Here you will find more detailed information about liquid biopsies and our ongoing projects.
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Medical research is permanently working on novel, more specific and efficient techniques to diagnose diseases, such as cancer at an early stage. A conventional tissue-based biopsy is still the golden-standard in tumor diagnostics. However, tissue biopsies are always an invasive intervention, harboring the risk of infection or unwanted side effects. Thus, frequent sampling is not feasible. However, a single biopsy will not deliver the required comprehensive data in order to monitor therapy response and tumor progression.
A good alternative is a “liquid biopsy”, which can be gained from all body fluids, such as blood, urine or liquor. A “liquid biopsy” is a minimal-invasive method, harboring less risk concerning side effects and allows serial sampling. Because of these features, liquid biopsies are a promising tool for early tumor diagnostics, especially before a tumor or metastasis is detectable by conventional clinical imaging techniques. Further, “liquid biopsies” enable immediate therapy adjustment, in the case of no or weak therapy response or tumor progression under therapy. These attributes are emphasizing the crucial role of liquid biopsy in personalized oncology.
Our group is aiming at improving the diagnostic value of blood-based “liquid biopsies” in head and neck squamous cell carcinoma (HNSCC). Blood contains many promising tumor markers, such as circulating tumor cells (CTCs), tumor-derived exosomes, miRNAs and circulating tumor DNA (ctDNA). [1, 2] We are using the liquid biopsies for biomarker analysis in the context of clinical trials. We are especially interested in the evaluation of the predictive and prognostic value of circulation cell free tumor DNA (ctDNA) and circulating tumor cells (CTC). For information that is more detailed, please see texts below.
1.Arneth, B., Update on the types and usage of liquid biopsies in the clinical setting: a systematic review. BMC Cancer, 2018. 18(1): p. 527.
2.Nonaka, T. and D.T.W. Wong, Liquid Biopsy in Head and Neck Cancer: Promises and Challenges. J Dent Res, 2018. 97(6): p. 701-708.
What is cell free circulating tumor DNA?
Cell free, circulating DNA (cfDNA), including cell free, circulating tumor DNA (ctDNA) in cancer patients, are non-encapsulated DNA-molecules which are released into the blood circulation either by apoptotic or necrotic normal/tumor cells or due to secretion. Since free, circulating DNA in the bloodstream is enzymatically degraded, for instance by macrophages, DNA fragments at a typical size of approx. 180 base pairs are formed. It was also shown that the concentration of ctDNA and its size is dependent on the tumor entity, tumor size, stage of disease and therapy response.
Due to new techniques for tumor diagnostic, it became more efficient to detect and analyze ctDNA in blood samples. The new methods are powerful highly sensitive tools in personalized oncology, and allow the monitoring of tumor progression and therapy response in real-time. ctDNA analysis may lead to a faster adaption of therapy in case of non- or poor response.
Currently, two projects in our group are focusing the molecular analysis of ctDNA isolated from patient blood samples. The analysis for concordance in periphery (blood) and tumor tissue regarding their mutational burden and profile are the primary objectives of both.
1. Chaudhuri, A.A., et al., Predicting Radiotherapy Responses and Treatment Outcomes Through Analysis of Circulating Tumor DNA. Seminars in radiation oncology, 2015. 25(4): p. 305-312.
cfDNA projects and their aims
1. EXLIQUID Trial
EXLIQUID is a multicenter DKTK Joint-finding study that places particular emphasis on improving personalized therapy selection, as well as monitoring the course of disease for patients with advanced solid tumors. This will be done through the exploitation of liquid biopsies that focus on the analysis of mutational variants and methylation signatures in cell free circulating DNA (ctDNA) obtained from patient derived plasma.
The primary endpoints of this liquid biopsy study are:
- Investigating the concordance of mutational profiles generated by the sequencing of tissue biopsies relative to those derived from ctDNA
- Assessing the significance of kinetic analysis of ctDNA as an early biomarker for determining the appropriateness of molecularly-stratified therapies
- Elucidating the role of ctDNA methylation signatures for the subclassification of tumors
- Determining the sensitivity and specificity of liquid biopsy based analysis for determining disease progression compared to standard imaging techniques.
Patients with advanced cancers that are being treated in the context of the Molecular Tumor Board of the Charité Comprehensive Cancer Center (MTB-CCCC), and therefore present for molecular pathological tumor diagnostics i.e. the development of individualized therapy concepts.
