Patient-Matched Tumor-Normal Sequencing: What It Is and Why It Matters

The basis is simple. Every person has unique DNA signatures. Across patient populations, these signatures share similarities within families and traits, but each person has unique variations, including benign mutations that multiply as we age. Unlike cancer-causing mutations, these genomic differences do not negatively impact cell behavior. When we sequence the DNA in a patient’s tumor to identify opportunities for targeted therapies, we need to rule out these benign mutations and focus on pathogenic mutations. We do this by subtracting “normal” mutations from the full set of mutations in the tumor.

That’s how PMTN sequencing adds value to therapy selection. Nothing is more accurate than using the patient's own biology as a reference,¹ so PMTN sequencing identifies normal germline DNA alterations in a patient’s blood sample, and then subtracts them from those found in the tumor sample to isolate the cancer-related mutations. This approach identifies the disease-associated variants with great certaintyand ensures that doctors can make better informed decisions for patients with advanced solid tumors. And the process is fast and simple—an integrated part of OncoExTra^®, a single test for DNA and RNA variants.

What makes PMTN sequencing different?

For years, the only way to isolate cancerous variants was the “tumor-only” approach, where we rule out common normal mutations based on public databases. But public databases do not account for each person’s unique DNA signatures, and they under-represent the normal mutations distinct to the genetics of many cultural backgrounds.² As a result, a patient’s benign mutations can be falsely reported as being cancer-related. Some algorithms designed to reduce such problems can actually increase the false positive rate or over-filter the results, missing cancerous mutations.³

The source of truth is and should be the patient’s own blood sample, which is what makes PMTN sequencing so exciting. PMTN sequencing is improving the accuracy of sequencing regardless of race, ethnicity, or sex because it references a patient’s own biology instead of a database.¹ As part of the OncoExTra test, PMTN sequencing provides wholly somatic results linked to targeted therapies, while ruling out benign germline mutations (false positives).4 With integrated PMTN sequencing, the OncoExTra test identifies actionable variants in 84% of cases with advanced solid tumors—90% of cases with common cancers such as colorectal, lung, bladder, and breast cancer.⁴

So how does PMTN sequencing work in the lab? Doctors submit the patient’s tumor sample and blood sample. DNA from white blood cells and tumor cells is extracted and sequenced. After sequencing is complete, the DNA data from both samples is paired and goes through a complex series of bioinformatic analyses that filter variants identified in the blood-based DNA from variants in the tumor DNA data. The final result is a somatically focused, patient-specific genomic report that’s highly accurate and easy to interpret for patient care.

Seamlessly integrated with added TMB accuracy

In addition to raising the accuracy of tumor analysis, PMTN sequencing offers added advantages for identifying immune checkpoint therapies—all in one integrated, accessible test:

• PMTN sequencing is the gold-standard method for calculating tumor mutational burden (TMB).¹ TMB is used to help determine eligibility for immune checkpoint therapies such as nivolumab, pembrolizumab, and ramucirumab. Because PMTN sequencing results consist of purely somatic mutations with fewer artifacts from benign mutations,1,4 it is more accurate for calculating TMB than the tumor-only approach, which can artificially inflate TMB, particularly for groups under-represented in public databases.¹
• PMTN sequencing is integrated, accessible, and easy. It isn’t an additional option—it’s part of every patient’s OncoExTra test. Doctors simply submit 1 tube of the patient’s blood in addition to the tumor sample, and they receive the OncoExTra test DNA and RNA sequencing report within 14 days after both samples are received.⁴ Our pathologists can even help if a tumor sample falls below the testing threshold of 20% tumor cell content, employing a process called macrodissection to enrich the sample and proceed with testing.

With these advantages, PMTN sequencing helps deliver a panoramic view of the tumor profile and actionable results. The OncoExTra test’s whole-exome approach focuses on analyzing the regions of DNA that ultimately code for proteins (exons) as well as adjacent areas where actionable structural variants can be located. Whole-transcriptome RNA analysis identifies therapeutically targetable fusion and transcript variants.

After one single, efficient test, doctors receive a report that translates the data into quick-reference results, including appropriate targeted therapies, immunotherapies, and clinical trials. Because we’ve incorporated advanced PMTN sequencing into this process, doctors can be assured that the results and therapeutic matches reflect the most accurate options for each patient’s unique DNA.

1. Asmann YW, Parikh K, Bergsagel PL, et al. Inflation of tumor mutation burden by tumor-only sequencing in under-represented groups. NPJ Precis Oncol. 2021;5(1):22.

2. Sirugo G, Williams SM, Tishkoff SA. The missing diversity in human genetic studies. Cell. 2019;177(1):26-31.

3. Shi W, Ng CKY, Lim RS, et al. Reliability of whole-exome sequencing for assessing intratumor genetic heterogeneity. Cell Rep. 2018;25(6):1446-1457.

4. White T, Szelinger S, LoBello J, et al. Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®. Oncotarget. 2021;12(8):726-739.

Janine LoBello, DO, is Senior Clinical Laboratory Medical Director at Exact Sciences in Phoenix, Arizona. Amin Mazloom, PhD, is Vice President, Bioinformatics, Biostatistics & Data Innovation at Exact Sciences in San Diego, California.

The OncoExTra test was developed, and the performance characteristics validated by Genomic Health, Inc., a wholly owned subsidiary of Exact Sciences Corporation following College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA) regulations. The OncoExTra test is performed at the Genomic Health Phoenix clinical laboratory. Exact Sciences clinical laboratories are accredited by CAP, certified under CLIA regulations, and qualified to perform high-complexity clinical laboratory testing. This test has not been cleared or approved by the US Food and Drug Administration or other notified regulatory authority.

Why does the OncoExTra test include patient-matched tumor-normal (PMTN) sequencing?

• PMTN distinguishes true somatic variants from likely benign germline variants by directly comparing the tumor sample to the patient’s own normal DNA in a blood sample
• PMTN is a gold-standard method for calculating tumor mutational burden (TMB), which is used to determine immune checkpoint eligibility¹
• PMTN addresses the health equity problem of racial and ethnic under-representation in normative databases by using each patient’s own DNA as the reference sample for germline subtraction
• PMTN’s integration in the OncoExTra test means that doctors get these advantages for every patient in an efficient timeframe. The test is covered for 98% of patients, and doctors receive results in under 14 days after both samples are received⁴