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Molecular Diagnostics Overview JAN A. NOWAK, PHD, MD PATHOLOGY AND LABORATORY MEDICINE MOLECULAR DIAGNOSTICS LABORATORY FEBRUARY 15, 2018

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Page 1: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Diagnostics

Overview JAN A. NOWAK, PHD, MD

PATHOLOGY AND LABORATORY MEDICINE

MOLECULAR DIAGNOSTICS LABORATORY

FEBRUARY 15, 2018

Page 2: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Some Key Points

Molecular Testing has applications in every section of the clinical laboratory

The advantages of molecular testing stem from its extreme specificity and

sensitivity, as well as its rapidity in producing results

Applications in oncology include diagnosis, classification, prognostication, and

prediction of response to targeted therapies

Clinical Laboratories in the US are regulated under CLIA

Laboratories may develop tests that are not FDA approved, under the

authority of the CLIA Medical Director

Payment for laboratory testing has two major components, coverage and

reimbursement

Page 3: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Exploiting the Specificity of Sequence Based

Testing

Assume genome is 50% GC – i.e. A,T,G,C ‘s are equally represented

Consider this 15 bp sequence: aatgcagtacctttg

The probability of A in position one is 0.25

The probability of A in position two is 0.25

The probability of A in position one and A in position two is (0.25)(0.25)=0.06

The probability of a T following AA is (.25)(.25)(.25)=0.0156

The probability of any specific 15 bp sequence is

(0.25)E15=9.3x10E-10

Page 4: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Consider a PCR reaction with two 15-mer

primers

Primer 1 has 9x10E-10 probability of finding perfect match site

Primer 2 has 9x10E-10 probability of finding perfect match site

Combined probability is 81x10E-20

This is further reduced by the expectation of a specific amplicon size

If your PCR reaction produces a product of the expected size, it is

extremely unlikely that this is due to chance

Page 5: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality
Page 6: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

PCR-RFLP (Restriction Fragment Length

Polymorphism)

Page 7: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Discovery of activating mutations in EGFR kinase domain in

lung cancers (2004)

Page 8: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Dramatic response to gefitinib

Clinical predictors

–Women

–East Asian

–Never smokers

Histologic predictors

–Adenocarcinomas with BAC features, TTF1+

–None completely reliable

Page 9: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality
Page 10: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

EGFR MUTATIONS based on MSKCC experience 2009-2010 (1131 cases

screened, 258 EGFR mutant, 23%)

Page 11: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

How do we test for EGFR mutations?

Exon 19 deletions

PCR product length

Sequencing

IHC (only good for 15 bp deletion)

Exon 21 (L858R)

PCR RFLP

Sequencing

Exon 20 (T790M)

Allele specific PCR

Sequencing

Page 12: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Homo sapiens epidermal growth factor receptor (EGFR), RefSeqGene on

chromosome 7

NCBI Reference Sequence: NG_007726.1

Exon 19

LREA deletion site shown in green.

TCTTTTATCTAAATAATCAGTGTGATTCGTGGAGCCCAACAGCTGCAGGGCTGCGGGGGCGTCACAGCCC

CCAGCAATATCAGCCTTAGGTGCGGCTCCACAGCCCCAGTGTCCCTCACCTTCGGGGTGCATCGCTGGTA

ACATCCACCCAGATCACTGGGCAGCATGTGGCACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTC

TCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGA

GAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCA

TGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCC

ACATCCTAAATGTTCACTTTCTATGTCTTTCCCTTTCTAGCTCTAGTGGGTATAACTCCCTCCCCTTAGA

GACAGCACTGGCCTCTCCCATGCTGGTATCCACCCCAAAAGGCTGGAAACAGGCAATTACTGGCATCTAC

Exon 21

Sau96I (GGNCC) site shown in turquoise. Site of L858R (2573T>G) shown

in green.

GATGGAGAAAAGTTAATGGTCAGCAGCGGGTTACATCTTCTTTCATGCGCCTTTCCATTCTTTGGATCAG

TAGTCACTAACGTTCGCCAGCCATAAGTCCTCGACGTGGAGAGGCTCAGAGCCTGGCATGAACATGACCC

TGAATTCGGATGCAGAGCTTCTTCCCATGATGATCTGTCCCTCACAGCAGGGTCTTCTCTGTTTCAGGGC

ATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACAC

CGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGC

AGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACCAGGGACCAGGCTGCC

TTCCCACTAGCTGTATTGTTTAACACATGCAGGGGAGGATGCTCTCCAGACATTCTGGGTGAGCTCGCAG

CAGCTGCTGCTGGCAGCTGGGTCCAGCCAGGGTCTCCTGGTAGTGTGAGCCAGAGCTGCTTTGGGAACAG

Page 13: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality
Page 14: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality
Page 15: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

BRAF PCR with FRET Probe Temperature Melt

Analysis

Page 16: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Quantitative RT-PCR

Page 17: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Overview of Molecular Diagnostic Testing

Landscape of Molecular Diagnostic Tests

Regulatory Issues

Coding

Coverage and Reimbursement

Page 18: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Categories of Molecular Diagnostic Tests

Infectious Disease

Identity Testing

Genetics

Pharmacogenomics

Oncology

Predictive Medicine

Page 19: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Infectious Disease Testing High volume tests

HIV quantitation and genotyping

HCV screening, quantitation and typing

GC / Chlamydia screening

HPV screening and genotyping

Page 20: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Infectious Disease Testing Moderate volume screening tests

Influenza A (H1, H3, 2009 H1N1) and Influenza B

MRSA

B. pertussis / B. parapertussis

Page 21: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Infectious Disease Testing Lower volume tests

CMV detection

CMS quantitation

VZV detection

HSV 1 & 2 detection

HSV 1 & 2 quantitation

RSV detection

Adenovirus detection

Parvovirus detection

Enterovirus detection

S. aureus screening

C. difficile detection

Malaria detection and typing

M. tuberculosis detection

Mycobacterial speciation

Sequenced based ID

Respiratory pathogen panel

GI pathogen panel

Page 22: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Infectious Disease Testing Antibiotic resistance tests

Methicillin

Vancomycin

Ganciclovir

Rifampin

ESBL

Carbapenem resistant Enterobacteriaceae

Page 23: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Identity Testing

Transfusion Medicine

RBC and platelet antigen typing

HLA typing

Transplantation

Disease association

Forensics

Paternity

Page 24: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Pharmacogenomic Applications

Warfarin CYP 2C9 / VKOR

Plavix CYP 2C19

Irinotecan UGT1A1

Abacovir HLA-B*5710

SSRI CYP2D6

Page 25: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Applications in Genetics

Common heritable conditions

Sickle cell disease

Hemoglobinopathies

Hemochromatosis

Thrombophilia

Heritable diseases

WES – diagnostic odysseys

Inherited predisposition

Prenatal testing

CF screening

SMA screening

Inherited disease panels

Chromosomal abnormalities

Page 26: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Testing in Oncology

Hematologic malignancies

Classification

Prognostication

Therapy selection

Monitoring

Minimal Residual Disease

Inherited predisposition

BRCA1 and 2, FAP, Lynch, et al.

Page 27: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Testing in Oncology

Solid tumor

Single analyte

Detection and diagnosis

Prognosis

Therapy

Resistance

Liquid biopsy

Panels

Page 28: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Molecular Testing in Oncology

Solid Tumor

Classification

Prognosis

Targeted therapies

Colon Cancer

NSCLC

CNS tumors

GIST

Malignant melanoma

Page 29: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Predictive Medicine

Cardiovascular disease

Alzheimer’s Disease

Page 30: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Regulatory Issues – All clinical laboratories in

the US are certified by CMS under CLIA

CLIA – Clinical Laboratory Improvement Act of 1988

Specifies personnel requirements

Quality Assurance and Proficiency Testing Standards

Laboratory Inspection

SOP and validation requirements

Deemed Agents

CAP – College of American Pathologists

NY CLEP – New York State Clinical Laboratory Evaluation Program

Page 31: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

CLIA vs. FDA

FDA evaluates medical devices for safety and effectiveness

FDA monitors Severe Adverse Events, but not ongoing Quality

Assurance

The Laboratory Medical Director, under CLIA, is responsible for day-

to-day QC and proficiency testing

The laboratory, under the direction of the Medical Director, can

develop, validate, and implement tests for clinical use – Laboratory

Developed Tests (LDT)

Page 32: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Regulatory Issues

CLIA vs FDA

Lab tests as medical procedures or as medical devices

LDT – Laboratory Developed Test

Companion Diagnostics

Complementary Diagnostics

FDA Cleared and FDA Approved

3rd Party Review

Page 33: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Coding Issues

Current Procedural Terminology (CPT) Codes

Molecular Pathology Codes

Tier 1

Tier 2

Genomic Sequencing Procedure Codes

81479 – Unlisted Molecular Pathology Procedure

MAAA – Multianalyte Assay with Algorithmic Analyses - Category I

MAAA – Multianalyte Assay with Algorithmic Analysis - Administrative

PAMA

Proprietary Laboratory Assay (PLA) codes

Mol Dx

Palmetto required

Page 34: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Payer Issues

Private payers vs. Medicare

CMS – Center for Medicare and Medicaid Services

Coverage Policies

Does a procedure or service influence treatment decisions or outcome?

In what clinical context is the service performed?

Medicare restrictions (age, screening, etc)

Reimbursement

Inpatient vs. outpatient vs. non-patient

Page 35: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Current Reimbursement Environment

Panels

CPT GSP Codes

NCD Pending

Excludes LDTs

Linked to Companion/Complimentary Diagnostics

PAMA

Palmetto

LCDs vs statements

Page 36: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Future Applications

Crisper/Cas 9 Gene editing

Idiosyncratic testing

Immune response editing

“engraftment” monitoring

Immune response prediction

TMB vs MSI

Page 37: Molecular Diagnostics Overview - Cancer TreatmentCLIA vs. FDA FDA evaluates medical devices for safety and effectiveness FDA monitors Severe Adverse Events, but not ongoing Quality

Some Key Points

Molecular Testing has applications in every section of the clinical laboratory

The advantages of molecular testing stem from its extreme specificity and sensitivity, as well as its rapidity in producing results

Applications in oncology include diagnosis, classification, prognostication, and prediction of response to targeted therapies

Molecular genetic testing can also inform about inherited predisposition to certain malignancies

Clinical Laboratories in the US are regulated under CLIA

Laboratories may develop tests that are not FDA approved, under the authority of the CLIA Medical Director

Payment for laboratory testing has two major components, coverage and reimbursement


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