Clinicogenomic associations in childhood Langerhans cell histiocytosis: an international cohort study.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder caused by somatic genetic alterations in hematopoietic precursor cells differentiating into CD1a+/CD207+ histiocytes. LCH clinical manifestation is highly heterogeneous. BRAF and MAP2K1 mutations account for ∼80% of genetic driver alterations in neoplastic LCH cells. However, their clinical associations remain incompletely understood. Here, we present an international clinicogenomic study of childhood LCH, investigating 377 patients genotyped for at least BRAFV600E. MAPK pathway gene alterations were detected in 300 (79.6%) patients, including 191 (50.7%) with BRAFV600E, 54 with MAP2K1 mutations, 39 with BRAF exon 12 mutations, 13 with rare BRAF alterations, and 3 with ARAF or KRAS mutations. Our results confirm that BRAFV600E associates with lower age at diagnosis and higher prevalence of multisystem LCH, high-risk disease, and skin involvement. Furthermore, BRAFV600E appeared to correlate with a higher prevalence of central nervous system (CNS)-risk bone lesions. In contrast, MAP2K1 mutations associated with a higher prevalence of single-system (SS)-bone LCH, and BRAF exon 12 deletions seemed to correlate with more lung involvement. Although BRAFV600E correlated with reduced event-free survival in the overall cohort, neither BRAF nor MAP2K1 mutations associated with event-free survival when patients were stratified by disease extent. Thus, the correlation of BRAFV600E with inferior clinical outcome is (primarily) driven by its association with disease extents known for high rates of progression or relapse, including multisystem LCH. These findings advance our understanding of factors underlying the remarkable clinical heterogeneity of LCH but also question the independent prognostic value of lesional BRAFV600E status.

The sensitivity of pan-TRK immunohistochemistry in solid tumours: A meta-analysis.

INTRODUCTION: Since the approval of neurotrophic tropomyosin receptor kinase (NTRK) tyrosine kinase inhibitors for fist-line advanced stage pan-cancer therapy, pathologists and molecular biologists have been facing a complex question: how should the large volume of specimens be screened for NTRK fusions? Immunohistochemistry is fast and cheap, but the sensitivity compared to RNA NGS is unclear. METHODS: We performed RNA-based next-generation sequencing on 1,329 cases and stained 24 NTRK-rearranged cases immunohistochemically with pan-TRK (ERP17341). Additionally, we performed a meta-analysis of the literature. After screening 580 studies, 200 additional NTRK-rearranged cases from 13 studies, analysed with sensitive molecular diagnostics as well as pan-TRK IHC, were included. RESULTS: In the included 224 NTRK-rearranged solid tumours, the sensitivity for pan-TRK IHC was 82% and the false-negative rate was 18%. NTRK3 fusions had more false negatives (27%) compared to NTRK1 (6%) and NTRK2 (14%) (p = 0.0006). Membranous, nuclear and peri-nuclear staining patterns strongly correlated with different fusion products, with membranous staining being more prevalent in NTRK1 and NTRK2, nuclear in NTRK3, and perinuclear in NTRK1. CONCLUSION: Despite a reduction in the number of molecular analysis, using pan-TRK immunohistochemistry as a prescreening method to detect NTRK fusions in solid tumours will miss 18% of all NTRK-fused cases (especially involving NTRK3). Therefore, the most comprehensive and optimal option to detect NTRK fusions is to perform molecular testing on all eligible cases. However, in case of financial or logistical limitations, an immunohistochemistry-first approach is defensible in tumours with a low prevalence of NTRK fusions.

Functional Analysis Identifies Damaging CHEK2 Missense Variants Associated with Increased Cancer Risk.

Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell-based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 were found to impair protein function to a similar extent as truncating variants, while 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging CHEK2 variants as a group [OR 2.23; 95% confidence interval (CI), 1.62-3.07; P < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21-2.20; P = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87-1.46; P = 0.378). Finally, a damaging VUS in CHEK2, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer. SIGNIFICANCE: Quantitative assessment of the functional consequences of CHEK2 variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers.

The RECAP Test Rapidly and Reliably Identifies Homologous Recombination-Deficient Ovarian Carcinomas.

Recent studies have shown that the efficacy of PARP inhibitors in epithelial ovarian carcinoma (EOC) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2 deficient EOC. A robust method with which to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we investigated the proficiency of a functional HR assay based on the detection of RAD51 foci, the REcombination CAPacity (RECAP) test, in identifying HRD tumors in a cohort of prospectively collected epithelial ovarian carcinomas (EOCs). Of the 39 high-grade serous ovarian carcinomas (HGSOC), the RECAP test detected 26% (10/39) to be HRD, whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Of the HRD tumors that could be sequenced, 8/9 showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation, indicating that the RECAP test reliably identifies HRD, including but not limited to tumors related to BRCA1/2 deficiency. Furthermore, we found a trend towards better overall survival (OS) of HGSOC patients with RECAP-identified HRD tumors compared to patients with HRP tumors. This study shows that the RECAP test is an attractive alternative to DNA-based HRD tests, and further development of a clinical grade RECAP test is clearly warranted.

Performance of the pattern-based interpretation of p53 immunohistochemistry as a surrogate for TP53 mutations in vulvar squamous cell carcinoma.

