ß3-Adrenoreceptor Blockade Reduces Hypoxic Myeloid Leukemic Cells Survival and Chemoresistance.

ß-adrenergic signaling is known to be involved in cancer progression; in particular, beta3-adrenoreceptor (ß3-AR) is associated with different tumor conditions. Currently, there are few data concerning ß3-AR in myeloid malignancies. Here, we evaluated ß3-AR in myeloid leukemia cell lines and the effect of ß3-AR antagonist SR59230A. In addition, we investigated the potential role of ß3-AR blockade in doxorubicin resistance. Using flow cytometry, we assessed cell death in different in vitro myeloid leukemia cell lines (K562, KCL22, HEL, HL60) treated with SR59230A in hypoxia and normoxia; furthermore, we analyzed ß3-AR expression. We used healthy bone marrow cells (BMCs), peripheral blood mononuclear cells (PBMCs) and cord blood as control samples. Finally, we evaluated the effect of SR59230A plus doxorubicin on K562 and K562/DOX cell lines; K562/DOX cells are resistant to doxorubicin and show P-glycoprotein (P-gp) overexpression. We found that SR59230A increased cancer cell lines apoptosis especially in hypoxia, resulting in selective activity for cancer cells; moreover, ß3-AR expression was higher in malignancies, particularly under hypoxic condition. Finally, we observed that SR59230A plus doxorubicin increased doxorubicin resistance reversion mainly in hypoxia, probably acting on P-gp. Together, these data point to ß3-AR as a new target and ß3-AR blockade as a potential approach in myeloid leukemias.

Next generation sequencing technologies for a successful diagnosis in a cold case of Leigh syndrome.

BACKGROUND: Leigh Syndrome (LS, OMIM 256000) is an early-onset, progressive neurodegenerative disorder characterized by broad clinical and genetic heterogeneity; it is the most frequent disorder of mitochondrial energy production in children. LS inheritance is complex because patients may present mutations in mitochondrial DNA (mtDNA) or in nuclear genes, which predominantly encode proteins involved in respiratory chain structure and assembly or in coenzyme Q10 biogenesis. However, during the last 15 years, the discovery of several genetic mutations and improved knowledge of the natural history of LS has significantly increased our understanding of this mitochondrial disorder. CASE PRESENTATION: Here we describe a 19-year-old male with clinical and neuroimaging LS diagnosed at 3 years of age. Genetic analyses of the whole mtDNA for maternally inherited LS (MILS) and neuropathy ataxia retinitis pigmentosa (NARP) syndrome failed to reveal any pathogenic mutations. CONCLUSIONS: Recently, a missense mutation in ECHS1 and a ~ 35 kb deletion in 10q26.3 involving the region including the gene were identified by WES (whole exome sequencing), uncovering the genetic diagnosis clinically hypothesized for 15 years. We also report the long-term follow-up of this patient, showing a comparison with classical LS or other Leigh-like pictures.

Glioblastoma Chemoresistance: The Double Play by Microenvironment and Blood-Brain Barrier.

For glioblastoma, the tumor microenvironment (TME) is pivotal to support tumor progression and therapeutic resistance. TME consists of several types of stromal, endothelial and immune cells, which are recruited by cancer stem cells (CSCs) to influence CSC phenotype and behavior. TME also promotes the establishment of specific conditions such as hypoxia and acidosis, which play a critical role in glioblastoma chemoresistance, interfering with angiogenesis, apoptosis, DNA repair, oxidative stress, immune escape, expression and activity of multi-drug resistance (MDR)-related genes. Finally, the blood brain barrier (BBB), which insulates the brain microenvironment from the blood, is strongly linked to the drug-resistant phenotype of glioblastoma, being a major physical and physiological hurdle for the delivery of chemotherapy agents into the brain. Here, we review the features of the glioblastoma microenvironment, focusing on their involvement in the phenomenon of chemoresistance; we also summarize recent advances in generating systems to modulate or bypass the BBB for drug delivery into the brain. Genetic aspects associated with glioblastoma chemoresistance and current immune-based strategies, such as checkpoint inhibitor therapy, are described too.

Topoisomerase 1 Promoter Variants and Benefit from Irinotecan in Metastatic Colorectal Cancer Patients.

OBJECTIVE: Topoisomerase 1 (topo-1) is an important target for the treatment of metastatic colorectal cancer (CRC). The aim of the present study was to evaluate the correlation between topo-1 single-nucleotide polymorphisms (SNPs) and clinical outcome in metastatic CRC (mCRC) patients. METHODS: With the use of specific software (PROMO 3.0), we performed an in silico analysis of topo-1 promoter SNPs; the rs6072249 and rs34282819 SNPs were included in the study. DNA was extracted from 105 mCRC patients treated with FOLFIRI ± bevacizumab in the first line. SNP genotyping was performed by real-time PCR. Genotypes were correlated with clinical parameters (objective response rate, progression-free survival, and overall survival). RESULTS: No single genotype was significantly associated with clinical variables. The G allelic variant of rs6072249 topo-1 SNP is responsible for GC factor and X-box-binding protein transcription factor binding. The same allelic variant showed a nonsignificant trend toward a shorter progression-free survival (GG, 7.5 months; other genotypes, 9.3 months; HR 1.823, 95% CI 0.8904-3.734; p = 0.1). CONCLUSION: Further analyses are needed to confirm that the topo-1 SNP rs6072249 and transcription factor interaction could be a part of tools to predict clinical outcome in mCRC patients treated with irinotecan-based regimens.

Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer.

NRAS mutations occur in 3-5% of colorectal cancer. Differently from KRAS and BRAF mutations, the role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer (mCRC) has been investigated to a lesser extent. A retrospective series suggested the role of NRAS mutations as predictors of resistance to anti-EGFR monoclonal antibodies (MoAbs) in chemo-refractory patients with mCRC. In our study, KRAS codons 12, 13, 61 and BRAF codon 600 mutational status were evaluated in mCRCs referred to our Institution from 2009 to 2012. NRAS codons 12, 13 and 61 mutational status was analyzed in KRAS/BRAF wt patients. We collected pathological and clinical features in the overall population and outcome data in a subset of NRAS mutated chemo-refractory patients treated with anti-EGFR MoAbs in advanced lines. NRAS was mutated in 47/786 (6%) mCRCs. NRAS and KRAS mutated tumors did not show significant differences in terms of clinical and pathological characteristics, except for a lower prevalence of mucinous histology (p = 0.012) and lung metastases (p = 0.012) among NRAS mutated tumors. In the uni- and multivariate model, NRAS mutations were associated with shorter overall survival (OS) compared to all wt patients (median OS 25.6 vs 42.7 months; univ: HR = 1.91, 95% CI 1.39-3.86, p = 0.0013; multiv: HR = 1.75, 95% CI 1.1.3-2.72, p = 0.013). None of the chemo-refractory NRAS mutated patients evaluable for response to anti-EGFRs achieved response. In conclusion, NRAS mutations have a relevant incidence in patients with mCRC and showed an association with specific clinical and pathological features. NRAS mutations affect mCRC patients' prognosis and predict lack of response to anti-EGFRs.

Molecular and pathological characterization of the EZH2 rs3757441 single nucleotide polymorphism in colorectal cancer.

BACKGROUND: The enhancer of zeste-homolog 2 (EZH2) is involved in cancer development through gene silencing by trimethylation of lysine 27 of histone 3 (H3K27me3). The C/C genotype for the EZH2 rs3757441 single-nucleotide polymorphism (SNP) is linked with poor prognosis in metastatic colorectal cancer (CRC), but molecular and pathological characterization of this SNP is lacking. METHODS: 119 primary CRCs were analyzed. SNP was evaluated by real-time PCR from colonic healthy tissue, while EZH2 and H3K27me3 expression were studied by immunohistochemistry. We primarily looked for correlation between EZH2 rs3757441 genotypes and EZH2/H3K27me3 expression. Potential associations between EZH2/H3K27me3 expression and clinico-pathological features or KRAS exon 2 and BRAF exon 15 mutations were secondary endpoints. Statistical analysis was performed by chi-square test, T-test or ANOVA. RESULTS: The C/C genotype was significantly associated with higher EZH2 (100 vs. 44 %; P = 0.019) and H3K27me3 (100 vs. 38 %; P = 0.009) staining intensity compared with C/T and T/T. EZH2 3+ staining significantly correlated with stronger H3K27me3 expression (P = 0.039). KRAS and BRAF mutations were not associated with EZH2 or H3K27me3 expression. CONCLUSION: EZH2 rs3757441 C/C genotype is associated with stronger EZH2 and H3K27me3 immunoreactivity in primary CRC: this SNP may serve as a promising biomarker for EZH2-targeting agents and may add independent information to KRAS and BRAF testing.

Identification of the novel HLA-DPA1*01:195 allele by next-generation sequencing.

The novel HLA-DPA1*01:195 allele differs from HLA-DPA1*01:03:01:01 by one nucleotide substitution in exon 4.

Identification of the novel HLA-DPB1 allele, HLA-DPB1*1326:01, in an Italian bone marrow donor.

The novel HLA class II allele HLA-DPB1*1326:01 is described.

Identification of a new HLA-DPB1 allele, HLA-DPB1*1485:01, in an Italian bone marrow donor.

The novel HLA class II allele HLA-DPB1*1485:01 is described.

ß3-Adrenoceptors as Putative Regulator of Immune Tolerance in Cancer and Pregnancy.

