Are circulating hematopoietic stem and progenitor cells a potential biomarker for therapy response and disease progression in patients with squamous cell carcinoma of the head and neck?

Hematopoietic stem cells and their downstream specialized progenitor cells
(HSPCs) are responsible for the renewal of blood cells. Although bone marrow is
the principle resident site of HSPCs, they are also present in blood and other
tissues. Small numbers of HSPCs continually egress from bone marrow into blood,
circulate throughout the body and back to the blood via lymph1. HSPCs cannot
only replace progeny pools, but also act as pivotal primary immune responders
to various pathological conditions. For example, HSPCs can remain in infected
tissue and differentiate to replenish local supplies of immune cells1.

Cancer is associated with profound perturbations in hematopoiesis as well. Wu
et al. reported that the composition of circulating HSPCs was significantly
altered in patients with breast cancer, lung cancer, esophageal cancer,
gastrointestinal cancer, hepatocellular carcinoma, ovarian cancer, and cervical
cancer2. The frequencies of circulating granulocyte-monocyte progenitors
(GMPs), a highly proliferative subset of HSPCs with committed lineage
potential, were increased four- to sevenfold in all types of tumors examined.
Furthermore, the circulating hematopoietic precursors exhibited myeloid bias
with a skew toward granulocytic differentiation in patients with solid tumors.
More importantly, these myeloid precursors were found to be selectively
enriched in tumor tissues and positively correlated with disease progression2.
Adding granulocyte colony-stimulating factor (GM-CSF) and IL-6, substances
produced by many solid tumors, to an in vitro culture of umbilical cord
blood-derived HPSCs promoted GMP expansion and myeloid-derived suppressor cell
(MDSC) differentiation, which recapitulated the in vivo observation in patients
with solid tumors2. MDSCs are key regulators of immune dysfunctions. They can
enhance the *stemness* of cancer cells, facilitate tumor progression,
metastasis and angiogenesis3-6.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer
in the world. Despite significant advances in the treatment modalities
involving surgery, radiotherapy, and concomitant chemoradiotherapy, the 5-year
survival rate remained below 50% for the past 30 years. This poor prognosis is
likely associated with the fact that HNSCC suppresses the host immune system in
several ways7. It has also been reported that the percentage of both
monocytic-MDSCs (M-MDSCs) and granulocytic-MDSCs (G-MDSCs) are increased in the
peripheral blood of patients HNSCC8. Furthermore, the level of circulating
G-MDSCs significantly correlated with the tumor stage and the overall survival
rate of HNSCC patients8. However, the connection between MDSCs and circulating
HSPCs has not been investigated yet for HNSCC.

Cancer and the immune system interact with each other throughout the process of
tumorigenesis, progression and metastasis, which influences the therapy
response and clinical outcome. Since HSPCs possess the most plastic and
proliferative potential, patrol constantly via peripheral blood and act as
primary responders to cancer, investigating the frequency and composition of
circulating HSPCs and their commitments for differentiation will help us
understand their role in immunopathogenesis of HNSCC.

Several studies have suggested that the level of immune inhibitory CD34+
progenitor cells was associated with GM-CSF secretion by the tumor and was
elevated in both the peripheral blood9 and tumors10 of HNSCC patients when
compared to healthy controls. Furthermore, higher levels of GM-CSF and CD34+
cells correlated with more advanced disease and nodal involvement, and
depletion of CD34+ cells in disassociated cancer cell suspension lead to
increased IL-2 production by intratumoral T cells9,10. However, since these
studies were performed before advanced multicolor flow cytometry was developed,
the so-called CD34+ progenitor cells were not phenotypically described in
detail. Actually, researchers only used Lin*CD34+ as definition markers, which
are far from adequate to tell the identity of these cells. Although some
studies showed that under certain in vitro cytokine treatments, these *CD34+
progenitor cells* can differentiate into myeloid cells and even into dendritic
cells (DCs) with antigen-presenting capability9,11, poor population purity has
dampened the credibility of these findings. Hence, the frequency, composition
and clinical value of circulating HSPCs in HNSCC patients need to be reassessed
in more detail.

