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The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease lacking specific therapies. This study reports on the identification, characterization, and immunotherapeutic application of HLA-presented neoantigens specific for the DNAJB1-PRKACA fusion transcript in fibrolamellar hepatocellular carcinoma. DNAJB1-PRKACA-derived HLA class I and HLA class II ligands induce multifunctional cytotoxic CD8 + and T-helper 1 CD4 + T cells, and their cellular processing and presentation in DNAJB1-PRKACA expressing tumor cells is demonstrated by mass spectrometry-based immunopeptidome analysis. Single-cell RNA sequencing further identifies multiple T cell receptors from DNAJB1-PRKACA-specific T cells. Vaccination of a fibrolamellar hepatocellular carcinoma patient, suffering from recurrent short interval disease relapses, with DNAJB1-PRKACA-derived peptides under continued Poly (ADP-ribose) polymerase inhibitor therapy induces multifunctional CD4 + T cells, with an activated T-helper 1 phenotype and high T cell receptor clonality. Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion. The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease with limited therapeutic options. Here, the authors identify the DNAJB1-PRKACA protein as a source for immunogenic neoepitopes and a potential target of T cell-based immunotherapy.

Immune checkpoint inhibitors (ICI) have significantly improved overall survival in melanoma patients. However, 60% experience severe adverse events and early response markers are lacking. Circulating tumour DNA (ctDNA) is a promising biomarker for treatment-response and recurrence detection. The prospective PET/LIT study included 104 patients with palliative combined or adjuvant ICI. Tumour-informed sequencing panels to monitor 30 patient-specific variants were designed and 321 liquid biopsies of 87 patients sequenced. Mean sequencing depth after deduplication using UMIs was 6000x and the error rate of UMI-corrected reads was 2.47×10 −4 . Variant allele fractions correlated with PET/CT MTV (rho=0.69), S100 (rho=0.72), and LDH (rho=0.54). A decrease of allele fractions between T1 and T2 was associated with improved PFS and OS in the palliative cohort (p = 0.008 and p < 0.001). ctDNA was detected in 76.9% of adjuvant patients with relapse (n = 10/13), while all patients without progression (n = 9) remained ctDNA negative. Tumour-informed liquid biopsies are a reliable tool for monitoring treatment response and early relapse in melanoma patients with ICI. Tumour-informed liquid biopsies can be used for treatment monitoring and detection of relapse in melanoma patients during or after immunotherapy. Here, the authors utilise targeted sequencing to monitor variants in advanced melanoma patients in ctDNA and show ctDNA could be detected in adjuvant patients.

Angioimmunoblastic T-cell lymphoma is a peripheral T-cell lymphoma derived from follicular T-helper cells. High-throughput genomic sequencing studies have shown that angioimmunoblastic T-cell lymphoma carries frequent mutations in RHOA G17V and IDH2 R172 genes. The clinico-pathological features of angioimmunoblastic T-cell lymphoma cases with RHOA G17V mutations have been addressed; however, similar studies for IDH2 mutated cases are lacking. Therefore, the aim of the present study was to evaluate the pathological features of angioimmunoblastic T-cell lymphoma with IDH2 mutations. In order to identify cases with IDH2 mutations, 50 cases previously diagnosed as angioimmunoblastic T-cell lymphoma were subjected to next-generation sequencing analysis using a custom panel covering four genes frequently mutated in angioimmunoblastic T-cell lymphoma including DNMT3A , TET2 , IDH2 and RHOA . All cases were analyzed for PD1, ICOS, CXCL13, CD10, BCL6, CD21, CD23 and EBER in situ hybridization. Mutational analysis recognized three groups. Group 1: IDH2 R172 mutations were identified in 20 cases (40%). All cases carried RHOA G17V mutations. Group 2: RHOA G17V mutations without IDH2 R172 mutation were identified in 16 cases (32%), and Group 3: 14 cases (28%) without RHOA G17V or IDH2 R172 mutations. Morphologically, angioimmunoblastic T-cell lymphoma cases with IDH2 R172 mutations were characterized by the presence of medium to large clear cells ( p  = 0.00001), and a follicular T-helper phenotype with the particular feature of strong CD10 ( p  = 0.0268) and CXCL13 expression ( p  = 0.0346). Interestingly, TET2 mutations were identified in 32 of 33 (97%) cases with IDH2 R172 and/or RHOA G17V mutations whereas only 55% of angioimmunoblastic T-cell lymphoma cases wild-type for these two genes carried TET2 mutations ( p  = 0.0022). In contrast, DNMT3A mutations were found in 48% of the cases and were equally distributed in the three groups. In conclusion, our results support the results of gene expression profiling studies suggesting that IDH2 R172 mutations define a unique subgroup within angioimmunoblastic T-cell lymphoma with strong follicular T-helper-like phenotype and characteristic morphological features.

