We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular –minimal residual disease studies can lead to reliable identification of lineage switch.

Simultaneous multilineage hematologic malignancies are uncommon and associated with poorer prognosis than single-lineage leukemia or lymphoma. Here, we describe a concomitant malignant neoplasm in a 4-year-old boy. The child presented with massive lymphoproliferative syndrome, nasal breathing difficulties, and snoring. Morphological, immunocytochemical, and flow cytometry diagnostics showed coexistence of acute myeloid leukemia (AML) and peripheral T-cell lymphoma (PTCL). Molecular examination revealed a rare t(9;9)(q34;q34)/SET::NUP214 translocation as well as common TCR clonal rearrangements in both the bone marrow and lymph nodes. The disease showed primary refractoriness to both lymphoid and myeloid high-dose chemotherapy as well as combined targeted therapy (trametinib + ruxolitinib). Hence, HSCT was performed, and the patient has since been in complete remission for over a year. This observation highlights the importance of molecular techniques for determining the united nature of complex SET::NUP214-positive malignant neoplasms arising from precursor cells with high lineage plasticity.

While somatic mutations are known to be enriched in genome regions with non-canonical DNA secondary structure, the impact of particular mutagens still needs to be elucidated. Here, we demonstrate that in human cancers, the APOBEC mutagenesis is not enriched in direct repeats, mirror repeats, short tandem repeats, and G-quadruplexes, and even decreased below its level in B-DNA for cancer samples with very high APOBEC activity. In contrast, we observe that the APOBEC-induced mutational density is positively associated with APOBEC activity in inverted repeats (cruciform structures), where the impact of cytosine at the 3’-end of the hairpin loop is substantial. Surprisingly, the APOBEC-signature mutation density per TC motif in the single-stranded DNA of a G-quadruplex (G4) is lower than in the four-stranded part of G4 and in B-DNA. The APOBEC mutagenesis, as well as the UV-mutagenesis in melanoma samples are absent in Z-DNA regions, due to depletion of their mutational signature motifs.

Background: The advent of next-generation sequencing (NGS) has revolutionized the analysis of genetic data, enabling rapid identification of pathogenic variants in patients with inborn errors of immunity (IEI). Sometimes, the use of NGS-based technologies is associated with challenges in the evaluation of the clinical significance of novel genetic variants. Methods: In silico prediction tools, such as SpliceAI neural network, are often used as a first-tier approach for the primary examination of genetic variants of uncertain clinical significance. Such tools allow us to parse through genetic data and emphasize potential splice-altering variants. Further variant assessment requires precise RNA assessment by agarose gel electrophoresis and/or cDNA Sanger sequencing. Results: We found two novel heterozygous variants in the coding region of the LYST gene (c.10104G>T, c.10894A>G) in an individual with a typical clinical presentation of Chediak–Higashi syndrome (CHS). The SpliceAI neural network predicted both variants as probably splice-altering. cDNA assessment by agarose gel electrophoresis revealed the presence of abnormally shortened splicing products in each variant’s case, and cDNA Sanger sequencing demonstrated that c.10104G>T and c.10894A>G substitutions resulted in a shortening of the 44 and 49 exons by 41 and 47 bp, respectively. Both mutations probably lead to a frameshift and the formation of a premature termination codon. This, in turn, may disrupt the structure and/or function of the LYST protein. Conclusions: We identified two novel variants in the LYST gene, predicted to be deleterious by the SpliceAI neural network. Agarose gel cDNA electrophoresis and cDNA Sanger sequencing allowed us to verify inappropriate splicing patterns and establish these variants as disease-causing.

