The Application of Next-generation Sequencing Tumor Molecular Profiling in the Diagnosis and Management of a Case of Acute Myelogenous Leukemia With MLL-PTD in a Pediatric Heart Transplant Recipient.

A 6-year-old girl with a history of heart transplantation was diagnosed with myelodysplastic syndrome, which progressed to acute myelogenous leukemia. Comprehensive genomic profiling of her tumor discovered an MLL-PTD (partial tandem duplication) and she received chemotherapy and a hematopoietic stem cell transplant (HSCT). She subsequently relapsed and tumor molecular profiling was repeated, revealing 2 new potentially targetable mutations (FLT3 and IDH2). A novel treatment regimen targeting these mutations with sorafenib and azacitidine without using cytotoxic chemotherapy produced remission and she subsequently pursued a second HSCT. She remains disease-free 17 months after HSCT. This case report demonstrates how repeated tumor molecular profiling provided novel actionable information for the diagnosis and management at 2 timepoints.

Oral vitamin C supplementation to patients with myeloid cancer on azacitidine treatment: Normalization of plasma vitamin C induces epigenetic changes.

BACKGROUND: Patients with haematological malignancies are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), the first step in active DNA demethylation. Here, we investigate whether oral vitamin C supplementation can correct vitamin C deficiency and affect the 5hmC/5mC ratio in patients with myeloid cancers treated with DNA methyltransferase inhibitors (DNMTis). RESULTS: We conducted a randomized, double-blinded, placebo-controlled pilot trial (NCT02877277) in Danish patients with myeloid cancers performed during 3 cycles of DNMTi-treatment (5-azacytidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n = 10) or placebo (n = 10) daily during the last 2 cycles. Fourteen patients (70%) were deficient in plasma vitamin C (< 23 µM) and four of the remaining six patients were taking vitamin supplements at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (< 11.4 µM; 4.997 vs 4.656% 5mC relative to deoxyguanosine, 95% CI [0.126, 0.556], P = 0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (mean increase 34.85 ± 7.94 µM, P = 0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in mononuclear myeloid cells from patients receiving oral vitamin C compared to placebo (0.037% vs - 0.029%, 95% CI [- 0.129, - 0.003], P = 0.041). CONCLUSIONS: Normalization of plasma vitamin C by oral supplementation leads to an increase in the 5hmC/5mC ratio compared to placebo-treated patients and may enhance the biological effects of DNMTis. The clinical efficacy of oral vitamin C supplementation to DNMTis should be investigated in a large randomized, placebo-controlled clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02877277 . Registered on 9 August 2016, retrospectively registered.

Successful treatment of post-transplant relapsed acute myeloid leukemia with FLT3 internal tandem duplication using the combination of induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Report of three cases.

Acute myeloid leukemia is a hematopoietic stem cell neoplastic disease associated with high morbidity and mortality. The presence of FLT3 internal tandem duplication mutations leads to high rates of relapse and decreased overall survival. Patients with FLT3 internal tandem duplication are normally treated with hematopoietic stem cell transplantation in first complete remission. Nevertheless, the incidence of post-transplant relapse is considerable in this group of patients, and the management of this clinical condition is challenging. The report describes the outcomes of patients with FLT3 internal tandem duplication positive acute myeloid leukemia who relapsed after allogeneic hematopoietic stem cell transplantation and were treated with the combination of re-induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Three cases are described and all patients achieved prolonged complete remission with the combined therapy. The combination of induction chemotherapy followed by donor lymphocyte infusion, and the maintenance with azacitidine and sorafenib can be effective approaches in the treatment of post-hematopoietic stem cell transplant and relapsed FLT3 internal tandem duplication positive acute myeloid leukemia patients. This strategy should be further explored in the context of clinical trials.

Integrative Genomics Identifies the Molecular Basis of Resistance to Azacitidine Therapy in Myelodysplastic Syndromes.

Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders.

Sorafenib and azacitidine as salvage therapy for relapse of FLT3-ITD mutated AML after allo-SCT.

OBJECTIVE: Patients with acute myeloid leukemia (AML) carrying FLT3-ITD mutations (FLT3-ITD+) who relapse after allogeneic transplantation (allo-SCT) have a very dismal prognosis with the currently available treatment options. METHODS: We treated eight patients with FLT3-ITD+ AML who had relapsed in median 91 d (range, 28-249) following allo-SCT with a combination of the multikinase inhibitor sorafenib and the DNA methyltransferase inhibitor azacitidine (Aza). RESULTS: Patients received a median of five cycles of Aza (range, 2-9) and sorafenib with a median daily dosage of 750 mg (range 400-800) for 129 d (range, 61-221). Six of eight patients received donor lymphocyte infusions (DLI) with a median number of two DLI per patient (range, 1-4). Following this treatment, four patients (50%) achieved a complete remission and three of them a complete molecular remission. Median duration of CR was 182 d (range, 158-406), and two patients remain in ongoing remission for 406 and 168 d. Median overall survival was 322 d (range, 108-574 d) with three patients being currently alive. CONCLUSION: Taken together, the combination of sorafenib, Aza, and DLI shows promising efficacy and deserves further evaluation in larger patient groups.

Eficacia y seguridad de azacitidina en el tratamiento de leucemia mieloide aguda debut no elegibles para quimioterapia intensa / Efficacy and safety of azacitidine in the treatment of acute myeloid leukemia not eligible for intensive chemotherapy

INTRODUCCIÓN: Antecedentes: El presente dictamen presenta la evaluación de la eficacia y seguridad del uso de azacitidina en el tratamiento de pacientes con leucemia mieloide aguda debut, no tributarios a trasplante. Aspectos generales: La leucemia mieloide aguda (LMA) es un trastorno clonal heterogéneo que involucra una proliferación y diferenciación anormal de las células madre hematopoyéticas. La incidencia aumenta con la edad, siendo más frecuentemente diagnosticada entre los 65 y 74 años de edad, con una mediana de edad de diagnóstico de 67 años(1), y es la leucemia más frecuente en adultos. Tecnología Sanitaria de Interés: La Azacitidina es un nucleósido de pirimidina, análogo de citidina(16). Inhibe la ADN metiltransferasa, que es la enzima responsable de metilar ADN recientemente sintetizado, resultando en síntesis de ADN hipometilado y consecuentes cambios en la transcripción y expresión de genes. METODOLOGÍA: Se realizó una búsqueda de literatura publicada sobre Azacitidina en el tratamiento de LMA en las bases de datos: Medline y Tripdatabase. Adicionalmente, se realizaron búsquedas en los portales web de entidades que realizan revisiones sistemáticas, evaluaciones de tecnologías sanitarias y guías de práctica clínica: The Cochrane Library, National Institute for Health and Care Excellence (NICE) del Reino Unido, European Society For Medical Oncology (ESMO), National Guideline of Clearinghouse (NGC), y National Comprehensive Cancer Network (NCCN) de los Estados Unidos, y The Scottish Intercollegiate Guidelines Network (SIGN) de Escocia. RESULTADOS:Guias de Pratica Clinica: La guía refiere que la estrategia de quimioterapia de inducción está influenciada por las características individuales del paciente, como la edad, la presencia de condiciones de comorbilidad que afecten el estado funcional, e historia de mielodisplasia. Los pacientes cuyo estatus los hace malos candidatos para regímenes estándar de quimioterapia pueden aún ser aptos de participar en ensayos clínicos usando agentes epigenéticos diseñados para atender a esta población de pacientes. Si un ensayo clínico no es opción, entonces la terapia de baja intensidad o cuidados de soporte pueden ser la opción apropiada. Revisiones Sistemáticas y Meta-Análisis: No se encontraron revisiones sistemáticas ni meta-análisis de azacitidina en pacientes con LMA no elegibles para quimioterapia intensa. Evaluaciones de Tecnologías: El National Institute for Health and Clinical Excellence - NICE, del Reino Unido, realizó una evaluación de tecnología sobre azacitidina en leucemia mieloide aguda con más de 30% de blastos medulares. Esta evaluación se publicó el 27 de julio de 2016. La evaluación de tecnología se realizó en base a la información proveída por el fabricante, que consistió en un solo ensayo controlado: el AZA-AML-001, un estudio internacional multicéntrico, controlado, de fase III, de etiqueta abierta, con diseño de grupos paralelos. CONCLUSIONES: EDl Instituto de Evaluación de Tecnologías en Salud e Investigación (IETSI) aprueba el uso de azacitidina como alternativa de tratamiento en pacientes con leucemia mieloide aguda debut con conteo de blastos entre 20% y 30% y no elegibles para terapia intensa, según lo establecido en el anexo 1. El periodo de vigencia de este dictamen es de dos años y la continuación de dicha aprobación estará sujeta a los resultados obtenidos de los pacientes que se beneficien con dicho tratamiento y a nueva evidencia que pueda surgir en el tiempo.

