being considered» Midostaurin was approved by the Swissmedic and European
Commission for the treatment of adult patients with newly
diagnosed AML who are FLT3 mutation positive» Gilteritinib fumarate was approved by the European Commission
for the treatment of relapsed/ refractory FLT3mut+ AML

Test Name
LeukoStrat® CDx FLT3 Mutation Assay

Assay Type
Capillary Electrophoresis

Method Description
The LeukoStrat® CDx FLT3 Mutation Assay is designed to detect ITD
and TKD mutations in the FLT3 gene.
The assay is performed on DNA isolated from mononuclear cells
obtained from peripheral blood or bone marrow aspirates of patientsdiagnosed with AML.

Predictive test for the efficacy
of RYDAPT® (midostaurin) and XOSPATA®
(gilteritinib fumarate)

Primers targeting both in and around the juxtamembrane region
for ITD testing and kinase domain of the FLT3 gene are used toamplify DNA extracted from a patient sample. The TKD PCR productis further digested with a restriction enzyme. The ITD PCR products
and the digested TKD PCR products are analyzed on a capillaryelectrophoresis instrument.
FLT3 ITDs are detected by a change in the expected size of a wild
type fragment. An amplicon larger than the wild type fragment
indicates the presence of FLT3 ITD. The TKD digestion pattern
identifies loss of the normal gene sequences and ensures thatdigestion occurred.

Indications for Testing» At initial diagnosis or relapse of AML» In regions where midostaurin is available, the LeukoStrat CDx
FLT3 mutation assay is used as an aid in the assessment of AML
patients for whom midostaurin treatment is being considered.
» In regions where gilteritinib fumarate is available, the LeukoStrat
CDx FLT3 mutation assay is used as an aid in the assessmentof AML patients for whom gilteritinib fumarate treatment is being
considered.

Interpretation
An interpretive report will be issued indicating the absenceor presence of a FLT3 mutation and its corresponding signal ratio.
The report will further indicate whether the patient is eligible fora therapy with midostaurin or gilteritinib fumarate.

Turnaround Time
2 to 3 business days
>95% patient samples are reported within 48 hours of receipt

Specimen Requirements
Recommended Specimen Volume (Preservative)
2 mL of peripheral blood in Sodium Heparin or EDTA
0.5mL of bone marrow in Sodium Heparin or EDTA

Minimum Specimen Volume (Preservative)

1mL of peripheral blood in Sodium Heparin or EDT
0.25 mL of bone marrow in Sodium Heparin or EDTA

Shipping Conditions
2°C to 8°C up to 72 hours; do not freeze.

Specimen Stability
2°C to 8°C up to 7 days

22

Predictive test for the efficacy of Gilteritinib
Fumarate and Quizartinib Hydrochloride
Japan - MHLW/PMDA Approved

Intended Use
The LeukoStrat CDx FLT3 Mutation Assay is a PCR-based, in vitro
diagnostic test designed to detect internal tandem duplication (ITD)
mutations and tyrosine kinase domain (TKD) mutations D835 andI836 in the FLT3 gene in genomic DNA extracted from mononuclear
cells obtained from peripheral blood or bone marrow aspirates
of patients diagnosed with acute myelogenous leukemia.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in theassessment of patients with AML for whom Gilteritinib Fumarate
treatment is being considered.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in theassessment of patients with AML for whom Quizartinib Hydrochloride
treatment is being considered.
» Mutations in the FLT3 gene are the most common mutations foundin AML» Presence of a FLT3 activation mutation in patients with AML may
be prognostic and clinically actionable.
» The LeukoStrat® CDx FLT3 Mutation Assay is the first PMDA
approved test for assessment of AML patients eligible for treatment
with Gilteritinib Fumarate or Quizartinib Hydrochloride

FLT3 ITDs are detected by a change in the expected size of a wild
type fragment. An amplicon larger than the wild type fragment
indicates the presence of FLT3 ITD. The TKD digestion pattern
identifies loss of the normal gene sequences and ensures thatdigestion occurred.

Indications for Testing» At initial diagnosis or relapse of AML» As a tool for the assessment of AML patients for whom Gilteritinib
Fumarate treatment is being considered» As a tool for the assessment of AML patients for whom Quizartinib
Hydrochloride treatment is being consideredInterpretation
An interpretive report will be issued, indicating whether the patient iseligible for Gilterinib Fumarate treatment or Quizartinib Hydrochloride

Turnaround Time
2 to 3 business days
>95% patient samples are reported within 48 hours of receipt» Gilteritinib Fumarate received manufacturing and marketing
approval for the treatment of FLT3 mutation-positive relapse
or refractory AML in Japan

Specimen Requirements» Quizartinib Hydrochloride is MHLW/PMDA approved for thetreatment of relapsed/ refractory FLT3-ITD+ AML

Recommended Specimen Volume (Preservative)

Test Name

Minimum Specimen Volume (Preservative)

LeukoStrat® CDx FLT3 Mutation Assay

Assay Type
Capillary Electrophoresis

Method Description
The LeukoStrat® CDx FLT3 Mutation Assay is designed to detect ITD
and TKD mutations in the FLT3 gene.
The assay is performed on DNA isolated from mononuclear cells
obtained from peripheral blood or bone marrow aspirates of patientsdiagnosed with AML.
Primers targeting both in and around the juxtamembrane region
for ITD testing and kinase domain of the FLT3 gene are used toamplify DNA extracted from a patient sample. The TKD PCR productis further digested with a restriction enzyme. The ITD PCR products
and the digested TKD PCR products are analyzed on a capillaryelectrophoresis instrument.

2 mL of peripheral blood in Sodium Heparin or EDTA
0.5mL of bone marrow in Sodium Heparin or EDTA

1mL of peripheral blood in Sodium Heparin or EDT
0.25 mL of bone marrow in Sodium Heparin or EDTA

Shipping Conditions
2°C to 8°C up to 72 hours; do not freeze.

Specimen Stability
2°C to 8°C up to 7 days

Reimbursement Points
4200 points

Companion Diagnostic Testing 23

MolecularDiagnostic Tests
Somatic mutations play an increasingly important role in the risk stratificationand management of leukemia and lymphoma patients. Traditionally,classification and risk stratification have relied on cytogenetic studies;
however, molecular detection of gene mutations and gene rearrangements
are now central in the classification, risk stratification, and managementof lymphoproliferative diseases. Molecular testing also complements
cytogenetic testing results, which helps further refine stratification andprognosis, especially within specific disease subgroups.
All of LabPMM’s molecular tests conform to the Standard of Care as definedby the World Health Organization (WHO) and are recommended by membersof the National Comprehensive Cancer Network, LeukemiaNet, and otherworld opinion leaders in hematology.

24 Molecular Diagnostic Tests

NPM1 Mutation Assay
Clinical Information

Interpretation

The Nucleophosmin (NPM1) gene is one of the most commonly
mutated genes in acute myeloid leukemia (AML), occurring in
about 35% of AML patients at diagnosis.1 The vast majority of NPM1
mutations are insertions in exon 12 occurring near the C-terminusof the protein that result in cytoplasmic localization.2 Currently there
are over 40 known NPM1 mutations, most of which will be detectedwith our assay.

