Biomarker identification of radionuclide therapy-induced radiation responses.

PRRT with 177Lu-DOTATATE is a form of internal radiation treatment for patients
with NETs to reduce tumor growth and stabilize disease. Unfortunately,
objective response rates are limited and fewer than 1% of the patients can
achieve complete response following PRRT. Administering a higher cumulative
dose than currently applied might induce more toxicity in healthy tissues and
probably will be detrimental to patients. To be able to eventually improve this
therapy it is necessary to define its underlying molecular mechanisms, such as
the cellular response of healthy cells to the radiation. A better understanding
of these responses, such as transcriptional- and DNA damage responses, could
contribute to identification of biomarkers for toxicity and/or efficacy
prediction. This was previously shown for external beam radiation in which they
identified radiation responsive genes in ex vivo irradiated blood cells. These
genes were also up- or downregulated following in vivo exposure to total-body
irradiation of patients. Besides identification of novel radiation biomarkers,
this shows that ex vivo data can provide good prediction of in vivo exposures.

PRRT induces different types of DNA damage in both cancer cells and healthy
tissue, of which double-strand breaks (DSBs) are the most cytotoxic.
Radiation-induced foci (RIF) at the site of a DSB and can be visualized using
the biomarkers γ-H2AX and 53BP1. Different studies have shown a good
correlation between radiation dose to the blood and DSB level detection in
peripheral blood mononuclear cells (PBMCs) for various PRRT-like treatments.
Since a blood draw is a relatively non-invasive manner, it is an ideal way to
provide this information .

In PBMCs of 16 patients treated with 177Lu-DOTATATE the average number of RIF
per cell increased in the first hours after treatment and significantly
deceased 5 hours after administration of the radiopharmaceutical. This
illustrates the effect of ionizing radiation on PBMCs after PRRT as a function
of the absorbed dose to the blood and provides a clear understanding of the
correlation between the average number of RIF per cell and the absorbed dose to
the blood. Furthermore, this enables the use of the RIF assay as an in vivo
dosimeter.

Schumann et al. looked into DNA damage and repair in PBMCs of patients treated
with [131I]NaI. They show that DNA repair after internal radiation with
low-dose rates follows similar patterns as ex vivo irradiation with high-dose
rates. Since lutetium-177 and Iodine-131 are both beta-emitters, DSB analysis
in PBMCs seem to be an ideal method to analyze dose response mechanisms with
PRRT as well.

Exposure to ionizing radiation (IR) leads to complex cellular responses
including changes in gene expression which can differ between individuals. It
is shown that in vivo exposure to x-ray showed a transcriptional radiation
induced response after 24 hours for the genes APOBEC3H and FDXR, together with
a strong dose-dependent response in blood irradiated ex vivo. The expression of
the gene FDXR was significantly up-regulated 24 hours after radiotherapy and no
significant difference was seen between in vivo and ex vivo irradiated blood.
This indicates the possibility to identify radiation response biomarkers in
PMBCs.

Altogether, these results confirm that ex vivo irradiation can mimic the in
vivo transcriptional regulation and DNA damage, and these events can be
measured in PBMCs. Our study for PRRT with 177Lu-DOTATATE can teach us more
about the radiation effects and might give us information on biomarkers for
effectiveness and toxicity. Therefore, we will analyze the effects of PRRT with
177Lu-DOTATATE on transcriptional regulation in white blood cells and how is
this regulation related to radiation dose and DNA damage induction.

Next to healthy cells, we will also investigate the tumor cell response to PRRT
using ctDNA. We want to explore if it is possible to detect ctDNA in patients
with NET as currently only one group has reported on this biomarker in NET.
Since limited is known about this. Similar to what has been observed in other
malignancies, we expect that the detection of ctDNA can provide information
about therapeutic response, assessment of tumor burden and disease progression.
ctDNA contains genetic information from tumor cells in multiple regions
(primary tumor and metastases), making it thus more representative than a
single-lesion tissue biopsy to assess the tumor DNA. In addition, blood draw is
less invasive compared to a tissue biopsy. The presence of ctDNA in blood of
NET patients has been associated with poor prognosis in small, heterogeneous
patient populations. In addition, ctDNA analysis is a promising tool to monitor
treatment response. ctDNA presence in the blood is a result of cancer cell
death and can therefore provide information on the radiation response. Indeed,
tumor irradiation led to temporarily amplification of the release of ctDNA in
lung cancer mouse models treated with external beam radiation (7). This
indicates that changes in ctDNA levels early after radiotherapy may predict
treatment outcome and allow clinicians to modify the therapy if needed (14).
Indeed, Azad et al. showed in a group of 45 patients with esophageal cancer who
received chemoradiotherapy that the presence of ctDNA was associated with tumor
progression, formation of distant metastases and shorter disease-specific
survival times.

