Citations

Abstract Objectives The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL. Methods We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed. Results Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression. Conclusions Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL.

Abstract Background Approximately 25% of patients with chronic lymphocytic leukemia (CLL) experience a flare of disease following ibrutinib discontinuation. A critical question is whether this phenomenon may also occur when ibrutinib is temporarily held. This study aimed to determine the frequency and characteristics of disease flares in this setting and assess risk factors and clinical outcomes. Materials and Methods We identified all patients with CLL seen at Mayo Clinic between October 2012 and March 2019 who received ibrutinib. Temporary interruptions in treatment and associated clinical findings were ascertained. Results Among the 372 patients identified, 143 (38%) had at least one temporary interruption (median 1 hold, range 1–7 holds) in treatment. The median duration of interruption was 8 days (range 1–59 days) and the most common indication was periprocedural. Among the 143 patients with ≥1 hold, an associated disease flare was seen in 35 (25%) patients: mild (constitutional symptoms only) in 21 patients and severe (constitutional symptoms with exam/radiographic findings or laboratory changes) in 14 patients. Disease flare resolved with resuming ibrutinib in all patients. Predictive factors of disease flare included progressive disease at time of hold and ≥ 24 months of ibrutinib exposure. The occurrence of disease flare with an ibrutinib hold was associated with shorter event-free survival (hazard ratio 2.3; 95% confidence interval 1.3–4.1; p = .007) but not overall survival. Conclusion Temporary interruptions in ibrutinib treatment of patients with CLL are common, and one quarter of patients who held ibrutinib in this study experienced a disease flare. Resolution with resuming ibrutinib underscores the importance of awareness of this phenomenon for optimal management. Implications for Practice Ibrutinib is a very effective treatment for chronic lymphocytic leukemia (CLL) but needs to be taken continuously. Side effects, such as increased bleeding risk with procedures, require temporary interruptions in this continuous treatment. Rapid CLL progression following ibrutinib discontinuation has been increasingly recognized. This study demonstrates that similar flares in disease signs or symptoms may occur during ibrutinib holds as well. Importantly, management with restarting ibrutinib led to quick clinical improvement. Awareness of this phenomenon among clinicians is critical to avoid associated patient morbidity and premature cessation of effective treatment with ibrutinib if the flare is misidentified as true progression of disease.

Key Points An incidental histologic diagnosis of DLBCL was identified during temporary interruption of ibrutinib treatment in patients with CLL. In contrast to an aggressive clinical course typical of Richter transformation, these patients responded to reinitiation of ibrutinib alone.

Abstract Background: Atezolizumab (atezo) and bevacizumab (bev) combination therapy has demonstrated robust clinical activity in patients with unresectable HCC who have not received prior systemic therapy (Lee et al., APPLE 2019; Cheng et al., ESMO Asia 2019). In this exploratory analysis, we aimed to identify tumor-based molecular biomarkers that may be associated with clinical response or resistance to atezo + bev. We also investigated how VEGF blockade with bev could potentiate PD-L1 checkpoint inhibition with atezo in pts with advanced HCC. Methods: Archival tumor tissues or fresh biopsies taken prior to treatment were collected from HCC pts enrolled in the Phase 1b trial GO30140 (NCT02715531). Arm A (n = 104) was a single-arm evaluation of atezo + bev; Arm F (n = 119) was a randomized arm comparing atezo + bev with atezo. Whole-exome sequencing was carried out on these tumor tissues to determine tumor mutation burden (TMB). Gene expression in tumors was profiled by RNAseq analysis. The association between biomarker expression and clinical response (responders [R] vs. non-responders [NR]) or PFS was assessed by t-tests or Cox regression models, respectively. All p-values are descriptive. Results: In Arm A, 90/104 pts were biomarker evaluable. TMB was not associated with response to atezo + bev or PFS. In contrast, analysis of baseline tumor gene expression showed that pre-existing immunity appeared to be associated with clinical response and longer PFS, which included high expression of CD274 (PD-L1) (R vs. NR, p < 2.1 × 10−5; PFS: HR = 0.42 [0.25-0.72]), and T effector signature (GZMB, PRF1, CXCL9) (R vs. NR, p < 0.0004; PFS: HR = 0.46 [0.27-0.78]). Gene expression related to Notch pathway activation (i.e. high expression of HES1) appeared to be associated with lack of response to atezo + bev (p < 0.039) and shorter PFS (HR = 2.1 [1.3-3.6]). In Arm F, 91/119 pts were biomarker evaluable. High expression of VEGF receptor 2 (VEGFR2; HR = 0.36 [0.16-0.81]), Treg (HR = 0.35 [0.15-0.82]), myeloid inflammation (HR = 0.43 [0.19-0.95]), and TREM1/MDSC signatures (HR = 0.43 [0.19-0.94]) was associated with longer PFS in patients treated with atezo + bev than in those treated with atezo alone. Analysis of 12 serial biopsy pairs confirmed reduced levels of VEGFR2 and Treg signatures after atezo + bev treatment. Conclusion: We identified candidate biomarkers for predicting response to atezo + bev in HCC. Furthermore, the findings in Arm F are consistent with previous preclinical studies supporting a multi-faceted role of VEGF/VEGFR signaling in promoting immune suppression in addition to angiogenesis. Overall, the data presented here further support the mechanistic hypotheses on how anti-VEGF may combine with immune checkpoint blockade to increase its clinical benefit. As these results are exploratory, future study is needed to confirm these findings in a larger population. Citation Format: Andrew X. Zhu, Yinghui Guan, Alexander R. Abbas, Hartmut Koeppen, Shan Lu, Chih-Hung Hsu, Kyung-Hun Lee, Michael S. Lee, Aiwu Ruth He, Amit Mahipal, Beiying Ding, Jessica Spahn, Wendy Verret, Baek-Yeol Ryoo, Yulei Wang. Genomic correlates of clinical benefits from atezolizumab combined with bevacizumab vs. atezolizumab alone in patients with advanced hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT044.

Abstract Immune dysfunction is a hallmark of CLL/MBL; ~25% individuals with newly diagnosed CLL have hypogammaglobulinemia at diagnosis. In this study of newly diagnosed CLL/MBL, we sought to a) investigate the association between serum immunoglobulins and specific mutations identified by next generation sequencing, and b) determine if serum immunoglobulin levels predict TFT, independent of the CLL-International Prognostic Index (CLL-IPI) and tumor mutational load (TML). We used the Mayo Clinic CLL Resource to identify newly diagnosed CLL/MBL individuals consented within 2 years of diagnosis. Baseline clinical characteristics and the CLL-IPI score was ascertained on all individuals. DNA was extracted from peripheral blood CD5+/CD19+ clonal B-cells; and the entire coding regions of 60 somatically recurring mutated CLL driver genes were enriched using a customized Agilent SureSelect gene panel and sequenced in Illumina Hiseq4000. Serum immunoglobulins were quantitated by radial immunodiffusion using Immunoplates. The relationship between serum immunoglobulins was assessed: a) with individual gene mutations (mutated vs. not); and b) using the TML score (using a value of 0, 1, and 2 or greater [2+] mutated genes). The impact of serum immunoglobulin levels on TFT was analyzed for all individuals. Of 356 individuals identified, 80 (22%) had MBL, median age was 63 years (28-87), 261 (73%) were male, and the CLL-IPI distribution was: low risk (n=149, 42%), intermediate risk (n=112, 32%), and high/very high risk (n=94, 26%). We observed 43%, 39%, and 18% individuals with 0, 1, or 2+ mutated genes, respectively. The most commonly mutated genes were NOTCH1 (13%), SF3B1 (13%), TP53 (8%), ATM (8%), BIRC3 (7%), and NFKBIE (7%). Using standard cutoffs, 135 (38%) individuals had low serum IgG, 73 (21%) had low serum IgA, and 154 (44%) had low serum IgM at diagnosis. There were no differences in individual gene mutation frequencies between individuals with low and normal serum IgG. In contrast, individuals with low serum IgA were more likely to have mutations in NOTCH1 (25% vs 10%) and ATM (14% vs 7%) compared to those with normal IgA, and individuals with low serum IgM were more likely to have mutations in NOTCH1 (19% vs 9%) and BIRC3 (11% vs 4%) compared to those with normal IgM (all p-values <0.05). There were no significant differences in individuals with low serum IgG based on TML scores of 0, 1, and 2+ (31%, 35%, 40%, respectively, p=0.28). However, individuals with low serum IgA and low serum IgM were more likely to have a higher TML (8%, 20%, 36% for IgA, p<0.0001; and 35%, 45%, 54% for IgM, p<0.0001, for TML scores of 0, 1, and 2+, respectively). After a median follow-up of 6.9 years, 157 individuals required CLL therapy. After adjusting for CLL-IPI and TML, low serum IgG, low serum IgA, and low serum IgM (hazard ratio [HR]: 1.8, 2.3, and 2.4, respectively, all p-values <0.05) were independently associated with a shorter TFT. Our study shows that individuals with CLL/MBL who harbor mutations in NOTCH1, ATM and BIRC3 are more likely to have low serum IgA and IgM at diagnosis. Serum IgA and IgM (but not serum IgG) levels decrease with an increase in the TML score. Finally, low serum immunoglobulins can predict TFT, independent of the CLL-IPI and TML, which has important implications for risk stratification in newly diagnosed CLL/MBL. Citation Format: Sameer A. Parikh, Cristine Allmer, Geffen Kleinstern, Nicholas J. Boddicker, Shulan Tian, Cecilia B. De Campos, Laura A. Bruins, Daniel R. o"brien, Brian F. Kabat, Kari G. Rabe, Aaron D. Norman, Huihuang Yan, Xing Li, Timothy G. Call, Wei Ding, Jose F. Leis, Saad J. Kenderian, Celine M. Vachon, James R. Cerhan, Neil E. Kay, Susan L. Slager, Esteban Braggio. Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6466.

Abstract Glycogen synthase kinase-3 (GSK-3) is overexpressed and accumulates in the nucleus in several human malignancies including pancreatic cancer. We recently demonstrated that GSK-3 inhibition sensitizes pancreatic cancer cells to chemotherapy by abrogating the TopBP1/ATR-mediated DNA damage response. Specifically, GSK-3 kinase activity is required to stabilize TopBP1 protein. Herein, we investigated the mechanism by which GSK-3 phosphorylation of TopBP1 prevents its degradation. Interestingly, GSK-3 inhibition not only decreased the total levels of TopBP1, but also reduced the ability of TopBP1 to bind chromatin following gemcitabine treatment. Mutation of a putative GSK-3 phosphorylation site in TopBP1 (S860A) resulted in decreased TopBP1 stability and chromatin recruitment, whereas a phosphomimetic mutant (S860E) had the opposite effect. Furthermore, we found enhanced TopBP1 ubiquitination after GSK-3 inhibition as well as an increased interaction between TopBP1 and hHYD, an ubiquitin ligase that has been shown to ubiquitylate TopBP1 resulting in its degradation. Significantly, the S860A mutation showed a stronger interaction with hHYD compared to WT or the S860E mutant of TopBP1. In conclusion, our study identifies GSK-3 phosphorylation of TopBP1 as a mechanism to prevent the interaction with and ultimate ubiquitylation-mediated degradation by hHYD. Citation Format: Li Ding, Kaely R. Roeck, Olga Alekhina, Daniel M. Schmitt, Daniel D. Billadeau. Glycogen synthase kinase-3 inhibition impairs DNA damage response through ubiquitin ligase hHYD-mediated TopBP1 degradation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4138.