Detection of recirculating tumor-associated tissue macrophages and circulating melanoma cells as a tool for monitoring melanoma patients: a pilot study.

Every year, over 6,000 patients are diagnosed with cutaneous melanoma in The
Netherlands. In the case of a stage IB or higher, a sentinel lymph node
examination is performed to determine the stage of melanoma. After the initial
diagnosis, periodic physical check-ups, and imaging in the case of suspected
metastatic disease, are performed to detect disease progression and response to
(adjuvant) treatment. These regular physical check ups are performed by a
dermatologist or surgeon and include skin and lymph node station examination.
In the case of (suspected) metastasized disease, the medical oncologist is
involved. Visual skin examination is performed with the aid of dermoscopy (a
handheld microscope), which can increase diagnostic accuracy, but still has a
limited specificity, resulting in a high number of benign melanocytic lesions
being unnecessarily excised. To lower this number, a new diagnostic tool is
needed to help distinguish between benign moles and cutaneous melanoma, which
should be fast, minimally invasive, and cost-effective. Such new tool would
help to diagnose and monitor melanoma patients, and could assist in determining
the best treatment for individual patients. Furthermore, there are specific
groups of individuals who have a higher risk of developing melanoma in life,
including people with hereditary melanoma due to mutations in the CDKN2A gene.
In addition, patients with a history of multiple melanomas, or those with
multiple atypical melanocytic nevi are at increased risk of melanoma
development. These high-risk groups undergo regular periodic surveillance,
consisting of once or twice yearly skin examination to investigate the skin for
possible melanomas. These patients could benefit from a newly developed
screening/diagnostic tool, which might assist the physician in examination of
the skin and possible diagnosis of melanoma.
One possible approach to improve the screening, staging, and monitoring could
be the detection of circulating tumor cells (CTCs) in peripheral blood. Some
studies already showed that the presence of CTCs is related to relapse after
treatment and treatment response. However, these CTCs usually have a low
frequency, which implies substantial volumes of peripheral blood need to be
analyzed. In general, the analysis of CTCs requires 8-10 mL of peripheral
blood, in which 0-291 CTCs are detected, varying per CTC method and melanoma
stage. Another possible approach involves the analysis of immune cells, which
continuously perform sensitive intra-tissue scanning. In particular monocytes
and tissue macrophages (TiMas) are interesting, since these cells phagocytize
and digest apoptotic cells and tissue debris, including dying cancer cells.
Recent evidence suggests that at least part of these TiMas can recirculate via
lymph nodes to the bloodstream, where they might be detected and scanned for
their phagolysosomal content. Supporting data for this recirculation concept
has been obtained in glioma patients, where brain-specific proteins were
identified in recirculating TiMas in peripheral blood. If this TiMa
recirculation concept is true, the tissues involved in this recirculation
pathway, being primary tissue (e.g. skin), lymph node, and peripheral blood,
should contain (recirculating) TiMas, containing tissue-specific and/or
tumor-associated peptides in their phagolysosomes. This would imply that
detection of recirculating TiMas and antibody-based scanning of these TiMas for
the presence of tissue-specific and/or tumor-associated peptides has the
potential to be a powerful tool for screening and monitoring of cancer
patients. Considering that melanomas originate from melanocytes, the
to-be-detected tissue-specific and/or tumor-associated peptides in melanoma
patients should be melanocyte specific and/or melanoma-associated. Since
melanocytes are most abundantly present in the skin, and to a much lesser
extent in the retina, inner ear, the meninges and mucosal surfaces, detection
of melanocyte-specific and/or melanoma-associated peptides in peripheral blood
most likely indicates aberrant processes in the skin. Since melanocytes are a
distinct cell type, melanocyte-specific proteins like GP100, MART 1, and
tyrosinase could be suitable candidates to identify melanocyte-specific and/or
melanoma-associated peptides.
In this study, we want to investigate the presence of possible recirculation of
tumor-associated TiMas via lymph nodes to peripheral blood and circulating
melanoma cells in melanoma patients, and evaluate the potential usefulness of
flow cytometric detection of these TiMas and circulating melanoma cells in
lymph nodes and peripheral blood of cutaneous melanoma patients to support
screening for early diagnosis in high-risk groups, support diagnosis and
staging of melanoma, and to monitor patients over time to assess treatment
effectiveness and potential disease progression.

Primary Objective:
- Investigate whether recirculating tumor-associated tissue macrophages
(TiMas), as well as circulating melanoma cells can be detected using high-end
flow cytometry methods in peripheral blood from melanoma patients.

Secondary Objective(s):
- Explore which proteins are present in melanoma cells and identify potential
post-digestion peptides.
- Investigate whether recirculating tumor-associated tissue macrophages
containing post-digestion peptides can be found in the recirculation pathway,
being primary tissue (skin), lymph nodes, and peripheral blood, to proof
recirculation of tissue macrophages.
- Investigate whether flow cytometric detection of recirculating
tumor-associated tissue macrophages containing post-digestion peptides and
circulating melanoma cells might assist in staging methodologies performed by
the pathologist in involved lymph nodes.
- Investigate the *normal background* of melanoma/melanocyte peptide positive
TiMas in blood samples of healthy individuals and patients with non-melanoma
skin diseases, such as inflammatory skin diseases.

This is a prospective cohort study performed in four phases, where Phase 1 is
focused on the identification of melanoma/melanocyte-specific proteins and the
corresponding post digestion peptides for development of the diagnostic tool;
Phase 2 aims at evaluating the recirculation of macrophages from the tissue
(skin) to the lymph nodes and peripheral blood using antibodies raised against
the melanoma/melanocyte-specific protein fragments (as defined in Phase 1), and
will be divided in Phase 2a, 2b and an optional 2c; Phase 3 will assess the
*background levels* of the newly developed diagnostic tool in healthy subjects
and patients with non-melanoma skin diseases.

Blood will be drawn via venipuncture in the arm. This can be an uncomfortable
experience and can cause local pain of hematoma. Otherwise, no risks or
interventions are part of this study. Since all visits are concurrent with
diagnostic visits, participants will not lose much time participating in this
study.