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Telomeres are specialized nucleoprotein complexes, localized at the physical ends of chromosomes, that contribute to the maintenance of genome stability. One of the features of chronic myeloid leukemia (CML) cells is a reduction in telomere length which may result in increased genomic instability and progression of the disease. Aberrant telomere maintenance in CML is not fully understood and other mechanisms such as the alternative lengthening of telomeres (ALT) are involved. In this work, we employed five BCR-ABL1-positive cell lines, namely K562, KU-812, LAMA-84, MEG-A2, and MOLM-1, commonly used in the laboratories to study the link between mutation, copy number, and expression of telomere maintenance genes with the expression, copy number, and activity of BCR-ABL1. Our results demonstrated that the copy number and expression of BCR-ABL1 are crucial for telomere lengthening. We observed a correlation between BCR-ABL1 expression and telomere length as well as shelterins upregulation. Next-generation sequencing revealed pathogenic variants and copy number alterations in major tumor suppressors, such as TP53 and CDKN2A, but not in telomere-associated genes. Taken together, we showed that BCR-ABL1 kinase expression and activity play a crucial role in the maintenance of telomeres in CML cell lines. Our results may help to validate and properly interpret results obtained by many laboratories employing these in vitro models of CML.

Endometriosis is an inflammatory condition manifested by the presence of endometrial-like tissue outside of the uterine cavity. The most common clinical presentations of endometriosis are dysmenorrhea, infertility, and severe pelvic pain. Few hypotheses attempt to explain the pathogenesis of endometriosis; however, none of the theories have been fully confirmed or considered universal. We examined somatic mutations in eutopic endometrium samples, deep endometriotic nodules and peripheral blood from 13 women with deep endometriosis of the rectovaginal space. Somatic variants were identified in laser microdissected samples using next-generation sequencing. A custom panel of 1296 cancer-related genes was employed, and selected genes representing cancer drivers and non-drivers for endometrial and ovarian cancer were thoroughly investigated. All 59 detected somatic variants were of low mutated allele frequency (<10%). In deep ectopic lesions, detected variants were significantly more often located in cancer driver genes, whereas in eutopic endometrium, there was no such distribution. Our results converge with other reports, where cancer-related mutations were found in endometriosis without cancer, particularly recurrent KRAS mutations. Genetic alterations located in ectopic endometriotic nodules could contribute to their formation; nevertheless, to better understand the pathogenesis of this disease, more research in this area must be performed.

Hypopharyngeal cancer is a poorly characterized type of head and neck squamous cell carcinoma (HNSCC) with bleak prognosis and only few studies focusing specifically on the genomic profile of this type of cancer. We performed molecular profiling of 48 HPV (Human Papilloma Virus)-negative tumor samples including 23 originating from the hypopharynx and 25 from the larynx using a targeted next-generation sequencing approach. Among genes previously described as significantly mutated, TP53, FAT1, NOTCH1, KMT2C, and CDKN2A were found to be most frequently mutated. We also found that more than three-quarters of our patients harbored candidate actionable or prognostic alterations in genes belonging to RTK/ERK/PI3K, cell-cycle, and DNA-damage repair pathways. Using previously published data we compared 67 hypopharyngeal cancers to 595 HNSCC from other sites and found no prominent differences in mutational frequency except for CASP8 and HRAS genes. Since we observed relatively frequent mutations of KTM2C (MLL3) in our dataset, we analyzed their role, in vitro, by generating a KMT2C-mutant hypopharyngeal cancer cell line FaDu with CRISPR-Cas9. We demonstrated that KMT2C loss-of-function mutations resulted in increased colony formation and proliferation, in concordance with previously published results. In summary, our results show that the mutational profile of hypopharyngeal cancers might be similar to the one observed for other head and neck cancers with respect to minor differences and includes multiple candidate actionable and prognostic genetic alterations. We also demonstrated, for the first time, that the KMT2C gene may play a role of tumor suppressor in HNSCC, which opens new possibilities in the search for new targeted treatment approaches.

