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1.
Int J Mol Sci ; 25(13)2024 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-39000262

RESUMEN

Radiotherapy in the head-and-neck area is one of the main curative treatment options. However, this comes at the cost of varying levels of normal tissue toxicity, affecting up to 80% of patients. Mucositis can cause pain, weight loss and treatment delays, leading to worse outcomes and a decreased quality of life. Therefore, there is an urgent need for an approach to predicting normal mucosal responses in patients prior to treatment. We here describe an assay to detect irradiation responses in healthy oral mucosa tissue. Mucosa specimens from the oral cavity were obtained after surgical resection, cut into thin slices, irradiated and cultured for three days. Seven samples were irradiated with X-ray, and three additional samples were irradiated with both X-ray and protons. Healthy oral mucosa tissue slices maintained normal morphology and viability for three days. We measured a dose-dependent response to X-ray irradiation and compared X-ray and proton irradiation in the same mucosa sample using standardized automated image analysis. Furthermore, increased levels of inflammation-inducing factors-major drivers of mucositis development-could be detected after irradiation. This model can be utilized for investigating mechanistic aspects of mucositis development and can be developed into an assay to predict radiation-induced toxicity in normal mucosa.


Asunto(s)
Mucosa Bucal , Humanos , Mucosa Bucal/efectos de la radiación , Rayos X/efectos adversos , Traumatismos por Radiación/etiología , Traumatismos por Radiación/patología , Masculino , Mucositis/etiología , Mucositis/patología , Femenino , Relación Dosis-Respuesta en la Radiación , Estomatitis/etiología , Estomatitis/patología , Adulto , Persona de Mediana Edad
2.
Cancers (Basel) ; 16(4)2024 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-38398132

RESUMEN

Tumors with a pathogenic BRCA1/2 mutation are homologous recombination (HR)-deficient (HRD) and consequently sensitive to platinum-based chemotherapy and Poly-[ADP-Ribose]-Polymerase inhibitors (PARPi). We hypothesized that functional HR status better reflects real-time HR status than BRCA1/2 mutation status. Therefore, we determined the functional HR status of 53 breast cancer (BC) and 38 ovarian cancer (OC) cell lines by measuring the formation of RAD51 foci after irradiation. Discrepancies between functional HR and BRCA1/2 mutation status were investigated using exome sequencing, methylation and gene expression data from 50 HR-related genes. A pathogenic BRCA1/2 mutation was found in 10/53 (18.9%) of BC and 7/38 (18.4%) of OC cell lines. Among BRCA1/2-mutant cell lines, 14/17 (82.4%) were HR-proficient (HRP), while 1/74 (1.4%) wild-type cell lines was HRD. For most (80%) cell lines, we explained the discrepancy between functional HR and BRCA1/2 mutation status. Importantly, 12/14 (85.7%) BRCA1/2-mutant HRP cell lines were explained by mechanisms directly acting on BRCA1/2. Finally, functional HR status was strongly associated with COSMIC single base substitution signature 3, but not BRCA1/2 mutation status. Thus, the majority of BRCA1/2-mutant cell lines do not represent a suitable model for HRD. Moreover, exclusively determining BRCA1/2 mutation status may not suffice for platinum-based chemotherapy or PARPi patient selection.

3.
NPJ Breast Cancer ; 9(1): 80, 2023 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-37777518

RESUMEN

We developed a functional ex vivo anthracycline-based sensitivity test. Surgical resection material of primary breast cancer (BC) was used to determine criteria for the ex vivo sensitivity assay based on morphology, proliferation and apoptosis. Subsequently, a proof-of-concept study was performed correlating results of this assay on primary BC biopsies with in vivo response after treatment with anthracycline-containing neoadjuvant chemotherapy (NAC). Cut off values for the ex vivo anthracycline-based sensitivity test were established based on analysis of 21 primary breast tumor samples obtained after surgery. In the proof-of-concept study based on a new set of tumor biopsies, 41 patients were included. Eight biopsies did not contain tumor cells and three patients could not be biopsied for various reasons. In the remaining 30 biopsies, the success rate of the ex vivo test was 77% (23/30); six out of seven failed tests were due to excessive apoptosis, our pre-specified test criteria. Of the 23 patients with a successful ex vivo test result, three patients did not undergo NAC after the biopsy. Here we report the ex vivo anthracycline-based sensitivity assay is feasible on biopsy material and shows 75% concordance between ex vivo outcomes and in vivo MRI response. Unfortunately, the percentage of unsuccessful tests is rather high. This study provides the foundation for further development of ex vivo sensitivity assays.

