Expression and subcellular localization of the bromodomain-containing protein 7 is a prognostic biomarker in breast cancer.

Bromodomain-containing protein 7 (BRD7) is a member of the bromodomain-containing protein family. Previous studies suggest that BRD7 is predominantly localized in the nucleus, wherein it functions as a transcriptional regulator. Several lines of evidence imply a tumour suppressor function for BRD7. However, the importance of BRD7 in the pathogenesis of breast cancer is not well understood. We have investigated the expression, CpG island methylation and subcellular localization of BRD7 in breast cancer cell lines and clinical cases and thereby assessed its prognostic significance by correlating with clinical-pathological features and time-dependent clinical outcomes. We show that nuclear exclusion of BRD7 occurs commonly in breast cancer and is strongly associated with cases expressing wild-type p53. Moreover, clinical outcomes are significantly less favourable in cases with nuclear exclusion or loss of expression than those in which there is nuclear expression of BRD7. Methylation of the CpG island of BRD7 increases in breast cancer relative to normal breast tissue, but there is not an obvious correlation between methylation and reduced expression or between methylation and clinical outcomes. Overall, our results suggest that nuclear exclusion, rather than transcriptional silencing, is a common mechanism by which the tumour suppressor function of wild-type p53 is inhibited in breast cancer, and show that BRD7 is a promising candidate biomarker in breast cancer.

MicroRNAs role as potential biomarkers and key regulators in melanoma.

Malignant melanoma (MM) is a highly aggressive skin cancer with high incidence worldwide. It originates from melanocytes and is characterized by invasion, early metastasis and despite the use of new drugs it is still characterized by high mortality. Since an early diagnosis determines a better prognosis, it is important to explore novel prognostic markers in the management of patients with MM. microRNAs (miRNAs) are small (∼22 nucleotides) single-stranded non-coding RNAs that negatively regulate the expression of more than 60% of human genes.miRNAs alterations are involved in several cancers, including MM, where a differential expression for some of them has been reported between healthy controls and MM patients. Moreover, since miRNAs are stable and easily detectable in body fluids, they might be considered as robust candidate biomarkers useful to identify risk of MM, to diagnose an early lesion and/or an early metastatic disease. This review highlights the importance of miRNAs as risk factors, prognostic factors and their role as molecular regulator in the development and progression of MM. © 2016 Wiley Periodicals, Inc.

Elevated basal antibody-dependent cell-mediated cytotoxicity (ADCC) and high epidermal growth factor receptor (EGFR) expression predict favourable outcome in patients with locally advanced head and neck cancer treated with cetuximab and radiotherapy.

BACKGROUND: Antibody-dependent cell-mediated cytotoxicity (ADCC) may contribute to the antitumor activity of cetuximab. However, the extent of this contribution is unclear. In this study, we investigated the impact of baseline ADCC on the outcome of patients with locally advanced squamous cell carcinoma treated with cetuximab and radiotherapy. METHODS: We determined baseline ADCC in 28 patients treated with cetuximab and radiotherapy and in 15 patients treated with chemoradiation. We linked the values observed with complete response and with overall survival. We also considered the role of epidermal growth factor receptor (EGFR) expression and studied the combined effect of EGFR and ADCC. RESULTS: We observed a wide range of baseline values of ADCC. Complete response did not correlate with either ADCC or EGFR expression. However, when ADCC and EGFR were considered together using a mixed score, they significantly correlated with achieving a complete response (p = 0.04). High baseline ADCC significantly correlated with outcome compared to low (p = 0.03), but not in patients treated without cetuximab. Patients showing high baseline levels of both ADCC and EGFR3+ achieved the best outcome compared to the others (p = 0.02). CONCLUSIONS: In this study, patients treated with cetuximab and radiotherapy, showing high baseline of both ADCC and EGFR3+, have significant higher probability of achieving a complete response and a long overall survival compared to the others.

Schedule-dependent interaction between temsirolimus and cetuximab in head and neck cancer: a preclinical study.

Aberrant epidermal growth factor receptor (EGFR) signaling is associated with tumor growth in head and neck squamous cell carcinoma (HNSCC) and is a major focus of targeted therapy. The phosphatidylinositol-3-kinase/AKT/mammalian target of the rapamycin (PI3K/AKT/mTOR) signaling pathway is frequently mutated in HNSCC and is involved in disease progression and resistance to EGFR inhibitors. The aim of this study was to assess the antiproliferative effects of mTOR inhibition (temsirolimus) combined with the anti-EGFR monoclonal antibody cetuximab, administered according to different combination schedules. Antiproliferative effects of the combination of temsirolimus and cetuximab were determined on the representative HNSCC CAL33 cell line (PI3KCA H1047R mutated and K-RAS wild-type). In addition, key proteins related to the EGFR pathway (pEGFR/EGFR, pAKT/AKT) and the mTOR pathway (p-p70S6K1, p4E-BP1) were determined to explain the cytotoxic effects. Temsirolimus and cetuximab showed a synergistic effect when administered in combination. Supra-additive effect was lost when the two drugs were administered sequentially, irrespective of which drug was administered first. Synergistic effect of the combination was corroborated by a marked downregulation of pEGFR, significant downregulation of pAKT expression, and a marked diminution of p70S6K1 and p4E-BP1 expression. Our study demonstrated a synergistic effect of temsirolimus and cetuximab administered in combination, well illustrated by a simultaneous blockade of intracellular signaling pathways regulating cell proliferation and survival. These results establish the notion of a schedule dependency for the combined treatment, which can be of importance at the clinical level.

Treatment effect of buparlisib, cetuximab and irradiation in wild-type or PI3KCA-mutated head and neck cancer cell lines.

Introduction In complement to anti-EGFR therapy, the targeting of PI3K/AKT/mTOR signaling pathway is of particular interest in the management of Head and Neck Squamous Cell Carcinoma (HNSCC). Here, we assess the effects of PI3K inhibition combined with anti-EGFR monoclonal antibody cetuximab and/or irradiation (RT). Material and methods Anti-proliferative effects of the combination of buparlisib (a specific PI3K inhibitor), cetuximab and RT was determined in two HNSCC cell lines (CAL33, PI3KCA H1047R-mutated and CAL27, PI3KCA wild-type). We examined biochemical factors related to proliferation, apoptosis (caspases), DNA repair (ERCC1, XRCC1) and the PI3K pathway (pEGFR/EGFR, pAKT/AKT, p-p70, p4EBP1). Results The best synergistic combined treatment in inhibiting cell proliferation was sequence 2 (cetuximab followed by buparlisib) in both cell lines. Addition of RT increased sequence 2 anti-proliferative effect only in CAL27. Data on protein expression indicated a possible activation of mTORC2 complex and caspases proteins in CAL27 not seen in CAL33. In CAL33, the synergistic anti-proliferative effect of the two drugs may derive from the higher sensitivity of mutated cells to PI3K targeting. Conclusions Our study demonstrates a synergistic effect of cetuximab followed by buparlisib in both PI3KCA wild-type and mutated cells, even with different intracellular signaling cross-talk depending on mutational status.

First Human Usutu Virus Reported in Asti (Piedmont, Italy, August 2022) and Early Follow-Up.

The Usutu virus (USUV) has recently attracted the attention of scientists because of its rapid spread across Europe and its growth over the previous seasons in Italy. Here, we describe the first case of USUV infection in Asti, Piedmont region, Italy. The patient remained asymptomatic in the acute phase and during the early follow-up, despite a mild increase in liver enzymes. The prompt diagnosis in this patient was due to positive qualitative PCR for WNV blood-donor screening with negative RT-PCR of WNV and positive USUV-RNA following the confirmation test. Blood-donor screening and transmission risk monitoring are pivotal in following the spread of this Flavivirus in non-endemic countries, due to the high percentage of asymptomatic carriers.

A functional common polymorphism in the vitamin D-responsive element of the GH1 promoter contributes to isolated growth hormone deficiency.

CONTEXT: Causal mutations have been detected only in a minority of isolated GH deficiency (IGHD) patients. Idiopathic IGHD might be the result of the interaction between several low-penetrance genetic factors and the environment. OBJECTIVE: The aim of this study was to test the contribution to IGHD of genetic variations in the GH1 gene regulatory regions. DESIGN AND PATIENTS: A case-control association study was performed including 118 sporadic IGHD patients with a nonsevere phenotype (height -4/-1 sd score and partial GH deficiency) and two control groups, normal stature (n=200) and short-stature individuals with normal GH secretion (n=113). Seven single-nucleotide polymorphisms in the GH1 promoter, one in the IVS4 region, and two in the locus control region were analyzed. RESULTS: The -57T allele within the vitamin D-responsive element showed a positive significant association when comparing patients with normal (P=0.006) or short stature (P=0.0011) controls. The genotype -57TT showed an odds ratio of 2.93 (1.44-5.99) and 2.99 (1.42-6.31), respectively. The functional relevance of the -57 variation was demonstrated by the luciferase assay in the presence of vitamin D. The vitamin D-induced inhibition of luciferase activity was significantly (P=0.012) stronger for the promoter haplotype carrying the associated variation -57T [haplotype #1 (hp#1)] with respect to hp#2, bearing -57G. Replacement of the T with a G at -57 on hp#1 abolished the repression, demonstrating that the T at position -57 is necessary to determine the greater vitamin D-induced inhibitory effect of hp#1. EMSA experiments showed a different band-shift pattern of the T and G sequences. CONCLUSION: The common -57G-->T polymorphism contributes to IGHD susceptibility, indicating that it may have a multifactorial etiology.

Clarithromycin resistance of Helicobacter pylori strains isolated from children' gastric antrum and fundus as assessed by fluorescent in-situ hybridization and culture on four-sector agar plates.

AIM: To assess validity of culture on four-sector agar plates and fluorescent in-situ hybridization (FISH) test, and clarithromycin resistance rate in Helicobacter pylori strains isolated from children in the last 10 years. METHODS: In the last 5 years, gastric biopsy specimens from antrum and fundus were taken from 89 consecutive children (median age 9 years) with H. pylori gastritis and from 21 controls. Culture was performed on 176 gastric biopsies (89 from antrum, 87 from fundus) on four-sector agar plates, and FISH test with DNA ProbeMix. After its validity was evaluated, FISH test was applied on additional 119 biopsies from 68 children (68 from the antrum, 51 from the fundus) stored in the Pathology archive in the previous 5 years. RESULTS: Culture was positive in 157 of 176 biopsies (sensitivity: 89.2%, 95% confidence interval (CI) 85-94). In 33 of 89 children (37%) resistant strains were found in one or both gastric sites. FISH test was positive in 148 of 176 biopsies from infected children (sensitivity 84.1%, 95%CI 79-89) and in none of 42 biopsies from controls (specificity 100%). When applied on archive biopsies, FISH test was positive in 96 of 119 (80.7%, 95%CI 74-88). Total children harboring resistant strains in the last 10 years, as assessed by FISH test, were 66 of 157 (42%). Mixed infection with both sensitive and resistant strains were found in 40 children (25%) and in 12 of them resistant strains were in the fundus only. CONCLUSIONS: Culture on four-sector agar plates and FISH test had a high sensitivity and specificity and showed co-presence of sensitive and resistant strains. In one-third of children with mixed infection, the resistant strains were in the fundus only. Clarithromycin resistance should be assessed in biopsies both from the antrum and the fundus, utilizing antral biopsies only can underestimate its prevalence.

DUSP2 methylation is a candidate biomarker of outcome in head and neck cancer.

BACKGROUND: Biomarkers predictive of response to chemoradiotherapy (CRT) regimens for locally advanced head and neck squamous cell carcinoma (LA-HNSCC) are urgently required to identify patients in whom this approach is likely to be effective. TP53 mutations and epidermal growth factor (EGFR) overexpression are common markers of disease. Dual-specificity-phosphatase-2 (DUSP2) has an essential role in cell proliferation, cancer and immune responses. METHODS: Aberrant DUSP2 methylation was investigated by pyrosequencing in 5 HNSCC cell lines, 112 LA-HNSCC tumours. EGFR was investigated by immunohistochemistry and TP53 was analysed by sequencing. RESULTS: We demonstrate methylation-dependent transcriptional silencing of DUSP2 in HNSCC cell lines. In LA-HNSCC patients, aberrant methylation in the DUSP2 CpG island was present in 51/112 cases (45.5%). LA-HNSCC cases with wild-type TP53, overexpression of EGFR and unmethylated DUSP2 had the worst overall survival (P≤0.001). CONCLUSIONS: DUSP2 methylation, when combined with EGFR and TP53, is a candidate biomarker of clinical outcome in LA-HNSCC treated with CRT.

Increased interleukin-10 in Helicobacter pylori infection could be involved in the mechanism protecting from allergy.

BACKGROUND: A protective effect of Helicobacter pylori infection against allergic diseases has been reported. The increasing incidence of childhood allergy in developed countries may be a result of reduced stimulation of the immune system by early chronic infections, with the protective effect of gastrointestinal microbes being mediated by regulatory T lymphocytes and production of interleukin (IL)-10. To elucidate a possible mechanism involved in protecting against the development of atopy, we measured expression of IL-10 in gastric mucosa of children with H pylori gastritis. PATIENTS AND METHODS: Gastric biopsies were performed during endoscopy in 48 children (median age, 9 years), 32 of whom had H pylori gastritis and 16 of whom served as controls. Interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), and IL-10 were measured in tissue homogenate by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The amounts of IFN-gamma, IL-1beta, and IL-10 transcripts were quantified via competitive RT-PCR with use of dilution series of specific competitors. RESULTS: Expression of IFN-gamma and IL-10 were significantly higher in H pylori-infected children. No direct correlation with age was found, but a further increase in IL-10 expression was found in H pylori-infected children older than 4 years, whereas in control subjects, IL-10 expression tended to be lower in older children. IL-1beta expression was similar in infected children and control subjects. In H pylori-infected children, the prevalence of allergy was significantly higher in children with lower cytokine expression in gastric mucosa. CONCLUSIONS: In children, H pylori-induced inflammatory response is associated with development of cell-mediated immunity of T-helper 1 type, as demonstrated by increased IFN-gamma expression. The significantly increased expression of gastric IL-10 in H pylori-infected children and its further increase in older children suggest that this chronic infection may influence IL-10 production even beyond the age of 4 years. H pylori may be one of the infections with the potential to modulate immune responses.

Prognostic Value of Different Allelic Polymorphism of Aldosterone Synthase Receptor in a Congestive Heart Failure European Continental Ancestry Population.

Aldosterone synthase (CYP11B2) is as an 9-exon gene on chromosome 8q22 and exists as a common single nucleotide polymorphism C-T transition for position -344. The aim of this study was to assess the -344T/C polymorphism of the aldosterone synthase promoter in a European continental ancestry congestive heart failure (CHF) population. METHODS: Patients discharged after an acute decompensation were enrolled and underwent echocardiography, determination of BNP, evaluation of non-invasive cardiac outputs and determination of -344 T/C SNP in the aldosterone synthase gene. RESULTS: 175 patients (137 male; age 69.9 ± 10.2 years) were enrolled. The genotype distribution of -344 T/C SNP demonstrated a TT genotype in 61 patients (34.9%), CT in 80 (45.7%) and finally CC in 34 (19.4%) CHF patients. According to presence of C allele, CHF patients were divided into C group (-CT/CC genotype, 114 subjects) and T Group (-TT genotype, 61 subjects). The two groups did not differ in term of age, non-invasive cardiac output at rest, creatinine level or end-systolic or diastolic left ventricle diameter, LVEF and BNP. In group C patients in comparison than in group T a higher degree of disability (Barthel Index p = 0.004), NYHA class (p = 0.02) and a lower cardiac index (p = 0.01) emerged. Moreover, the two groups showed a similar clinical outcome (death for any cause/hospital readmission for CHF) at 48 month follow-up (p = 0.16; log-rank 1.99). CONCLUSIONS: In European continental ancestry patients the C allele (CC or CT) at -344T/C SNP in the aldosterone synthase gene does not significantly influence clinical prognosis of CHF.

A novel deletion in the GH1 gene including the IVS3 branch site responsible for autosomal dominant isolated growth hormone deficiency.

CONTEXT: The majority of mutations responsible for isolated GH type II deficiency (IGHD II) lead to dominant negative deleteriously increased levels of the GH1 exon 3 skipped transcripts. OBJECTIVE: The aim of this study was the characterization of the molecular defect causing a familial case of IGHD II. PATIENTS: A 2-yr-old child and her mother with severe growth failure at diagnosis (-5.8 and -6.9 sd score, respectively) and IGHD were investigated for the presence of GH1 mutations. RESULTS: We identified a novel 22-bp deletion in IVS3 (IVS3 del+56-77) removing the putative branch point sequence (BPS). Analysis of patients' lymphocyte mRNA showed an excess exon 3 skipping. The mutated allele transfected into rat pituitary cells produced four differently spliced products: the exon 3 skipped mRNA as the main product and lower amounts of the full-length cDNA and of two novel mRNA aberrant isoforms, one with the first 86 bases of exon 4 deleted and the other lacking the entire exon 4. A mutagenized construct lacking exclusively the 7 bp of the BPS only generated the exon 4 skipped and the full-length isoforms. The presence of the full-length transcript in the absence of the canonical BPS points to an alternative BPS in IVS3. CONCLUSION: The IVS3 del+56-77 mutation, causing IGHD II in this family, has two separate effects on mRNA processing: 1) exon 3 skipping, analogous to most described cases of IGHD II, an effect likely caused by the reduction in size of the IVS3, and 2) partial or total exon 4 skipping, as a result of the removal of the BPS.

Prognostic and predictive biomarkers in metastatic colorectal cancer anti-EGFR therapy.

AIM: To reviewing genetic and epigenetic make-up of metastatic colorectal cancers (mCRCs) addicted to epidermal growth factor receptor (EGFR) signalling. METHODS: The present study summarizes the potential value of prognostic and predictive biomarkers in selecting mCRC patients treated with anti-EGFR therapy. A meta-analysis was performed using a systematic search of PubMed, Medline and Web of Science to identify eligible papers until March 21(st), 2016 using these following terms: ''colorectal cancer'', "predictive biomarkers'', "anti-EGFR therapy", "KRAS", "NRAS'', "PIK3CA", "TP53", "PTEN", ''EGFR", "MET", "HER2", "epiregulin", "amphiregulin", "prognostic biomarkers", "BRAF", "miRNA" and "antibody-dependent cell-mediated cytotoxicity (ADCC) activity". Two investigators independently evaluated and extracted data from each identified studies based on selected criteria of inclusion and exclusion. RESULTS: The introduction of agents targeting EGFR such as cetuximab and panitumumab increased overall survival of mCRCs. Nevertheless, it has firstly became evident that response rates to cetuximab regimens in unselected patient populations were typically lower than 30%. Clinical data confirmed the predictive value of RAS mutations for resistance to cetuximab and panitumumab leading to the license of these monoclonal antibodies exclusively for the management of patients with RAS-wild type colorectal cancers. So far the identification of predictive biomarkers have generated interesting, though preliminary and, at times, conflicting data on the importance of tumour mRNA levels of EGFR ligands, of activating mutations in other genes such as NRAS and PIK3CA. The prognostic value of selected microRNAs level and ADCC activity is under investigation, while the prognostic impact of BRAF status remains controversial. CONCLUSION: This review focuses on the personalized treatment of mCRC and discusses the potential of new prognostic and predictive biomarkers in selecting patients treated with anti-EGFR therapy.

E1 detection as prognosticator in human papillomavirus-positive head and neck cancers.

PURPOSE: HPV-related locally advanced head and neck cancers (LA-HNCs) show a good prognosis. This study aimed to investigate the HPV prevalence in LA-HNCs and compare the prognostic value of E1, E6 and L1 genomic viral fragments and p16, individually and in combination, in order to find the best prognosticator in terms of overall survival (OS) and progression-free survival (PFS). PATIENTS AND METHODS: HPV16 was searched in 255 LA-HNC formalin-fixed paraffin-embedded tumor tissues, 89 oropharyngeal cancers (OPCs), and 166 non-OPCs by DNA-PCR with 3 primer pairs. p16 was analyzed by immunohistochemistry in 235 patients. RESULTS: The prevalence of positive samples decreased constantly from E6 to L1 and E1 in both OPCs and non-OPCs. Each LA-HNC patient highlighted variable positivity for each fragment. OPCs showed a higher prevalence of positive samples compared to non-OPCs.Positive coexistence of all the fragments was more common in OPCs (31.5%) than non-OPCs (4.2%), and E1 detection was always associated with E6 and L1. E1-positive OPCs showed improved OS (p = 0.012) and PFS (p = 0.036), while L1- or E6-positive ones did not. p16-positive patients were more prevalent in the OPC (29.8%) than the non-OPC group (7.3%) (p<0.0001) and its prognostic value was not superior to that of E1. However, the multivariate Cox analysis which included E1, L1, E6 status and p16 expression did not show a significant p value. CONCLUSIONS: Though HPV16 positivity measured by DNA-PCR was higher for L1 and E6, they performed weakly as prognosticators; E1 might become a strong prognostic marker for OS and PFS in OPCs.

p16 cutoff in head and neck squamous cell carcinoma: correlation between tumor and patient characteristics and outcome.

BACKGROUND: p16 has been indicated as a suitable surrogate biomarker of HPV infection. The prognosis of p16-positive oropharynx tumors (OTs) of squamous cell carcinoma (SCC) histology is better than that of p16-negative tumors. METHODS: We analyzed 209 samples of head and neck SCC to establish a predictive cutoff for p16 and determine the role of p16 positivity in OTs versus non-OTs. We compared the outcomes of tumors harboring any percentage of p16-positive cells (≥1%) with those of p16-negative OTs. We then considered 3 cutoffs (10%, 50% and 70% positive cells) to evaluate the outcome of OTs/non-OTs with similar p16 expression and p16-positive versus p16-negative tumors stratified by patient age. RESULTS: p16-negative tumors among OTs and non-OTs were 29% and 49%, respectively (p = 0.0054). The cumulative distribution showed that the positive values were located around 2 focus points: 2% and 96%. Subgroup analysis showed that only OTs occurred in young patients (aged <65 years) and that there was a ≥70% gain in survival in cases with p16-positive cells. CONCLUSIONS: p16 positivity influences outcome only in young patients and OTs (p = 0.048).

Correlation of TP53 and MDM2 genotypes and clinical outcome in platinum-treated head and neck cancer patients with more than 10 years' follow-up.

PURPOSE: Adequate biomarkers are still required to optimize therapy in patients with locally advanced head and neck squamous carcinomas (HNSCC) treated with chemoradiotherapy (CRT). METHODS: We updated the follow-up of 66 HNSCC patients treated with CRT we described more than 10 years ago, focusing on SNP Arg/Pro (R/P) at codon 72 and somatic mutations in TP53 and on SNP309 in the MDM2 gene. RESULTS: In wild-type TP53 cases, overall survival (OS) was longer in 72RR and less favorable in 72PP (p = 0.005); when TP53 was mutated, OS was longest in 72PP and less favorable in 72RR and 72RP (p = 0.058). Median OS was significantly shorter in patients with MDM2 SNP309 GG or GT genotypes compared with the TT genotype (p = 0.002). CONCLUSIONS: TP53 SNP72 may be useful in selecting patients for CRT, but has to be related to somatic TP53 mutations. The MDM2 SNP309, easily determined in peripheral blood, might be more convenient as a predictive biomarker.

Evaluation of antibody-dependent cell-mediated cytotoxicity activity and cetuximab response in KRAS wild-type metastatic colorectal cancer patients.

AIM: To investigate the prognostic role of invariant natural killer T (iNKT) cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in wild type KRAS metastatic colorectal cancer (mCRC) patients treated with cetuximab. METHODS: Forty-one KRAS wt mCRC patients, treated with cetuximab and irinotecan-based chemotherapy in II and III lines were analyzed. Genotyping of single nucleotide polymorphism (SNP)s in the FCGR2A, FCGR3A and in the 3' untranslated regions of KRAS and mutational analysis for KRAS, BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprep-peripheral blood mononuclear cell and iNKT cells were defined by co-expression of CD3, TCRVα24, TCRVß11. ADCC was evaluated as ex vivo NK-dependent activity, measuring lactate dehydrogenase release. RESULTS: At basal, mCRC patients performing ADCC activity above the median level (71%) showed an improved overall survival (OS) compared to patients with ADCC below (median 16 vs 8 mo; P = 0.026). We did not find any significant correlation of iNKT cells with OS (P = 0.19), albeit we observed a trend to a longer survival after 10 mo in patients with iNKT above median basal level (0.382 cells/microliter). Correlation of OS and progression-free survival (PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2A and TT in FCGR3A presented a trend of longer PFS (median 9 vs 5 mo; P = 0.064). Chemotherapy impacted both iNKT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment. CONCLUSION: Our results suggest a link between iNKT cells, basal ADCC activity, genotypes in FCGR2A and FCGR3A, and efficacy of cetuximab in KRAS wt mCRC patients.

Reoxygenation Reverses Hypoxia-related Radioresistance in Head and Neck Cancer Cell Lines.

BACKGROUND/AIM: Head and neck cancer (HNC) is characterized by epidermal growth factor receptor (EGFR) overexpression and radiotherapy (RT) resistance. Cancer cells are able to survive and proliferate in hypoxic conditions. Hypoxia can be transiently interrupted by phases of reoxygenation. This work aimed to analyze the reoxygenation effect on proliferation in response to radiation in HNC cells. MATERIALS AND METHODS: HNC cell lines CAL33 and CAL166 were subjected to an 8-Gy radiation dose in hypoxia and/or after reoxygenation. Cell proliferation and molecular factors involved in response to treatments were studied. RESULTS: Cytotoxicity test confirmed radioresistance in hypoxia and highlighted that reoxygenation before RT restores sensitivity in both cell lines. Our results showed a similar proliferation inhibition effect and EGFR modulation but a different cell death mechanism in the two cell lines after treatment. CONCLUSION: Reoxygenation before RT rescued radiosensitivity in HNC cells.

The relevance of ADCC for EGFR targeting: A review of the literature and a clinically-applicable method of assessment in patients.

BACKGROUND: Advances in the understanding of tumor biology have led to the development of targeted therapies as monoclonal antibodies (MoAbs) in clinical oncology. Among their suggested mechanisms of action monoclonal antibodies (IgG1) selectively directed against tumor membrane receptors mediate of antibody-dependent cellular cytotoxicity (ADCC) by triggering Fc-γRIII on natural killer (NK) cells. METHODS: This study reviews the clinical context of ADCC measurement with a particular focus on EGFR targeting and describes an ex vivo ADCC method applied to MoAbs (cetuximab and panitumumab), against epidermal growth factor receptor (EGFR). The test performance was evaluated on different target cells lines (CAL166, A431, HNO91, CAL27), with different effector cells (peripheral blood mononuclear cells or natural killers -NK-) and in various experimental conditions, in order to establish a truly clinically applicable method. RESULTS: Using the experience available in the published literature, we optimized all variables involved in the experimental design: target cells type, numbers and ratio target cells and NK cells (effector cells) per well, time of exposure and repeatability. CONCLUSION: ADCC measurement may be of clinical relevance in the context of treatment with MoAbs. This study describes a non-radioactive method which has proven satisfactory in terms of sensitivity, reproducibility, feasibility and cost effectiveness for the measurement of ADCC activity mediated by NK with an orientation towards the EGFR target.

MGMT promoter methylation and glioblastoma: a comparison of analytical methods and of tumor specimens.

It is already well known that hypermethylation of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is a predictive biomarker of response to temozolomide treatment and of favorable outcomes in terms of overall survival (OS) and progression-free survival (PFS) in glioblastoma (GBM) patients. Nevertheless, MGMT methylation status has not currently been introduced into routine clinical practice, as the choice of the ideal technique and tissue sample specimen is still controversial. The aim of this study was to compare 2 analytical methods, methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), and their use on 2 different tissue type samples, snap-frozen and formalin-fixed paraffin-embedded (FFPE), obtained from a single-center and uniformly treated cohort of 46 GBM patients. We obtained methylation data from all frozen tissues, while no results were obtained for 5 FFPE samples. The highest concordance for methylation was found on frozen tissues (88.5%, 23/26 samples), using PSQ (76.7%, 23/30 samples). Moreover, we confirmed that OS and PFS for patients carrying methylation of the MGMT promoter were longer than for patients with an unmethylated promoter. In conclusion, we considered MSP a limited technique for FFPE tissues due to the high risk of false-positive results; in contrast, our data indicated PSQ as the most powerful method to stratify methylated/unmethylated patients as it allows reaching quantitative results with high sensitivity and specificity. Furthermore, frozen tumor tissues were shown to be the best specimens for MGMT methylation analysis, due to the low DNA degradation and homogeneity in methylation throughout the tumor.