An interpretive report will be issued indicating whether a NPM1
mutation was detected

Clinical studies have found that NPM1 mutations are associatedwith increased blast counts, higher extramedullary involvement andincreased platelet counts in AML.3 Furthermore, in the absenceof a FLT3 ITD mutation (or FLT3 ITD with a low ratio), NPM1 mutations
are associated with a favorable prognosis.4

Specimen Requirements

It has been suggested that the identification of mutations in both
NPM1 and FLT3 genes allows for the stratification of the AML patients
into three different prognostic groups:

Turnaround Time
1-3 business days

1-3 mL peripheral blood in EDTA, ACD or Heparin0.25-1 mL bone marrow in EDTA, ACD or Heparin
250 ng of previously isolated DNA

Shipping Conditions

» Favorable prognosis: NPM1 mutation without FLT3 ITD
or with FLT3 ITDlow

Ambient or Cool; do not freeze (peripheral blood or bone marrow
Ambient or frozen on dry ice (isolated DNA)» Intermediate prognosis: NPM1 mutation and FLT3 ITDhigh; NPM1without FLT3 ITD or with FLT3 ITDlow (without adverse-risk genetic
lesions)

Specimen Stability» Poor prognosis: NPM1 wild-type and FLT3 ITDhigh

Room Temp up to 72 hours
2-8 °C up to 7 days

It is recommended that AML patients be screened for NPM1
mutations as an effort to assess prognosis and aid in treatment
decisions. Results from NPM1 and FLT3 mutational screening should
be available within 48 to 72 hours (at least in patients eligible forintensive chemotherapy). Utilizing both NPM1 and FLT3
(mutant:wild-type ratio) mutation status is the most common
molecular method for stratification of the AML population.

Test Name
NPM1 mutation analysis (qualitative)

Assay Type
Capillary Electrophoresis

Method Description
Primers targeting exon 12 on the NPM1 gene are used to amplify thepatient’s DNA. The size of the NPM1 PCR product is determinedby capillary electrophoresis.
LabPMM offers the only internationally harmonized assay for NPM1
mutations and testing is performed pursuant to patents licensed from
Cardiff Oncology Inc. of San Diego, CA.

Indications for Testing At initial diagnosis of AML»» Stratification high and low risk AML» R ecurrence of leukemia after induction therapy on patients notinitially screened for NPM1 mutations

Molecular Diagnostic Tests 25

Clonality Tests
The unique process of genetic rearrangements in the immunoglobulin (Ig)and T-cell receptor (TCR) gene loci during B- and T-cell development andmaturation generates a vast pool of genetically distinct cells.
During early lymphoid differentiation, genes encoding the Ig and TCR molecules
are formed by stepwise rearrangement of variable (V), diversity (D), and joining(J) gene segments. During this V-D-J recombination process, nucleotides aredeleted and randomly inserted at the joining sites, resulting in an enormous
diversity of unique antigen receptors. As Ig/TCR gene rearrangements occur
sequentially in the earliest stages of lymphoid differentiation, they are present
in almost all immature and mature lymphoid cells.
Since lymphoma is a cancer of the lymphatic or the immune system,
the vast majority of lymphomas exhibit rearrangements in Ig and/or TCR genes.
Lymphoid malignancies are characterized by the reduced population diversityof these gene loci originating from the proliferative transformation of anindividual lymphoid cell. The associated cellular population typically shares one
or more cell-specific or “clonal” antigen-receptor gene rearrangements.
The detection of these clonal cells provides the basis for clonality assessmentin leukemia, lymphoma, and hematologic disease diagnosis.
Invivoscribe (LabPMM’s parent company) is an industry pioneer with nearly 3
decades of experience in providing clonality test solutions. Our expertisein clonality testing assures the highest rates of detection of clonal populations
as well as international standardization of results.

26 Clonality Tests

B-Cell Clonality Assay
Clinical Information

Assay Type

Lymphoid cells are different from the other somatic cells in the
body as during development, the antigen receptor genes in thesecells undergo somatic gene rearrangement.1 Since leukemia andlymphomas originate from the malignant transformation of individual
lymphoid cells, all leukemias and lymphomas generally share one ormore cell-specific or “clonal” antigen receptor gene rearrangements.
Clonality does not always imply malignancy; all results must be
interpreted in the context of all of the other available diagnostic
criteria. Tests that detect Ig clonal rearrangements are usefulin the characterization, monitoring, and treatment of B- andT-cell malignancies.

Next-Generation Sequencing (NGS)

Immunoglobulin Heavy (IGH)

Indications for Testing

The human IGH gene locus on chromosome 14 (14q32.3) includes
46-52 functional and 30 non-functional variable (VH) gene segments,
27 functional diversity (DH) gene segments, and 6 functional
joining (JH) gene segments spread over 1250 kilobases. During
B-cell development, genes encoding the human IGH proteins areassembled from multiple polymorphic gene segments that undergo
rearrangements and selection, generating VH-DH-JH combinations thatare unique in both length and sequence for each cell.2-3» Identify clonality in atypical lymphoproliferative disorders

Somatic Hypermutation (SHM)
An additional level of diversity is generated by point mutationsin the variable regions, also known as SHM. Immunoglobulin
variable heavy chain gene hypermutation status provides important
prognostic information for patients with chronic lymphocytic leukemia(CLL) and small lymphocytic lymphoma (SLL). The presence ofIGH SHM is defined as greater or equal to 2% difference from thegermline VH gene sequence, whereas less than 2% difference isconsidered evidence of no SHM. The status of SHM for clone(s) has
clinical relevance, as there is a clear distinction in the median survivalof patients with and without SHM. Hypermutation of the IGH variable
region is strongly predictive of a good prognosis, while lack ofmutation predicts a poor prognosis.4 In addition, this assay identifies
clonal rearrangements involving the V3-21 gene, which has been
associated with a poor prognosis in CLL independent of SHM status.
This assay has been shown to further stratify CLL patients.5

Method Description
Specimens received for B-cell clonality are tested for therecommended immunoglobulin gene target(s) based on clinicalindications. Next-generation sequencing of the PCR products is used
to identify DNA sequences specific to clonal gene rearrangements.
Bioinformatics tools facilitate the identification and characterizationof significant clonal rearrangements. These sequences can be used
to track specific clonal populations in follow-up testing.

» Support a differential diagnosis between reactive lesions andhematologic malignancies» Assign presumptive lineage in mature monoclonal
lymphoproliferative disorders» Monitor and evaluate disease recurrence

Interpretation
An interpretive report will be issued indicating the level of clonality
along with the rearrangement locus for the dominant clones and thespecific sequence for the dominant clone.

Turnaround Time
12 to 14 business days

Specimen Requirements
1-3 mL of peripheral blood in EDTA , ACD or Heparin0.25-1 mL of bone marrow in Heparin, EDTA or ACD
500 ng of previously isolated DNA

Shipping Conditions
Immunoglobulin Kappa (IGK)The human IGK locus on chromosome 2 (2p11.2) includes 7 variable(Vƙ) region gene segments and 5 joining (Jƙ) gene segments
upstream of the constant (Cƙ) region. The Kappa deleting element(Kde), approximately 24 kb downstream of the Jƙ-Cƙ region, can also
rearrange with Vƙ gene segments and the isolated recombination
signal sequence in the Jƙ-Cƙ intronic region.6 Specifically during B-celldevelopment, genes encoding IGK molecules are assembled frommultiple polymorphic gene segments that undergo rearrangements
generating gene receptors unique in both length and sequence.

 mbient or Cool; do not freeze (peripheral blood or bone marrow)A
Ambient or frozen on dry ice (isolated DNA)Specimen Stability
2-8 °C up to 7 days prior to testing

References
1. Tonegawa, S (1983) Nature. 302:575-581.
2. Trainor, KJ et al. (1990) Blood. 75:2220-222 2.

