The potential of triplet combination therapies for patients with FLT3-ITD -mutated acute myeloid leukemia.

INTRODUCTION: Acute myeloid leukemia (AML) encompasses a heterogeneous group of aggressive myeloid malignancies, where FMS-like tyrosine kinase 3 (FLT3) mutations are prevalent, accounting for approximately 25-30% of adult patients. The presence of this mutation is related to a dismal prognosis and high relapse rates. In the lasts years many FLT3 inhibitors have been developed. AREAS COVERED: This review provides a comprehensive overview of FLT3mut AML, summarizing the state of art of current treatment and available data about combination strategies including an FLT3 inhibitor. EXPERT OPINION: In addition, the review discusses the emergence of drug resistance and the need for a nuanced approaches in treating patients who are ineligible for or resistant to intensive chemotherapy. Specifically, it explores the historical context of FLT3 inhibitors (FLT3Is) and their impact on treatment outcomes, emphasizing the pivotal role of midostaurin, as well as gilteritinib and quizartinib, and providing detailed insights into ongoing trials exploring the safety and efficacy of novel triplet combinations involving FLT3Is in different AML settings.

Gemtuzumab ozogamicin plus midostaurin in combination with standard '7 + 3' induction therapy in newly diagnosed AML: Results from the SAL-MODULE phase I study.

We conducted a phase I trial in newly diagnosed acute myeloid leukaemia (AML) to investigate the combination of two novel targeted agents, gemtuzumab ozogamicin (GO) and midostaurin, with intensive chemotherapy in FLT3-mutated AML and CBF leukaemia. Three dose levels of midostaurin and one to three sequential doses of 3 mg/m2 GO in combination with '7 + 3' induction were evaluated. Based on safety findings in 12 patients, our results show that 3 mg/m2 GO on Days 1 + 4 and 100 mg midostaurin on Days 8-21 can be safely combined with IC in newly diagnosed AML.

[Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD+ Acute Myeloid Leukemia Cell Line and Its Mechanism].

OBJECTIVE: To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD+ acute myeloid leukemia (AML) cell line and its potential regulatory mechanism. METHODS: By knocking out miR-155 gene in FLT3-ITD+ AML cell line MV411 through CRISPR/Cas9 gene-editing technology, monoclonal cells were screened. The genotype of these monoclonal cells was validated by PCR and Sanger sequencing. The expression of mature miRNA was measured by RT-qPCR. The treatment response of doxorubicin, quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity. Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout, gene set enrichment analysis to analyze change of signaling pathway, and Western blot to verify expressions of key molecules in signaling pathway. RESULTS: Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing. RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones. MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD+ AML drugs increased significantly after miR-155 knockout compared with wild-type clones. RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout. Western blot showed that the expressions of key molecules p-mTOR, Wnt5α and ß-catenin in signaling pathway were down-regulated. CONCLUSION: Drug sensitivity of MV411 cells to doxorubicin, quizartinib and midostaurin can be enhanced significantly after miR-155 knockout, which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.

Day-21 bone marrow findings incorrectly designate residual leukaemia in FLT3-mutated acute myeloid leukaemia treated with intensive induction plus midostaurin: a morphology-focused study.

Early induction response assessment with day-21 bone marrow (D21-BM) is commonly performed in patients with FLT3-mutated acute myeloid leukaemia (AML), where detection of residual leukaemia (RL; blasts ≥5%) typically results in the administration of a second induction course. However, whether D21-BM results predict for RL at the end of first induction has not been systematically assessed. This study evaluates the predictive role of D21-BM morphology in detecting RL following first induction. Between August 2018 and March 2022, all patients with FLT3-AML receiving 7+3 plus midostaurin, with D21-BM performed, were identified. Correlation between D21-BM morphology vs D21-BM ancillary flow/molecular results, as well as vs D28-BM end of first induction response, were retrospectively reviewed. Subsequently, D21-BMs were subjected to anonymised morphological re-assessments by independent haematopathologists (total in triplicate per patient). Of nine patients included in this study, three (33%) were designated to have RL at D21-BM, all of whom entered complete remission at D28-BM. Furthermore, only low-level measurable residual disease was detected in all three cases by flow or molecular methods at D21-BM, hence none proceeded to a second induction. Independent re-evaluations of these cases failed to correctly reassign D21-BM responses, yielding a final false positive rate of 33%. In summary, based on morphology alone, D21-BM assessment following 7+3 intensive induction plus midostaurin for FLT3-AML incorrectly designates RL in some patients; thus correlating with associated flow and molecular results is essential before concluding RL following first induction. Where remission status is unclear, repeat D28-BMs should be performed.

