Inferior Relapse-free Survival Research Articles

Mediator kinase module proteins, genetic alterations and expression of super-enhancer regulated genes in colorectal cancer.

Genetic alterations are well characterized as contributors to the pathogenesis of cancers. Epigenetic abnormalities can lead to perturbations of the expression of genes in cancer cells without structural defects. Deregulation of proteins of the transcription machinery may result in perturbations of target genes. Mediator, a multiprotein component of the transcription machinery facilitates the function of RNA polymerase II, which transcribes most human genes. A part of the mediator with kinase activity, called the Mediator kinase module shows genetic alterations in a sub-set of colorectal cancers. Data from publicly available genomic series of colorectal cancer patients were examined to determine alterations of Mediator kinase module component genes, including MED12, MED12L, MED13, MED13L, CDK8, CDK19, and CCNC. The prevalence of alterations in genomically defined colorectal cancer sub-sets was also interrogated. The effect of Mediator kinase module member gene expression on colorectal cancer relapse-free survival was investigated. Mutations in genes of the Mediator kinase module were present in a small percentage of colorectal cancers, ranging between 2 to 10% for MED12 and MED13 and alternative units MED12L and MED13L and below 2% for kinases CDK8 and CDK19 and cyclin C. Amplifications of the CDK8 gene were observed in 3% to 5% of colorectal cancers. The highest prevalence of mutations was observed in MSI cancers and the equivalent CMS1 group, with other genomic groups showing much lower frequency. An association of higher expression of MED12 with inferior relapse-free survival was observed. In contrast, higher expression of cyclin C was associated with improved survival. Colorectal cancer cell lines with CDK8 amplifications displayed sensitivity to several small molecule inhibitors of the KRAS/PI3K pathway but not to BET inhibitors. The Mediator kinase module is deregulated in a sub-set of colorectal cancers with differences observed in genomically defined groups. These variations may result in differences in sensitivity to targeted therapies and may have to be taken into consideration as such therapies are developed.

Impact of NOTCH1 expression in primary breast adenoid cystic carcinoma

AimsAdenoid cystic carcinoma (AdCC) originates from salivary-type like glands in the head and neck, lung, and breast. AdCC shows chromosomal translocation, resulting in MYB::NFIB fusion and overexpression of MYB. Recently,...

Relevance of CD20 antigen expression among paediatric patients with B-lineage acute lymphoblastic leukaemia.

Literature regarding prognostic relevance of CD20 antigen expression among paediatric B-lineage acute lymphoblastic leukaemia (B-ALL) patients is sparse and contradictory. We analysed clinical laboratory parameters and survival characteristics pertinent to CD20 expression among 224 treatment-naïve paediatric B-ALL patients. 50% patients had CD20 expression (CD20+ B-ALL). There was no difference in the clinical & laboratory presentation and end of induction measurable residual disease (EOI-MRD) status according to CD20 expression. As compared to CD20- B-ALL patients, CD20+ B-ALL patients had two times more relapse (16% vs. 29%, p = 0.034), inferior relapse-free survival (79% vs. 66%, p = 0.025) but no difference in overall survival (75% vs. 69%, p = 0.126). Similar to high-risk NCI status and EOI-MRD positivity, CD20 expression was an independent predictor for inferior relapse-free survival (HR: 1.860, 95% CI: 1.008-3.432, p = 0.047). Compared to baseline, there was a significant increase in CD20-expressing EOI-residual blasts among CD20- B-ALL patients (5% vs. 13%, p = 0.001). EOI residual blasts of both CD20+ and CD20- patients had three times increased normalized CD20 expression intensity (nCD20), with the intensity among CD20- B-ALL patients reaching the pretreatment nCD20 of CD20+ B-ALL patients (4.9 vs. 3.6, p = 0.666). Rituximab can be considered in managing EOI-MRD-positive CD20- B-ALL patients as the residual blasts of these patients have quantitative and qualitative increases in CD20 expression.

Allogenic hematopoietic stem cell transplantation outcomes of patients aged ≥ 55 years with acute myeloid leukemia or myelodysplastic syndromes in China: a retrospective study

BackgroundElderly patients with acute myeloid leukemia or myelodysplastic syndromes (AML/MDS) have historically had poor prognoses. However, there has been a recent increase in the use of allogenic hematopoietic stem cell transplantation (allo-HSCT) are in this patient population. Nevertheless, the optimal choice of donor type for the patients remains an unmet need. Limited data exist on the use of allo-HSCT in elderly patients with AML/MDS from China. To better understand and optimize the selection of donor type for the elderly patients, particularly for those with refractory or relapsed disease, in comparison with the previous studies in the US and Europe.MethodsOur retrospective study enrolled 259 patients aged over 55 years who underwent their first allo-HSCT between April 2015 and August 2022. These patients were divided into three groups based on donor type: haploidentical related donor group (haploidentical related donor transplantation [HID], n = 184), matched sibling donor group (matched sibling donor transplantation [MSD], n = 39), and matched unrelated donor group (matched unrelated donor transplantation [MUD], n = 36). Statistics were performed with the chi-square test, the log-rank and Fine-Gray tests.ResultsThe median age of the cohort was 57 years (range: 55–75) and 26.25% of patients were over 60 years old. Younger patients had a higher incidence of acute graft-versus-host disease (HR = 1.942, P = 0.035), faster neutrophil recovery (HR = 1.387, P = 0.012), and better overall survival (HR = 0.567, P = 0.043) than patients aged ≥ 60 years across the entire cohort. Patients with refractory or relapsed (R/R) diseases had delayed neutrophil engraftment (P = 0.010, HR = 0.752) and platelet engraftment (P < 0.001, HR = 0.596), higher incidence of relapses (HR = 2.300, P = 0.013), and inferior relapse-free survival (RFS) (HR = 1.740, HR = 0.016) regardless of donor type. When it came to graft-versus-host-disease-free, relapse-free survival (GRFS), MUDs turned out to be superior to HIDs (HR = 0.472, P = 0.026) according to the multivariable analysis. In contrast, we found MSDs had an inferior GRFS to HIDs in parallel (HR = 1.621, P = 0.043).ConclusionThe choice of donor type did not significantly affect the outcomes of allo-HSCT. However, when considering the quality of post-transplant life, MUDs or HIDs from younger donors may be the optimal choice for elderly patients.

CD38 Is a Functional Partner of CD9 in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: We previously showed that CD9 was expressed in ~80% of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases and its positivity was associated with an inferior relapse-free survival (Leung et al, Leukemia, 2020), suggesting its oncogenic role in this disease entity. Yet, the mechanisms underlying how CD9 affects leukemia progression remain completely unknown. As a prototypic member of the tetraspanin superfamily proteins, CD9 is able to organize with transmembrane or intracellular proteins to form the “tetraspanin web” to exert its modulatory functions. In BCP-ALL, though there has been a standalone study reporting 28 CD9-binding proteins in NALM-6 cells, the results are unlikely representative of the entire disease spectrum due to tumor heterogeneity. Here, we perform an in-depth proteomic study to dissect the CD9 web composition and downstream protein partners to drive disease progression in BCP-ALL. Methods: Immunoprecipitation coupled mass spectrometry (IP-MS) was performed to map the overall composition of CD9 web in five CD9 + BCP-ALL cell lines with distinct cytogenetic backgrounds, including 697 ( TCF3-PBX1+), BV-173 ( BCR-ABL1+), HAL-01 ( TCF3-HLF+), KASUMI-2 ( TCF3-PBX1+) and RS4;11 ( KMT2A-AFF1+). Overlapping protein partners were then validated by IP-Western blot (IP-WB) in 13 patient samples spanning major BCP-ALL subtypes. Shortlisted CD9 partners were further characterized by antibody blockade and CRISPR/Cas9 loss-of-function experiments to evaluate their impact on leukemia proliferation in vitro and leukemia progression in vivo, coupled with enzymatic and phenotypic assays to evaluate the direct impact of CD9 on partner functions. Results: A total of 278 CD9-binding proteins comprising 24 known and 254 novel partners were identified by IP-MS. Venn analysis revealed a marked heterogeneity of CD9-binding proteins across leukemia subtypes, with only 10 being intersected representing the “universal CD9 partners” in BCP-ALL. These proteins consisted of 5 established CD9 partners: ADAM10, CD19, CD38, CD81 and IGSF8 as well as 5 novel partners: ANXA11, HBG2, TMEM109, SLC44A1 and STXBP3. Two universal CD9 partners, namely CD38 and CD81, were consistently detected in all pediatric BCP-ALL samples. Neutralizing antibodies against CD9, CD38 or CD81 unanimously inhibited proliferation of 697 cells in vitro ( P&amp;lt;0.05). However, in the background of CD9 neutralization, CD81, but not CD38, offered an additional impact on leukemia suppression ( P&amp;lt;0.05). Likewise, CD38 knockout cells treated with CD9 antibody did not appear to inhibit cell proliferation in an additive manner. Concordantly, in the 697 xenografts, CD9 or CD38 antibodies alone prolonged animal survival by 2.1- and 1.6-fold, but combinational treatment did not produce the projected additive effect (2.5-fold only). In addition, CD9 neutralization significantly reduced the enzymatic activity of CD38 by 52.5% ( P&amp;lt;0.05) without altering its expression. Conclusions: This work presents the composition of the CD9 protein web in BCP-ALL, revealing homogenous and heterogeneous partners across the leukemia subtypes. We also provide definitive evidence showing that CD9 binds to and regulates CD38 activity to mediate leukemia progression, representing the first universal protein partner being identified to have a functional consequence in BCP-ALL, thereby providing a mechanistic basis to support the development of CD9-targeted therapies for this life-threatening malignancy.

Prognostic Role of Multiparameter Flow Cytometry-Based Measurable Residual Disease Assessment in Acute Myeloid Leukemia Patients with FMS3-like Tyrosine Kinase-3 Internal Tandem Duplication (FLT3-ITD)

Introduction Measurable residual disease (MRD) monitoring is predictive in acute myeloid leukemia (AML). Assessment of FMS3-like tyrosine kinase-3 in-frame internal tandem duplications (FLT3-ITD) is usually performed at diagnosis by polymerase chain reaction (PCR). Due to its relative lower sensitivity of 2%, FLT3-ITD PCR is not routinely used during response assessment. Next-generation sequencing is similarly limited by technical difficulties in capturing tandem duplications using the short base pair-based sequencing method. Long base pair-based sequencing has been reported, but its use is limited by financial restrictions. Multiparameter flow cytometry (MFC) can be a useful tool for MRD monitoring in this AML subtype until such time that molecular techniques for detecting FLT3-ITD MRD are optimized. Patients and methods The study evaluated the outcomes of FLT3-ITD mutated AML patients diagnosed and treated from 2018 to 2022 at Princess Margaret Cancer Centre. We compared outcomes according to MFC-MRD post-induction and FLT3-ITD allele frequency (AF) status at diagnosis. MRD cut-off was 0.1%. Data were locked as of June 30, 2023. Clinical outcomes evaluated include overall survival (OS) and relapse-free survival (RFS). The cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated considering competing risk. The Kaplan-Meier method using a log-rank test and a multivariate Cox proportional hazard model was used for analyses, while the Gray test and Fine-Grey model were used for uni- and multivariate analysis for CIR and NRM. Results A total of 111 patients with a mean age of 63.5 years were included, of whom 90 received treatment. Secondary AML accounted for 12.7% of patients. Risk stratification according to European LeukemiaNet (ELN) 2022 was favorable in 2 (1.8%), intermediate in 67 (60.4%), and adverse in 42 patients (37.8%). Nucleophosmin 1 (NPM1) co-mutation was observed in 55 patients (49.5%). Seventy-nine patients (87.8%) could be assessed for overall response, including 69 (76.7%) who achieved complete remission (CR) or CR with incomplete count recovery (CRi). Of these, 54 achieved first CR/CRi (CR1) with 1 induction cycle. MFC-MRD data were available in 61 patients, of whom 44 (72.1%) were MRD negative, while 17 (27.9%) were MRD positive. With a median follow-up of 437 days, 50 patients (45%) were still alive. Median OS and RFS were 3.42 years and 1.05 years, respectively. Among patients who achieved CR1, post-induction MFC-MRD positivity correlated with an inferior OS (HR 2.35 [1.06-5.26], p=0.037) and a trend for a shorter RFS (HR 2.08 [0.99-4.35], p=0.052). We examined the impact of FLT3-ITD AF at diagnosis on long-term outcomes. By applying a binary recursive partitioning method, the cut-off of FLT3-ITD AF with the best risk stratification power for RFS, was defined at 54.6%. The group with a higher FLT3-ITD AF showed inferior OS (HR 1.86 [1.01-3.44], p=0.047) and RFS (HR 1.91 [1.09-3.33], p=0.023). Taking together FLT3-ITD AF at diagnosis and MFC-MRD status at CR1, patients with low FLT3-ITD AF and negative MRD had the highest OS rate of 86.2% at 12 months (p=0.023), and the highest RFS at 72.4% (p=0.096), while the corresponding values for low FLT3-ITD AF/positive MRD patients were 87.5% and 50%, respectively. In contrast, those with high FLT3-ITD AF/negative MRD had an OS of 72.7% and a RFS of 45.5%, while those with high FLT3-ITD AF/positive MRD showed the lowest OS (16.7%) and the shortest RFS (16.7%). There was no statistical difference in CIR and NRM among groups. Multivariate analysis with stepwise selection was performed, considering age at diagnosis, ELN 2022 risk, MFC-MRD at CR1, FLT3-ITD AF at diagnosis, cytogenetics, and NPM1 co-mutation. Predictive factors for OS were MFC-MRD post-induction (HR 2.49 [1.11-5.61], p=0.027) and FLT3-ITD AF (HR 2.29 [1.03-5.11], p=0.043). For RFS, age at diagnosis (HR 1.03 [1.00-1.06], p=0.038) and FLT3-ITD AF (HR 2.40 [1.15-5.01], p=0.019) were predictive, while MFC-MRD was not significant (HR 1.59 [0.72-3.53], p=0.25). Conclusion Our data demonstrate that MFC-based MRD assessment is feasible in AML with FLT3-ITD. Although better outcomes are expected in patients with a lower FLT3-ITD AF, patients who failed to achieve MRD negativity at CR1 showed inferior outcomes. The presence of both poor risk factors, a high AF of FLT3-ITD at diagnosis and MRD positivity at CR1, correlated with the worst treatment outcomes.

Activated Akt expression is associated with the recurrence of primary melanomas and further refines the prognostic and predictive values for relapse in acral melanomas.

A PTEN deficiency leads to the activation of phospho-Akt at serine 473 (p-Akt) and promotes the tumorigenesis of melanomas by coupling with NUAK2 amplification. We tested the prognostic impact of p-Akt and/or NUAK2 expression on the relapse-free survival (RFS) and overall survival (OS) of melanoma patients. Primary tumors from patients with acral melanomas (112), Low-cumulative sun damage (CSD) melanomas (38), and High-CSD melanomas (18) were examined using immunohistochemistry and their prognostic significance was analyzed statistically. The expression of p-Akt was found in 32.1%, 68.4%, and 55.6% of acral, Low-CSD, and High-CSD melanomas, while NUAK2 expression was found in 46.4%, 76.3%, and 50.0%, respectively. Either p-Akt or NUAK2 expression was inversely correlated with the RFS of primary melanoma patients and acral melanoma patients (p-Akt: p < .0001, p < .0001; NUAK2; p = .0005, p < .0001, respectively). Strikingly, multivariate analyses revealed that p-Akt had a significant impact on RFS (Hazard ratio = 4.454; p < .0001), while NUAK2 did not. Further subset analyses revealed that p-Akt expression had an inferior RFS of patients with acral melanomas (Hazard ratio = 4.036; p = .0005). We conclude that the expression of p-Akt has a significant impact on RFS of patients with primary melanomas and can predict the relapse of patients with acral melanomas.

Impact of Early Natural Killer Cell Reconstitution on the Outcomes of T Cell-Replete Allogeneic Hematopoietic Stem Cell Transplantation.

Early immune reconstitution is crucial to successful outcomes after allogeneic stem cell transplantation (allo-HSCT). However, in T cell-replete HSCT, the impact of natural killer (NK) cells on transplantation outcome and the factors influencing early NK cell reconstitution remain unclear. In this retrospective study, we analyzed 128 patients with hematological malignancies who received the first T cell-replete allo-HSCT between May 2019 and September 2021. After application of a conditioning regimen, prophylaxis for graft versus host disease (GVHD), and engraftment, the patients received prevention and treatment procedures for cytomegalovirus (CMV) reactivation. NK cells, T lymphocytes and B lymphocytes in peripheral blood were collected and analyzed at 30, 60, 90, 135 and 180 days after transplantation to observe immune cell reconstitution. Overall survival (OS), relapse-free survival (RFS), minimal residual disease (MRD), relapse, and non-relapse mortality (NRM) were evaluated. SPSS 25.0 and R version 4.2.1 were used for statistical analysis. In patients with rapid NK recovery (NK cell count at 30 days post-HSCT [NK30] >165/μL and 60 days post-HSCT [NK60] >265/μL), we observed lower rates of NRM, CMV reactivation and acute GVHD (aGVHD). Multivariate analysis indicated that a lower NK30 (≤165/μL) was an independent factor associated with inferior OS and RFS. The NK30 and NK60 in patients with CMV reactivation and aGVHD after transplantation were significantly lower than those in patients without these complications. In addition, CD107a expression in NK cells was also significantly lower in patients who experienced aGVHD. Correlation analysis did not find an inhibitory effect of T-lymphocyte subset reconstitution on NK cells in the early stage after transplantation. Rapid NK cell reconstitution early after allo-HSCT had protective effects on NRM and survival. Promoting early NK cell reconstitution represents a new approach to improving the outcomes of allo-HSCT.

Corticosteroids as graft-versus-host disease prophylaxis for allogeneic hematopoietic cell transplant recipients with calcineurin inhibitor intolerance

Background aimsAlthough calcineurin inhibitors (CNIs) have a well-established role in the prevention of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), their use can be limited by significant toxicities, which may result in premature treatment discontinuation. The optimal management of patients with CNI intolerance is unknown. The objective of this study was to determine the effectiveness of corticosteroids as GVHD prophylaxis for patients with CNI intolerance. MethodsThis retrospective single-center study included consecutive adult patients with hematologic malignancies who underwent myeloablative peripheral blood allogeneic HCT with anti-thymocyte globulin, CNI, and methotrexate GVHD prophylaxis in Alberta, Canada. Multivariable competing-risks regression was used to compare cumulative incidences of GVHD, relapse, and non-relapse mortality between recipients of corticosteroid versus continuous CNI prophylaxis, and multivariable Cox proportional hazards regression was applied to compare overall survival, relapse-free survival (RFS) and moderate-to-severe chronic GVHD and RFS. ResultsAmong 509 allogeneic HCT recipients, 58 (11%) patients developed CNI intolerance and were switched to corticosteroid prophylaxis at median 28 days (range 1-53) after HCT. Compared with patients who received continuous CNI prophylaxis, recipients of corticosteroid prophylaxis had significantly greater cumulative incidences of grade 2-4 acute GVHD (subhazard ratio [SHR] 1.74, 95% confidence interval [CI] 1.08–2.80, P = 0.024), grade 3–4 acute GVHD (SHR 3.22, 95% CI 1.55–6.72, P = 0.002), and GVHD-related non-relapse mortality (SHR 3.07, 95% CI 1.54–6.12, P = 0.001). There were no significant differences in moderate-to-severe chronic GVHD (SHR 0.84, 95% CI 0.43–1.63, P = 0.60) or relapse (SHR 0.92, 95% CI 0.53–1.62, P = 0.78), but corticosteroid prophylaxis was associated with significantly inferior overall survival (hazard ratio [HR] 1.77, 95% CI 1.20–2.61, P = 0.004), RFS (HR 1.54, 95% CI 1.06–2.25, P = 0.024), and chronic GVHD and RFS (HR 1.46, 95% CI 1.04–2.05, P = 0.029). ConclusionsAllogeneic HCT recipients with CNI intolerance are at increased risks of acute GVHD and poor outcomes despite institution of corticosteroid prophylaxis following premature CNI discontinuation. Alternative GVHD prophylaxis strategies are needed for this high-risk population.

Prognostic value of chest x-ray- and CT-defined large mediastinal adenopathy in high-risk pediatric Hodgkin lymphoma: A report from the Children's Oncology Group Study AHOD0831.

We compared the prognostic value of chest radiograph (CXR)- and computed tomography (CT)-derived definition of large mediastinal adenopathy (LMA) in pediatric Hodgkin lymphoma (HL). Total 143 patients treated for stage IIIB/IVB HL on COG AHOD0831 were included in this study. Six definitions of LMA were investigated: (i) mediastinal mass ratio on CXR (MRCXR )>1/3; (ii) mediastinal mass ratio on CT (MRCT )>1/3; (iii) mediastinal mass volume on CT (MVCT )>200mL; (iv) normalized mediastinal mass volume (MVCT /thoracic diameter [TD])>1mL/mm; (v) mediastinal mass diameter on CT (MDCT )>10cm; and (vi) normalized mediastinal mass diameter (MDCT /TD)>1/3. Median age at diagnosis was 15.8years (range: 5.2-21.3years). In patients with a slow early response (SER) to chemotherapy, MVCT >200mL, MDCT >10cm, and MDCT /TD>1/3 were associated with worse relapse-free survival (RFS) on MVA, while MRCXR >1/3, MRCT >1/3, and MVCT /TD>1mL/mm trended toward worse RFS; MDCT /TD was the most strongly prognostic for inferior RFS, with a hazard ratio of 6.41 for MDCT /TD>1/3 versus ≤1/3 on MVA (p=.02). LMA according to MVCT >200mL, MDCT >10cm, and MDCT /TD>1/3 is associated with poor prognosis in advanced-stage HL patients with SER. The normalized mediastinal diameter, MDCT /TD>1/3 appears to be the strongest predictor of inferior RFS.

Abstract 2127: TERT C228T and KDM6A alterations are potential predictive biomarkers in non-muscle-invasive bladder cancer treated with intravesical Bacillus Calmette-Guérin instillation

Abstract Introduction: Bacillus Calmette-Guérin (BCG) is the standard of care for high-risk non-muscle-invasive bladder cancer (NMIBC) following transurethral resection. However, patients often have heterogeneous responses. Even among those who initially respond well to BCG, 10-20% relapse. Identification of reliable biomarkers predicting the efficacy of BCG remains an unmet need. En bloc resection is a novel technique representing a substantial advancement in the surgical management of NMIBC. We sought to investigate genomic and tumor microenvironmental (TME) profiles in NMIBC and explore potential predictive markers for BCG treatment following en-block resection. Methods: A total of 40 patients with high-risk NMIBC (cTis-T1N0M0) were retrospectively enrolled who underwent en bloc resection followed by BCG instillation. Surgical samples were subjected to NGS sequencing using a 520-gene panel (Burning Rock Biotech, Guangzhou) and multiplex immunofluorescence (mIF) assay. Results: The cohort had a median age of 63 years, and 80% were male. After a median follow-up of 21.8 months, 19/40 patients relapsed with a one-year relapse-free survival (RFS) rate of 57.5%. All tumors were microsatellite stable and showed a median TMB of 7.98muts/Mb. Genomic profiling revealed a high prevalence of alterations in TERT (55%), KDM6A (32.5%), KMT2D (32.5%), FGFR3(30%), PIK3CA (30%), TP53(27.5%), KMT2C (25%), and ARID1A (20%). TME analysis showed higher proportions of M1 macrophages and CD56 dim NK cells in the tumoral compartment and more intense infiltration of CD8+ T cells, exhausted CD8+T, CD56 bright NK cells, and M2 macrophages in the stromal compartment. Multivariate analysis identified TERT C228T mutation (HR=3.28 [95%CI:1.225-8.79], p=0.0181) and alteration in KDM6A (HR=2.94 [95%CI:1.040-8.29], p=0.042) as two independent factors associated with inferior RFS. Patients with concomitant TERT C228T and KDM6A alteration had the shortest RFS (median RFS:5.83months) compared with those who were free of (median RFS: NR) or harbored either one of the two alterations (median RFS:9.13months) (p=0.0022). We also found that tumoral infiltration of CD8+T cells was positively associated with RFS (HR=0.29 [95%CI:0.097-0.885], p=0.0208). Conclusion: The study comprehensively depicted the genomic and TME profiles in NMIBC and identified potential predictive biomarkers for BCG treatment. Our findings may facilitate the stratification of patients and better guide the clinical decision-making on the management of NMIBC. Citation Format: Qi-Dong Xia, Yao-Bing Chen, Jian-Xuan Sun, Chen-Qian Liu, Jin-Zhou Xu, Zhi-Peng Yao, Ye An, Meng-Yao Xu, Si-Han Zhang, Xing-Yu Zhong, Na Zeng, Si-Yang Ma, Hao-Dong He, Heng-Long Hu, Jia Hu, Yi Lu, Lin Shao, Si-Qi Li, Zheng Liu, Shao-Gang Wang. TERT C228T and KDM6A alterations are potential predictive biomarkers in non-muscle-invasive bladder cancer treated with intravesical Bacillus Calmette-Guérin instillation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2127.

Abstract P3-05-36: Prognostic factors in non-metastatic hormone receptor-positive HER2-negative mucinous breast cancer: an international multicentre cohort study

Abstract Background: Mucinous carcinoma is the third most common histological subtype of breast cancer after ductal and lobular carcinomas, accounting for approximately 3% of invasive breast cancers. Although considered a favourable subtype with de-escalation of treatment recommended in the National Comprehensive Cancer Network guidelines, recurrence can occur and supporting data is limited. We thus examined prognostic factors of pure mucinous breast cancer (PMBC) in an international multicentre cohort study. Methods: Patients diagnosed between January 2000 to December 2015 with hormone receptor-positive HER2-negative stage I to III PMBC, invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) at 6 centers in Singapore, Taiwan, Korea and Japan were evaluated. Cox proportional hazards regression analyses were used to compare relapse-free survival (RFS), distant relapse-free survival (DRFS) and overall survival (OS) by histological subtypes, and to identify prognostic factors for PMBC. Results: Of 23,105 women eligible for analysis, 20,684 had IDC, 1,475 had ILC and 946 had PMBC. The median follow-up was 6.6 years; 5-year RFS, DRFS and OS for PMBC were 94.6%, 96.5% and 98.1% respectively. On multivariable cox regression analyses, PMBC demonstrated superior RFS (hazard ratio [HR] = 0.70, 95% CI: 0.56 - 0.88), DRFS (HR = 0.69, 95% CI: 0.53 - 0.89) and OS (HR = 0.70, 95% CI: 0.52 - 0.93) compared to IDC, while ILC had comparable survival outcomes as IDC. When restricted to only PMBC, significant independent prognostic factors for RFS included ethnicity (vs Chinese, “Others” [non-Chinese/Japanese/Korean, mainly Malay and Indian]: HR = 2.62, 95% CI 1.23 – 5.57), older age (vs &amp;lt; 40 years, &amp;gt;70 years: HR = 3.53, 95% CI 1.67 – 7.46), tumor size (vs T1, T3-4: HR = 2.79, 95% CI 1.45 – 5.37), positive lymph nodes (HR = 2.04, 95% CI: 1.10 – 3.77), use of radiotherapy (HR = 0.54, 95% CI 0.33 – 0.91) and endocrine therapy (HR = 0.31, 95% CI 0.12 – 0.77). On further analysis, the inferior RFS, DRFS and OS in older patients (&amp;gt;70 years) were driven largely by non-breast cancer deaths rather than relapses. Use of endocrine therapy was also associated with superior DRFS (HR = 0.26, 95% CI 0.09 – 0.73) but not OS. In a subgroup analysis, use of chemotherapy was associated with improved DRFS (HR = 0.25, 95% CI 0.08 – 0.82) and OS (HR = 0.07, 95% CI 0.01 – 0.37) with a trend in RFS (HR = 0.41, 95% CI 0.14 – 1.24) for lymph node-positive PMBC; no differences in outcomes were observed for the lymph node-negative subgroup. Conclusions: Larger tumor size, lymph node positivity and ethnicity were significant factors for RFS in PMBC. Use of endocrine therapy was associated with superior RFS and DRFS, while chemotherapy was associated with better DRFS and OS for lymph-node positive PMBC. Citation Format: Ryan Tan, Whee Sze Ong, Kyung-Hun Lee, Seri Park, Jabed Iqbal, Yeon Hee Park, Jeong-Eon Lee, Jong Han Yu, Ching-Hung Lin, Yen-Shen Lu, Makiko Ono, Takayuki Ueno, Yoichi Naito, Tatsuya Onishi, Geok hoon Lim, Su-Ming Tan, Han-Byoel Lee, Jiwon Koh, Han Suk Ryu, Wonshik Han, Veronique Kiak Mien Tan, Fuh-Yong Wong, Seock-Ah Im, Puay Hoon Tan, Yoon-Sim Yap. Prognostic factors in non-metastatic hormone receptor-positive HER2-negative mucinous breast cancer: an international multicentre cohort study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-36.

Clinical Significance of bZIP In-Frame CEBPA-Mutated Normal Karyotype Acute Myeloid Leukemia.

We evaluated the characteristics of CCAAT/enhancer-binding protein α (CEBPA) mutations and the significance of a basic leucine zipper in-frame mutation (bZIPin-f) of CEBPA in patients with acute myeloid leukemia with a normal karyotype. Based on updated knowledge of CEBPA mutations, we conducted next-generation sequencing analyses in a previously established real-world cohort. Among 78 of a total of 395 patients (19.7%), 50 had bZIPin-f CEBPA, and 28 had non-bZIPin-f CEBPA. In the multivariate analysis, patients with NPM1mut, those with bZIPin-f CEBPA, and those who underwent allogeneic hematopoietic cell transplantation (allo-HCT) had favorable overall survival (OS), but FLT3-ITDmut was a poor prognostic indicator. For relapse-free survival (RFS) and cumulative incidence of relapse, bZIPin-f CEBPA, and allo-HCT were associated with favorable outcomes; FLT3-ITDpos was associated with worse outcomes. In the CEBPA double-mutated group (CEBPAdm), bZIPin-f CEBPA was associated with superior outcomes in terms of OS (p=0.007) and RFS (p=0.007) compared with non-bZIPin-f CEBPA. Of 50 patients with bZIPin-f CEBPA, 36 patients had at least one mutation. When grouped by the presence of mutations in chromatic/DNA modifiers (C), cohesion complex (C), and splicing genes (S) (CCS mutations), CCS-mutated bZIPin-f CEBPA was associated with poor OS (p=0.044; hazard ratio [HR], 2.419) and a trend in inferior RFS (p=0.186; HR, 1.838). Only bZIPin-f CEBPA was associated with favorable outcomes in patients with CEBPAdm. However, some mutations accompanying bZIPin-f CEBPA showed inferior OS; thus, further studies with larger numbers of patients are required for clear conclusions of the significance of bZIPin-f CEBPA.

Restratifying the prognosis of acute myeloid leukemia patients with CEBPA double mutations based on CSF3R mutations and measurable residual disease

Objective: This investigation aims to assess the impact of CSF3R mutations and the presence of measurable residual disease (MRD) on the prognosis of patients with CEBPA double mutations who have acute myeloid leukemia (AML) . Methods: The prognostic significance of these two factors was examined in the present study, which included 66 patients with complete genetic mutations and sequential MRD information. Results: Following the second course of chemotherapy, the MRD status and CSF3R mutations of these patients were linked to their long-term prognosis. CSF3R mutated patients showed inferior relapse-free survival (RFS) (5-year RFS: 15.2% vs 38.7% , P=0.006) and overall survival (OS) (5-year OS: 18.2% vs 60.6% , P=0.038) compared with those with wild-type CSF3R. After the second course of chemotherapy, patients with negative MRD had an RFS of 64 months and an OS of not reaching, which was significantly longer than that of patients with positive MRD (15 and 48 months, and the P value were 0.004 and 0.050, respectively) . CSF3R mutations (HR=0.317, 95% CI 0.129-0.779, P=0.012) , WT1 mutations (HR=0.304, 95% CI 0.115-0.804, P=0.016) , and NRAS mutations (HR=0.153, 95% CI 0.061-0.385, P<0.001) were all independently associated with a poor prognosis for RFS, and CSF3R mutations and positive MRD tended to be independently associated with a poor prognosis for OS, according to the results of a Cox proportional-hazards model analysis (P values were 0.071 and 0.088, respectively) . The patients were divided into three groups based on their CSF3R mutation status and MRD status following treatment: wide-type CSF3R and negative MRD, mutated CSF3R or positive MRD, and mutated CSF3R and positive MRD, which showed significantly different RFS (P<0.001) and OS (P=0.006) . Conclusion: Both CSF3R mutations and positive MRD were associated with poor outcome in AML patients with CEBPA double mutations. An integrity model based on these two factors may be beneficial for accurately evaluating the prognosis of these patients.

Preclinical Efficacy of Novel Drug Combination Against AML with 3q26 Lesions and EVI1 Overexpression

The EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc finger transcription factor (TF). EVI1 protein is expressed in leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. As a result of the chromosome translocation t(3;3) or inv(3) at 3q26, the distal enhancer of the TF and tumor suppressor GATA2 is dislocated and repositioned to induce expression of EVI1, while concomitantly repressing GATA2. EVI1 overexpression results in therapy refractoriness and inferior relapse-free and overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor has activity against AML cells with inv(3)/t(3;3). Among the targets of EVI1 are the pro-growth and pro-survival proteins c-Myc and Bcl-xL, expressions of which are also regulated by enhancer activity that is undermined by BRD4 inhibition. We have recently reported that the nuclear TBL1/R1-β-catenin-TCF7L2 complex transcriptionally regulates c-Myc, cyclin D and Survivin and sustains AML stem cells, such that the undermining of this transcriptional axis by the TBL1/R1-β-catenin-binding disruptor tegavivint (BC-2059, Iterion) represses MYC, cyclin D1 and Survivin and inhibits growth and survival of AML stem-progenitor cells. Based on these observations, we interrogated the pre-clinical efficacy of pan-BET inhibitor OTX015 and/or tegavivint (TV) against AML cells with inv(3)/t(3;3) and overexpression of EVI1. Treatment with BET inhibitor OTX015 dose-dependently (100 to 1000 nM) induced apoptosis of AML cell lines (UCSD-AML1, OCI-AML20, MUTZ-3 and HNT-34) and patient-derived (PD) AML cells (AML191 and AML194) with inv(3)/t(3;3) and with or without monosomy 7. OTX015 treatment was associated with repression of c-Myc, c-Myb, CDK4, MECOM, and MCL1 as well as induction of HEXIM1 and cleaved PARP levels. We also determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions with/without monosomy 7. Furthermore, TV treatment resulted in attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, while inducing p21, CD11b, BIM and cleaved PARP levels. Next, utilizing H3K27Ac antibody Hi-ChIP (Arima Hi-ChIP), we interrogated the active gene regulatory interactions through a combined Hi-C and ChIP workflow, followed by Tn5 transposase-mediated on-bead library construction and NGS. We observed that in untreated AML191 cells numerous loop interactions were present between the super-enhancers and promoters respectively of MYC and BCL2. Following TV treatment (100 nM for 16 hours), differential loop calling revealed a loss of these interactions. RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of TGFβ, inflammatory response, TNFα and interferon signaling, and apoptosis signaling, but negative enrichment of gene sets of MYC, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in the phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99) harboring inv(3) and EVI1 overexpression. Co-treatment with TV and OTX015 or the BCL2 inhibitor venetoclax synergistically induced in vitro apoptosis (determined by SynergyFinder 2.0) in the AML cell lines and the PD AML cells. Notably, in the flank-implanted PD AML191 or tail vein AML242 models in NSG mice, treatment with TV and OTX015 for 2 to 5 weeks, compared to vehicle control or each drug alone, significantly reduced AML growth (p < 0.01). Furthermore, in the tail-vein infused and engrafted PD AML191 PDX model, treatment with TV and OTX015 or venetoclax for 6 weeks, as compared to vehicle control or each drug alone, significantly improved overall survival of the NSG mice (p < 0.01), without inducing any toxicity. These findings highlight the promising pre-clinical efficacy of novel combinations of TV and BET protein or venetoclax against AML models harboring 3q26 lesions and EVI1 overexpression.

The Prognostic Impact of HMGCLL1 Gene Variant on Treatment Outcomes in Chronic Myeloid Leukemia (CML) Patients: Adverse Impact on the Response, Failure, and Progression with Imatinib Which Can be Abrogated By the Use of 2nd Generation Tyrosine Kinase Inhibitor (TKI) Upfront Therapy

BackgroundHMGCLL1 is characterized as a lyase activity enzyme located in the extramitochondrial region. Its biological and physiological role is not fully elucidated. Our previous study (Leukemia 2019) demonstrated that: 1) The HMGCLL1 gene variant, ACGTAATG haplotype (A-haplotype) is associated with increased expression of overall levels and isoform 3 (IS3) of the HMGCLL1 gene and predicts a decreased likelihood of deep molecular response after Imatinib (IM) therapy, 2) blockade of HMGCLL1 IS3 results in G0/G1 cell cycle arrest, leading to increased eradication of CML cell lines and CML leukemic stem cells, 3) blockade of HMGCLL1 isoform 3 significantly suppressed CD34+ fractions in CML patients (pts). It was unclear whether a second-generation tyrosine kinase inhibitor (2G-TKI) can reverse the adverse impact of the HMGCLL1 gene variant in CML pts. The present study assessed the prognostic impact of the HMGCLL1 gene variant in CML pts treated with IM or 2G-TKI, and its impact on treatment-free remission (TFR) after TKI discontinuation. Patients and methods Samples from 387 pts were sequenced. Group 1 included 252 pts from 2 centers whose samples were archived retrospectively. These pts were treated with IM (n=190, 75.3%) or second-generation TKIs (n=62) including Dasatinib (n=15, 6.0%), Nilotinib (n=37, 14, 7%), and Bosutinib (n=10; 4.0%). Group 2 included 135 patient samples from 4 countries that attempted to discontinue TKI for TFR. We applied our internal CML-specific smMIP panel with 332 amplicon probes including 8 HMGCLL1 SNPs (rs10948926, rs10948927, rs9370435, rs4546489, rs4275061, rs9475323, rs9475327, rs9296791). Linkage disequilibrium (LD) was assessed using Haploview software (Bioinformatics 2005). For Group 1, treatment outcomes were evaluated concerning major molecular response (MMR), molecular response (MR) with 2 log/deeper (MR2) or 4 log/deeper (MR4), failure-free survival (FFS), progression, and overall survival (OS). Disease characteristics and treatment outcomes were evaluated and compared according to the HMGCLL1 variants using a dominant model. For Group 2, molecular relapse-free survival (RFS) was calculated from the date of TKI discontinuation to the date of confirmed loss of molecular response (either loss of major MMR or two consecutive episodes of MR loss). Kaplan-Meier survival estimate was used to evaluate RFS and Cox's proportional hazard regression model was applied using the dominant model. Results HMGCLL1genotype and haplotype As shown in Fig A, the LD plot showed no recombination among the 8 SNPs, leading to one block for the 8 SNPs to construct a haplotype. Two haplotypes were constructed as CTCAGGCA (C-) or ACGTAATG (A-haplotype) with MAF of 0.674:0.279 in Group 1, and 0.703:0.216 in Group 2, respectively. Summary of outcomes in table 1. Treatment outcomes following TKI therapy according to the HMGCLL1 haplotype Homozygous A-haplotype (A/A) was associated with worse outcomes, showing a lower MR2 (65.9% vs 78.9% at 12 months, p=0.012), lower MMR (64.4% vs 86.8% at 3 years, p=0.00029), lower MR4 (34.7% vs 62.3% at 5 years, p=0.0001; worse FFS (62.8% vs 77.3% at 3 years, p=0.0014), higher risk of progression (16.2% vs 3.4% at 5 years, p=0.000678), and worse OS (56.1% vs 77.7% at 10 years, p=0.0021). The adverse impact of A/A-haplotype was more prominent in the pts treated with Imatinib: A/A-haplotype pts showed a 30-50% lower chance of MR2, MMR, and MR4 achievement, and 2-3 times higher risk of TFR and progression in comparison to those having C-haplotype when treated with Imatinib. However, the use of 2G-TKIs could abrogate the adverse impact of the A/A-haplotype. Impact of HMGCLL1 variant on treatment-free remission following TKI discontinuation While the 12-month RFS rate was 59.9% in overall pts (n=135), an inferior 12-month RFS rate was noted in A/A haplotype (43.8% vs 62.3%) but with borderline significance (p=0.116). Conclusion: The present study demonstrated that the HMGCLL1 gene variant is confirmed as predictive of CML outcomes. Moreover, our result suggests that the adverse impact of the HMGCLL1 genotype can be abrogated by using 2G-TKIs as upfront therapy. We aim to validate this data in a larger cohort of pts treated with 2G-TKI therapy to reach a clearer conclusion on this result. HMGCLL1 genotype information would be helpful to guide CML therapy given that pts with adverse HMGCLL1 genotypes could benefit from 2G-TKI therapy as front-line therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Obesity Associates with Inferior Outcomes and Toxicity in Pediatric and Young Adult Patient with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia Treated with Commercially Manufactured Tisagenlecleucel

Introduction: The incidence of obesity continues to rise with over 50% of the world's population predicted to be overweight/obese by the year 2030. There is emerging epidemiological evidence suggesting that obesity is a risk factor for developing hematological malignancies, and previous data shows acute lymphoblastic leukemia (ALL) treatment is associated with the development of obesity. Alarmingly, the survival outcomes for obese children/young adults with B-ALL are historically inferior to those observed in healthy weight peers when treated with traditional chemotherapy regimens leading to the need for alternative therapeutic approaches. Chimeric antigen receptor T-cell therapy redirects the cytotoxicity of a T-cell towards a cancer cell and has been shown to induce remission in patients who have previously been treatment-refractory or multiply relapsed. Unfortunately, T-cell dysfunction has been reported in obesity. The impact of obesity on T-cell immunotherapies is currently unknown. Objective: Establish real world disease response and toxicity outcomes experienced by overweight/obese pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) treated with chimeric antigen receptor T-cell therapy (tisagenlecleucel), compared to their healthy weight peers. Design/Methods: Retrospective data was collected from Pediatric Real World CAR Consortium (PRWCC) institutions (n=15) on 168 pediatric/young adult patients (age 2-22), evaluable for BMI measure, who underwent infusion of commercially manufactured tisagenlecleucel for r/r B-ALL. Weight categories were established using CDC growth charts with gender/age specific BMI cutoffs (healthy 5th to 85th percentile; overweight/obese >85th percentile). Efficacy outcomes include relapse-free survival (RFS) and overall survival (OS). Toxicity endpoints include cytokine release syndrome (CRS) and neurotoxicity (ICANS). Demographic, baseline characteristics, and toxicities were summarized as median (range) for continuous variables and number and percentage for categorical variables. Comparisons between groups were done using Chi-square tests for categorical variables, or Wilcoxon test for continuous variables. The Kaplan-Meier method was used to analyze RFS and OS using the log rank test to compare weight groups. Hazard ratios with 95% confidence intervals were calculated. Univariable and multivariable analyses were performed. Results: Of 168 patients included in the analysis, 88 were in the healthy weight category and 80 were in the overweight/obese category. There was no difference in baseline characteristics between groups (age at infusion, gender, race/ethnicity, Down syndrome, marrow morphology prior to infusion (M1 vs >M1 marrow), CNS status prior to infusion, or transplant following infusion). Overweight/obese patients had statistically significant inferior RFS at 6 months (61.9% vs 81.5%), 12 months (51.4% vs 70%), and 24 months (31.1% vs 64.7%). Relapse hazard ratio for obese/overweight patients was 2.04 (95% CI 1.2, 3.45: p = 0.006). Overall survival of overweight/obese patients was not statistically different than healthy weight patients but trended towards decreased OS in overweight/obese patients at 6 months (82.4% vs 89.6%), 12 months (70.3% vs 77.5%) and 24 months (46.4% vs 64.7%). OS hazard ratio for obese/overweight patients was 1.54 (95% CI 0.85, 2.78: p = 0.149). Additionally, overweight/obese patients showed a trend towards higher grade toxicity than healthy weight patients with CRS grade ≥ 3 seen in 22.8% vs 14.8% (p = 0.272) and ICANS grade ≥ 3 seen in 11.4% vs 3.4% (p = 0.108), respectively. Conclusion: This retrospective, multi-institutional analysis highlights the high incidence of obesity among patients who are candidates for tisagenlecleucel. Furthermore, it shows that overweight/obese patients treated with tisagenlecleucel for r/r B-ALL have inferior outcomes (particularly RFS) and potential for increased toxicity compared to their healthy weight peers. Additional research is needed to interrogate generalizability of findings to immunotherapy recipients of all ages, across cancer subtypes, and illicit the mechanism of obesity induced immune dysfunction and related mitigating strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A glimpse into translocation (8;21) in acute myeloid leukemia: Profile and therapeutic outcomes from a tertiary care hematology center from East India

