Peripheral PD-1+CD56+ T-cell frequencies correlate with outcome in stage IV melanoma under PD-1 blockade

Identification of a rare SEPT9 variant in a family with autosomal dominant Charcot-Marie-Tooth disease.

Tumor Mutational Burden and Other Biomarkers Help to Assess the Efficacy of Immunotherapies

Immunotherapy has revolutionized the treatment of many types of cancer by taking advantage of the immune system’s ability to fight cancer cells. In the last few years, a growing number of immunotherapy-based clinical trials were launched to increase success rates further. Specific biomarkers help to stratify patients for the best treatment choice, and also to support the promising development of new immunotherapy combinations.

The center for Dermatooncology at the University hospital Tübingen conducted a prospective biomarker study (Forschner 2019) in cooperation with CeGaT GmbH. This study aimed to identify reliable biomarkers, which predict the patient’s response to anti-CTLA-4 and anti-PD-1 immunotherapy.

Tumor mutational burden (TMB) is a biomarker that has been described as particularly useful for immunotherapy and was thus assessed as part of this study. TMB refers to the number of mutations present in a cancer patients’ tumor and is quantified as mutations per million base pairs (mut/Mbp). Furthermore, repetitive liquid biopsies were collected to determine the concentration of circulating tumor DNA (ctDNA) during treatment. Several studies have shown that ctDNA levels are associated with treatment response and prognosis and could therefore serve as non-invasive predictive markers.

Within this study, we demonstrated that patients with a high TMB (>23.1) showed a significantly better response to therapy and, consequently, a better outcome compared to patients with TMB-low or TMB-intermediate tumors. Similarly, undetectable levels of ctDNA, or a >50% decrease of the cell-free DNA (cfDNA) concentration at the time of first follow-up (3w post treatment initiation) was significantly associated with therapy response and better overall survival.

The results of this study strengthen the increasing importance of the analysis of biomarkers such as TMB, ctDNA, or cfDNA in the prediction and early assessment of the effectiveness of immunotherapies.


CeGaT Research and Pharma Solution offers different methods of TMB analysis to meet the most diverse study requirements


At CeGaT, the TMB score can be evaluated based on different sequencing approaches. The best choice for a specific project depends on the underlaying scientific question and availability of patient material. Typically, the TMB score is assessed through whole-exome sequencing of the tumor tissue, as well as the corresponding normal tissue (e.g. blood sample). By comparing the variants detected in both samples, we can discriminate between tumor specific (somatic) mutations, and mutations present in every tissue of the patient.

However, recent studies have shown that the TMB score can also be effectively estimated if only a panel of genes gets sequenced, provided the sequencing panel covers a genomic region of at least 1,5 Mb (Buchhalter 2018). Thus, for many cases, it is not only sufficient but even more expedient to analyze a selected panel of genes. Therefore, we make use of our CeGaT somatic tumor panel which comprises more than 700 selected, cancer-related genes.

Clinical samples are very precious material and availability is often limited. A comparison of tumor and normal tissue might not always be possible. To meet this challenge, CeGaT uses the solution provided by Illumina, in which TMB score is evaluated based on tumor tissue analysis only: TruSight Oncology 500 (TSO500) comprises the next-generation sequencing based analysis of 523 cancer-relevant genes and their standardized bioinformatic assessment. No normal tissue is required.

For the most comprehensive results, CeGaT’s customers always receive information about the tumor’s microsatellite instability (MSI) in addition to TMB analysis – regardless of whether it is based on TSO500, whole-exome sequencing, or CeGaT somatic tumor panel sequencing. Besides TMB, MSI is another important biomarker for the patient’s response to immunotherapy.

Contact us in order to find out which TMB product is the best choice for your study.


Combine your TMB analysis with the following services offered by CeGaT Research and Pharma Solutions:

  • Detection and sequencing of certain immune subpopulations (e.g. CD4+, CD8+, PD-1+ T cells)
  • Detection of (neo)antigen-specific T cells
  • Microbiome analysis
  • T cell receptor (TCR) sequencing
  • Human leukocyte antigen (HLA) analysis
  • Transcriptome sequencing

Further information can be found here.


