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Context.—Lower Anogenital Squamous Terminology (LAST) standardization recommended p16INK4a immunohistochemistry (p16 IHC) for biopsies diagnosed morphologically as cervical intraepithelial neoplasia (CIN) grade 2 (CIN2) to classify them as low-grade or high-grade squamous intraepithelial lesions (HSILs).Objective.—To describe the relationships of p16 IHC and other biomarkers associated with cervical cancer risk with biopsy diagnoses.Design.—A statewide, stratified sample of cervical biopsies diagnosed by community pathologists (CPs), including 1512 CIN2, underwent a consensus, expert pathologist panel (EP) review (without p16 IHC results), p16 IHC interpretation by a third pathology group, and human papillomavirus (HPV) genotyping, results of which were grouped hierarchically according to cancer risk. Antecedent cytologic interpretations were also available.Results.—Biopsies were more likely to test p16 IHC positive with increasing severity of CP diagnoses, overall (Ptrend ≤ .001) and within each HPV risk group (Ptrend ≤ .001 except for low-risk HPV [Ptrend < .010]). All abnormal grades of CP-diagnosed biopsies were more likely to test p16 IHC positive with a higher HPV risk group (Ptrend < .001), and testing p16 IHC positive was associated with higher HPV risk group than testing p16 IHC negative for each grade of CP-diagnosed biopsies (P < .001). p16 IHC–positive, CP-diagnosed CIN2 biopsies were less likely than CP-diagnosed CIN3 biopsies to test HPV16 positive, have an antecedent HSIL+ cytology, or to be diagnosed as CIN3+ by the EP (P < .001 for all). p16 IHC–positive, CP-diagnosed CIN1 biopsies had lower HPV risk groups than p16 IHC–negative, CP-diagnosed CIN2 biopsies (P < .001).Conclusions.—p16 IHC–positive, CP-diagnosed CIN2 appears to be lower cancer risk than CP-diagnosed CIN3. LAST classification of “HSIL” diagnosis, which includes p16 IHC–positive CIN2, should annotate the morphologic diagnosis (CIN2 or CIN3) to inform all management decisions, which is especially important for young (

Background The severity of the 2017–2018 influenza season in the United States was high, with influenza A(H3N2) viruses predominating. Here, we report influenza vaccine effectiveness (VE) and estimate the number of vaccine-prevented influenza-associated illnesses, medical visits, hospitalizations, and deaths for the 2017–2018 influenza season. Methods We used national age-specific estimates of 2017–2018 influenza vaccine coverage and disease burden. We estimated VE against medically attended reverse-transcription polymerase chain reaction–confirmed influenza virus infection in the ambulatory setting using a test-negative design. We used a compartmental model to estimate numbers of influenza-associated outcomes prevented by vaccination. Results The VE against outpatient, medically attended, laboratory-confirmed influenza was 38% (95% confidence interval [CI], 31%–43%), including 22% (95% CI, 12%–31%) against influenza A(H3N2), 62% (95% CI, 50%–71%) against influenza A(H1N1)pdm09, and 50% (95% CI, 41%–57%) against influenza B. We estimated that influenza vaccination prevented 7.1 million (95% CrI, 5.4 million–9.3 million) illnesses, 3.7 million (95% CrI, 2.8 million–4.9 million) medical visits, 109 000 (95% CrI, 39 000–231 000) hospitalizations, and 8000 (95% credible interval [CrI], 1100–21 000) deaths. Vaccination prevented 10% of expected hospitalizations overall and 41% among young children (6 months–4 years). Conclusions Despite 38% VE, influenza vaccination reduced a substantial burden of influenza-associated illness, medical visits, hospitalizations, and deaths in the United States during the 2017–2018 season. Our results demonstrate the benefit of current influenza vaccination and the need for improved vaccines.