Hematopoiesis is tightly regulated by a number of circulating hematopoietic
growth factors, which can also be secreted by solid tumors. In a previous study
involving solid tumors other than HNSCC, tumor-secreted GM-CSF and IL-6 could
induce the differentiation of HSPCs into MDSCs2. We therefore hypothesize that
some cytokines produced by HNSCC may influence the hematopoiesis in patients.
Considering the increasing MDSCs in patients with HNSCC, we speculate that the
hematopoiesis might be altered with a myeloid bias and a skew towards
granulocytic differentiation. Thus, we hypothesize that the perturbation of
hematopoiesis by HNSCC can be restored by removing the tumor. In order to
answer this question, we will investigate the hematopoiesis of HNSCC patients
before and after local treatments, which are surgery and/or radiotherapy. Since
systemic chemotherapy may cause myelosuppression or egress of the HSPCs from
bone marrow, the present study excludes the patients receiving concomitant
chemotherapy or other medication known to affect immune system, except for
patients who undergo immunotherapy with immune checkpoint inhibitors prior to
surgery or radiotherapy.

We hypothesize that the cytokines secreted by HNSCC may perturb the
hematopoiesis in patients. We wonder whether this perturbation can be restored
after local treatment of the tumor.

Primary objective: In this pilot study, we aim to prospectively investigate the
frequency, composition and differentiation commitments of circulating and
tumor-resident HSPCs in treatment-naïve HNSCC patients, and compare them with
the results from healthy controls. Next, we aim to assess whether treatment
consisting of surgery, immunotherapy, radiotherapy or a combination of these
modalities could restore the perturbed hematopoiesis by comparing the sample
profile before and after treatment. We will correlate our findings to clinical
parameters such as disease progression and 2-year disease specific and overall
survival data.

Secondary objective: We will set up an in vitro culture study aiming to
investigate the mechanism of altered HSPC composition, frequency and
differentiation in patients with HNSCC when compared to healthy, age-matched
controls. Additionally, we aim to check whether we can experimentally
recapitulate the findings we observed in patients with HNSCC.

This is a single-center, prospective study. 30 patients who meet all relevant
criteria will be included. Patients will be asked to donate 26ml blood for
study purposes at baseline, if possible during a routine blood draw. During a
routine biopsy procedure (in case of primary HNSCC or recurrence during 2 years
follow up), patients will be asked to donate an additional, non-mandatory 5mm
tumor punch biopsy for cytokine detection, flow cytometry and cryopreservation
in order to run future analyses. Hereafter, patients will undergo
standard-of-care treatment consisting of surgery and/or radiotherapy. Ten
patients who receive neoadjuvant immunotherapy as a consequence of
participation in the N16IMC trial will be included. All patients will enter
regular post-treatment follow-up, as deemed appropriate by the Head and Neck
Oncology board or, for immunotherapy patients, as described in the N16IMC
protocol. For the purpose of this study, survival and disease progression data
will be collected for up to two years after treatment.

Five weeks after neoadjuvant immunotherapy and ten to twelve weeks after
surgery or (postoperative) radiotherapy, another 26ml of blood will be drawn.
In the case of recurrent disease after treatment, another venous puncture will
be performed during which additional 26ml will be requested for study purposes.

As a control group, we aim to analyze blood samples from 20 healthy,
age-matched controls. To obtain these, the researchers will approach potential
volunteers from within their patients will be kindly requested to bring a
healthy friend or relative of matched age who is willing to offer a control
blood sample. If this proves unfeasible, the investigators will complement the
control sample pool by asking blood from their own age-matched colleagues,
friends and relatives.

Risk
Participating patients will be asked to donate an additional 26ml of blood
during routine venous puncture performed for diagnostic purposes. No morbidity
is expected from this procedure Five weeks after neoadjuvant immunotherapy, ten
to twelve weeks after surgery and/or radiotherapy and in the case of HNSCC
recurrence, patients will be asked to donate 26ml once again. Wherever
possible, this will be planned to coincide with a venous puncture performed for
diagnostic/follow-up purposes, as to minimize patient discomfort. Since all
necessary patient tumor tissues will be obtained from the surgical specimen in
collaboration with the pathologist, there will be no added burden for the
patient. A non-mandatory, additional biopsy will be requested from patients,
preferably taken during routine biopsy for diagnostic purposes. Apart from
slight pain and discomfort, no additional morbidity is expected from this
biopsy.

Benefit
There are no direct benefits for our participants.