Targeted gene next-generation sequencing panel in patients with advanced lung adenocarcinoma: Paving the way for clinical implementation

Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (¿ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.We acknowledge the work of the members of our department, especially doctors from the thoracic oncology unit, oncology and pulmonology nurses and, patients and their relatives. N. Martins would like to thank the Portuguese Foundation for Science and Technology (FCT-Portugal) for the Strategic project ref. UID/BIM/04293/2013 and "NORTE2020—Northern Regional Operational Program" (NORTE-01-0145-FEDER-000012)

Distribution of HPV infection and tumour markers in cervical intraepithelial neoplasia from cone biopsies of Mozambican women

Aims: To evaluate human papillomavirus (HPV) infection in whole cervical cone specimens with cervical intraepithelial neoplasia (CIN). In addition, to evaluate the relation between the presence of CIN lesions and HPV infection and the expression of Ki-67, p53, cytokeratins, Gp230 glycoprotein, and simple mucin-type carbohydrates. Methods: Cervical cone specimens from five patients with CIN were studied. For each specimen, serial sections encompassing the whole cone were collected (52 samples). HPV infection and HPV types were detected by the polymerase chain reaction and enzyme immunoassay. The expression of Ki-67, p53, cytokeratins, Gp230, and simple mucin-type carbohydrates was examined immunohistochemically. Results: All cases showed high risk HPV types, namely types 16, 33, 35, and 58. Four of the five patients were infected by multiple viral types. HPV-58 was always seen in CIN III, whereas HPV-35 was more frequent in CIN I. The expression of Ki-67 and p53 was higher in CIN III lesions. The expression of cytokeratins 8 and 17 showed complete or almost complete overlap with CIN III. Altered expression of Gp230, Tn, and sialyl-T was often seen in all grades of CIN. Conclusions: When whole cervical cone specimens are evaluated the rate of multiple HPV infection is very high. The expression of cytokeratins 8 and 17 is a useful marker of CIN III

How the COVID-19 Pandemic Impacted Oncological Molecular Diagnosis: A Picture from a National Reference Center for Molecular Pathology

Introduction. The Portuguese healthcare system had to adapt at short notice to the COVID-19 pandemic. We implemented workflow changes to our molecular pathology laboratory, a national reference center, to maximize safety and productivity. We assess the impact this situation had on our caseload and what conclusions can be drawn about the wider impact of the pandemic in oncological therapy in Portugal. Material and Methods. We reviewed our database for all oncological molecular tests requested between March and April of 2019 and 2020. For each case, we recorded age, sex, region of the country, requesting institution, sample type, testing method, and turnaround time (TAT). A comparison between years was made. Results. The total number of tests decreased from 421 in 2019 to 319 in 2020 (p=0.0027). The greatest reduction was in clinical trial-related cases. Routine cases were similar between years (267 vs. 256). TAT was higher in 2019 (mean 15 days vs. 12.3 days; p=0.0003). Medium- to large-sized public hospitals in the north of the country were mostly responsible for the reduction in cases (p=0.0153). Conclusions. Case reduction was observed at hospitals that have mostly been involved in the treatment of COVID-19 and in the north of the country, the region worst-hit by the pandemic. Similar to other studies, our TAT decreased, even with a similar number of routine cases. Thus, we conclude that it is possible to successfully adapt the workflow of a molecular pathology laboratory to new safety standards without losing efficiency

Characterization of the P373L E-cadherin germline missense mutation and implication for clinical management

Aim: Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by E-cadherin germline mutations. One-third of these mutations are of the missense type, representing a burden in genetic counselling. A new germline missense mutation (P373L) was recently identified in a HDGC Italian family. The present work aimed at addressing the disease-causative nature of the P373L mutant.Methods: Assessment of the P373L mutation effect was based on cell aggregation and invasion assays. LOH analysis at the E-cadherin locus, search for somatic E-cadherin mutations and for promoter hypermethylation were performed to identify the mechanism of inactivation of the E-cadherin wild-type allele in the tumour.Results: In vitro the P373L germline mutation impaired the E-cadherin functions. E-cadherin promoter hypermethylation was observed in the tumour of the P373L mutation carrier.Conclusion: We conclude that the combination of clinical, in vitro and molecular genetic data is helpful for establishing an accurate analysis of HDGC-associated CDH1 germline missense mutations and subsequently for appropriate clinical management of asymptomatic mutation carriers. (c) 2007 Elsevier Ltd. All rights reserved

MSI-L gastric carcinomas share the hMLH1 methylation status of MSI-H carcinomas but not their clinicopathological profile

Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in target genes and display a particular clinicopathological profile. In SGC the MSI phenotype has been associated with hMLH1 promoter hypermethylation. Fifty-seven SGC, classified as high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), were analyzed for hMLH1 promoter methylation status and clinicopathological features. hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and to compare the molecular and clinicopathological features of MSI-L, MSI-H, and MSS tumors in an attempt to clarify the place of MSI-L tumors in the mismatch repair (MMR) pathway. Hypermethylation of hMLH1 promoter occurred in 27 of 57 SGC (47.3%) and was significantly associated with MSI status, target gene mutations, and expansive pattern of growth of the tumors. Seventy-five percent of the MSI-H and 50% of MSI-L carcinomas showed hypermethylation (Met+) of hMLH1 in contrast to 0% in MSS carcinomas. No hMLH1 expression was observed in MSI-L/Met+ and MSI-H/Met+ cases. MSS and MSI-L tumors share the same clinicopathological profile regardless of the methylation status of the latter and are distinct from MSI-H tumors. We conclude that mutations in target genes, more than hypermethylation or absence of expression of hMLH1, are the link between MSI status and most of the clinicopathological features of SGC

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer

TP53 germline mutations in Portugal and genetic modifiers of age at cancer onset

The Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant disease caused by TP53 germline mutations. This study aimed to characterize the TP53 mutational spectrum in patients suspected to have LFS in Portugal and to evaluate the influence of the MDM2-SNP309 and TP53-72Arg variants and of telomere length on age of tumor onset. Probands were primarily selected using the classical LFS criteria (two cases) or the more sensitive Chompret Li-Fraumeni-like (LFL) criteria (13 cases), but 12 additional patients that did not comply with those LFS or LFL criteria were included in the analysis based on clinical suspicion (LFS suspects). Nine of the 27 probands (33.3%) presented germline TP53 mutations, two of them occurring de novo and two of them being novel. Three of the nine TP53 mutations were found in families that did not comply with any of the commonly used criteria for TP53 testing, leaving room to recommend the use of less stringent criteria. An association was found between the presence of the TP53-72Arg (but not the MDM2-SNP309) variant and earlier age of onset in TP53 carriers. A negative correlation between telomere length and age of cancer onset was found in patients with germline TP53 mutation, whereas no such correlation was found in controls or in patients with wild-type TP53.We gratefully acknowledge Franclim R. Ribe-iro for statistical assistance. This study was supported by the Portuguese Health Ministry (Project no. 18/2007) and the Liga Portuguesa Contra o Cancro, Núcleo Regional do Norte