Next-Generation Sequencing in Tumor Diagnosis and Treatment II

A special issue of Diagnostics (ISSN 2075-4418). This special issue belongs to the section "Pathology and Molecular Diagnostics".

Deadline for manuscript submissions: closed (30 March 2022) | Viewed by 20457

Special Issue Editors

Special Issue Information

Dear Colleagues,

Next-generation sequencing (NGS) allows for the sequencing of multiple genes at a very high depth of coverage. The principle of targeted therapy consists in the application of drugs targeted against well-defined molecules that play a key role in tumor progression and/or survival. Considering the continuous discovery of new molecules as a putative target or that are responsible for treatment resistance mechanisms, single-gene diagnostics is becoming less effective. At present, precision medicine requires multigene characterization. The introduction of NGS to molecular diagnostics has allowed us to combine high analytical sensitivity with multigene analysis. The aim of this Special Issue is to focus on the application of NGS in characterizing molecular alterations in solid tumors for diagnostic, prognostic, or predictive purposes. We invite researchers and authors to submit original research on the application of NGS in the characterization of molecular alterations in solid tumors. Review articles that describe the state-of-the-art on this topic are also encouraged.

Dr. Dario de Biase
Dr. Umberto Malapelle
Prof. Dr. Matteo Fassan
Guest Editor

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Keywords

  • Next-generation sequencing
  • Lung tumors
  • Brain tumors
  • Pancreatic tumors
  • Liquid biopsy
  • Thyroid tumors
  • Colorectal carcinomas
  • Melanoma
  • Molecular techniques

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Published Papers (9 papers)

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Editorial

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4 pages, 197 KiB  
Editorial
Special Issue: Next-Generation Sequencing in Tumor Diagnosis and Treatment II
Diagnostics 2022, 12(8), 2017; https://doi.org/10.3390/diagnostics12082017 - 20 Aug 2022
Viewed by 1155
Abstract
Next-generation sequencing (NGS) allows for the sequencing of multiple genes at a very high depth of coverage [...] Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)

