Impact of genetic alterations on central nervous system progression of primary vitreoretinal lymphoma.

Primary vitreoretinal lymphoma (PVRL) is a rare malignant lymphoma subtype with an unfavorable prognosis due to frequent central nervous system (CNS) progression. Thus, identifying factors associated with CNS progression is essential for improving the prognosis of PVRL patients. Accordingly, we conducted a comprehensive genetic analysis using archived vitreous humor samples of 36 PVRL patients diagnosed and treated at our institution and retrospectively examined the relationship between genetic alterations and CNS progression. Whole-exome sequencing (n = 2) and amplicon sequencing using a custom panel of 107 lymphomagenesis-related genes (n = 34) were performed to assess mutations and copy number alterations. The median number of pathogenic genetic alterations per case was 12 (range: 0- 22). Pathogenic genetic alterations of CDKN2A, MYD88, CDKN2B, PRDM1, PIM1, ETV6, CD79B, and IGLL5, as well as aberrant somatic hypermutations, were frequently detected. The frequency of ETV6 loss and PRDM1 alteration (mutation and loss) was 23% and 49%, respectively. Multivariate analysis revealed ETV6 loss (hazard ratio [HR]: 3.26, 95% confidence interval [CI]: 1.08-9.85) and PRDM1 alteration (HR: 2.52, 95% CI: 1.03-6.16) as candidate risk factors associated with CNS progression of PVRL. Moreover, these two genetic factors defined slow-, intermediate-, and rapid-progression groups (0, 1, and 2 factors, respectively), and the median period to CNS progression differed significantly among them (52 vs. 33 vs. 20 months, respectively). Our findings suggest that genetic factors predict the CNS progression of PVRL effectively, and the genetics-based CNS progression model might lead to stratification of treatment.

[FLT3-ITD mutation-positive acute myeloid leukemia undergoing clonal transition with PTPN11 mutation at relapse].

A 28-year-old man was diagnosed with acute myelomonocytic leukemia. He achieved complete remission (CR) after two cycles of induction therapy. However, after consolidation therapy, bone marrow aspiration performed to prepare for allogeneic hematopoietic stem cell transplantation revealed disease relapse. Companion diagnostics confirmed the presence of the FLT3-ITD mutation. The patient received gilteritinib monotherapy and achieved CR. Subsequently, he underwent unrelated allogeneic bone marrow transplantation. One year after transplantation, the patient relapsed, and gilteritinib was resumed. However, the leukemia progressed, and panel sequencing using a next-generation sequencer showed that the FLT3-ITD mutation disappeared. A mutation in PTPN11, which regulates the RAS/MAPK signaling pathway, was also detected. Gilteritinib was discontinued, and the patient achieved CR with salvage chemotherapy. He underwent related haploidentical peripheral blood stem cell transplantation but died of relapse. This was a case in which genetic analysis revealed clonal transition and acquisition of resistance to treatment.

Azacitidine and gemtuzumab ozogamicin as post-transplant maintenance therapy for high-risk hematologic malignancies.

Disease recurrence remains the principal cause of treatment failure after allogeneic hematopoietic stem cell transplantation. Post-transplant maintenance therapy with azacitidine (AZA) is promising to prevent relapse but the outcomes are unsatisfactory in patients at high risk of recurrence. Herein, we evaluated the outcome in patients who received AZA and gemtuzumab ozogamicin (GO), anti-CD33 antibody-calicheamicin conjugate, as post-transplant maintenance therapy. Twenty-eight patients with high-risk hematologic malignancies harboring CD33-positive leukemic blasts received the maintenance therapy. AZA (30 mg/m2) was administered for 7 days, followed by GO (3 mg/m2) on day 8. The maximum number of cycles was 4. At transplant, 21 patients (75.0%) had active disease. Their 2-year overall survival, disease-free survival, relapse, and non-relapse mortality rates were 53.6%, 39.3%, 50.0%, and 10.7%, respectively. Of these patients, those with minimal residual disease at the start of maintenance therapy (n = 9) had a higher recurrence rate (66.7% vs. 42.1% at 2 years, P = 0.069) and shorter disease-free survival (11.1% vs. 52.6% at 2 years, P = 0.003). Post-transplant maintenance therapy with AZA and GO was generally tolerable but more than half of the patients eventually relapsed. Further improvements are needed to prevent relapse after transplantation in patients with high-risk hematologic malignancies.

Impact of gene alterations on clinical outcome in young adults with myelodysplastic syndromes.

Young adults with myelodysplastic syndrome (MDS) are rare, and the clinical significance of driver mutations has not yet been analysed. We analysed the gene mutations and copy number alterations (CNAs) in younger MDS patients using next-generation sequencing, targeting 68 genes that were recurrently mutated in myeloid malignancies, to investigate the correlation between their genetic alterations and clinical outcomes. We enrolled 55 patients retrospectively (aged < 50 years). At least one mutation was detected in 56% of the patients. The most frequently mutated genes were ASXL1 and RUNX1, 13% each. We defined higher-risk patients as those with ≥ 2 mutations, except for SF3B1 mutation, and/or CNA. The 3-year overall survival (OS) in patients with a higher-risk was lower than that in those with a lower-risk (50.8% vs. 71.8%, P = 0.024). Among the 44 transplant recipients, patients with higher-risk had a significantly lower OS and tended to have a higher cumulative incidence of relapse (CIR) than those with a lower-risk (3-year OS: 38.0% vs. 64.4%, P = 0.039; 3-year CIR: 44.0% vs. 24.1%, P = 0.076). Our results showed that genetic aberrations can predict clinical outcomes in younger MDS patients, despite the low rate of genetic mutations.

