ALBERT

Description: Background Helicobacter pylori, a bacterium that infects the human stomach, has high genetic diversity. Because its evolution is parallel to human, H. pylori is used as a tool to trace human migration. However, there are few studies about the relationship between phylogeography of H. pylori and its host human. Methods We examined both H. pylori DNA and the host mitochondrial DNA and Y-chromosome DNA obtained from a total 119 patients in the Dominican Republic, where human demography consists of various ancestries. DNA extracted from cultured H. pylori were analyzed by multi locus sequence typing. Mitochondrial DNA and Y-chromosome DNA were evaluated by haplogroup analyses. Results H. pylori strains were divided into 2 populations; 68 strains with African group (hpAfrica1) and 51 strains with European group (hpEurope). In Y-chromosomal haplogroup, European origin was dominant, whereas African origin was dominant both in H. pylori and in mtDNA haplogroup. These results supported the hypothesis that mother-to-child infection is predominant in H. pylori infection. The Amerindian type of mtDNA haplogroup was observed in 11.8% of the patients; however, Amerindian type (hspAmerind) of H. pylori was not observed. Although subpopulation type of most hpAfrica1 strains in Central America and South America were hybrid (hspWAfrica/hpEurope), most Dominican Republic hpAfrica1 strains were similar to those of African continent. Conclusions Genetic features of H. pylori, mtDNA, and Y haplogroups reflect the history of colonial migration and slave trade in the Dominican Republic. Discrepancy between H. pylori and the host human genotypes support the hypothesis that adaptability of hspAmerind H. pylori strains are weaker than hpEurope strains. H. pylori strains in the Dominican Republic seem to contain larger proportion of African ancestry compared to other American continent strains.

Description: Background: Pretreatment status assessment at diagnosis in patients with chronic myeloid leukemia in chronic phase (CML-CP) is more important for successful treatment in the tyrosine kinase inhibitors (TKIs) era. For instance, the Charlson comorbidity index (CCI) score is considered the useful tool for predicting overall survival (OS) in patients with CML treated with imatinib (IM) (Saussele S et al., 2015). However, the effect of CCI in patients with CML treated with second-generation TKI (2nd TKI) and clinical practice setting was unknown. This study aimed to evaluate the effect of pretreatment statuses, including CCI, on OS of participants of the New TARGET observational study 1 conducted by the Japanese Society of Hematology (JSH). Methods: Patients newly diagnosed with CML-CP were registered to the New TARGET observational study 1 from April 2010 to March 2013. They were treated with TKIs (IM, nilotinib [NIL], or dasatinib [DAS]) after registration. Exclusion criteria were defined as follows: (1) accelerated phase/blast crisis (AP/BC) CML and (2) pretreatment with interferon-alfa, any TKIs, or hydroxyurea for more than 3 months, or allogeneic hematopoietic stem cell transplantation before registration. Other details of the protocol were previously reported (Kizaki et al., 2019). Patients were classified into CCI risk groups of 2, 3, and ≥4 for analysis. The New TARGET observational study 1 was supported by research fundings from Novartis Pharmaceuticals and Bristol-Myers Squibb to JSH. This subgroup analysis was approved by the institutional review board of the Hamamatsu University School of Medicine. The chi-square test was used to compare clinical characteristics for categoricaldata and the Wilcoxon rank-sum test for continuous data. OS was calculated using the Kaplan-Meier method and compared by the log-rank test. Gray's test was used to compare cumulative incidence curves. Cox proportional hazard analyses were performed to determine prognostic indicators of OS. The Wald test was used to assess the prognostic significance of a candidate variable. Statistical analyses were performed using EZR, a graphical user interface for R (Kanda Y, 2013). Results: Among 506 enrolled patients, 475 with a median age of 56 years were assessable. The median follow-up period was 5.4 years. In total, 103 patients (21.7%) had various types of comorbidities, with diabetes mellitus, mild liver disease, peripheral vascular disease, myocardial infarction, renal disease, and peptic ulcer disease (7.3%, 4.1%, 3.2%, 2.6%, 2.2%, and 2.2%, respectively) as the most common. The lowest CCI score was 2 owing to CML. CCI scores were stratified as follows: 2, 372 patients (78%); 3, 74 patients (16%); and ≥4, 29 patients (6%). Higher CCI scores were significantly associated with older age (P

Description: Background: Bosutinib, a Src/Abl tyrosine kinase inhibitor, is approved at a starting dose of 500 mg once daily (QD) in many countries, including Japan, for patients with Philadelphia chromosome-positive (Ph+) chronic phase (CP), accelerated phase (AP), or blast phase (BP) chronic myeloid leukemia (CML) after prior therapy. The indication for bosutinib was expanded to patients with newly diagnosed CP CML, at a starting dose of 400 mg QD, in 2017 by the US Food and Drug Administration and in 2018 by the European Medicines Agency. Approval of first-line bosutinib for CP CML was based on data from the global phase 3 BFORE trial, which demonstrated a significantly higher major molecular response (MMR) rate at Month 12 with bosutinib vs imatinib in patients with Ph+ CP CML and e13a2/e14a2 transcripts (primary endpoint; 47.2% vs 36.9%; 2-sided P=0.02). We conducted a phase 2 study to evaluate the efficacy, safety, and pharmacokinetics (PK) of bosutinib in Japanese patients with newly diagnosed CP CML. Methods: In this open-label, single-arm study (NCT03128411), Japanese patients ≥20 years of age with a molecular diagnosis of CP CML within 6 months, Eastern Cooperative Oncology Group performance status 0 or 1, adequate renal and hepatic function, and no prior treatment for CML (hydroxyurea within 6 months permitted) received bosutinib at a starting dose of 400 mg QD. The primary endpoint was MMR at Month 12 in the modified as-treated population, which included patients who were Ph+ and had e13a2/e14a2 transcripts. A total of 60 patients was required in the modified as-treated population for the study to have 〉82% power to reject the null hypothesis (25% MMR rate at Month 12) and accept the alternative hypothesis (40% MMR rate at Month 12) with a 1-sided ∝-level of 5%. Secondary endpoints included MMR and complete cytogenetic response (CCyR) by Month 12, event-free survival (EFS), overall survival, safety, and PK. Results: In all, 60 Japanese patients with CP CML were treated with bosutinib; all patients were Ph+ and had e13a2/e14a2 transcripts and were included in the modified as-treated population analyzed for efficacy. Median age was 55 years (range 20-83), 60.0% of patients were male, and 45.0%, 43.3%, and 11.7% had low-, intermediate-, and high-risk Sokal scores, respectively. Median duration of follow-up was 16.6 months (range 11.1-21.9), and median duration of bosutinib treatment was 15.3 months (range 0.3-21.9). After 12 months of follow-up, 42 (70.0%) patients remained on bosutinib; 17 (28.3%) discontinued due to adverse events (AEs) and 1 (1.7%) due to physician decision. Median dose intensity was 354.7 mg/day (range 95.3-494.1). The MMR rate at Month 12 was 55.0% (2-sided 90% confidence interval [CI] 44.4-65.6); the test of the null hypothesis was rejected (1-sided P

Description: Rituximab has greatly improved the prognosis of B cell malignancies (BCM). However, several resistance mechanisms have been reported, including the inhibition of apoptosis, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) as well as modulation of CD20 antigen and increased complement-related cytotoxicity inhibition factor (CD55). The calicheamicin-conjugated anti-CD22 monoclonal antibody (mAb), CMC544, is one of the new promising agents, and effective on several types of BCM in vitro, especially when used in combination with rituximab. However, details regarding the cytotoxic effects of CMC544 and mechanisms triggering cell death, particularly when it is combined with rituximab, have not been elucidated. We studied cell cycle features of CMC544 on BCM used alone or in combination with rituximab, and analyzed quantitative alteration of target molecules such as CD20, CD22 and CD55.(Materials and Methods) The cell lines used in this study were CD22-positive Daudi and Raji cells, and CD22-negative Jurkat and NB4 cells. Cells obtained from 8 patients with BCM were also used. CMC544, unconjugated anti-CD22 mAb (G5/44), and free NAC-calicheamicin DMH were provided by Wyeth Pharmaceutical Co., Ltd. The effects of CMC544 were analyzed by incubating the cells at the concentrations of 0–100μg/ml for 0–96h, then by measuring cell count, cell viability, 3H-thymidine incorporation and cell cycle distribution on flow cytometry (FCM) as well as by video microscopic observation. The amounts of CD20, CD22 and CD55 antigens were analyzed by FCM, laser scanning microscope. The process of CMC544-induced cell death was assessed by Apocyto®. Three possible mechanisms of the combined effect of CMC544 and rituximab were investigated separately by direct effect of the mAb, CDC and ADCC. (Results) Analysis of the cell cycle distribution indicated that CD22-positive cells were temporally arrested at the G2/M phase after 6–12h incubation with CMC544, and the hypodiploid proportion increased after 24–72h. CD22-positive cells enlarged after 12–24h incubation with CMC544. Apoptotic morphological changes such as bleb formation and cell shrinkage were observed in 35–57% of the cells. These changes were not observed after incubation with G5/44. Apocyto® stain showed a continuous pattern from early to late apoptosis. Similar effects were observed in clinical samples that expressed CD22. The amounts of CD22 and CD55 were significantly reduced 3h after incubation with CMC544, while the CD20 expression was maintained 24h after incubation with CMC544. Incubation of the cells for 12–24h with CMC544 and then for 12hr with rituximab induced 1.2–2.1 times more anti-proliferation, apoptosis, CDC and ADCC than simultaneous incubation.(Conclusion) The effect of CMC544 was clearly observed as G2/M arrest following increase of the hypodiploid proportion in the cell cycle distribution, and this was a useful method to investigate the effect of CMC544. Combination of CMC544 and rituximab enhanced the cytotoxic effect, and sequential administration was more effective. The reduction of CD55 and continued expression of CD20 after the incubation with CMC544 supported the above observation. Further investigations are required to find the most effective treatment regimen to overcome drug resistance of B cell malignancies.