Research

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14 pages, 2164 KiB  
Article
Comprehensive Development and Implementation of Good Laboratory Practice for NGS Based Targeted Panel on Solid Tumor FFPE Tissues in Diagnostics
Diagnostics 2022, 12(5), 1291; https://doi.org/10.3390/diagnostics12051291 - 23 May 2022
Cited by 6 | Viewed by 2646
Abstract
The speed, accuracy, and increasing affordability of next-generation sequencing (NGS) have revolutionized the advent of precision medicine. To date, standardized validation criteria for diagnostic accreditation do not exist due to variability across the multitude of NGS platforms and within NGS processes. In molecular [...] Read more.
The speed, accuracy, and increasing affordability of next-generation sequencing (NGS) have revolutionized the advent of precision medicine. To date, standardized validation criteria for diagnostic accreditation do not exist due to variability across the multitude of NGS platforms and within NGS processes. In molecular diagnostics, it is necessary to ensure that the primary material of the FFPE sample has good quality and optimum quantity for the analysis, otherwise the laborious and expensive NGS test may result in unreliable information. Therefore, stringent quality control of DNA and RNA before, during, and after library preparation is an essential parameter. Considering the various challenges with the FFPE samples, we aimed to set a benchmark in QC metrics that can be utilized by molecular diagnostic laboratories for successful library preparation and high-quality NGS data output. In total, 144 DNA and 103 RNA samples of various cancer types with a maximum storage of 2 years were processed for 52 gene focus panels. During the making of DNA and RNA libraries, extensive QC check parameters were imposed at different checkpoints. The decision tree approach can be set as a benchmark for FFPE samples and as a guide to establishing a good clinical laboratory practice for targeted NGS panels. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)
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11 pages, 1183 KiB  
Article
Molecular Characterization of Pancreatic Ductal Adenocarcinoma Using a Next-Generation Sequencing Custom-Designed Multigene Panel
Diagnostics 2022, 12(5), 1058; https://doi.org/10.3390/diagnostics12051058 - 23 Apr 2022
Cited by 4 | Viewed by 1813
Abstract
Despite the efforts made in the management of PDAC, the 5-year relative survival rate of pancreatic ductal adenocarcinoma (PDAC) still remains very low (10%). To date, precision oncology is far from being ready to be applied in cases of PDAC, although studies exploring [...] Read more.
Despite the efforts made in the management of PDAC, the 5-year relative survival rate of pancreatic ductal adenocarcinoma (PDAC) still remains very low (10%). To date, precision oncology is far from being ready to be applied in cases of PDAC, although studies exploring the molecular and genetic alterations have been conducted, and the genomic landscape of PDAC has been characterized. This study aimed to apply a next-generation sequencing (NGS) laboratory-developed multigene panel to PDAC samples to find molecular alterations that could be associated with histopathological features and clinical outcomes. A total of 68 PDACs were characterized by using a laboratory-developed multigene NGS panel. KRAS and TP53 mutations were the more frequent alterations in 75.0% and 44.6% of cases, respectively. In the majority (58.7%) of specimens, more than one mutation was detected, mainly in KRAS and TP53 genes. KRAS mutation was significantly associated with a shorter time in tumor recurrence compared with KRAS wild-type tumors. Intriguingly, KRAS wild-type cases had a better short-term prognosis despite the lymph node status. In conclusion, our work highlights that the combination of KRAS mutation with the age of the patient and the lymph node status may help in predicting the outcome in PDAC patients. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)
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18 pages, 6694 KiB  
Article
Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer
Diagnostics 2021, 11(12), 2375; https://doi.org/10.3390/diagnostics11122375 - 16 Dec 2021
Cited by 3 | Viewed by 2580
Abstract
The detection of actionable mutations in tumor tissue is a prerequisite for treatment customization in patients with metastatic colorectal cancer (mCRC). Analysis of circulating tumor DNA (ctDNA) for the identification of such mutations in patients’ plasma is an attractive alternative to invasive tissue [...] Read more.
