Initial Experience With Single-Isocenter Radiosurgery to Target Multiple Brain Metastases Using an Automated Treatment Planning Software: Clinical Outcomes and Optimal Target Volume Margins Strategy

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Stereotactic MR-guided adaptive radiotherapy (SMART) for primary rectal cancer: evaluation of early toxicity and pathological response

Background: The purpose of this study is to measure the effects of stereotactic MR-guided adaptive radiotherapy (SMART) for rectal cancer patients in terms of early toxicity and pathological response. Materials and methods: For this prospective pilot study, patients diagnosed with locally advanced rectal cancer (LARC) with positive lymph node clinical staging underwent SMART on rectal lesion and mesorectum using hybrid MR-Linac (MRIdian ViewRay). Dose prescription at 80% isodose for the rectal lesion and mesorectum was 40 Gy (8 Gy/fr) and 25 Gy (5 Gy/fr), respectively, delivered on 5 days (3 fr/week). Response assessment by MRI was performed 3 weeks after SMART, then patients fit for surgery underwent total mesorectal excision. Primary endpoint was evaluation of adverse effect of radiotherapy. Secondary endpoint was pathological complete response rate. Early toxicity was graded according to the Common Terminology Criteria for Adverse Events (CTCAE v5.0). Results: From October 2020 to January 2022, twenty patients underwent rectal SMART. No grade 3–5 toxicity was recorded. Twelve patients were eligible for total mesorectal excision (TME). Mean interval between the completion of SMART and surgery was 4 weeks. Pathological downstaging occurred in all patients; rate of pathological complete response (pCR) was 17%. pCR occurred with a prolonged time to surgery (> 7 weeks). Conclusion: To our knowledge, this is the first study to use stereotactic radiotherapy for primary rectal cancer. SMART for rectal cancer is well tolerated and effective in terms of tumor regression, especially if followed by delayed surgery.

p53 Stabilization induces cell growth inhibition and affects IGF2 pathway in response to radiotherapy in adrenocortical cancer cells.

Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.(wt)p53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases

Induction of apoptosis by ionizing radiation following p53 stabilization.

<p>H295R, SW-13 and SK-OV-3 cell lines were transfected with empty vector (mock) or p53-vector (WT) and irradiated at a dose of 6 Gy. (A) TUNEL assay was performed at 48 and 72 after transfection. In all cell lines, TUNEL-positive cells are detectable only in irradiated samples expressing the wild type form of <i>TP53</i>. Cells were also stained with Hoechst 33342 and then visualized with a fluorescence microscope at a magnification of 40×. Figure shown is representative of three experiments performed in duplicate. (B) Percentage of apoptosis in H295R, SW-13 and SK-OV-3 was calculated by comparing TUNEL-positive cells with total cells, stained with Hoechst 33342. Apoptosis increases in cells transfected with p53 vector compared with mock-irradiated samples, starting from 48 h since transfection and the effect strengthens after 72 h. (*, p<0.05; #, p<0.01). (C) <i>BCL2</i> expression was evaluated in H295R cell line by RT-PCR. p53 restoration induces a significant reduction in <i>BCL2</i> expression at 48 and 72 h after irradiation. (D) Inverted images relative to semi-quantitative RT-PCR analysis of <i>BCL2</i> gene expression performed on SW-13 cells, showing a significant reduction in <i>BCL2</i> signal in cells transfected with p53-vector following ionizing radiations treatment. (E) The effect of p53 restoration on <i>BCL2</i> mRNA levels in response to irradiation was evaluated in SK-OV-3 cell lines. p53 inhibits <i>BCL2</i> expression and the effect is stronger at 72 h after transfection. Images are representative of at least three independent experiments. <i>GAPDH</i> was used for normalization.</p

RT-PCR analysis of ^wtp53 effect on <i>IGF2</i> expression.

<p>(A) Inverted images relative to semi-quantitative RT-PCR analysis of <i>IGF2</i> gene expression performed on H295R cells, showing a massive loss of <i>IGF2</i> signal in irradiated cells at 72 h after transfection of p53-vector (WT). (B) RT-PCR analysis of <i>IGF2</i> mRNA levels performed on SW-13 cells. A significant reduction in <i>IGF2</i> expression is detectable in cells transfected with p53-vector (WT) following ionizing radiations treatment. Images are representative of three independent experiments. <i>GAPDH</i> expression was used for normalization and bands' intensities were quantified using ImageJ. Band densitometry is shown in the right panels. (*, p<0.05; #, p<0.01).</p

Effect of ionizing radiations on p53 stabilization.

<p>Expression levels of <i>TP53</i> and p53 protein evaluated by Real Time RT-PCR and Western blotting in H295R, SW-13 and SK-OV-3 cells 72 h after transfection with empty vector (mock) or p53-vector (WT). Samples were treated with IR at a dose of 6 Gy where indicated. (A) RT-PCR (top) and Real Time RT-PCR (bottom) analysis of <i>TP53</i> expression in H295R cells. Samples transfected with pBABE-neo-p53 vector show a 5-fold increase in <i>TP53</i> mRNA compared with mock. (B) Western blotting analysis of p53 in H295R cells (top) revealed a major band of ∼53 kDa (^wtp53) and a minor band of ∼44 kDa, corresponding to the truncated form of p53 (p53^Δ8–9). Densitometry shows that ^wtp53 is stabilized by IR treatment (bottom). (C) RT-PCR analysis of <i>TP53</i> mRNA in SW-13 cells shows over-expression of the gene in samples transfected with pBABE-neo-p53 vector (top). Real Time RT-PCR analysis revealed a 3-fold increase in <i>TP53</i> transcript in samples transfected with p53 vector. The expression of <i>TP53</i> is not modulated by irradiation (bottom). (D) Levels of p53 protein in SW-13 cells significantly increase after IR treatment in cells transfected with pBABE-neo-p53 vector, while no change is detectable in samples transfected with empty vector. (E) In SK-OV-3 cells, no <i>TP53</i> transcript was detectable by RT-PCR in samples transfected with empty vector, while in cells transfected with pBABE-neo-p53 vector a strong signal, corresponding to <i>TP53</i> mRNA, is observable (top). Real Time RT-PCR analysis shows no modulation of <i>TP53</i> expression after irradiation (bottom). (F) Western blotting analysis of p53 in SK-OV-3 cells revealed a detectable band only in samples transfected with pBABE-neo-p53 vector (top). Densitometry shows that p53 levels increase after irradiation, indicating the stabilization of the protein (bottom). Results are representative of at least three independent experiments. <i>GAPDH</i> and vinculin were used for normalization. (*, p<0.05; #, p<0.01).</p

Sequencing analysis of <i>TP53</i> coding sequence in H295R and SW-13 cell lines.

<p>(A) Representation of <i>TP53</i> mutations identified in H295R and SW-13 cell lines, showing a large deletion affecting 211 base pairs in H295R cells (top) and a homozygous point mutation in exon 6 (<i>r.577c>u</i>) in SW-13 cell line (bottom). SW-13 cells also exhibit a polymorphic site located in exon 4 (<i>r.215c>g</i>). (B) Inverted image relative to RT-PCR analysis of exons 7 to 10 of <i>TP53</i> gene. Electrophoresis on a 2% agarose gel revealed a major band of ∼575 bp, corresponding to SW-13 transcript, and a minor band of ∼364 bp, corresponding to H295R transcript (DNA ladder, 1 Kb plus). (C) Predicted amino-acid sequence of p53 in H295R and SW-13 cell lines. Deletion of exons 8 and 9 in H295R determines a frameshift starting at codon 261, creating a subsequent stop codon at position 274 (top). In SW-13 cells, a homozygous point mutation at codon 193 determines an amino-acid substitution (histidine to tyrosine) in the DBD of the protein (bottom). (D) Western blotting analysis of p53 in H295R and SW-13 cells, showing the presence of a shorter protein in H295R cell line, which molecular weight is ∼44 kDa.</p

Effect of p53 stabilization on Akt activation after ionizing radiation treatment.

<p>(A) Western blotting analysis of total Akt and phosphorylated Akt levels, performed on irradiated H295R cells transfected with empty vector (mock) and p53-vector (WT) at 48 and 72 h after transfection. Densitometric analysis of Akt normalized to vinculin and p-Akt <i>versus</i> Akt shows that Akt expression is not influenced by p53 status, while the activated form of the protein is significantly reduced in ^wtp53-expressing cells. (B) Total cell lysates obtained from SW-13 cell line were subjected to Western blotting analysis using antibodies targeting Akt and Akt-pSer473. Phosphorylation levels of Akt are significantly reduced by p53 stabilization in response to ionizing radiation treatment. Results shown are representative of at least three experiments. Bands' intensities were quantified using ImageJ and bands' densitometry is shown in the right panels. (*, p<0.05; #, p<0.01).</p

Effect of p53 over-expression and irradiation on cell proliferation in H295R, SW-13 and SK-OV-3 cells.

<p>Cell growth was evaluated in H295R, SW-13 and SK-OV-3 cell lines transfected with empty vector (mock) or p53-vector (WT), either irradiated or not. (A) In H295R cell lines (left), ionizing radiation (IR) treatment induces a significant inhibition of cell growth in cells expressing wild type p53, starting from 48 h after transfection (−18% compared to mock irradiated cells; p<0.05) and reaching −25% after 72 h (p<0.01). Irradiation does not induce a stable effect in SW-13 cells (right) transfected with empty vector, whereas cells expressing wild type p53 are significantly inhibited at 48 h after transfection (−28%; p<0.05) until the end of the experiment (−31%; p<0.01). In SK-OV-3 cells (bottom), p53 restoration induces an strong effect on cell growth, which is evident since 48 from transfection (−20% of growth inhibition in WT+IR sample compared with mock +IR; p<0.01) and endures up to 72 h (−28%; p<0.01). Data are mean ± S.D. of at least three independent experiments carried out by duplicates. (B) Western blotting analysis of cyclin E and cyclin D1 expression in H295R cells at 72 h after transfection revealed a down-regulation of these proteins in cells expressing ^wtp53 treated with IR at a dose of 6 Gy. (C) Analysis of cyclin E and cyclin D1 expression in SW-13 cell line by Western blotting shows a decrease in protein levels in samples transfected with p53-vector after irradiation. Bands' intensities were quantified with ImageJ software, using vinculin for normalization. (*, p<0.05).</p