Test Name
B-cell Clonality Assay

3. van Dongen, JJM et al. (2003) Leukemia. 17:2257–2317.
4. Ghia, P et al. (2007) Leukemia. 21:1-3.
5. Stamatopoulos, B et al. (2017) Leukemia. 31(4):837-845.
6. Miller, JE et al. (2013, 2nd ed.) Springer Science & Business Media. 2.7.13 and 30.2.7.18.

Clonality Tests 27

T-Cell Clonality Assay
Clinical Information

Method Description

Lymphoid cells are different from the other somatic cells in the
body. During development the antigen receptor genes in lymphoidcells (including gene segments within TRG and TRB), undergo
somatic gene rearrangement.1 These developmentally regulated,
programmed gene rearrangements generate Vγ-Jγ combinations thatare unique for each cell.2

Specimens received for T-cell clonality are tested for therecommended T-cell receptor gene target(s) based on clinicalindications. Next-generation sequencing of the PCR products is used
to identify DNA sequences specific to clonal gene rearrangements.
Bioinformatics tools facilitate the characterization of sequences
present at greater than 2.5% of the population. These sequences canbe used to track specific clonal populations
in follow-up testing.

Since leukemias and lymphomas originate from the malignant
transformation of individual lymphoid cells, which means that all
leukemias and lymphomas generally share one or more cell-specificor “clonal” antigen receptor gene rearrangements. Clonality does
not always imply malignancy; all results must be interpreted in thecontext of all of the other available diagnostic criteria. Tests that
detect TCR clonal rearrangements can be used to help identify T-celland certain B-cell malignancies.

T-cell Receptor Gamma (TRG)
The human TRG locus on chromosome 7 (7q14) includes 14 variable(Vγ) genes (Group I, II, III, and IV), 5 joining (Jγ) gene segments, and 2
constant (Cγ) genes spread over 200 kilobases.3
During T-cell ontogeny rearrangement of the TRG locus occurs before
rearrangement of the alpha beta loci. So, clonal rearrangements of
TRG are often present, commonly detected, and can be tracked inT-cell malignancies involving alpha-beta T-cells. This makes TRG apowerful tool for both clonal and MRD analysis of T-cell and someB-cell tumors.

T-cell Receptor Beta (TRB)
The human TRB gene locus on chromosome 7 (7q34) includes 64-67
variable (Vß) gene segments (belonging to 30 subgroups), 2 diversity(Dß) gene segments, and 13 joining (Jß) gene segments, spreadover 685 kilobases, making this locus far more complex than others.
Nevertheless, accurate molecular analysis of the TRB genes is an
important tool for the assessment of clonality in suspected T-cell andsome B-cell proliferations, as TRB gene rearrangements occur not
only in almost all mature T-cell malignancies, but also in about onethird of precursor B-acute lymphoblastic leukemias (B-ALL).3

Test Name
T-cell Clonality Assay

Assay Type
Next-Generation Sequencing (NGS)

Indications for Testing» Identify clonality in atypical lymphoproliferative disorders» Support a differential diagnosis between reactive lesions andhematologic malignancies» Assign presumptive lineage in mature monoclonal
lymphoproliferative disorders» Monitor and evaluate disease recurrence

Interpretation
An interpretive report will be issued indicating whether evidenceof clonality was detected. The report further provides a summaryof the top 5 merged sequences, including the % total reads, therearrangement class and the sequence.

Turnaround Time
12 to 14 business days

Specimen Requirements1 -3 mL of peripheral blood in Heparin, EDTA or ACD
0.25-1 mL of bone marrow in Heparin, EDTA or ACD
500 ng of previously isolated DNA

Shipping Conditions
 mbient or Cool; do not freeze (peripheral blood or bone marrow)A
Ambient or frozen on dry ice (isolated DNA)Specimen Stability
2-8 °C up to 7 days prior to testing

References
1. Tonegawa, S. (1983) Nature. 302:575-581.
2. Miller, JE et al. (2013, 2nd ed.) Springer Science & Business Media. 302.2.7.13 and 30.2.7.18.
3. Lawnickie, LC et al. (2003) Journal of Molecular Diagnostics. 5:82-87.

28 Clonality Tests

Measurable Residual
Disease Tests
Measurable also known as Minimal Residual Disease (MRD) testing has shown strong
potential for the optimization of therapeutic management of lymphoproliferative
diseases. Currently, MRD tests complement and leverage the information obtainedat diagnosis. Due to their increased sensitivity, these measurements are most usefulat time points where they are compared and contrasted with more traditional
methods. An example of this is before transplant, when MRD levels have been shown
to be predictive of transplantation success.
Several patient-specific PCR-based (e.g. ASO-PCR) and flow cytometric technologies
have been developed by regional test centers in order to routinely assess MRD levels
during the course of therapy. However, ASO-PCR requires patient- andtumor-specific primer and probe sets, making it cost prohibitive and impossible
to offer as a standardized method. Flow cytometry assays are also subjective anddifficult to standardize between testing centers. Even innovative multiparametricflow cytometry methods are difficult to standardize and therefore do not meet thestandards required to take them through the regulatory agencies.
Next-Generation Sequencing (NGS) methods have recently been developed forthe detection and monitoring of MRD. These forefront technologies use regulatorycompliant chemistries, run on regulatory-compliant instruments, and canbe interpreted using regulatory compliant, and design-controlled bioinformatics
software. Due to the read depth of this non-biased patient agnostic testing approach,ultra deep sequencing overcomes virtually all of the shortcomings of other MRD
technologies, providing internationally harmonized MRD testing for virtually any
targeted biomarker.
LabPMM’s MRD tests are NGS-based assays that can be used to detect clonal generearrangements identified at diagnosis within the B- and T-cell antigen receptor loci.
Once a specific rearrangement sequence (the clonotype) has been identified
in a primary sample, bioinformatics tools allow for objective longitudinal tracking
of clonal populations with a sensitivity up to 1 x 10-6, provided sufficient DNA
is tested.
LabPMM also offers FLT3 ITD and NPM1 MRD assays, which are used for the detectionof targeted mutations. These sensitive NGS-based assays reliably detect sequences
present at 5 x 10-5.

Measurable Residual Disease Tests 29

AML -FLT3 ITD MRD Assay
Clinical Information

Interpretation

Measurable residual disease (MRD) detection in patients withleukemia has proven to be useful in the clinical managementof disease and can facilitate the development of new therapies.
Mutations in the fms-like tyrosine kinase 3 (FLT3) gene are the mostprevalent mutations found in acute myeloid leukemia (AML)1 and arecharacterized by an aggressive phenotype with a high prevalenceof relapse. Internal tandem duplication (ITD) mutations within thejuxtamembrane domain are the most common mutations of FLT3 .2
The development of a sensitive and specific assay for FLT3 ITD
mutations represents a significant advancement in guiding
treatment decisions.

An interpretive report will be issued indicating whether FLT3 ITD MRD
was detected

LabPMM’s FLT3 ITD MRD test is an NGS-based, targeted, deepsequencing assay that detects ITDs ranging from 3 bp to over 200 bp
in size. Once a specific ITD (length and sequence) has been identified
in a primary sample, it can easily be tracked in subsequent samples
at a sensitivity of 5x10-5, provided sufficient DNA quantity is tested.
The treatment of AML has become a paradigm for precision
medicine. This MRD assay is at least two orders of magnitude
more sensitive than other commercially available FLT3 assays.
It detects the persistence of a driver mutation, FLT3 ITD, in patientswith no overt evidence of disease, allowing clinicians to identify
those patients that can benefit from continuation or modificationof treatment.3
MRD detection by Next-Generation Sequencing has demonstrated
utility in predicting clinical outcomes and in generating clinicallyactionable results, allowing early intervention, confirmationof disease status prior to transplant, and increased confidence
in remission status.

