Cancer panel CZECANCA

The aim of the testing is to identify patients/families with a congenital predisposition to certain types of cancer, to specify the cancer risk for members of families with a positive cancer history and to recommend appropriate prevention in these families. Mutations in the coding regions and adjacent non-coding regions of the genes contained in the CZECANCA panel are detected as clinically indicated. 

The testing is performed using massively parallel sequencing on the Illumina platform (NovaSeq Xplus) from whole-exome sequencing (WES) data using the CZECANCA virtual panel. This panel includes 226 genes associated with hereditary cancers. Minimum sequencing depth of 30x is achieved at >97% of target regions. The obtained data are analysed according to our own bioinformatic evaluation, which includes mapping to the GRCh38 genome reference sequence, variant calling, annotation with the current version of Ensembl and filtering of clinically significant variants according to a number of annotation outputs from population, clinical and prediction databases. Each of the above steps is supplemented by quality control. Intron variants are assessed for the effect on pre-mRNA splicing in the range of +/- 15bp from the exon boundary. Variants in 5' and 3' UTR regions are reported if there is evidence of their pathogenic impact in clinical databases. Results are reported only in samples that have passed bioinformatic quality control (e.g. phred score quality, depth of sequencing, uniformity of coverage). 

Clinical interpretation of genetic variants detected is performed by experts based on the indication for testing, a review of relevant scientific literature, external databases, our internal clinical genetic database and the manual checking of sequencing data in Alamut Visual or IGV. All available evidence of identified variants is classified according to the MGL GENNET Variant Interpretation Procedure based on IACR* standards, ACMG guidelines (PMID: 25741868) and specifying procedures (PMID: 28492532, doi: 10.1136/jmedgenet-2020-107248) and HGVS genetic nomenclature. The clinical significance status of the ClinVar database is periodically checked with the release of a new version of Ensembl and an updated laboratory report for the affected patients is issued when pathogenicity increases (to class 4/5) in the already reported variant. Evaluations are performed according to SOP-MGL-013, and the analysis is focused mainly on indicated genes or group of genes. If a class 4/5 variant is found in one of the genes listed in the Flexibility Record, such a variant is always reported regardless of indication. Only class 4/5 variants are confirmed and reported for BMPR1A and SDHA genes where the sensitivity of the method is reduced due to large homologous pseudogeneous sequences. If necessary, the analysis can be extended to other desired genes contained in the panel. The detection of exon deletions and duplications and major gene rearrangements in ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD51C, RAD51D, STK11, TP53, EPCAM, MLH1, MSH2, MSH6, PMS2, APC, MUTYH, POLD1, POLE and indicated accredited genes in samples meeting the qualitative criteria is performed by analysing NGS data coverage of the target regions of the CZECANCA panel. Confirmation of CNV findings is performed by MLPA analysis, if available.

When genetic testing is appropriate in cancer patients (or their relatives): 

• in the case of a diagnosis of cancer at an unusually young age, 
• in people with different types of cancer,
•  in the case of bilateral tumours in paired organs, 
• in the case of recurrence of the same type of cancer (e.g. breast cancer or colorectal cancer) in the family, 
• in the combination of certain types of tumours in close relatives (breast cancer and ovarian cancer, intestinal and uterine cancer), especially in cases where the manifestation of the disease occurred at a younger age. 

The indication of genetic testing is decided by a clinical geneticist based on an analysis of the patient’s personal and family history, if the so-called ‘indication criteria’ for the testing of any hereditary cancer syndromes, such as hereditary breast and ovarian cancer, Lynch syndrome, familial adenomatous polyposis, Li-Fraumeni syndrome, tuberous sclerosis, etc. 

The indication criteria were developed as a consensus of the professional cancer societies and Society for Medical Genetics and Genomics, J. E. Purkyne Czech Medical Society and were published in the Supplements of Klinická onkologie in 2009, 2012 and 2016 (www.mou.cz). For the testing of hereditary predisposition to cancer by NGS using the CZECANCA cancer panel, the laboratory is granted flexibility, it is an accredited regimen for validation of the procedure for testing a new gene within cancer diagnosis. This regimen includes notification of new versions of testing procedures, monitoring of the results of internal and external quality control and, in the event of identified deficiencies, withdrawal and revision of test results, including close communication with referring physicians.