Liquid Biopsy

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:

1. Investigating the concordance of mutational profiles generated by the sequencing of tissue biopsies relative to those derived from ctDNA
2. Assessing the significance of kinetic analysis of ctDNA as an early biomarker for determining the appropriateness of molecularly-stratified therapies
3. Elucidating the role of ctDNA methylation signatures for the subclassification of tumors
4. Determining the sensitivity and specificity of liquid biopsy based analysis for determining disease progression compared to standard imaging techniques.

Inclusion criteria

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:  

1. 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***

1. Shortly before the start of the molecularly targeted therapy (t0b)
2. At each subsequent clinical follow-up appointment (t1, ….tn-1)
3. 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

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