NL NVALT2017 / Translational research for immune modulating and targeted therapy in non-small cell lung cancer, an exploratory NVALT study

An effective and sustainable immune response against NSCLC is dependent on
several immunological processes described by the cancer immune cycle. This
cascade begins with the release of cancer cell antigens, followed by dendritic
cell antigen capture and processing, T cell priming in the lymph node, T cell
trafficking to tumors, and finally by CD8 positive T cells recognition and
killing of cancer cells. In this last process the PD-1/PDL-1 checkpoint axis is
important and recently effective check point inhibitors have become available
for the treatment of lung cancer. Phase III studies showed that immunotherapy
with nivolumab, pembrolizumab and atezolizumab provide a prolonged survival
compared with docetaxel as second line treatment in advanced non-small cell
lung cancer (NSCLC). Pembrolizumab also showed activity as first line treatment
in advanced NSCLC in those patients who had *50% PD-L1 positive tumor cells and
improved survival compared to chemotherapy.
Another group of patients of interest are those with advanced NSCLC that have a
targetable gene mutation and are being treated with specific drugs. The
incidence of these DNA aberrations in the total population of NSCLC is less
than 5%. Resistant EGFR mutations such as T790M, HER-2 amplifications or
mutations, cMET amplifications, mutations, exon 14 skipping, and translocations
such as ALK, ROS1, RET are just examples where targeted drugs are biologically
active and need specific knowledge how to treat and follow these patients.

Checkpoint and first line targeted drug resistant tumor cells will be present
at the start of treatment or may develop during treatment. Many resistance
mechanisms have been described on the basis of associations in only small
resistant patient cohorts.
To determine patterns of signal transduction or changes in gate keeper
mutations, blood samples at baseline, in week 2 or 3, 6, 12 and at progression
or recurrence of disease and tumor biopsies at baseline, optional at 6 weeks
and at progression or recurrence of disease are gathered from patients that are
registered in the NVALT *Dure Geneesmiddelen* Registry.

Moreover, results from especially immunotherapy trials show that we are in need
of better biomarkers. The drugs are expensive and identification of a patient
group that does not benefit could reduce healthcare expenses and auto-immune
related toxicity. Similarly, the contribution of drug-induced resistance
mechanisms in patients treated with targeted therapy should be further explored
with comprehensive methods.

* To explore baseline predictive biomarkers for tumor response to immune
modulating and targeted treatment in patients with NSCLC.
* To develop biomarkers of therapy resistance to anti-PD-1 treatment in
patients with NSCLC.
* To explore patterns of resistance in patients with NSCLC.

For this study, the PRoBE design principles will be followed. The PRoBE
guidelines are an application of standard principles of good population science
to the special case of biomarker research. In this descriptive study, somatic
tumor phenotypic changes and their underlying genotype will be explored.
Phenotypes are described by patient and tumor characteristics using clinical
observations e.g. responders versus non-reponders and genotypes by targeted DNA
and RNA seq methods. Patients will be treated on an outpatient basis with
immune checkpoint inhibitors according to label and targeted treatment
according to *Stand van de Wetenschap*. These patients will be registered at
NVALT *Dure Geneesmiddelen* Registry.
Baseline examination will include a (PET-) CT scan of the thorax and upper
abdomen for tumor size and textural measurements, blood sampling for ctDNA,
PBMC, plasma, protein and thrombocyte extraction and a tissue sample for IHC,
DNA and RNA analyses.
During treatment blood will be drawn according to a standard protocol (week 2
or 3, 6, 12 and at progression of disease) and CT scan of the thorax and upper
abdomen every six to eight weeks to evaluate tumor response. Tumor biopsy will
be performed before, at 6 weeks (optional) and upon progression of disease
and/or disease recurrence. Origin of the biopsy, i.e. whether it is taken from
the primary tumor or from a metastasis, will be reported. Except for the extra
blood samples and tumor biopsies, all investigations are standard diagnostic
procedures for the evaluation of a NSCLC patient.

Baseline blood sampling, tissue biopsy and (PET-)CT imaging is part of routine
diagnostic workup of non-small cell lung cancer before treatment initiation. As
is follow-up with a CT scan every twelve weeks upon treatment. For the purpose
of this study extra blood will be drawn at regularly planned visits (week 0, 2
or 3, 6, 12 and at progression or recurrence of disease). Two EDTA blood tubes
(20 ml), one serum tube (10 ml) and 1 tube of whole blood (10 ml) will be taken
for PBMC isolation.
The tumor biopsy at baseline and upon disease progression is routine clinical
practice, six weeks after the first PD-1/PD-L1 infusion tumor or start of TKI,
biopsy is optional but recommended and should be discussed with patients.
Biopsies will be taken from the most easily accessible tumor site and will be
performed by means of bronchoscopy, CT or ultrasound guidance biopsy (no
cytology) by an experienced radiologist or pulmonologist.
In this study, tumor and germline DNA will be analyzed using sequencing
techniques. Therefore, there is a small possibility of detection of unsolicited
findings, i.e. germline DNA variants that confer an increased risk of
developing malignancies or other diseases both for the patient and his/her
family. Patients should be informed in case of clinically relevant and
medically actionable genetic alterations. We will consult a medical genetist
for counseling. The analysis of ctDNA / ctRNA is not part of an established
workflow and methods have not been validated yet. Therefore, we will not report
such data to the patient.