Targeting ABCA1 via Extracellular Vesicle-Encapsulated Staurosporine as a Therapeutic Strategy to Enhance Radiosensitivity.

Cancer stem cells (CSCs) are essential for tumor initiation, recurrence, metastasis, and resistance. However, targeting CSCs as a therapeutic approach remains challenging. Here, a stemness signature based on 22-gene is developed to predict prognosis in esophageal squamous cell carcinoma (ESCC). Staurosporine (STS) is identified as a radioresistance suppressor by high-throughput screening of a library of 2131 natural compounds, leading to dramatically improved radiotherapy efficacy in subcutaneous tumor models. Mechanistically, STS inhibits cell proliferation through the mTOR/AKT signaling pathway and suppressed stemness by targeting ATP-binding cassette A1 (ABCA1), which is transcriptionally regulated by liver X receptor alpha (LXRα). STS can selectively bind to the nucleotide-binding domain (NBD) of ABCA1 and compete for ATP, blocking ABCA1-mediated drug efflux and facilitating intracellular accumulation of STS. Considering the cytotoxicity of STS, an extracellular vesicle-encapsulated STS system (EV-STS) is established for effective STS delivery. EV-STS shows remarkable tumor growth inhibition, even at half the dose of STS, with superior safety and efficacy. These findings indicate that ABCA1 may serve as a predictor of response to neoadjuvant chemotherapy and/or radiotherapy in ESCC patients. EV-STS has shown improved antitumor efficacy and low systemic toxicity, offering a promising therapeutic approach for ESCC.

Quantification of midostaurin in plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry: Application to a cohort of patients with acute myeloid leukemia.

OBJECTIVES: Midostaurin is an oral multitargeted tyrosine kinase inhibitor for the treatment of acute myeloid leukemia (AML). Therapeutic drug monitoring of midostaurin may support its safe use when suspecting toxicity or combined with strong CYP3A4 inhibitors. METHODS: A stable isotope dilution liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and quantification of midostaurin in human plasma and serum. Midostaurin serum concentrations were analyzed in 12 patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML during induction chemotherapy with cytarabine, daunorubicin, and midostaurin. Posaconazole was used as prophylaxis of invasive fungal infections. RESULTS: Linear quantification of midostaurin was demonstrated across a concentration range of 0.01-8.00 mg/L. Inter- and intraday imprecisions of the proposed method were well within ±10%. Venous blood samples were taken in nine and three patients in the first and second cycle of induction chemotherapy. Median (range) midostaurin serum concentration was 7.9 mg/L (1.5-26.1 mg/L) as determined in 37 independent serum specimens. CONCLUSION: In a real-life cohort of AML patients, interindividual variability in midostaurin serum concentrations was high, highlighting issues concerning optimal drug dosing in AML patients. A personalized dosage approach may maximize the safety of midostaurin. Prospective studies and standardization of analytical methods to support such an approach are needed.

Midostaurin drug interaction profile: a comprehensive assessment of CYP3A, CYP2B6, and CYP2C8 drug substrates, and oral contraceptives in healthy participants.