The detection of actionable mutations in tumor tissue is a prerequisite for treatment customization in patients with metastatic colorectal cancer (mCRC). Analysis of circulating tumor DNA (ctDNA) for the identification of such mutations in patients’ plasma is an attractive alternative to invasive tissue biopsies. Despite having the high analytical sensitivity required for ctDNA analysis, digital polymerase chain reaction (dPCR) technologies can only detect a very limited number of hotspot mutations, whilst a broader mutation panel is currently needed for clinical decision making. Recent advances in next-generation sequencing (NGS) have led to high-sensitivity platforms that allow screening of multiple genes at a single assay. Our goal was to develop a small, cost- and time-effective NGS gene panel that could be easily integrated in the day-to-day clinical routine in the management of patients with mCRC. We designed a targeted panel comprising hotspots in six clinically relevant genes (KRAS, NRAS, MET, BRAF, ERBB2 and EGFR) and validated it in a total of 68 samples from 30 patients at diagnosis, first and second disease progression. Results from our NGS panel were compared against plasma testing with BEAMing dPCR regarding the RAS gene status. The overall percent of agreement was 83.6%, with a positive and negative percent agreement of 74.3% and 96.2%, respectively. Further comparison of plasma NGS with standard tissue testing used in the clinic showed an overall percent agreement of 86.7% for RAS status, with a positive and negative percent agreement of 81.2% and 92.8%, respectively. Thus, our study strongly supports the validity and efficiency of an affordable targeted NGS panel for the detection of clinically relevant mutations in patients with mCRC. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)
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14 pages, 1330 KiB  
Article
Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients
Diagnostics 2021, 11(8), 1468; https://doi.org/10.3390/diagnostics11081468 - 13 Aug 2021
Cited by 8 | Viewed by 2643
Abstract
Genetic screening of somatic mutations in circulating free DNA (cfDNA) opens up new opportunities for personalized medicine. In this study, we aim to illustrate the implementation of NGS-based liquid biopsy in clinical practice for the detection of somatic alterations in selected genes. Our [...] Read more.
Genetic screening of somatic mutations in circulating free DNA (cfDNA) opens up new opportunities for personalized medicine. In this study, we aim to illustrate the implementation of NGS-based liquid biopsy in clinical practice for the detection of somatic alterations in selected genes. Our work is particularly relevant for the diagnosis and treatment of NSCLC. Beginning in 2020, we implemented the use of Roche’s Avenio ctDNA expanded panel in our diagnostic routine. In this study, we retrospectively review NGS-based clinical genetic tests performed in our laboratory, focusing on key analytical parameters. Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at >0.5% variant allele frequency (VAF), and high consistency in reproducibility. Since 2020, we performed cfDNA genotyping test in 86 NSCLC patients, and we successfully sequenced 96.5% (83/86) of samples. We observed consistency in sequencing performance based upon sequencing depth and on-target rate. At least one gene variant was identified in 52 samples (63%), and one or more actionable variants were detected in 21 out of 83 (25%) of analysed patients. We demonstrated the feasibility of implementing an NGS-based liquid biopsy assay for routine genetic characterization of metastatic NSCLC patients. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)
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10 pages, 268 KiB  
Article
Feasibility of BRCA1/2 Testing of Formalin-Fixed and Paraffin-Embedded Pancreatic Tumor Samples: A Consecutive Clinical Series
Diagnostics 2021, 11(6), 1046; https://doi.org/10.3390/diagnostics11061046 - 07 Jun 2021
Cited by 5 | Viewed by 2121
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer, with most patients diagnosed at advanced stages. First-line treatment based on a combined chemotherapy (FOLFIRINOX or gemcitabine plus nab-paclitaxel) provides limited benefits. Olaparib, a PARP inhibitor, has been approved as maintenance for PDAC patients harboring [...] Read more.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer, with most patients diagnosed at advanced stages. First-line treatment based on a combined chemotherapy (FOLFIRINOX or gemcitabine plus nab-paclitaxel) provides limited benefits. Olaparib, a PARP inhibitor, has been approved as maintenance for PDAC patients harboring germline BRCA1/2 pathogenic mutations and previously treated with a platinum-based chemotherapy. BRCA1/2 germline testing is recommended, but also somatic mutations could predict responses to PARP inhibitors. Analysis of tumor tissues can detect both germline and somatic mutations and potential resistance alterations. Few data are available about BRCA1/2 testing on pancreatic tumor tissues, which often include limited biological material. We performed BRCA1/2 testing, by an amplicon-based Next Generation Sequencing (NGS) panel, on 37 consecutive PDAC clinical samples: 86.5% of cases were adequate for NGS analysis, with a success rate of 81.2% (median DNA input: 10 nanograms). Three BRCA2 mutations were detected (11.5%). Failed samples were all from tissue macrosections, which had higher fragmented DNA than standard sections, biopsies and fine-needle aspirations, likely due to fixation procedures. BRCA1/2 testing on pancreatic tumor tissues can also be feasible on small biopsies, but more cases must be analyzed to define its role and value in the PDAC diagnostic algorithm. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)

