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1.
Cancers (Basel) ; 16(8)2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38672643

RESUMO

Background: Precision oncology treatments are being applied more commonly in breast and gynecological oncology through the implementation of Molecular Tumor Boards (MTBs), but real-world clinical outcome data remain limited. Methods: A retrospective analysis was conducted in patients with breast cancer (BC) and gynecological malignancies referred to our center's MTB from 2018 to 2023. The analysis covered patient characteristics, next-generation sequencing (NGS) results, MTB recommendations, therapy received, and clinical outcomes. Results: Sixty-three patients (77.8%) had metastatic disease, and forty-four patients (54.3%) had previously undergone three or more lines of systemic treatment. Personalized treatment recommendations were provided to 50 patients (63.3%), while 29 (36.7%) had no actionable target. Ultimately, 23 patients (29.1%) underwent molecular-matched treatment (MMT). Commonly altered genes in patients with pan-gyn tumors (BC and gynecological malignancies) included TP53 (n = 42/81, 51.9%), PIK3CA (n = 18/81, 22.2%), BRCA1/2 (n = 10/81, 12.3%), and ARID1A (n = 9/81, 11.1%). Patients treated with MMT showed significantly prolonged progression-free survival (median PFS 5.5 vs. 3.5 months, p = 0.0014). Of all patients who underwent molecular profiling, 13.6% experienced a major clinical benefit (PFSr ≥ 1.3 and PR/SD ≥ 6 months) through precision oncology. Conclusions: NGS-guided precision oncology demonstrated improved clinical outcomes in a subgroup of patients with gynecological and breast cancers.

2.
Cancers (Basel) ; 14(18)2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36139590

RESUMO

BACKGROUND: Increasing knowledge of cancer biology and an expanding spectrum of molecularly targeted therapies provide the basis for precision oncology. Despite extensive gene diagnostics, previous reports indicate that less than 10% of patients benefit from this concept. METHODS: We retrospectively analyzed all patients referred to our center's Molecular Tumor Board (MTB) from 2018 to 2021. Molecular testing by next-generation sequencing (NGS) included a 67-gene panel for the detection of short-sequence variants and copy-number alterations, a 53- or 137-gene fusion panel and an ultra-low-coverage whole-genome sequencing for the detection of additional copy-number alterations outside the panel's target regions. Immunohistochemistry for microsatellite instability and PD-L1 expression complemented NGS. RESULTS: A total of 109 patients were referred to the MTB. In all, 78 patients received therapeutic proposals (70 based on NGS) and 33 were treated accordingly. Evaluable patients treated with MTB-recommended therapy (n = 30) had significantly longer progression-free survival than patients treated with other therapies (n = 17) (4.3 vs. 1.9 months, p = 0.0094). Seven patients treated with off-label regimens experienced major clinical benefits. CONCLUSION: The combined focused sequencing assays detected targetable alterations in the majority of patients. Patient benefits appeared to lie in the same range as with large-scale sequencing approaches.

3.
Ann Hematol ; 101(4): 811-824, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35106639

RESUMO

With the advent of novel, highly effective therapies for multiple myeloma (MM), classical serologic monitoring appears insufficient for response assessment and prediction of relapse. Moreover, serologic studies in MM are hampered by interference of therapeutic antibodies. The detection of malignant plasma cell clones by next generation sequencing (NGS) or multiparameter flow cytometry (MFC) circumvents these difficulties and can be performed in the peripheral blood (pB) by targeting circulating cell-free DNA (cfDNA) or circulating plasma cells (CPCs), thus also avoiding an invasive sampling procedure. Here, we applied NGS of VJ light chain (LC) rearrangements in cfDNA and MFC of magnetically-enriched CD138-positive CPCs (me-MFC) to investigate disease burden in unselected MM patients. Sequencing was successful for 114/130 (87.7%) cfDNA samples and me-MFC results were analyzable for 196/205 (95.6%) samples. MM clones were detectable in 38.9% of samples taken at initial diagnosis or relapse (ID/RD), but only in 11.8% of samples taken during complete remission (CR). Circulating MM plasma cells were present in 83.3% of ID/RD samples and 9.9% of CR samples. Residual disease assessment by NGS or me-MFC in samples taken during very good partial remission or CR was 80% concordant. Notably, 4/4 (NGS) and 5/8 (me-MFC) positive CR samples were from patients with oligo- or non-secretory myeloma. The time to progression was shorter if there was evidence of residual myeloma in the pB. Together, our findings indicate that our two novel analytical approaches accurately indicate the course of MM and may be particularly valuable for monitoring patients with serologically non-trackable disease.


