Impact of COVID-19 pandemic on a daily-based outpatient treatment routine: experience of a radiotherapy department of a tertiary public/university hospital in Brazil.

OBJECTIVES: To report the impact of the COVID-19 pandemic on patient attendance at a radiotherapy department two months after the implementation of specific policies regarding the pandemic. METHODS: The proposed treatment schemes, favoring hypofractionated schedules, and COVID-19 management strategies regarding irradiation are presented. Attendance after two months of implementation of these policies was measured and compared with that during the same period in 2019. RESULTS: A 10% reduction in the number of treated patients and a 26% reduction in the number of sessions was observed. The main impact was a decrease in the treatment of benign diseases and gastrointestinal tumors, with a general increase in breast cancer treatments. Eighteen (1.7%) patients were confirmed as having COVID-19 during radiotherapy in April and May 2020, three of whom were hospitalized, and one patient died because of COVID-19. Among the 18 patients, 12 had their treatments interrupted for at least 15 days from symptom appearance. CONCLUSION: There was a decrease in the number of treated patients in our radiotherapy department, with a greater decrease in the total number of sessions. This indicated, overall, a smaller number of fractions/patients treated, despite our efforts to maintain the treatment routine. We had several patients who were infected with COVID-19 and one related death during treatment in the first few months of the pandemic in São Paulo Brazil.

Impact of COVID-19 pandemic on a daily-based outpatient treatment routine: experience of a radiotherapy department of a tertiary public/university hospital in Brazil

OBJECTIVES: To report the impact of the COVID-19 pandemic on patient attendance at a radiotherapy department two months after the implementation of specific policies regarding the pandemic. METHODS: The proposed treatment schemes, favoring hypofractionated schedules, and COVID-19 management strategies regarding irradiation are presented. Attendance after two months of implementation of these policies was measured and compared with that during the same period in 2019. RESULTS: A 10% reduction in the number of treated patients and a 26% reduction in the number of sessions was observed. The main impact was a decrease in the treatment of benign diseases and gastrointestinal tumors, with a general increase in breast cancer treatments. Eighteen (1.7%) patients were confirmed as having COVID-19 during radiotherapy in April and May 2020, three of whom were hospitalized, and one patient died because of COVID-19. Among the 18 patients, 12 had their treatments interrupted for at least 15 days from symptom appearance. CONCLUSION: There was a decrease in the number of treated patients in our radiotherapy department, with a greater decrease in the total number of sessions. This indicated, overall, a smaller number of fractions/patients treated, despite our efforts to maintain the treatment routine. We had several patients who were infected with COVID-19 and one related death during treatment in the first few months of the pandemic in São Paulo Brazil.

DNMT3A mutations mediate the epigenetic reactivation of the leukemogenic factor MEIS1 in acute myeloid leukemia.

This corrects the article DOI: 10.1038/onc.2015.359.

Tumor MGMT promoter hypermethylation changes over time limit temozolomide efficacy in a phase II trial for metastatic colorectal cancer.

BACKGROUND: Objective response to dacarbazine, the intravenous form of temozolomide (TMZ), in metastatic colorectal cancer (mCRC) is confined to tumors harboring O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter hypermethylation. We conducted a phase II study of TMZ enriched by MGMT hypermethylation in archival tumor (AT), exploring dynamic of this biomarker in baseline tumor (BT) biopsy and plasma (liquid biopsy). PATIENTS AND METHODS: We screened 150 mCRC patients for MGMT hypermethylation with methylation-specific PCR on AT from FFPE specimens. Eligible patients (n = 29) underwent BT biopsy and then received TMZ 200 mg/m(2) days 1-5 q28 until progression. A Fleming single-stage design was used to determine whether progression-free survival (PFS) rate at 12 weeks would be ≥35% [H0 ≤ 15%, type I error = 0.059 (one-sided), power = 0.849]. Exploratory analyses included comparison between MGMT hypermethylation in AT and BT, and MGMT methylation testing by MethylBEAMing in solid (AT, BT) and LB with regard to tumor response. RESULTS: The PFS rate at 12 weeks was 10.3% [90% confidence interval (CI) 2.9-24.6]. Objective response rate was 3.4% (90% CI 0.2-15.3), disease control rate 48.3% (90% CI 32.0-64.8), median OS 6.2 months (95% CI 3.8-7.6), and median PFS 2.6 months (95% CI 1.4-2.7). We observed the absence of MGMT hypermethylation in BT in 62.7% of tumors. CONCLUSION: Treatment of mCRC with TMZ driven by MGMT promoter hypermethylation in AT samples did not provide meaningful PFS rate at 12 weeks. This biomarker changed from AT to BT, indicating that testing BT biopsy or plasma is needed for refined target selection.