Course of study
Blood samples (2 x 10 ml Streck tubes) are collected from participants at the following time points:
- During the initial visit to the MTB-CCCC general consultation hours for all patients (t0a)
***in the case that a molecularly targeted therapy recommendation is given by the MTB-CCCC***
- Shortly before the start of the molecularly targeted therapy (t0b)
- At each subsequent clinical follow-up appointment (t1, ….tn-1)
- At disease progression (tn)
All plasma samples will be stored in the Charité central biobank (ZeBanc). In addition to the study specific analyses that are to be carried out in the context of EXLIQUID, the liquid biopsy samples that are hereby generated will also serve as an important foundation for future research projects.
2. Establishing a ddPCR assay for detection of Cyclin D1 amplification in cfDNA isolated from patients plasma
In about one third of all head and neck squamous cell carcinoma (HNSCC) patients an amplification of chr. 11q13 is observed. The 11q13 amplicon includes among others the CCND1 gene which encodes for cyclin D1 protein. By upregulating cyclin D1 expression, this amplification induces enhanced cell proliferation resulting in an aggressive tumor growth. Further, it is associated with a high potential to form metastasis, which also correlates with a poor prognosis.
Aims of the project:
We aim to establish a ddPCR based detection method, to detect CCND1 amplified cases based on patient-derived cfDNA samples. After establishing the assay using spiking of CCND1-amplified cell lines into blood samples from healthy donors, plasma samples from different clinical trials in HNSCC which have been collected at different time points of the treatment course will be used to evaluate the diagnostic accuracy of the assay. Aims of the project:
- Establishment of ddPCR assay for detection of CCND1 amplification in cfDNA derived from plasma
- To evaluate whether detection of CCND1 amplifications can be used for prediction of response to standard as well as novel molecular treatment
3. Sequencing of cfDNA and correlating DNA from tumor tissue samples collected in the context of the CeFCID trial
In a previous project, a 327-gene panel for targeted-sequencing was established and used to analyze the mutational profile and to estimate to total tumor mutational burden (TMB) in head and neck squamous cell carcinoma (HNSCC). The panel targets both genes, which are frequently altered in HNSCC as well as driver genes commonly mutated in the majority of solid tumor entities.
In the CeFCID trial the treatment efficacy of EXTREME protocol in combination with Docetaxel and alone was evaluated. In our translational project, cfDNA and the correlating DNA, isolated from tumor FFPE- tissue, will be sequenced by using this HNSCC-specific 327-gene panel in collaboration with the BIH core facility Genomics (Dr. Tomasz Zemojtel).
Aim of the Project:
- determine the degree of concordance between the mutational profile / burden of bulk tumor and plasma ctDNA
What are circulating tumor cells?
It is well known that solid tumors can release malignant cells into the blood circulation. These cells, designated as circulating tumor cells (CTCs) have to undergo phenotypic changes in order to detach from the bulk tumor tissue and invade the bloodstream. The majority of CTCs die, but a small subpopulation survives and can form metastasis at distant niches or reseed at the primary tumor site.  The analysis of CTCs as blood-based tumor biomarker has attracted much attention within the last years. A large number of clinical trials have evaluated the prognostic value of CTCs. These trials consistently showed that enumeration of CTCs in patients with solid tumors can be used to monitor disease progression and therapy response.
The number of CTCs depends on tumor entity and stage of disease, as well as the markers and device used for their detection. Although strong efforts were made to improve the efficiency of CTC detection, it is still challenging to detect the small number of CTCs in the overwhelming background of blood cells.
In our lab, we are using the AMNIS ImageStream®X platform, an imaging flow cytometer, for CTC analysis. This device allows quantitative as well as qualitative analysis of CTCs. We are currently using this platform for the expression analysis of the PD-1/PD-L1 immune checkpoint in CTCs. This checkpoint is frequently used by tumors in order to evade immune surveillance by T-cells and NK cells. Current therapeutic strategies are targeting the PD-1 immune checkpoint by antibodies directed to PD-1 or its ligands PD-L1 and PD-L2, thereby attenuating the immune suppression exerted by tumors. Expression of PD-L1 in tumor tissue has been established as predictive marker for the efficacy of immune checkpoint inhibitors while the predictive role of PD-L1 expression in CTCs remains unclear.
1. Dasgupta, A., A.R. Lim, and C.M. Ghajar, Circulating and disseminated tumor cells: harbingers or initiators of metastasis? Molecular oncology, 2017. 11(1): p. 40-61.
