The clonal relation of primary upper urinary tract urothelial carcinoma and paired urothelial carcinoma of the bladder.

The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor-specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities.

Somatic aberrations of mismatch repair genes as a cause of microsatellite-unstable cancers.

Lynch syndrome (LS) is caused by germline mutations in mismatch repair (MMR) genes, resulting in microsatellite-unstable tumours. Approximately 35% of suspected LS (sLS) patients test negative for germline MMR gene mutations, hampering conclusive LS diagnosis. The aim of this study was to investigate somatic MMR gene aberrations in microsatellite-unstable colorectal and endometrial cancers of sLS patients negative for germline MMR gene mutations. Suspected LS cases were selected from a retrospective Clinical Genetics Department diagnostic cohort and from a prospective multicentre population-based study on LS in The Netherlands. In total, microsatellite-unstable tumours of 40 sLS patients (male/female 20/20, median age 57 years) were screened for somatic MMR gene mutations by next-generation sequencing. In addition, loss of heterozygosity (LOH) of the affected MMR genes in these tumours as well as in 68 LS-associated tumours and 27 microsatellite-unstable tumours with MLH1 promoter hypermethylation was studied. Of the sLS cases, 5/40 (13%) tumours had two pathogenic somatic mutations and 16/40 (40%) tumours had a (likely) pathogenic mutation and LOH. Overall, LOH of the affected MMR gene locus was observed in 24/39 (62%) tumours with informative LOH markers. Of the LS cases and the tumours with MLH1 promoter hypermethylation, 39/61 (64%) and 2/21 (10%) tumours, respectively, demonstrated LOH. Half of microsatellite-unstable tumours of sLS patients without germline MMR gene mutations had two (likely) deleterious somatic MMR gene aberrations, indicating their sporadic origin. Therefore, we advocate adding somatic mutation and LOH analysis of the MMR genes to the molecular diagnostic workflow of LS.

Characterization of the T-cell-mediated immune response against the Aspergillus fumigatus proteins Crf1 and catalase 1 in healthy individuals.

Invasive aspergillosis is a serious infectious complication after allogeneic stem cell transplantation. One of the strategies to improve the management of aspergillosis is the adoptive transfer of antigen-specific T cells, the success of which depends on the development of a broad repertoire of antigen-specific T cells. In this study, we identified CD4+ T cells specific for the Aspergillus proteins Crf1 and catalase 1 in 18 of 24 healthy donors by intracellular staining for interferon γ and CD154. Crf1- and catalase 1-specific T cells were selected on the basis of CD137 expression and underwent single-cell expansion. Aspergillus-specific T-cell clones mainly exhibited a T-helper cell 1 phenotype and recognized a broad variety of T-cell epitopes. Five novel Crf1 epitopes, 2 previously described Crf1 epitopes, and 30 novel catalase 1 epitopes were identified. Ultimately, by using overlapping peptides of Aspergillus fumigatus proteins, Aspergillus-specific T-cell lines that have a broad specificity and favorable cytokine profile and are suitable for adoptive T-cell therapy can be generated in vitro.

RET Fluorescence In Situ Hybridization Analysis Is a Sensitive but Highly Unspecific Screening Method for RET Fusions in Lung Cancer.

INTRODUCTION: RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions. METHODS: Between January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci. RESULTS: FISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci. CONCLUSIONS: FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC.

Prevalence of c-KIT mutations in gonadoblastoma and dysgerminomas of patients with disorders of sex development (DSD) and ovarian dysgerminomas.

Activating c-KIT mutations (exons 11 and 17) are found in 10-40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ~3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY.