Longitudinal exploratory analysis of tumor-specific T-cell immunity and microbiome/calprotectin testing in patients with solid tumors

There is now widespread evidence that tumor-specific T-cell responses can
contribute to the control of solid tumors. As an example, treatment of patients
with the checkpoint inhibitors ipilimumab (an FDA-registered anti-CTLA4
antibody) or nivolumab (an FDA -registered anti-PD1 antibody) has shown a
survival benefit in patients with different metastatic cancers, in particular
melanoma, lung cancer, renal cancer and colorectal cancer. Likewise, treatment
of patients with metastatic cancer with ex-vivo expanded tumor-infiltrating
lymphocytes has been shown to result in clinical responses in our and other
centers.
At the same time, little is known about the presence and longitudinal
development of tumor-specific T-cell immunity upon immunotherapeutic treatment.
Are there biomarkers that can predict the response? Does the breadth or
strength of the therapy-induced T-cell response predict clinical course? Does
reactivity against certain tumor-associated antigens correlate with tumor
regression or with treatment-induced autoimmune disease (e.g. vitiligo in case
of melanoma). Better knowledge on the T-cell responses both in peripheral blood
and at the tumor site is likely to offer leads for early monitoring of
treatment response, a better understanding of the mechanisms underlying the
response and for the rational development of more targeted immunotherapies.
Furthermore, it has been postulated that also other therapeutic strategies that
have been developed or are currently in development, may exert their effect in
part through stimulation of the immune system. For example, the release of
melanoma-associated antigens upon inhibition of BRAF may promote the induction
of T-cell responses against these antigens. At present, not enough data are
available on the relationship between treatment of solid tumors with these
types of drugs and the development of tumor-specific T-cell responses, either
in peripheral blood or at the tumor site. In addition, we will do multiplex
kinase activity profiling for investigation of prediction of responses in
collaboration with PamGene®.
Preliminary reports have suggested the microbiome in faeces may have an impact
on response of melanoma patients to immunotherapy. However, the sample size of
these studies is small. We want to investigate the use of shotgun metagenomic
sequencing to study microbial compositions on a longitudinal basis in a larger
patient cohort undergoing immunotherapy. We will, in conjunction with Vedanta®,
isolate individual bacterial strains from faecal samples, pinpoint their
identities by genetic analysis and determine their biological properties. It
has already been shown that commensal organisms can stimulate CD8 T cells in
vivo. An immediate objective of analysis of the faecal samples is determination
of whether these T cell stimulatory strains are similar to strains associated
with benefit in patients. Furthermore, the composition of the gut microbiome of
cancer patients can be used to predict which subjects are likely to develop
colitis. This will also be investigated by calprotectin testing, which is
already used in inflammatory bowel patents to predict the occurrence of colitis.

Previous studies have shown that the immune system changes when patients age.
For example, the numbers of dendritic cells and CD4+ naive T cells decline
while the pool of terminally-differentiated CD8+ T-cells increases. In
addition, the number of circulating and intratumoral myeloid derived suppressor
cells (MDSC*s) increase. Hence, T-cell function may decrease and lead to
impaired responsiveness to therapies aiming to boost tumor immunity. It is
known that these factors are associated with clinical frailty as identified
with geriatric tests that are performed in a geriatric assessment. However, no
previous studies have investigated the relation between immunological ageing
(so-called immunosenescence), biological ageing and immunotherapy efficacy and
toxicity.

To study the effect of immune modulating therapies, including anti-CTLA4,
anti-PD-1, anti-PD-L1checkpoint inhibitors, and TIL therapy, on the composition
and activation of systemic and local immunity, with an emphasis on the size and
diversity of tumor-specific T-cell populations, in serial blood and tumor
samples, measured by several complimentary immune analyses.

To study additional immune related parameters such as CRP, LDH, human leukocyte
antigen (HLA) typing, autoantibodies, plasma cytokine levels and other factors
in serum/ plasma.

To examine the impact of baseline immune parameters on treatment outcome and
toxicity.

To examine faecal and mucosal samples for analysis of the microbiome together
with calprotectin, serum and peripheral immune cells for predicton of toxicity
(including calprotectin testing for prediction of colitis) and response.

To study the effects of immunotherapy-associated adverse events on quality of
life.

To study the relation between geriatric parameters and immunosenescence in a
subcohort of patients aged 65 years and older

To study the effect of immune modulating therapies on the composition of the B
cell receptor repertoire in peripheral blood

All patients with solid tumors who start treatment with therapies that based on
literature are expected to stimulate the immune system can be enrolled in this
study.

All patients will be informed about this study consisting of two parts: I. To
allow peripheral blood sampling for longitudinal analysis of tumor-reactive
immune responses, and II. To allow tissue collection through serial tumor
biopsies. Patients will be asked to sign an informed consent (ICF) for each
part of the study with a separate signature form. After having signed the ICF,
peripheral blood samples will be drawn prior to, during and after the treatment
and at progression and isolated peripheral blood mononuclear cells (PBMCs) will
be cryopreserved immediately for research purposes. If metastases are easily
accessible and the patient signed the ICF for tumor biopsies, a biopsy will be
taken before treatment, during and after therapy and at progression.

Sampling of blood and tumor tissue before, during and after therapy will be
partly dependent on the type of treatment. The blood sampling of patients on
immune stimulating therapy will be done before baseline (20 ml), at baseline
(100 ml), at 1, and 3 months after initiation of treatment (60 ml per time
point), thereafter 3 monthly (60 ml) and before switching to another immune
stimulating therapy (60 ml; coinciding with response evaluation). The tumor
samples of patients will be collected at start, at 3 months, at the time of
signs of regression and at progression before switching to another therapy.

Faecal samples will be collected at baseline, after 6, 12 and 24 weeks for
microbiome analysis. For calprotectin testing, faecal samples will be collected
at baseline and after 3, 6, 9 and 12 weeks. Part of these samples will be send
to Vedanta®.

The idea behind these time points for follow-up samplings is based on the mode
of action of the drug and the knowledge or expectation of the time to response.
For instance immunotherapy conducted with ipilimumab results in a rather slow
clinical response and therefore longer follow-up time is warranted.

In all patients aged 65 years and older, we will additionally perform a short
geriatric assessment (10-15 minutes). This will take place in addition to an
appointment that is already scheduled, in order to keep the burden for patients
as low as possible. International guidelines advice to perform a geriatric
assessment in all older patients with cancer, so this will likely become
standard of care for all older patients in the near future.
During follow-up, patients will receive short follow-up consults via telephone
after 6 months, 1 year and 2 years.

In patients with colitis > grade 1, we will perform extra biopsies during
standard endoscopy and 2 extra questionnaires. Also 2 extra bloodsamples and
feacal samples will be collected.

No toxicity is expected from drawing blood samples. However, patients may have
discomfort due to taking biopsies or excisions. Biopsies will be taken from
metastases that are easily accessible (mainly subcutaneous and lymph node
metastases) and will in general be performed by means of ultrasonic guidance by
an experienced radiologist or during endoscopy by an experienced
gastroenterologist (only in case of regular endoscopies for colitis); excisions
will be performed by an experienced surgeon. No toxicity is expected from
faecal sample collection.

In a subgroup of patients aged 65 years and older, we will perform a short
geriatric assessment (10-15 minutes). This will take place in addition to an
appointment that is already scheduled, in order to keep the burden for patients
as low as possible. International guidelines advice to perform a geriatric
assessment in all older patients with cancer, so this will likely become
standard of care for all older patients in the near future.
During follow-up, patients will receive short follow-up consults via telephone
after 6 months, 1 year and 2 years.