DNMT3A mutations mediate the epigenetic reactivation of the leukemogenic factor MEIS1 in acute myeloid leukemia.

Close to half of de novo acute myeloid leukemia (AML) cases do not exhibit any cytogenetic aberrations. In this regard, distortion of the DNA methylation setting and the presence of mutations in epigenetic modifier genes can also be molecular drivers of the disease. In recent years, somatic missense mutations of the DNA methyltransferase 3A (DNMT3A) have been reported in ~20% of AML patients; however, no obvious critical downstream gene has been identified that could explain the role of DNMT3A in the natural history of AML. Herein, using whole-genome bisulfite sequencing and DNA methylation microarrays, we have identified a key gene undergoing promoter hypomethylation-associated transcriptional reactivation in DNMT3 mutant patients, the leukemogenic HOX cofactor MEIS1. Our results indicate that, in the absence of mixed lineage leukemia fusions, an alternative pathway for engaging an oncogenic MEIS1-dependent transcriptional program can be mediated by DNMT3A mutations.

Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

The PDGFRß-AKT pathway contributes to CDDP-acquired resistance in testicular germ cell tumors.

PURPOSE: We examined whether PI3K-AKT or extracellular signal-regulated kinase (ERK) signaling pathways could play a role in the development of cisplatin (CDDP) resistance in testicular germ cell tumor (TGT) cells. EXPERIMENTAL DESIGN: We compared AKT and ERK activation levels in CDDP-sensitive testicular tumor cells and in their corresponding CDDP-resistant-derived cells. We also analyzed these pathways in orthotopic testicular tumors and human patient samples. RESULTS: Our results indicated that there was overactivation of AKT in CDDP-resistant cells compared with sensitive cells, but no effect on activated ERK levels. We observed an increase in mRNA and protein levels for platelet-derived growth factor (PDGF) receptor ß and PDGF-B ligand. These were responsible for AKT overactivation in CDDP-resistant cells. When PDGFRß levels were decreased by short hairpin RNA (shRNA) treatment or its activation was blocked by pazopanib, CDDP-resistant cells behaved like sensitive cells. Moreover, CDDP-resistant cells were more sensitive to incubation with PDGFRß inhibitors such as pazopanib or sunitinib than sensitive cells, a finding consistent with these cells being dependent on this signaling pathway. We also found overexpression of PDGFRß and pAKT in CDDP-resistant choriocarcinoma orthotopic tumor versus their CDDP-sensitive counterparts. Finally, we found high PDGFRß levels in human testicular tumors, and overexpression in CDDP-resistant testicular choriocarcinomas compared with the CDDP-sensitive and nontreated tumors. CONCLUSIONS: The PDGFRß-AKT pathway plays a critical role in the development of CDDP resistance in testicular tumoral cells.

Gene amplification of the histone methyltransferase SETDB1 contributes to human lung tumorigenesis.

Disruption of the histone modification patterns is one of the most common features of human tumors. However, few genetic alterations in the histone modifier genes have been described in tumorigenesis. Herein we show that the histone methyltransferase SETDB1 undergoes gene amplification in non-small and small lung cancer cell lines and primary tumors. The existence of additional copies of the SETDB1 gene in these transformed cells is associated with higher levels of the corresponding mRNA and protein. From a functional standpoint, the depletion of SETDB1 expression in amplified cells reduces cancer growth in cell culture and nude mice models, whereas its overexpression increases the tumor invasiveness. The increased gene dosage of SETDB1 is also associated with enhanced sensitivity to the growth inhibitory effect mediated by the SETDB1-interfering drug mithramycin. Overall, the findings identify SETDB1 as a bona fide oncogene undergoing gene amplification-associated activation in lung cancer and suggest its potential for new therapeutic strategies.

Dynamic epigenetic regulation of the microRNA-200 family mediates epithelial and mesenchymal transitions in human tumorigenesis.

Epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transitions occur in the development of human tumorigenesis and are part of the natural history of the process to adapt to the changing microenvironment. In this setting, the miR-200 family is recognized as a master regulator of the epithelial phenotype by targeting ZEB1 and ZEB2, two important transcriptional repressors of the cell adherence (E-cadherin) and polarity (CRB3 and LGL2) genes. Recently, the putative DNA methylation associated inactivation of various miR-200 members has been described in cancer. Herein, we show that the miR-200ba429 and miR-200c141 transcripts undergo a dynamic epigenetic regulation linked to EMT or MET phenotypes in tumor progression. The 5'-CpG islands of both miR-200 loci were found unmethylated and coupled to the expression of the corresponding miRNAs in human cancer cell lines with epithelial features, such as low levels of ZEB1/ZEB2 and high expression of E-cadherin, CRB3 and LGL2, while CpG island hypermethylation-associated silencing was observed in transformed cells with mesenchymal characteristics. The recovery of miR-200ba429 and miR-200c141 expression by stable transfection in the hypermethylated cells restored the epithelial markers and inhibited migration in cell culture and tumoral growth and metastasis formation in nude mice. We also discovered, using both cell culture and animal models, that the miR-200 epigenetic silencing is not an static and fixed process but it can be shifted to hypermethylated or unmethylated 5'-CpG island status corresponding to the EMT and MET phenotypes, respectively. In fact, careful laser microdissection in human primary colorectal tumorigenesis unveiled that in normal colon mucosa crypts (epithelia) and stroma (mesenchyma) already are unmethylated and methylated at these loci, respectively; and that the colorectal tumors undergo selective miR-200 hypermethylation of their epithelial component. These findings indicate that the epigenetic silencing plasticity of the miR-200 family contributes to the evolving and adapting phenotypes of human tumors.

Fatty acid profile of human milk of Portuguese lactating women: prospective study from the 1st to the 16th week of lactation.

BACKGROUND/AIMS: Fatty acid (FA) composition varies over the course of the day and during lactation. The aim of this study was to evaluate FA composition and its compositional stability in human milk, from day 7 to week 16 of lactation. METHODS: Human milk was collected from all feedings over 24 h at day 7 and weeks 4, 8, 12 and 16 of lactation in 31 lactating women. FAs were analyzed through gas chromatography. Comparisons were made with analysis of variance. RESULTS: Total monounsaturated FAs decreased from 33.04 +/- 2.58% wt/wt at day 7 to 31.48 +/- 3.32% wt/wt at week 16 of lactation, much at the expenses of the decrease in the major monounsaturated FA found in human milk, oleic acid. Main polyunsaturated FAs n-6 and n-3 showed fluctuations from day 7 up to week 16 of lactation, but with no statistical significance. Arachidonic acid significantly decreased from transitional to mature milk. CONCLUSIONS: The FA profile obtained throughout the study time points presented very low levels of oleic acid and very high linoleic acid/alpha-linoleic acid ratios which reflect recent changes in Portuguese women's food patterns. Despite this, the arachidonic acid/docosahexaenoic acid ratio [corrected] remained constant during the study, suggesting a protective metabolic mechanism.

KRAS and BRAF oncogenic mutations in MSS colorectal carcinoma progression.

In sporadic colorectal cancer (CRC), KRAS are alternative to BRAF mutations and occur, respectively, in 30 and 10% of cases. Few reports addressed the association between KRAS-BRAF mutations and tumour progression specifically in sporadic microsatellite-stable (MSS) CRC. We screened KRAS and BRAF in 250 MSS primary CRC and 45 lymph node (LN) metastases and analysed the pathological features of the cases to understand the involvement of KRAS-BRAF activation in progression and metastasis. Forty-five per cent of primary MSS CRCs carried mutations in at least one of these genes and mutations were associated with wall invasion (P=0.02), presence and number of LN metastases (P=0.02 and P=0.03, respectively), distant metastases (P=0.004) and advanced stage (P=0.01). We demonstrated that KRAS and BRAF are alternative events in Tis and T1 MSS CRC and, KRAS rather than BRAF mutations, contributed to the progression of MSS CRC. The frequency of KRAS and/or BRAF mutations was higher in LN metastases than in primary carcinomas (P=0.0002). Mutated LN metastases displayed KRAS associated or not with BRAF mutations. BRAF mutations were never present as a single event. Concomitant KRAS and BRAF mutations increased along progression of MSS CRCs, suggesting that activation of both genes is likely to harbour a synergistic effect.