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Determining Prognostic Significance of Biomarkers for Risk Stratification of Early Stage Lung Cancer

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During this year’s AACR Annual Meeting we spoke to Dr. Delphine Lissa and Dr. Ana Robles from the National Cancer Institute to learn more about the team’s most recent study which evaluated the prognostic significance of two biomarkers, HOXA9 promoter methylation and blood vessel invasion (BVI), for risk stratification of stage I lung adenocarcinoma patients using droplet digital PCR (ddPCR).

Dr. Lissa and Dr. Robles tell us more about the study and the two biomarkers evaluated, highlight the key benefits of using ddPCR for biomarker assessment, and discuss future studies.

Laura Mason (LM): Could you provide a brief summary of the study you are presenting at AACR?

Dr. Ana Robles (AR):
At this year’s AACR we presented a continuation of our study where we looked at early stage lung cancer through genome-wide DNA methylation analysis and identified a particular methylation site associated with prognostic features of lung adenocarcinomas.

This particular methylation site is on the HOXA9 gene promoter, which is part of the homeobox gene family. These genes are associated with development, but also methylated in lung and other cancers.

We wanted to bring this particular methylation site into the droplet digital PCR (ddPCR) format and apply it for the analysis of samples collected through routine pathology (as opposed to fresh frozen tumor samples that we had used in our earlier study –  that is what’s most commonly used for the initial identification of biomarkers in general).

Dr. Delphine Lissa (DL): Our study addresses the critical need of developing novel prognostic biomarkers for stage I lung cancer. The current model to evaluate lung cancer prognosis is based on clinico-pathological parameters. However, within five years after surgery, 30% of patients relapse and die of their disease. This demonstrates that the current method is not accurate enough to predict survival. In addition, with the implementation of Low Dose Computed Tomography (LDCT) scan for lung cancer screening, the number of lung cancer cases detected at an early stage is expected to rise.

AR: To expand on that last point, clinically, lung cancer is generally diagnosed at an advanced stage, with very poor prognosis. When the patient enters the clinic with symptoms, the disease may have already metastasized, and at that point it is often resectable, but requires aggressive treatment– with chemotherapy or immunotherapy, for example.

One very important advance in recent years, is the development of a new strategy to screen for lung cancers that is based on LDCT. When individuals at high risk are screened, (older heavy smokers or people who are former smokers who have quit within 15 years or so), very small lesions can be picked up using LDCT. In most cases these lesions are benign, but when the screening actually picks up cancer, it will most likely be stage I lung cancers (about 60% of the time). This has produced a ‘stage shift’ for diagnosis. As implementation of screening increases, the rate at which early stage lung cancer is diagnosed increases. Early stage lung cancer is mostly treated by surgery, and in few cases followed by adjuvant chemotherapy. Stage IA is treated by surgery alone, and chemotherapy may be recommended for stage IB, if some markers of high risk are present (considerations include size and other features of the tumor).

We are interested in those cases in which surgery with curative intent is the main or sole treatment option. Molecular markers will give clinicians clues as to what to expect for a particular patient, so that as newer immunotherapies or more developed therapeutic strategies are coming in, they can be more informed in terms of how to select patients that can benefit the most from therapy, or on the flip side spare those at low risk from aggressive treatment. So, on one side, high risk patients can be followed up with a more stringent regime and low risk patients can be spared potential side effects from chemotherapy.

LM: Could you tell me a bit more about the two biomarkers evaluated during the study?

The first marker we evaluated was HOXA9 promoter methylation, which is the one we identified in our earlier study. In this study, we moved into a different technology (ddPCR) and biospecimens (formalin-fixed paraffin-embedded tissues, or FFPE) that are more readily available to the clinician. Because we were using FFPE tissues, we also decided to incorporate a biomarker that has already been established within the lung cancer community as a good prognostic biomarker for early stage lung cancer, based on immunohistochemical staining: blood vessel invasion, or BVI. As the name suggests, BVI gives us information about neovascularization of the tumor, and therefore potential features of early micrometastasis.

