In localized prostate cancer (PCa), Grade Group (GG) and Gleason Score (GS) have a well-established prognostic role. In metastatic castration resistant prostate cancer (mCRPC), the prognostic role of ...GS and GG is less defined. In first-line treatment of mCRPC, androgen receptor (AR)-directed drugs (abiraterone acetate, enzalutamide) and docetaxel represent the referring options. There is no evidence that the GS/GG systems can add information to guide the choice between AR-directed drugs and docetaxel in the first-line setting of mCRPC. Nowadays there are no validated biomarkers, which define patients who may benefit or not from hormonal treatments or chemotherapy. Androgen receptor (AR) copy number variations (CNV) are predictive factors of poor response to abiraterone and enzalutamide. There are no available data about the association between AR CNV and GG. In this retrospective study, we analysed the association of the highest GG score with AR CNV and their impact on the clinical outcome of AR-directed drugs and docetaxel as first-line therapy for mCRPC patients. Patients benefit from docetaxel, abiraterone or enzalutamide regardless the GG. However, the presence of GG5 and AR CNV gain identifies a subgroup of patients with poor prognosis, which could benefit from front-line docetaxel instead of AR-directed drugs.
Animal experiments are necessary for a better understanding of diseases and for developing new therapeutic strategies. The mouse (Mus musculus) is currently the most popular laboratory animal in ...biomedical research. Experimental procedures on animals often require anesthesia and/or analgesia to obtain adequate immobilization and to reduce stress or pain. Mice anesthesia is challenging for several reasons including the animals' size, metabolic rate, and the high risk of hypothermia and hypoglycemia. Moreover, anesthetic agents influence physiological parameters, further interfering with experimental results. Small animal imaging procedures are increasingly used in biomedical research both because the animals allow in vivo monitoring and because they are readily available for longitudinal and noninvasive studies as well as investigations into the evolution of diseases and the effects of new therapies. Anesthesia must adapt to the imaging technique, the procedure length, and the aim of the study. The purpose of this article is to review the existing literature on anesthetic protocols adopted in mice for molecular imaging studies and to report our experience.
Purpose
To evaluate the feasibility and sensitivity of multimodality PET/CT and MRI imaging for non-invasive characterization of brain microglial/macrophage activation occurring during the acute ...phase in a mouse model of relapsing remitting multiple sclerosis (RR-MS) using
18
FDPA-714, a selective radioligand for the 18-kDa translocator protein (TSPO), superparamagnetic iron oxide particles (SPIO), and ex vivo immunohistochemistry.
Methods
Experimental autoimmune encephalomyelitis (EAE) was induced in female SJL/J mice by immunization with PLP
139–151
. Seven symptomatic EAE mice and five controls underwent both PET/CT and MRI studies between 11 and 14 days post-immunization. SPIO was injected i.v. in the same animals immediately after
18
FDPA-714 and MRI acquisition was performed after 24 h. Regional brain volumes were defined according to a mouse brain atlas on co-registered PET and SPIO-MRI images.
18
FDPA-714 standardized uptake value (SUV) ratios (SUVR), with unaffected neocortex as reference, and SPIO fractional volumes (SPIO-Vol) were generated. Both SUVR and SPIO-Vol values were correlated with the clinical score (CS) and among them. Five EAE and four control mice underwent immunohistochemical analysis with the aim of identifying activated microglia/macrophage and TSPO expressions.
Results
SUVR and SPIO-Vol values were significantly increased in EAE compared with controls in the hippocampus (
p
< 0.01;
p
< 0.02, respectively), thalamus (
p
< 0.02;
p
< 0.05, respectively), and cerebellum and brainstem (
p
< 0.02), while only SPIO-Vol was significantly increased in the caudate/putamen (
p
< 0.05). Both SUVR and SPIO-Vol values were positively significantly correlated with CS and among them in the same regions. TSPO/Iba1 and F4/80/Prussian blue staining immunohistochemistry suggests that increased activated microglia/macrophages underlay TSPO expression and SPIO uptake in symptomatic EAE mice.
Conclusions
These preliminary results suggest that both activated microglia and infiltrated macrophages are present in vulnerable brain regions during the acute phase of PLP-EAE and contribute to disease severity. Both
18
FDPA-714-PET and SPIO-MRI appear suitable modalities for preclinical study of neuroinflammation in MS mice models.
To evaluate the feasibility and sensitivity of (18)F-DPA-714 for the study of microglial activation in the brain and spinal cord of transgenic SOD1(G93A) mice using high-resolution PET/CT and to ...evaluate the Iba1 and TSPO expression with immunohistochemistry.
Nine symptomatic SOD1(G93A) mice (aged 117 ± 12.7 days, clinical score range 1 - 4) and five WT SOD1 control mice (aged 108 ± 28.5 days) underwent (18)F-DPA-714 PET/CT. SUV ratios were calculated by normalizing the cerebellar (rCRB), brainstem (rBS), motor cortex (rMCX) and cervical spinal cord (rCSC) activities to that of the frontal association cortex. Two WT SOD1 and six symptomatic SOD1(G93A) mice were studied by immunohistochemistry.
In the symptomatic SOD1(G93A) mice, rCRB, rBS and rCSC were increased as compared to the values in WT SOD1 mice, with a statistically significantly difference in rBS (2.340 ± 0.784 vs 1.576 ± 0.287, p = 0.014). Immunofluorescence studies showed that TSPO expression was increased in the trigeminal, facial, ambiguus and hypoglossal nuclei, as well as in the spinal cord, of symptomatic SOD1(G93A) mice and was colocalized with increased Iba1 staining.
Increased (18)F-DPA-714 uptake can be detected with high-resolution PET/CT in the brainstem of transgenic SOD1(G93A) mice, a region known to be a site of degeneration and increased microglial activation in amyotrophic lateral sclerosis, in agreement with increased TSPO expression in the brainstem nuclei shown by immunostaining. Therefore, (18)F-DPA-714 PET/CT might be a suitable tool to evaluate microglial activation in the SOD1(G93A) mouse model.
•Two new ene-reductases cloned and characterized.•Reduction shown for a variety of enones, dimethylesters, aldehyde and maleimdes.•Asymmetric reduction of the isomers of carvone, ketoisophorone and ...2-methylcyclohexenone.•NAD(P)H-free light-driven biocatalysis.
The Old Yellow Enzyme (OYE) homologues or ene-reductases (ER) from Deinococcus radiodurans (DrER) and Ralstonia metallidurans (RmER) were cloned and characterized. Sequence and phylogenetic analysis revealed both these enzymes to belong to the YqjM-like or “thermophilic-like” group of OYEs, both sharing more than 60% sequence similarity to the ER from Thermus scotoductus. This group of OYEs is characterized by a conserved cysteine residue modulating the redox potential of the flavin cofactor as well as a conserved tyrosine residue located at the N-terminus region involved in binding certain ligands. The genes were recombinantly expressed in Escherichia coli as functional soluble proteins. Both ERs have monomer molecular weights of approximately 40kDa, with DrER a homodimer in solution and RmER a monomer. DrER and RmER are optimally active at pH 7–7.5 at 30°C and 35°C respectively. Although the enzymes showed comparable affinities towards the ubiquitous ER substrate 2-cyclohexenone, the specific activity and catalytic efficiency of DrER were more than twice those observed for RmER. Similar to other members of this subclass of ERs, no conversion was detected with cyclic Cβ substituted enones, and only DrER was able to convert citral. Both DrER and RmER were highly active at reducing N-phenyl substituted maleimides. The selectivity of the ERs was assessed using both the isomers of carvone, which were converted with high diastereomeric excesses. Ketoisophorone and 2-methylcyclopentenone were converted to their (R)- and (S)-enantiomeric products respectively. Finally, a light-driven cofactor regeneration system was used to drive enzymatic reduction in the absence of NAD(P)H.
Molecular characterization of an Italian series of sporadic GISTs Origone, P.; Gargiulo, S.; Mastracci, L. ...
Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association,
10/2013, Letnik:
16, Številka:
4
Journal Article
Recenzirano
Purpose
Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract. Most (80 %) contain activating mutations in the KIT receptor tyrosine kinase, roughly 10 % in ...platelet-derived growth factor receptor-alpha (PDGFRA). In a small subset, BRAF mutations are an alternative molecular pathway. GISTs respond well to imatinib, but low response is seen in patients with wild-type KIT or PDGFRA. Resistance has also been reported as a result of mutations in downstream effectors such as BRAF.