Objectives: Translocation (8;21) is a RUNX1-RUNX1T1 fusion transcript, a favorable risk cytogenetic abnormality with a variable clinicopathological profile. However, there is a paucity of data on the outcomes of acute myeloid leukemia (AML) with t(8;21) from East India. This report is an analysis of data of AML with t(8;21) at our center. Material and Methods: De novo AML patients with the presence of t(8;21) cytogenetic abnormality from 2015 to 2019 were analyzed for clinical, pathological, and molecular characteristics and were compared with treatment outcomes. Relapse-free survival (RFS) and overall survival (OS) were determined using Kaplan–Meier curves. Results: Twenty-nine patients (10%) with de novo AML had t(8;21) with 18 male patients and a median age of 20 years. Aberrant expression of CD19, CD56, and CD7 expressions was noted in 44.8%, 17.24%, and 10.29% of patients, respectively. Additional cytogenetic abnormality was observed in 31.03%. CD19 had an 80% correlation with the occurrence of C-kit status. High-dose induction therapy had complete remission rates of 100%. The median duration of follow-up was 287.5 days. The presence of myeloid sarcoma (MS) and C-kit positivity had inferior OS and RFS (P &lt; 0.05). The dose of cytosine arabinoside, given in consolidation of 3 g/m2 and 1.5 g/m2, had a median OS of 758 and 479 days (P = 0.661) and median RFS of 348 and 150 days (P = 0.002), respectively. In the group that received intensive therapy, by the end of 3 years, only 15.7% of patients remain in remission. Conclusion: AML with t(8;21) is seen in young patients with a positive correlation between CD 19 with C-kit positivity. The presence of MS and C-kit positivity endowed inferior OS and RFS. Cytosine arabinoside consolidation in a dose of 3 g/m2 offered an advantage in median RFS.

Frequency and clinical impact of WT1 mutations in the context of CEBPA-mutated acute myeloid leukemia

ABSTRACT Introduction Several studies have confirmed that mutations in the Wilms tumor 1 (WT1) gene occur in adult acute myeloid leukemia (AML). However, few data are available regarding the incidence of WT1 mutations in CEBPAmut AML and their impact. Methods We retrospectively analyzed the frequency and clinical impact of WT1 mutations in 220 newly diagnosed AML patients with CEBPA mutations(CEBPAmut). Chromosome karyotype analysis was performed by R or G banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT–PCR). Mutations were detected with a panel of 112mutational genes using next-generation sequencing (NGS). Results Overall, 30 WT1 mutations were detected in 29 of the 220 CEBPAmut AML patients (13.18%) screened. These mutations clustered overwhelmingly in exon 7 (n=16). WT1 mutations were found to be significantly more frequent in AML patients with double-mutated CEBPA (CEBPAdm) than in AML patients with single-mutated CEBPA (17.36%vs. 8.08%, P = 0.043). Among WT1-mutated patients, the most common co-mutation was FLT3-ITD (n = 7, 24.14%), followed by NRAS (n = 5, 17.24%), CSF3R (n = 4, 13.79%), GATA2 (n = 4, 13.79%), and KIT (n = 4, 13.79%). The most frequent functional pathway was signaling pathways inas many as 62.07% of cases. Notably,the concomitant mutations in epigenetic regulatorswere inversely correlated with WT1 mutations(P = 0.003). CEBPAdm AML patients with WT1 mutations had inferior relapse-free survival, event-free survival and overall survival compared with patients CEBPAdm AML without WT1 mutations (P = 0.002, 0.004, and 0.010, respectively). Conclusion Our data showed that WT1 mutations are frequently identified in CEBPAmut AML, especially in CEBPAdm AML. CEBPAmut AML patients with WT1 mutations show distinct spectrum of comutations. In the context of CEBPAdm AML, WT1 mutations predict a poor prognosis.

Abstract 2648: Novel combination therapies against AML with 3q26 lesions and EVI1 overexpression

Abstract The EVI1 gene is located on 3q26.2 and encodes a zinc fingers-containing transcription factor. Nuclear β-catenin-TBL1/R1-TCF7L2 axis activity and EVI1 expression characterize leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. EVI1 is overexpressed in AML with chromosome translocation t(3;3) or inv(3) at 3q26, where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression confers poor response to therapy and inferior relapse-free and overall survival in AML. Tegavivint (BC-2059, Iterion) targets TBL1/R1 and disrupts its binding to β-catenin and TCF7L2, repressing MYC, cyclin D1 and Survivin, leading to apoptosis of AML cells. Here, we determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines (UCSD-AML1, MUTZ3 and OCI-AML20) and patient-derived (PD) AML cells (AML191 and AML194) with 3q26.2 lesions with/without monosomy 7. This was associated with attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but upregulation of CD11b, BIM and cleaved PARP levels. Additionally, by reducing BRD4 occupancy at GATA2 enhancer and repressing EVI1, the pan-BET protein inhibitor OTX015 (100-1000 nM) also dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of inflammatory response, TNFα and interferon signaling, TGFβ and apoptosis signaling, but negative enrichment of gene sets of c-Myc, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in phenotypically characterized AML stem cells harboring t(3:3)/inv(3) and EVI1 overexpression (with high expression of CLEC12A, CD123, CD244, CD99). In vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (determined by SynergyFinder) of AML cell lines and the PD AML cells. In the tail-vein infused and engrafted PD AML191 cell model in NSG mice, treatment with TV and/or venetoclax or OTX015 for 6 weeks significantly reduced AML burden and improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. These findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 axis combined with BET protein or BCL2 inhibition is effective therapy against AML models harboring 3q26 lesions and EVI1 overexpression. Citation Format: Christine Birdwell, Warren C. Fiskus, Christopher P. Mill, John A. Davis, Qi Jin, Courtney D. DiNardo, Koichi Takahashi, Stephen Horrigan, Tapan M. Kadia, Naval Daver, Kapil N. Bhalla. Novel combination therapies against AML with 3q26 lesions and EVI1 overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2648.