Assuntos
Ácidos Nucleicos Livres , Mieloma Múltiplo , Citometria de Fluxo/métodos , Humanos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Recidiva Local de Neoplasia , Neoplasia Residual/diagnóstico , Plasmócitos/patologia
4.
Haematologica ; 104(2): 277-287, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30190345

RESUMO

Differential induction therapy of all subtypes of acute myeloid leukemia other than acute promyelocytic leukemia is impeded by the long time required to complete complex and diverse cytogenetic and molecular genetic analyses for risk stratification or targeted treatment decisions. Here, we describe a reliable, rapid and sensitive diagnostic approach that combines karyotyping and mutational screening in a single, integrated, next-generation sequencing assay. Numerical karyotyping was performed by low coverage whole genome sequencing followed by copy number variation analysis using a novel algorithm based on in silico-generated reference karyotypes. Translocations and DNA variants were examined by targeted resequencing of fusion transcripts and mutational hotspot regions using commercially available kits and analysis pipelines. For the identification of FLT3 internal tandem duplications and KMT2A partial tandem duplications, we adapted previously described tools. In a validation cohort including 22 primary patients' samples, 9/9 numerically normal karyotypes were classified correctly and 30/31 (97%) copy number variations reported by classical cytogenetics and fluorescence in situ hybridization analysis were uncovered by our next-generation sequencing karyotyping approach. Predesigned fusion and mutation panels were validated exemplarily on leukemia cell lines and a subset of patients' samples and identified all expected genomic alterations. Finally, blinded analysis of eight additional patients' samples using our comprehensive assay accurately reproduced reference results. Therefore, calculated karyotyping by low coverage whole genome sequencing enables fast and reliable detection of numerical chromosomal changes and, in combination with panel-based fusion-and mutation screening, will greatly facilitate implementation of subtype-specific induction therapies in acute myeloid leukemia.


Assuntos
Biomarcadores Tumorais , Estudos de Associação Genética , Predisposição Genética para Doença , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Aberrações Cromossômicas , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Feminino , Estudos de Associação Genética/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , Adulto Jovem
5.
PLoS One ; 10(4): e0123181, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25901794

RESUMO

RAS mutations are frequently found among acute myeloid leukemia patients (AML), generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA) and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1) in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC) driven differentiation. Taken together, our findings show that AML with inv(16) and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.


Assuntos
Diferenciação Celular , GTP Fosfo-Hidrolases/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Membrana/metabolismo , Idoso , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Estudos de Coortes , Citarabina/farmacologia , Citarabina/uso terapêutico , Feminino , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas ras/genética , Proteínas ras/metabolismo
6.
Water Res ; 46(15): 4665-76, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22763289

RESUMO

An ever-growing need exists for rapid, quantitative and meaningful methods to quantify and characterize the effect of different treatment steps on the microbiological processes and events that occur during drinking water treatment and distribution. Here we compared cultivation-independent flow cytometry (FCM) and adenosine tri-phosphate (ATP) analysis with conventional cultivation-based microbiological methods, on water samples from two full-scale treatment and distribution systems. The two systems consist of nearly identical treatment trains, but their raw water quality and pre-treatment differed significantly. All of the drinking water treatment processes affected the microbiological content of the water considerably, but once treated, the finished water remained remarkably stable throughout the distribution system. Both the FCM and ATP data were able to describe the microbiology of the systems accurately, providing meaningful process data when combined with other parameters such as dissolved organic carbon analysis. Importantly, the results highlighted a complimentary value of the two independent methods: while similar trends were mostly observed, variations in ATP-per-cell values between water samples were adequately explained by differences in the FCM fingerprints of the samples. This work demonstrates the value of alternative microbial methods for process/system control, optimization and routine monitoring of the general microbial quality of water during treatment and distribution.


Assuntos
Trifosfato de Adenosina/análise , Desinfecção , Citometria de Fluxo/métodos , Abastecimento de Água , Aeromonas/isolamento & purificação , Bactérias/crescimento & desenvolvimento , Biomassa , Contagem de Colônia Microbiana , Filtração , Microbiologia da Água
7.
Appl Environ Microbiol ; 77(3): 1041-8, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21148700

RESUMO

Pellet softening reactors are used in centralized and decentralized drinking water treatment plants for the removal of calcium (hardness) through chemically induced precipitation of calcite. This is accomplished in fluidized pellet reactors, where a strong base is added to the influent to increase the pH and facilitate the process of precipitation on an added seeding material. Here we describe for the first time the opportunistic bacterial colonization of the calcite pellets in a full-scale pellet softening reactor and the functional contribution of these colonizing bacteria to the overall drinking water treatment process. ATP analysis, advanced microscopy, and community fingerprinting with denaturing gradient gel electrophoretic (DGGE) analysis were used to characterize the biomass on the pellets, while assimilable organic carbon (AOC), dissolved organic carbon, and flow cytometric analysis were used to characterize the impact of the biological processes on drinking water quality. The data revealed pellet colonization at concentrations in excess of 500 ng of ATP/g of pellet and reactor biomass concentrations as high as 220 mg of ATP/m(3) of reactor, comprising a wide variety of different microorganisms. These organisms removed as much as 60% of AOC from the water during treatment, thus contributing toward the biological stabilization of the drinking water. Notably, only a small fraction (about 60,000 cells/ml) of the bacteria in the reactors was released into the effluent under normal conditions, while the majority of the bacteria colonizing the pellets were captured in the calcite structures of the pellets and were removed as a reusable product.


Assuntos
Bactérias/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Carbonato de Cálcio/química , Purificação da Água/métodos , Abrandamento da Água/métodos , Abastecimento de Água , Bactérias/química , Bactérias/genética , Biomassa , Biotecnologia/métodos , Carbono/análise , Ingestão de Líquidos , Água/química , Microbiologia da Água
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