Shingo Okuno

A new prognostic index to make short-term prognoses in MDS patients treated with azacitidine: A combination of p53 expression and cytogenetics

TP53 mutation is associated with various hematological malignancies and immunohistochemistry of p53 has been used as a simple method to establish the presence of a TP53 mutation. Since the significance of p53 expression is controversial in myelodysplastic syndrome (MDS) patients treated with azacitidine (Aza), we analyzed the prevalence of p53 expression as a prognostic factor in 60 MDS patients treated with Aza. To assess p53 expression, immunohistochemical analyses of bone marrow clot sections were performed. Overall survival (OS) was significantly lower in p53-positive patients compared with p53-negetive patients (59% vs. 85% at 12 months; P=0.006). Multivariate analysis demonstrated that p53-positive was a significant prognostic factor for OS along with poor cytogenetics. Here, we propose a new prognostic index to make short-term prognoses of MDS patients in the era of Aza treatment; high: p53-positive and poor cytogenetics, low: p53-negative and absence of poor cytogenetics, and intermediate: the others. OS was significantly different among the three groups according to this index (Low 92%, Intermediate 65% and High 27% at 12 months; P<0.0001). In conclusion, p53 expression was a significant prognostic factor in MDS patients treated with Aza. In combination with cytogenetic abnormalities, it is possible to make short-term prognoses.

High incidence of extensive chronic graft-versus-host disease in patients with the REG3A rs7588571 non-GG genotype

Regenerating islet-derived protein 3 alpha (REG3A) is a biomarker of lower gastrointestinal graft-versus-host disease (GVHD); however, the biological role of REG3A in the pathophysiology of GVHD is not understood. Here, we examined the association between a single nucleotide polymorphism in the REG3A gene, rs7588571, which is located upstream and within 2 kb of the REG3A gene, and transplant outcomes including the incidence of GVHD. The study population consisted of 126 adult Japanese patients who had undergone bone marrow transplantation from a HLA-matched sibling. There was no association between rs7588571 polymorphism and the incidence of acute GVHD. However, a significantly higher incidence of extensive chronic GVHD was observed in patients with the rs7588571 non-GG genotype than in those with the GG genotype (Odds ratio 2.6; 95% confidence interval, 1.1–6.0; P = 0.029). Semi-quantitative reverse transcription PCR demonstrated that the rs7588571 non-GG genotype exhibited a significantly lower REG3A mRNA expression level than the GG genotype (P = 0.032), and Western blot analysis demonstrated that the rs7588571 non-GG genotype exhibited a trend toward lower REG3A protein expression level than the GG genotype (P = 0.053). Since REG proteins have several activities that function to control intestinal microbiota, and since intestinal dysbiosis is in part responsible for the development of GVHD, our findings lead to the novel concept that REG3A could have some protective effect in the pathogenesis of GVHD through the regulation of gut microbiota.

Clinical efficacy of fecal occult blood test and colonoscopy for dasatinib-induced hemorrhagic colitis in CML patients

A positive fecal occult blood test (FOBT) is occasionally observed in some chronic myeloid leukemia (CML) patients treated with a tyrosine kinase inhibitor (TKI), and hemorrhagic colitis in patients treated with dasatinib has been reported. To clarify the frequency of TKI-induced hemorrhagic colitis and the screening efficacy of an FOBT followed by a colonoscopy, we prospectively enrolled CML patients treated with a TKI. FOBTs were performed in all patients and colonoscopy was performed in patients with positive FOBTs. When TKI-induced hemorrhagic colitis was pathologically identified, the TKI was interrupted, and the FOBTs were reevaluated. The first FOBT was positive in 10 of 30 patients. All patients with positive FOBTs were treated with dasatinib and developed no symptoms. Dasatinib-induced hemorrhagic colitis was confirmed in 6 of 18 patients treated with dasatinib (33%). Its endoscopic feature was a red flare and/or erosion. Immunohistological analyses showed CD3+, CD8+, CD56+, and Granzyme B+ cytotoxic T lymphocyte infiltration. After dasatinib discontinuation, the FOBTs became negative in all but one patient who had concurrent colorectal polyps. Dasatinib-induced hemorrhagic colitis was observed in one third of asymptomatic patients treated with dasatinib. An FOBT followed by a colonoscopy can be a useful strategy to detect the disease.

Introduction of Genetically-Modified CD3ζ Improves Proliferation and Persistence of Antigen-specific CTLs