Various studies have been performed in which potential genetic abbreviations in
the ctDNA can be detected. ctDNA analysis research for NETs so far, however,
has been hampered by the absence of a highly recurrent genetic variation in
this population (such as TP53). The whole genome data generated in NET patients
in the CPCT study were recently analyzed in depth, showing that it is still a
challenge to identify a (set of) markers which can be detected in all NET
subtypes. Alternatively, methylation sequencing could provide an opportunity to
look at epigenetic marks in a larger part of the tumor genome compared to
mutation-based approaches, and these marks in general have a higher penetrance
throughout the tumor. Anticancer treatments can influence these methylation
patterns and could thus be used to monitor treatment response. Liao et al.
investigated 41 patient with hepatocellulair carcinoma (HCC) before and after
surgery. In 8 of those patients, they successfully analyzed presented
tumor-associated mutations in ERT, CTNNB1 and TP53 genes in ctDNA. Patients
with mutations in ctDNA were more likely to relapse. A prospective clinical
trial evaluated ctDNA methylation markers (WIF1 and NPY) in 805 patients with
colorectal carcinoma (CRC) postoperatively. The 2-year disease-free survival
rate was significantly lower in the ctDNA-positive group (64%) than in the
ctDNA-negative group (82%). In 87 patients with breast cancer treated with
neoadjuvant chemotherapy, methylated ctDNA was detected based on
hypermethylation of the RASSF1A promoter. Methylated ctDNA levels were
significantly correlated with the extent of residual tumor burden. Altogether,
next to PBMCs, ctDNA could be a way to monitor PRRT response in a minimal
invasive manner.

This is a pilot study to validate the possibility of determining the effect of
PRRT with 177Lu-DOTATATE on transcriptional regulation and DNA damage
induction in PBMCs and how this is related to the radiation dose.
We will also want to explore if we can detect ctDNA in NET patients to
investigate the effect of PRRT to ctDNA.

This concerns a prospective pilot study in patients.
Twenty subjects with advanced NETs and an indication for PRRT will be
prospectively enrolled following informed consent. For purposes of this study,
all subjects will undergo venous blood sampling at four time points. Blood
samples will undergo isolation of cell-free DNA for ctDNA analysis.

Study subjects will undergo 4 venipunctures for blood collection at different
time-points surrounding the first cycle of PRRT. Those time-points are:
- At baseline.
- 4h hours after the administration of PRRT
- 24 hours after the administration of PRRT
- Before the 2nd cycle of PRRT.
In our routine practice PRRT is given in approximately 8 week cycles.
Study subjects will have completed the study after collection of the 4th sample.

DNA damage and transcriptional profiles in PBMCs will be analyzed and
correlated to the radiation dose found in the blood. Moreover, ctDNA levels and
methylation profiles will be analyzed and correlated to the radiation dose
found in the blood.
For PBMC analysis, blood will be collected at the 4 time points. Cell free
ctDNA will be isolated from blood collected at baseline and before the 2nd
cycle of PRRT. For all analyses, laboratory findings will be correlated to
clinical outcome of the patients.
Patient inclusion and samples collection will take place at the ENETS Center of
Excellence, Erasmus Medical Center, Rotterdam, The Netherlands.

Radioactive dose will be determined by measuring blood samples in the
gamma-counter. Gamma-counter measurements will be performed as technical
triplicate per sample.

DNA damage will be assessed by immunofluorescent stainings and microscopic
detection of γ-H2AX and 53BP1 RIF. RIF numbers of at least 50 cells from at
least 4 fields of view per blood sample will be quantified using an automated
quantification macro in ImageJ.

Transcriptional profiling will be assessed by nanopore sequencing. RNA
isolation, sequencing, analysis and qPCR validation will be done at Radiation
Effects Department of UK Health Security in Oxfordshire, United Kingdom.
Validation of the identified differentially expressed genes will be performed
in triplicate by qPCR. For sequencing, we will use an unique in-house analysis
method using a Snakemake pipeline. RIF and sequencing analysis will be
performed on blinded samples to perform unbiased analy

For the assessment of circulating biomarkers of the tumor cells we will measure
ctDNA levels in the blood before PRRT and 8 weeks after the first cycle. DNA
methylation sequencing method will be used to analyze the ctDNA. The MeD-seq
assay will be used for genome-wide DNA methylation profiling on cell-free DNA
(cfDNA).

The action being done consists of blood samples at four time points during
admission for the PRRT. The burden and risks are both low since no
interventions are done and a blood sample is a minor burden.