Alterations in Plasma Lipid Profiles Associated with Melanoma and Therapy Resistance.

Dysfunctions of lipid metabolism are associated with tumor progression and treatment resistance of cutaneous melanoma. BRAF/MEK inhibitor resistance is linked to alterations of melanoma lipid pathways. We evaluated whether a specific lipid pattern characterizes plasma from melanoma patients and their response to therapy. Plasma samples from patients and controls were analyzed for FASN and DHCR24 levels and lipidomic profiles. FASN and DHCR24 expression resulted in association with disease condition and related to plasma cholesterol and triglycerides in patients at different disease stages (n = 144) as compared to controls (n = 115). Untargeted lipidomics in plasma (n = 40) from advanced disease patients and controls revealed altered levels of different lipids, including fatty acid derivatives and sphingolipids. Targeted lipidomics identified higher levels of dihydroceramides, ceramides, sphingomyelins, ganglioside GM3, sphingosine, sphingosine-1-phosphate, and dihydrosphingosine, saturated and unsaturated fatty acids. When melanoma patients were stratified based on a long/short-term clinical response to kinase inhibitors, differences in plasma levels were shown for saturated fatty acids (FA 16:0, FA18:0) and oleic acid (FA18:1). Our results associated altered levels of selected lipid species in plasma of melanoma patients with a more favorable prognosis. Although obtained in a small cohort, these results pave the way to lipidomic profiling for melanoma patient stratification.

Combination of Deauville score and quantitative positron emission tomography parameters as a predictive tool of anti-CD19 chimeric antigen receptor T-cell efficacy.

BACKGROUND: Autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is an effective treatment for approximately 40% of relapsed/refractory large B cell lymphomas (LBCL), and early identification of patients at risk for relapse or progression after CAR T-cell therapy represents a clinical need. METHODS: The authors conducted a single-center prospective study on 47 relapsed/refractory LBCL receiving CAR T-cell therapy to evaluate the prognostic value of baseline and after infusion 18 F-fluorodeoxyglucose positron emission tomography (PET)-computed tomography. Qualitative and quantitative metabolic parameters were evaluated before lymphodepletion, at day 30 and 90 post-infusion. RESULTS: Deep variation of standardized uptake value (SUV)mean between baseline and day 30 correlated with response at day 90 (hazard ratio [HR], 1.49; 95% confidence interval [CI], 1.01-2.2); p = .04) and better progression-free survival (PFS) (HR, 0.63; 95% CI, 0.41-0.97); p = .04). In the overall population, 1-year PFS was 63% for Deauville score (DS)1-3 and 39% for DS4-5 patients, respectively (p = .02), however, the prognostic role of DS was lost when survivals are analyzed by considering 38 patients not progressing at 30 days. In these patients, in partial response or stable disease, the combination of DS and variation of SUVmean allowed identification of three groups with different prognosis: patients with DS1-3 and those with DS4-5 and decreased SUVmean had similar 1-year PFS of 62% and 61%, whereas patients with DS4-5 and increased SUVmean had a poorer 1-year PFS of 33% (p = .04). CONCLUSIONS: PET parameters and association of DS and variation of SUVmean at 30 days could help in identify patients at high risk of CAR T-cell failure. LAY SUMMARY: This is a single-center prospective study on 47 lymphoma patients receiving commercial chimeric antigen receptor T-cell therapy aimed to evaluate the prognostic value of baseline and after infusion 18 F-fluorodeoxyglucose positron emission tomography. Among patients in partial remission or stable disease at day 30, the authors observed two subgroups with significantly different prognosis; patients with Deauville score (DS)4-5 and a concomitant reduction of standardized uptake value (SUV)mean had higher probability of long-lasting response than those with DS4-5 and an increase of SUVmean .

Quality of life after prophylactic surgery for colorectal adenomatous polyposis.

PURPOSE: Colorectal adenomatous polyposis is characterized by the onset of tens to thousands of adenomas in the colorectal epithelium and, if not treated, leads to a lifetime increased risk of developing colorectal cancer compared to the general population. Thus, prophylactic surgery is recommended. This study aims to investigate the quality of life of colorectal adenomatous polyposis patients following prophylactic surgery and indirectly compares these findings with those of healthy adults of the normative sample. METHODS: All patients who underwent prophylactic surgery for polyposis and were in follow-up at the hereditary digestive tract tumors outpatient department of our institute were eligible for the study. The Short Form-36 questionnaire and 21 ad hoc items were used at the time of clinical evaluation. RESULTS: A total of 102 patients were enrolled. For the SF-36 domains, mean values ranged from 64.18 for vitality to 88.49 for physical functioning, with the highest variability for role-physical limitations; the minimum value of functioning was reached for role-physical limitations, role-emotional limitations, and social functioning. The maximum value of functioning was reached for role-emotional limitations (73.96%) and role-physical limitations (60.42%). In total, 48.96% and 90.63% of patients reported no fecal or urinary incontinence episodes, respectively; 69.79% of patients did not have problems in work/school resumption or the personal sexual sphere. CONCLUSION: Quality of life following prophylactic surgery for these patients seems to be good when indirectly compared to HP-normative samples'. Young adult patients appear to quickly manage and adapt to changes in bowel functioning. A minority of patients may experience social and sexual issues.

T-cell immune response after mRNA SARS-CoV-2 vaccines is frequently detected also in the absence of seroconversion in patients with lymphoid malignancies.

Patients affected by lymphoid malignancies (LM) are frequently immune-compromised, suffering increased mortality from COVID-19. This prospective study evaluated serological and T-cell responses after complete mRNA vaccination in 263 patients affected by chronic lymphocytic leukaemia, B- and T-cell lymphomas and multiple myeloma. Results were compared with those of 167 healthy subjects matched for age and sex. Overall, patient seroconversion rate was 64·6%: serological response was lower in those receiving anti-cancer treatments in the 12 months before vaccination: 55% vs 81·9% (P < 0·001). Anti-CD20 antibody plus chemotherapy treatment was associated with the lowest seroconversion rate: 17·6% vs. 71·2% (P < 0·001). In the multivariate analysis conducted in the subgroup of patients on active treatment, independent predictors for seroconversion were: anti-CD20 treatment (P < 0·001), aggressive B-cell lymphoma diagnosis (P = 0·002), and immunoglobulin M levels <40 mg/dl (P = 0·030). The T-cell response was evaluated in 99 patients and detected in 85 of them (86%). Of note, 74% of seronegative patients had a T-cell response, but both cellular and humoral responses were absent in 13·1% of cases. Our findings raise some concerns about the protection that patients with LM, particularly those receiving anti-CD20 antibodies, may gain from vaccination. These patients should strictly maintain all the protective measures.

Hereditary colorectal cancer syndromes and the COVID-19 pandemic: results from a survey conducted in patients enrolled in a dedicated registry.

PURPOSE: The coronavirus 2019 (COVID-19) pandemic has had profound consequences also for non-infected patients. This study aimed to evaluate the impact of the pandemic on the quality of life of a population with hereditary gastrointestinal cancer predisposition syndromes and on the surveillance/oncological care program of patients enrolled in a dedicated registry. METHODS: The study was conducted by means of an online self-report survey during the first Italian national lockdown. The survey comprised four sections: demographics; perception/knowledge of COVID-19; impact of the COVID-19 pandemic on surveillance and cancer care; health status (SF-12 questionnaire). RESULTS: 211 complete questionnaires were considered. 25.12% of respondents reported being not at all frightened by COVID-19, 63.98% felt "not at all" or "a little" more fragile than the healthy general population, and 66.82% felt the coronavirus to be no more dangerous to them than the healthy general population. 88.15% of respondents felt protected knowing they were monitored by a team of dedicated professionals. CONCLUSION: Patients with hereditary gastrointestinal cancer predisposition syndromes reported experiencing less fear related to COVID-19 than the healthy general population. The study results suggest that being enrolled in a dedicated registry can reassure patients, especially during health crises.

Potential of the Stromal Matricellular Protein Periostin as a Biomarker to Improve Risk Assessment in Prostate Cancer.

Prostate cancer (PCa) ranges from indolent to aggressive tumors that may rapidly progress and metastasize. The switch to aggressive PCa is fostered by reactive stroma infiltrating tumor foci. Therefore, reactive stroma-based biomarkers may potentially improve the early detection of aggressive PCa, ameliorating disease classification. Gene expression profiles of PCa reactive fibroblasts highlighted the up-regulation of genes related to stroma deposition, including periostin and sparc. Here, the potential of periostin as a stromal biomarker has been investigated on PCa prostatectomies by immunohistochemistry. Moreover, circulating levels of periostin and sparc have been assessed in a low-risk PCa patient cohort enrolled in active surveillance (AS) by ELISA. We found that periostin is mainly expressed in the peritumoral stroma of prostatectomies, and its stromal expression correlates with PCa grade and aggressive disease features, such as the cribriform growth. Moreover, stromal periostin staining is associated with a shorter biochemical recurrence-free survival of PCa patients. Interestingly, the integration of periostin and sparc circulating levels into a model based on standard clinico-pathological variables improves its performance in predicting disease reclassification of AS patients. In this study, we provide the first evidence that circulating molecular biomarkers of PCa stroma may refine risk assessment and predict the reclassification of AS patients.

Plasma miRNA-based signatures in CRC screening programs.

Colorectal cancer (CRC) screening programs help diagnose cancer precursors and early cancers and help reduce CRC mortality. However, currently recommended tests, the fecal immunochemical test (FIT) and colonoscopy, have low uptake. There is therefore a pressing need for screening strategies that are minimally invasive and consequently more acceptable to patients, most likely blood based, to increase early CRC identification. MicroRNAs (miRNAs) released from cancer cells are detectable in plasma in a remarkably stable form, making them ideal cancer biomarkers. Using plasma samples from FIT-positive (FIT+) subjects in an Italian CRC screening program, we aimed to identify plasma circulating miRNAs that detect early CRC. miRNAs were initially investigated by quantitative real-time PCR in plasma from 60 FIT+ subjects undergoing colonoscopy at Fondazione IRCCS Istituto Nazionale dei Tumori, then tested on an internal validation cohort (IVC, 201 cases) and finally in a large multicenter prospective series (external validation cohort [EVC], 1121 cases). For each endoscopic lesion (low-grade adenoma [LgA], high-grade adenoma [HgA], cancer lesion [CL]), specific signatures were identified in the IVC and confirmed on the EVC. A two-miRNA-based signature for CL and six-miRNA signatures for LgA and HgA were selected. In a multivariate analysis including sex and age at blood collection, the areas under the receiver operating characteristic curve (95% confidence interval) of the signatures were 0.644 (0.607-0.682), 0.670 (0.626-0.714) and 0.682 (0.580-0.785) for LgA, HgA and CL, respectively. A miRNA-based test could be introduced into the FIT+ workflow of CRC screening programs so as to schedule colonoscopies only for subjects likely to benefit most.

Short-term and long-term outcomes after preventive surgery in adolescent patients with familial adenomatous polyposis.

BACKGROUND: APC gene pathogenic variants are characterized by a lifetime risk of nearly 100% to develop a colorectal carcinoma. International guidelines suggest a prophylactic surgery in the second decade. METHODS: A descriptive analysis was performed evaluating a surgical series of adolescent patients with familial adenomatous polyposis (FAP) enrolled in the prospectively maintained hereditary polyposis registry. RESULTS: Thirty-eight adolescent patients (median age 16 years; range, 7-19) underwent laparoscopic prophylactic surgery. APC gene pathogenic variants were detected in all patients, and six patients were proband. No patients were converted to open surgery. Median postoperative stay was five days (4-16). Early postoperative complications were one dural puncture and one anastomotic leakage. Regarding late complications, we observed one patient having small bowel obstruction 56 months after surgery. Pathological reports showed one patient with pTis adenocarcinoma in two separate sites; 33 patients with low-grade dysplasia, four with high-grade dysplasia. One patient developed a desmoid tumor 37 months after surgery. After a median follow-up of 40.5 months, no patients died or had a second abdominal surgery because of cancer in rectal stump. CONCLUSIONS: Rectal sparing surgery was the first choice in the major respect of patients' quality of life. Laparoscopic prophylactic surgery for FAP is well accepted from adolescents. It represents a safe option due to the low incidence of post-surgical desmoids and quick postoperative recovery.

Early Modulation of Circulating MicroRNAs Levels in HER2-Positive Breast Cancer Patients Treated with Trastuzumab-Based Neoadjuvant Therapy.

Circulating microRNA (ct-miRNAs) are able to identify patients with differential response to HER2-targeted therapy. However, their dynamics are largely unknown. We assessed 752 miRNAs from 52 NeoALTTO patients with plasma pairs prior and two weeks after trastuzumab. Increased levels of ct-miR-148a-3p and ct-miR-374a-5p were significantly associated with pathological complete response (pCR) (p = 0.008 and 0.048, respectively). At a threshold ≥ the upper limit of the 95%CI of the mean difference, pCR resulted 45% (95%CI 24%-68%), and 44% (95%CI 22%-69%) for ct-miR-148a-3p and ct-miR-374a-5p, respectively. Notably, ct-miR-148a-3p retained its predictive value (OR 3.42, 95%CI 1.23-9.46, p = 0.018) in bivariate analysis along with estrogen receptor status. Combined information from ct-miR-148a-3p and ct-miR140-5p, which we previously reported to identify trastuzumab-responsive patients, resulted in greater predictive capability over each other, with pCR of 54% (95%CI 25%-81%) and 0% (95%CI 0%-31%) in ct-miR-148a/ct-miR-140-5p high/present and low/absent, respectively. GO and KEGG analyses showed common enriched terms between the targets of these ct-miRNAs, including cell metabolism regulation, AMPK and MAPK signaling, and HCC progression. In conclusion, early modulated ct-miR-148-3p may inform on the functional processes underlying treatment response, integrate the information from already available predictive biomarkers, and identify patients likely to respond to single agent trastuzumab-based neoadjuvant therapy.

Different pixel pitch and maximum luminance of medical grade displays may result in different evaluations of digital radiography images.

AIM: To evaluate the effects of display pixel pitch and maximum luminance on intra- and inter-observer reproducibility and observer performance when evaluating chest lesions and bone fractures. MATERIALS AND METHODS: This was a multi-institutional study for a retrospective interpretation of selected digital radiography images. Overall, 82 images were selected by senior radiologists, including 50 cases of chest lesions and 32 cases of bone fractures. These images were displayed at two pixel pitches (0.212 and 0.165 mm size pixels) and two maximum luminance values (250 and 500 cd/m2) and reviewed twice by senior and junior radiologists. All the observers had to indicate the likelihood of the presence of the lesions and to rate the relative confidence of their assessment. Cohen Kappa statistic was computed to estimate the reproducibility in correctly identifying lesions; for multi-reader-multi-case (MRMC) analysis, weighted Jackknife Alternative Free-response Receiver Operating Characteristic (wJAFROC) statistical tools was applied. RESULTS: The intra-radiologist and inter-observer reproducibility values were the highest for the 0.165 mm size pixel at 500 cd/m2 display, for both chest lesions and bone fractures evaluations. As regards chest lesions, observer performances were significantly greater with 0.165 mm size pixel display at 500 cd/m2 than with lower maximum luminance and/or larger pixel pitch displays. Concerning bone fractures, the performance obtained with 0.212 mm size pixel display at 250 cd/m2 was statistically lower than that obtained with 0.165 mm sixe pixel display at 500 cd/m2. CONCLUSION: Our results indicate that an increased maximum luminance level and a decreased pixel pitch of medical-grade display improve the accuracy of detecting both chest lesions and bone fractures.

Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors.

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

Plasma hepcidin in early-stage breast cancer patients: no relationship with interleukin-6, erythropoietin and erythroferrone.

OBJECTIVE: Hepcidin-25 production is stimulated by systemic inflammation, and it interferes with iron utilization, leading to anemia. This study aimed to investigate the relationships between the plasma levels of hepcidin, interleukin-6 (IL-6), erythropoietin (EPO) and erythroferrone (ERFE) in patients with benign breast disease or cancer. METHODS: Plasma samples from a cohort of 131 patients (47 with benign breast disease and 84 with breast cancer) were subjected to the evaluation of hepcidin, IL-6, EPO and ERFE using SELDI-TOF-MS or immunoassays. RESULTS: An elevated hepcidin was observed in malignant breast tumors compared to benign ones. No correlation was observed between hepcidin and IL-6, EPO or ERFE. CONCLUSION: Since the study included a cohort of patients (87%) with breast cancers smaller than 2 cm, these results may support our previous evidence about the potential role of hepcidin in breast cancer disease.

Peptide-nanoparticle ligation mediated by cutinase fusion for the development of cancer cell-targeted nanoconjugates.

The relationship between the positioning of ligands on the surface of nanoparticles and the structural features of nanoconjugates has been underestimated for a long time, albeit of primary importance to promote specific biological recognition at the nanoscale. In particular, it has been formerly observed that a proper molecular orientation can play a crucial role, first optimizing ligand immobilization onto the nanoparticles and, second, improving the targeting efficiency of the nanoconjugates. In this work, we present a novel strategy to afford peptide-oriented ligation using genetically modified cutinase fusion proteins, which combines the presence of a site-directed "capture" module based on an enzymatic unit and a "targeting" moiety consisting of the ligand terminal end of a genetically encoded polypeptide chain. As an example, the oriented presentation of U11 peptide, a sequence specific for the recognition of urokinase plasminogen activator receptor (uPAR), was achieved by enzyme-mediated conjugation with an irreversible inhibitor of cutinase, an alkylphosphonate p-nitrophenol ester linker, covalently bound to the surface of iron oxide nanoparticles. The targeting efficiency of the resulting protein-nanoparticle conjugates was assessed using uPAR-positive breast cancer cells exploiting confocal laser scanning microscopy and quantitative fluorescence analysis of confocal images. Ultrastructural analysis of transmission electron micrographs provided evidence of a receptor-mediated pathway of endocytosis. Our results showed that, despite the small average number of targeting peptides presented on the nanoparticles, our ligand-oriented nanoconjugates proved to be very effective in selectively binding to uPAR and in promoting the uptake in uPAR-positive cancer cells.

Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: The SPIDIA experience.

In this note, we present an ad hoc procedure that combines qualitative (visual evaluation) and quantitative (ImageJ software) evaluations of Pulsed-Field Gel Electrophoresis (PFGE) images to assess the genomic DNA (gDNA) integrity of analyzed samples. This procedure could be suitable for the analysis of a large number of images by taking into consideration both the expertise of researchers and the objectiveness of the software. We applied this procedure on the first SPIDIA DNA External Quality Assessment (EQA) samples. Results show that the classification obtained by this ad hoc procedure allows a more accurate evaluation of gDNA integrity with respect to a single approach.

Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience.

BACKGROUND: Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the pre-analytical phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for pre-analytical phase studies. METHODS: Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). RESULTS: The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. CONCLUSIONS: The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

Receptor tyrosine kinase profiles and human papillomavirus status in oropharyngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinomas (OSCCs) are two distinct entities. We defined the molecular profiles of druggable receptor tyrosine kinases (RTKs) in both groups. MATERIALS AND METHODS: E5 expression and RTK alterations were studied in 17 HPV-positive and 59 HPV-negative formalin-fixed OSCCs. RTK activation was explored in further 12 frozen OSCCs. RESULTS: The HPV-positive OSCCs showed E5 expression and 33.3% expressed low level of HER2. The HPV-negative OSCCs showed HER2 expression (31.2%), increased HER2 gene copy number (46.51%, P = 0.045) and HER2 activation through HER2/EGFR heterodimerisation; HER3 (51.06%, P = 0.008) and neuregulin (65.63%; P = 0.03) expression, HER3 activation and HER3/EGFR heterodimerisation; and increased IGF-1R copy number (40.50%, P = 0.021), high IGF-1R cDNA values (P = 0.002), IGF-1R activation and expression of IGF1/2 and amphiregulin. PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases. CONCLUSION: Human papillomavirus-positive and HPV-negative OSCC showed different RTK profiles. In HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRß. In HPV-negative cases, HER3 may be a promising druggable biomarker that deserves further investigation. PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCC, particularly in HPV-positive/PI3KCA-mutated OSCCs because they may be driven by PI3KCA mutation alone.

PALB2 sequencing in Italian familial breast cancer cases reveals a high-risk mutation recurrent in the province of Bergamo.

PURPOSE: Monoallelic germ-line deleterious mutations of PALB2 (partner and localizer of BRCA2) are associated with breast cancer risk and have been found in several populations, with carrier frequencies of ~1-2%. Initially, these mutations were considered to have moderate penetrance, but accumulating evidence now indicates that they are associated with much higher risk. METHODS: In this study, we sequenced the PALB2 coding regions unlinked to BRCA (breast cancer) genes in 575 probands from Italian breast cancer families recruited in Milan. RESULTS: We found 12 carriers (2.1%) of deleterious mutations, and none of the mutations was found in 784 controls collected in Milan. One of these mutations, the c.1027C>T (p.Gln343X), was found to be recurrent in the province of Bergamo in northern Italy, being detected in 6/113 (5.3%) familial breast cancer cases and 2/477 (0.4%) controls recruited in this area (Fisher's exact test: P < 0.01). CONCLUSIONS: Our data provide confirmatory findings that, in the Italian population also, deleterious mutations of PALB2 are relatively frequent predisposing factors for breast cancer and may be associated with high risk of the disease.

NqA: an R-based algorithm for the normalization and analysis of microRNA quantitative real-time polymerase chain reaction data.

In this note, we propose an R function named NqA (Normalization qPCR Array, where qPCR is quantitative real-time polymerase chain reaction) suitable for the identification of a set of microRNAs (miRNAs) to be used for data normalization in view of subsequent validation studies with qPCR data. NqA is available through the website of the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan (http://www.istitutotumori.mi.it/modules.php?name=Content&pa=showpage&pid=812) with a dedicated user's guide. We applied our function on a qPCR dataset downloaded from the Gene Expression Omnibus (GEO) database. Results show that NqA provides a functional subset of reference miRNAs and a set of promising significantly modulated miRNAs for subsequent validation studies.

Antiproliferative effect of ASC-J9 delivered by PLGA nanoparticles against estrogen-dependent breast cancer cells.

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with "multidrug" resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the in vitro drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed ad hoc. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer.