Primary central nervous system lymphomas (PCNSL) are aggressive non-Hodgkin lymphomas affecting the central nervous system (CNS). Although immunophenotyping studies suggested an uniform activated B-cell (ABC) origin, more recently a spectrum of ABC and germinal center B-cell (GC) cases has been proposed, with the molecular subtypes of PCNSL still being a matter of debate. With the emergence of novel therapies demonstrating different efficacy between the ABC and GC patient groups, precise assignment of molecular subtype is becoming indispensable. To determine the molecular subtype of 77 PCNSL and 17 secondary CNS lymphoma patients, we used the NanoString Lymphoma Subtyping Test (LST), a gene expression-based assay representing a more accurate technique of subtyping compared with standard immunohistochemical (IHC) algorithms. Mutational landscapes of 14 target genes were determined using ultra-deep next-generation sequencing. Using the LST-assay, a significantly lower proportion (80% vs 95%) of PCNSL cases displayed ABC phenotype compared with the IHC-based characterization. The most frequently mutated genes included MYD88, PIM1, and KMT2D. In summary, we successfully applied the LST-assay for molecular classification of PCNSL, reporting higher proportion of cases with GC phenotype compared with IHC analyses, leading to a more precise patient stratification potentially applicable in the diagnostic algorithm of PCNSL.

Recent advances in molecular technologies enable sensitive and quantitative assessment of circulating tumor DNA, offering a noninvasive disease monitoring tool for patients with malignant disorders. Here, we demonstrated on four follicular lymphoma cases that circulating tumor DNA based EZH2 mutation analysis performed by a highly sensitive droplet digital PCR method may be a valuable treatment monitoring approach in EZH2 mutant follicular lymphoma. EZH2 variant allele frequencies changed in parallel with the volume of metabolically active tumor sites observed on 18F-fluorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. Variant allele frequencies of EZH2 mutations decreased or were eliminated rapidly upon successful treatment, with treatment failure being associated with elevated EZH2 variant allele frequencies. We also demonstrated spatial heterogeneity in a patient with two different EZH2 mutations in distinct anatomical sites, with both mutations simultaneously detected in the liquid biopsy specimen. In summary, circulating tumor DNA based EZH2 mutation analysis offers a rapid, real-time, radiation-free monitoring tool for sensitive detection of EZH2 mutations deriving from different anatomical sites in follicular lymphoma patients receiving immunochemotherapy.

Primary cutaneous follicle center lymphoma (PCFCL) is an indolent variant of follicular lymphoma (FL) with limited information available on the genetic background of the disease. The genetic hallmark of nodal FL, the t(14;18) translocation, affecting the BCL2 gene, is rare in PCFCL. Loss of 1p36, the most common secondary chromosomal abnormality in nodal FL, has been recently reported in 16.7% of PCFCL cases. In order to further characterize PCFCL, 21 cases were analyzed using interphase fluorescence in situ hybridization with BCL2 break apart and 1p36/1q25 dual color probes. Sanger sequencing was used to investigate TNFRSF14 and EZH2 mutations and immunohistochemistry to assess BCL2, EZH2 protein expressions.1p36 deletion occurred in 22% (5/21), BCL2 gene break in 10% (2/20) of the PCFCL cases. Mutations of the candidate tumor suppressor gene of the 1p36 region, TNFRSF14 mutations were detected in 4/17 (23.5%) cases with 2 cases presenting with concurrent 1p36 deletion. EZH2 hotspot mutations at Y641, A682, and A692 were not found. High EZH2 protein expression associated with a BCL2 negative phenotype was observed in 43% (9/21) of the cases. BCL2 gene break or 1p36 deletion did not impact the prognosis; however, they showed association with advanced stages at diagnosis (p = 0.016) and a tendency with shorter event free survival (p = 0.052).In conclusion, 1p36 deletion co-occurs with acquired TNFRSF14 mutations, suggesting a role of this tumor suppressor gene in the development of a subgroup of PCFCL. High EZH2 protein expression associated with BCL2 negative phenotype is common and might represent an ideal therapeutic target.

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Absztrakt: A molekuláris biológiai technikák utóbbi évtizedekben látott fejlődésének köszönhetően a vérplazmában keringő biomolekuláknak, így például a tumor eredetű sejtmentes DNS-fragmentumoknak is korábban elképzelhetetlen mélységű vizsgálata vált elérhetővé. A minimális invazivitással járó folyadékbiopsziás mintavétel metasztatikus tumorok esetében lehetővé teszi eltérő anatómiai lokalizációval megjelenő genetikai aberrációk egyidejű feltérképezését, azok nyomon követését, illetve a klonális evolúció következtében megjelenő további abnormalitások kimutatását is. Ebből következően számos, szolid tumorokat vizsgáló, folyadékbiopsziás mintavételt alkalmazó tanulmány jelent meg az utóbbi évtizedben, néhány entitás vonatkozásában pedig az eljárás már a rutindiagnosztikában is alkalmazásra került. Onkohematológiában az ebből a szempontból legintenzívebben kutatott megbetegedések közé a diffúz nagy B-sejtes limfóma, a Hodgkin-limfóma, illetve a plazmasejtes mielóma tartozik. Saját, illetve irodalmi adatok alapján a tumor eredetű sejtmentes DNS vizsgálata hasznos lehet a kezelést megelőző prognózisbecslésben, a célozható molekuláris eltérések azonosításában, a terápiás hatékonyság és a minimális reziduális betegség monitorozásában, valamint a terápiarezisztens klónok kimutatásában. Jelen összefoglaló közleményünkben a folyadékbiopszia-vizsgálatok onkohematológiai alkalmazásait tekintjük át.