4.
Theranostics ; 13(10): 3117-3130, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37351169

RESUMEN

Background: Peptide receptor radionuclide therapy (PRRT) increases progression-free survival and quality of life of neuroendocrine tumor (NET) patients, however complete cures are rare and dose-limiting toxicity has been reported. PRRT induces DNA damage of which DNA double strand breaks (DSBs) are the most cytotoxic. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key player in DSB repair and its inhibition therefore is a potential way to enhance PRRT efficacy without increasing the dosage. Methods: We analyzed effects of combining PRRT and DNA-PKcs inhibitor AZD7648 on viability, cell death and clonogenic survival on SSTR2-expressing cell lines BON1-SSTR2, GOT1 and NCI-H69. Therapy-induced DNA damage response was assessed by analyzing DSB foci levels and cell cycle distributions. In vivo efficacy was investigated in BON1-SSTR2 and NCI-H69 xenografted mice and hematologic and renal toxicity were monitored by blood counts, creatinine levels and analyzing renal morphology. Results: Combining PRRT and AZD7648 significantly decreased viability of BON1-SSTR2, GOT1 and NCI-H69 cells and induced cell death in GOT1 and BON1-SSTR2 cells. A strong effect of AZD7648 on PRRT-induced DSB repair was found. In GOT1 cells, this was accompanied by induction of cell cycle blocks. However, BON1-SSTR2 cells were unable to fully arrest their cell cycle and polyploid cells with high DNA damage levels were detected. In vivo, AZD7648 significantly sensitized BON1-SSTR2 and NCI-H69 xenograft models to PRRT. In addition, combination therapy did not induce significant changes in body weight, blood composition, plasma creatinine levels and renal morphology, indicating the absence of severe acute hematologic and renal toxicity. Conclusion: These results highlight that the potentiation of the therapeutic effect of PRRT by DNA-PKcs inhibition is a highly effective and well-tolerated therapeutic strategy. Based on our findings, we recommend initiation of phase I/II studies in patients to find a safe and effective combination regimen.


Asunto(s)
Tumores Neuroendocrinos , Humanos , Ratones , Animales , Tumores Neuroendocrinos/tratamiento farmacológico , Tumores Neuroendocrinos/radioterapia , Proteína Quinasa Activada por ADN/metabolismo , Creatinina , Calidad de Vida , Radioisótopos/metabolismo , ADN
5.
Cancers (Basel) ; 15(3)2023 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-36765883

RESUMEN

Peptide receptor radionuclide therapy (PRRT), a form of internal targeted radiation treatment using [177Lu]Lu [DOTA0-Tyr3]octreotate, is used to treat patients with metastasized neuroendocrine tumors (NETs). Even though PRRT is now the second line of treatment for patients with metastasized NETs, the majority of patients will not be cured by the treatment. PRRT functions by inducing DNA damage upon radioactive decay and inhibition of DNA damage repair proteins could therefore be used as a strategy to potentiate PRRT. Previous work has shown promising results on the combination of PRRT with the PARP inhibitor olaparib in cell lines and mice and we have been taken the next step for further in vivo validation using two different xenografted mouse models. We observed that this combination therapy resulted in increased therapeutic efficacy only in one model and not the other. Overall, our findings indicate a tumor-type dependent anti-tumor response to the combination of PRRT and olaparib. These data emphasize the unmet need for the molecular stratification of tumors to predetermine the potential clinical value of combining PARP inhibition with PRRT.

6.
Cancers (Basel) ; 15(2)2023 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-36672427

RESUMEN

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) displays a large heterogeneity in treatment response, and consequently in patient prognosis. Despite extensive efforts, no clinically validated model is available to predict tumor response. Here we describe a functional test for predicting tumor response to radiation and chemotherapy on the level of the individual patient. METHODS: Resection material of 17 primary HNSCC patients was cultured ex vivo, irradiated or cisplatin-treated, after which the effect on tumor cell vitality was analyzed several days after treatment. RESULTS: Ionizing radiation (IR) affected tumor cell growth and viability with a clear dose-response relationship, and marked heterogeneity between tumors was observed. After a single dose of 5Gy, proliferation in IR-sensitive tumors dropped below 30% of the untreated level, while IR-resistant tumors maintained at least 60% of proliferation. IR-sensitive tumors showed on average a twofold increase in apoptosis, as well as an increased number and size of DNA damage foci after treatment. No differences in the homologous recombination (HR) proficiency between IR-sensitive and -resistant tumors were detected. Cisplatin caused a decrease in proliferation, as well as induction of apoptosis, again with marked variation between the samples. CONCLUSIONS: Our functional ex vivo assay discriminated between IR-sensitive and IR-resistant HNSCC tumors, and may also be suitable for predicting response to cisplatin. Its predictive value is currently under investigation in a prospective clinical study.