2. Nicolazzo, C., et al., Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab. Sci Rep, 2016. 6: p. 31726.
CTC projects and their aims
1. Evaluation of the predictive value of PD-L1 expressing CTCs
In order to determine the predictive value of PD-L1 expressing CTCs, we analyze patient samples for CTCs by using our established assay. Since various clinical trials are ongoing aiming the investigation of the efficiency of immune checkpoint inhibitors in locally advanced HNSCC, we are able to collect patient blood samples. For this we are collecting serial blood samples before therapy (T0) and under treatment (T1). Using the Amnis ImageStream platform, we determine the absolute numbers of CTCs in 7.5 ml blood and assess their PD-L1/PD-L2 expression. CTC numbers and their PD-L1/PD-L2 expression status will be correlated with outcome of patients.
Aims of the Project:
- Analyze the CTCs for immune checkpoint marker in patient blood samples being under immune checkpoint inhibition
- Evaluate the predictive value of PD-L1 expression in CTCs as early marker for therapy response
- Determine the prognostic value of PD-L1+ CTCs in HNSCC
- Compare the predictive value of PD-L1 expression in CTCs versus tumor tissue (FFPE-tumor samples)
2. Establishment of a CTC score
A further CTC-based project in our lab is the establishment of a “digital CTC Score”. For this purpose, digital droplet PCR, a highly sensitive molecular-biological method, will be used for quantification of tumor-related transcripts in blood samples of HNSCC patients. The diagnostic performance of the digital CTC score will be compared with our previous CTC detection assay based on EGFR transcripts only.
Aim of this project:
- Establish a new method for CTC detection in blood samples of patients with HNSCC
general project aims
- establish a method to isolate cfDNA from patient-derived plasma samples
- quantification of the extracted cfDNA
- analyze the mutation profile of both primary tumor and ctDNA and analyze for any concordance
- establish CTC enrichment protocols
- analyze CTCs for their stem cell properties and their metastasizing potential
- determine therapy response or resistance at an early stage
- Authors:Arneth, B.Journal:BMC Cance Year:2018; Volume:18Issue:(1):Pages:527.
- Authors:Nonaka, T.
Wong, D. T. W.Journal:J Dent Res Year:2018; Volume:97Issue:(6):Pages:701-708.
- Authors:Chaudhuri, A. A.; Binkley, M. S.; Osmundson, E. C.; Alizadeh, A. A.; Diehn, M.
Journal:Semin Radiat Oncol Year:2015; Volume:25Issue:(4):Pages:305-12.
Title:Predicting Radiotherapy Responses and Treatment Outcomes Through Analysis of Circulating Tumor DNA
- Authors:Noorlag, R.; Boeve, K.; Witjes, M. J.; Koole, R.; Peeters, T. L.; Schuuring, E.; Willems, S. M.; van Es, R. J.
Journal:Head Neck Year:2017; Volume:39Issue:(2):Pages:326-333.
Title:Amplification and protein overexpression of cyclin D1: Predictor of occult nodal metastasis in early oral cancer
- Authors:Krebs, M. G.; Hou, J. M.; Ward, T. H.; Blackhall, F. H.; Dive, C.Journal:Ther Adv Med Oncol Year:2010; Volume:2Issue:(6):Pages:351-65.
- Authors:Nicolazzo, C.; Raimondi, C.; Mancini, M.; Caponnetto, S.; Gradilone, A.; Gandini, O.; Mastromartino, M.; Del Bene, G.; Prete, A.; Longo, F.; Cortesi, E.; Gazzaniga, P.
Journal:Sci Rep Year:2016; Volume:6:Pages:31726.
Title:Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab
- Authors:Dasgupta, A.
Lim, A. R.
Ghajar, C. M.Journal:Mol Oncol Year:2017; Volume:11Issue:(1):Pages:40-61.
- Authors:Diaz LA Jr, Bardelli AJournal:Clin Oncol Year:2014; Volume:32Issue:(6):Pages:579-586.
- Authors:Wang Y, Springer S, Mulvey CL, Silliman N, Schaefer J, Sausen M, James N, Rettig EM, Guo T, Pickering CR, Bishop JA, Chung CH, Califano JA, Eisele DW, Fakhry C, Gourin CG, Ha PK, Kang H, Kiess A, Koch WM, Myers JN, Quon H, Richmon JD, Sidransky D, Tufano RP, Westra WH, Bettegowda C, Diaz LA Jr, Papadopoulos N, Kinzler KW, Vogelstein B, Agrawal N
Journal:Sci Transl Med. Year:2015; Volume:7Issue:(293)
Title:Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas