Introduction

Molecular Testing Pearls

In Part 4 of our Heme Path series, we thoroughly examine the details of molecular testing and how it relates to hematologic and oncologic malignancies

I. Molecular Testing Basics

A. Provides a means of assessing patient’s genotypes, specifically at smaller changes in the genetic information

B. How is it performed?

1. Polymerase chain reaction (PCR)-based testing, which involves using a specific primer that is complementary to the area of interest on the patient’s DNA

2. PCR can allow for both amplification and quantification of gene of interest

C. Can look for either single gene mutations (faster) or a panel of mutations (slower but more information) also known as NGS

II. Clinical Utility of Molecular Testing

A. Very useful in risk stratification based on the mutations noted (some mutations are unfavorable and some are favorable)

B. Certain genetic mutations have drugs that are effective against them, therefore provides information about targeted therapeutic options

C. In hematologic malignancies, can be used to also assess response to treatment

1. You can determine minimal residual disease or MRD

2. Can look for a gene mutation that was present in the original cancer clone and see if there is any amount of residual cancer left over on the order of 1 in a million cells

D. In solid cancers, used to determine presence of genetic changes that have prognostic and targeted treatment implications

1. BRAF V600E mutation in melanoma → BRAF inhibitor pill treatment

2. EGFR mutation in lung cancer → EGFR inhibitor pill treatment

III. How is molecular testing different than FISH?

A. Both require choosing probes and understanding what you are looking for before running the test

B. FISH (discussed in part 3!) reports out of 200 cells and provides information about only larger kilobase sized genetic changes (translocations, inversions, deletions)

C. Molecular testing analyzes a much larger number of cells and can detect changes at the single base pair level. Much more detailed and microscopic evaluation of genetic changes

IV. Single Gene Molecular Testing

A. Look for a specific gene mutation (i.e. EGFR for lung cancer, BRAF for melanoma, FLT3-ITD for AML)

B. Pros:

1. Faster turnaround time

2. Has a higher resolution and effective for detecting MRD

B. Cons:

1. Only looks for one genetic mutation as opposed to a panel like in NGS

2. Some diseases ideally require understanding of multiple mutations not just one for prognostication and treatment planning

V. Next Generation Sequencing (NGS)

A. Allows to sift through a larger part of the genome to identify a panel of mutations

B. Panel of mutations chosen is based on the clinical context

1. For example: NGS for acute myeloid leukemia is much different than NGS testing for lung cancer as each cancer has a much different genetic mutation profile

C. Overview of technical aspects of running NGS

1. Massively parallel sequencing meaning that many tiny primers are used and the areas that primers encode may be overlapping

2. A computer takes all of the smaller pieces and puts them together to determine the correct sequence

D. Pros:

1. Gives us an understanding of many different mutations present based on the panel chosen

2. Again, this has both prognostic and predictive treatment implications

E. Cons:

1. May find mutations of undetermined significance meaning we currently do not understand how these mutations will affect prognosis and treatment decisions

2. Very time consuming (~2-4 week turnaround time)

3. Costly

References:

1. https://jamanetwork.com/journals/jamaoncology/fullarticle/2734828 - Quick overview of NGS

2. https://ashpublications.org/blood/article/125/26/3996/34323/Minimal-residual-disease-diagnostics-in-acute - Look at table 1 to see the difference in sensitivity for MRD testing

3. https://www.oncotarget.com/article/27602/text/ - Emphasizes prognostic relevance of EGFR mutations in NSCLC

4. https://www.nejm.org/doi/full/10.1056/NEJMoa1612674 - Phase 3 trial showed that targeted treatment for EGFR mutation in NSCLC was superior to chemotherapy

5. https://www.nejm.org/doi/full/10.1056/nejmoa1614359 - Phase 3 trial showed that targeted treatment of FLT3 mutation in AML improved outcomes

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