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Clinical resistance to tyrosine kinase inhibitors (TKI) remains a significant problem in the therapy of patients with chronic myeloid leukemia (CML). Although BCR-ABL1-kinase domain mutations are the major cause of resistance to TKI, some patients manifest primary or develop secondary resistance to TKI without detectable BCR-ABL1 mutations. We aimed to assess the prevalence of additional gene mutations in a group of 50 patients with primary (n = 26) or secondary resistance (n = 24) to TKI, in most cases to imatinib (n = 49) and in one to dasatinib. We employed ~1000 genes custom target enrichment kit and next-generation sequencing on Illumina platform, as well as Sanger sequencing. In 21 patients, we were able to match and analyze paired samples collected at diagnosis (before treatment) and at the time of resistance and in five other cases we analyzed additional samples collected later during TKI-resistant chronic phase or in blast crisis. The most frequent genetic aberrations detected at the time of TKI-resistance were mutations in ASXL1 and BCR-ABL1 kinase domain coding sequence, present in 28% (14/50) and 26% (13/50) of patients, respectively. Both genes were mutated in 12% of patients (6/50), while 30% (15/50) had either ASXL1 or BCR-ABL1 mutations alone. Non-recurrent genetic aberrations in other genes were also noted in single patients (e.g. DNMT3A mutations). According to COSMIC database, all but three ASXL1 mutations detected in this study were previously described in hematologic malignancies and all (including the novel ones) are predicted to introduce stop codons or cause frameshifts in codon range of 512-943, thus truncating the protein before the C-terminal PHD domain. Among patients with available paired samples three had more than one ASXL1 mutation at various time points and in all of them we observed dynamics of specific ASXL1 mutations, related partially to the leukemic cell content in the sample but also providing evidence of clonal evolution (Table 1). In most of patients with concurrent ASXL1 and BCR-ABL1 mutations, we observed that both mutations were present with similar variant allele frequency (VAF), suggesting that ASXL1 mutations occur in Philadelphia-positive leukemic clones (Table 2). ASXL1 mutation frequency was significantly higher in TKI-resistant patients, than in our previously characterized group of CML patients without resistance, who achieved major molecular response (28% ; 14/50 vs 5.6% ; 2/36, respectively, p = 0.0105, Fisher exact test). All ASXL1-mutated patients with secondary (n = 1) or primary resistance (n = 5) for whom diagnostic sample was available, carried ASXL1 mutation also at the time of diagnosis. No significant difference in mutation frequency was found between patients with primary and secondary resistance (ASXL1 mutation, 26.9% vs 29.2% ; BCR-ABL1 mutation, 15.4% vs 37.5%). Our results provide evidence that preexisting ASXL1 mutations in BCR-ABL1-positive leukemic clone present at diagnosis may have impact on clinical response to imatinib and may be useful in assessing the risk of treatment failure. Disclosures Niesiobedzka-Krezel: Novartis: Honoraria. Seferynska:Novartis: Honoraria. Gora Tybor:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sacha:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Background Ibrutinib, an inhibitor of the Bruton's kinase (BTK), is characterized by high efficacy in the therapy of patients with relapsed and refractory chronic lymphocytic leukemia (RR‐CLL). Aims To analyze the potential significance of the mutational status of selected 30 genes on the disease outcome in 45 patients with RR‐CLL using custom‐made gene panel and sequencing on Illumina MiSeq FGx platform. Results The highest rate of mutations was observed in TP53 (n = 18; 40.0%), NOTCH1 (n = 13; 28.8%), SF3B1 (n = 11; 24.4%), ATM (n = 7; 15.6%), MED12 (n = 6, 13.3%), CHD2 (n = 5; 11.1%), XPO1 (n = 5; 11.1%), NFKBIE (n = 5; 11.1%), BIRC3 (n = 4; 8.9%), SPEN (n = 4; 8.9%), POT1 (n = 4; 8.9%), EGR2 (n = 3; 6.7%), and RPS15 (n = 3; 6.7%). With a median observation time of 45.9 months, the median progression‐free survival (PFS) and overall survival (OS) were not reached. The 36‐month estimated rate of PFS and OS were 64% and 68.2%, respectively. The overall response rate was noted in 23 patients (51.1%), while twenty (44.4%) patients achieved stability. Progression was noted in 2 (4.5%) cases. Analyzed molecular factors had no impact on PFS and OS. Conclusion Despite accumulation of several poor prognostic factors in our real‐life cohort of heavily pretreated patients with CLL, ibrutinib treatment showed long‐term clinical benefit.