Methods
We provide here a molecular characterization of a series of primary GISTs from Italian patients. Of 121 GIST cases diagnosed between 2000 and 2012, 83 were evaluated by PCR amplification and direct sequencing for mutations in KIT exons 8, 9, 11, 13, and 17, PDGFRA exons 12, 14, and 18, and BRAF exon 15. Eighty-one GISTs also underwent K-RAS testing.
Results
Sixty-four GISTs were positive: 55 had mutations in KIT and 9 in PDGFRA; 16 patients were mutation negative. Three samples came from NF1 patients and were KIT- and PDGFRA negative. Overall, we identified six novel mutations in KIT (p.K550_M552delinsL, p.Q556_W557delinsG p.Q556_G575del, p.W557_V559delinsQ p.P573_R588dup, p.G592_K593dup) and one novel mutation in PDGFRA (p.D842_N848delinsVDV), thus contributing to widening the spectrum of known mutations in GIST tumors and confirming the most frequently altered regions underlying GIST development.
Conclusions
Among the 64 KIT- and PDGFRA-positive sporadic patients in our series, no BRAF or KRAS mutations were identified, suggesting that co-occurrence of these mutations is likely to be rare in the northwestern Italian population and not a frequent cause of primary resistance to imatinib in KIT-positive GIST patients.
Summary
We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital‐based study of single (SPM, n = 398) and multiple primary melanoma ...(MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four‐fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43–7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non‐coding variants with unknown functional significance were also found (5′UTR −25C > T, −21C > T, −67G > C, IVS1 +37G > C); the novel 5′UTR −21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2‐fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.
Pancreatic adenocarcinoma (PC) is the third most common cancer associated with
BRCA
mutations. Most notice has been given to
BRCA2
, while the association between
BRCA1
and PC is less widely ...reported. Recently,
PALB2
has been implicated in both PC and breast cancer (BC) susceptibility. We selected 29 Italian PC patients from a case–control study of PC according to their personal and family history of both PC and breast/ovarian cancer (BC/OC) and tested them for presence of germline mutations in
BRCA1
,
BRCA2
and
PALB2.
We identified no germline mutations or deletions in
PALB2
, but detected 7 BRCA mutations (4 in BRCA1 and 3 in BRCA2). These findings suggest that
PALB2
does not play a major role in PC susceptibility in our population. As we found an almost equal frequency of germline mutations in
BRCA1
and
BRCA2,
germline alterations in either of these genes may explain a subset of Italian families presenting both PC and BC/OC. Moreover, as we began the observation of these families from probands who are affected by PC, we provide here a direct assessment of the role of
PALB2
and
BRCA
mutations in PC susceptibility.
Lynch syndrome is an inherited cancer syndrome caused by germline mutations in mismatch repair (MMR) genes
MLH1
,
MSH2
,
MSH6
and
PMS2.
LS predisposes to high risk of early-onset colorectal, ...endometrial and other tumors. Patients with Lynch syndrome have also been shown to have an elevated risk for pancreatic cancer (PC). In this study, we aimed to estimate the frequency of suspected Lynch syndrome among a series of 135 PC patients. Further, we wanted to determine the frequency of MMR gene mutations in the suspected Lynch syndrome cases. We also aimed to verify the pathogenicity of any novel non-truncating variants we might detect with a functional assay. Based on personal and/or familial cancer history, 19 patients were classified as suspected Lynch syndrome cases. DNA material for mutation analysis was available for eleven of them. Four patients were found to carry a total of five
MLH1
or
MSH2
variants. Of these,
MSH2
-Q402X,
MSH2
-G322D, and
MLH1
-K618A had been previously reported, while the
MSH2
-E205Q and
MSH2
-V367I variants were novel.
MSH2
-Q402X is a known stop mutation and reported here for the first time here in association with PC.
MLH1
-K618A was found in the unaffected branch of a kindred, suggesting that it may be a polymorphism or a low penetrance variant.
MSH2
-G322D likely does not cause a MMR defect, although this variant has also been associated with breast cancer as indeed seen in our patient. The novel variants
MSH2
-E205Q and
MSH2
-V367I were found in the same patient. Both novel variants were however functional in the applied MMR assay. Our findings suggest that only a small subset of pancreatic cancer patients carry pathogenic MMR mutations.
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