Introduction. Risk stratification in pediatric acute myeloid leukemia (AML) is traditionally based on the detection of chromosomal rearrangements using conventional molecular cytogenetic methods. However, these approaches are limited in detecting structurally complex and new genomic alterations. Whole-genome sequencing (WGS) overcomes these limitations, ensuring detection of all types of structural variants and providing their comprehensive molecular characterization. Aim: to evaluate the capabilities of WGS in identifying chromosomal rearrangements in children with AML treated under the AML-MRD-2018 protocol. Materials and methods. Out of 723 patients enrolled in the AML-MRD-2018 protocol, 335 (46%) children aged 0–18 years (median age – 9 years) were included in the study. WGS was performed on the DNBSEQ-T7 platform (150 × 2), with a median sequencing depth of 112×. Structural variants were identified using the MANTA algorithm with subsequent verification using a genome visualizer and confirmation by polymerase chain reaction and Sanger sequencing. Results. The results of WGS and standard testing methods were completely concordant in 228 (68%) out of 335 patients. WGS identified key stratifying translocations: KMT2Ar (n = 137; 41%), RUNX1::RUNX1T1 (n = 34; 10%), CBFB::MYH11 (n = 27; 8%), NUP98r (n = 19; 6%). In patients in whom standard methods had failed to detect markers, WGS revealed 26 rearrangements, including cryptic variants of KMT2A (n = 7), NUP98 (n = 5) and ERG rearrangements (n = 2), as well as rare fusion genes. Three novel fusion genes were identified. Overall, WGS provided additional clinically significant information in 40 cases, with an added diagnostic yield of 12%. Conclusion. WGS effectively detects both recurrent and rare, cryptic, and structurally complex chromosomal rearrangements, while providing their thorough molecular characterization. The method allows for improved risk stratification and can serve as a basis for minimal residual disease monitoring, increasing diagnostic accuracy and the potential for treatment personalization.

Введение. Саркома с аберрацией гена BCOR представляет собой относительно недавно охарактеризованную группу опухолей, характеризующуюся аберрациями гена BCOR, приводящих к его онкогенной активации, в результате чего они имеют разнообразное клиническое поведение, отличное от саркомы Юинга (СЮ), с которой данные опухоли имеют определенное морфологическое сходство. Морфологический диагноз в подавляющем большинстве случаев оказывается верен, однако для его подтверждения порой приходится прибегать к многообразию методик молекулярной диагностики для определения прямых или суррогатных диагностических маркеров. Материалы и методы. В исследование вошло 23 пациента с молекулярно-подтвержденным диагнозом саркомы с аберрацией гена BCOR при помощи ОТ-ПЦР, секвенирования РНК или метода цифрового баркодирования РНК Nanostring. Всем пациентам проведена оценка клинических данных и показателей общей и бессобытийной выживаемости в зависимости от проведенного лечения методом Каплана — Майера. Результаты. У 22 (96 %) пациентов из 23 входящим морфологическим диагнозом являлась саркома с аберрацией гена BCOR, у одного (4 %) пациента — недифференцированная мелкокруглоклеточная саркома (НМКкС). Морфологический диагноз был подтвержден в 17 из 23 случаев методом ОТ-ПЦР, в двух случаях — с пограничным качеством выделенной РНК методом цифрового баркодирования РНК Nanostring, у четверых — методом высокопроизводительного секвенирования с обнаружением более редких аберраций — BCOR ITD (ex15), BCOR::MAML3 (ex15::ex2) и YWHAE::NUTM2B (ex5::ex2). Медиана возраста на момент заболевания составила 10,5 (0,25–16,4) года. Локализованная форма была диагностирована в 17 (74 %) случаях, в 6 (26 %) определялись отдаленные метастазы. 13 (57 %) пациентов получали лечение в соответствии с протоколами Euro Ewing-2008 и 2012, 10 (43 %) — CWS-2009. Рецидив развился у восьми человек (35 %), у шести из восьми — локальный, у двух — системный. Общая трехлетняя выживаемость среди 23 пациентов составила 96,0 ± 0,04 %, бессобытийная выживаемость — 66,8 ± 0,12 %. Выводы. Для пациентов с наиболее часто встречаемой перестройкой BCOR::CCNB3 характерны индолентное течение, тропность к костям осевого скелета и преобладание у лиц мужского пола. При сравнении бессобытийной выживаемости в зависимости от использования юинг-ориентированного протокола лечения или протокола лечения для мягкотканых сарком достоверной разницы не получено.