We found that higher methylation of HOXA9 promoter is associated with poor prognosis. Together with BVI, as a combined biomarker approach, they have very strong prognostic features. When a particular tumor is BVI positive and highly methylated for HOXA9, then the prognosis is exceedingly poor.

The other thing that’s interesting is that, BVI and micrometastasis are features of a more advanced tumor even though it might be categorized by pathology as stage I. In the case of the HOXA9 methylation, by looking at other molecular analyses that we have for these tumors, we were able to show that the stage I tumors that have high HOXA9 methylation also have a higher frequency of TP53 mutations.

TP53 is a tumor suppressor gene that is frequently mutated in lung cancer but its mutation is associated with heavier smokers and also with a higher stage. Our finding is that the stage I or even stage IA subset of tumors that have high HOXA9 methylation have this additional molecular feature: presence of TP53 mutations at a frequency that you would really expect in a more advanced stage of the disease.

Other interesting molecular features of this particular set of tumors include expression of certain onco-microRNAs like miR-9 which have been independently associated in the literature with advanced lung cancer.

LM: Your abstract mentions that ‘the type of biospecimens and the choice of assay platform are key issues to foster translation of biomarkers to the clinic.’ Could you expand on this?

These two elements are critical to facilitate the implementation of any biomarker in the clinic and should be considered from the early stage of assay development to speed up the process. We chose FFPE biospecimens as they are routinely generated in pathology and therefore easily accessible. We used the ddPCR system developed by Bio-Rad because the workflow is simple, and it received CE-IVD marking in 2016 for use as an in vitro diagnostic in the EU. 

AR: Essentially, once FFPE blocks are cut into sections for pathology, generally one or more sections will be used for the initial staining for staging, and then the rest of the block is either not used, or used for other assays, like immunohistochemical assays. And in our case, a single slice of this block will yield enough DNA to run the HOXA9 methylation assay.

LM: What are the key clinical benefits of droplet digital PCR?

There are several advantages to using this particular technology for biomarker assessment. As I mentioned, the amount of input DNA that is required is minute, and ddPCR can analyze fragmented DNA as well. For example, the DNA in FFPE sections may not be of good integrity, but because the amplicons are short, it’s still good enough quality for ddPCR analysis.

DL: The technology is rapid and some hospitals already have this kind of system implemented. Another benefit is that the technique doesn’t require much training. The analysis is much simpler than sequencing, for example. All these factors in combination make it advantageous.

LM: The study results suggest that there is a clear benefit to using ddPCR to identify patients at high risk of recurrence. Could you tell us more about next steps, are there plans to validate these findings in a larger independent study?

AR: In terms of next steps, the implementation of LDCT will lead to the detection of more stage I lung cancers, so for us, a natural progression from this study would be to validate these findings in a cohort of lung cancer patients that were diagnosed via LDCT as opposed to the cohort that we’re showing in this study, in which patients were clinically diagnosed with lung cancer, not screening based diagnoses.

DL: We are also collaborating with other groups, because one of the most important pieces of biomarker translation is to be sure that when the parameters of an assay are ‘locked down’, the biomarker will perform well in different sample cohorts, that were obtained, for instance, in a regular clinical lab or even a different population (e.g. lung cancer from Japan where 50% of lung cancers will be from non-smokers). This will allow us to confirm global applicability of this assay. We already have indication that it will be the case from our earlier study where our second validation cohort included lung cancers from Japan and showed that HOXA9 was prognostic within that population as well.

AR: And then there are other more long-term ideas, some of them related to topics discussed at AACR 2018, like immunotherapy. We see that immunotherapy is now being applied to earlier stages of lung cancer, whereas at the beginning it was only implemented for advanced lung cancer. But there is a lack of molecular markers, of prediction and prognosis for early stage, that might actually support the implementation of this therapeutic strategy. We think that there can be a space there where biomarkers such as the ones discussed here can really help.

Dr. Delphine Lissa and Dr. Ana Robles were speaking to Laura Elizabeth Mason, Science Writer for Technology Networks.