Eficacia y seguridad del uso de azacitidina en el tratamiento de síndrome mielodisplásico de bajo riesgo o intermedio-i con alto requerimiento transfusional con falla al tratamiento con primera línea / Efficacy and safety of the use of azacitidine in the treatment of low-risk or intermediate-i myelodysplastic syndrome with high transfusion requirement with failure of first-line treatment

INTRODUCCIÓN: Antecedentes: El presente dictamen expone la evaluación de tecnología de la eficacia y seguridad de azacitidna en el tratamiento de síndrome mielodisplásico de bajo riesgo o internedio-i con alto requerimientp transfusional con falla al tratamientop con primera linea. Aspectos Generales: En el sistema de clasificación de cánceres hematológicos de la Organización Mundial de la Salçud, los síndromes mielodisplásicos representam una de las cinco categorais principales de neoplasias mieloides. Es un trastorno hematológico caracteriazado por hematopoyesis ineficaz, una o más citopenias, y con frecuencia médula ósea hipercelular, además de una predisposición inherente a transformarse en leucemia mieloide aguda. Tecnologia Sanitaria de Interés: La Azacitidina es un cucleósido de pirimidina, análogo de citidina. Inhibe la ADN metiltransferada, que es la enzima responsable de metilar el ADN recientemente sintetizado, la hipometilación resultante en el ADN conlleva cambios en la transcripción y expressión de algunos genes. METODOLOGIA: Estrategia de Búsqueda: Se realizó una busqueda de literatura publicada sobre Azacitidina en el tratamiento de Síndrome Mielodisplásico de riesgo bajo o intermedio-I en la bases de datos: Medline y Tripdatabase. Adicionalmente, se realizaron búsquedas en los portales web de entidades que realizan revisiones sistemáticas, evaluaciones de tecnologías sanitarias y guías de práctica clínica: The Cochrane Library, National Institute for Health and Care Excelence (NICE) del Reino Unido, national Guideline of Clearinghouse, y National Comprehensive Cancer Network (NCCN) de los Estados Unidos, y The Scottish Intgercollegiate Network (SIGN) de Escocia. RESULTADOS: Sinopsis de la evidencia: Se realizó la búsqueda y la revisión de la evidencia acorde a lo estipulado en la pregunta PICO formulada. Así se consideraron los estudios que tuvieran como intervención azacitidina en el tratamiento de síndrome mielodisplásico de bajo riesgo o intermedio-i con alto requerimiento transfusional con falla al tratamiento con primera línea. CONCLUSIONES: La presente evaluación de tecnología tuvo por objeitvo la evaluación de la eficacia y seguridad del uso de azacitidina en el tratamiento de síndrome mielodisplásico de bajo riesgo o intermedio-i con alto requerimiento transfusional con falla al tratamiento con primera línea. No se ha encontrado en la presente evaluación de tecnología sanitaria evidencia consistente que establezca cual es el beneficio neto atribuible al uso de azacitidina por sobre la mejor terapia de soporte en pacientes con síndrome mielodisplásico, con IPSS de riesgo bajo o intermedio-1, alto requerimiento transfusional, y resistencia o falla a los agentes estimulantes de hematopoyesis. Considerando que la principales guías recomiendan en estos pacientes a la terapia de soporte, las transfusiones, los agentes estimulantes de eritropoyesis, inmunosupresores (en candidatos apropiados), y lenalodomida en pacientes con deleción 5a; se debe mantener estas recomendaciones de tratamiento hasta que futuros ensayos clínicos que comparen azacitidina versus terapia de soporte en esta población, informen concluyentemente sobre la eficacia y seguridad de su uso. El Instituto de Evaluación de Tecnologías e Investigación - IETSI no aprueba el uso de azacitidina como una alternativa de tratamiento para pacientes con síndrome mielodisplásico, con IPSS de riesgo bajo o intermedio-1, alto requerimiento transfusional, y resistencia o falla a los agentes estimulantes de hematopoyesis.

Upregulation of Key Molecules for Targeted Imaging and Therapy.

Targeted diagnosis and therapy enable precise tumor detection and treatment. Successful examples for precise tumor targeting are diagnostic and therapeutic radioligands. However, patients with tumors expressing low levels of the relevant molecular targets are deemed ineligible for such targeted approaches. METHODS: We performed a screen for drugs that upregulate the somatostatin receptor subtype 2 (sstr2). Then, we characterized the effects of these drugs on transcriptional, translational, and functional levels in vitro and in vivo. RESULTS: We identified 9 drugs that act as epigenetic modifiers, including the inhibitor of DNA methyltransferase decitabine as well as the inhibitors of histone deacetylase tacedinaline and romidepsin. In vitro, these drugs upregulated sstr2 on transcriptional, translational, and functional levels in a time- and dose-dependent manner. Thereby, their combinations revealed synergistic effects. In vivo, drug-based sstr2 upregulation improved the tumor-to-background and tumor-to-kidney ratios, which are the key determinants of successful sstr2-targeted imaging and radiopeptide therapy. CONCLUSION: We present an approach that uses epigenetic modifiers to improve sstr2 targeting in vitro and in vivo. Translation of this method into the clinic may potentially convert patients ineligible for targeted imaging and therapy to eligible candidates.

Enhanced inhibition of clonogenic survival of human medulloblastoma cells by multimodal treatment with ionizing irradiation, epigenetic modifiers, and differentiation-inducing drugs.

BACKGROUND: Medulloblastoma (MB) is the most common pediatric brain tumor. Current treatment regimes consisting of primary surgery followed by radio- and chemotherapy, achieve 5-year overall survival rates of only about 60 %. Therapy-induced endocrine and neurocognitive deficits are common late adverse effects. Thus, improved antitumor strategies are urgently needed. In this study, we combined irradiation (IR) together with epigenetic modifiers and differentiation inducers in a multimodal approach to enhance the efficiency of tumor therapy in MB and also assessed possible late adverse effects on neurogenesis. METHODS: In three human MB cell lines (DAOY, MEB-Med8a, D283-Med) short-time survival (trypan blue exclusion assay), apoptosis, autophagy, cell cycle distribution, formation of gH2AX foci, and long-term reproductive survival (clonogenic assay) were analyzed after treatment with 5-aza-2'-deoxycytidine (5-azadC), valproic acid (VPA), suberanilohydroxamic acid (SAHA), abacavir (ABC), all-trans retinoic acid (ATRA) and resveratrol (RES) alone or combined with 5-aza-dC and/or IR. Effects of combinatorial treatments on neurogenesis were evaluated in cultured murine hippocampal slices from transgenic nestin-CFPnuc C57BL/J6 mice. Life imaging of nestin-positive neural stem cells was conducted at distinct time points for up to 28 days after treatment start. RESULTS: All tested drugs showed a radiosynergistic action on overall clonogenic survival at least in two-outof-three MB cell lines. This effect was pronounced in multimodal treatments combining IR, 5-aza-dC and a second drug. Hereby, ABC and RES induced the strongest reduction of clongenic survival in all three MB cell lines and led to the induction of apoptosis (RES, ABC) and/or autophagy (ABC). Additionally, 5-aza-dC, RES, and ABC increased the S phase cell fraction and induced the formation of gH2AX foci at least in oneout-of-three cell lines. Thereby, the multimodal treatment with 5-aza-dC, IR, and RES or ABC did not change the number of normal neural progenitor cells in murine slice cultures. CONCLUSION: In conclusion, the radiosensitizing capacities of epigenetic and differentiation-inducing drugs presented here suggest that their adjuvant administration might improve MB therapy. Thereby, the combination of 5-aza-dC/IR with ABC and RES seemed to be the most promising to enhance tumor control without affecting the normal neural precursor cells.

A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

Azacitidine and Sorafenib Therapy in a Pediatric Patient With Refractory Acute Myeloid Leukemia With Monosomy 7 and Somatic PTPN11 Mutation.

Monosomy 7 is a well-documented cytogenetic aberration in pediatric acute myeloid leukemia (AML) and may occur in combinations with molecular abnormalities including PTPN11 mutation. PTPN11 mutations contribute to leukemogenesis through upregulation of Ras pathway signaling. We present the case of a 3-year-old female with AML with monosomy 7 and somatic PTPN11 mutation who was refractory to conventional AML chemotherapy but responded to a novel regimen of azacitidine and sorafenib followed by stem cell transplantation. Combination therapy with azacitidine and sorafenib may be an effective therapeutic strategy for patients with AML with Ras pathway abnormalities.

