E-cadherin variants associated with oral facial clefts trigger aberrant cell motility in a REG1A-dependent manner.

BACKGROUND: Germline mutations of E-cadherin contribute to hereditary diffuse gastric cancer (HDGC) and congenital malformations, such as oral facial clefts (OFC). However, the molecular mechanisms through which E-cadherin loss-of-function triggers distinct clinical outcomes remain unknown. We postulate that E-cadherin-mediated disorders result from abnormal interactions with the extracellular matrix and consequent aberrant intracellular signalling, affecting the coordination of cell migration. METHODS: Herein, we developed in vivo and in vitro models of E-cadherin mutants associated with either OFC or HDGC. Using a Drosophila approach, we addressed the impact of the different variants in cell morphology and migration ability. By combining gap closure migration assays and time-lapse microscopy, we further investigated the migration pattern of cells expressing OFC or HDGC variants. The adhesion profile of the variants was evaluated using high-throughput ECM arrays, whereas RNA sequencing technology was explored for identification of genes involved in aberrant cell motility. RESULTS: We have demonstrated that cells expressing OFC variants exhibit an excessive motility performance and irregular leading edges, which prevent the coordinated movement of the epithelial monolayer. Importantly, we found that OFC variants promote cell adhesion to a wider variety of extracellular matrices than HDGC variants, suggesting higher plasticity in response to different microenvironments. We unveiled a distinct transcriptomic profile in the OFC setting and pinpointed REG1A as a putative regulator of this outcome. Consistent with this, specific RNAi-mediated inhibition of REG1A shifted the migration pattern of OFC expressing cells, leading to slower wound closure with coordinated leading edges. CONCLUSIONS: We provide evidence that E-cadherin variants associated with OFC activate aberrant signalling pathways that support dynamic rearrangements of cells towards improved adaptability to the microenvironment. This proficiency results in abnormal tissue shaping and movement, possibly underlying the development of orofacial malformations.

Next-Generation Sequencing of Breast Cancer in the Neoadjuvant Setting.

INTRODUCTION: Many patients with locally advanced breast cancer are proposed to neoadjuvant chemotherapy (NAT) before surgery. Only some of them achieve a pathological complete response (pCR). The determination of gene somatic alterations using next-generation sequencing (NGS) in the non-pCR tumors is important, in order to identify potential opportunities of treatment for the patients, if targeted therapies are available. METHODS: Breast cancer tissue samples of 31 patients, collected before NAT, were analyzed by NGS using the Oncomine™ Comprehensive Assay Plus (OCA-Plus) panel. RESULTS: Twelve patients achieved pCR after NAT. ERBB2 gene alterations were the most frequent in this cohort of pCR patients, followed by BRCA 1 and 2, MYC, TP53, PIK3CA, and MET alterations. Tumors that did not achieve a pCR were mainly triple negative. In this subgroup some BRCA 1 and 2 and PIK3CA gene alterations were identified, as well as TP53 mutations. The NGS panel employed in this study also allowed for the determination of tumor mutation burden (TMB). CONCLUSION: This study showcases the significance of employing comprehensive genomic testing in breast cancer cases, primarily due to the scarcity of specific target assays. The detection of somatic mutations, coupled with the availability of targeted therapies, holds promise as a potential therapeutic avenue to enhance tumor response rates during NAT, or as a complementary treatment following surgery. Moreover, evaluating the TMB in non-pCR samples could serve as a valuable criterion for selecting patients suitable for immunotherapy. Further exploration through clinical trials is imperative to investigate these prospects.

European Real-World Assessment of the Clinical Validity of a CE-IVD Panel for Ultra-Fast Next-Generation Sequencing in Solid Tumors.