Understanding the mechanisms of immune tolerance is currently one of the most important challenges of scientific research. Pregnancy affects the immune system balance, leading the host to tolerate embryo alloantigens. Previous reports demonstrated that ß-adrenergic receptor (ß-AR) signaling promotes immune tolerance by modulation of NK and Treg, mainly through the activation of ß2-ARs, but recently we have demonstrated that also ß3-ARs induce an immune-tolerant phenotype in mice bearing melanoma. In this report, we demonstrate that ß3-ARs support host immune tolerance in the maternal microenvironment by modulating the same immune cells populations as recently demonstrated in cancer. Considering that ß3-ARs are modulated by oxygen levels, we hypothesize that hypoxia, through the upregulation of ß3-AR, promotes the biological shift toward a tolerant immunophenotype and that this is the same trick that embryo and cancer use to create an aura of immune-tolerance in a competent immune environment. This study confirms the analogies between fetal development and tumor progression and suggests that the expression of ß3-ARs represents one of the strategies to induce fetal and tumor immune tolerance.

A microRNA profile of pediatric glioblastoma: The role of NUCKS1 upregulation.

MicroRNAs (miRNAs/miRs) are a novel class of gene regulators that may be involved in tumor chemoresistance. Recently, specific miRNA expression profiles have been identified in adult glioblastoma (aGBM), but there are only limited data available on the role of miRNAs in pediatric GBM (pGBM). In the present study, the expression profile of miRNAs was examined in seven pGBMs and three human GBM cell lines (U87MG, A172 and T98G), compared with a non-tumoral pool of pediatric cerebral cortex samples by microarray analysis. A set of differentially expressed miRNAs was identified, including miR-490, miR-876-3p, miR-876-5p, miR-448 and miR-137 (downregulated), as well as miR-501-3p (upregulated). Through bioinformatics analysis, a series of target genes was predicted. In addition, similar gene expression patterns in pGBMs and cell lines was confirmed. Of note, drug resistant T98G cells had upregulated nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) expression, a protein overexpressed in many tumors that serves an important role in cell proliferation and progression. On the basis of the present preliminary report, it could be intriguing to further investigate the relationship between each of the identified differentially expressed miRNAs and NUCKS1, in order to clarify their involvement in the multi-drug resistance mechanism of pGBMs.

Aldoxorubicin and Temozolomide combination in a xenograft mice model of human glioblastoma.

Glioblastoma Multiforme (GBM) is still an incurable disease. The front-line Temozolomide (TMZ)-based therapy suffers from poor efficacy, underlining the need of new therapies. Preclinically, Aldoxorubicin (Aldox), a novel prodrug of Doxorubicin (Dox), has been successfully tested against GBM, encouraging the study of its association with other agents. For the first time, we evaluated the effectiveness of Aldox combined to TMZ in preclinical models of GBM. Our in vitro results demonstrated that the anti-glioma effect of Aldox was more marked than TMZ and their combination increased the killing effect of the anthracycline in TMZ-resistant GBM cells. Moreover, unlike Dox, Aldox was able to accumulate in P-glycoprotein (P-gp)-overexpressed cells due to a negative regulation of the P-gp function. We also compared efficacy and safety of weekly administrations of Aldox (16 mg/kg), with or without TMZ (0.9 mg/kg, daily injections), in the U87 xenograft mouse model. Aldox therapy induced a moderate tumor volume inhibition (TVI) and an increased survival rate (+12.5% vs vehicle). On the other hand, when combined to TMZ, Aldox caused a significant TVI (P=0.0175 vs vehicle) and delayed the mortality during the experimental period, although TVI and endpoint survival percentage (+37.5% vs vehicle) were not significantly different from TMZ alone. Our preliminary data showed that Aldox exerts anti-glioma effects in vitro and in vivo. It also enhances its antitumor activity when combined with TMZ, resulting in a superior efficacy compared to the single agents, without adverse side effects.

Whole-exome analysis in osteosarcoma to identify a personalized therapy.

Osteosarcoma is the most common pediatric primary non-hematopoietic bone tumor. Survival of these young patients is related to the response to chemotherapy and development of metastases. Despite many advances in cancer research, chemotherapy regimens for osteosarcoma are still based on non-selective cytotoxic drugs. It is essential to investigate new specific molecular therapies for osteosarcoma to increase the survival rate of these patients. We performed exomic sequence analyses of 8 diagnostic biopsies of patients with conventional high grade osteosarcoma to advance our understanding of their genetic underpinnings and to correlate the genetic alteration with the clinical and pathological features of each patient to identify a personalized therapy. We identified 18,275 somatic variations in 8,247 genes and we found three mutated genes in 7/8 (87%) samples (KIF1B, NEB and KMT2C). KMT2C showed the highest number of variations; it is an important component of a histone H3 lysine 4 methyltransferase complex and it is one of the histone modifiers previously implicated in carcinogenesis, never studied in osteosarcoma. Moreover, we found a group of 15 genes that showed variations only in patients that did not respond to therapy and developed metastasis and some of these genes are involved in carcinogenesis and tumor progression in other tumors. These data could offer the opportunity to get a key molecular target to identify possible new strategies for early diagnosis and new therapeutic approaches for osteosarcoma and to provide a tailored treatment for each patient based on their genetic profile.