Test Name
FLT3 ITD MRD Assay

Assay Type
Next-Generation Sequencing (NGS)
CLIA-validated assay

Method Description
To track and identify previously detected FLT3 ITD mutations in posttreatment follow-up samples, a multiplex master mix targeting thejuxtamembrane domain of the FLT3 gene is used to amplify DNA
extracted from a patient sample.
Next-generation sequencing of the PCR products is used to identify
DNA sequences specific to previously identified mutations detected at
diagnosis. Bioinformatics tools facilitate the detection of these specificsequences present at an allelic sensitivity level of 5x10-5.

Indications for Testing» Identify tumor-specific markers for post-treatment monitoring» Monitor and evaluate disease recurrence

30 Measurablel Residual Disease Tests

Turnaround Time
7 to 10 business days

Specimen Requirements
1-3 mL of peripheral blood in EDTA
0.25-1 mL of bone marrow in EDTA
1 µg of previously isolated DNA

Shipping Conditions
Ambient or Cool; do not freeze (peripheral blood or bone marrow)
Ambient or frozen on dry ice (isolated DNA)Specimen Stability
2-8 °C up to 7 days prior to testing

References
1. The Cancer Genome Atlas Research Network (2013) Genomic and Epigenomic Landscapesof Adult De Novo Acute Myeloid Leukemia. N Engl J Med. 368: 2059–2074.
2. Konig H. et al. (2015) Targeting FLT3 to treat leukemia. Expert Opin Ther Targets 19:37-54.
3. Levis, M. J. et al (2018) A next-generation sequencing–based assay for minimal residual
disease assessment in AML patients with FLT3-ITD mutations. Blood Advances, 2: 825-831.

AML - NPM1 MRD Assay
Clinical Information

Specimen Requirements

Measurable residual disease (MRD) detection in patients withleukemia is useful for the clinical management of disease, and canfacilitate the development of new therapies.

1-3 mL of peripheral blood in EDTA
0.25-1 mL of bone marrow in EDTA
1 µg of previously isolated DNA

Mutations in the nucleophosmin (NPM1) gene represent someof the most prevalent gene mutations in AML.1 NPM1 mutations
predominantly occur in AML with normal cytogenetics and areof prognostic value, especially within the context of FLT3 ITD
mutations. Furthermore, because NPM1 displays a homogeneous
mutation pattern, this gene represents an attractive target for
MRD monitoring.2

Shipping Conditions

LabPMM’s NPM1 MRD test is a NGS-based, targeted, deepsequencing assay that can be used to detect NPM1 mutations thatwere previously identified in a primary sample. The sensitive assay
reliably detects sequences present at 5 x 10-5.

2-8 °C up to 7 days prior to testing

MRD detection by Next-Generation Sequencing has demonstrated
utility in predicting clinical outcomes and in generating clinicallyactionable results, allowing early intervention, confirmationof disease status prior to transplant, and increased confidence
in remission status.

Ambient or Cool; do not freeze (peripheral blood or bone marrow)
Ambient or frozen on dry ice (isolated DNA)Specimen Stability
References
1. Falini B. et al. (2005) Cytoplasmic nucleophosmin in acute myelogenous leukemiawith a normal karyotype. N Engl J Med 352:254–266.
2. Krönke J. et al. (2011) Monitoring of minimal residual disease in NPM1-mutated acute
myeloid leukemia: a study from the German-Austrian acute myeloid leukemia study group.
J Clin Oncol 29:2709-2716.

Test Name
NPM1 MRD Assay

Assay Type
Next-Generation Sequencing (NGS)
CLIA-validated assay

Method Description
To track and identify previously detected NPM1 mutations in posttreatment follow-up samples, a multiplex master mix targetingexon 12 on the NPM1 gene is used to amplify DNA extracted froma patient sample. Next-generation sequencing of the PCR products
is used to identify DNA sequences specific to previously identified
mutations detected at diagnosis. Bioinformatics tools facilitate thedetection of these specific sequences present at an allelic sensitivity
level of 5x10-5.

Indications for Testing» Identify tumor-specific markers for post-treatment monitoring» Monitor and evaluate disease recurrence

Interpretation
An interpretive report will be issued indicating whether
NPM1 ITD MRD was detected

Turnaround Time
7 to 10 business days

Measurable Residual Disease Tests 31

B-cell MRD Assay
Clinical Information
Combinations of chemotherapy, radiation therapy and bone marrowtransplantation are potentially curative for several hematologic
malignancies. However, in some patients, occult tumor cells exist
and are thought to increase the patient’s risk of relapse.1
These subclinical levels of residual leukemia are termed measurable
residual disease (MRD) and can be evaluated using more sensitive
assays. The tracking of antigen-receptor gene rearrangements forclonality analyses and MRD monitoring can be applied to virtually all
patients. During early B-cell development, the germline variable (VH),
diverse (DH), and joining (JH) fragments of the human immunoglobulin
heavy chain (IGH) locus become rearranged through the random
deletion or insertion of nucleotides within the junctional region,
generating specific and unique sequences within each lymphocyte.
Cancer cells that arise from alterations in single lymphoid precursors
acquire clonal IGH junctional regions, which can be used as tumorspecific markers.2,3 MRD detection by Next-Generation Sequencing
has demonstrated utility in predicting clinical outcomes andin generating clinically actionable results, allowing early intervention,
confirmation of disease status prior to transplant, and increased
confidence in remission status.

Indications for Testing

Test Name

Specimen Requirements

B-cell MRD Assay

1-2 mL of peripheral blood in EDTA or Heparin
1-2 mL of bone marrow in EDTA or Heparin
0.5 - 20 µg of previously isolated DNA depending on levelof sensitivity required

Assay Type
Next-Generation Sequencing (NGS)CAP/CLIA-Validated

Method Description
Clonal B-cell gene rearrangement sequences identified at diagnosis,
are used to track post-treatment / follow-up samples for measurable
residual disease (MRD) status. A multiplex master mix targeting theconserved framework region 1 (FR1) or framework region 3 (FR3),
and the joining region is used for PCR amplification. Next generation
sequencing of the PCR products is used to identify and track FR1
and/or FR3 clonal sequences that were previously identified
at diagnosis. Bioinformatics tools facilitate the detection of these
specific sequences present at MRD levels up to 1 x 10-6 with sufficient
DNA input.
The assay typically requires a sample taken at diagnosis as wellas the post-treatment follow-up samples. If the patient has previously
been tested by LabPMM for IGH clonality, no diagnostic sample
is needed.

» Identify tumor-specific gene rearrangement sequences forpost-treatment monitoring in:- Multiple Myeloma- Chronic Lymphocytic Leukemia (CLL)- Mantle Cell Lymphoma (MCL)- Acute Lymphocytic Leukemia (ALL)» Monitor and evaluate for refractory and relapsed disease

Interpretation
An interpretive report will be issued indicating whether
IGH MRD was detected.

Turnaround Time
14 to 21 business days

Shipping Conditions
Peripheral Blood: Ambient or Cool; do not freeze
Bone Marrow**: Ambient, cool or frozen on dry ice
Isolated DNA: Ambient or frozen on dry ice

Specimen Stability
15 to 30 °C for up to 3 days
2 to 8 °C up to 7 days
-15 to -30 °C for up to 3 months

References
1. Rezuke, WN et al. (1997) Clinical Chemistry. 43:1814-23.
2. Gazzola, A et al. (2014) Therapeutic Advances in Hematology. 5:35-47.
3. González, D et al. (2007) Blood. 110:3112-21.