Molecular profiling of solid tumors facilitates personalized, targeted therapeutic interventions. The ability to perform next-generation sequencing (NGS), especially from small tissue samples, in a short turnaround time (TAT) is essential to providing results that enable rapid clinical decisions. This multicenter study evaluated the performance of a CE in vitro diagnostic (IVD) assay, the Oncomine Dx Express Test, on the Ion Torrent Genexus System for detecting DNA and RNA variants in solid tumors. Eighty-two archived formalin-fixed paraffin embedded (FFPE) tissue samples from lung, colorectal, central nervous system, melanoma, breast, gastric, thyroid, and soft tissue cancers were used to assess the presence of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variations (CNVs), gene fusions, and splice variants. These clinical samples were previously characterized at the various academic centers using orthogonal methods. The Oncomine Dx Express Test showed high performance with 100% concordance with previous characterization for SNVs, indels, CNVs, gene fusions, and splice variants. SNVs and indels with allele frequencies as low as 5% were correctly identified. The test detected all the expected ALK, RET, NTRK1, and ROS1 fusion isoforms and MET exon 14-skipping splice variants. The average TAT from extracted nucleic acids to the final variant report was 18.3 h. The Oncomine Dx Express Test in combination with the Ion Torrent Genexus System is a CE-IVD-compliant, performant, and multicenter reproducible method for NGS detection of actionable biomarkers from a range of tumor samples, providing results in a short TAT that could support timely decision- making for targeted therapeutic interventions.

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression.

OBJECTIVE: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. DESIGN: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). RESULTS: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. CONCLUSION: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.

European Registry on Helicobacter pylori management (Hp-EuReg): patterns and trends in first-line empirical eradication prescription and outcomes of 5 years and 21 533 patients.

OBJECTIVE: The best approach for Helicobacter pylori management remains unclear. An audit process is essential to ensure clinical practice is aligned with best standards of care. DESIGN: International multicentre prospective non-interventional registry starting in 2013 aimed to evaluate the decisions and outcomes in H. pylori management by European gastroenterologists. Patients were registered in an e-CRF by AEG-REDCap. Variables included demographics, previous eradication attempts, prescribed treatment, adverse events and outcomes. Data monitoring was performed to ensure data quality. Time-trend and geographical analyses were performed. RESULTS: 30 394 patients from 27 European countries were evaluated and 21 533 (78%) first-line empirical H. pylori treatments were included for analysis. Pretreatment resistance rates were 23% to clarithromycin, 32% to metronidazole and 13% to both. Triple therapy with amoxicillin and clarithromycin was most commonly prescribed (39%), achieving 81.5% modified intention-to-treat eradication rate. Over 90% eradication was obtained only with 10-day bismuth quadruple or 14-day concomitant treatments. Longer treatment duration, higher acid inhibition and compliance were associated with higher eradication rates. Time-trend analysis showed a region-dependent shift in prescriptions including abandoning triple therapies, using higher acid-inhibition and longer treatments, which was associated with an overall effectiveness increase (84%-90%). CONCLUSION: Management of H. pylori infection by European gastroenterologists is heterogeneous, suboptimal and discrepant with current recommendations. Only quadruple therapies lasting at least 10 days are able to achieve over 90% eradication rates. European recommendations are being slowly and heterogeneously incorporated into routine clinical practice, which was associated with a corresponding increase in effectiveness.

Adverse Event Profile During the Treatment of Helicobacter pylori: A Real-World Experience of 22,000 Patients From the European Registry on H. pylori Management (Hp-EuReg).

INTRODUCTION: The safety of Helicobacter pylori eradication treatments and to what extent adverse events (AEs) influence therapeutic compliance in clinical practice are hardly known. Our aim was to assess the frequency, type, intensity, and duration of AEs, and their impact on compliance, for the most frequently used treatments in the "European Registry on Helicobacter pylori management." METHODS: Systematic prospective noninterventional registry of the clinical practice of European gastroenterologists (27 countries, 300 investigators) on the management of H. pylori infection in routine clinical practice. All prescribed eradication treatments and their corresponding safety profile were recorded. AEs were classified depending on the intensity of symptoms as mild/moderate/severe and as serious AEs. All data were subject to quality control. RESULTS: The different treatments prescribed to 22,492 patients caused at least 1 AE in 23% of the cases; the classic bismuth-based quadruple therapy was the worst tolerated (37% of AEs). Taste disturbance (7%), diarrhea (7%), nausea (6%), and abdominal pain (3%) were the most frequent AEs. The majority of AEs were mild (57%), 6% were severe, and only 0.08% were serious, with an average duration of 7 days. The treatment compliance rate was 97%. Only 1.3% of the patients discontinued treatment due to AEs. Longer treatment durations were significantly associated with a higher incidence of AEs in standard triple, concomitant, bismuth quadruple, and levofloxacin triple or quadruple therapies. DISCUSSION: Helicobacter pylori eradication treatment frequently induces AEs, although they are usually mild and of limited duration. Their appearance does not interfere significantly with treatment compliance.