Mutation profiles of diffuse large B-cell lymphoma transformation of splenic B-cell lymphoma/leukemia, unclassifiable on whole-exome sequencing.

A 58-year-old male was diagnosed with splenic B-cell lymphoma/leukemia, unclassifiable (SPLL-U). The lymphoma transformed into diffuse large B-cell lymphoma (DLBCL), and multidrug chemotherapy and autologous stem cell transplantation achieved complete remission. Two years later, the lymphoma relapsed as SPLL-U. Serial whole-exome sequencing indicated that the mutation profiles were similar between the onset and relapsed samples while those in DLBCL were partially distinctive, which was in line with the clinical course. Hierarchical clustering revealed that an IGLL5 mutation was the founder mutation proceeding the development of the diseases and suggested that KRAS and other mutations might contribute to the transformation.

Archival bone marrow smears are useful in targeted next-generation sequencing for diagnosing myeloid neoplasms.

Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.

Prognostic impact of TP53 mutation, monosomal karyotype, and prior myeloid disorder in nonremission acute myeloid leukemia at allo-HSCT.

Outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in nonremission acute myeloid leukemia (AML) are dismal [2-year overall survival (OS): 20-30%]. Though several risk classifications have been used, some factors are unavailable until the start of conditioning or transplantation. We analyzed prognostic gene mutations by targeted next-generation sequencing to identify predisposing factors for predicting OS at 1 month before transplantation. We enrolled 120 patients with nonremission AML who underwent first allo-HSCT between 2005 and 2018. Mutations were found in 98 patients; frequently mutated genes were FLT3-ITD, TP53, RUNX1, and WT1. TP53 mutation was detected in 21 patients and was the only predictor of poor OS. Multivariate analysis using Cox regression hazard model revealed primary AML, monosomal karyotype (MK), and TP53 mutation as independent factors for predicting poor OS. Based on these, patients were stratified into three groups. The low-risk group included patients with prior myeloid disorder without MK (n = 26). Among the rest, patients with TP53 mutation were assigned to the high-risk group (n = 19) and the rest into the intermediate-risk group (n = 75). Two-year OS in low-, intermediate-, and high-risk groups differed significantly (50.0%, 24.9%, and 0%, respectively). This suggests that the indication of allo-HSCT should be carefully judged for high-risk patients.

Potential prognostic impact of EBV RNA-seq reads in gastric cancer: a reanalysis of The Cancer Genome Atlas cohort.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC), whose prognosis remains controversial, is diagnosed by in situ hybridization of EBV-derived EBER1/2 small RNAs. In The Cancer Genome Atlas (TCGA) Stomach Adenocarcinoma (STAD) project, the EBV molecular subtype was determined through a combination of multiple next-generation sequencing methods, but not by the gold standard in situ hybridization method. This leaves unanswered questions regarding the discordance of EBV positivity detected by different approaches and the threshold of sequencing reads. Therefore, we reanalyzed the TCGA-STAD RNA sequencing (RNA-seq) dataset including 375 tumor and 32 normal samples, using our analysis pipeline. We defined a reliable threshold for EBV-derived next-generation sequencing reads by mapping them to the EBV genome with three different random arbitrary alignments. We analyzed the prognostic impact of EBV status on the histopathological subtypes of gastric cancer. EBV-positive cases identified by reanalysis comprised nearly half of the cases (49.6%) independent from infiltrating lymphocyte signatures, and showed significantly longer overall survival for adenocarcinomas of the 'not-otherwise-specified' type [P = 0.016 (log-rank test); hazard ratios (HR): 0.476; 95% CI: 0.260-0.870, P = 0.016 (Cox univariate analysis)], but shorter overall survival for the tubular adenocarcinoma type [P = 0.005 (log-rank test); HR: 3.329; 95% CI: 1.406-7.885, P = 0.006 (Cox univariate analysis)]. These results demonstrate that the EBV positivity rates were higher when determined by RNA-seq than when determined by EBER1/2 in situ hybridization. The RNA-seq-based EBV positivity demonstrated distinct results for gastric cancer prognosis depending on the histopathological subtype, suggesting its potential to be used in clinical prognoses.

Discovery of Novel Spiroindoline Derivatives as Selective Tankyrase Inhibitors.

The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/ß-catenin signal by preventing poly ADP-ribosylation-dependent degradation of AXIN, a negative regulator of Wnt/ß-catenin signaling. With the goal of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out to find small-molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor with >7000-fold selectivity against the PARP1 enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.