Quizartinib with donor lymphocyte infusion for post-transplant relapse of FLT3-ITD-positive acute myeloid leukemia.

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-positive acute myeloid leukemia (AML) has a poor prognosis, particularly with DNMT3A and NPM1 mutations. Quizartinib, a FLT3 inhibitor showing clinical benefit in FLT3-ITD-positive AML, has unclear safety and efficacy when combined with donor lymphocyte infusion (DLI). We report a case of FLT3-ITD-positive AML with DNMT3A and NPM1 mutations that relapsed after allogeneic hematopoietic stem cell transplantation (allo-HCT) and was treated with quizartinib and DLI. A 49-year-old man was diagnosed with AML. Target-sequencing analysis of the bone marrow revealed FLT3-ITD, DNMT3A R882, and NPM1 mutations. Although the patient achieved complete remission (CR) through induction therapy and received allo-HCT, he relapsed on day 71. Quizartinib was initiated on day 79, and the patient achieved CR with incomplete recovery on day 106. He did not desire a second allo-HCT and continued quizartinib in combination with DLI, which was started on day 156 and administered eight times every 2 to 3 months. The patient achieved hematological CR on day 163 and remained in molecular CR 3 years after allo-HCT without adverse effects. Quizartinib combined with DLI may be a feasible treatment for early relapse of FLT3-ITD-positive AML after allo-HCT, even with concurrent DNMT3A and NPM1 mutations.

Case Report: Structurally Rare EML4-ALK Identified by Next Generation Sequencing in a Patient with NSCLC with Bilateral Ovarian Metastases.

The EML4-ALK oncogene is a fusion of the EML4 and ALK genes and is found in approximately 5-6% of the cases of non-small cell lung cancer (NSCLC). Herein, we present a unique case of lung adenocarcinoma with metastases to the bilateral ovaries harboring a rare EML4-ALK fusion gene variant in a 52-year-old patient. The patient had initially received a diagnosis of ovarian cancer, then had undergone neo-adjuvant chemotherapy followed by a surgical resection. Despite two cycles of adjuvant chemotherapy consisting of carboplatin and gemcitabine, CT revealed that the pleural effusion had increased from it before chemotherapy, and the shortness of breath worsened. Molecular profiling revealed an EML4-ALK rearrangement containing ALK -EML4 and ALK -NPR2 fusion genes. The diagnosis was changed to primary lung adenocarcinoma with metastases to the bilateral ovaries based on a pathological reevaluation. Treatment with alectinib, a second-generation ALK-tyrosine kinase inhibitor, led to a partial response of 18 months' duration, and the shortness of breath improved. No adverse events related to the alectinib therapy occurred. To assess the unique structure of the fusion genes, RNA sequencing was performed. An intronic sequence from both ALK and EML4 was found between ALK and EML4 exon, possibly because of an unusual insertion of a gene fragment derived from NRP2, indicated by the panel sequencing results. Variations in the drug response among EML4-ALK fusion variants highlight the importance of understanding their molecular structure. Further investigation is warranted to refine fusion gene detection methods and assess the therapeutic implications of rare fusion variants.

RNA-DNA hybrids on protein coding genes are stabilized by loss of RNase H and are associated with DNA damages during S-phase in fission yeast.

RNA-DNA hybrid is a part of the R-loop which is an important non-standard nucleic acid structure. RNA-DNA hybrid/R-loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA-DNA hybrids and map their genomic locations in fission yeast cells. The RNA-DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild-type. The majority of RNA-DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S-phase and about 20% of the RNA-DNA hybrids overlap with Rad52 loci. During S-phase, more robust association of Rad52 with RNA-DNA hybrids was observed in the protein coding region than in M-phase. These results suggest that persistent RNA-DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S-phase, potentially through collision with DNA replication forks.

Successful treatment of two cases with Philadelphia-chromosome positive acute lymphoblastic leukemia who relapsed after allogeneic stem cell transplantation and the treatments with novel immunotherapies and ponatinib.

The outcomes of relapsed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) resistant to new drugs such as tyrosine kinase inhibitors, inotuzumab ozogamicin (InO) and blinatumomab are dismal. We treated two cases of Ph+ALL resistant to these drugs that achieved long-term survival after treatment with chimeric antigen receptor (CAR)-T cell therapy or a second allogeneic hematopoietic stem cell transplantation (HCT) with a sequential conditioning regimen. Case 1: A 15-year-old boy was diagnosed with Ph+ALL. Despite the second HCT after the treatment of ponatinib and blinatumomab, hematological relapse occurred. InO was ineffective and he was transferred to a CAR-T center. After the CAR-T cell therapy, negative measurable residual disease (MRD) was achieved and maintained for 38 months without maintenance therapy. Case 2: A 21-year-old man was diagnosed with Ph+ALL. Hematological relapse occurred after the first HCT. Despite of the treatment with InO, ponatinib, and blinatumomab, hematological remission was not achieved. The second HCT was performed using a sequential conditioning regimen with clofarabine. Negative MRD was subsequently achieved and maintained for 42 months without maintenance therapy. These strategies are suggestive and helpful to treat Ph+ALL resistant to multiple immunotherapies.