Description: Several new agents have been introduced for the treatment for B cell malignancies (BCM) to overcome resistance to rituximab. Inotuzumab ozogamicin (CMC544), a humanized anti-CD22 mAb conjugated to N-acetyl-g-calicheamicin demethyl hydrazide (NAC-calicheamicin DMH), binds CD22, leading to internalization and delivery of calicheamicin inside the cells. We have been studying gemtuzumab ozogamicin (CMA676), another calicheamicin-conjugated mAb targeting CD33, and we have reported several new findings regarding multi-drug resistance (MDR) and modification of surface antigens. In this study, we attempted to clarify the effect of CMC544 on BCM cells in relation to MDR, and we investigated the restoration effect of the MDR modifier. We also analyzed the effect of CMC544 in relation to CD22 and P-glycoprotein (P-gp) in the samples from BCM. (Materials and Methods) The cell lines used in this study were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR, respectively. CD22-negative Jurkat, K562 and NB4 cells, and cells obtained from 11 patients with BCM, were also used. CMC544, unconjugated anti-CD22 mAb (G5/44), CMA676, and unconjugated NAC-calicheamicin DMH were kindly provided by Wyeth Pharmaceutical Co., Ltd. The amount of cell surface antigens and P-gp were analyzed by flow cytometry (FCM). For P-gp analysis, cells underwent a reaction with biotinylated MRK16 mAb or with a subclass-matched control mAb and were then stained with streptavidin-Cy-7. P-gp function was determined by intracellular rhodamine-123 (Rh123) accumulation and its enhancement by MDR modifiers, PSC-833 (Novartis) and MS209 (Mitsui), as previously described. The effect of CMC544 was analyzed by cell count, cell viability, and cell cycle distribution on FCM. It was also determined with or without a MDR modifier. Relationships between the CMC544 effect and the amount of P-gp or CD22 were examined statistically. (Results) A dose-dependent, selective cytotoxic effect of CMC544 was observed in cell lines that expressed CD22. CMC544 is not effective on P-gp-expressing MDR sublines, compared with parental cell lines. MDR modifiers restored the cytotoxic effect of CMC544 in P-gp-expressing sublines. In clinical samples, the cytocidal effect of CMC544, estimated from the fraction of cells in the hypo-diploid portion of the cell cycle, was inversely related to the amount of P-gp estimated by MRK16 mAb (P=0.04), and to the P-gp function assessed by intracellular Rh123 accumulation in the presence of PSC833 or MS209 as MDR modifier (P=0.02 and P=0.01, respectively). Additionally, these MDR modifiers reversed CMC544 resistance in P-gp-expressing CD22-positive cells. On the other hand, the cytotoxic effect of CMC544 was positively correlated with the amount of CD22 (P=0.01). (Conclusion) This study demonstrates that the CMC544 effect depends on the amounts of P-gp and CD22. We might be able to predict the clinical effects of this drug based on these factors. The treatment of BCM has progressed extremely rapidly, and we can now select particular drugs more specifically among many promising agents. Our results contribute to the development of new strategies to treat BCM. In CD22-positive BCM with P-gp, combined use of CMC544 and MDR modifiers may be more beneficial.