AIMS: The most commonly mutated gene in vulvar squamous cell carcinoma (VSCC) is TP53 and its prognostic value, particularly in HPV-independent VSCC, is uncertain. In other tumours, p53 immunohistochemistry (IHC) is an excellent surrogate marker for TP53 mutations. In order to study this in VSCC, we assigned six p53 IHC patterns into two final classes: 'wild-type' or 'mutant'. We determined the performance and interobserver variability of this pattern-based p53 IHC approach. METHODS AND RESULTS: Two experienced gynaecological pathologists scored the predefined p53 IHC patterns of 59 VSCC, independently and blinded for molecular data. Agreement was calculated by Cohen's kappa. All disagreements regarding p53 IHC patterns were resolved by a consensus meeting. After DNA isolation, the presence of pathogenic TP53 variants was determined by next-generation sequencing (NGS). Sensitivity, specificity and accuracy of p53 IHC as a surrogate marker for TP53 mutation status were calculated. Initial p53 IHC pattern interpretation showed substantial agreement between both observers (k = 0.71, P < 0.001). After consensus, 18 cases (30.5%) were assigned a final p53 IHC class as TP53 wild-type and 41 cases (69.5%) as mutant. The accuracy between the p53 IHC class and TP53 mutation status, after the consensus meeting, was 96.6%. Moreover, the sensitivity and specificity were high 95.3% [95% confidence interval (CI) = 82.9-99.1% and 100% (95% CI = 75.9-100%)]. CONCLUSIONS: Pattern-based p53 IHC classification is highly reproducible among experienced gynaecological pathologists and accurately reflects TP53 mutations in VSCC. This approach to p53 IHC interpretation offers guidance and provides necessary clarity for resolving the proposed prognostic relevance of final p53 IHC class within HPV-independent VSCC.

Optimizing Mutation and Fusion Detection in NSCLC by Sequential DNA and RNA Sequencing.

INTRODUCTION: Frequently, patients with locally advanced or metastatic NSCLC are screened for mutations and fusions. In most laboratories, molecular workup includes a multitude of tests: immunohistochemistry (ALK, ROS1, and programmed death-ligand 1 testing), DNA sequencing, in situ hybridization for fusion, and amplification detection. With the fast-emerging new drugs targeting specific fusions and exon-skipping events, this procedure harbors a growing risk of tissue exhaustion. METHODS: In this study, we evaluated the benefit of anchored, multiplexed, polymerase chain reaction-based targeted RNA sequencing (RNA next-generation sequencing [NGS]) in the identification of gene fusions and exon-skipping events in patients, in which no pathogenic driver mutation was found by DNA-based targeted cancer hotspot NGS (DNA NGS). We analyzed a cohort of stage IV NSCLC cases from both in-house and referral hospitals, consisting 38.5% cytology samples and 61.5% microdissected histology samples, mostly core needle biopsies. We compared molecular findings in a parallel workup (DNA NGS and RNA NGS, cohort 1, n = 198) with a sequential workup (DNA NGS followed by RNA NGS in selected cases, cohort 2, n = 192). We hypothesized the sequential workup to be the more efficient procedure. RESULTS: In both cohorts, a maximum of one oncogenic driver mutation was found per case. This is in concordance with large, whole-genome databases and suggests that it is safe to omit RNA NGS when a clear oncogenic driver is identified in DNA NGS. In addition, this reduced the number of necessary RNA NGS to only 53% of all cases. The tumors of never smokers, however, were enriched for fusions and exon-skipping events (32% versus 4% in former and current smokers, p = 0.00), and therefore benefited more often from the shorter median turnaround time of the parallel approach (15 d versus only 9 d in the parallel workup). CONCLUSIONS: We conclude that sequentially combining DNA NGS and RNA NGS is the most efficient strategy for mutation and fusion detection in smoking-associated NSCLC, whereas for never smokers we recommend a parallel approach. This approach was shown to be feasible on small tissue samples including for cytology tests, can drastically reduce the complexity and cost of molecular workup, and also provides flexibility in the constantly evolving landscape of actionable targets in NSCLC.

Targeted Treatment Options of Recurrent Radioactive Iodine Refractory Hürthle Cell Cancer.

Objective: To evaluate the efficacy and treatment rationale of Hürthle cell carcinoma (HCC) following a patient with progressive and metastatic HCC. HCC was recently shown to harbor a distinct genetic make-up and the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kiase (PI3K)/AKT signaling pathways are potential targets for anti-cancer agents in the management of recurrent HCC. The presence or absence of gene variants can give a rationale for targeted therapies that could be made available in the context of drug repurposing trials. Methods: Treatment included everolimus, sorafenib, nintedanib, lenvatinib, and panitumumab. Whole genome sequencing (WGS) of metastatic tumor material obtained before administration of the last drug, was performed. We subsequently evaluated the rationale and efficacy of panitumumab in thyroid cancer and control cell lines after epidermal growth factor (EGF) stimulation and treatment with panitumumab using immunofluorescent Western blot analysis. EGF receptor (EGFR) quantification was performed using flow cytometry. Results: WGS revealed a near-homozygous genome (NHG) and a somatic homozygous TSC1 variant, that was absent in the primary tumor. In the absence of RAS variants, panitumumab showed no real-life efficacy. This might be explained by high constitutive AKT signaling in the two thyroid cancer cell lines with NHG, with panitumumab only being a potent inhibitor of pEGFR in all cancer cell lines tested. Conclusions: In progressive HCC, several treatment options outside or inside clinical trials are available. WGS of metastatic tumors might direct the timing of therapy. Unlike other cancers, the absence of RAS variants seems to provide insufficient justification of single-agent panitumumab administration in HCC cases harboring a near-homozygous genome.

Apparent Lack of BRAFV600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8+ T Cells in Langerhans Cell Histiocytosis.

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAFV600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAFV600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAFV600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAFV600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAFV600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAFV600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATEK was not detected in the HLA class I peptidomes of two distinct BRAFV600E transduced cell lines with confirmed expression of HLA-A*03:01 or HLA-A*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAFV600E protein are not presented by HLA class I molecules. Given that the BRAFV600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.