Autologous peripheral blood stem cell transplantation for Philadelphia chromosome‐positive acute lymphoblastic leukemia is safe but poses challenges for long‐term maintenance of molecular remission: Results of the Auto‐Ph17 study

Abstract Autologous hematopoietic stem cell transplantation (SCT) is not a standard treatment option for Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL); however, its position has been reassessed since the introduction of tyrosine kinase inhibitors (TKIs). We prospectively analyzed the efficacy and safety of autologous peripheral blood SCT (auto‐PBSCT) for Ph+ALL patients aged between 55 and 70 years who had achieved complete molecular remission. Melphalan, cyclophosphamide, etoposide, and dexamethasone were used for conditioning. A total of 12 courses of maintenance therapy, including dasatinib, were performed. The required number of CD34+ cells was harvested in all five patients. No patient died within 100 days after auto‐PBSCT, and no unexpected serious adverse events were observed. Although 1‐year event‐free survival was 100%, hematological relapse was observed in three patients at a median of 801 days (range, 389–1088 days) after auto‐PBSCT. Molecular progressive disease was observed in the other two patients, although they maintained their first hematological remission at the last visit. Auto‐PBSCT can be safely performed for Ph+ALL with TKIs. A limitation of auto‐PBSCT was suggested, despite the increase in the intensity of a single treatment. The development of long‐term therapeutic strategies by including new molecular targeted drugs is warranted to maintain long‐term molecular remission.

Scientific Research, Grant/Award Numbers: 22H00455, 17H06162 auto-PBSCT, and no unexpected serious adverse events were observed. Although 1-year event-free survival was 100%, hematological relapse was observed in three patients at a median of 801 days (range, 389-1088 days) after auto-PBSCT. Molecular progressive disease was observed in the other two patients, although they maintained their first hematological remission at the last visit. Auto-PBSCT can be safely performed for Ph+ALL with TKIs. A limitation of auto-PBSCT was suggested, despite the increase in the intensity of a single treatment. The development of long-term therapeutic strategies by including new molecular targeted drugs is warranted to maintain long-term molecular remission.

K E Y W O R D S
autologous peripheral blood stem cell transplantation, dasatinib, Philadelphia chromosomepositive acute lymphoblastic leukemia, regulatory T cell

INTRODUCTION
High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (SCT) is a standard treatment for several hematological malignancies [1,2]. Advances in testing techniques and treatments have led to using measurable residual disease (MRD) as a prognostic indicator for leukemia. As a result, the positioning of autologous SCT for acute leukemia has changed [3].

Allogeneic SCT (allo-SCT) is a standard treatment option for
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) [4], despite the dramatic improvement in the outcome of chemotherapy dramatically with the introduction of tyrosine kinase inhibitors (TKIs) [5]. In recent prospective studies, autologous SCT was performed in 19 and 35 patients using imatinib [6,7]. Patient survival among those who underwent autologous SCT was equivalent to those who underwent allo-SCT in both studies. Similarly, a large retrospective study, including 67 autologous SCTs, reported comparable results between autologous and allo-SCTs [8].
Because autologous SCT has not been recommended in Japan since the introduction of TKIs, there is no data on autologous SCT for Japanese Ph+ALL patients. In this study, we planned to perform autologous SCT for Ph+ALL patients (aged 55 years or older) with complete molecular remission (CMR) who had transplant toxicity concerns for allo-SCT, with the expectation of a more potent antileukemic effect than conventional chemotherapy.

Study design
This was a multicenter prospective exploratory study with a target sample size of 5. The primary end point was the proportion of deaths within 100 days of autologous peripheral blood SCT (auto-PBSCT).
Secondary end points examining treatment efficacy included the pro-

Treatment schedule
The study protocol has been described in detail elsewhere [9]. Briefly, the study regimes consisted of three phases: peripheral blood stem cell harvest (PBSCH), conditioning prior to auto-PBSCT, and maintenance ( was not increased again, and the treatment continued at the reduced dose. MRD using RQ-PCR for the detection of BCR-ABL1 chimeric gene was planned to be analyzed at designated time points: before conditioning for auto-PBSCT, 30 days after auto-PBSCT, before maintenance courses #4, #8, and #12, and 3 years after auto-PBSCT.

Immunological analysis of T cells
We assessed the numerical and phenotypic features of peripheral T cells at the same time as MRD analyses using multiparametric flow cytometry (FCM).
Peripheral blood mononuclear cells were isolated by density gradient with Ficoll-Paque (GE HealthCare) and were stored in liquid nitrogen until analysis. Cells were washed with phosphate-buffered saline containing 2% fetal calf serum (Biosera, Orange, CA, USA) and subjected to staining with surface antibodies, shown in Table S1. Intra-

Definitions
CMR was defined by the absence of detectable MRD with a sensitivity of at least 0.01%. Molecular relapse was defined when the

Statistical analysis
The Kaplan-Meier method was used to estimate OS and EFS [10]. OS was measured from the date of auto-PBSCT to the date of death from any cause, and surviving patients were censored at the date of the last contact. EFS was measured from the date of auto-PBSCT until hematological relapse or death from any cause. The cumulative incidences of relapse and NRM were calculated using Gray's method [11,12]. For relapse rate, death without relapse was the competing event; for NRM, relapse was the competing event.

Patient characteristics
Between September 2017 and February 2019, five patients were enrolled in this study. The patient flowchart is shown in Figure 1. The median age at registration was 62 years (range, 59-68), the white blood cell count at diagnosis was 7100 (range, 600-168,700), and additional chromosomal abnormalities were identified in three patients (Table 2). CMR was achieved after one course of chemotherapy in three patients and after two courses of chemotherapy in two patients.
The median time from diagnosis to CMR was 46 days (range, 43-76 days).

PBSCH
The target number of CD34 + cells (2 × 10 6 /kg) or more was harvested during the first PBSCH regimen in three patients, a median of 2 days of the procedure (range, 1-3 days non-hematological adverse events were grades 1 or 2, and no specific adverse events were observed in more than half of the patients ( Figure 2).

Auto-PBSCT
The median number of infused CD34 + cells was 3.03 × No unexpected serious adverse events were observed.

Maintenance therapy
Maintenance therapy was initiated at a median of 59 days after auto-PBSCT (range, 30-69 days). All but one patient were able to start dasatinib at 50 mg/day as planned and gradually increased to 100 mg/day. Only 1 patient was able to receive dasatinib at 100 mg/day throughout the 12 courses; the other patients needed a dose reduction ( Figure 3). Neutropenia and thrombocytopenia were frequently observed as hematological adverse events, but more than half of the cases were grades 1-2. On the other hand, non-hematological adverse events were infrequently observed due to the appropriate dose reduction of dasatinib ( Figure 4). All patients were able to continue dasatinib until the planned number of maintenance courses or relapse.

Long-term outcomes
The median follow-up period was 1100 days after auto-PBSCT (range, 837-1519 days). The secondary end points are summarized in Table 3.

T-cell immunity
Serial changes in peripheral T cells after auto-PBSCTs were assessed with FCM at the designated sampling points. The percentages of CD4 + and CD8 + cells among T cells were almost constant after auto-PBSCT, and the CR4/CD8 ratio remained low throughout the observation period ( Figure 6A). On the other hand, the percentage of effector Tregs F I G U R E 2 Non-hematological adverse events during peripheral blood stem cell harvest (PBSCH) and autologous peripheral blood stem cell transplantation (auto-PBSCT).
The CD8/eTreg ratio was higher in the two patients who survived without hematological relapse (AP-01 and AP-03) and was particularly high in AP-03, who received a lower dose of dasatinib ( Figure 6C). In addition, PD-1 and CTLA-4 expressions on CD8 + T cells were relatively low in patient AP-03 ( Figure 6D).

DISCUSSION
This study prospectively evaluated the safety and efficacy of auto-PBSCT for Ph+ALL. For PBSCH, the required number of CD34 + cells was collected in all cases. All patients survived for at least 100 days after auto-PBSCT with no unexpected serious adverse events, indicating that there were no safety concerns. However, all patients eventually experienced molecular progressive disease or hematological relapse. If the goal was to develop a cure, the long-term results were not satisfactory.
As NRM in allo-SCT becomes higher as patient age increases, especially for patients aged 55 years or older [14], the age of eligibility for this study was set at 55 years or older. Although auto-PBSCT is a more potent treatment than conventional chemotherapy, the fact that no treatment-related deaths were observed suggested that the safety considerations were adequate in this study.
The 3-year OS after allo-SCT with reduced intensity conditioning was reported to be 64.2% in a study that analyzed Japanese registry data [15]. As for chemotherapy without allo-SCT in CR1, the 4-year OS of patients who achieved CMR after 3 months was reported to be 63% [16]. Although it should be evaluated carefully due to the small number of patients, the 3-year OS of 75% in this study was not disappointing, considering that the median age was relatively high. Given that the median time from auto-PBSCT to molecular progressive disease was as long as 731 days, it is possible that MRD became negative for a long period due to the deep molecular remission obtained by auto-PBSCT.
This study was planned with the concept of aiming for a cure by eliminating leukemia with high-dose chemotherapy followed by auto-PBSCT. However, the antileukemic effect achieved by donor immunity could not be obtained, unlike allo-SCT. Relapse after a long period since auto-PBSCT suggested a limit to increasing the intensity of a single-chemotherapy treatment. Recently, TKI plus blinatumomab showed excellent outcomes for de novo Ph+ALL patients [17,18].
The efficacy of blinatumomab maintenance after allo-SCT for B-ALL   has also been reported [19]. In addition, it has been reported that ponatinib, which is a third-generation TKI, improves treatment results for Ph+ALL patients [20]. Long-term use of a novel TKI and other molecular targeted drugs without allo-SCT could be a new therapeutic strategy.
It is interesting that the patient with the earliest relapse (AP-02) had higher eTreg cells and a lower CD8/eTreg ratio about 4 months before relapse. Increased eTreg cells may have led to early relapse by suppressing the antileukemic immune response. However, a careful interpretation of this outcome is required due to the small number of cases in this analysis. An increase in eTreg cells after autologous SCT is reportedly associated with early relapse in multiple myeloma [21] and attempts to deplete Treg cells in autologous SCT have been performed [22].
A high CD8/Treg ratio in solid tumor tissue correlates with a favorable prognosis [23]. In the present study, patients who survived without hematological relapse after auto-PBSCT showed a higher CD8/eTreg ratio, suggesting the existence of an immune response. As for dasatinib, it has been reported that dasatinib has a potential impact on immune cells through its off-target effects on various tyrosine kinase molecules.
The proliferation of CD8 + T cells was reported to be inhibited by dasatinib in a dose-dependent manner [24], which suggested high doses of In conclusion, auto-PBSCT can be safely performed for patients with Ph+ALL. In all cases, molecular remission was sustained for more than a year after auto-PBSCT, but disease progression was eventually observed. Long-term treatment strategies, such as continued maintenance therapy incorporating new molecular targeted drugs, could be promising. Squibb, Otsuka Pharmaceutical Co., Ltd., Perseus Proteomics Inc., Daiichi Sankyo Co., Ltd., AbbVie Inc., CURED, Inc., Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Zenyaku Kogyo Co., Ltd., Nippon Shinyaku Co., Ltd., Eisai Co., Ltd., Takeda Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Novartis Pharma K.K., and Sanofi K.K., and honoraria from Astellas Pharma Inc., AbbVie Inc., Chugai Pharmaceutical Co., Ltd., and Novartis Pharma K.K. outside of this study. The remaining authors declare no competing financial interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
This study was approved by the institutional review board of each participating hospital.

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.