Francesca Pirini

Cold atmospheric plasma treatment selectively targets head and neck squamous cell carcinoma cells

The treatment of locoregional recurrence (LRR) of head and neck squamous cell carcinoma (HNSCC) often requires a combination of surgery, radiation therapy and/or chemotherapy. Survival outcomes are poor and the treatment outcomes are morbid. Cold atmospheric plasma (CAP) is an ionized gas produced at room temperature under laboratory conditions. We have previously demonstrated that treatment with a CAP jet device selectively targets cancer cells using in vitro melanoma and in vivo bladder cancer models. In the present study, we wished to examine CAP selectivity in HNSCC in vitro models, and to explore its potential for use as a minimally invasive surgical approach that allows for specific cancer cell or tumor tissue ablation without affecting the surrounding healthy cells and tissues. Four HNSCC cell lines (JHU-022, JHU-028, JHU-029, SCC25) and 2 normal oral cavity epithelial cell lines (OKF6 and NOKsi) were subjected to cold plasma treatment for durations of 10, 30 and 45 sec, and a helium flow of 20 l/min−1 for 10 sec was used as a positive treatment control. We showed that cold plasma selectively diminished HNSCC cell viability in a dose-response manner, as evidenced by MTT assays; the viability of the OKF6 cells was not affected by the cold plasma. The results of colony formation assays also revealed a cell-specific response to cold plasma application. Western blot analysis did not provide evidence that the cleavage of PARP occurred following cold plasma treatment. In conclusion, our results suggest that cold plasma application selectively impairs HNSCC cell lines through non-apoptotic mechanisms, while having a minimal effect on normal oral cavity epithelial cell lines.

Nuclear and Mitochondrial DNA Alterations in Newborns with Prenatal Exposure to Cigarette Smoke

Newborns exposed to maternal cigarette smoke (CS) in utero have an increased risk of developing chronic diseases, cancer, and acquiring decreased cognitive function in adulthood. Although the literature reports many deleterious effects associated with maternal cigarette smoking on the fetus, the molecular alterations and mechanisms of action are not yet clear. Smoking may act directly on nuclear DNA by inducing mutations or epigenetic modifications. Recent studies also indicate that smoking may act on mitochondrial DNA by inducing a change in the number of copies to make up for the damage caused by smoking on the respiratory chain and lack of energy. In addition, individual genetic susceptibility plays a significant role in determining the effects of smoking during development. Furthermore, prior exposure of paternal and maternal gametes to cigarette smoke may affect the health of the developing individual, not only the in utero exposure. This review examines the genetic and epigenetic alterations in nuclear and mitochondrial DNA associated with smoke exposure during the most sensitive periods of development (prior to conception, prenatal and early postnatal) and assesses how such changes may have consequences for both fetal growth and development.

Global and gene-specific DNA methylation pattern discriminates cholecystitis from gallbladder cancer patients in Chile

Aim The aim of the study was to evaluate the use of global and gene-specific DNA methylation changes as potential biomarkers for gallbladder cancer (GBC) in a cohort from Chile. Material & methods DNA methylation was analyzed through an ELISA-based technique and quantitative methylation-specific PCR. Results Global DNA Methylation Index (p = 0.02) and promoter methylation of SSBP2 (p = 0.01) and ESR1 (p = 0.05) were significantly different in GBC when compared with cholecystitis. Receiver curve operator analysis revealed promoter methylation of APC, CDKN2A, ESR1, PGP9.5 and SSBP2, together with the Global DNA Methylation Index, had 71% sensitivity, 95% specificity, a 0.97 area under the curve and a positive predictive value of 90%. Conclusion Global and gene-specific DNA methylation may be useful biomarkers for GBC clinical assessment.