If less than 20 µg is provided, the sensitivity of the assay may be impacted.
** Ambient bone marrow may limit the sensitivity that can be achieved.

32 Measurablel Residual Disease Tests

T-cell MRD Assay
Clinical Information
Combinations of chemotherapy, radiation therapy and bone marrowtransplantation are potentially curative for several hematologic
malignancies. However, in some patients, occult tumor cells exist andare thought to increase the patient’s risk of relapse.1 These subclinicallevels of residual leukemia are termed measurable residual disease(MRD) and can be evaluated using sensitive assays. The trackingof antigen-receptor gene rearrangements for clonality analyses
and MRD monitoring can be applied to virtually all patients. During
early T-cell development, the germline variable (Vγ), constant (Cγ),
and joining (Jγ) fragments of the T-cell receptor gamma (TRG) andT-cell receptor beta loci become rearranged through the random
deletion or insertion of nucleotides within the junctional region,
generating specific and unique sequences within each lymphocyte.
Cancer cells that arise from alterations in single lymphoid precursors
acquire clonal TRB and/or TRG junctional regions which can be used
as tumor-specific markers.2,3 MRD detection by Next-Generation
Sequencing has demonstrated utility in predicting clinical outcomes
and in generating clinically actionable results, allowing earlyintervention, confirmation of disease status prior to transplant,
and increased confidence in remission status.

Indications for Testing» Identify tumor-specific markers for post-treatment monitoring» Monitor and evaluate for refractory and relapsed disease

Specimen Requirements
1-3 mL of peripheral blood in EDTA or Heparin0.25-1 mL of bone marrow in EDTA or Heparin
0.7 - 20 µg of previously isolated DNA depending on levelof sensitivity required

Shipping Conditions
Ambient or Cool; do not freeze (peripheral blood or bone marrow)
Ambient or frozen on dry ice (isolated DNA)Specimen Stability

Test Name

2 to 8 °C up to 7 days prior tot testing

T-cell MRD Assay

References

Assay Type

1. Rezuke, WN et al. (1997) Clinical Chemistry. 43:1814-23.
2. Gazzola, A et al. (2014) Therapeutic Advances in Hematology. 5:35-47.
3. González, D et al. (2007) Blood. 110:3112-21.

Next-Generation Sequencing (NGS)
Research Use Only (RUO)

Method Description* If less than 20 µg is provided, the sensitivity of the assay may be impacted.

Clonal T-cell (TRG or TRB) gene rearrangement sequences identified
at diagnosis, will be used to track post-treatment / follow-up samples
for measurable residual disease (MRD) status. Multiplex master mixes
targeting the Vγ and the Jγ region are used for PCR amplification.
Next-generation sequencing of the PCR products is used to identify
and track TRG and/or TRB clonal frequencies that were previously
identified. Share clonal rearrangements detected at diagnosis.
Bioinformatics tools facilitate the detection of these specific sequences
present at MRD levels up to 1 x 10-6 with sufficient DNA input.
The assay typically requires a sample taken at diagnosis as wellas the post-treatment follow-up samples. If the patient has previously
been tested by LabPMM for clonality, no diagnostic sample is needed.

Measurable Residual Disease Tests 33

NGS Cancer
PanelsCytogenetic identification of chromosome abnormalities has become essential for theclinical management of patients with leukemia, and it is currently used to help classify
patients into risk groups. With the development of novel genomics technologies, suchas Next-Generation Sequencing, numerous new mutations and gene expression
signatures have been identified. These breakthroughs allow us to better understandthe molecular heterogeneity of hematologic diseases and to better stratify and assess
risk for cancer patients.
Using these molecular tools, it has become evident that leukemias, lymphomas,
and hematologic diseases are characterized by a remarkable amount of genetic
heterogeneity, with individual patients presenting distinct and almost unique
combinations of chromosome changes and somatically-acquired gene mutations.
LabPMM offers comprehensive NGS gene panels for AML and other hematologicalmalignancies. Our MyAML® cancer panel is designed to analyze and interpretsequence information in genes known or suspected to be involved in AML and otherhematologic diseases. This comprehensive assay is capable of detecting single
nucleotide substitutions, insertions, deletions, and gene rearrangements.
Our MyMRD® panel was designed to sensitively capture all classes of variants
identified in a precisely defined set of targets that commonly drive myeloid
malignancies including AML, MPN, and MDS.
Our MyAML and MyMRD cancer panels are aimed at promoting a broader
understanding of patients' clinical responses and outcomes. Panels run at the timeof diagnosis identify both clinically-actionable driver mutations associated with theprimary tumor, as well as the subclonal architecture that may be present. Temporal
specimens collected and tested during the course of treatment identify the loss or
elimination of driver mutations, as well as emergence or re-emergence of new clones
and new potential therapeutic targets.

34 NGS Cancer Panels

Clinical Information

Method Description

Understanding the clonal architecture of AML patients is vitalfor successful treatments.1 Many different mutations, epigenetic
aberrations, or downstream abnormalities can generate the same
clinical treatment plan. However, these differences are responsible
for the variable responses observed with therapy, which is a major
feature in patients with AML.2 Therefore, since varied somatic
mutations affect patient outcomes, conventional genotyping
is no longer the most suitable method for screening patients.

Using proprietary design, the coding regions and potential genomic
breakpoints within known somatic gene fusions are sequenced
to an average depth of coverage of 1000x. By utilizing long read
lengths, the assay accurately detects and characterizes thebreakpoints of structural variants and gene fusions, often with single
base-pair precision. In addition, these long reads enhance the ability
to identify both the insertion site and DNA content of large internal
tandem duplications. Coupling comprehensive gene coverage withenhanced depth of coverage, long read lengths, and the powerof our robust annotation software and bioinformatics database,
MyAML identifies the underlying somatic mutations that are present
as low as 5% allelic frequency. The data and report include single
base resolution of the genomic breakpoint and sequencesof mutations, facilitating optimized treatment plans and longitudinal
tracking of measurable residual disease.

MyAML is a CLIA-validated assay that identifies clinically actionable,
pathogenic, and potentially pathogenic mutations in 194 genes
associated with AML. Using the latest version in Next-Generation
Sequencing chemistry,
MyAML identifies all somatic mutations, large and small insertions/deletion, and translocations under NCCN/ELN guidelines, as well as
novel somatic variants that may have prognostic significance for AML.
Screening with MyAML allows informed treatment decisions
to be made once all the relevant mutations are known, both in theprevalent clones, as well as the 'secondary' or 'tertiary' clones, which
could become the new prominent clones leading to refractory andrelapsed disease.

