Developing liquid biopsy diagnostic testing for cancer immunotherapy selection in NSCLC patients

More than 125,000 people are diagnosed yearly with lung cancer in the United States, and more than half die from the disease.¹ Non-small cell lung cancer (NSCLC) is one the most common forms of lung cancer, and it is most frequently diagnosed during advanced stages. Timely and appropriate precision diagnosis and treatment is critical for clinical management and patient survival. Blood-based liquid biopsy is gaining traction in clinical testing as an alternative to more invasive tissue biopsy for diagnosis and monitoring in patients with NSCLC.

Advances in immunotherapy

Advances in cancer immunotherapy research have enabled significant progress in the development of targeted therapies, with encouraging success in the clinic. Newly available immunotherapies, some referred to as checkpoint inhibitors, are today increasingly selected for treatment of advanced-stage NSCLC patients who cannot undergo surgical resection, as well as those who have failed to respond to platinum-based chemotherapy. Among the most promising targets of these checkpoint inhibitors are immune checkpoint proteins, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1).

Cancer cells can express PD-L1, which binds PD-1 on the surface of T cells and suppresses their ability to attack cancer cells. Inhibitors of PD-1 and PD-L1 have been designed to break this communication, thus restoring T cell anti-tumor activity. Positive PD-L1 expression of cancer cells is now considered a predictive and prognostic biomarker by clinicians, and it is used to determine which patients can benefit from PD-1/PD-L1 immunotherapy.² A variety of antibody-based inhibitors of PD-1/PD-L1 have obtained approval by the U.S. Food and Drug Administration (FDA), including nivolumab, pembrolizumab, and atezolizumab, and have shown great promise in patients whose tumor biopsies have tested positive for PD-L1.³

Drawbacks of IHC

Immunohistochemistry (IHC) of biopsied tumor tissue is currently the standard method to test for the presence of PD-L1 expression in cancer, to determine whether patients are likely to derive benefit from anti-PD-1/anti-PD-L1 immunotherapies over chemotherapy. IHC is a powerful tool. However, it can be variable in performance and requires invasive surgical procedures. The four IHC-based tissue assays approved by the FDA for PD-L1 use different antibodies, detection platforms,cutoff points, and scoring systems, which complicates standardization for clinical management.^4,5

Researchers have also shown that there can be as much as 20 percent variability among pathologists in scoring when the cutoff is set at ≥1 percent positive tumor cells.⁶ Furthermore, re-testing using IHC requires performing more than one surgical biopsy, which increases the burden on the patient and the cost of care, and may present additional clinical risks. Last, IHC test results may take weeks to be delivered, which could delay the administration of a targeted therapy.⁷

Two alternative test methods for measuring biomarkers relevant in immunotherapy that are drawing widespread interest are droplet digital PCR (ddPCR) and next-generation sequencing (NGS). In January 2018, a molecular testing company announced a partnership with PD-L1 IHC expert Fred R. Hirsch, MD, PhD, laboratory director of the Hirsch Biomarker Laboratory at the University of Colorado Cancer Center.⁸ The company presented results on a newly-developed blood-based PD-L1 research assay at the 2018 American Society of Clinical Oncology and the Society for Immunotherapy of Cancer (ASCO-SITC) Clinical Immuno-Oncology Symposium.9

Improving PD-L1 diagnostics

Preliminary results compared the use of ddPCR technology to IHC for PD-L1 expression. The ddPCR test detected PD-L1 mRNA successfully in plasma and formalin-fixed paraffin-embedded (FFPE) tissue resections from the same individuals.⁹

The researchers used ddPCR technology, which partitions each sample into thousands of nanoliter-sized droplets, and enables absolute quantification of PD-L1 transcripts in each droplet, without need for standard curves as in traditional RT-PCR methods. ddPCR can be used to detect circulating nucleic acids from plasma, thus allowing the use of blood samples, which are much more easily obtained than tissue biopsies.

The results of this study concluded that this mRNA assay may require establishment of independent cutoff values, as IHC may not be the best validation measure for these assays. The ddPCR-based product concept was able to detect PD-L1 copies in the range of 6-1,272 copies from tissue, and 32-138 copies from plasma.⁹ Overall, the study demonstrated the feasibility of testing for PD-L1 expression by ddPCR in plasma and in FFPE tissue sections.

Following these early results, a prospective study was initiated to continue to assess IHC and ddPCR testing for PD-L1, which includes performance measures of NSCLC patients after receiving PD-1/PD-L1 immunotherapy.