Decitabine and Sorafenib Therapy in FLT-3 ITD-Mutant Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) characterized by Feline McDonough Sarcoma-like tyrosine kinase-3 (FLT-3) internal tandem duplication (ITD) mutations have poor outcomes. Treatment options are limited, because these mutations confer resistance to conventional chemotherapy. FLT-3 inhibitors such as sorafenib have been studied as a single agent and in combination with conventional chemotherapy or azacytidine with fair responses. PATIENTS AND METHODS: Here we describe our preclinical and clinical experience with the combination of the DNA hypomethylating agent, decitabine and sorafenib for the treatment of FLT-3 ITD-mutant AML. RESULTS: In vitro treatment of the human FLT-3 ITD-mutant AML cell line, MV4-11, with both drugs significantly improved growth inhibition over single-agent therapy and resulted in synergistic antitumor effects (combination index < 1). A case series of 6 patients treated with off protocol combination of decitabine and sorafenib demonstrated overall responses in 5 patients (83%) with a median survival of 155 days. Four of the 5 patients (80%) with relapsed/refractory AML achieved complete responses with incomplete count recovery. The combination was also well tolerated. CONCLUSION: Further investigation is warranted to confirm these responses.

Decitabine Enhances Lymphocyte Migration and Function and Synergizes with CTLA-4 Blockade in a Murine Ovarian Cancer Model.

The lack of second-line treatment for relapsed ovarian cancer necessitates the development of improved combination therapies. Targeted therapy and immunotherapy each confer clinical benefit, albeit limited as monotherapies. Ovarian cancer is not particularly responsive to immune checkpoint blockade, so combination with a complementary therapy may be beneficial. Recent studies have revealed that a DNA methyl transferase inhibitor, azacytidine, alters expression of immunoregulatory genes in ovarian cancer. In this study, the antitumor effects of a related DNA methyl transferase inhibitor, decitabine (DAC), were demonstrated in a syngeneic murine ovarian cancer model. Low-dose DAC treatment increases the expression of chemokines that recruit NK cells and CD8(+) T cells, promotes their production of IFNγ and TNFα, and extends the survival of mice bearing subcutaneous or orthotopic tumors. While neither DAC nor immune checkpoint blockade confers durable responses as a monotherapy in this model, the efficacy of anti-CTLA-4 was potentiated by combination with DAC. This combination promotes differentiation of naïve T cells into effector T cells and prolongs cytotoxic lymphocyte responses as well as mouse survival. These results suggest that this combination therapy may be worthy of further consideration for improved treatment of drug-resistant ovarian cancer.

Potentiation of growth inhibition and epigenetic modulation by combination of green tea polyphenol and 5-aza-2'-deoxycytidine in human breast cancer cells.

Epigenetic therapy by DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza 2'dC) is clinically effective in acute myeloid leukemia; however, it has shown limited results in treatment of breast cancer and has significant toxicity to normal cells. Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has anti-cancer and DNA demethylating properties with no significant toxicity toward normal cells. Therefore, the objective of this study was to evaluate the therapeutic efficacy of a combination of non-toxic, low dose of 5-aza 2' dC with EGCG, on growth inhibition of breast cancer cells. Human breast cancer cell lines (MCF-7, MDA-MB 231) and non-tumorigenic MCF-10A breast epithelial cells were treated with either 5-aza 2' dC, EGCG, or their combination for 7 days. Cell growth inhibition was determined by cell count, cell viability, cell cycle, and soft agar assay, whereas genes expression changes were determined by quantitative real-time PCR and/or Western blot analysis. Histone modifications and global DNA methylation changes were determined by Western blot and RAPD-PCR, respectively. The results revealed significantly greater inhibition of growth of breast cancer cells by co-treatment with 5-aza 2' dC and EGCG compared to individual treatments, whereas it has no significant toxicity to MCF-10A cells. This was further confirmed by gene expression analysis. Changes in DNA methylation and histone modifications were also greater in cells with combination treatment. Findings of this study suggest that potentiation of growth inhibition of breast cancer cells by 5-aza 2' dC and EGCG combination treatment, at least in part, is mediated by epigenetic mechanism.

Phase 2 study of azacytidine plus sorafenib in patients with acute myeloid leukemia and FLT-3 internal tandem duplication mutation.

Patients received 5-azacytidine (AZA) 75 mg/m(2) intravenously daily for 7 days and sorafenib 400 mg orally twice daily continuously; cycles were repeated at ~1-month intervals. Forty-three acute myeloid leukemia (AML) patients with a median age of 64 years (range, 24-87 years) were enrolled; 37 were evaluable for response. FMS-like tyrosine kinase-3 (FLT3)-internal tandem duplication (ITD) mutation was detected in 40 (93%) patients, with a median allelic ratio of 0.32 (range, 0.009-0.93). They had received a median of 2 prior treatment regimens (range, 0-7); 9 had failed prior therapy with a FLT3 kinase inhibitor. The response rate was 46%, including 10 (27%) complete response with incomplete count recovery (CRi), 6 (16%) complete responses (CR), and 1 (3%) partial response. The median time to achieve CR/CRi was 2 cycles (range, 1-4), and the median duration of CR/CRi was 2.3 months (range, 1-14.3 months). Sixty-four percent of patients achieved adequate (defined as >85%) FLT3 inhibition during their first cycle of therapy. The degree of FLT3 inhibition correlated with plasma sorafenib concentrations. FLT3 ligand levels did not rise to levels seen in prior studies of patients receiving cytotoxic chemotherapy. The combination of AZA and sorafenib is effective for patients with relapsed AML and FLT-3-ITD. This trial was registered at clinicaltrials.gov as #NCT01254890.

Clinical and pharmacodynamic activity of bortezomib and decitabine in acute myeloid leukemia.

We recently reported promising clinical activity for a 10-day regimen of decitabine in older AML patients; high miR-29b expression associated with clinical response. Subsequent preclinical studies with bortezomib in AML cells have shown drug-induced miR-29b up-regulation, resulting in loss of transcriptional activation for several genes relevant to myeloid leukemogenesis, including DNA methyltransferases and receptor tyrosine kinases. Thus, a phase 1 trial of bortezomib and decitabine was developed. Nineteen poor-risk AML patients (median age 70 years; range, 32-84 years) enrolled. Induction with decitabine (20 mg/m(2) intravenously on days 1-10) plus bortezomib (escalated up to the target 1.3 mg/m(2) on days 5, 8, 12, and 15) was tolerable, but bortezomib-related neuropathy developed after repetitive cycles. Of previously untreated patients (age ≥ 65 years), 5 of 10 had CR (complete remission, n = 4) or incomplete CR (CRi, n = 1); 7 of 19 overall had CR/CRi. Pharmacodynamic analysis showed FLT3 down-regulation on day 26 of cycle 1 (P = .02). Additional mechanistic studies showed that FLT3 down-regulation was due to bortezomib-induced miR-29b up-regulation; this led to SP1 down-regulation and destruction of the SP1/NF-κB complex that transactivated FLT3. This study demonstrates the feasibility and preliminary clinical activity of decitabine plus bortezomib in AML and identifies FLT3 as a novel pharmacodynamic end point for future trials.

A phase I study of 5-azacytidine and erlotinib in advanced solid tumor malignancies.

INTRODUCTION: The epidermal growth factor receptor (EGFR) is a validated target in malignancy; however, patients with wild type EGFR obtain little sustained benefit from anti-EGFR monotherapy. Epigenetic therapy to reactivate tumor suppressor genes may enhance the anti-proliferative effect of erlotinib. This phase I study evaluated the combination of erlotinib and 5-azacytidine for safety and maximal tolerated dose (MTD). METHODS: Thirty patients with advanced solid tumors were treated in a standard 3 + 3 cohort design. Erlotinib was dosed at 150 mg daily, and 5-azacytidine was escalated by increasing the number of daily doses of 75 mg/m(2) per cycle. Patients were followed for dose-limiting toxicity (DLT). Efficacy was assessed by RECIST criteria. RESULTS: Common non-hematologic toxicities included rash, diarrhea, nausea, and fatigue; the majority was ≤ Grade 2. DLTs included conjunctivitis in cohort 1 and infusion reaction in cohort 2. No DLTs occurred in cohorts 3, 4, or 5; however, 2 serious neutropenic infections arose in cohort 5 after cycle 1. Cohort 4 was expanded to 6 patients and was the MTD. Partial response (lung, ovarian) and stable disease occurred in 2 and 11 patients, respectively. Median progression-free survival was 2 months. Two patients with lung and larynx cancer had prolonged stable disease. CONCLUSION: The combination of erlotinib and 5-azacytidine was well tolerated with interesting clinical activity in lung, head and neck, and ovarian cancer. The recommended dose for phase II study is erlotinib 150 mg daily and 5-azacytidine 75 mg/m(2) daily on days 1-4 and 15-18 of a 28-day cycle.