Alterations in the function of K + channels such as the voltage- and Ca 2+ -activated K + channel of large conductance (BK Ca ) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BK Ca splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBK Ca ). Through a combination of genetic knockdown and knockout along with a cell permeable BK Ca channel blocker, we show that mitoBK Ca modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the ‘Warburg effect’, thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBK Ca and BK Ca transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBK Ca could represent a treatment strategy for selected BC patients in future.

Extranodal NK/T-cell lymphoma (ENKTL) is an Epstein-Barr virus (EBV) associated lymphoma, prevalent in Asia and Latin America. Studies in Asian cohorts have identified some recurrent gene mutations in ENKTL; however, the mutational landscape of ENKTL in Latin America is unknown. In this study, we investigated the mutational profile and EBV strains of 71 ENKTL cases from Latin America (42 from Mexico, 17 from Peru, and 12 from Argentina) and compared it with Asian cohorts. The mutational analysis was performed by next generation sequencing (NGS) using an Ion AmpliSeq™ custom panel covering for the most frequently mutated genes identified in ENKTL. STAT3 was the most frequent mutated gene (16 cases: 23%), followed by MSN (10 cases; 14%), BCOR (9 cases; 13%), DDX3X (6 cases; 8%), TP53 (6 cases; 8%), MGA (3 cases; 4%), JAK3 (2 cases; 3%), and STAT5B (1 case; 1%). Mutations in STAT3 , BCOR , and DDX3X were nearly mutually exclusive, suggesting different molecular pathways involved in the pathogenesis of ENKTL; whereas mutations in MGA , MSN , and TP53 were concomitant with other mutations. Most cases (75%) carried Type A EBV without the 30-bp LMP1 gene deletion. The overall survival was significantly associated with serum LDH level, Eastern Cooperative Oncology Group (ECOG) performance status, International Prognostic Index (IPI) score, and therapy ( p  < 0.05), but not associated with any mutation, EBV strain or deletion in EBV LMP1 gene. In conclusion, mutational analysis of ENKTL from Latin America reveals frequent gene mutations leading to activation of the JAK-STAT pathway (25%), mostly STAT3 . Compared to Asian cohorts, BCOR, DDX3 X   and TP53 mutations were also identified but with different frequencies. None of these mutations were associated with prognosis.

Plasmablastic lymphoma (PBL) represents a rare and aggressive lymphoma subtype frequently associated with immunosuppression. Clinically, patients with PBL are characterized by poor outcome. The current understanding of the molecular pathogenesis is limited. A hallmark of PBL represents its plasmacytic differentiation with loss of B-cell markers and, in 60% of cases, its association with Epstein-Barr virus (EBV). Roughly 50% of PBLs harbor a MYC translocation. Here, we provide a comprehensive integrated genomic analysis using whole exome sequencing (WES) and genome-wide copy number determination in a large cohort of 96 primary PBL samples. We identify alterations activating the RAS-RAF, JAK-STAT, and NOTCH pathways as well as frequent high-level amplifications in MCL1 and IRF4 . The functional impact of these alterations is assessed using an unbiased shRNA screen in a PBL model. These analyses identify the IRF4 and JAK-STAT pathways as promising molecular targets to improve outcome of PBL patients. Plasmablastic lymphoma (PBL) is an aggressive lymphoma subtype characterized by poor prognosis but the molecular knowledge of the disease is limited. Here, the authors perform whole exome sequencing and copy number determination of primary samples highlighting IRF4 and JAK-STAT pathways as therapeutic targets for PBL.

