Next-Generation Sequencing Applied to the Cancer Landscape

Hossein Borghaei, DO, MS

The use of next-generation sequencing (NGS) to screen patients with advanced-stage lung cancers has opened a new world of treatment options and has undoubtedly contributed to the recent, continued decline in lung cancer death rates.¹

Quite simply, NGS provides oncologists with accurate information in a cost-effective manner, thereby affording treatment options we would not otherwise have. Because NGS is a deeper dive into DNA, oncologists can identify more targets for treatment than more limited panels can.

Targeted therapies in general are associated with better overall patient outcomes compared with chemotherapy. Moreover, new targets that don’t have an FDA-approved drug available, but might be included in clinical trials, are being identified. By relying solely on a limited panel that gives us only FDA- approved targets, we could miss the opportunity for a clinical trial that a patient might be able to access.

Consider RET mutations, which by all accounts are present in 1% to 2% of all patients with lung cancer. At the Fox Chase Cancer Center, we identified 2 or 3 patients with these alterations. When a study opened at an academic institution close to us, we were able to refer those patients to this institution. That clinical trial led to the approval of the drug selpercatinib (Retevmo), which is now the standard of care for patients with that alteration.

At Fox Chase, we conduct many clinical trials— some with targets that are not FDA approved at this time. However, after our NGS panel underwent a comprehensive overhaul 3 years ago, most targets can be identified within our panel.

When a target is found using NGS, there can be up to 8 or 9 FDA-approved drugs that go with those specific targets. In looking at the data, I find that patients who are treated up front with a targeted therapy experience better outcomes. These drugs are easier to take because most are oral agents—and the adverse events are easier to tolerate compared with most IV medications. Furthermore, in my view, clinical efficacy seems to be optimal if the target is detected right at the time of initial diagnosis. This invites many other treatment options for consideration for various mutations we can define.

Obtaining adequate tumor samples has always been a little more difficult with lung cancers because of the locations of the tumors. A larger tissue sample than we are used to obtaining is generally required, and traditionally, a minimal amount of tissue would allow a pathologist to determine the presence of lung cancer, and that would be the end of the investigation. Now, we need a core level biopsy to determine the exact subtype of lung cancer; we want the molecular data NGS provides. For this reason, an adequate sample is required to perform a DNA isolation and conduct appropriate sequencing, which is why the use of “liquid biopsy” has become more popular. Essentially, with 2 tubes of blood drawn in an office, we can detect almost all of the alterations that we need to formulate a treatment plan.

Our Success Story

At Fox Chase Cancer Center, we have found great success using NGS. For example, in 2019 we announced that our researchers found a fusion gene signature in primary tracheobronchial adenoid cystic carcinoma, a rare lung tumor.²

This study is the first time the MYB/ MYBL1 fusion genes have been systematically investigated for this cancer type, as we studied 7 specimens of the carcinoma and detected fusions of either MYB or MYBL1 genes in all 7.3 Three cases had MYB-NFIB, and 3 had MYBL1-NFIB. The remaining case showed a rare MYBL1-RAD51B fusion, which had never been reported in tracheobronchial adenoid cystic carcinoma. These findings suggest that rearrangement involving MYB or MYBL1 is a hallmark of tracheobronchial adenoid cystic carcinoma.

This finding could also be used as a diagnostic tool and can be used for potential targeted therapy. NGS RNA sequencing is a powerful approach for identifying translocation/rearrangement of solid tumors or hematological malignancies. The targeted RNA fusion panel used in this study consists of 507 well-known cancer-related fusion genes, which covers 7690 exotic regions that are targeted with a total of 21,283 probes.

This targeted RNA fusion NGS panel is in its development stage for clinical use and will be provided for clinicians soon. In the clinical setting, we perform a separate RNA fusion panel, which covers roughly 500 genes— assuring us that we’re not missing rare fusions. In my practice over the past few months, I’ve begun offering a liquid biopsy at the time of initial diagnosis in addition to the tumor-based NGS and RNA fusion testing to broaden the results I can get.