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.

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.

[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.

CHD5 gene variant predicts leptomeningeal metastasis after surgical resection of brain metastases of breast cancer.

PURPOSE: Leptomeningeal metastasis (LM) is a complication of surgery for brain metastasis and is a risk factor of poor prognosis. The risk of LM is particularly high after surgery for a breast cancer metastasis to the brain. If the risk of LM after surgical resection for a brain metastasis were predictable, appropriate adjuvant therapy could be administered to individual patients to improve their prognosis. The present study aimed to reveal the genetic characteristics of brain metastases as means of predicting LM in breast cancer patients. METHODS: Ten patients with brain metastases of breast cancer presented LM after surgical resection were analyzed by whole-exome sequencing. RESULTS: A chromodomain-helicase-DNA-binding protein 5 (CHD5) gene alteration was detected in nine cases (90%), including a nonsynonymous variant in four cases and copy number deletion in five cases. CHD5 protein expression was lost in nine cases and had decreased in one case. The frequency of CHD5 gene alteration in brain metastases with LM was significantly higher than in primary breast cancer (2.3%) or in brain metastases of breast cancer (0%) (p < 0.0001). CONCLUSIONS: These results suggested that the CHD5 gene alteration was associated with LM after surgical resection of breast cancer brain metastases. Searching for the gene alteration might predict the LM risk after surgical resection.

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.

Attenuated humoral response against SARS-CoV-2 mRNA vaccination in allogeneic stem cell transplantation recipients.

Antibody persistence several months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccination in allogeneic stem cell transplantation recipients remains largely unknown. We sequentially evaluated the humoral response to two doses of mRNA vaccines in 128 adult recipients and identified the risk factors involved in a poor response. The median interval between stem cell transplantation and vaccination was 2.7 years. The SARS-CoV-2 S1 Ab became positive after the second vaccination dose in 87.6% of the recipients, and the median titer was 1235.4 arbitrary units (AU)/ml. In patients on corticosteroid treatment, the corticosteroid dose inversely correlated with Ab titer. Multivariate analysis identified risk factors for poor peak response such as an interval from stem cell transplantation ≤1 year, history of clinically significant CMV infection, and use of >5 mg/day prednisolone at vaccination. Six months after vaccination, the median titer decreased to 185.15 AU/ml, and use of >5 mg/day prednisolone at vaccination was significantly associated with a poor response. These results indicate that early vaccination after stem cell transplantation (<12 months) and CMV infection are risk factors for poor peak response, while steroid use is important for a peak as well as a persistent response. In conclusion, although humoral response is observed in many stem cell transplantation recipients after two doses of vaccination, Ab titers diminish with time, and factors associated with persistence and a peak immunity should be considered separately.

Establishment and characterization of a new activated B-cell-like DLBCL cell line, TMD12.

We report the establishment of a novel activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, from a patient with highly refractory DLBCL. ABC-DLBCL is a subtype with a relatively unfavorable prognosis that was originally categorized using gene expression profiling according to its cell of origin. TMD12 cells were isolated from the pleural effusion of the patient at relapse and passaged continuously in vitro for >4 years. The cells displayed cluster of differentiation (CD)19, CD20, CD22, CD38, human leukocyte antigen-DR isotype, and κ positivity and CD5, CD10, CD23, and λ negativity, as detected using flow cytometric analysis. The chromosomal karyotypic analysis, including the spectral karyotyping method, confirmed t(1;19)(q21:q13.1), del(6q23), gain of chromosome 18, and other abnormalities. Mutation analyses, including whole-exome sequencing, revealed that TMD12 cells harbored mutations in MYD88 and CD79B, indicating an ABC subtype. TMD12 cells exhibited chronic active B-cell receptor signaling and constitutive activation of the nuclear factor κB pathway, which is typically associated with sensitivity to a specific Bruton tyrosine kinase inhibitor, ibrutinib. Intriguingly, TMD12 cells displayed moderate resistance to ibrutinib and lacked activation of Janus kinase/signal transducers and activators of transcription 3 signaling, another hallmark of this DLBCL subtype. Treatment with an inhibitor against tumor progression locus 2 (TPL2), a multifunctional intracellular kinase that is activated particularly downstream of Toll-like receptors or MYD88 and IκB kinase α/ß (IKKα/ß), suppressed the proliferation of TMD12 cells, implying the possible involvement of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. Because only a limited number of ABC-DLBCL cell lines are currently available, TMD12 cells might provide a useful tool in the search for novel druggable targets for this intractable lymphoma.

INT6/eIF3e represses E-cadherin expression through HIF2α in lung carcinoma A549 cells.