List of Genes on the MyAML Panel
Structural Rearrangements: Inv(16) t(16;16) t(8;21) t(15;17) t(9;11)
inv(3) t(3;3) t(6;9) t(9;22)

Genes: CEBPA DNMT3A FLT3 IDH1 IDH2 KIT NPM1
Other Fusions and Gene Rearrangements: ABL1 ADGRG7 AFF1
BCR CBFB CREBBP DEK EIF4E2 ELL ETV6 GAS6 GAS7 KAT6A KAT6B
KMT2A MECOM MKL1 MLLT10 MLLT1 MLLT3 MLLT4 MYH11 NSD1
NUP214 NUP98 PICALM PML RARA RBM15 RPN1 RUNX1 RUNX1T1
SEPT5 SET TFG TMEM255B

Other Genes: ABCC1 ACVR2B ADRBK1 AKAP13 ANKRD24 ARID2

ARID4B ASXL1 ASXL2 ASXL3 BCOR BCORL1 BRINP3 BRPF1 BUB1 CACNA1E
CBL CBX5 CBX7 CDC73 CEP164 CPNE3 CSF1R CSTF2T CTCF CYLD DCLK1
DDX1 DDX23 DHX32 DIS3 DNAH9 DNMT1 DNMT3B DYRK4 EED EGFR
EP300 EPHA2 EPHA3 ETV3 EZH2 FANCC GATA1 GATA2 GFI1 GLI1 HDAC2
HDAC3 HNRNPK HRAS IKZF1 JAK1 JAK2 JAK3 JMJD1C KDM2B KDM3B
KDM6A KDM6B KMT2B KMT2C KRAS MAPK1 METTL3 MST1R MTA2
MTOR MXRA5 MYB MYC MYLK2 MYO3A NF1 NOTCH1 NOTCH2 NRASNRK OBSCN PAPD5 PAX5 PDGFRA PDGFRB PDS5B PDSS2 PHF6 PKD1L2
PLRG1 POLR2A PRDM16 PRDM9 PRKCG PRPF3 PRPF40B PRPF8 PTEN
PTPN11 PTPN14 PTPRT RAD21 RBBP4 RBMX RPS6KA6 SAP130 SCML2SETBP1 SETD2 SF1 SF3A1 SF3B1 SMC1A SMC3 SMC5 SMG1 SNRNP200
SOS1 SPEN SRRM2 SRSF2 SRSF6 STAG2 STK32A STK33 STK36 SUDS3
SUMO2 SUPT5H SUZ12 TCF4 TET1 TET2 THRB TP53 TRA2B TRIO TTBK1
TYK2 TYW1 U2AF1 U2AF1L4 U2AF2 UBA3 WAC WAPAL WEE1 WNK3 WNK4WT1 ZBTB33 ZBTB7B ZRSR2Test Name

A completed patient consent form must be submitted for eachsample sent to LabPMM.

Indications for Testing» At initial diagnosis of AML» Stratifying risk for AML» Monitor and evaluate for refractory and relapsed disease

Interpretation
An interpretive report will be issued indicating the SNVs, indels,
inversions and translocations identified

Turnaround Time
14 to 21 business days

Specimen Requirements
3 mL of peripheral blood in Heparin, EDTA or ACD
1 mL of bone marrow in Heparin, EDTA or ACD
Cell Pellets in cell culture media or buffered solutions without fixatives
1 µg of purified, high quality genomic DNA

Shipping Conditions
Ambient or Cool; do not freeze

Specimen Stability
Room Temp up to 72 hours
2-8 °C up to 7 days

References
1. Döhner K et al. (2014) Intermediate-risk acute myeloid leukemia therapy: current and future.
Hematology Am Soc Hematol Educ Program 1,34-43.
2. Estey EH (2014) Acute myeloid leukemia: 2014 update on risk-stratification andmanagement. Am J Hematol 89:1063-1081.

MyAML - NGS Gene Panel Assay

Assay Type
Next-Generation Sequencing (NGS)
CLIA-validated assay

NGS Cancer Panels 35

Clinical Information
Measurable residual disease (MRD) detection has proven to be usefulin the clinical management of patients with leukemia and can facilitate
the development of new therapies.
Patients with myeloid neoplasms are typically divided into different
prognostic groups based upon both cytogenetics and traditional
molecular profiles;1 however, this may not reflect the heterogeneityof disease2 that can be exploited using MRD assessment. Moreover,multiple sampling is not feasible for many patients and thus thedevelopment of a sensitive and reliable assay to detect several
mutations within one sample represents a significant advancementin guiding treatment decisions.
The MyMRD is a hotspot panel that detects all classes of variants
identified in a precisely defined set of targets that commonly drive
myeloid malignancies including AML, MPN and MDS. It can detect SNVs,
indels and translocations to the genomic base pair, yielding unparalleled
precision and detection of low level mutations in patients. The MyMRD
assay, detects at least one driver mutation in 90%-95% of all AMLs.
This gene panel is validated to a 5x10-3 level of detection for all
targeted sites.

gene coverage with enhanced depth of coverage, long read lengths,
and the power of our robust MyInformatics® annotation softwareand bioinformatics database, MyMRD confidently and reproducibly
detects mutations with a mutant allele frequency of 5x10-3, whilesome mutations, such as FLT3 ITDs, are detected at mutation allele
frequencies as low at 1x10-3.
A completed patient consent form must be submitted for eachsample sent to LabPMM.

Indications for Testing» Identify tumor-specific markers for post-treatment monitoring» Monitor and evaluate for refractory and relapsed disease

Interpretation
An interpretive report will be issued indicating the detected
pathogenic mutations and their frequencies in the interrogatedsample.

Turnaround Time
List of Genes on the MyMRD Panel

14 to 21 business days

SNV and Indel Targets in Genes (Exons) (23 genes)

Specimen Requirements

ASXL1 BRAF CALR CEBPA CSF3R DNMT3A FLT3 IDH1 IDH2 JAK2 KIT KMT2A
KRAS MPL MYH11 NPM1 NRAS PTPN11 RUNX1 SF3B1 SRSF2 TP53 ZRSR2Structural Variants (Translocations and Partial Tandem Duplicationsin Intronic Structures)
CBFB-MYH11 KMT2A RUNX1-RUNX1T1

Test NameMyMRD - NGS Gene Panel Assay

Assay Type
Next-Generation Sequencing (NGS)
CLIA-validated assay

Method Description
Indexed whole-genome libraries are hybridized with MyMRD probes
targeting mutation hotspots in a total of 23 genes (ASXL1 BRAF CALRCEBPA CSF3R DNMT3A FLT3 IDH1 IDH2 JAK2 KIT KRAS MPL NPM1
NRAS PTPN11 RUNX1 SF3B1 SRSF2 TP53 ZRSR2 CBFB-MYH11 KMT2A
RUNX1-RUNX1T1). In addition to targeting single nucleotide variants(SNVs) and indels in the first 21 genes, 5 structural variant breakpoints
within the final 3 genes are also targeted. Coupling comprehensive

36 NGS Cancer Panels

3 mL of peripheral blood in Heparin, EDTA or ACD
1 mL of bone marrow in Heparin, EDTA or ACD
1 µg of purified, high quality genomic DNA

Shipping Conditions
Ambient or Cool; do not freeze

Specimen Stability
Room Temp up to 72 hours
2-8 °C up to 7 days

References
1. Arber, DA et al. (2016). The 2016 revision to the World Health Organization classificationof myeloid neoplasms and acute leukemia. Blood, 127(20):2391-2405.
2. Sperling, AS et al. (2017). The genetics of myelodysplastic syndrome: from clonal
hematopoiesis to secondary leukemia. Nature Reviews. Cancer, 17(1):5–19.

LabPMM Assay
Guidelines

NGS Cancer Panels 37

MyAML is a targeted gene panel that analyzes the coding
and non-coding exons of nearly 200 genes, as well as thebreakpoint hotspots within 36 genes. MyAML combines
long read chemistry and deep sequencing with an
optimized and validated custom bioinformatics pipeline,
MyInformatics®, to specifically examine genomic variants
in AML patients.

