Molecular-biological Diagnostics in Oncology

Malignant neoplasms are characterized by autonomous invasive growth of tumor cells and their ability to form distant metastases. Transformation of a normal cell into a tumor cell usually depends on the presence of a number of mutations in genes of different classes. Ionizing radiation, chemicals or biological agents (oncogenic viruses) are carcinogenic. Inherited mutations in specific genes (eg. tumor suppressor genes) can also cause the development of malignant cancer.

Sporadic and Familial Occurrence of Cancer
Sporadic occurrence, which is not related to genetic predisposition but to the action of external factors, significantly predominates in most tumors. Hereditary tumor syndromes, the development of which is mainly due to hereditary mutations in tumor suppressor genes, include, for example:
 * Familial adenomatous polyposis (FAP) with hereditary APC gene mutations;
 * Lynch syndrome (hereditary nonpolyposis colorectal cancer - HNPCC) with germline mutations in genes MSH2, MLH1, MSH6, PMS1 (mismatch repair genes);
 * hereditary breast and ovarian cancer syndrome with BRCA1 and BRCA2 gene mutations;
 * hereditary retinoblastoma with RB gene mutations;
 * Li-Fraumeni syndrome with p53 gene mutations.

Hereditary tumors often develop at a younger age than sporadic tumors, are often bilateral, and tumor duplications are also typical. A higher number of genes may be responsible for the development of hereditary cancer. For example, predisposition to non-polyposis colorectal cancer occurs in inherited mutations in a number of genes that interfere with DNA repair processes.

You can find more detailed information on the Hereditary cancer syndromes page.

Tissue specificity varies significantly between mutations in individual tumor suppressor genes. For example, germline mutations in the RB gene are particularly associated with a risk (> 90%) of developing retinoblastoma (often bilateral in early childhood); in contrast, families with hereditary mutations of the p53 gene have various tumors: breast tumors account for 28.1% of all malignancies, brain tumors 15.1%, soft tissue sarcomas 12.8%, osteosarcomas 12.3%, adrenal tumors 7.1%, hematological malignancies 3.2%, other tumors 21.4%.

In patients with a familial cancer, genetic testing confirms its hereditary origin. It then provides asymptomatic family members with information about the tumor predisposition (the risk of developing cancer). In people at high risk of cancer (mutation carriers), a preventive screening program can be designed for its early diagnosis.

Analyzed Material
The genetic material analyzed is usually genomic DNA; sometimes RNA is also tested. Genetic material is most often obtained from peripheral non-coagulated blood leukocytes or tumor tissue (obtained immediately after surgery or archived in paraffin blocks). DNA obtained from both tumor and normal tissue is analyzed to demonstrate microsatellite instability (MSI) or loss of heterozygosity (LOH) in tumor cells.

Types of Mutations
You can find more detailed information on the Mutations page.

Mutations can be defined as stable changes in the nucleotide sequence or arrangement of DNA. When germ cells are affected, they are passed on to offspring and can cause hereditary tumors. Somatic mutations are then important in the genesis of sporadic tumors. Depending on the type, mutations can be divided into substitutions, which represent transitions (A ↔ G or C ↔ T) or transversions (C/T ↔ A/G), deletions, insertions, duplications, and extensive genomic deletions and rearrangements. Extensive deletions or chromosomal translocations can lead to microscopically detectable structural changes in chromosomes. Mutations can also be divided according to the effect:
 * Synonymous mutations do not change codon meaning and do not cause amino acid substitutions.
 * Missense mutations change the sense of the codon, resulting in the incorporation of a different amino acid.
 * Nonsense mutations result in a stop codon and therefore terminate translation process.
 * Shift mutations lead to a frameshift and usually to a premature termination of protein synthesis.

Mutation Analysis


In the analysis of mutations, suitable gene fragments are usually first amplified by PCR (or RT-PCR) and their sequence analysis is then used to determine the mutation. When using prescreening assays in mutation analysis, subsequent sequencing serves to confirm and characterize the mutation. A number of methods can be used to prescreen mutations in genes responsible for tumorigenesis. Restriction fragment length polymorphism analysis (RFLP-analysis) can be used to detect known point mutations (interfering with restriction enzyme recognition sequences), which typically occur, for example, in ras oncogenes. Electrophoretic methods, such as DGGE (denaturing gradient gel electrophoresis) and SSCP (single strand conformation polymorphism), are standard techniques that detect changes in the nucleotide sequence of an amplified DNA fragment. In addition to pathogenic mutations, these methods also detect non-pathogenic polymorphisms or sequence variants whose significance may be unclear (some missense mutations).

