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Lung Cancer Diagnostic Approach Takes Aim at Identifying Malignancy, Guiding Immunotherapy From Single Sample


NEW YORK (GenomeWeb) – Australian researchers are developing an approach to advance the diagnostic utility of molecular testing of unfixed, small bronchoscopy specimens.

In a study published recently in The Journal of Molecular Diagnostics, researchers at RMIT University in Victoria, described collecting bronchial brushings that may enable diagnosing non-small cell lung cancer patients, determining the stage of their diseases, and guiding management of immunotherapies based on PD-LI expression.

A key component of the study is the discovery and preliminary validation of a biomarker — matrix metalloproteinase-9 (MMP-9) and TIMP3, its endogenous inhibitor — whose expression levels are implicated in the diagnosis of non-small-cell lung cancer (NSCLC), Steven Bozinovski, a study author and associate professor of clinical sciences at RMIT University, said in an interview.

The approach could provide a commercial approach made available within a few years to clinicians looking to make the most of a single lung cancer tissue sample, Bozinovski said.

In its preliminary study, which will require further validation using hundreds of patient samples, the research group demonstrated that the new marker could accurately differentiate a malignant from a nonmalignant tissue specimen without the need to fix tissue for histologic assessment, Bozinovski said.

The approach also quickly and accurately evaluated PD-L1 transcript levels, according to the researchers, and detected several oncogenic mutations with the use of high-quality nucleic acid from a single bronchoscopy specimen.

"I'm encouraged that people are looking into this because the big picture is that we want to be able to complete a diagnosis, staging, and molecular testing for lung cancer in one biopsy so as not to expose the patient to more invasive procedures and their associated risks," said Clarke Low, a doctor of hematology and oncology at Intermountain Healthcare. That is among the most important characteristics of the test approach being developed by the Australian researchers, he said.

Low, who is not involved in the development of the new approach, said that he routinely uses fixed bronchoscopy specimens in his practice to obtain a diagnosis of non-small cell lung cancer and to determine the stage of the cancer at the same time.

"I'm increasingly hoping to have enough tissue from that biopsy to do some molecular testing to help guide therapy or determine if the patient is eligible for certain clinical trials," he said. "Fixed bronchoscopy specimens can be quite small and can have a lot of unviable cells that cannot be used for diagnosis, staging, or doing therapy-guiding molecular testing."

The study's bronchoscopy-based specimen collection and molecular testing approach "shows promise in being able to identify viable cells and get a good sense in real time of how much viable tissue is present to, hopefully, maximize the chance of getting enough tissue to do all three of those broad aims," Low said.

Use of MMP-9:TIMP3 expression levels is not part of routine practice and its use by the Australian researchers is probably the first time it has been reported, Low said. Its use to differentiate tissue with tumors from healthy tissue is another important aspect of the molecular method being developed by the team, he noted.

Precision therapies targeting the PD-1/PD-L1 pathway have the potential to improve response in some patients with cancer. However, according to the Australian researchers, this therapeutic approach requires more reliable and faster methods than are currently available for identifying patients whose cancers are particularly likely to respond.

PD-L1 protein expression detected by immunohistochemistry testing is becoming a widely used method to determine which patients may benefit from anti-PD-1/ PD-L1 therapies, but it is a complex undertaking and can have challenges related to variability in pathologists' interpretation.

At the same time, the growth of lung cancer screening trials is driving greater demand for defining the molecular nature of suspect lung nodules.

The approach being developed by the Australian team could be used as an alternative to existing methods within three years to rapidly discriminate malignant from benign tumors and to identify mutational status as well as guide and refine therapeutic decisions, Bozinovski said.

In the study, the authors used radial probe endobronchial ultrasound to obtain bronchoscopy brushings from 28 patients enrolled at the Royal Melbourne Hospital. Following rapid onsite evaluation of the malignant tissue, they preserved the specimen in nucleic acid stabilization buffer, which enabled conducting downstream analyses on the preserved, non-fixed tissue, Bozinovski said.

They used RT-qPCR to screen the specimens for biomarkers that could have diagnostic utility.

"We identified the MMP9:TIMP3 transcription ratio that we described in the study as a biomarker of the presence of malignant cells," Bozinovski said, adding that the biomarker proved effective in differentiating benign from malignant tissue.

In one instance, a specimen was initially considered nonmalignant by pathology, but they found that the MMP9:TIMP3 ratio had increased more than three-hundredfold. A subsequent specimen collected nine months later confirmed malignancy by pathology, and the MMP9:TIMP3 ratio at that time remained above the cutoff to determine that the specimen was malignant.

"We also wanted to know whether we could take the approach a step further and measure PD-L1 levels and ascertain how the new approach performed relative to [US Food and Drug Administration]-approved immunohistochemistry assays," Bozinovski added.

The researchers observed a high concordance between measurement of PD-L1 levels using RT-qPCR and using immunohistochemistry.

"That gave us a streamlined, rapid approach to achieving both goals — identifying PD- L1 levels to guide managing immune checkpoint inhibitors and simultaneously determining the presence of lung cancer cells," Bozinovski said.

Because the tissue is unfixed and preserves the integrity of DNA and RNA, enough genomic DNA is available to allow multi-panel targeted next-generation sequencing to assess the total mutational burden of a tumor, the investigators said.

Further, in the current study, the investigators found TP53 gene mutations in 10 of 15 NSCLC samples — information that could be used to refine patient selection for a treatment regimen.

In future versions of its test, the researchers may move from the use of RT-qPCR to a more automated droplet digital PCR as a testing platform. That would further enhance the platform's clinical utility by enabling testing for additional biomarkers. Clinicians could include PD-L1 markers along with EGFR and ALK rearrangements as lung cancer markers in a multiplex test, Bozinovski said.

He noted that the group is also looking to automate its process — from purification of nucleic acids to generating and analyzing PCR data — to provide results in around 24 hours.

Bozinovski said that the study findings need to be validated in a large cohort, and the researchers are seeking funding that they anticipate will support a blinded, multicenter study with a few hundred patients. The group also hopes to commercialize the approach so it can be available to clinicians in hospitals on a broader scale.

To achieve that, the team is looking to collaborate with an industry partner with expertise in how to take the test through regulatory approvals, he said. Bozinovski said the research team intends to assess regulatory options in collaboration with an industry partner.

Extensive validation trials that are required for regulatory approvals are also used by government and private payors to assess reimbursements for tests.

In 2017, Veracyte's Percepta bronchial genomic classifier became the first genomic test to be covered by Medicare for improved lung cancer screening and diagnosis, and in January the firm entered a collaborative agreement with Johnson & Johnson to focus on further developing its lung cancer tests.