7.
Int J Mol Sci ; 23(14)2022 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-35887398

RESUMEN

Prostate specific membrane antigen targeted radionuclide therapy (PSMA-TRT) is a promising novel treatment for prostate cancer (PCa) patients. However, PSMA-TRT cannot be used for curative intent yet, thus additional research on how to improve the therapeutic efficacy is warranted. A potential way of achieving this, is combining TRT with poly ADP-ribosylation inhibitors (PARPi), which has shown promising results for TRT of neuroendocrine tumor cells. Currently, several clinical trials have been initiated for this combination for PCa, however so far, no evidence of synergism is available for PCa. Therefore, we evaluated the combination of PSMA-TRT with three classes of PARPi in preclinical PCa models. In vitro viability and survival assays were performed using PSMA-expressing PCa cell lines PC3-PIP and LNCaP to assess the effect of increasing concentrations of PARPi veliparib, olaparib or talazoparib in combination with PSMA-TRT compared to single PARPi treatment. Next, DNA damage analyses were performed by quantifying the number of DNA breaks by immunofluorescent stainings. Lastly, the potential of the combination treatments was studied in vivo in mice bearing PC3-PIP xenografts. Our results show that combining PSMA-TRT with PARPi did not synergistically affect the in vitro clonogenic survival or cell viability. DNA-damage analysis revealed only a significant increase in DNA breaks when combining PSMA-TRT with veliparib and not in the other combination treatments. Moreover, PSMA-TRT with PARPi treatment did not improve tumor control compared to PSMA-TRT monotherapy. Overall, the data presented do not support the assumption that combining PSMA-TRT with PARPi leads to a synergistic antitumor effect in PCa. These results underline that extensive preclinical research using various PCa models is imperative to validate the applicability of the combination strategy for PCa, as it is for other cancer types.


Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Neoplasias de la Próstata , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Próstata/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/radioterapia , Radioisótopos/uso terapéutico
8.
Oncogene ; 41(26): 3498-3506, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35662281

RESUMEN

Germline BRCA1/2 mutation status is predictive for response to Poly-[ADP-Ribose]-Polymerase (PARP) inhibitors in breast cancer (BC) patients. However, non-germline BRCA1/2 mutated and homologous recombination repair deficient (HRD) tumors are likely also PARP-inhibitor sensitive. Clinical validity and utility of various HRD biomarkers are under investigation. The REpair CAPacity (RECAP) test is a functional method to select HRD tumors based on their inability to form RAD51 foci. We investigated whether this functional test defines a similar group of HRD tumors as DNA-based tests. An HRD enriched cohort (n = 71; 52 primary and 19 metastatic BCs) selected based on the RECAP test (26 RECAP-HRD; 37%), was subjected to DNA-based HRD tests (i.e., Classifier of HOmologous Recombination Deficiency (CHORD) and BRCA1/2-like classifier). Whole genome sequencing (WGS) was carried out for 38 primary and 19 metastatic BCs. The RECAP test identified all bi-allelic BRCA deficient samples (n = 15) in this cohort. RECAP status partially correlated with DNA-based HRD test outcomes (70% concordance for both RECAP-CHORD and RECAP-BRCA1/2-like classifier). RECAP selected additional samples unable to form RAD51 foci, suggesting that this functional assay identified deficiencies in other DNA repair genes, which could also result in PARP-inhibitor sensitivity. Direct comparison of these HRD tests in clinical trials will be required to evaluate the optimal predictive test for clinical decision making.


Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Antineoplásicos/uso terapéutico , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , ADN , Femenino , Recombinación Homóloga/genética , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Poli(ADP-Ribosa) Polimerasas/genética , Reparación del ADN por Recombinación/genética
9.
Cancers (Basel) ; 14(5)2022 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-35267560

RESUMEN

Background chemotherapy is part of most breast cancer (BC) treatment schedules. However, a substantial fraction of BC tumors does not respond to the treatment. Unfortunately, no standard biomarkers exist for response prediction. Therefore, we aim to develop ex vivo sensitivity assays for two types of commonly used cytostatics (i.e., platinum derivates and taxanes) on organotypic BC tissue slices. METHODS: Ex vivo cisplatin sensitivity assays were established using organotypic tissue slices derived from the surgical resection material of 13 primary BCs and 20 fresh histological biopsies obtained from various metastatic sites. Furthermore, tissue slices of 10 primary BCs were used to establish a docetaxel ex vivo sensitivity assay. RESULTS: Cisplatin sensitivity was assessed by tissue morphology, proliferation and apoptosis, while the relative increase in the mitotic index was discriminative for docetaxel sensitivity. Based on these read-outs, a scoring system was proposed to discriminate sensitive from resistant tumors for each cytostatic. We successful completed the cisplatin sensitivity assay on 12/16 (75%) biopsies as well. CONCLUSIONS: We developed an ex vivo cisplatin and docetaxel assay on BC slices. We also adapted the assay for biopsy-sized specimens as the next step towards the correlation of ex vivo test results and in vivo responses.

10.
Proc Natl Acad Sci U S A ; 119(10): e2113233119, 2022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35235448

RESUMEN

SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.


Asunto(s)
Daño del ADN , Reparación del ADN por Unión de Extremidades , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN , Humanos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética
11.
Cancer Res ; 81(24): 6171-6182, 2021 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-34548335

RESUMEN

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. SIGNIFICANCE: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.See related commentary by Mishra et al., p. 6080.


Asunto(s)
Proteína BRCA1/fisiología , Proteína del Grupo de Complementación N de la Anemia de Fanconi/fisiología , Regulación Neoplásica de la Expresión Génica , Recombinación Homóloga , Neoplasias Mamarias Animales/patología , Reparación del ADN por Recombinación , Animales , Apoptosis , Proteína BRCA2/fisiología , Proliferación Celular , Femenino , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/fisiología
12.
Theranostics ; 11(2): 491-505, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33391488

RESUMEN

Patients with neuroendocrine tumors (NETs) can be treated with peptide receptor radionuclide therapy (PRRT). Here, the somatostatin analogue octreotate radiolabeled with lutetium-177 is targeted to NET cells by binding to the somatostatin receptor subtype 2 (SST2). During radioactive decay, DNA damage is induced, leading to NET cell death. Although the therapy proves to be effective, mortality rates remain high. To appropriately select more optimal treatment strategies, it is essential to first better understand the radiobiological responses of tumor cells to PRRT. Methods: We analyzed PRRT induced radiobiological responses in SST2 expressing cells and xenografted mice using SPECT/MRI scanning and histological and molecular analyses. We measured [177Lu]Lu-DOTA-TATE uptake and performed analyses to visualize induction of DNA damage, cell death and other cellular characteristics. Results: The highest accumulation of radioactivity was measured in the tumor and kidneys. PRRT induced DNA damage signaling and repair in a time-dependent manner. We observed intra-tumor heterogeneity of DNA damage and apoptosis, which was not attributed to proliferation or bioavailability. We found a strong correlation between high DNA damage levels and high SST2 expression. PRRT elicited a different therapeutic response between models with different SST2 expression levels. Heterogeneous SST2 expression levels were also confirmed in patient NETs. Conclusion: Heterogeneous SST2 expression levels within NETs cause differentially induced DNA damage levels, influence recurrent tumor phenotypes and impact the therapeutic response in different models and potentially in patients. Our results contribute to a better understanding of PRRT effects, which might impact future therapeutic outcome of NET patients.


Asunto(s)
Complejos de Coordinación/uso terapéutico , Octreótido/análogos & derivados , Neoplasias Pancreáticas/radioterapia , Radiofármacos/uso terapéutico , Receptores de Somatostatina/metabolismo , Animales , Apoptosis , Proliferación Celular , Complejos de Coordinación/farmacocinética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Octreótido/farmacocinética , Octreótido/uso terapéutico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Radiofármacos/farmacocinética , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Int J Mol Sci ; 21(18)2020 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-32937838

RESUMEN

The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5' extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.