Caveats With Using NGS

Although NGS is a necessity in driving progress in treating lung cancer, its sustainability will depend on implementing thorough informatics and infrastructure that can support the integration of genomic results into electronic medical records.⁴

We have also learned that in some instances, when patients are evaluated solely with an NGS panel, certain targets could be missed because these are not necessarily genetic mutations but fusions, or translocations, that require a different diagnostic technique. Therefore, the concept of using RNA to find some of these targets has become very appealing. Many academic institutions now do a separate RNA fusion panel, and there’s at least 1 commercial entity that performs an RNA analysis and a DNA analysis and reports the findings of each.

The simple act of an oncologist declaring a desire to perform NGS has significant down-stream and upstream implications. Whoever is obtaining the tissue needs to obtain more at the potential risk of complications for the patient. The pathologist must be made aware, so tissue is conserved, and then there’s this issue of chain of custody: Who orders the molecular tests? If we wait until the oncologist sees the patient, that could be 2 or 3 weeks from the biopsy.

Another layer of complexity was added with the introduction of liquid biopsy, essentially, drawing a couple of tubes of blood and sending it for the same NGS sequencing based on DNA of the tumor that’s shed into the bloodstream that can be detected and sequenced.

The advantage of a liquid biopsy is that results are obtained quickly—in 7 to 10 days on average—whereas for tissue, under the optimal conditions, it can take up to 2 and a half weeks. Of course, conditions are rarely optimal; moreover, some of our colleagues have published results indicating that a liquid panel and a tumor panel can be complementary; the false negatives and positives in both tests can be offset if performed at the same time.

Make no mistake: These are also relatively expensive tests. Oncologists must be aware that these tests have limits. A completely negative liquid biopsy test does not mean a patient doesn’t have a mutation—it just could mean that it was not detected. Therefore, performing a complementary tumor biopsy allows the physician to be as close to 100% comfortable as can be that there are no new mutations found.

It is worth noting that although academic centers like Fox Chase have committed to establishing molecular laboratories stocked with the proper equipment and staffed by experienced personnel, not all private practices have access to such facilities, which are expensive to set up and maintain. Commercial entities exist to perform NGS for practices that don’t have ready access to molecular testing in house; those businesses remain current on the literature and provide the requisite expertise to interpret patient reports.

Deciding on the Right Treatment

To be clear, the right treatment for non-squamous NSCLC in the advanced stage is to perform molecular testing and to offer appropriate treatment based on this testing.

If the molecular testing doesn’t reveal anything we can act upon right now, there is a different pathway for the treatment of that patient. We can only trigger that pathway once we know for sure there are no actionable genetic alterations that can be treated.

In short, the clinical implications of NGS are significant. Toxicity management, quality of life, and overall, better survival rates based on data from retrospective studies. The advantages of performing molecular testing are well established in the literature; it is important to perform NGS, and data are solid in supporting the correlation between good clinical outcomes for patients who receive the right drugs.

_REFERENCES

_{1. Annual report to the nation: Rapid decrease in lung cancer and melanoma deaths lead overall continued decline in cancer death rate. News release. National Cancer Institute. July 8, 2021. Accessed Feb- ruary 9, 2022. https://bit.ly/3oxzpMQ}

<sub>2. Targeted RNA-sequencing detects fusion gene signature in rare form of lung cancer. News release. Fox Chase Cancer. April 29, 2019. Accessed February 9, 2022. https://bit.ly/3rBWkII
3. Pei J, Flieder DB, Patchefsky A, et al. Detecting MYB and MYBL1 fu- sion genes in tracheobronchial adenoid cystic carcinoma by targeted RNA-sequencing. Mod Pathol. 2019;32(10):1416-1420. doi:10.1038/ s41379-019-0277-x</sub>

_{4. Seymour C. Key advances showcase the potential of precision medicine in NSCLC. OncLive. October 5, 2021. Accessed February 9, 2022. https://bit.ly/3gAqSEy}