Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Next-generation sequencing (NGS), comprising targeted panels (TP), exome sequencing (ES), and genome sequencing (GS) became robust clinical tools for diagnosing hereditary ataxia (HA). Determining their diagnostic yield (DY) is crucial for optimal clinical decision-making. We conducted a comprehensive systematic literature review on the DY of NGS tests for HA. We searched PubMed and Embase databases for relevant studies between 2016 and 2022 and manually examined reference lists of relevant reviews. Eligible studies described the DY of NGS tests in patients with ataxia as a significant feature. Data from 33 eligible studies showed a median DY of 43% (IQR = 9.5-100%). The median DY for TP and ES was 46% and 41.9%, respectively. Higher DY was associated with specific phenotype selection, such as episodic ataxia at 68.35% and early and late onset of ataxia at 46.4% and 54.4%. Parental consanguinity had a DY of 52.4% (p = 0.009), and the presumed autosomal recessive (AR) inheritance pattern showed 62.5%. There was a difference between the median DY of studies that performed targeted sequencing (tandem repeat expansion, TRE) screening and those that did not (p = 0.047). A weak inverse correlation was found between DY and the extent of previous genetic investigation (rho = - 0.323; p = 0.065). The most common genes were CACNA1A and SACS. DY was higher for presumed AR inheritance pattern, positive family history, and parental consanguinity. ES appears more advantageous due to the inclusion of rare genes that might be excluded in TP.

In the past two decades since the first sequencing of the human genome, clinical genomics has undergone a renaissance with the advent of next generation sequencing (NGS) technologies. Whole genome sequencing is now increasingly available in the clinical setting at a fraction of the cost of the human genome reference sequence, and with turnaround times as short as 7-10days. Clinical genetics-based management has become more tractable with improved payer reimbursement and increased availability of targeted therapies, especially in the oncology space. Precision diagnostics in cancer allow clinicians to assess a patient\'s risk of developing cancers, detect and classify tumors, predict prognoses, select targeted therapies when available, and monitor their patient\'s disease burden longitudinally. This approach ushers in a new era of predictive and preventive oncology care that can be personalized to the genetics of the individual patient, the patient\'s tumor, and their clinical presentation. Within oncology, there are global collaborative efforts including the International Cancer Genome Consortium (ICGC), Pan-Cancer Analysis of Whole Genomes (PCAWG) and The Cancer Genome Atlas (TCGA) that have archived thousands of cancer genomes. In collaboration with databases including but not limited to cancer cataloging variant association with disease risk (ClinVar and others) and drug responses (PharmGKB and others), the study of somatic and germline genomic variation provides a wealth of information on cancer development, evolution, heterogeneity, and treatment response. Although many challenges still exist for defining all possible actionable genomic variants, we have unprecedented opportunity to gain mechanistic and clinical insights into human neoplastic diseases.

Detection of copy number variants from targeted sequencing, including whole-exome sequencing, can be particularly difficult since the break points of the CNV are not always captured. Here we describe DECoN, a software tool which uses changes in read depth to identify CNVs that affect whole exons. It is optimized for clinical use and allows for interactive visualization of CNVs identified.

Molecular profiling of tumor samples has acquired importance in cancer research, but currently also plays an important role in the clinical management of cancer patients. Rapid identification of genomic aberrations improves diagnosis, prognosis and effective therapy selection. This can be attributed mainly to the development of next-generation sequencing (NGS) methods, especially targeted DNA panels. Such panels enable a relatively inexpensive and rapid analysis of various aberrations with clinical impact specific to particular diagnoses. In this review, we discuss the experimental approaches and bioinformatic strategies available for the development of an NGS panel for a reliable analysis of selected biomarkers. Compliance with defined analytical steps is crucial to ensure accurate and reproducible results. In addition, a careful validation procedure has to be performed before the application of NGS targeted assays in routine clinical practice. With more focus on bioinformatics, we emphasize the need for thorough pipeline validation and management in relation to the particular experimental setting as an integral part of the NGS method establishment. A robust and reproducible bioinformatic analysis running on powerful machines is essential for proper detection of genomic variants in clinical settings since distinguishing between experimental noise and real biological variants is fundamental. This review summarizes state-of-the-art bioinformatic solutions for careful detection of the SNV/Indels and CNVs for targeted sequencing resulting in translation of sequencing data into clinically relevant information. Finally, we share our experience with the development of a custom targeted NGS panel for an integrated analysis of biomarkers in lymphoproliferative disorders.