Other

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7 pages, 970 KiB  
Case Report
Utility of Next-Generation Sequencing in the Reconstruction of Clonal Architecture in a Patient with an EGFR Mutated Advanced Non-Small Cell Lung Cancer: A Case Report
Diagnostics 2022, 12(5), 1266; https://doi.org/10.3390/diagnostics12051266 - 19 May 2022
Cited by 2 | Viewed by 1754
Abstract
EGFR tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, targeted therapies impose a strong selective pressure against the coexisting tumor populations that lead to the emergence of resistant clones. Molecular characterization [...] Read more.
EGFR tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, targeted therapies impose a strong selective pressure against the coexisting tumor populations that lead to the emergence of resistant clones. Molecular characterization of the disease is essential for the clinical management of the patient, both at diagnosis and after progression. Next-generation sequencing (NGS) has been established as a technique capable of providing clinically useful molecular profiling of the disease in tissue samples and in non-invasive liquid biopsy samples (LB). Here, we describe a case report of a patient with metastatic NSCLC harboring EGFR mutation who developed two independent resistance mechanisms (EGFR-T790M and TP53 + RB1 mutations) to dacomitinib. Osimertinib given as a second-line treatment eliminated the EGFR-T790M population and simultaneously consolidated the proliferation of the TP53 + RB1 clone that eventually led to the histologic transformation to small-cell lung cancer (SCLC). Comprehensive NGS profiling revealed the presence of the TP53 + RB1 clone in the pretreatment biopsy, while EGFR-T790M was only detected after progression on dacomitinib. Implementation of NGS studies in routine molecular diagnosis of tissue and LB samples provides a more comprehensive view of the clonal architecture of the disease in order to guide therapeutic decision-making. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)
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7 pages, 1563 KiB  
Case Report
Multiple Genetic Alterations as Resistance Mechanism during Second-Line Lorlatinib for Advanced ALK-Rearranged Lung Adenocarcinoma: A Case Report
Diagnostics 2022, 12(3), 682; https://doi.org/10.3390/diagnostics12030682 - 11 Mar 2022
Cited by 2 | Viewed by 1839
Abstract
Second and third-generation ALK-TKI inhibitors have showed better activity and have replaced crizotinib in most of cases of advanced ALK-rearranged lung adenocarcinoma. The emergence of resistance adversely affects also the activity of these newer drugs; in particular, lorlatinib often shows multiple and complex [...] Read more.
Second and third-generation ALK-TKI inhibitors have showed better activity and have replaced crizotinib in most of cases of advanced ALK-rearranged lung adenocarcinoma. The emergence of resistance adversely affects also the activity of these newer drugs; in particular, lorlatinib often shows multiple and complex resistance mechanisms. The case reported here highlights the importance of reassessing the biomolecular profile during the disease course, both by tissutal and liquid biopsy, with the aim of improving the knowledge of these resistance mechanisms, and so identifying new drugs or sequences able to optimize the management of these patients. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)
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9 pages, 4177 KiB  
Case Report
Concomitant Mutations G12D and G13D on the Exon 2 of the KRAS Gene: Two Cases of Women with Colon Adenocarcinoma
Diagnostics 2021, 11(4), 659; https://doi.org/10.3390/diagnostics11040659 - 06 Apr 2021
Cited by 4 | Viewed by 2068
Abstract
Colorectal cancer (CRC) is rapidly increasing representing the second most frequent cause of cancer-related deaths. From a clinical-molecular standpoint the therapeutically management of CRC focuses on main alterations found in the RAS family protein, where single mutations of KRAS are considered both the [...] Read more.
Colorectal cancer (CRC) is rapidly increasing representing the second most frequent cause of cancer-related deaths. From a clinical-molecular standpoint the therapeutically management of CRC focuses on main alterations found in the RAS family protein, where single mutations of KRAS are considered both the hallmark and the target of this tumor. Double and concomitant alterations of KRAS are still far to be interpreted as molecular characteristics which could potentially address different and more personalized treatments for patients. Here, we firstly describe the case of two patients at different stages (pT2N0M0 and pT4cN1cM1) but similarly showing a double concurrent mutations G12D and G13D in the exon 2 of the KRAS gene, normally mutually exclusive. We also evaluated genetic testing of dihydropyrimidine dehydrogenase (DPYD) and microsatellite instability (MSI) by real-time PCR and additional molecular mutations by next generation sequencing (NGS) which resulted coherently to the progression of the disease. Accordingly, we reinterpreted and discuss the clinical history of both cases treated as single mutations of KRAS but similarly progressing towards a metastatic asset. We concluded that double mutations of KRAS cannot be interpreted as univocal genomic alterations and that they could severely impact the clinical outcome in CRC, requiring a tighter monitoring of patients throughout the time. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Tumor Diagnosis and Treatment II)
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