An Interim 4-Year Analysis of Prospective Multicenter Observational Study of PNH-Type Cells in Japanese Patients with Bone Marrow Failure Syndrome (OPTIMA study)
Authors: Shirasugi Yukari , Noji Hideyoshi , Shichishima Tsutomu , Sugimori Chiharu , Obara Naoshi , Chiba Shigeru , Ninomiya Haruhiko , Nakamura Yoshihiko , Ando Kiyoshi , Yonemura Yuji , Kawaguchi Tatsuya , Hosokawa Kohei , Ueda Yasutaka , Nishimura Jun-Ichi , Kanakura Yuzuru , Nakao Shinji 
Published: October 2019

Abstract
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder caused by the clonal expansion of the phosphatidylinositol glycan class A (PIGA) mutant hematopoietic stem cells, which results in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Through the high-resolution flow cytometry-based method, GPI-APs deficient blood cells (i.e., PNH-type cells) are often detectable in patients with bone marrow failure syndromes (BMF), such as aplastic anemia (AA) and low-risk types of myelodysplastic syndromes (MDS). Sugimori et al reported that when BMF patients possessed increased PNH-type cells, the patients had a good prognosis and showed a high response rate to immunosuppressive therapies, suggesting that detection of PNH-type cells is potentially useful in determining an optimal treatment for BMF patients. Thus, we conducted a nationwide, multi-center prospective observational investigation, the OPTIMA study.
Methods: From July 2011, we start recruiting the patients with BMF that were diagnosed at various hematology clinics throughout Japan to the OPTIMA study. The primary endpoint of this study was to determine the prevalence of BMF patients with PNH-type cells and to clarify the clinical significance of the presence and quantitative changes of these cells with regard to the clinical features. Six different university laboratories were assigned as regional analyzing centers. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted twice a year to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively.
Results
Between July 2011 and May 2015, a total of 2328 patients were enrolled to this study, and we analyzed 2212 patients who were eligible for the interim analysis. Of these patients, 74 (3.3%) were diagnosed with PNH, 690 (31.2%) with AA, 592 (26.8%) with MDS, and 856 (38.7%) with undiagnosed BMF. Using high-resolution flow cytometry-based method, 755 (34.1%; 95.9% in PNH, 52.8% in AA, 18.2% in MDS, and 24.8% in undiagnosed BMT) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. Overall, 181(8.2%) patients had ≥1% of both PNH-type erythrocytes and granulocytes; the prevalence in each disease subset was 68/74 (91.9%) in PNH, 67/690 (9.7%) in AA, 22/592 (3.7%) in MDS, and 24/856 (2.8%) in undiagnosed BMF. Regarding FAB and WHO classifications of MDS subtype, no patients with RARS (0/22), RAEB-1 (0/37) or RAEB-2 (0/23) had PNH-type cells. In contrast, 20.4% (56/275) patients with RCMD, 18.3% (26/153) patients with RCUD and 50% (2/4) patients with del (5q) MDS possessed increased PNH-type cells. Blood samples from 75 (65 with and 10 without PNH-type cells) patients were analyzed three years after the first examination. Of 65 PNH (+) patients, PNH-type cells disappeared in 4 (6.2%), while the percentage remained stable in 61 (93.8%). All of the 10 PNH (-) at the enrollment were also negative for PNH-type cells in 3 years.
Conclusions: A high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells below 0.01% was successfully transferred from Kanazawa University to other laboratories in Japan. Our interim analysis confirmed previous findings that PNH-type cells were detectable in patients with 52.8% of AA and 18.2% of MDS patients. Regarding FAB and WHO classifications of MDS subtype, PNH-type cells were not detected in any of MDS RARS, RAEB-1 or RAEB-2 patients. Further analysis are required to determine the clinical significance of the minimal level of PNH-type cells as well as chronological changes in the PNH-type cell percentage, especially in terms of their relation to response to immunosuppressive therapy.

Disclosures
Ninomiya: Alexion Pharmaceuticals: Honoraria. Ando:Eisai Co., Ltd.: Honoraria, Research Funding. Yonemura:1. Chugai Pharma, 2. Alexion Pharma, 3. Japan Blood Products Organization, 4. OHARA Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Ueda:Alexion Pharma: Research Funding. Nishimura:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.