Pilot Study of Nilogen 3D-EX and Its Ability to Predict Therapeutic Response to Anti-PD1 or Anti-PDL1 in NSCLC

Pilot Study of the Nilogen Ex-Vivo Assay (3D-EX) and Its Ability to Predict Therapeutic Response to Anti-PD1 (Nivolumab, Embrolizumab) or Anti-PDL1(Atezolizumab) in Advanced Non-small Cell Lung Cancer (NSCLC)

Subjects will be eligible for this study if they are about to start on a drug called nivolumab for lung cancer. Some patients' cancers respond to nivolumab but a majority of patients do not. To better determine which patients will most likely respond to nivolumab or not, the investigators are testing an assay that tests biopsy tissue to determine if the subject's tumor will likely respond to nivolumab.

The main purpose of this research study is to see if this specialized test can help identify people with locally advanced or metastatic non-small cell lung cancer who are more likely to benefit from treatment with nivolumab. The results of the tests will not affect whether or not subjects receive nivolumab but may help identify future patients who are more likely to benefit from nivolumab. The study assay is extra and experimental.

Overview of PD-1 inhibition for locally advanced or metastatic NSCLC Lung cancer (LC) is the most common malignancy-related death in the United States (causing deaths in ~ 86,380 men and ~71,660 women in 2015. Fifteen percent of LC patients present with Stage I disease, which is treated primarily with surgery, leading to a 5-year survival rate of 54%. Approximately 57% of LC patients are initially diagnosed with metastatic disease (Stage IV) with a corresponding 5-year survival rate of 4.2%.

More recently, major treatment responses have been observed with the use of immune checkpoint inhibitor proteins expressed on the surface of lymphocytes and other immune cells, most notably on cytotoxic T-cells. When bound to their specific ligand, often another surface-bound protein on a neighboring cell, they can transmit stimulatory or inhibitory signals to activate or inhibit the cellular adaptive immune response. Several studies showed that the predominant mechanism by which NSCLC evades detection and elimination by the immune system is by exploiting one such inhibitory pathway through the expression of programmed death ligand 1 (PD-L1, B7-H1). PD-L1 then binds to its receptor, programmed cell death protein 1 (PD1), on surveilling lymphocytes and initiates a signaling cascade which leads to lymphocyte exhaustion, a state of impaired function. The most successful immune checkpoint inhibitors so far are anti PD1 or anti-PD-L1 monoclonal antibodies that prevent PD1-PD-L1 interaction at the tumor-immune interface. In this pilot study, the investigators will investigate if Nilogen's functional ex vivo bioassay can predict therapeutic response to anti PD1 (nivolumab, pembrolizumab) or anti PD-L1 (atezolizumab) in advanced non-small cell lung cancer (NSCLC).

Functional bioassay as a potential predictive biomarker. The intent of using anti-PD-1/PD-L1 as an anti-cancer therapeutic is to block one of the ways that tumors inhibit T cell function within the tumor microenvironment, allowing restoration of T cell function. Therefore, the investigators hypothesize that a bioassay that directly measures restoration of T cell function in the context of all of the elements of the tumor microenvironment could accurately predict whether or not a patient will be clinically responsive to a particular immunotherapeutic agent that is designed to target the tumor microenvironment. A tumor biopsy from a patient who will be clinically responsive to anti-PD-1 will contain inactive T cells that may have their functional activity restored by the provision of anti-PD-1/PD-L1 ex vivo, much like what is seen in the ex vivo experiments performed at Nilogen using the 3D-EX platform (www.nilogen.com). To assess tumor response to nivolumab, pembrolizumab or atezolizumab the investigators will use their proprietary immune cell function analysis in tumor samples obtained by FNA biopsy upon treatment with nivolumab, pembrolizumab or atezolizumab, pembrolizumab or atezolizumab ex vivo.

Hypothesis

1. In 80% of patients, FNA samples will provide a sufficient number of viable tumor cells to perform ex vivo functional bioassays with nivolumab, pembrolizumab or atezolizumab.
2. Patients whose tumor FNA samples fail to respond to nivolumab, pembrolizumab or atezolizumab, in the 3D Ex-Vivo assays will likely not show a clinical response to systemic nivolumab, pembrolizumab or atezolizumab treatment.

Study Objectives

Primary Objective To test whether it is feasible to perform the 3D-EX functional response bioassay in the context of patients with advanced NSCLC receiving treatment with nivolumab, pembrolizumab or atezolizumab in a standard clinical setting.