The value of cell-free circulating tumour DNA profiling in advanced non-small cell lung cancer (NSCLC) management.

Liquid biopsy (LB) has boosted a remarkable change in the management of cancer patients by contributing to tumour genomic profiling. Plasma circulating cell-free tumour DNA (ctDNA) is the most widely searched tumour-related element for clinical application. Specifically, for patients with lung cancer, LB has revealed valuable to detect the diversity of targetable genomic alterations and to detect and monitor the emergence of resistance mechanisms. Furthermore, its non-invasive nature helps to overcome the difficulty in obtaining tissue samples, offering a comprehensive view about tumour diversity. However, the use of the LB to support diagnostic and therapeutic decisions still needs further clarification. In this sense, this review aims to provide a critical view of the clinical importance of plasma ctDNA analysis, the most widely applied LB, and its limitations while anticipating concepts that will intersect the present and future of LB in non-small cell lung cancer patients.

The Adaptive Immune Landscape of the Colorectal Adenoma-Carcinoma Sequence.

BACKGROUND: The tumor immune microenvironment exerts a pivotal influence in tumor initiation and progression. The aim of this study was to analyze the immune context of sporadic and familial adenomatous polyposis (FAP) lesions along the colorectal adenoma-carcinoma sequence (ACS). METHODS: We analyzed immune cell counts (CD3+, CD4+, CD8+, Foxp3+, and CD57+), tumor mutation burden (TMB), MHC-I expression and PD-L1 expression of 59 FAP and 74 sporadic colorectal lesions, encompassing adenomas with low-grade dysplasia (LGD) (30 FAP; 30 sporadic), adenomas with high-grade dysplasia (22 FAP; 30 sporadic), and invasive adenocarcinomas (7 FAP; 14 sporadic). RESULTS: The sporadic colorectal ACS was characterized by (1) a stepwise decrease in immune cell counts, (2) an increase in TMB and MHC-I expression, and (3) a lower PD-L1 expression. In FAP lesions, we observed the same patterns, except for an increase in TMB along the ACS. FAP LGD lesions harbored lower Foxp3+ T cell counts than sporadic LGD lesions. A decrease in PD-L1 expression occurred earlier in FAP lesions compared to sporadic ones. CONCLUSIONS: The colorectal ACS is characterized by a progressive loss of adaptive immune infiltrate and by the establishment of a progressively immune cold microenvironment. These changes do not appear to be related with the loss of immunogenicity of tumor cells, or to the onset of an immunosuppressive tumor microenvironment.

Review: Gastric cancer: Basic aspects.

Gastric cancer is still one of the most prevalent and deadliest cancers in the world. Although our knowledge about the disease has progressed extraordinarily, this has not been accompanied by our capacity to effectively treat the disease. In the last years, immunotherapy made its way into the cancer field and was responsible for major changes in the treatment success rates for several cancer types. Although gastric cancer was not among the first successful targets of this type of therapy, the relationship between this type of cancer, immunosurveillance and immunotherapy is now being actively researched. In this article, we review the literature of the past year regarding the relationship between gastric cancer, its immune microenvironment and response to immunotherapy. Published data indicate that the immune microenvironment influences the clinical behaviour of gastric cancer, and is correlated with its histologic and molecular subtypes with an emphasis on the microsatellite- and EBV-positive tumour subgroups. Although the literature regarding response to immunotherapy is scarce, there is good evidence that patient stratification for immunotherapy is going to become a reality in gastric cancer.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

New massive parallel sequencing approach improves the genetic characterization of congenital myopathies.

