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Blood Tests in Tumor Diagnostics – Circulating Tumor Cells (CETC/CTC) and Circulating Tumor DNA (ctDNA)

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Liquid Biopsy refers to the collection and analysis of body fluids such as blood, urine, cerebrospinal fluid and pleural or peritoneal effusions for the purpose of diagnosing or monitoring diseases. This method is gaining increasing importance, particularly in oncology, as malignant tumors release whole cells, cell-free tumor DNA, exosomes and other components into surrounding tissues or the bloodstream.

Tumor cells (CETC/CTC) and tumor DNA (ctDNA) can circulate in the blood and their analysis can provide valuable information about the tumor and its activity - without the need for an invasive tissue extraction as required in a traditional biopsy. From a pathological perspective, the term "liquid biopsy" is somewhat misleading when referring to ctDNA detection, as this involves purely molecular-analytical methods rather than a biopsy in the classical pathological sense.

What are circulating tumor cells and circulating tumor DNA?

Circulating tumor cells (CETC/CTC) are intact tumor cells that have detached from a tumor and entered the bloodstream. As complete cells, they provide important information about the cellular components of the primary tumor and can be further analyzed in the lab. However, they are typically present in very low numbers in the blood and are therefore difficult to detect. Detection rates vary depending on the method used. A special subgroup of these are circulating tumor cells with stem cell-like properties, known as circulating cancer stem cells (cCSCs). These play a crucial role in metastasis and disease progression, as they can differentiate into various cell types and may cause relapse even years later.

Circulating tumor DNA (ctDNA) refers to DNA fragments of varying lengths that originate from tumor cells and can be detected in the blood. When tumor cells - either within the tumor or those that have already detached - die, their DNA is released into the bloodstream. The genetic alterations of the tumor may be present in these fragments. For ctDNA to be specifically detected and monitored, the relevant mutations must first have been identified in the primary tumor. ctDNA is part of the total cell-free DNA (cfDNA) found in blood which largely derives from the breakdown of normal body cells, especially blood cells. Since the proportion of ctDNA within the total cfDNA is very low, highly sensitive molecular analysis methods are required for its reliable detection.

How are circulating tumor cells and circulating tumor DNA detected?

Detection is done via a minimally invasive blood draw. Depending on the method used, the blood sample is collected in tubes with fixative (CellSearch™ test for CTC), in EDTA blood collection tubes (maintrac® method for CETC/CTC) or in specialized stabilization tubes (for ctDNA) and then analyzed in the laboratory.

Circulating tumor cells (CETC/CTC) are detected based on specific surface markers, such as the EpCAM antigen - a cell adhesion molecule expressed by epithelial carcinomas. In the CellSearch™ method, tumor cells are enriched using magnetic antibodies targeting EpCAM, followed by magnetic separation. The maintrac® method labels tumor cells directly with a fluorescence-conjugated EpCAM antibody. thereby avoiding tumor cell loss. An additional dye is used to distinguish viable from non-viable tumor cells. The labeled cells are then automatically identified and counted using a fluorescence microscope. Since neither fixation nor filtration is required, the tumor cells remain intact and can be further characterized both cytochemically and functionally. By analyzing serial blood samples, changes in tumor cell numbers can be monitored in real time over the course of the disease.

To detect circulating tumor DNA (ctDNA), either serum or plasma is obtained from the blood sample which contains the cell-free DNA (cfDNA). The next step involves targeted detection of tumor-specific genetic alterations that distinguish ctDNA from normal cfDNA. Common methods for this include molecular biology techniques such as polymerase chain reaction (PCR) or next-generation sequencing (NGS). These techniques enable the identification of both common and individual mutations within the tumor genome. Since ctDNA is present in the bloodstream only in very small quantities, highly sensitive methods are required for reliable detection. When a tumor biopsy is not feasible or a recurrence is suspected after treatment, the detection of ctDNA can be considered. Moreover, the analysis of ctDNA allows for the identification of newly aquired genetic alterations that may have emerged during therapy - such as EGFR mutations in lung cancer - and thus supports the targeted adaption of treatment strategies to more effective therapeutic options.

Opportunitites and Limitations in comparison

Both CETC/CTC and ctDNA analyses offer valuable insights into the progression of cancer and enable more personalized therapy planning, close monitoring of disease progression and early detection of relapses.

Circulating tumor cells		Circulating tumor DNA
CETC/CTC maintrac®	CTC CellSearch™
Opportunitites: Independent of genetic mutations - not dependent on specific mutations or genetic microheterogeneity Scientifically validated - the methods are supported by extensive research and clinical studies		Opportunitites: Enables access to genetic information about the tumor Allows real-time monitoring of genetic changes during therapy, e.g. resistance mutations High specificity in detecting recurrences Primarily applicable in the metastatic stage
Multivalent biomarker:Enables a wide range of tests directly on intact, viable tumor cells, e.g. cell count, drug sensitivity testing, tumor cell characterization High sensitivity for early detection of recurrence, even in early stages Assessment of aggressiveness and metastatic risk via stemtrac® (circulating cancer stem cells)	Prognostic value for recurrence detection Particularly useful in metastatic tumors FDA-approved method for the quantitative detection of circulating tumor cells
*Limitations:* Low specificity In poorly differentiated tumors or during epithelial-mesenchymal transition (EMT), loss of surface markers can occur making detection more difficult Heterogeneously standardized CTC analysis techniques Technically complex and labor-intenisve sample processing as cells are present in low numbers in blood		*Limitations:* Requires prior knowledge of the relevant mutations in the primary tumor for targeted analysis Provides no cellular information - functional testing is not possible Very low concentration in early tumor stages with risk of false-negative results due to low ctDNA release Dilution by cfDNA during increased cell turnover Requires high technical precision and expertise

Clinical significance of circulating tumor cells and circulating tumor DNA

The clinical relevance of circulating tumor cells (CTCs/CETCs) and circulating tumor DNA (ctDNA) is steadily increasing in modern oncology. Both liquid biopsy components provide vital information about tumor dynamics and enable non-invasive, real-time monitoring of cancer progression. While CETCs/CTCs as intact cells offer valuable insights into the cellular properties and functional characteristics of the tumor (e.g. through immunophenotyping or drug testing), ctDNA provides a base-specific molecular-genetic perspective on tumor mutations.

Numerous clinical studies have confirmed the benefits of both approaches. CETC/CTC analysis has proven to be a valuable predictor of metastasis risk and overall survival. At the same time, ctDNA analysis is being successfully used to detect therapy-associated mutations and to monitor therapy resistance. Despite remaining methodological challenges, it is clear that liquid biopsy methods are gaining increasing importance in clinical practice. With further expansion of clinical evidence and ongoing technological advancements, both CETC/CTC and ctDNA analyses are expected to become integral components of personalized treatment regimens - serving as key technologies in modern precision oncology.

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