It may be safer to improve adoptive T-cell therapy through increased signaling rather than increased antigen affinity of the TCRs. Modifying intracellular signaling enhanced the proliferation and persistence of CTLs while improving antitumor efficacy. The clinical efficacy of T-cell therapies based on T cells transduced with genes encoding tumor-specific T-cell receptors (TCR-T) is related to the in vivo persistence of the T cells. To improve persistence without modifying TCR affinity, we instead modified intracellular signaling, using artificial T cell–activating adapter molecules (ATAM), generated by inserting the intracellular domain (ICD) of activating T-cell signaling moieties into CD3ζ. ATAMs with the ICD of either CD28 or 4-1BB were generated, assembled into the TCR complex as a part of CD3ζ, and enhanced downstream signaling from the supramolecular activation cluster. ATAMs were retrovirally introduced into human CMV-specific or NY-ESO-1–specific TCR-transduced CD8+ T lymphocytes, and downstream functionality was then examined. ATAM-transduced NY-ESO-1 TCR-T cells were also investigated using the U266-xenograft mouse model. ATAMs were successfully transduced and localized to the cell membrane. ATAM-transduced CMV-specific T cells retained their cytotoxic activity and cytokine production against peptide-pulsed target cells without altering antigen-specificity and showed resistance to activation-induced cell death. Upon both single and repeated stimulation, CD3ζ/4-1BB–transduced T cells had superior proliferation to the CD3ζ-transduced T cells in both the CMV-specific and the NY-ESO-1 TCR-T models and significantly improved antitumor activity compared with untransduced T cells both in vitro and in a mouse xenograft model. ATAM-transduced TCR-T cells demonstrated improved proliferation and persistence in vitro and in vivo. This strategy to control the intracellular signaling of TCR-T cells by ATAM transduction in combination with various tumor-specific TCRs may improve the efficacy of TCR-T therapy. Cancer Immunol Res; 6(6); 733–44. ©2018 AACR.

Quantitative assessment of T cell clonotypes in human acute graft-versus-host disease tissues

Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-β deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated.

Successful treatment with allogeneic stem cell transplantation followed by DLI and TKIs for e6a2 BCR-ABL-positive acute myeloid leukaemia: A case report and literature review

Rationale: Patients with the e6a2 BCR-ABL transcript, 1 of the atypical transcripts, have been reported to have a poor prognosis, and allogeneic stem cell transplantation (ASCT) can be considered as additional therapy. However, long-term survival after ASCT for this disease is rare. Patient concerns: This report concerns a 55-year-old female patient with e6a2 BCR-ABL-positive acute myeloid leukemia including the outcome of ASCT followed by donor lymphocyte infusion (DLI). Diagnoses: The breakpoint was confirmed by direct sequencing. Her minimal residual disease could be detected by nested reverse-transcription polymerase chain reaction using primers for the minor BCR-ABL (e1a2) transcript. Interventions: Treatment with tyrosine kinase inhibitors (TKIs) and ASCT followed by DLI. Outcomes: Despite multiple cytogenetic and molecular relapses after ASCT, she remains in molecular remission at 46 months after ASCT. Lessons: This case indicates the efficacy of the combination of the graft-versus-leukemia effect and TKIs for e6a2 BCR-ABL-positive acute leukemia. When the Philadelphia chromosome with an unusual chromosomal breakpoint is suggested, we should clarify the breakpoint because that information can aid molecular assessments and decisions to provide an additional or alternative therapy.

Impact of Synchronous Multiple Primary Malignant Tumors on Newly Diagnosed Hematological Malignancies

Micro‐Abstract The existence of synchronous multiple primary malignant tumors was not a significant risk factor for patients with newly diagnosed hematological malignancies. It is important to provide adequate treatment to both hematological malignancies and solid tumors appropriately. Background: Hematological malignancies are occasionally observed with synchronous multiple primary malignant tumors (sMPMTs) at diagnosis. We aimed to clarify the impact of sMPMTs on newly diagnosed hematological malignancies and determine the optimal treatment strategies. Patients and Methods: We analyzed the outcomes of 649 patients with hematological malignancies, including 19 patients with sMPMTs (2.9%), and compared the outcomes between patients with and without sMPMTs. Results: The overall survival (OS) and disease‐free survival (DFS) rates for patients with sMPMTs were 77% and 70%, respectively, at 2 years; these rates were not statistically different from those for patients without sMPMTs (P = .17 and P = .64, respectively). Multivariate analysis showed that the presence of sMPMTs was not a significant prognostic factor for OS, DFS, or relapse (hazard ratio [HR] 1.48, 95% confidence interval [CI] 0.65‐3.38, P = .35; HR 0.97, 95% CI 0.46‐2.10, P = .97; and HR 0.79, 95% CI 0.29‐2.14, P = .65). In patients with sMPMTs, the order of treatment was not a significant prognostic factor. However, discontinuation of treatment was a marginally favorable factor and might reflect a selection bias. Conclusion: The presence of sMPMTs was not a significant risk factor for patients with newly diagnosed hematological malignancies. It is important to provide adequate treatment for both hematological malignancies and solid tumors at the physicians discretion.

Cervical epidural hematoma in a healthy donor presenting stroke mimic symptoms: a rare adverse event following peripheral blood stem cell apheresis

Peripheral blood stem cell apheresis from a healthy donor is indispensable for allogeneic peripheral blood stem cell transplantation. Here, we report a rare adverse event following peripheral blood stem cell apheresis. A female sibling donor, aged 61 years with an unremarkable medical history, complained of pain in the left neck and shoulder and numbness in the left upper limb 1 h after the end of peripheral blood stem cell apheresis. Paralysis of the left upper and lower limbs appeared consecutively. Computed tomography and magnetic resonance imaging of the head showed no abnormalities. Anticoagulant therapy was initiated according to the standard treatment of atherothrombotic brain infarction. Magnetic resonance imaging of the cervical cord on the following day revealed a cervical epidural hematoma. An emergency C4-C5 laminectomy was performed, and the paralysis was improved immediately after surgery. This report is the first case of cervical epidural hematoma in a healthy donor who underwent peripheral blood stem cell apheresis and presented symptoms confusingly similar to those of brain infarction.