Microsatellite instability (MSI) is one of the most important molecular characteristics of a tumor, which occurs among various tumor types. In this review article, we examine the molecular characteristics of MSI tumors, both sporadic and Lynch-associated. We also overview the risks of developing hereditary forms of cancer and potential mechanisms of tumor development in patients with Lynch syndrome. Additionally, we summarize the results of major clinical studies on the efficacy of immune checkpoint inhibitors for MSI tumors and discuss the predictive role of MSI in the context of chemotherapy and checkpoint inhibitors. Finally, we briefly discuss some of the underlying mechanisms causing therapy resistance in patients treated with immune checkpoint inhibitors.

Next generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved. DNA was extracted from 26 formalin fixed paraffin embedded (FFPE) specimens and processed with the TrueSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) targeting 48 cancer-related genes and sequenced in single run. Sequencing data were comparatively analyzed by several bioinformatics pipelines. Libraries yielded sufficient coverage to detect even low prevalent mutations. We found that the number of FFPE sequence artifacts significantly correlates with pre-normalization concentration of libraries (rank correlation −0.81; p < 1e−10), thus, contributing to sample-specific variant detection cut-offs. Surprisingly, extensive validation of EGFR mutation calls by a combination of aligners and variant callers resulted in identification of two false negatives and one false positive that were due to complexity of underlying genomic change, confirmed by Sanger sequencing. Additionally, the study of the non-EGFR amplicons revealed 33 confirmed unique mutations in 17 genes, with TP53 being the most frequently mutated. Clinical relevance of these finding is discussed. Reporting of entire mutational spectrum revealed by targeted sequencing is questionable, at least until the clinically-driven guidelines on reporting of somatic mutations are established. The standardization of sequencing protocols, especially their data analysis components, requires assay-, disease-, and, in many cases, even sample-specific customization that could be performed only in cooperation with clinicians.

Introduction: Immune checkpoint inhibitors are highly effective in treating various cancers. We analyzed the significance of the KRAS/STK11 co-mutation in relation to the efficacy of immune checkpoint inhibitors in pan-cancer patient cohort. Methods: We analyzed data from open-access research: MSK-IMPACT (molecular profiling data from patients receiving systemic antitumor therapy) and MSK-TMB (molecular profiling data from patients receiving immune checkpoint inhibitors). In both studies, high throughput sequencing was used for molecular profiling. Results: A total of 10,336 patients receiving antitumor therapy (MSK-IMPACT study) and 1661 patients receiving immune checkpoint inhibitors (MSK-TMB study) were included in the analysis. Co-mutation STK11/KRAS was found in 156 (1.5%) and 46 (2.8%) patients in the two studies, respectively. Most patients with the STK11/KRAS co-mutation had non-small cell lung cancer (83% and 85% in the two studies, respectively). Among non-small cell lung cancer patients, the STK11 mutation was associated with a worse outcome for patients receiving systemic antitumor therapy, but not immune checkpoint inhibition therapy (HR for OS 1.90 [95% CI 1.36-2.65] and 1.44 [95% CI 0.88-2.37]). Co-mutation STK11/KRAS was also not associated with patient outcome in any of the studies (HR for OS 0.93 [95% CI 0.56-1.52] and 1.09 [95% CI 0.54-2.19]). High tumor mutational burden was associated with better outcome in the cohort of patients receiving immune checkpoint inhibitors. An analogous analysis among patients in the pan-cancer cohort (excluding patients with non-small cell lung cancer) showed STK11 mutations and high tumor mutational burden have a predictive role for the efficacy of immune checkpoint inhibitors, but not STK11/KRAS co-mutation. Conclusions: Co-mutation STK11/KRAS is common among patients with non-small cell lung cancer and is not an independent predictive marker for the efficacy of immune checkpoint inhibitors. Further studies are required to clarify the role of STK11 mutations in immune checkpoint inhibitor treatment response.

Cystic fibrosis (CF) is one of the most common life-threatening genetic disorders. Around 2000 variants in the CFTR gene have been identified, with some proportion known to be pathogenic and 300 disease-causing mutations have been characterized in detail by CFTR2 database, which complicates its analysis with conventional methods. We conducted next-generation sequencing (NGS) in a cohort of 89 adult patients negative for p.Phe508del homozygosity. Complete clinical and demographic information were available for 84 patients. By combining MLPA with NGS, we identified disease-causing alleles in all the CF patients. Importantly, in 10% of cases, standard bioinformatics pipelines were inefficient in identifying causative mutations. Class IV-V mutations were observed in 38 (45%) cases, predominantly ones with pancreatic sufficient CF disease; rest of the patients had Class I-III mutations. Diabetes was seen only in patients homozygous for class I-III mutations. We found that 12% of the patients were heterozygous for more than two pathogenic CFTR mutations. Two patients were observed with p.[Arg1070Gln, Ser466*] complex allele which was associated with milder pulmonary obstructions (FVC 107 and 109% versus 67%, CI 95%: 63-72%; FEV 90 and 111% versus 47%, CI 95%: 37-48%). For the first time p.[Phe508del, Leu467Phe] complex allele was reported, observed in four patients (5%). NGS can be a more information-gaining technology compared to standard methods. Combined with its equivalent diagnostic performance, it can therefore be implemented in the clinical practice, although careful validation is still required.

Genotyping of cell-free DNA (cfDNA) in plasma samples has the potential to allow for a noninvasive assessment of tumor biology, avoiding the inherent shortcomings of tissue biopsy. Next generation sequencing (NGS), a leading technology for liquid biopsy analysis, continues to be hurdled with several major issues with cfDNA samples, including low cfDNA concentration and high fragmentation. In this study, by employing Ion Torrent PGM semiconductor technology, we performed a comparison between two multi-biomarker amplicon-based NGS panels characterized by a substantial difference in average amplicon length. In course of the analysis of the peripheral blood from 13 diagnostic non-small cell lung cancer patients, equivalence of two panels, in terms of overall diagnostic sensitivity and specificity was shown. A pairwise comparison of the allele frequencies for the same somatic variants obtained from the pairs of panel-specific amplicons, demonstrated an identical analytical sensitivity in range of 140 to 170 bp amplicons in size. Further regression analysis between amplicon length and its coverage, illustrated that NGS sequencing of plasma cfDNA equally tolerates amplicons with lengths in the range of 120 to 170 bp. To increase the sensitivity of mutation detection in cfDNA, we performed a computational analysis of the features associated with genome-wide nucleosome maps, evident from the data on the prevalence of cfDNA fragments of certain sizes and their fragmentation patterns. By leveraging the support vector machine-based machine learning approach, we showed that a combination of nucleosome map associated features with GC content, results in the increased accuracy of prediction of high inter-sample sequencing coverage variation (areas under the receiver operating curve: 0.75, 95% CI: 0.750–0.752 vs. 0.65, 95% CI: 0.63–0.67). Thus, nucleosome-guided fragmentation should be utilized as a guide to design amplicon-based NGS panels for the genotyping of cfDNA samples.