Anaplastic large cell lymphoma (ALCL) represents a group of malignant T-cell lymphoproliferations that share morphological and immunophenotypical features, namely strong CD30 expression and variable loss of T-cell markers, but differ in clinical presentation and prognosis. The recognition of anaplastic lymphoma kinase (ALK) fusion proteins as a result of chromosomal translocations or inversions was the starting point for the distinction of different subgroups of ALCL. According to their distinct clinical settings and molecular findings, the 2016 revised World Health Organization (WHO) classification recognizes four different entities: systemic ALK-positive ALCL (ALK+ ALCL), systemic ALK-negative ALCL (ALK− ALCL), primary cutaneous ALCL (pC-ALCL), and breast implant-associated ALCL (BI-ALCL), the latter included as a provisional entity. ALK is rearranged in approximately 80% of systemic ALCL cases with one of its partner genes, most commonly NPM1, and is associated with favorable prognosis, whereas systemic ALK− ALCL shows heterogeneous clinical, phenotypical, and genetic features, underlining the different oncogenesis between these two entities. Recognition of the pathological spectrum of ALCL is crucial to understand its pathogenesis and its boundaries with other entities. In this review, we will focus on the morphological, immunophenotypical, and molecular features of systemic ALK+ and ALK− ALCL. In addition, BI-ALCL will be discussed.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress into primary central nervous system lymphoma (PCNSL). To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed B-cell receptors (BCR) from PVRL patients and tested for binding against human auto-antigens. SEL1L3, a protein with multiple glycosylation sites, was identified as the BCR target in 3/20 PVRL cases. SEL1L3 induces proliferation and BCR pathway activation in aggressive lymphoma cell lines. Moreover, SEL1L3 conjugated to a toxin killed exclusively lymphoma cells with respective BCR-reactivity. Western Blot analysis indicates the occurrence of hyper-N-glycosylation of SEL1L3 at aa 527 in PVRL patients with SEL1L3-reactive BCRs. The BCR of a PVRL patient with serum antibodies against SEL1L3 was cloned from a vitreous body biopsy at diagnosis and of a systemic manifestation at relapse. VH4-04*07 was used in both lymphoma manifestations with highly conserved CDR3 regions. Both BCRs showed binding to SEL1L3, suggesting continued dependence of lymphoma cells on antigen stimulation. These results indicate an important role of antigenic stimulation by post-translationally modified auto-antigens in the genesis of PVRL. They also provide the basis for a new treatment approach targeting unique lymphoma BCRs with ultimate specificity.

Oncoprotein Y‐box‐binding protein‐1 (YB‐1) is involved in all cancer hallmarks. One of the most studied post‐translational modifications of YB‐1 is phosphorylation on Serine 102 (S102), which is involved in cancer progression. KRAS mutations are frequent, have been associated with poor prognosis and therapy resistance, and they are considered a major stimulator of S102 YB‐1 in vitro. In this study, a relationship between S102 YB‐1 phosphorylation in subcellular fractions and KRAS mutation was investigated in CRC tissues, and its association with clinicopathological parameters was analyzed. Immunohistochemistry on 36 patient samples and 5 normal tissue samples highlighted nuclear S102 YB‐1 was specific to cancer tissues. Nuclear S102 YB‐1 was expressed in 47.2% of tumor tissues, which was positively correlated with KRAS mutation (P = 0.017). There was no significant association between cytoplasmic S102 YB‐1 with KRAS mutation status (P = 0.391). Further studies in larger cohorts are needed to validate the observed results. The significant association between S102 YB‐1 in the nucleus and KRAS mutation may suggest YB‐1 as an effective target to improve survival of CRC patients with KRAS‐mutated tumors. This study identifies nuclear YB‐1 S102 phosphorylation as a marker associated with KRAS and FBXW7 mutations in colorectal cancer. Mutated KRAS correlates specifically with nuclear, not cytoplasmic, S102 YB‐1. These findings provide the first ex vivo evidence of this link in CRC and suggest future studies should assess the prognostic and therapeutic relevance of its subcellular localization.

The detection of somatic driver mutations by next-generation sequencing (NGS) is becoming increasingly important in the care of advanced melanoma patients. In our study, we evaluated the NGS results of 82 melanoma patients from clinical routine in 2017. Besides determining the tumor mutational burden (TMB) and annotation of all genetic driver alterations, we investigated their potential as a predictor for resistance to immune checkpoint inhibitors (ICI) and as a distinguishing feature between melanoma subtypes. Melanomas of unknown primary had a similar mutation pattern and TMB to cutaneous melanoma, which hints at its cutaneous origin. Besides the typical hotspot mutation in BRAF and NRAS, we frequently observed CDKN2A deletions. Acral and mucosal melanomas were dominated by CNV alterations affecting PDGFRA, KIT, CDK4, RICTOR, CCND2 and CHEK2. Uveal melanoma often had somatic SNVs in GNA11/Q and amplification of MYC in all cases. A significantly higher incidence of BRAF V600 mutations and EGFR amplifications, PTEN and TP53 deletions was found in patients with disease progression while on ICI. Thus, NGS might help to characterize melanoma subtypes more precisely and to identify possible resistance mechanisms to ICI therapy. Nevertheless, NGS based studies, including larger cohorts, are needed to support potential genetic ICI resistance mechanisms.