PURPOSE: Midostaurin, approved for treating FLT-3-mutated acute myeloid leukemia and advanced systemic mastocytosis, is metabolized by cytochrome P450 (CYP) 3A4 to two major metabolites, and may inhibit and/or induce CYP3A, CYP2B6, and CYP2C8. Two studies investigated the impact of midostaurin on CYP substrate drugs and oral contraceptives in healthy participants. METHODS: Using sentinel dosing for participants' safety, the effects of midostaurin at steady state following 25-day (Study 1) or 24-day (Study 2) dosing with 50 mg twice daily were evaluated on CYP substrates, midazolam (CYP3A4), bupropion (CYP2B6), and pioglitazone (CYP2C8) in Study 1; and monophasic oral contraceptives (containing ethinylestradiol [EES] and levonorgestrel [LVG]) in Study 2. RESULTS: In Study 1, midostaurin resulted in a 10% increase in midazolam peak plasma concentrations (Cmax), and 3-4% decrease in total exposures (AUC). Bupropion showed a 55% decrease in Cmax and 48-49% decrease in AUCs. Pioglitazone showed a 10% decrease in Cmax and 6% decrease in AUC. In Study 2, midostaurin resulted in a 26% increase in Cmax and 7-10% increase in AUC of EES; and a 19% increase in Cmax and 29-42% increase in AUC of LVG. Midostaurin 50 mg twice daily for 28 days ensured that steady-state concentrations of midostaurin and the active metabolites were achieved by the time of CYP substrate drugs or oral contraceptive dosing. No safety concerns were reported. CONCLUSION: Midostaurin neither inhibits nor induces CYP3A4 and CYP2C8, and weakly induces CYP2B6. Midostaurin at steady state has no clinically relevant PK interaction on hormonal contraceptives. All treatments were well tolerated.

Combination of midostaurin and ATRA exerts dose-dependent dual effects on acute myeloid leukemia cells with wild type FLT3.

BACKGROUND: Midostaurin combined with chemotherapy is currently used to treat newly diagnosed acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 (FLT3)-mutations. However, midostaurin acts as an antagonist to some chemotherapeutic agents in leukemia cell lines without FLT3 mutations. All-trans retinoic acid (ATRA) induces apoptosis when used in combination with midostaurin in FLT3-mutated AML cells. This combination has been shown to be safe in AML patients. However, the effect of this combination has not been investigated in AML without FLT3 mutations. METHODS: Cell proliferation was assessed by a cell counting assay. Cell death was evaluated by cell viability and Annexin-V assays. Cell differentiation was assessed by CD11b expression profiling and morphological analysis. To explore the underlying mechanisms, we studied the role of caspase3/7, Lyn, Fgr, Hck, RAF, MEK, ERK, AKT, PU.1, CCAAT/enhancer binding protein ß (C/EBPß) and C/EBPε by Western blot analysis and immunoprecipitation assays. Antitumor activity was also confirmed in mouse xenograft models established with AML cells. RESULTS: In this study, 0.1 - 0.25 µM midostaurin (mido(L)) combined with ATRA induced differentiation while 0.25 - 0.5 µM midostaurin (mido(H)) combined with ATRA triggered apoptosis in some AML cell lines without FLT3-mutations. Midostaurin combined with ATRA (mido-ATRA) also exhibited antitumor activity in mouse xenograft models established with AML cells. Mechanistically, mido(H)-ATRA-induced apoptosis was dependent on caspase-3/7. Mido(L)-ATRA inhibited Akt activation which was associated with decreased activity of Lyn/Fgr/Hck, resulted in dephosphorylation of RAF S259, activated RAF/MEK/ERK, along with upregulating the protein levels of C/EBPß, C/EBPε and PU.1. A MEK specific inhibitor was observed to suppress mido(L)-ATRA-induced increases in the protein levels of C/EBPs and PU.1 and mido(L)-ATRA-induced differentiation. Furthermore, inhibition of Akt activity promoted mido(L)-ATRA-induced downregulation of RAF S259 phosphorylation and mido(L)-ATRA-induced differentiation. Therefore, Lyn/Fgr/Hck-associated Akt inhibition activated RAF/MEK/ERK and controlled mido(L)-ATRA-induced differentiation by upregulation of C/EBPs and PU.1. Mido(L)-ATRA also promoted assembly of the signalosome, which may facilitate RAF activation. CONCLUSIONS: Midostaurin combined with ATRA exerts antitumor activity against AML with wild-type FLT3 mutations in vitro and in vivo. These findings may provide novel therapeutic strategies for some AML patients without FLT3 mutations and imply a new target of midostaurin.

Evaluation of drug-drug interactions between midostaurin and strong CYP3A4 inhibitors in patients with FLT-3-mutated acute myeloid leukemia (AML).

PURPOSE: Midostaurin, approved for the treatment of newly diagnosed, FLT3-mutated acute myeloid leukemia (AML), is metabolized by cytochrome P450 3A4 (CYP3A4). Midostaurin with concomitant strong CYP3A4 inhibitors use (e.g., antifungal azoles) may result in drug-drug interactions. This post hoc analysis of RATIFY phase 3 study data evaluated effects of strong CYP3A4 inhibitor use on the exposure and safety of midostaurin. METHODS: Trough concentrations were used to assess midostaurin and metabolite exposure in the presence and absence of strong CYP3A4 inhibitors. Adverse event (AE) frequency was assessed in patients who received concomitant strong CYP3A4 inhibitors vs those who did not. Time to first clinically notable AE (CNAE) was also assessed in patients with high midostaurin plasma exposure vs those of matched placebo controls. RESULTS: Use of concomitant strong CYP3A4 inhibitors was most frequent during the induction phase (60.8%). A 1.44-fold increase in midostaurin plasma exposure was observed in patients with concomitant strong CYP3A4 inhibitor use vs those without. Midostaurin-treated patients who received concomitant strong CYP3A4 inhibitors experienced grade 3/4 infection-related AEs more frequently vs those who did not. Patients with high levels of midostaurin exposure had a shorter median time to first grade 3/4 CNAE vs placebo controls (36 vs 41 days, respectively; P = .012). CONCLUSION: Although concomitantly administered strong CYP3A4 inhibitors increased midostaurin exposure 1.44-fold, no clinically relevant differences in safety were noted. Midostaurin dose adjustment is not necessary with concomitant strong CYP3A4 inhibitors in patients with FLT3-mutated AML; however, caution is advised, and patients should be closely monitored.

The FLT3 Y842D mutation may be highly sensitive to midostaurin: a case report.

A Y842D mutation within the activation loop of fms-like tyrosine kinase 3 (FLT3) has been shown to confer strong resistance to sorafenib in vitro. Whether this type of mutation exerts clinically significant effects in patients with acute myeloid leukaemia (AML) remains unclear. Here, a novel Y842D activating mutation within the kinase domain of FLT3, in a pregnant patient with de novo hyperleucocyte acute myeloid leukaemia, is described. Following induction failure with standard dose idarubicin and cytarabine (IA), the patient received re-induction combined with midostaurin, a promising agent targeting mutant-FLT3, and IA regimen. Fortunately, morphological remission was achieved. During the period of midostaurin treatment, the patient exhibited a symptom that was characteristic of differentiation syndrome, which disappeared following treatment with methylprednisolone. The present case revealed that Y842D, an uncommon activating mutation in the activation loop of FLT3, may be a midostaurin-sensitive mutation type in patients with acute myeloid leukaemia.

Clinical outcomes in patients with relapsed/refractory FLT3-mutated acute myeloid leukemia treated with gilteritinib who received prior midostaurin or sorafenib.

The fms-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib is indicated for relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML), based on its observed superior response and survival outcomes compared with salvage chemotherapy (SC). Frontline use of FLT3 tyrosine kinase inhibitors (TKIs) midostaurin and sorafenib may contribute to cross-resistance to single-agent gilteritinib in the R/R AML setting but has not been well characterized. To clarify the potential clinical impact of prior TKI use, we retrospectively compared clinical outcomes in patients with R/R FLT3-mutated AML in the CHRYSALIS and ADMIRAL trials who received prior midostaurin or sorafenib against those without prior FLT3 TKI exposure. Similarly high rates of composite complete remission (CRc) were observed in patients who received a FLT3 TKI before gilteritinib (CHRYSALIS, 42%; ADMIRAL, 52%) and those without prior FLT3 TKI therapy (CHRYSALIS, 43%; ADMIRAL, 55%). Among patients who received a prior FLT3 TKI in ADMIRAL, a higher CRc rate (52%) and trend toward longer median overall survival was observed in the gilteritinib arm versus the SC arm (CRc = 20%; overall survival, 5.1 months; HR = 0.602; 95% CI: 0.299, 1.210). Remission duration was shorter with prior FLT3 TKI exposure. These findings support gilteritinib for FLT3-mutated R/R AML after prior sorafenib or midostaurin.

Midostaurin plus intensive chemotherapy for younger and older patients with AML and FLT3 internal tandem duplications.

We conducted a single-arm, phase 2 trial (German-Austrian Acute Myeloid Leukemia Study Group [AMLSG] 16-10) to evaluate midostaurin with intensive chemotherapy followed by allogeneic hematopoietic-cell transplantation (HCT) and a 1-year midosta urin maintenance therapy in adult patients with acute myeloid leukemia (AML) and fms-related tyrosine kinase 3 (FLT3) internal tandem duplication (ITD). Patients 18 to 70 years of age with newly diagnosed FLT3-ITD-positive AML were eligible. Primary and key secondary endpoints were event-free survival (EFS) and overall survival (OS). Results were compared with a historical cohort of 415 patients treated on 5 prior AMLSG trials; statistical analysis was performed using a double-robust adjustment with propensity score weighting and covariate adjustment. Results were also compared with patients (18-59 years) treated on the placebo arm of the Cancer and Leukemia Group B (CALGB) 10603/RATIFY trial. The trial accrued 440 patients (18-60 years, n = 312; 61-70 years, n = 128). In multivariate analysis, EFS was significantly in favor of patients treated within the AMLSG 16-10 trial compared with the AMLSG control (hazard ratio [HR], 0.55; P < .001); both in younger (HR, 0.59; P < .001) and older patients (HR, 0.42; P < .001). Multivariate analysis also showed a significant beneficial effect on OS compared with the AMLSG control (HR, 0.57; P < .001) as well as to the CALGB 10603/RATIFY trial (HR, 0.71; P = .005). The treatment effect of midostaurin remained significant in sensitivity analysis including allogeneic HCT as a time-dependent covariate. Addition of midostaurin to chemotherapy was safe in younger and older patients. In comparison with historical controls, the addition of midostaurin to intensive therapy led to a significant improvement in outcome in younger and older patients with AML and FLT3-ITD. This trial is registered at clinicaltrialsregistry.eu as Eudra-CT number 2011-003168-63 and at clinicaltrials.gov as NCT01477606.

ABCB1 as a potential beneficial target of midostaurin in acute myeloid leukemia.

Low curability of patients diagnosed with acute myeloid leukemia (AML) must be seen as a call for better understanding the disease's mechanisms and improving the treatment strategy. Therapeutic outcome of the crucial anthracycline-based induction therapy often can be compromised by a resistant phenotype associated with overexpression of ABCB1 transporters. Here, we evaluated clinical relevance of ABCB1 in a context of the FMS-like tyrosine kinase 3 (FLT3) inhibitor midostaurin in a set of 28 primary AML samples. ABCB1 gene expression was absolutely quantified, confirming its association with CD34 positivity, adverse cytogenetic risk, and unachieved complete remission (CR). Midostaurin, identified as an ABCB1 inhibitor, increased anthracycline accumulation in peripheral blood mononuclear cells (PBMC) of CD34+ AML patients and those not achieving CR. This effect was independent of FLT3 mutation, indicating even FLT3- AML patients might benefit from midostaurin therapy. In line with these data, midostaurin potentiated proapoptotic processes in ABCB1-overexpressing leukemic cells when combined with anthracyclines. Furthermore, we report a direct linkage of miR-9 to ABCB1 efflux activity in the PBMC and propose miR-9 as a useful prognostic marker in AML. Overall, we highlight the therapeutic value of midostaurin as more than just a FLT3 inhibitor, suggesting its maximal therapeutic outcomes might be very sensitive to proper timing and well-optimized dosage schemes based upon patient's characteristics, such as CD34 positivity and ABCB1 activity. Moreover, we suggest miR-9 as a predictive ABCB1-related biomarker that could be immensely helpful in identifying ABCB1-resistant AML phenotype to enable optimized therapeutic regimen and improved treatment outcome.

Superior Efficacy of Midostaurin Over Cladribine in Advanced Systemic Mastocytosis: A Registry-Based Analysis.

PURPOSE: On the basis of data from the German Registry on Disorders of Eosinophils and Mast Cells, we compared the efficacy of midostaurin and cladribine in patients with advanced systemic mastocytosis (AdvSM). PATIENTS AND METHODS: Patients with AdvSM (n = 139) were treated with midostaurin only (n = 63, 45%), cladribine only (n = 23, 17%), or sequentially (midostaurin-cladribine, n = 30, 57%; cladribine-midostaurin, n = 23, 43%). Prognosis was assessed through the Mutation-Adjusted Risk Score (MARS). Besides the comparison of efficacy between midostaurin and cladribine on response (eg, organ dysfunction, bone marrow mast cell [MC] infiltration, and tryptase), overall survival (OS), and leukemia-free survival, we focused on the impact of treatment on involved non-MC lineages, for example, monocytes or eosinophils, and the KIT D816V expressed allele burden. RESULTS: Midostaurin only was superior to cladribine only with effects from responses on MC and non-MC lineages conferring on a significantly improved OS (median 4.2 v 1.9 years, P = .033) and leukemia-free survival (2.7 v 1.3 years, P = .044) on the basis of a propensity score-weighted analysis of parameters included in MARS. Midostaurin compensated the inferior efficacy of cladribine in first- and second-line treatment. On midostaurin in any line, response of eosinophilia did not improve its baseline adverse prognostic impact, whereas response of monocytosis proved to be a positive on-treatment parameter. Multivariable analysis allowed to establish three risk categories (low/intermediate/high) through the combination of MARS and the reduction of the KIT D816V expressed allele burden of ≥ 25% at month 6 (median OS not reached v 3.0 years v 1.0 year; P < .001). CONCLUSION: In this registry-based analysis, midostaurin revealed superior efficacy over cladribine in patients with AdvSM. In midostaurin-treated patients, the combination of baseline MARS and molecular response provided a compelling three-tier risk categorization (MARSv2.0) for OS.

Practical tips for managing FLT3 mutated acute myeloid leukemia with midostaurin.

INTRODUCTION: FLT3 inhibitors have been recently introduced as novel treatment targets in patients with FLT3-mutated acute myeloid leukemia (AML). Midostaurin is an oral multikinase inhibitor that targets multiple receptor tyrosine kinases including FLT3 and has been approved for the treatment of AML with FLT3 mutations in patients candidates for intensive chemotherapy. This article presents an updated overall overview of the use of midostaurin in clinical practice. AREAS COVERED: Tests and examinations to be performed before the use of midostaurin, antifungal and antimicrobial treatment, as well as antifungal and antimicrobial prophylaxis are discussed. Practical tips for the treatment of QTc interval prolongation and heart failure are also presented. EXPERT OPINION: Midostaurin is the first agent showing significant survival benefit when combined with chemotherapy in FLT3-mutated AML patients. Optimal use of midostaurin should be a priority, being essential to know the interactions with other drugs like strong CYP3A4 inhibitors or inducers, which are particularly used in the concomitant treatment of AML patients and may increase toxicity or decrease therapeutic benefit. The active role of hematologists and nursing teams is crucial to ensure patient adherence to midostaurin treatment and to minimize adverse effects by administrating the optimal dose for each situation.

p53 oligomerization status as an indicator of sensitivity of p53-wildtype neuroblastomas to the combination of DNA damaging agent and Chk1 inhibitor.

Neuroblastomas are one of the most common types of solid tumors in infants and children and are responsible for approximately 15% of childhood cancer deaths. Neuroblastomas rarely have mutations in p53, with less than 2% of NB containing mutations in p53, compared to up to 60% for other tumor classes. Previous studies on the therapeutic combination of a DNA damaging agent and checkpoint kinase 1 (Chk1) inhibitor have shown that DNA damage-induced cell cycle arrest can be specifically abrogated in p53-defective tumors. However, some p53-wildtype tumors have also been shown to be sensitive to this therapeutic combination, suggesting that these cells have other defects in the p53 response that can be exploited for therapeutic purposes. In the current study, we investigated the response to the combination of a DNA damaging agent (SN38) and a Chk1 inhibitor (UCN-01) of four p53-wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, SK-N-AS, and Lan-5. When the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml, all four cell lines accumulated p53 which was phosphorylated on serines 15 and 20. However, only the SK-N-SH were found to activate p21waf1 and repress cyclin B. In order to assess sensitivity to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 10 ng/ml SN38 for 24 h, followed by 25 nM UCN-01 for 6 and 24 h. The SK-N-SH showed no sensitivity to UCN-01 treatment whereas the SH-SY5Y, SK-N-AS, and Lan-5 abrogated G2 arrest within 24 h. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers, so we analyzed the oligomerization status of p53 in all four cell lines using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53-wildtype MCF10A cells. The SH-SY5Y, SK-N-AS, and Lan-5 however, had extremely low to undetectable levels of dimers and tetramers. Our study also showed no cytoplasmic accumulation of p53 in these cells contrary to some previous reports. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53-wildtype tumors to the therapeutic combination of DNA damaging agent and Chk1 inhibitor.

Midostaurin therapy for advanced systemic mastocytosis: Mayo Clinic experience in 33 consecutive cases.

We retrospectively examined our experience with midostaurin therapy in 33 consecutive patients (median age 68 years; 58% females) with advanced systemic mastocytosis (adv-SM): aggressive SM (ASM; n = 17), SM associated with another hematologic neoplasm (SM-AHN; n = 14) and mast cell leukemia (MCL; n = 2). KITD816V mutation was detected in 84% of the patients and C findings in 91%. Eleven (33%) patients were previously treated with other cytoreductive drugs, including cladribine (n = 4) and imatinib (n = 3). Median time from diagnosis to initiation of midostaurin therapy was 2.2 months (range 0.3-41). Using modified valent criteria, overall response was 42% (53% ASM, 29% SM-AHN, 50% MCL; p = .22), all classified as being major. Responses included ≥50% reduction in bone marrow mast cells in 40% and normalization of serum tryptase in 29%, of evaluated cases. After a median follow-up of 14.6 months from initiation of midostaurin therapy, 7 (21%) deaths, 1 (3%) leukemic progression, and 18 (55%) treatment discontinuations were documented; median duration of midostaurin treatment was 7.9 months (range 0.5-123) and response duration 21.5 months (range 2.9-123). Most frequent side effect was gastrointestinal (51%) while grade 3/4 neutropenia or thrombocytopenia occurred in 12% of patients. Response to treatment was not predicted by KIT mutation (p = .67) or exposure to prior cytoreductive therapy (p = .44). Median survival was longer in midostaurin responders but not significantly (median 26.5 vs. 16 months; p = .15). Findings from the current study are broadly consistent with previously published clinical trial observations.