Congenital myopathies (CMs) are a heterogeneous group of muscle diseases characterized by hypotonia, delayed motor skills and muscle weakness with onset during the first years of life. The diagnostic workup of CM is highly dependent on the interpretation of the muscle histology, where typical pathognomonic findings are suggestive of a CM but are not necessarily gene specific. Over 20 loci have been linked to these myopathies, including three exceptionally large genes (TTN, NEB and RYR1), which are a challenge for molecular diagnosis. We developed a new approach using massive parallel sequencing (MPS) technology to simultaneously analyze 20 genes linked to CMs. Assay design was based on the Ion AmpliSeq strategy and sequencing runs were performed on an Ion PGM system. A total of 12 patients were analyzed in this study. Among the 2534 variants detected, 14 pathogenic mutations were successfully identified in the DNM2, NEB, RYR1, SEPN1 and TTN genes. Most of these had not been documented and/or fully characterized, hereby contributing to expand the CM mutational spectrum. The utility of this approach was demonstrated by the identification of mutations in 70% of the patients included in this study, which is relevant for CMs especially considering its wide phenotypic and genetic heterogeneity.

Pathogenesis of Gastric Cancer.

Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. GC is a heterogeneous disease and the endpoint of a long multistep process largely influenced by Helicobacter pylori infection, genetic susceptibility, and environmental factors. In a subset of GC cases, infection with the Epstein-Barr virus (EBV) may also be involved. The development of GC is the consequence of the accumulation of multiple epi/genetic changes during the patient's lifetime that will result in oncogenic activation and/or tumor suppressor pathways' inactivation. This review will focus on the most recent updates on the characterization of the molecular phenotypes of sporadic and hereditary GC. This article will also update the most recent findings on the relationship between H. pylori infection and stem cells at the origin of GC. The understanding of the molecular genetics underlying gastric carcinogenesis is of paramount importance to identify novel potential targets for the development of screening and prognostic markers that can be clinically valuable for the management of GC patients and for the design of clinical trials.

Genetic variants in the IL1A gene region contribute to intestinal-type gastric carcinoma susceptibility in European populations.

The most studied genetic susceptibility factors involved in gastric carcinoma (GC) risk are polymorphisms in the inflammation-linked genes interleukin 1 (IL1) B and IL1RN. Despite the evidence pointing to the IL1 region, definite functional variants reproducible across populations of different genetic background have not been discovered so far. A high density linkage disequilibrium (LD) map of the IL1 gene cluster was established using HapMap to identify haplotype tagSNPs. Eighty-seven SNPs were genotyped in a Portuguese case-control study (358 cases, 1,485 controls) for the discovery analysis. A replication study, including a subset of those tagSNPs (43), was performed in an independent analysis (EPIC-EurGast) containing individuals from 10 European countries (365 cases, 1284 controls). Single SNP and haplotype block associations were determined for GC overall and anatomopathological subtypes. The most robust association was observed for SNP rs17042407, 16Kb upstream of the IL1A gene. Although several other SNP associations were observed, only the inverse association of rs17042407 allele C with GC of the intestinal type was observed in both studies, retaining significance after multiple testing correction (p = 0.0042) in the combined analysis. The haplotype analysis of the IL1A LD block in the combined dataset revealed the association between a common haplotype carrying the rs17042407 variant and GC, particularly of the intestinal type (p = 3.1 × 10(-5) ) and non cardia localisation (p = 4.6 × 10(-3) ). These results confirm the association of IL1 gene variants with GC and reveal a novel SNP and haplotypes in the IL1A region associated with intestinal type GC in European populations.

E-cadherin impairment increases cell survival through Notch-dependent upregulation of Bcl-2.