Hypoxia-inducible factor 2 α (HIF2α), a transcription factor playing a vital role in hypoxia, promotes cancer metastasis. We had previously reported that the cancer-related gene integration site 6/eukaryotic translation initiation factor 3 subunit e (INT6/eIF3e) negatively regulates the protein stability of HIF2α in an oxygen-independent manner. Presently, the downstream targets for INT6/eIF3e-regulated HIF2α are unknown. Given the roles of HIF2α and INT6/eIF3e in epithelial-mesenchymal transition (EMT) that promotes cancer metastasis, we hypothesized that INT6/eIF3e-regulated HIF2α controls EMT. This study shows that INT6/eIF3e knockdown in lung carcinoma A549 cells led to increased expression of HIF2α protein and an EMT-like phenotypic change. The increased HIF2α subsequently repressed the E-cadherin gene. Mechanistically, HIF2α interacts with the twist family bHLH transcription factor 1 (TWIST1) known to regulate EMT process, and binds to the proximal promoter region of E-cadherin, repressing it. Collectively, our work demonstrates that HIF2α, regulated by INT6/eIF3e, represses the E-cadherin gene through TWIST1 to enhance EMT, suggesting a role of the INT6/eIF3e-HIF2α axis in cancer metastasis.

[Klinefelter's syndrome diagnosed at the onset of acute myeloid leukemia with inv (16) following treatment for germ cell tumor].

A 22-year-old man with a history of mediastinal germ cell tumor, which was diagnosed at age 20 and remained disease-free after chemotherapy, was diagnosed with acute myeloid leukemia (AML) M2 in January 2020. Karyotype analysis of bone marrow (BM) specimen at diagnosis detected 47,XXY, inv (16) in all cells. Following induction treatment, he achieved complete remission with a remarkable decrease in the minimal residual disease marker. Although considered related to therapy, the AML had a prognostically favorable karyotype, and the initial treatment response was very good. He had no human leukocyte antigen-matched sibling donor candidate. Thus, allogeneic hematopoietic stem cell transplantation was not scheduled at the first complete remission. After three cycles of consolidation therapy, he remained disease-free for over one year. Karyotype analysis of BM during remission revealed that all analyzed cells harbored 47,XXY, and Klinefelter syndrome (KS) was diagnosed. Although the patient experienced an adjustment disorder on KS diagnosis, he had overcome the difficulty with the assistance of psycho-oncologists, clinical psychologists, and genetic counselors. Herein, we report this rare case of KS that manifested after AML diagnosis following mediastinal germ cell tumor treatment.

Mitochondrial Fragmentation Triggers Ineffective Hematopoiesis in Myelodysplastic Syndromes.

Ineffective hematopoiesis is a fundamental process leading to the pathogenesis of myelodysplastic syndromes (MDS). However, the pathobiological mediators of ineffective hematopoiesis in MDS remain unclear. Here, we demonstrated that overwhelming mitochondrial fragmentation in mutant hematopoietic stem cells and progenitors (HSC/P) triggers ineffective hematopoiesis in MDS. Mouse modeling of CBL exon deletion with RUNX1 mutants, previously unreported comutations in patients with MDS, recapitulated not only clinically relevant MDS phenotypes but also a distinct MDS-related gene signature. Mechanistically, dynamin-related protein 1 (DRP1)-dependent excessive mitochondrial fragmentation in HSC/Ps led to excessive reactive oxygen species production, induced inflammatory signaling activation, and promoted subsequent dysplasia formation and impairment of granulopoiesis. Mitochondrial fragmentation was generally observed in patients with MDS. Pharmacologic inhibition of DRP1 attenuated mitochondrial fragmentation and rescued ineffective hematopoiesis phenotypes in mice with MDS. These findings provide mechanistic insights into ineffective hematopoiesis and indicate that dysregulated mitochondrial dynamics could be a therapeutic target for bone marrow failure in MDS. SIGNIFICANCE: We demonstrated that excessive mitochondrial fragmentation is a fundamental pathobiological phenomenon that could trigger dysplasia formation and ineffective hematopoiesis in MDS. Our findings provide mechanistic insights into ineffective hematopoiesis and suggest dysregulated mitochondrial dynamics as a therapeutic target for treating MDS.This article is highlighted in the In This Issue feature, p. 1.

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.

Successful Cord Blood Transplantation for Idiopathic CD4+ Lymphocytopenia.

Idiopathic CD4+ lymphocytopenia (ICL) is the depletion of CD4+ lymphocytes to <300 cells/mm3 without human immunodeficiency virus infection or other causes of lymphocytopenia. ICL causes fatal infections; its etiology remains unclear and it lacks consensus regarding therapeutic options. We report the first patient with ICL who had a successful clinical course following a cord blood transplant (CBT). A 45-year-old woman was diagnosed with ICL and underwent partial hepatectomy for an abscess caused by the Mycobacterium avium complex. No specific gene alterations were detected through next generation sequencing-based evaluation. Following a reduced-intensity conditioning (RIC) regimen consisting of fludarabine, busulfan, and 4 Gy total body irradiation, a single-unit CBT was performed. Neutrophils were engrafted on day +14. CD4+ lymphocyte counts increased to over 300 cells/mm3 on day +436. After 75 months, she was alive without any sequelae. CBT with an RIC regimen could be a curable treatment option for ICL.