Asunto(s)
Proteínas de Unión al ADN/genética , ADN/genética , Autoantígeno Ku/genética , Proteínas Nucleares/genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Proteína Quinasa Activada por ADN/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones , Fosforilación/genética
15.
JCO Precis Oncol ; 3: 1-12, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-35100677

RESUMEN

PURPOSE: Biomarkers that predict response to poly (ADP-ribose) polymerase inhibitors (PARPis) are required to detect PARPi sensitivity beyond germline BRCA-mutated (gBRCAm) cancers and PARPi resistance among reverted gBRCAm cancers. Therefore, we previously developed the Repair Capacity (RECAP) test, a functional homologous recombination (HR) assay that exploits the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer tissue. The aim of the current study was to validate the feasibility of this test on histologic biopsy specimens from metastatic breast cancer and to explore the utility of the RECAP test as a predictive tool for treatment with DNA-damaging agents, such as PARPis. METHODS: Fresh tissue biopsies from easily accessible metastatic lesions from patients with locally advanced or metastatic breast cancer were irradiated with 5 Gy and cultured for 2 hours followed by detection of RAD51 foci presence (HR proficient) or absence (HR deficient [HRD]). HRD biopsy specimens as well as platinum/PARP-resistant specimens were subjected to BRCA1/2 sequencing. RESULTS: RECAP had a success rate of 93% on biopsy specimens from metastatic breast cancer lesions (n = 44). Although HRD was detected in 13 (32%) of 41 specimens, only five showed a gBRCAm. In three patients with gBRCAm, post-treatment RECAP tests showed HR phenotype reversion after in vivo progressive disease on platinum and PARPi treatment, which was explained in one patient by a secondary BRCA1 mutation. CONCLUSION: The RECAP test, which reflects real-time HR status regardless of BRCA mutations, is feasible in metastatic breast cancer biopsy specimens. Compared with gBRCA analysis, it may identify twice as many candidates for PARPi treatment.

16.
Clin Cancer Res ; 24(24): 6277-6287, 2018 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-30139880

RESUMEN

PURPOSE: Tumors of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double-strand break (DSB) repair and high sensitivity to PARP inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. In this study, we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancer tissue: the recombination REpair CAPacity (RECAP) test. EXPERIMENTAL DESIGN: Fresh samples of 170 primary breast cancer were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring BRCA deficiencies, mutational signatures, tumor-infiltrating lymphocytes (TIL), and microsatellite instability (MSI). RESULTS: RECAP was completed successfully in 148 of 170 samples (87%). Twenty-four tumors showed HRD (16%), whereas six tumors were HR intermediate (HRi; 4%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas seven HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts (P = 0.023) compared with HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in breast cancer (1%; P = 0.017). CONCLUSIONS: RECAP is a robust functional HR assay detecting both BRCA1/2-deficient and BRCA1/2-proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.

17.
Blood ; 128(5): 650-9, 2016 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-27281794

RESUMEN

Repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining pathway (NHEJ) is important not only for repair of spontaneous breaks but also for breaks induced in developing lymphocytes during V(D)J (variable [V], diversity [D], and joining [J] genes) recombination of their antigen receptor loci to create a diverse repertoire. Mutations in the NHEJ factor XLF result in extreme sensitivity for ionizing radiation, microcephaly, and growth retardation comparable to mutations in LIG4 and XRCC4, which together form the NHEJ ligation complex. However, the effect on the immune system is variable (mild to severe immunodeficiency) and less prominent than that seen in deficiencies of NHEJ factors ARTEMIS and DNA-dependent protein kinase catalytic subunit, with defects in the hairpin opening step, which is crucial and unique for V(D)J recombination. Therefore, we aimed to study the role of XLF during V(D)J recombination. We obtained clinical data from 9 XLF-deficient patients and performed immune phenotyping and antigen receptor repertoire analysis of immunoglobulin (Ig) and T-cell receptor (TR) rearrangements, using next-generation sequencing in 6 patients. The results were compared with XRCC4 and LIG4 deficiency. Both Ig and TR rearrangements showed a significant decrease in the number of nontemplated (N) nucleotides inserted by terminal deoxynucleotidyl transferase, which resulted in a decrease of 2 to 3 amino acids in the CDR3. Such a reduction in the number of N-nucleotides has a great effect on the junctional diversity, and thereby on the total diversity of the Ig and TR repertoire. This shows that XLF has an important role during V(D)J recombination in creating diversity of the repertoire by stimulating N-nucleotide insertion.