The role of E-cadherin in tumorigenesis has been attributed to its ability to suppress invasion and metastization. However, E-cadherin impairment may have a wider impact on tumour development. We have previously shown that overexpression of mutant human E-cadherin in Drosophila produces a phenotype characteristic of downregulated Notch. Hence, we hypothesized that Notch signalling may be influenced by E-cadherin and may mediate tumour development associated with E-cadherin deficiency. De novo expression of wild-type E-cadherin in two cellular models led to a significant decrease in the activity of the Notch pathway. In contrast, the ability to inhibit Notch-1 signalling was lost in cells transfected with mutant forms of E-cadherin. Increased Notch-1 activity in E-cadherin-deficient cells correlated with increased expression of Bcl-2, and increased resistance to apoptotic stimuli. After Notch-1 inhibition, E-cadherin-deficient cells were re-sensitized to apoptosis in a similar degree to wild-type E-cadherin cells. We also show that Notch-inhibiting drugs are able to significantly inhibit the growth of E-cadherin-deficient cells xenografted into nude mice. This effect was comparable with the one observed in animals treated with the chemotherapeutic agent taxol, a chemical inducer of cell death. In conclusion, our results show that aberrant Notch-1 activation, Bcl-2 overexpression and increased cell survival are likely to play a crucial role in neoplastic transformation associated with E-cadherin impairment. These findings highlight the possibility of new targeted therapeutical strategies for the treatment of tumours associated with E-cadherin inactivation.

Detection of common and less frequent EGFR mutations in cytological samples of lung cancer.

OBJECTIVE: Lung cancer represents the leading cause of cancer death. EGFR mutations, detected in 10-40% of lung adenocarcinomas, are an essential key to therapeutic management. EGFR-activated mutations comprise mainly deletions in exon 19 and point mutations in exon 21. Although histology is the traditional method of detection, we investigated the role of cytology in EGFR mutations. STUDY DESIGN: A total of 774 lung cancers were studied for EGFR mutations (676 histological and 98 cytological samples), including 424 adenocarcinomas, 326 non-small cell lung carcinomas not otherwise specified, and 24 squamous cell carcinomas. RESULTS: We had a total of 164 (21.2%) cases of mutations. Common mutations were short in-frame deletions in exon 19 (53.7%) and single-nucleotide substitutions in exon 21 (34.1%); less frequent mutations included single-nucleotide substitutions in exon 18 (3.7%) and in-frame insertions/deletions in exon 20 (8.5%). Histologically, EGFR mutations in exons 19 and 21 occurred in 19.4% and in exons 18 and 20 in 2.2%, while the rates cytologically were 13.3% for exons 19 and 21 and 5.1% for exons 18 and 20. CONCLUSIONS: The sensitivity for the detection of EGFR mutations in cytological samples overlaps histology, so the use of cytological material constitutes an adequate approach for treatment selection in patients with locally advanced or metastatic lung cancer.

Implementation of a Pilot Study to Analyze Circulating Tumor DNA in Early-Stage Lung Cancer.

INTRODUCTION: Liquid biopsies based on plasma circulating tumour deoxyribonucleic acid (ctDNA) have shown promise in monitoring lung cancer evolution. The expression of ctDNA across time, its relationship with clinicopathological parameters and its association with lung cancer progression through imaging allow us to weigh how useful ctDNA could be in monitoring surgically resectable lung cancer. The aim of this study was to assess the impact of ctDNA analysis implementation in early-stage lung cancer. METHODS: A cohort of 47 patients was sequentially recruited. Only 34 patients with early-stage lung cancer were included. All patients had a tissue specimen and five blood samples drawn: at the preoperative stage, from the pulmonary vein, at surgical discharge, at the first follow-up and at the last follow-up. All blood samples were evaluated for ctDNA expression. RESULTS: On average, the maximum yield of ctDNA was obtained in liquid biopsies at the surgical discharge of patients when compared with PO, PV, and F1 (p < 0.0001, p < 0.0001, p < 0.0001 respectively). No statistically significant differences were found when comparing the last follow-up to surgical discharge ctDNA expression (p = 0.851). The correlation between ctDNA concentration according to five-time points and the four clinicopathological characteristics showed that patients younger than 70 years had a statistically significant reduction of the concentration of ctDNA at the preoperative and surgical discharge time point [ß = -16 734 (-27 707; - 5760); p = 0.003; ß = -21 785 (-38 447; -5123); p = 0.010], as opposed to an increase of the concentration of ctDNA at the pulmonary vein and last follow-up time points [ß = 8369 (0.359; 16 378); p = 0.041; ß = 34 402 (12 549; 56 254); p = 0.002] all with a confidence level of 95%. In the cases where actionable mutations were identified in tissue biopsies, the expected mutation was found in five out of six patients plasma samples at the pre-operatory time point and in two out of six patients plasma samples at the pulmonary vein time point. Two out of six patients with actionable mutations had disease progression. CONCLUSION: The results of this pilot study suggest that the maximum yield of ctDNA is obtained at the surgical discharge of the patients and that the pre-operatory timepoint is the one offering the highest sensitivity for the detection of actionable mutations in ctDNA in early-stage lung cancer.

Future perspective for the application of predictive biomarker testing in advanced stage non-small cell lung cancer.

For patients with advanced stage non-small cell lung cancer (NSCLC), treatment strategies have changed significantly due to the introduction of targeted therapies and immunotherapy. In the last few years, we have seen an explosive growth of newly introduced targeted therapies in oncology and this development is expected to continue in the future. Besides primary targetable aberrations, emerging diagnostic biomarkers also include relevant co-occurring mutations and resistance mechanisms involved in disease progression, that have impact on optimal treatment management. To accommodate testing of pending biomarkers, it is necessary to establish routine large-panel next-generation sequencing (NGS) for all patients with advanced stage NSCLC. For cost-effectiveness and accessibility, it is recommended to implement predictive molecular testing using large-panel NGS in a dedicated, centralized expert laboratory within a regional oncology network. The central molecular testing center should host a regional Molecular Tumor Board and function as a hub for interpretation of rare and complex testing results and clinical decision-making.

Developments in predictive biomarker testing and targeted therapy in advanced stage non-small cell lung cancer and their application across European countries.

In the past two decades, the treatment of metastatic non-small cell lung cancer (NSCLC), has undergone significant changes due to the introduction of targeted therapies and immunotherapy. These advancements have led to the need for predictive molecular tests to identify patients eligible for targeted therapy. This review provides an overview of the development and current application of targeted therapies and predictive biomarker testing in European patients with advanced stage NSCLC. Using data from eleven European countries, we conclude that recommendations for predictive testing are incorporated in national guidelines across Europe, although there are differences in their comprehensiveness. Moreover, the availability of recently EMA-approved targeted therapies varies between European countries. Unfortunately, routine assessment of national/regional molecular testing rates is limited. As a result, it remains uncertain which proportion of patients with metastatic NSCLC in Europe receive adequate predictive biomarker testing. Lastly, Molecular Tumor Boards (MTBs) for discussion of molecular test results are widely implemented, but national guidelines for their composition and functioning are lacking. The establishment of MTB guidelines can provide a framework for interpreting rare or complex mutations, facilitating appropriate treatment decision-making, and ensuring quality control.

Corrigendum: Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

[This corrects the article DOI: 10.3389/fmolb.2023.1082915.].

Nonoptical massive parallel DNA sequencing of BRCA1 and BRCA2 genes in a diagnostic setting.

The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer and respective analytical validation. The methodology relies on a multiplex PCR amplification of the BRCA1 and BRCA2 genes combined with a variant prioritization pipeline, designed to minimize the number of false-positive calls without the introduction of false-negative results. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. Performing the study in a blind manner relative to the variants in the samples and using conventional Sanger sequencing as standard, the workflow resulted in a strategy with a maximum analytical sensitivity ≥98.6% with a confidence of 95% and a specificity of 96.9%. Importantly, no true variant was missed. This study presents a comprehensive massive parallel sequencing-Sanger sequencing based strategy, which results in a high analytical sensitivity assay that provides a time- and cost-effective strategy for the identification of mutations in the BRCA1 and BRCA2 genes.