Multiple-gene panel analysis in a case series of 255 women with hereditary breast and ovarian cancer

As new genes predisposing to breast (BC) and ovarian cancer (OC) are constantly emerging, the use of panels of genes analyzed by Next-Generation Sequencing (NGS) is increasing in clinical diagnostics. The identification of a large number of new germline mutations allows for deeper knowledge of cancer predisposition, although raising many questions about patient management. BC and OC patients recruited by our counseling service between 2012-2015 were included in this study. DNA was extracted from peripheral blood and a panel of 94 genes involved in hereditary tumors was analyzed by NGS. Patient clinical features of BC and OC and cancer family history were collected and compared to the patient genetic profile. A total of 255 women were analyzed, 57 of whom had a pathogenic mutation in BRCA1/2 genes, and 17 carried pathogenic mutations in other genes, such as PALB2, ATM, BRIP1, RAD51D, MSH6, PPM1D, RECQL4, ERCC3, TSC2, SLX4 and other Fanconi anemia genes. Patients with a pathogenic mutation in genes other than BRCA1 and BRCA2 showed no significant difference from the BRCA1/2-mutated carriers with respect to age at diagnosis and clinical features, suggesting that mutations in other genes could pose a high risk of cancer development. These patients had a much higher percentage of bilateral breast cancer (BBC) and a lower rate of OC than BRCA-mutated patients and patients with no pathogenic mutations: as a consequence, the surveillance protocol should be customized to the patient genetic characteristics.

Early detection of gastric cancer using global, genome-wide and IRF4, ELMO1, CLIP4 and MSC DNA methylation in endoscopic biopsies

Clinically useful molecular tools to triage gastric cancer patients are not currently available. We aimed to develop a molecular tool to predict gastric cancer risk in endoscopy-driven biopsies obtained from high-risk gastric cancer clinics in low resource settings. We discovered and validated a DNA methylation biomarker panel in endoscopic samples obtained from 362 patients seen between 2004 and 2009 in three high-risk gastric cancer clinics in Lima, Perú, and validated it in 306 samples from the Cancer Genome Atlas project (“TCGA”). Global, epigenome wide and gene-specific DNA methylation analyses were used in a Phase I Biomarker Development Trial to identify a continuous biomarker panel that combines a Global DNA Methylation Index (GDMI) and promoter DNA methylation levels of IRF4, ELMO1, CLIP4 and MSC. We observed an inverse association between the GDMI and histological progression to gastric cancer, when comparing gastritis patients without metaplasia (mean = 5.74, 95% CI, 4.97−6.50), gastritis patients with metaplasia (mean = 4.81, 95% CI, 3.77−5.84), and gastric cancer cases (mean = 3.38, 95% CI, 2.82−3.94), respectively (p < 0.0001). Promoter methylation of IRF4 (p < 0.0001), ELMO1 (p < 0.0001), CLIP4 (p < 0.0001), and MSC (p < 0.0001), is also associated with increasing severity from gastritis with no metaplasia to gastritis with metaplasia and gastric cancer. Our findings suggest that IRF4, ELMO1, CLIP4 and MSC promoter methylation coupled with a GDMI>4 are useful molecular tools for gastric cancer risk stratification in endoscopic biopsies.

Prenatal exposure to tobacco smoke leads to increased mitochondrial DNA content in umbilical cord serum associated to reduced gestational age

Abstract We investigated if prenatal exposures to tobacco smoke lead to changes in mitochondrial DNA content (mtDNA) in cord serum and adversely affect newborns’ health. Umbilical cord serum cotinine levels were used to determine in utero exposure to smoking. Cord serum mtDNA was measured by quantitative polymerase chain reaction analysis of the genes coding for cytochrome c oxidase1 (MT-CO1) and cytochrome c oxidase2 (MT-CO2). Log transformed levels of mtDNA coding for MT-CO1 and MT-CO2 were significantly higher among infants of active smokers with higher serum level of cotinine (p < 0.05) and inversely associated with gestational age (p = 0.08; p = 0.02). Structural equation modeling results confirmed a positive association between cotinine and MT-CO1 and2 (p < 0.01) and inverse associations with gestational age (p = 0.02) and IGF-1 (p < 0.01). We identified a dose-dependent increase in the level of MT-CO1 and MT-CO2 associated to increased cord serum cotinine and decreased gestational age.