Asunto(s)
Enzimas Reparadoras del ADN/deficiencia , Proteínas de Unión al ADN/deficiencia , Nucleótidos/metabolismo , Recombinación V(D)J/genética , Animales , Antígenos/metabolismo , Regiones Determinantes de Complementariedad/genética , ADN Nucleotidilexotransferasa/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Reordenamiento Génico/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoglobulinas/genética , Ratones , Radiación Ionizante , Receptores de Antígenos de Linfocitos T/genética
18.
BMC Cancer ; 16: 78, 2016 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-26860465

RESUMEN

BACKGROUND: The high incidence of breast cancer has sparked the development of novel targeted and personalized therapies. Personalization of cancer treatment requires reliable prediction of chemotherapy responses in individual patients. Effective selection can prevent unnecessary treatment that would mainly result in the unwanted side effects of the therapy. This selection can be facilitated by characterization of individual tumors using robust and specific functional assays, which requires development of powerful ex vivo culture systems and procedures to analyze the response to treatment. METHODS: We optimized culture methods for primary breast tumor samples that allowed propagation of tissue ex vivo. We combined several tissue culture strategies, including defined tissue slicing technology, growth medium optimization and use of a rotating platform to increase nutrient exchange. RESULTS: We could maintain tissue cultures for at least 7 days without losing tissue morphology, viability or cell proliferation. We also developed methods to determine the cytotoxic response of individual tumors to the chemotherapeutic treatment FAC (5-FU, Adriamycin [Doxorubicin] and Cyclophosphamide). Using this tool we designated tumors as sensitive or resistant and distinguished a clinically proven resistant tumor from other tumors. CONCLUSION: This method defines conditions that allow ex vivo testing of individual tumor responses to anti-cancer drugs and therefore might improve personalization of breast cancer treatment.


Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales , Técnicas de Cultivo de Tejidos/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/patología , Ciclofosfamida/administración & dosificación , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos/genética , Femenino , Fluorouracilo/administración & dosificación , Humanos , Medicina de Precisión , Células Tumorales Cultivadas/efectos de los fármacos
19.
PLoS One ; 10(4): e0126029, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25927440

RESUMEN

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors.


Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Vejiga Urinaria/genética , Vejiga Urinaria/patología , Xerodermia Pigmentosa/genética , ADN/genética , Proteínas de Unión al ADN/análisis , Humanos , Células Tumorales Cultivadas , Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Xerodermia Pigmentosa/patología
20.
J Clin Endocrinol Metab ; 100(5): E789-98, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25742519

RESUMEN

CONTEXT: Severe short stature can be caused by defects in numerous biological processes including defects in IGF-1 signaling, centromere function, cell cycle control, and DNA damage repair. Many syndromic causes of short stature are associated with medical comorbidities including hypogonadism and microcephaly. OBJECTIVE: To identify an underlying genetic etiology in two siblings with severe short stature and gonadal failure. DESIGN: Clinical phenotyping, genetic analysis, complemented by in vitro functional studies of the candidate gene. SETTING: An academic pediatric endocrinology clinic. PATIENTS OR OTHER PARTICIPANTS: Two adult siblings (male patient [P1] and female patient 2 [P2]) presented with a history of severe postnatal growth failure (adult heights: P1, -6.8 SD score; P2, -4 SD score), microcephaly, primary gonadal failure, and early-onset metabolic syndrome in late adolescence. In addition, P2 developed a malignant gastrointestinal stromal tumor at age 28. INTERVENTION(S): Single nucleotide polymorphism microarray and exome sequencing. RESULTS: Combined microarray analysis and whole exome sequencing of the two affected siblings and one unaffected sister identified a homozygous variant in XRCC4 as the probable candidate variant. Sanger sequencing and mRNA studies revealed a splice variant resulting in an in-frame deletion of 23 amino acids. Primary fibroblasts (P1) showed a DNA damage repair defect. CONCLUSIONS: In this study we have identified a novel pathogenic variant in XRCC4, a gene that plays a critical role in non-homologous end-joining DNA repair. This finding expands the spectrum of DNA damage repair syndromes to include XRCC4 deficiency causing severe postnatal growth failure, microcephaly, gonadal failure, metabolic syndrome, and possibly tumor predisposition.


Asunto(s)
Estatura/genética , Proteínas de Unión al ADN/genética , Hipogonadismo/genética , Síndrome Metabólico/genética , Polimorfismo de Nucleótido Simple , Adulto , Exoma , Femenino , Humanos , Masculino , Mutación , Hermanos
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