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Novel Biomarkers for the Treatment of Non-Small Cell Lung Cancer

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Non-small cell lung cancer (NSCLC) is currently treated with surgery, radiotherapy, immunotherapy or chemotherapy, depending on the stage of disease. Immunotherapy has been found to be effective in the treatment of NSCLC and can improve the life expectancy of some patients, however, immunotherapy is not effective in all patients. Current biomarkers are not always able to predict therapeutic response.

Technology Networks
spoke to Dr. Alberto Cruz Bermúdez, postdoctoral investigator at Puerta de Hierro Majadahonda University Hospital to learn more about the current state of NSCLC therapies. Bermudez also discusses his involvement in characterizing T cell receptor (TCR) repertoires and the identification of two novel biomarkers for NSCLC.

Kate Robinson 
(KR): What is non-small cell lung cancer (NSCLC) and how is it currently treated?

Alberto Cruz Bermúdez
Non-small cell lung cancer is the most common type of lung cancer, representing 80-85% of lung cancers, and is one of the leading causes of cancer-related death in the world. (Cancer.org)

The treatment and prognosis of NSCLC depends on the extent of disease at diagnosis, which is determined by the tumor stage, based on tumor size, lymph node involvement and the presence or absence of distant metastases.

Early stages (I-II) are usually treated with surgery. Locally advanced or intermediate stages (stage III) that are considered resectable are treated with chemotherapy (denominated neoadjuvant) and subsequent surgery, while those that are unresectable are usually treated with chemotherapy and radiotherapy. Finally, patients with advanced or metastatic disease (stage IV) are treated with platinum-based chemotherapy, targeted therapies against specific "driver" pathways, or immunotherapy depending on the expression of the PD-L1 protein in the tumor cells.

Immunotherapy has been a revolution in treatments, increasing the life expectancy of patients in advanced stages. These positive results have led researchers to test the efficacy of these immunotherapy-based treatments in early stages through clinical trials, such as the NADIM trial promoted by the Spanish Lung Cancer Group. The NADIM trial tested the activity and safety of chemoimmunotherapy in resectable, locally advanced NSCLC.

KR: Why are predictive biomarkers and T cell receptor (TCR) repertoires important in NSCLC?

Despite the efficacy of immunotherapy, not all patients respond favorably to it. Knowing a priori whether the patient will benefit or not from the treatment by determining some predictive parameter can advance the application of precision medicine. In this case, the patient is given the treatment that is likely to give the best result, avoiding unnecessary toxicities, improving the prognosis of patients and allowing the search for other therapeutic alternatives.

Immunotherapy consists of reactivating the antitumor response exerted by the patient's immune system, in which the adaptive immune system and T-cells plays a key role.

In this sense, high levels of tumor mutational burden (TMB) (which would theoretically generate more neoantigens to be recognized by T cells) and PD-L1 (that would prevent the activation of T-cells) have been used as predictive biomarkers for anti-PD1 immunotherapy and chemoimmunotherapy response. However, these parameters are not perfect; existing patients with elevated TMB or PD-L1 do not respond to treatment, while other patients end up benefitting from therapy who do not have these biomarkers elevated.

The mechanism by which the adaptive immune system responds to immunotherapy and recognizes tumor antigens relies mainly on the highly polymorphic T-cell receptors (TCRs) present in an individual. That is why the characterization of the TCR repertoire in terms of clones, diversity and antigen specificity by sequencing the CDR3 hypervariable region, seems to be a promising approach that reflects the response to immunotherapy. In addition, the study of the TCR repertoire could better reflect the immune status of the patient, as compared to earlier points in the immune cycle such as TMB, neoantigens or immunosuppressive factors such as PD-L1 levels.

KR: Can you outline the methods utilized in the current study to characterize TCR repertoires?

Next-generation TCR sequencing was performed in peripheral blood and tissue samples of 40 NSCLC patients, before and after neoadjuvant chemoimmunotherapy. Gene libraries were generated from total RNA extracted from tumor biopsies of paraffin-embedded tissue (100 ng) as well as from PBMCs (25 ng) (peripheral blood mononuclear cells) isolated from peripheral blood. RNA was used instead of DNA to decrease material input requirements and maximize valid samples for sequencing. From 77 tissue samples collected, we obtained valid TCR data for 60 (78%); 3 samples did not have sufficient tissue for RNA extraction and 14 did not yield enough RNA quantity for further analysis, however, all samples with enough RNA quantity generated valid NGS data. The high success rate in tissue samples is important given that tissue quantity is a major limitation in NSCLC. On the contrary, from 65 blood samples collected, we were able to obtain valid TCR data in all cases (100%).

KR: What is chemoimmunotherapy and how does it compare to other cancer treatments?

Chemoimmunotherapy combines the use of platinum-based chemotherapy, which promotes the death of dividing tumor cells, with immunotherapy based on blocking the interaction of PD-1 proteins with PD-L1 using monoclonal antibodies (in our case nivolumab, an anti-PD1 drug).

Under basal conditions, the PD-1 protein expressed in lymphocytes inhibits activation following its interaction with the PD-L1 ligand, which is frequently overexpressed in tumors. Blocking this interaction reactivates the antitumor capacity of lymphocytes.

Recent clinical trials seem to show the superiority of chemoimmunotherapy combination compared to chemotherapy alone in locally advanced stages. Due to the novelty of combination therapy, many of the mechanisms involved in the response are unknown – increasing the understanding of these mechanisms was a core objective of our project.

KR: What were the main findings from the current study and what implications do these findings have for future treatment of NSCLC?

We have identified two biomarkers (top 1% clonal space and TCR evenness) associated with T-cell repertoire imbalance that outperform the established biomarkers PD-L1 and TMB, regarding prediction of patients that will be free of disease after chemoimmunotherapy (i.e., that achieve complete pathological response (CPR) in surgery). To identify these CPR patients is key since achieving CPR is associated with improved survival.

Tumors with higher top 1% clonal space presented a higher pro-inflammatory profile reflected by up-regulation of biological processes such as leukocyte-mediated immunity, cell killing and T cell activation, among others – despite having similar levels of PD-L1, TMB or tumor infiltrating populations. This stronger immunogenic profile could explain the repertoire imbalance and greater pathologic responses, since in such a permissive microenvironment the specific activation and clonal expansion after antigen recognition of some anti-tumoral T cell clones would be more easily allowed.

The study of the pre-treatment tissue top 1% clonal space as a potential biomarker is encouraging – it reflects tumor immunogenicity and is strongly associated with tumor response being technically robust and affordable considering turnaround times and costs. Thus, if its value is confirmed in larger cohorts with longer follow-ups for survival analysis, we believe that the analysis of the top 1% clonal space could be implemented in the clinic. This could allow for the personalization of follow-up care and the treatment of patients with low top 1% clonal space, who would presumably not achieve CPR, and would enable studies to determine the value of surgery in patients that will achieve CPR, that likely would present high top 1% clonal space at diagnosis.

Alberto Cruz Berm
údez was speaking to Kate Robinson, Editorial Assistant for Technology Networks.