Related LDTs

The company has also developed and validated two prior, complementary laboratory-developed tests (LDTs), one of which is a ddPCR genomic test, and the other a Mass Spec proteomic test. These tests are used by clinicians to aid in the treatment of patients with NSCLC.

The genomic test detects cancer-driving mutations from liquid biopsy samples, including EGFR sensitizing (L858R and del19 E746-A750) and resistant (T790M) mutations, KRAS and BRAF mutations, and EML4-ALK, ROS1, and RET gene fusions.⁷ Patients testing positive for any of these mutations may benefit from targeted therapies directed specifically toward these mutations or fusions.

The proteomics liquid biopsy test measures the chronic activation of complex proteomic pathways known to be associated with a poor prognosis and potentially limited response to therapy. A positive result provides prognostic information that a patient can derive maximum benefit from standard-of-care treatment options, such as platinum-based therapy, EGFR-TKIs, single-agent therapy, and immunotherapy. This may correlate with longer progression-free survival and overall survival after treatment.¹⁰ A negative test result provides valuable clinical information that may be used to support patient-physician conversations regarding poor prognosis, as well as identifying a subset of patients who might benefit from other treatment strategies such as best supportive care or clinical trials.

Future outlook

Although further testing is required, PD-L1 in liquid biopsy that uses ddPCR technology has the potential to be a simpler and faster procedure that can return results in under 72 hours. Clinicians can use ddPCR-based LDTs as a rapid screening tool to look for tumor mutations or other biomarkers. PD-L1 testing by ddPCR could be especially favorable for patients with aggressive tumors, who may not tolerate tissue biopsy and could benefit from PD-1/PD-L1 immunotherapy.

As we continue to expand our understanding of the complex pathways cancers use to subdue the immune system, and to create increasingly targeted therapies against these molecules, we expect to see development of more sensitive and specific laboratory-developed tests based on liquid biopsy.

REFERENCES

1. U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999-2014 Incidence and Mortality Web-based Report. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Cancer Institute. https://www.cdc.gov/cancer/lung/statistics/index.htm.
2. Lin G, Fan X, Zhu W, et al. Prognostic significance of PD-L1 expression and tumor infiltrating lymphocyte in surgically resectable non-small cell lung cancer. Oncotarget. 2017;8:83986-83994.
3. Gong J, Chehrazi-Raffle A, Reddi S, Salgia R. Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations. J Immunother Cancer. 2018;6:8. doi: 10.1186/s40425-018-0316-z.
4. Liu D, Wang S, Bindeman W. Clinical applications of PD-L1 bioassays for cancer immunotherapy. J Hematol Oncol. 2017;10:110. doi: 10.1186/s13045-017-0479-y.
5. Hirsch FR, McElhinny A, Stanforth D, et al. PD-L1 Immunohistochemistry assays for lung cancer: results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project. J Thorac Oncol. 2017;12:208-222. doi: 10.1016/j.jtho.2016.11.2228.
6. Brunnström H, Johansson A, Westbom-Fremer S, et al. PD-L1 immunohistochemistry in clinical diagnostics of lung cancer: inter-pathologist variability is higher than assay variability. Mod Pathol. 2017;30(10):1411-1421. doi: 10.1038/modpathol.2017.59.
7. Mellert H, Foreman T, Jackson L, et al. Development and clinical utility of a blood-based test service for the rapid identification of actionable mutations in non-small cell lung carcinoma. J Mol Diagn. 2017;19(3):404-416. doi: 10.1016/j.jmoldx.2016.11.004.
8. Genome Web. Biodesix, University of Colorado Partner on Lung Cancer Assay. https://www.genomeweb.com/molecular-diagnostics/biodesix-university-colorado-partner-lung-cancer-assay?utm_source=Sailthru&utm_medium=email&utm_campaign=GWDN Wed PM 2018-01-31&utm_term=GW Daily News Bulletin.
9. Mellert H, Jackson, L, Pestano, G. Performance verification of a plasma-based PD-L1 test that reliably measures mRNA expression from patients with NCSLC. https://meetinglibrary.asco.org/record/156613/abstract.
10. Taguchi F, Solomon B, Gregorc V, et al. Mass spectrometry to classify non–small-cell lung cancer patients for clinical outcome after treatment with epidermal growth factor receptor tyrosine kinase inhibitors: a multicohort cross-institutional study. J Natl Cancer Inst. 2007;99(11):838-846.

Gary Pestano, PhD, serves as Vice President of Development and Operations at Biodesix, Inc., and led development of the company’s GeneStrat test, a genomic test for patients with non-small cell lung cancer.

Lisa Jensen-Long serves as Vice President of Marketing for Digital Biology Group at Bio-Rad Laboratories.