Using PTT (protein truncation test) it is possible to detect pathogenic mutations leading to premature translation termination (nonsense mutations, shift mutations). Fragments amplified by PCR (RT-PCR) carrying the promoter phage sequence and the sequence necessary to initiate eukaryotic translation (Kozak sequence) serve in this technique as a template for in vitro RNA synthesis and subsequent translation (performed in the presence of a labeled amino acid). The synthesized proteins are further separated by polyacrylamide electrophoresis in the presence of SDS (SDS PAGE) and detected autoradiographically. Depending on the size of the translation products, it is possible to approximate the mutation. The method allows the analysis of long gene fragments (1000–2000 bp) and is used in the analysis of genes that are inactivated in most cases by mutations leading to protein truncation (eg APC gene, BRCA1/BRCA2 genes).

MLPA and aCGH analysis
Rozsáhlé genomové delece a přestavby, které mohou v případě některých tumorsupresorových genů představovat více než 10 % patogenních mutací, se obvykle prokazují pomocí MLPA (multiple ligation-dependent probe amplification), eventuálně aCGH (oligonucleotide array-based comparative genomic hybridization) analýzy. Po ohraničení delece (přibližném určení míst zlomů) se zkrácený genový fragment nesoucí deleci amplifikuje pomocí PCR a delece (genomová přestavba) se charakterizuje na základě sekvenční analýzy.


 * U MLPA-analýzy se změny v počtu genových kopií detekují na základě rozdílů v hybridizaci gen specifických oligonukleotidových sond. Po ukončení hybridizace se sondy specifické vůči jednotlivým exonům spojí DNA ligázou a po následné amplifikaci se produkty PCR odlišné délky rozdělí v genetickém analyzátoru. Vzájemným porovnáním velikostí jednotlivých vrcholů lze provést kvantifikaci všech exonů příslušného genu a určit rozsah genové alterace.


 * U aCGH se určuje počet genových kopií na základě rozdílu v hybridizaci analyzované a referenční DNA. Při použití aCGH specifické pro určitý chromosom s vysokou hustotou oligonukleotidových sond je možné poměrně přesně lokalizovat genovou alteraci (deleci nebo duplikaci).

Loss of Heterozygosity
Demonstration of the loss of heterozygosity of intragenic or nearby microsatellite markers in tumor cells indicates a deletion of the allele of the respective gene. Typically, loss of heterozygosity can be demonstrated in hereditary tumor syndromes caused by germline mutations in tumor suppressor genes. The assay is used to demonstrate the inactivation of both alleles of the tumor suppressor gene in the tumor tissue. Microsatellite instability is typically seen in tumor cells in Lynch syndrome. The different length of microsatellite repetitive sequences in tumor tissue compared to normal tissue is due to a defect in DNA repair processes in hereditary mutations of mismatch repair genes.

Minimal Residual Disease
Jako minimální reziduální nemoc (minimal residual disease – MRD) se označuje stav kompletní klinické remise, kdy u pacienta nelze běžnými metodami (např. cytogenetickými) prokázat minimální počet přítomných nádorových buněk. Použitím molekulárně-biologických metod se podařilo výrazně zvýšit citlivost detekce nádorových buněk. Pomocí PCR nebo RT-PCR je možné detekovat jedinou nádorovou buňku ve vzorku s vysokým počtem (až 106) buněk normálních. Většinou se vysoké citlivosti detekce cílového fragmentu DNA (specifického pro nádorové buňky) dosahuje dvoustupňovou amplifikací (nested PCR).

Detekce MRD pomocí PCR analýz se využívá především u hematologických malignit, eventuálně aCGH analýzy. Například u CML lze zachytit pomocí RT-PCR transkript fúzního genu bcr-abl, který se vyskytuje v nádorových buňkách u více než 90 % onemocnění. Stav „kompletní molekulární remise“, kdy po terapii opakovaně nelze pomocí PCR nebo RT-PCR detekovat nádorové buňky, se považuje za příznivý prognostický faktor. V současnosti kvantitativní PCR metody umožňují sledovat v čase přírůstek či úbytek přežívajících nádorových buněk. {{Netisknout|

Související články

 * Polymerasová řetězová reakce
 * Vyhledávání mutací
 * Hereditární nádorové syndromy
 * Možnosti detekce minimálního reziduálního onemocnění

Reference
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