INSIG2 rs7566605 single nucleotide variant and global DNA methylation index levels are associated with weight loss in a personalized weight reduction program

Single nucleotide polymorphisms associated with lipid metabolism and energy balance are implicated in the weight loss response caused by nutritional interventions. Diet-induced weight loss is also associated with differential global DNA methylation. DNA methylation has been proposed as a predictive biomarker for weight loss response. Personalized biomarkers for successful weight loss may inform clinical decisions when deciding between behavioral and surgical weight loss interventions. The aim of the present study was to investigate the association between global DNA methylation, genetic variants associated with energy balance and lipid metabolism, and weight loss following a non-surgical weight loss regimen. The present study included 105 obese participants that were enrolled in a personalized weight loss program based on their allelic composition of the following five energy balance and lipid metabolism-associated loci: Near insulin-induced gene 2 (INSIG2); melanocortin 4 receptor; adrenoceptor β2; apolipoprotein A5; and G-protein subunit β3. The present study investigated the association between a global DNA methylation index (GDMI), the allelic composition of the five energy balance and lipid metabolism-associated loci, and weight loss during a 12 month program, after controlling for age, sex and body mass index (BMI). The results demonstrated a significant association between the GDMI and near INSIG2 locus, after adjusting for BMI and weight loss, and significant trends were observed when stratifying by gender. In conclusion, a combination of genetic and epigenetic biomarkers may be used to design personalized weight loss interventions, enabling adherence and ensuring improved outcomes for obesity treatment programs. Precision weight loss programs designed based on molecular information may enable the creation of personalized interventions for patients, that use genomic biomarkers for treatment design and for treatment adherence monitoring, thus improving response to treatment.

Abstract LB-247: Early detection of gastric cancer using global and gene-specific promoter DNA methylation in endoscopic biopsies

Clinically useful molecular tools to triage gastric cancer patients are not currently available. We aimed to develop a molecular tool to predict gastric cancer risk in endoscopy-driven biopsies obtained from high-risk gastric cancer clinics in low resource settings. We discovered and validated a DNA methylation biomarker panel in endoscopic samples obtained from 362 patients seen between 2004 and 2009 in three high-risk gastric cancer clinics in Lima, Peru, and validated it in 306 samples from the Cancer Genome Atlas project (“TCGA”). Global, genome wide and gene-specific DNA methylation analyses were used in a Phase I Biomarker Development Trial to identify a continuous biomarker panel that combines a Global DNA Methylation Index (GDMI) and promoter DNA methylation levels of IRF4, ELMO1, CLIP4 and MSC. We observed an inverse association between the GDMI and histological progression to gastric cancer, when comparing gastritis patients without metaplasia (mean= 5.74, 95% CI, 4.97-6.50), gastritis patients with metaplasia (mean= 4.81, 95% CI, 3.77-5.84), and gastric cancer cases (mean=3.38, 95% CI, 2.82-3.94), respectively (p 4, is a useful molecular tool for gastric cancer risk stratification in endoscopic biopsies. Citation Format: Francesca Pirini, Sassan Noazin, Martha Jahuira Arias, Sebastian Rodriguez-Torres, Leah Friess, Christina Michailidi, Jaime Cok, Juan Combe, Goria Vargas, William Prado, Ethan Soudry, Jimena Perez, Tikki Yudin, Andrea Mancinelli, Helen Unger, Carmen Ili, Priscilla Brebi, Douglas Berg, Masamichi Hayashi, David Sidransky, Robert Gilman, Rafael E. Guerrero-Preston. Early detection of gastric cancer using global and gene-specific promoter DNA methylation in endoscopic biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2017-LB-247

Abstract B11: Viral and host gene methylation in liquid prep: novel molecular screening and triage tools to reduce cervical cancer disparities

Latino women in the United States have the highest cervical cancer incidence rate, yet the highest death rate from cervical cancer is among Black women. The disproportionate burden of cervical cancer in the United States is primarily due to lack of screening, among the medically underserved, regardless of race or ethnicity. Rural and inner city women living in poverty have lower rates of screening and higher rates of cervical cancer in the US. The advent of molecular diagnostics provides an opportunity to develop novel cervical cancer screening and triage tools that can be packaged as point of care and self-testing solutions. We set out to identify a panel of methylated HPV DNA and human genes that can discriminate between CIN2+ and normal/CIN1 patients in liquid prep samples and in Transrenal DNA (TrDNA) isolated from urine. We used three independent cohorts, from Chile, Baltimore and Puerto Rico, to develop a method that can be used to triage into colposcopy, HPV+ women with abnormal cytology. Participants were women with no cervical intraepithelial lesions or malignancy and women with abnormal cervical biopsies– Cervical Intraepithelial Neoplasia (CIN), carcinoma in-situ and cervical cancer. Using DNA methylation arrays for Discovery and quantitative Methylation Specific PCR (qMSP) for Validation, we found that promoter methylation of ZNF516, FKBP6, and INST1 discriminates samples with CIN2+ lesions from samples with no intraepithelial lesions or malignancy (NILM): 88.3% sensitivity, 88.9% specificity, 93.2 Area Under the Curve (AUC), 86.9% positive predictive value (PPV) and 90.2% negative predictive value (NPV). Using custom sequence capture pools of baits, we pulled down genomic and bisulfite converted high-risk HPV DNA before library prep for NGS in 454 and MiSeq instruments, respectively. Using our NGS results, we optimized a Syber Greeen qPCR assay to detect high risk HPV DNA and a qMSP primer-probe set to detect methylated HPV. We replicated the results in liquid prep samples (n=67), adding HPV16 methylation to the panel: 90.9% sensitivity, 60.9% specificity, 90.1 (AUC), 52.6% positive predictive value (PPV) and 93.3% NPV. These results were verified in plasma DNA (AUC=80.7) and TrDNA (AUC=86.1) isolated from a subset of patients who provided the liquid prep samples. Our results suggest that our panel of viral and host gene methylation markers may be used as a reflex test in liquid prep to triage high risk HPV positive women into colposcopy, or as a screening and triage test in TrDNA, in combination with our high risk HPV test. There are several commercial co-testing options for identification of oncogenic high-risk HPV types (HPV+) in patients with abnormal cytology. However, there are currently no tests that can reliably identify the HPV+ patients with abnormal cytology that need to be referred for colposcopy. Consequently, more than half of the women that are referred to colposcopy either have a negative biopsy, or a grade 1 Cervical Intraepithelial Neoplasia (CIN1) diagnosis, which usually reverts to normal in 12-24 months. A triage test from cytology to colposcopy that can identify in the liquid prep sample those patients with a CIN grade more likely to progress to cervical cancer (CIN2+), would decrease the number of unnecessary cervical mucosa biopsies performed today, decreasing health care cost and improving the quality of health care delivery. These molecular tools can also be packaged as point of care and self-testing solutions to eliminate cervical cancer disparities in medically underserved women. Note: This abstract was not presented at the conference. Citation Format: Rafael Guerrero-Preston, Anne Jedlicka, Blanca L. Valle, Nitesh Turaga, Liliana Florea, Oluwasina Folawiyo, Francesca Pirini, Fahcina Lawson, Angelo Vergura, Maartje Noordhuis, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Diaz, Edgardo De Jesus, Teresa Diaz-Montes, Bruce Trock, Keimari Mendez, Josefina Romaguera, David Sidransky. Viral and host gene methylation in liquid prep: novel molecular screening and triage tools to reduce cervical cancer disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B11.

Abstract A1-50: Genomic and epigenomic alterations in human and high-risk HPV DNA can discriminate normal from cervical dysplasia patients in urine

Human Papilloma Virus (HPV) testing is increasingly used for cervical cancer screening in conjunction with cervical cytology. Although privacy, cultural, and infrastructure issues challenge the effective implementation of HPV testing for cervical cancer screening worldwide, several countries have already implemented HPV testing in their screening protocols. There are currently no tests that can reliably identify the patients with abnormal cytology and positive oncogenic HPV results (HPV+) that need to be referred for colposcopy. A useful triage test from cytology to colposcopy must discriminate the patients with a Cervical Intraepithelial Neoplasia (CIN) lesion that are more likely to progress to cervical cancer (CIN2+), from those that are less likely to progress. We set out to identify a panel of methylated HPV and human genes that can discriminate between CIN2+ and normal/CIN1 patients, first in cytology samples and then in urine samples. The Reverse Line Blot Assay identified high-risk HPV types in study participants from a Discovery cohort (n=96): 31 women with normal cervical cytology and 65 women with dysplasia (LSIL, n=43) and (HSIL, n=22). We developed custom sequence capture pools of baits to pull down genomic (Roche SeqCap EZ) and bisulfite converted high-risk HPV DNA (Agilent SureSelect) from urine, before library prep for NGS in a Roche 454 and MiSeq instruments, respectively. We also optimzed a qPCR assay to identify high risk HPV types, and quantitative Methylation Specific PCR (qMSP) assays to examine methylated HPV and human genes, in Pap smear DNA and TrDNA samples from women with normal Pap and women with CIN1, CIN2 and CIN3 lesions (n=80). The human genes selected for the qMSP assays, ZNF516, FKBP6, and INTS1, were identified in a previously published genome-wide analysis of the cevical cancer methylome using Nimblegen 385K CpG plus Promoters oligonucleotide tiling arrays. Presence of high-risk HPV in cervical epithelium correctly classified 37% of HSIL when compared with LSIL patients, with 61% sensitivity and 22% specificity. Promoter methylation of INTS1 correctly classified 67% (31% sensitivity, 92% specificity), promoter methylation of ZNF516 correctly classified 54% (11% sensitivity, 84% specificity), promoter methylation of FKBP6 correctly classified 59% (19% sensitivity, 88% specificity), and methylation of the HPVL1 gene correctly classified 61% (22% sensitivity, 90% specificity) of HSIL patients when compared with LSIL patients. A molecular panel comprised of HPV16-L1 methylation and promoter methylation of INTS1, ZNF516 and FKBP6 correctly classified 70% of HSIL patients with an Area Under the Curve of 0.66, 44% sensitivity, 88% specificity and 72% Positive Predictive value. While HPV16-L1 methylation discriminated patients with normal cytology from women with cervical dysplasia with close to 100% sensitivity and specificity, the high-risk HPV TrDNA qPCR assay had a pre-capture and post-capture sensitivity of 79% with 50% specificity. The detection of circulating HPV DNA in urine is a cost-effective and non-invasive alternative for cervical cancer screening. We have shown that a panel of genomic and epigenomic alterations in human and high risk HPV DNA can discriminate normal from cervical dysplasia in cervical epithelium DNA and TrDNA isolated from patients seen in a high risk cervical cancer screening setting. Citation Format: Rafael Guerrero-Preston, Anne Jedlicka, Oluwasina Folawiyo, Francesca Pirini, Blanca L. Valle, Fahcina Lawson, Angelo Vergura, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Diaz, Liliana Florea, Teresa Diaz-Montes, Josefina Romaguera, David Sidransky. Genomic and epigenomic alterations in human and high-risk HPV DNA can discriminate normal from cervical dysplasia patients in urine. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-50.