Overall study design Eligible patients will have a histologic or cytologic diagnosis of NSCLC; advanced-stage disease who are appropriate candidates for therapy with nivolumab, pembrolizumab or atezolizumab, and age greater than 18 years. Prior surgery, chemotherapy and/or radiotherapy are allowed. Inclusion and exclusion criteria are outlined in the appropriate section. All inclusion and exclusion criteria will be assessed within 6 weeks prior to the first dose of nivolumab, pembrolizumab or atezolizumab. Baseline radiographic studies should be performed within 30 days of screening. This clinical study will require central IRB approval. Informed consent will be obtained from each patient prior to screening and enrolling in the study.

Nivolumab, pembrolizumab or atezolizumab will be administered as per the established standard of care for the eligible population.

Prediction of Response to Nivolumab, pembrolizumab or atezolizumab by Nilogen's 3D-EX Bioassay.

In previous preclinical and clinical studies the investigators showed that FNA samples provide a sufficient number of viable tumor cells to perform drug sensitivity assays ex-vivo. The assay protocol is optimized to process tumor FNA samples, where the tumor microenvironment is intact and contains tumor cells and all other tumor-infiltrating inflammatory cells as well as specialized tumor stroma. Prior to initiation of therapy, tumor samples will be collected by FNA from accessible tumor tissue. For the ex vivo assays, aspirates will be obtained from each patient after collection of biopsy samples required for SOC. The FNA samples will be immediately shipped to the Nilogen Laboratory. All proposed assays will be performed at the Nilogen Oncosystems laboratory in Tampa, FL according to CLIA standards.

Patients who do not have a site of disease that will provide enough tumor cells for analyses will be taken off study. In adequate samples, tumor FNA samples will be treated nivolumab, pembrolizumab or atezolizumab at clinically relevant concentrations. At the end of incubation T-cell activation will be assessed by a proprietary approach including evaluation of drug-mediated changes in three parameters: (1) T-cell activation, (2) cytokine release and (3) gene expression profiles. The response rate to treatment will be scored for T-cell activity and correlated with patient outcome.

The investigators hypothesize that tumors that fail to respond ex vivo will not be likely to be responsive in vivo in cancer patients.

Inclusion Criteria:

In order to be eligible for participation in this trial, the subject must:

1. Be willing and able to provide written informed consent for the trial.

2. Be at least 18 years of age on the day of signing informed consent.

3. Have a histologic or cytologic diagnosis of Stage IV NSCLC.

4. Must be medically eligible to receive nivolumab, pembrolizumab or atezolizumab as the standard of care for the next line of therapy (must have previously received first line platinum doublet chemotherapy) as determined by their oncologist.

5. Have measurable disease based on RECIST 1.1 (see section 7.1 or appropriate number).

6. Be willing and medically fit to undergo a fresh (newly obtained) diagnostic biopsy of a metastatic lesion or primary site of disease before receiving nivolumab, pembrolizumab or atezolizumab. "Fresh (newly-obtained)" is defined as a specimen obtained up to 6 weeks prior to initiation of treatment with nivolumab, pembrolizumab or atezolizumab on Day 1.

7. Fit in either of these categories:

   1. Stage I-III NSCLC patients who develop metastatic disease within 6 months of receiving definitive (curative) treatment that includes platinum-based chemotherapy, and who require biopsy for either confirmation of diagnosis or further molecular or immunohistochemical testing to guide treatment.
   2. Stage IV NSCLC patients with progression of disease following platinum based chemotherapy, and who require biopsy for either confirmation of diagnosis or further molecular or immunohistochemical testing to guide treatment.
   3. Stage IV NSCLC patients who will receive nivolumab, pembrolizumab or atezolizumab as the standard of care in special circumstances (eg, when chemotherapy is contraindicated or if a patient declines to be treated with chemotherapy).

8. Be willing to undergo at least 4 fine needle aspirations for experimental purposes at the time of the standard of care biopsy.

9. Have a performance status of 0 or 1 on the ECOG Performance Scale.

Exclusion Criteria:

The subject must be excluded from participating in the trial if the subject:

1. Is currently participating in and receiving therapy as part of a different clinical trial.
2. Is not a candidate to receive nivolumab, pembrolizumab or atezolizumab as determined by the patient's oncologist.
3. Has an active infection requiring systemic therapy.
4. Has a history or current evidence of any condition, therapy, or laboratory abnormality that might; (1) confound the results of the trial, (2) that would substantially increase risk of incurring adverse events (AEs) from nivolumab, pembrolizumab or atezolizumab, (3) that would interfere with the subject's participation for the full duration of the trial, or (4) is not in the best interest of the subject to participate, in the opinion of the treating investigator.
5. Has known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial.
6. Is pregnant or breastfeeding.
7. Has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies).
8. Has known active Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA [qualitative] is detected).