Consider testing with MyAML as an entry point into
clinical trials or to optimize treatment
NPM1

FLT3 or LeukoStrat® CDx
FLT3 Mutation Assay

Our FLT3 ITD and NPM1 MRD tests
are amplicon-based NGS assays,
with a limit of detection of 5x10-5

The MyMRD is a NGS-based panel that assesses variants in hotspots from 23
genes that commonly drive myeloid malignancies, including AML, MPN and MDS.
It can detect SNVs, indels, and translocations to the genomic basepair, offering
unparalleled precision and detection of low level mutations in patients. The
MyMRD assay detects at least one driver mutation in 90%-95% of all AMLs.

B- and T-cell clonality testingaccording to disorder

Patient with suspected orconfirmed AML diagnosis

Follow-up with MRD assays unique for biomarkers
identified in each patient, such as FLT3 ITD MRD,
NPM1 MRD and MyMRD

Consider testing with MyMRD as an entry point
into clinical trials or to optimize treatment andmonitor tumor-specific markers

Patient with suspected orconfirmed hematologicalmalignancy

Follow-up with MRD assays unique for biomarkers
identified in each patient, such as FLT3 ITD MRD
and NPM1 MRDMyAML and MyMRD are CLIA validated assays.

38 NGS Cancer Panels

Multiparametric
Flow Cytometry
Multiparametric Flow Cytometry (MFC) is a diverse technology platform with many
clinical applications. One application of MFC is the ability to evaluate individual cells
in suspension for the presence and absence of specific antigens and can be used
to characterize disease in a clinical setting. The technology plays a critical rolein the evaluation of peripheral blood and bone marrow samples for screening,
diagnosis, prognosis and classification of many hematolymphoid neoplasia,
including AML. Immunophenotyping white blood cells allows for the identificationof normal and abnormal cell phenotypes which can identify normal cell populations,
lymphoid neoplasia, plasma cell dyscrasia, acute leukemia and other diseaseof various cell lineages. MFC in the clinical setting now allows for >12 parameters
to be simultaneously measured and analyzed and can provide rapid and actionable
results for clinical decision making.
Another application of MFC includes measurable residual disease (MRD) testing.
MRD testing by MFC can detect disease levels down below 10-4 and can provide
information about remission status, outcome prediction, early identification of relapse
and as a potential surrogate end point to accelerate drug testing and approvals.

Hematolymphoid
Screening Panel - MFC
Clinical Information

Turnaround TimeThe 10-color Hematolymphoid Screening Panel provides
a comprehensive approach for evaluating bone marrow andperipheral blood samples for the presence or absence ofhematolymphoid malignancies. The panel characterizes and identifies
all major white blood cell lineages and identifies all major typesof hematopoietic neoplasia. Biomarker selection follows the2006 Bethesda Consensus1 for immunophenotypic analysis ofhematolymphoid neoplasia with additions chosen by our internal
hematopathologist based on recent literature including the 2017 WHO
Classification of Tumours of Haematopoietic and Lymphoid Tissues.2-8

24-48 hours

This panel not only provides evaluation and monitoring of patients withhematological malignancy but provides a wide array of applications
due to its comprehensive biomarker selection.

Specimen Stability

Biomarkers in the Screening Panel
CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD13, CD14, CD15, CD16,
CD19, CD20, CD23, CD33, CD34, CD38, CD45, CD56, CD57, CD64, CD71,
CD117, CD123, HLA-DR, Kappa, Lambda, and TCR Gamma/Delta.

Test Name
Hematolymphoid Screening Panel

Assay TypeMultiparametric Flow Cytometry (10-color)
CAP/CLIA-validated

Method Description
Immunophenotyping-Lyse/Wash/Stain

Indications for Testing» Classifying Acute Leukemia» Diagnosing and Classifying B cell Disorders» Evaluating T cell and NK disorders» Evaluating Plasma Cell DyscrasiasInterpretation
An interpretive report will indicate the presence/absence of
normal and abnormal cell populations and their associatedimmunophenotypic profile.

40 Multiparametric Flow Cytometry Panels

Specimen Requirements -4 mL of peripheral blood in EDTA or Sodium Heparin2
2-4 mL of bone marrow in EDTA or Sodium Heparin

Shipping Conditions
Ambient or Cool; do not freeze

Specimens should be stored at 2-8°C and must be received
by the lab within 48 hours after draw

References
1. BL Wood et al., Cytometry B Clin Cytom. 72:S14-S22 (2007).
2. FE Craig et al., Blood. 111(8):3941-67 (2008).
3. BL Wood. Arch Pathol Lab Med. 130:680-90 (2006).
4. SH Swerdlow et al., WHO Classification of Tumours, Revised 4th Edition, Volume 2 (2017)
5. CLSI H43: Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells, 2nd
Edition
6. JJ Van Dongen et al., Leukemia. 26(9):1908-75 (2012).
7. A Porwit and A Rajab. Int. Jnl. Lab. Hem., 37:133-143 (2015).
8. Sheikholeslami et al., Methods in Molecular Biology.
378. 53-63. 10.1007/978-1-59745-323-3_4 (2007).

AML MRD Assay - MFC
Clinical Information

Interpretation

Measurable residual disease, also called “minimal residual disease”(MRD) refers to persistent leukemic cells in the blood or bone marrowof cancer patients during or after treatment. Undetected or untreated
MRD is a main cause of cancer recurrence hence sensitive MRD tests
are necessary for guiding optimal treatment programs and providing
a prognostic indicator for risk stratification of treated patients.

An interpretive report will indicate the presence/absence of AML
cell populations, level of detection in relation to the clinical cutoff,
percent and number of aberrant myeloblasts and their associatedimmunophenotypic profile.

Multi-parameter Flow Cytometry (MFC) is a widely accepted platform
for assessing MRD in patients with Acute Myeloid Leukemia (AML)
and can be performed within a short period of time. In our lab weutilize a comprehensive panel of antibody markers to characterizepotential AML blast cells using a LAIP based different from normal(DfN) approach. This approach takes into account information fromdiagnosis, if available but also identifies aberrant cells that havedifferentiated from normal maturation without previous patient history.

24-48 hours

Using a comprehensive selection of antibodies and a standardized
panel across all testing points, MRD populations can be characterized
and tracked down to 0.01% sensitivity. Utilizing up to 12 biomarkers pertube allows for the identification of more LAIPS with less sample thanpreviously was available before.

Turnaround Time
Specimen Requirements -4 mL of peripheral blood in EDTA or Sodium Heparin2
2-4 mL of bone marrow in EDTA or Sodium Heparin

Shipping Conditions
Ambient or Cool; do not freeze

Specimen Stability

This panel not only provides evaluation and monitoring of patients withhematological malignancy but provides a wide array of applications
due to its comprehensive biomarker selection.

