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.

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.

Background: Mucosal and acral melanoma respond worse to immune checkpoint inhibitors (ICI) than cutaneous melanoma. MDM2/4 as well as EGFR amplifications are supposed to be associated with hyperprogression on ICI in diverse cancers. We therefore investigated the response of metastatic acral and mucosal melanoma to ICI in regard to MDM2/4 or EGFR amplifications and melanoma type. Methods: We conducted a query of our melanoma registry, looking for patients with metastatic acral or mucosal melanoma treated by ICI. Whole exome sequencing, FISH and immunohistochemistry on melanoma tissue could be performed on 45 of the total cohort of 51 patients. Data were correlated with patients’ responses to ICI and survival. Results: 22 out of 51 patients had hyperprogressive disease (an increase in tumor load of >50% at the first staging). Hyperprogression occurred more often in case of MDM2/4 or EGFR amplification or <1% PD-L1 positive tumor cells. Nevertheless, this association was not significant. Interestingly, the anorectal melanoma type and the presence of liver metastases were significantly associated with worse survival. Conclusions: So far, we found no reliable predictive marker for patients who develop hyperprogression on ICI, specifically with regard to MDM2/4 or EGFR amplifications. Nevertheless, patients with anorectal melanoma, liver metastases or melanoma with amplified MYC seem to have an increased risk of not benefitting from ICI.

Vitreoretinal lymphoma (VRL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) considered a variant of primary central nervous system lymphoma (PCNSL). Diagnosis of VRL requires examination of vitreous fluid, but cytologic differentiation from uveitis remains difficult. Due to its rarity and difficulty in obtaining diagnostic material, little is known about the genetic profile of VRL. The aim of our study was to investigate the mutational profile of a large series of primary and secondary VRL. Targeted next generation sequencing using a custom panel containing the most frequent mutations in PCNSL was performed on 34 vitrectomy samples of 31 patients with VRL and negative controls with uveitis. In a subset of cases, genome-wide copy number alterations (CNA) were assessed using the Oncoscan platform. Mutations in MYD88 (74%), PIM1 (71%), CD79B (55%), IGLL5 (52%), TBL1XR1 (48%), ETV6 (45%) and 9p21/CDKN2A deletions (85%) were the most common alterations, with similar frequencies in primary (15), synchronous (3) or secondary (13) VRL. This mutational spectrum is similar to MYD88mut/CD79Bmut (MCD or cluster 5) DLBCL with activation of Toll-like and B-cell receptor pathways and CDKN2A loss, confirming their close relationship. Oncoscan analysis demonstrated a high number of CNAs (mean 18.6/case). Negative controls lacked mutations or CNAs. Using cell free DNA of vitreous fluid supernatant, mutations present in cellular DNA were reliably detected in all examined cases. Mutational analysis is a highly sensitive and specific tool for the diagnosis of VRL and can also be applied successfully to cell free DNA derived from the vitreous.

Background: HPV-related cervical cancer (CC) is the fourth most frequent cancer in women worldwide. Cell-free tumour DNA is a potent biomarker to detect treatment response, residual disease, and relapse. We investigated the potential use of cell-free circulating HPV-DNA (cfHPV-DNA) in plasma of patients with CC. Methods: cfHPV-DNA levels were measured using a highly sensitive next-generation sequencing-based approach targeting a panel of 13 high-risk HPV types. Results: Sequencing was performed in 69 blood samples collected from 35 patients, of which 26 were treatment-naive when the first liquid biopsy sample was retrieved. cfHPV-DNA was successfully detected in 22/26 (85%) cases. A significant correlation between tumour burden and cfHPV-DNA levels was observed: cfHPV-DNA was detectable in all treatment-naive patients with advanced-stage disease (17/17, FIGO IB3–IVB) and in 5/9 patients with early-stage disease (FIGO IA–IB2). Sequential samples revealed a decrease of cfHPV-DNA levels in 7 patients corresponding treatment response and an increase in a patient with relapse. Conclusions: In this proof-of-concept study we demonstrated the potential of cfHPV-DNA as a biomarker for therapy monitoring in patients with primary and recurrent CC. Our findings facilitate the development of a sensitive and precise, non-invasive, inexpensive, and easily accessible tool in CC diagnosis, therapy monitoring and follow-up.