Next generation sequencing (NGS) based technology has contributed enormously to our understanding of the biology of myeloid malignancies including acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Assessment of clinically important mutations by NGS is a powerful tool to define diagnosis, determine prognostic risk, monitor measurable residual disease and uncover predictive mutational markers/therapeutic targets, and is now a routine component in the workup and monitoring of haematological disorders. There are many technical challenges in the design, implementation, analysis and reporting of NGS based results, and expert interpretation is essential. It is vital to distinguish relevant somatic disease associated mutations from those that are known polymorphisms, rare germline variants and clonal haematopoiesis of indeterminate potential (CHIP) associated variants. This review highlights and addresses the technical and biological challenges that should be considered before the implementation of NGS based testing in diagnostic laboratories and seeks to outline the essential and expanding role NGS plays in myeloid malignancies. Broad aspects of NGS panel design and reporting including inherent technological, biological and economic considerations are covered, following which the utility of NGS based testing in AML and MDS are discussed. In current practice, patient care is now strongly shaped by the results of NGS assessment and is considered a vital piece of the puzzle for clinicians as they manage these complex haematological disorders.

Cancer immunotherapy represents a promising and rapidly developing approach for the treatment of oncological diseases. Among the methods of personalized adjuvant immunotherapy, neoantigenic peptide-based drugs have demonstrated substantial efficiency. These drugs are designed to target mutant proteins arising from somatic alterations in the genome of tumor cells and thus stimulate immune response against tumor tissues. The methods of individual screening for potentially immunogenic mutations are mostly based on next-generation exome sequencing of tumor samples, which is a complex and costly procedure for clinical application. Targeted gene sequencing panels limited to a certain set of genes represent a reasonable alternative to WES. Targeted sequencing is also more efficient when there is a low amount of the sample DNA available. We have estimated the potential efficiency of targeted oncological panels in terms of somatic neoantigen profiling in colorectal cancer (colon and rectal adenocarcinoma). The clinical practice of identification of frequent somatic variants does not provide enough data for designing an efficient personalized drug when applied to low and medium mutated cancers such as colorectal cancer. Our analysis of 11 commercially available panels containing different number of genes has shown that neither the larger size of a panel nor its initial customization for colorectal cancer provides a significantly better estimation of an individual somatic mutation profile. The optimal approach is to use the general-purpose medium-sized cancer panels (2300-11200 amplicons and/or 150-600 genes). These panels allow to detect a sufficient number of immunogenic epitopes (>3) per patient for over 30-50% of patients.
Immunoterapevticheskie podkhody k lecheniiu onkologicheskikh zabolevaniĭ iavliaiutsia mnogoobeshchaiushchim stremitel\'no razvivaiushchimsia napravleniem. Odnim iz metodov personalizirovannoĭ ad\"iuvantnoĭ immunoterapii iavliaetsia stimulirovanie immunnogo otveta na mutantnye belki opukholi s pomoshch\'iu preparatov na osnove neoantigennykh peptidov, sinteziruemykh opukholevymi kletkami v rezul\'tate poiavleniia somaticheskikh mutatsiĭ v ikh genome. Tekhnologii skrininga individual\'nykh dlia patsienta i potentsial\'no neoantigennykh mutatsiĭ osnovany na vysokoproizvoditel\'nom sekvenirovanii, chashche vsego opukholevogo ékzoma, chto iavliaetsia slozhnoĭ i dorogostoiashcheĭ protseduroĭ. Vozmozhnym al\'ternativnym resheniem iavliaetsia ispol\'zovanie dlia sekvenirovaniia targetnykh paneleĭ, ogranichennykh izbrannymi genami. Primenenie takikh paneleĭ mozhet byt\' bolee priemlemym i v tekh sluchaiakh, kogda po tem ili inym prichinam ne udaetsia poluchit\' dostatochnoe kolichestvo opukholevoĭ DNK. My otsenili potentsial primeneniia targetnykh paneleĭ onkologicheskoĭ napravlennosti dlia detektsii neoantigennykh somaticheskikh variantov pri kolorektal\'nom rake (adenokartsinoma tolstoĭ i priamoĭ kishki). Primeniaemye v klinicheskoĭ praktike metody detektirovaniia nekotorykh chastykh somaticheskikh variantov ne pozvoliaiut poluchit\' neoantigennyĭ profil\' opukholi, dostatochnyĭ dlia dizaĭna éffektivnogo preparata v sluchae malo- i srednemutirovannykh rakov, k kotorym otnositsia kolorektal\'nyĭ rak. Provedennyĭ nami analiz 11 dostupnykh targetnykh paneleĭ, otlichaiushchikhsia po chislu okhvatyvaemykh imi genov, pokazal, chto kak bol\'shoĭ razmer paneli, tak i spetsializatsiia sostava paneli pod konkretnoe zabolevanie (kolorektal\'nyĭ rak) ne daet printsipial\'nykh preimushchestv pri otsenke individual\'nogo neoantigennogo profilia. Optimal\'nym dlia dannoĭ zadachi iavliaetsia ispol\'zovanie onkopaneleĭ obshchego profilia, soderzhashchikh srednee (2300-11200) kolichestvo amplikonov i/ili okhvatyvaiushchie 150-600 genov. Takie paneli pozvoliaiut detektirovat\' dostatochnoe kolichestvo immunogennykh épitopov (>3) dlia doli patsientov, prevyshaiushcheĭ pokazatel\' v 30-50%.