Specimens should be stored at 2-8°C and must be received
by the lab within 48 hours after draw

Biomarkers in AML MRD Assay

References

CD2, CD4, CD5, CD7, CD11b, CD13, CD14, CD15, CD16, CD19, CD33,
CD34, CD36, CD38, CD45, CD 56, CD64, CD117, CD123, HLADR, 7AAD

1. Schuurhuis, Gerrit J et al. “Minimal/measurable residual disease in AML: a consensus
document from the European LeukemiaNet MRD Working Party.” Blood vol. 131,12 (2018):
1275-1291. doi:10.1182/blood-2017-09-801498NCCN
2. Wood BL. Acute Myeloid Leukemia Minimal Residual Disease Detection: The Difference
from Normal Approach.Curr Protoc Cytom. 2020 Jun;93(1):e73. doi: 10.1002/cpcy.73.PMID:
32311834
3. Cloos J, Harris JR, Janssen JJWM, Kelder A, Huang F, Sijm G, Vonk M, Snel AN, Scheick JR,
Scholten WJ, Carbaat-Ham J, Veldhuizen D, Hanekamp D, Oussoren-Brockhoff YJM,
Kaspers GJL, Schuurhuis GJ, Sasser AK, Ossenkoppele G. Comprehensive Protocol to
Sample and Process Bone Marrow for Measuring Measurable Residual Disease andLeukemic Stem Cells in Acute Myeloid Leukemia. J Vis Exp. 2018 Mar 5;(133):56386. doi:
10.3791/56386.PMID: 29553571
4. Short NJ, Ravandi F. How close are we to incorporating measurable residual disease into
clinical practice for acute myeloid leukemia? Haematologica. 2019 Aug;104(8):1532-1541.
doi: 10.3324/ haematol.2018.208454. Epub 2019 Jul 4. PMID: 31273094; PMCID:
PMC6669140.
5. Zeijlemaker W, Kelder A, Cloos J, Schuurhuis GJ. Immunophenotypic Detection of
Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia
Curr Protoc Cytom. 2019 Dec;91(1):e66. doi: 10.1002/cpcy.66.PMID: 31763792 .
6. Dix, C.; Lo, T.-H.; Clark, G.; Abadir, E. Measurable Residual Disease in Acute Myeloid
Leukemia Using Flow Cytometry: A Review of Where We Are and Where We Are Going. J.
Clin. Med. 2020, 9, 1714

Test NameAML MRD Assay by MFC

Assay TypeMultiparametric Flow Cytometry (12-color)
CAP/CLIA-validated

Method Description
12-color Multiparametric Flow Cytometry with a 0.01% sensitivity

Indications for Testing» Identify tumor-specific markers for post-treatment monitoring» Clinical trial enrollment and surrogate endpoint in trials» Monitor and evaluate for disease relapse and recurrence» Monitor response to therapy

Multiparametric Flow Cytometry Panels 41

CLL MRD Assay - MFC
Clinical Information

Method Description

The global burden of chronic lymphocytic leukemia (CLL) has increased
over the past 30 years by approximately 7%, with a higher incidence
among men and adults over 65.1,2,3 Moreover, CLL is the mostprevalent type of leukemia, comprising of 25% – 30% of all leukemiasin Western populations4,5, indicating potential heredity.6,7 While thisdisease has not experienced the same innovative therapeutic boon
as AML, new treatment strategies have recently become available,
including targeted inhibitors—which necessitate a sensitive, objective
confirmation of treatment response. MRD (measurable residual
disease, or often referred to as minimal residual disease) is a sensitive
indicator of disease burden during and after fixed-duration treatment.
MRD has been correlated with progression free survival (PFS) andoverall survival (OS)8 and is an independent prognostic indicatorin patients with CLL.

11-color Multiparametric Flow Cytometry with a sensitivityof 0.005% – 0.002%.

Flow cytometry has long been the standard of care for managementof many blood cancers including CLL9, however, achieving MRD levelsensitivity for detection of disease with limited sample requires
a technique with a higher level of complexity, such as multiparametricflow cytometry (MFC). MFC is a state-of-the-art method for MRD
assessment that is widely used because it is highly sensitive, fast andcost-effective.10
The CLL MRD Assay is an extensive 11-color MFC panel targeting
CLL-specific biomarkers designed to characterize potential CLL cellswith clear separation from other B-lineage cells. The design of thisassay allows several key functions:» Using a standardized panel across all time points, MRD populations
can be characterized and tracked to 0.005% sensitivity» Alignment with International Harmonized Approach9,10,11 and ERIC 		
protocol9,11» Designed to work with Peripheral Blood and Bone Marrow 		
specimens» Optimize shared/limited samples by co-testing with MFC and NGS

Biomarkers in CLL MRD Assay
CD81, CD79b, CD22, CD19, CD43, CD200, CD20, CD5, CD3,
CD38, CD40, 7AAD

Test Name
CLL MRD Assay by MFC

Assay TypeMultiparametric Flow Cytometry (11-color)
CAP/CLIA-validation Planned

42 Multiparametric Flow Cytometry Panels

Indications for Testing» Identify tumor-specific immunophenotypes for post-treatment
monitoring and evaluation for relapse» Stratify patient risk and streamline clinical trial enrollment» Use as a potential surrogate endpoint in clinical trials

Interpretation
An interpretive report will indicate the presence/absenceof CLL MRD in relation to the level of detection of assay and theclinical cut-off value.

Turnaround Time
24-48 hours

Specimen Requirements
2-4 mL of peripheral blood in EDTA or Sodium Heparin
2-4 mL of bone marrow in EDTA or Sodium Heparin

Shipping Conditions
Ambient or Cool; do not freeze

Specimen Stability
Specimens should be stored at 2-8°C and must be received
by the lab within 48 hours after draw

References
1. Y Dong et al. Exp Hematol Oncol 9, 14 (2020). https://doi.org/10.1186/s40164-020-00170-6
2. WG Wierda et al. Leukemia 35, 3059–3072 (2021). https://doi.org/10.1038/s41375-021-01241-1
3. Y Ou et al. Frontiers in Oncology 12, 840616 (2022). https://doi.org/10.3389/fonc.2022.840616
4. JL Batista et al. (2017) Pathology and Epidemiology of Cancer. Springer. Cham. https://doi
org/10.1007/978-3-319-35153-7_29
5. AC Rawstron et al. Leukemia 4, 929-36 (2016). doi: 10.1038/leu.2015.313
6. JR Cerhan and SL Slager SL. Blood. 2015;126(20):2265–73.
7. Y Yao et al. BioMedical Engineering 21, 4 (2022).
8. M Hallek et al. Am J Hematol. 96(12): 1679- 1705 (2021). doi:10.1002/ajh.26367
9. AC Rawstron et al. Leukemia 1,142-9 (2013). doi: 10.1038/leu.2012.216. Epub 2012 Jul 31.
PMID: 23041722.
10. MM Sartor et al. Cytometry Part B 2013; 84B: 96–103.
11. AC Rawstron et al. Leukemia. 2016; Apr;30(4):929-36. doi: 10.1038/leu.2015.313. Epub 2015 		
Dec 7. PMID: 26639181; PMCID: PMC4832072.

Invivoscribe's wholly-owned Laboratories for Personalized Molecular Medicine® (LabPMM) is a network of international reference laboratories thatprovide the medical and pharmaceutical communities with worldwide access to harmonized and standardized clinical testing services. We viewinternationally reproducible and concordant testing as a requirement for consistent stratification of patients for enrollment in clinical trials, and thefoundation for establishing optimized treatment schedules linked to patient’s individual profile.
LabPMM provides reliable patient stratification at diagnosis and monitoring, throughout the entire course of treatment in support of Personalized
Molecular Medicine® and Personalized Molecular Diagnostics®.
Invivoscribe currently operates four clinical laboratories to serve partners in the USA (San Diego, CA), Europe (Munich, Germany), and Asia (Kawasaki,
Japan and Shanghai, China). These laboratories use the same critical reagents and software which are developed consistently with ISO 13485
design control. Our cGMP reagents, rigorous standards for assay development & validation, and testing performed consistently under ISO 15189
requirements help ensure LabPMM generates standardized and concordant test results worldwide.
LabPMM is an international network of PersonalMed Laboratories® focused on flow cytometry and molecular oncology biomarker studies.

LabPMM Laboratories are wholly-owned subsidiaries of Invivoscribe.
The following are registered trademarks of Invivoscribe, Inc.: Laboratory for Personalized Molecular Medicine®, LabPMM®, Personalized MolecularDiagnostics®, Personalized Molecular Medicine® and PersonalMed Laboratories®.

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