Forschner A, Battke F, Hadaschik D, Schulze M, Weißgraeber S, Han CT, Kopp M, Frick M, Klumpp B, Tietze N, Amaral T, Martus P, Sinnberg T, Eigentler T, Keim U, Garbe C, Döcker D, Biskup S. Tumor mutation burden and circulating tumor DNA in combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma – results of a prospective biomarker study.
J Immunother Cancer. 2019 Jul 12;7(1):180. doi: 10.1186/s40425-019-0659-0.

Buchhalter I, Rempel E, Endris V, Allgäuer M, Neumann O, Volckmar AL, Kirchner M, Leichsenring J, Lier A, von Winterfeld M, Penzel R, Christopoulos P, Thomas M, Fröhling S, Schirmacher P, Budczies J, Stenzinger A. Size matters: Dissecting key parameters for panel-based tumor mutational burden analysis. Int J Cancer. 2019. Feb 15; 144(4):848-858. doi: 10.1002/ijc.31878.

Saving a Little Girl‘s Life

An exact diagnosis is the basis for an optimal treatment. Here is a case from our daily work where exome diagnostics saved a little girl’s life. 

Our patient had shown signs of severe illness at an early age. Her parents sought medical assistance when she was about six months old. Examining the baby, the physicians identified a bone marrow developmental defect. But the reasons for the deficiency remained unclear. However, the physicians knew that time was precious, since the little girl‘s older brother already died at the age of seven months, displaying similar symptoms. Facing this, the only option the physicians had was to prepare for bone marrow transplantation. The surgery posed a high risk for the girl‘s life because survival rates for this type of procedure for a baby are far from good, at around 50%. But the physicians also knew that if two siblings show such similar symptoms, there had to be a genetic cause.

Finding the needle in the haystack

To get results as fast as possible and to retain a chance to spare the girl the risky bone marrow transplantation, the treating physician approached CeGaT directly. CeGaT’s task was to analyze the DNA of the girl and both parents. At CeGaT, the DNA from the little girl and both parents was analyzed (so-called “trio exome diagnostics”).

Every human has thousands of genetic alterations and nearly all of them do not cause diseases. Finding the one genetic variant that causes a patient‘s disease is the wellknown “needle in a haystack problem”. This procedure requires a profound understanding of sequencing technology, molecular biology, and human genetics.

Gene analysis reveals a hidden condition

CeGaT was able to identify two variants in our patient, one inherited from each parent, in a gene called TCN2. The protein defined by this gene helps Vitamin B12 enter body cells, such as bone marrow cells. This is essential for bone marrow development. As the girl‘s blood values for vitamin B12 were normal, the physicians had no indication of this condition. In order to compensate for the reduced transfer of vitamin B12 into the bone marrow, the physicians decided to provide her with B12 supplements through medication and postponed the bone marrow transplantation. The treatment worked – the little girl recovered, bone marrow transplantation was not needed. She is developing well:
Today, she is a healthy, happy four-year-old girl.

For the many diseases that are inherited, genetic diagnostics give precise insights into their causes and pathomechanisms. Without genetic testing, the diagnosis very often remains vague and cannot be confirmed. Understanding a disease is the first step in choosing the best therapeutic option and thereby helping patients to get healthy or increase their quality of life drastically. Genetic diagnostics is a cost-efficient diagnostic tool that should be integrated early into the diagnostic plan of many patients – because an exact diagnosis is the basis for optimal treatment.

Case Overview

6 months old girl

Congenital bone marrow failure, recurrent infections, anemia, dystrophy, reduced neutrophil and thrombocyte count

Familial history:
Brother with same symptoms passed away at the age of seven months

Inital planned therapy strategy:
Bone marow transplantation. Chance of survival only approx. 50%

Genetic testing:
Trio exome diagnostics at CeGaT

Homozygous mutation in TCN2 (encodes for vitamin B12 carrier) leading to a defect in vitamin B12 metabolism (despite normal blood values)

No need for bone marrow transplantation. High-dose therapy with vitamin B12. Child can live a normal life

Visit the CeGaT Exome Xtra website

Two mutations in the nicotinic acetylcholine receptor subunit A4 (CHRNA4) in a family with autosomal dominant sleep-related hypermotor epilepsy.