ALBERT

Description: Ph-like acute lymphoblastic leukemia (also known as BCR-ABL1-like ALL) is a new disease entity of B-cell ALL (B-ALL) that exhibits a mRNA expression profile similar to that of Philadelphia chromosome-positive ALL (Ph+ ALL). Ph-like signature is presumably driven by kinase-activating gene alterations. Thus, both gene expression pattern and DNA mutational status should be assessed to make a definitive diagnosis for Ph-like ALL. A variety of approaches combining multiple methods, including RNA sequencing (RNA-seq), Taqman low-density array (LDA), fluorescence in situ hybridization (FISH) and targeted DNA sequencing, are being tested; however, such multi-omics approaches are available only in limited institutions. Since Ph-like ALL patients generally exhibit poor response to standard chemotherapy, and tyrosine kinase inhibitors (TKIs) may benefit them when used in a timely manner, a fast, accurate and generalizable diagnostic method is critically needed. In the present study, we have developed a nCounter-based diagnostic method for Ph-like ALL and validated it using a cohort of Japanese adult B-ALL cases. To identify genes that are uniquely expressed (or not expressed) in Ph+ B-ALL, we first obtained publicly-available gene expression datasets comprising 1146 B-ALL cases and identified 82 differentially-expressed genes in Ph+ ALL cases. We then assessed expression levels of those genes in an independent cohort using the nCounter, which enables fast, sensitive and accurate RNA detection. We also tested whether nCounter-based methods can detect fusion transcripts relevant for Ph-like ALL pathogenesis using probes targeting ABL1, ABL2, CSF1R, PDGFRB, and JAK2. We analyzed 123 samples (Ph+ = 42, Ph- = 81, age 16 to 67) obtained from newly-diagnosed adult B-ALL patients enrolled in two clinical trials conducted by the Fukuoka Blood and Marrow Transplantation Group (FBMTG) (Nagafuji et al. Eur J Haematol 2019). Unsupervised hierarchical clustering successfully stratified 123 cases into two disease clusters: Ph+ and Ph- subgroups. As expected, Ph+ subgroup included almost all Ph+ ALL cases (40 out of 42 cases), while 18 out of 81 Ph- ALL cases (22%) were categorized into the Ph+ subgroup. We defined these cases as Ph-like ALL. To validate the nCounter-based Ph-like ALL classification, we performed RNA-seq and target-capture DNA sequencing of all Ph- ALL cases. As expected, we detected kinase-activating fusions/rearrangements, including CRLF2 rearrangements (7 cases), PDGFRB fusions (3 cases), JAK2 fusions (2 cases), EPOR rearrangements (2 cases), ABL1 fusion (1 case), and FLT3 internal tandem duplication (1 case) in 16 Ph-like ALL cases, while no genetic alterations were detected in 2 cases. Fusion genes involving PDGFRB were consistently detected by nCounter (3/3); however, detection of those involving JAK2 (1/2) and ABL1 (0/1) were inconsistent. JAK2 and/or RAS mutations were detected in 5 of 7 Ph-like ALL cases harboring CRLF2 rearrangements. Of note, CRLF2 protein expression was detected by FACS in all CRLF2-rearranged cases. We next assessed significance of the Ph-like signature on clinical outcomes using a cohort of 40 Ph- ALL cases, in which minimal/measurable residual disease (MRD) status, assessed by IgH and/or TCR rearrangements, as well as clinical data were available (Nagafuji et al. Eur J Haematol 2019). Ph-like ALL cases exclusively exhibited MRD positivity after induction therapy as compared to non-Ph-like cases (p=0.04), indicative of the chemo-resistant nature of Ph-like ALL as previously reported (Roberts et al. N Engl J Med, 2014 and Roberts et al. J Clin Oncol, 2017). As expected, Ph-like ALL cases exhibited significantly poor disease-free survival compared with non-Ph-like ALL cases (p=0.04); however, no significant difference was evident in overall survival (p=0.62) presumably due to the fact that all MRD-positive cases were subjected to allo-HSCT after induction therapy. These data indicate that MRD-based therapy stratification could overcome chemo-resistant nature of Ph-like ALL. Our data suggest that nCounter-based diagnostic method is fast and accurate to identify Ph-like ALL. Since Ph-like signature generally dictates poor clinical outcomes, and upfront TKI therapy may improve them, our method could facilitate precision medicine in the treatment of Ph- B-ALL. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Description: Abstract 285 Background: KW-0761 is a defucosylated, humanized, monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC; Potelligent®) that binds to CC chemokine receptor 4 (CCR4). CCR4 is expressed on the surfaces of cells comprising several T-cell malignancies such as ATL, peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL). A phase I study of KW-0761 in patients with CCR4-positive ATL and PTCL demonstrated that 4 weekly intravenous infusions of KW-0761 were well tolerated up to 1.0 mg/kg and it showed encouraging clinical activity with an overall response rate (ORR) of 31.3% (4 of 13 ATL and 1 of 3 PTCL) in 16 patients (J Clin Oncol 2010;28:1591-8). Methods: A multicenter phase II study of KW-0761 has been conducted for relapsed patients with CCR4-positive ATL to evaluate its efficacy, pharmacokinetics (PK), safety and immunogenicity. Patients were planned to receive 8 weekly intravenous infusions of KW-0761 at 1.0 mg/kg. The primary endpoint was ORR. Objective responses were assessed after the 4th and 8th infusions of KW-0761 according to the response criteria for ATL (J Clin Oncol 2009;27:453-9) by each investigator and the independent efficacy assessment committee. The number of patients required was estimated to be 25, for 90% power to detect a lower limit of the 95% confidence interval (CI) exceeding the 5% threshold of ORR, based on the assumptions that the minimum required response ORR to a new drug for relapsed ATL is 5% and the expected ORR to KW-0761 is 30%. Results: Twenty-seven patients (12 males and 15 females) were enrolled and received KW-0761. The median age was 64 years (range: 49–83). The disease subtypes of ATL consisted of 14 acute-, 6 lymphoma-, and 7 chronic-types with unfavorable prognostic factors. Among the 27 patients enrolled, 14 patients (52%) completed the protocol treatment of 8 infusions. Eleven patients (41%) discontinued the protocol treatment because of progressive disease, and the remaining 2 discontinued because of skin rash or the concurrent colon tumor. The treatment-related grade (G) 2 or greater adverse events (AEs) were lymphopenia (96%), leukopenia (56%), skin rash (52%), neutropenia (33%), thrombocytopenia (26%), AST increase (26%), ALT increase (22%), hypoxemia (19%), anemia (15%), pruritus (15%), g-GTP increase (15%) and hypophosphatemia (15%). G2 or greater Infusion-related toxicities were observed in 22 of 27 patients (81%) including 1 G3, but immediately recovered after treatment with systemic steroids. Treatment-related severe AEs (SAEs) were observed in 5 patients, including a Stevens-Johnson syndrome (G3) and 4 skin rashes (each G3). All these AEs also improved by steroids. PK analysis demonstrated that Cmax and trough (C168h) after the 8th infusion was 38,853 ± 11,267 and 25,934 ± 10,193 ng/mL, respectively, and T1/2 after the 8th infusion were 457 ± 144 h. No anti-KW-0761 antibody has been detected. Among the 26 patients evaluable for efficacy, KW-0761 exhibited an ORR of 54% (14/26; 95% CI, 33 to 73) (acute: 6/14 patients, lymphoma: 3/6 patients, chronic: 5/6 patients) including 7 complete responses (CRs) (27%; 95% CI, 12 to 48) and 7 partial responses (PRs). These are remarkable results, considering that the ORR of relapsed or refractory patients with ATL to a single-agent chemotherapy has been reported to be low (7 to 39%). Response rates according to the affected disease lesion were 100% (13 patients, all CR), 71% (5 of 7 patients), and 38% (5 of 13 patients), respectively, for peripheral blood, skin, and lymph node disease. Conclusions: KW-0761 is a highly effective agent with acceptable toxicity profiles in relapsed patients with CCR4-positive ATL who have no standard therapies. A multicenter, randomized study for untreated ATL patients to compare mLSG15 (a dose-intensified multi-agent regimen, J Clin Oncol 2007;25:5458-64) + KW-0761 with mLSG15 alone has been initiated. Disclosures: Ogura: Kyowa Hakko Kirin Co Ltd: Consultancy. Akinaga:Kyowa Hakko Kirin Co Ltd: Employment.

Description: We have reported that S-phase kinase-associated protein 2 (Skp2) expression in tumor cells is an unfavorable prognostic factor in diffuse large B cell lymphoma (DLBCL) with CHOP-R. Therapeutic strategies other than CHOP-R should be needed for DLBCL patients exhibiting a high Skp2 expression at the time of diagnosis. High dose chemotherapy followed by autologous peripheral blood stem cell transplantation (APBSCT) is mainly adapted for DLBCL patients in high and high intermediate risk groups in IPI. However, the definite beneficial evidence for APBSCT in DLBCL remains unclear. In the present study, we investigated the clinical significance of Skp2 expression in the patients with DLBCL treated with CHOP-R plus high-dose chemotherapy followed by APBSCT. We retrospectively analyzed the outcomes of 93 patients (age range: 14-65). The patients were newly diagnosed as having DLBCL from 2002 to 2012, and were treated with either CHOP-R plus upfront APBSCT (n=31) or CHOP-R (n=62) in the19 hospitals in Kyushu, Japan. All patients had high and high intermediate risk in IPI. All biopsy specimens were immunohistopathologically reconfirmed by one pathologist with expertise before entering into this study. The median follow-up time was 2.3 y. Survival analyses were performed using the Kaplan-Meier method and the Cox proportional Hazard model with inverse probability of treatment weight (IPTW). In background of the patients, age was younger in transplant group (mean age 52) than in non-transplant group (mean age 59). CR rate was higher in non-transplant (30.7%) than in transplant (12.9%). Sex, stage, PS, LDH extranodal lesion and IPI showed no difference in both group. Overall survival (OS) for 3 years were 77.0% and 59% in transplant group and in non-transplant group (P=0.077), respectively. Progression-free survival (PFS) for 3 years, 65.7% and 53.2% in transplant and non-transplant group (P=0.233), respectively. In Skp2 high expression (positive rate 〉40%) group (n=37), 3y-OS was 44.9% and 16.4% in transplant (n=13) and non-transplant (n=24) group (P=0.065), respectively. 3y-PFS was 40.3% and 7.5% in transplant and non-transplant (P=0.032), respectively (Fig A). However, in low Skp2 expression group (n=56), OS was 100% and 92.7% (P=0.254), PFS was 83.6% and 82.6% (P=0.945)(Fig B)in transplant (n=18) and non-transplant (n=38) group, respectively. In propensity score analysis using center effect, age, extranodal lesion, IPI and CR rate as logistic regression model, transplant group showed the excellent benefit in OS ( OR= 0.469, 95%CI =0.266-0.825, P=0.009) and PFS (OR=0.456, 95%CI=0.255-0.815, P=0.008) in Skp2 high expression group (n=37).P value for transplant x Skp2 interaction was P=0.643 in OS and P=0.020 in PFS. In conclusion, Skp2 was a good biomarker for indication of ABSCT for high risk DLBCL patients. In low expression of Skp2, patients should not be treated with high dose chemotherapy followed by APBSCT, even though in patients with high risk in IPI. However, ABSCT have some advantage in DLBCL patients with high expression of Skp2. Disclosures: No relevant conflicts of interest to declare.

Description: We originally identified T-cell immunoglobulin mucin-3 (TIM-3) as a leukemic stem cells (LSCs)-specific surface molecule and a useful marker for discriminating LSCs from hematopoietic stem cells (HSCs). Furthermore, we recently identified an unique autocrine loop composed of TIM-3 and its ligand galectin-9 (Gal-9). This TIM-3/Gal-9 autocrine loop enhances self-renewal capacity of AML-LSCs and contributes to leukemia progression(Kikushige et al., Cell Stem Cell 2015). To clarify the molecular mechanism how TIM-3 signaling enhances stem cell properties of AML-LSCs, we performed shRNA-mediated knock-down(KD) of TIM-3 in KASUMI-3 (TIM-3+ AML cell line). As a result, TIM-3-KD significantly reduce the proliferation of KASUMI-3. To investigate how TIM-3-KD attenuated leukemia propagation, we compared gene expression profile of scramble-infected control and TIM-3-KD KASUMI-3 cells. Gene Set Enrichment Analysis (GSEA) revealed TIM-3-KD resulted in the significaltly attenuated expression of HSCs and LSCs-related genes (Eppert et al., Nature Med 2011) and canonical Wnt pathway-related genes (BioCarta). Since canonical Wnt pathway is known to regulate stem cell properties via inducing the nucleus accumulation ofβ-catenin in many types of stem cells including HSCs, we next examined the β-catenin level in TIM-3-KD-KASUMI-3 cells. Array scan analysis revealed that KD of TIM-3 significantly impaired the the nucleus accumulation of β-catenin in KASUMI-3 cells. In various types of cancers, the constitutive activation of β-catenin is one of the most common oncogenic signatures, and it is mainly driven by somatic mutations in the genes related to the canonical Wnt pathway. However, such mutations are rarely detected in AML.We, therefore, hypothesized that TIM-3/Gal-9 autocrine loop signaling might be a novel mutation-independent machinery for the constitutive activation of canonical Wnt pathway in primary AML. To test this hypothethis, we first evaluated the phosphorylation status of LDL receptor-related protein 6 (LRP6), because LRP6 phosphorylation is a crucial step for canonical Wnt pathway activation. Surprisingly, Gal-9 stimulation induced phosphorylation of LRP6 and subsequent β-catenin accumulation in primary TIM-3+ AML cells even in the absence of Wnt ligand. On the other hand, LRP6 phosphorylation induced by Gal-9 stimulation was canceled by the blocking antibody to TIM-3 (F38-2E2). These results indicate that Gal-9 stimulation should induce canonical Wnt pathway activation via TIM-3 dependent manner in primary AML cells. Since Gal-9 ligation to TIM-3 is known to induce phosphorylation of tyrosine residues of TIM-3 cytoplasmic tail, leading to the recruitment and activation of several Src family kinases (SFKs) for downstream signal transduction. We found that TIM-3/Gal-9 interaction recruited hematopoietic cell kinase (HCK), a member of SFKs, to TIM-3 and activated HCK by phosphorylation of the SH1 domain (Y410 in HCK) in TIM-3+ AML cells. Furthermore, we confirmed a specific HCK inhibitor A-419259 efficiently blocked the LRP6 phosphorylation and subsequent β-catenin accumulation induced by TIM-3/Gal-9 interaction in primary AML cells. These results suggest that the canonical Wnt pathway activation induced by Gal-9 ligation to TIM-3 is mainly mediated by HCK in primary AML cells. We next focused on p120-catenin as a bridging molecule between HCK activation and LRP6 phosphorylation because, as previously reported, this molecule can be directly activated by SFKs and the activation of this molecule is an initial step for canonical Wnt pathway activation preceding LRP6 phosphorylation. We found that Gal-9 ligation to TIM-3 immediately induced the direct association of HCK and p120-catenin, leading to the activation of p120-catenin in TIM-3+ AML cells. We also confirmed that specific inhibition of HCK completely abrogated the activation of p120-catenin and subsequent canonical Wnt pathway activation in primary TIM-3+ AML cells, indicating that the activation of p120-catenin mediated by HCK is a bridging moleculer machinery for connecting TIM-3 signaling and previously identified canonical Wnt pathway. Since TIM-3 as well as HCK is specifically expressed in LSCs but not in HSCs, we conclude that TIM-3/Gal-9 autocrine loop is a novel Wnt-ligands independent machinery for the constitutive activation of canonical Wnt pathway specific for AML-LSCs. (summarized in figure) Figure. Figure. Disclosures Akashi: Celgene: Research Funding, Speakers Bureau; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Taiho Pharmaceutical: Research Funding; MSD: Research Funding; Asahi-kasei: Research Funding; Chugai Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Eli Lilly Japan: Research Funding; Novartis pharma: Research Funding; Eisai: Research Funding; sanofi: Research Funding; Astellas Pharma: Research Funding.