Venetoclax synergizes with gilteritinib in FLT3 wild-type high-risk acute myeloid leukemia by suppressing MCL-1.

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.

Drug-microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL.

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited and systematic analyses are lacking. Using chronic lymphocytic leukaemia (CLL) as a model disease, we investigated the influence of 17 microenvironmental stimuli on 12 drugs in 192 genetically characterised patient samples. Based on microenvironmental response, we identified four subgroups with distinct clinical outcomes beyond known prognostic markers. Response to multiple microenvironmental stimuli was amplified in trisomy 12 samples. Trisomy 12 was associated with a distinct epigenetic signature. Bromodomain inhibition reversed this epigenetic profile and could be used to target microenvironmental signalling in trisomy 12 CLL. We quantified the impact of microenvironmental stimuli on drug response and their dependence on genetic alterations, identifying interleukin 4 (IL4) and Toll-like receptor (TLR) stimulation as the strongest actuators of drug resistance. IL4 and TLR signalling activity was increased in CLL-infiltrated lymph nodes compared with healthy samples. High IL4 activity correlated with faster disease progression. The publicly available dataset can facilitate the investigation of cell-extrinsic mechanisms of drug resistance and disease progression.

Comparing the value of mono- vs coculture for high-throughput compound screening in hematological malignancies.

Large-scale compound screens are a powerful model system for understanding variability of treatment response and discovering druggable tumor vulnerabilities of hematological malignancies. However, as mostly performed in a monoculture of tumor cells, these assays disregard modulatory effects of the in vivo microenvironment. It is an open question whether and to what extent coculture with bone marrow stromal cells could improve the biological relevance of drug testing assays over monoculture. Here, we established a high-throughput platform to measure ex vivo sensitivity of 108 primary blood cancer samples to 50 drugs in monoculture and coculture with bone marrow stromal cells. Stromal coculture conferred resistance to 52% of compounds in chronic lymphocytic leukemia (CLL) and 36% of compounds in acute myeloid leukemia (AML), including chemotherapeutics, B-cell receptor inhibitors, proteasome inhibitors, and Bromodomain and extraterminal domain inhibitors. Only the JAK inhibitors ruxolitinib and tofacitinib exhibited increased efficacy in AML and CLL stromal coculture. We further confirmed the importance of JAK-STAT signaling for stroma-mediated resistance by showing that stromal cells induce phosphorylation of STAT3 in CLL cells. We genetically characterized the 108 cancer samples and found that drug-gene associations strongly correlated between monoculture and coculture. However, effect sizes were lower in coculture, with more drug-gene associations detected in monoculture than in coculture. Our results justify a 2-step strategy for drug perturbation testing, with large-scale screening performed in monoculture, followed by focused evaluation of potential stroma-mediated resistances in coculture.

Ex vivo drug response profiling for response and outcome prediction in hematologic malignancies: the prospective non-interventional SMARTrial.

Ex vivo drug response profiling is a powerful tool to study genotype-drug response associations and is being explored as a tool set for precision medicine in cancer. Here we conducted a prospective non-interventional trial to investigate feasibility of ex vivo drug response profiling for treatment guidance in hematologic malignancies (SMARTrial, NCT03488641 ). The primary endpoint to provide drug response profiling reports within 7 d was met in 91% of all study participants (N = 80). Secondary endpoint analysis revealed that ex vivo resistance to chemotherapeutic drugs predicted chemotherapy treatment failure in vivo. We confirmed the predictive value of ex vivo response to chemotherapy in a validation cohort of 95 individuals with acute myeloid leukemia treated with daunorubicin and cytarabine. Ex vivo drug response profiles improved ELN-22 risk stratification in individuals with adverse risk. We conclude that ex vivo drug response profiling is clinically feasible and has the potential to predict chemotherapy response in individuals with hematologic malignancies beyond clinically established genetic markers.

Proteogenomics refines the molecular classification of chronic lymphocytic leukemia.

Cancer heterogeneity at the proteome level may explain differences in therapy response and prognosis beyond the currently established genomic and transcriptomic-based diagnostics. The relevance of proteomics for disease classifications remains to be established in clinically heterogeneous cancer entities such as chronic lymphocytic leukemia (CLL). Here, we characterize the proteome and transcriptome alongside genetic and ex-vivo drug response profiling in a clinically annotated CLL discovery cohort (n = 68). Unsupervised clustering of the proteome data reveals six subgroups. Five of these proteomic groups are associated with genetic features, while one group is only detectable at the proteome level. This new group is characterized by accelerated disease progression, high spliceosomal protein abundances associated with aberrant splicing, and low B cell receptor signaling protein abundances (ASB-CLL). Classifiers developed to identify ASB-CLL based on its characteristic proteome or splicing signature in two independent cohorts (n = 165, n = 169) confirm that ASB-CLL comprises about 20% of CLL patients. The inferior overall survival in ASB-CLL is also independent of both TP53- and IGHV mutation status. Our multi-omics analysis refines the classification of CLL and highlights the potential of proteomics to improve cancer patient stratification beyond genetic and transcriptomic profiling.

Comparison of changes in corneal volume and corneal thickness after myopia correction between LASIK and SMILE.

Myopia is the most common refractive error. Surgical correction with laser is possible. LASIK and SMILE are the techniques currently most used. Aim of the study was to compare changes in corneal volume and thickness after the respective laser treatment. 104 eyes of 52 patients were matched based on refractive error into two equally sized groups, either treated with LASIK or SMILE. Measurements were obtained from the Scheimpflug camera (Pentacam) preoperatively and at 3 and 12 months postoperatively. 3 months postoperatively, the flapless SMILE procedure resulted in a significant overall greater loss of corneal volume (P < 0.01) and corneal thickness (P < 0.01) compared to LASIK. No significant difference was found when comparing the 3 to 12-months values in each group. Within the currently used ranges of refractive error correction, loss in central corneal thickness and corneal volume with SMILE is higher in comparison to LASIK. As greater loss in corneal volume and thickness might contribute to higher level of corneal instability maximum ranges of refractive error correction with SMILE should not supersede those set currently for LASIK until more long-term results on corneal ectasia are available for SMILE.

Phagocytosis by stroma confounds coculture studies.

Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. Bone marrow stromal cell coculture models are frequently used to study the influence of the bone marrow niche on ex vivo drug response. Here, we show that mesenchymal stromal cells from selected donors and NKTert, a stromal cell line, which is commonly used for coculture studies with primary leukemia cells, extensively phagocytose apoptotic cells. This could lead to misinterpretation of results, especially if viability readouts of the target cells (e.g. leukemic cells) in such coculture models are based on the relative proportions of dead and alive cells. Future coculture studies which aim to investigate the impact of bone marrow stromal cells on drug response should take into account that stromal cells have the capacity to phagocytose apoptotic cells.

An autologous culture model of nodal B-cell lymphoma identifies ex vivo determinants of response to bispecific antibodies.

Bispecific antibodies (BsAbs) can induce long-term responses in patients with refractory and relapsed B-cell lymphoma. Nevertheless, response rates across patients are heterogeneous, and the factors determining quality and duration of responses are poorly understood. To identify key determinants of response to BsAbs, we established a primary, autologous culture model allowing us to mimic treatment with CD3xCD19 and CD3xCD20 BsAbs within the lymph node microenvironment ex vivo. T cell-mediated killing of lymphoma cells and proliferation of T cells varied significantly among patients but highly correlated between BsAbs targeting CD20 or CD19. Ex vivo response to BsAbs was significantly associated with expansion of T cells and secretion of effector molecules (eg, granzyme B, perforin) but not with expression of T-cell exhaustion (eg, PD1, TIM3) or activation markers (eg, CD25, CD69) or formation of intercellular contacts. In addition, we identified a distinct phenotype of regulatory T cells that was linked to ex vivo response independently from T-cell frequency at baseline. High expression levels of Aiolos (IKZF1), ICOS, and CXCR5 were positively associated with ex vivo response, whereas strong expression of Helios (IKZF2) had an unfavorable impact on ex vivo response to BsAbs. We further showed that lenalidomide, nivolumab, and atezolizumab improved ex vivo response to BsAbs by potentiating T-cell effector functions. In summary, our ex vivo study identified a distinct regulatory T-cell phenotype as a potential contributor to treatment failure of BsAbs and suggests drug combinations of high clinical relevance that could improve the efficacy of BsAbs.

Comparison of femtosecond laser-assisted cataract surgery and conventional cataract surgery: a meta-analysis and systematic review.

PURPOSE: To compare the efficacy and safety of femtosecond laser-assisted cataract surgery (FLACS) with conventional cataract surgery (CCS). SETTING: Department of Ophthalmology, Goethe-University, Frankfurt am Main, Germany. DESIGN: Meta-analysis. METHODS: PubMed, Cochrane Library, and EMBASE were systematically searched for studies comparing FLACS and CCS. Outcomes were efficacy and safety parameters. The effect measures were weighted mean differences or odds ratios with 95% CIs. RESULTS: A total of 73 studies (25 randomized controlled, 48 observational) were reviewed with a total of 12 769 eyes treated with FLACS and 12 274 eyes treated with CCS. In eyes treated with FLACS, uncorrected and corrected distance visual acuities and spherical equivalent after 1 month to 3 months (P = .04, P = .005, and P = .007, respectively) were better, total and effective phacoemulsification times were shorter (P < .001 each), cumulative dissipated energy was less (P < .001), circularity was more accurate (P < .001), central corneal thickness after 1 day and 1 month to 3 months was less (P < .001 and P = .004, respectively), and endothelial cell loss after 3 to 6 weeks and 3 months was less (P = .002 and P < .001, respectively) compared with CCS. Anterior capsule ruptures occurred more often with FLACS. No significant differences among groups were found in visual acuity at 1 week and after 6 months or in posterior capsule rupture rates and endothelial cell loss after 6 months. CONCLUSIONS: Both FLACS and CCS are effective and safe. FLACS required less ultrasound energy and a more precise treatment. However, mid-term visual acuity did not show any difference between both methods.

Comparison of standard and accelerated corneal cross-linking for the treatment of keratoconus: a meta-analysis.

PURPOSE: To compare results between standard and accelerated corneal collagen cross-linking (CXL) for the treatment of progressive keratoconus. METHODS: We performed literature searches in PubMed, Cochrane Library, Web of Science, ISRCTN registry, ClinicalTrials.gov, and EMBASE for studies comparing conventional Dresden (C-CXL) and accelerated CXL (A-CXL). Outcomes were clinical results and changes in corneal properties. Weighted mean differences were used to evaluate the effects. RESULTS: Here, 22 studies with 1158 eyes (C-CXL: 577 eyes; A-CXL: 581 eyes) were included. At the last follow-up, C-CXL was superior regarding minimum keratometry (p < 0.00001) and demarcation line depth (p < 0.00001), whereas A-CXL should be favoured when considering minimum corneal thickness (p = 0.0005). No differences in uncorrected and corrected distance visual acuity (p = 0.09 and 0.98), spherical equivalent (p = 0.11), spherical and cylindrical error (p = 0.29 and 0.32), maximal and average keratometry (p = 0.05 and 0.65), central corneal thickness (p = 0.15), corneal biomechanical properties (p ≥ 0.21 respectively), time of reepithelialization (p = 0.76), subbasal nerve density (p = 0.69), endothelial cell density (p = 0.30) and morphology (p ≥ 0.40 respectively) were found among both groups. CONCLUSION: Consideration of less corneal thinning favours A-CXL, whereas the deeper demarcation line and greater changes in minimum keratometric values in C-CXL may indicate a higher treatment efficacy. Altogether, C-CXL, as well as A-CXL, provides successful results in the strengthening of corneal tissue.

Characteristics of preoperative and postoperative astigmatism in patients having Descemet membrane endothelial keratoplasty.

PURPOSE: To evaluate the characteristics of preoperative and postoperative astigmatism in patients having Descemet membrane endothelial keratoplasty (DMEK). SETTING: Department of Ophthalmology, Goethe University, Frankfurt, Germany. DESIGN: Retrospective case series. METHODS: Measurements were obtained using a Scheimpflug camera (Pentacam AXL) preoperatively and 3 months and 12 months postoperatively. Values of front and back astigmatism and total astigmatism in the central 4.0 mm diameter zone (TCA4) were analyzed. RESULTS: Fifty-three eyes of 45 patients were included. The prevalence of TCA4 above 1.0 diopter (D) was considerably higher (79%) and with-the-rule astigmatism was less frequent in this cohort of European patients with Fuchs endothelial dystrophy (mean age 65 years) than that reported in a meta-analysis of healthy European eyes. The TCA4 values correlated with anterior astigmatism preoperatively and postoperatively (P < .001) and with posterior astigmatism at the 1-year follow-up (P < .01). Although, no correlation was found between the preoperative and 1-year results for anterior astigmatism (P = .12), posterior astigmatism (P = .35), or total corneal astigmatism (P = .47), the difference in vector analysis between the two measurements was only 0.01 at 109 degrees, 0.03 at 98 degrees, and 0.02 at 157 degrees, respectively. However, the greater the difference between the preoperative TCA3 and preoperative TCA5 values, the greater the decrease in corneal astigmatism (P < .001). CONCLUSIONS: The percentage of eyes with corneal astigmatism above 1.0 D was higher preoperatively and postoperatively in patients with Fuchs endothelial dystrophy than in a healthy population. Predicting postoperative astigmatism based on preoperative results is not possible; however, in eyes with a high difference between TCA3 and TCA5, a reduction in corneal astigmatism after DMEK is likely.

Comparison of 9 modern intraocular lens power calculation formulas for a quadrifocal intraocular lens.

PURPOSE: To evaluate the accuracy of 9 formulas (Barrett Universal II, Haigis, Hill-Radial Basis Function [RBF], Hoffer Q, Holladay 1, Holladay 2, Olsen, Sanders-Retzlaff-Kraff/theoretical [SRK/T], and T2) calculating the power of the quadrifocal Acrysof IQ Panoptix TFNT00 intraocular lens (IOL). SETTING: Department of Ophthalmology, Goethe University, Frankfurt, Germany. DESIGN: Retrospective case series. METHODS: The study included patients having cataract surgery with insertion of a quadrifocal IOL over 15 months. Preoperative biometry measurements were obtained from an IOLMaster 500. Optimized IOL constants were calculated to reduce the mean refractive prediction error. The primary outcomes were differences in mean absolute prediction error between the formulas. Median and maximum absolute prediction errors were evaluated as well as percentages of eyes within prediction errors of ±0.25 diopters (D), ±0.50 D, ±1.00 D, and ±2.00 D. RESULTS: The study comprised 75 eyes of 38 patients. The formulas were ranked by the mean absolute refractive prediction error as follows: Barrett Universal II (0.294 D), Hill-RBF (0.332 D), Olsen (0.339 D), T2 (0.351 D), Holladay 1 (0.381 D), Haigis (0.382 D), SRK/T (0.393 D), Holladay 2 (0.399 D), and Hoffer Q (0.410 D). The differences in absolute errors between the formulas were significant (P < .001). The lowest maximum absolute prediction error was obtained with the Barrett Universal II. CONCLUSION: The most accurate predictions of actual postoperative refraction were achieved using the Barrett Universal II, Hill-RBF, Olsen, or T2 formula. Thus, one of these formulas should be used for IOL power calculation of the quadrifocal IOL.

PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos.

In eukaryotes, Suv39h H3K9 trimethyltransferases are required for pericentric heterochromatin formation and function. In early mouse preimplantation embryos, however, paternal pericentric heterochromatin lacks Suv39h-mediated H3K9me3 and downstream marks. Here we demonstrate Ezh2-independent targeting of maternally provided polycomb repressive complex 1 (PRC1) components to paternal heterochromatin. In Suv39h2 maternally deficient zygotes, PRC1 also associates with maternal heterochromatin lacking H3K9me3, thereby revealing hierarchy between repressive pathways. In Rnf2 maternally deficient zygotes, the PRC1 complex is disrupted, and levels of pericentric major satellite transcripts are increased at the paternal but not the maternal genome. We conclude that in early embryos, Suv39h-mediated H3K9me3 constitutes the dominant maternal transgenerational signal for pericentric heterochromatin formation. In absence of this signal, PRC1 functions as the default repressive back-up mechanism. Parental epigenetic asymmetry, also observed along cleavage chromosomes, is resolved by the end of the 8-cell stage--concurrent with blastomere polarization--marking the end of the maternal-to-embryonic transition.