Objectives Human papillomavirus (HPV) testing for cervical screening has been shown to increase the yield of precancerous disease and reduce the incidence of cervical cancer more than cytology alone. Here we document the state-wide uptake of co-testing with HPV and cytology in women aged 30–64 years as recommended by national and international bodies. Methods Registry-based study of all screening cytology and HPV tests in New Mexico from 2008 to 2019 among women aged 21–64 years, with a focus on cytology negative tests to distinguish co-testing from reflex HPV testing to triage equivocal or mildly abnormal cytology. Results A total of 1,704,055 cervical screening tests from 681,440 women aged 21–64 years in the state of New Mexico were identified. The proportion of screening tests which were co-tests rose from 5.6% in 2008 to 84.3% in 2019 among women aged 30–64 years with a marked change from the near exclusive use of the Hybrid Capture II HPV test, (a signal amplified test method) to the use of target amplified HPV tests. The largest increases were seen between 2013 and 2015, reflecting the introduction and adoption of new clinical guidelines. Increases in co-testing were also seen in younger women. Conclusions Co-testing is now well established in women aged 30–64 years, but smaller increases have also been seen at younger ages, although this is not currently recommended. The impact of co-testing on cervical disease outcomes and number of colposcopies and biopsies in routine population settings remain important, especially in young women.

Biomarkers are chemical or biological markers that are indicators of internal exposures, disease status, or preclinical precursors to the onset of disease. In the last 30 years, major advances have been made in our ability to measure biomarkers that can indicate external exposures to environmental contaminants, track the progress of metabolic processes that convert them to toxins, and to determine the genetic susceptibility of certain populations to the impacts of environmental exposures. The use of biomarkers of exposure and biomarkers of disease in Medical Geology has been briefly disc in Chap. 2 of this book. The use of internal biomarkers in pharmacokinetic models to estimate external exposures is discussed in Chap. 9 by Bowers (2021) in this book. The purpose of this chapter is to introduce the theory and application of current techniques used in research and diagnostic laboratories in order to assess biological impact of environmental exposures. Environmental exposure examples which can be analyzed by each technique will also be discussed. Biomedical techniques are a broad group of scientific methods and assays, which have a wide clinical application across medicine. This chapter is designed to increase your theoretical, conceptual, and practical understanding of biomedical techniques used in Molecular Epidemiology.

Purpose The prevalence of Diabetes Type 2 is on the rise internationally. Currently, Fasting Plasma Glucose (FPG) and HbA1c are both used to determine if an individual is diabetic or prediabetic. We aimed to describe the prevalence of diabetes, prediabetes, and glycemic control in a population-based sample of elderly Hispanic and non-Hispanic White participants in New Mexico. Methods To do this, we compared HbA1c with FPG using Chi-Square analysis across gender and ethnicity to provide information for future health care policy. We also performed non-parametric regression using a locally weighted smoothing technique to investigate the relationship between FPG and HbA1c levels. Results Our analysis identifies a large variation between the sensitivity of HbA1c and FPG in the identification of both prediabetes and diabetes. Interestingly, 95% of diabetics defined by FPG are also defined by HbA1c, representing overlap between the two measures. When comparing the prevalence of prediabetes between the two measures, the overlap of FPG with HbA1c was only 30% and HbA1c identifies more individuals as prediabetic than FPG. Prevalence of diabetes was also higher when defined by HbA1c compared to FPG and the overall agreement between HbA1c and FPG appears to be poor particularly by sex and ethnicity (K=0.22-0.34). Glycemic control was poor overall with Hispanics displaying a larger amount of uncontrolled diabetes. Conclusion We compared HbA1c and FPG by gender and ethnicity and showed both measures of diabetes differ in their sensitivity across ethnic groups. Our results suggest that using HbA1c, rather than FPG, results in higher rates of prediabetes and diabetes, a finding with numerous implications for health care practice.

Purpose: Given rising rates of deadly melanoma skin cancer in Hispanics, the study objective was to examine skin cancer-related risk reduction behaviors and beliefs to dictate content for culturally targeted skin cancer prevention strategies for Hispanics. Methods/Data Source: An anonymous survey was administered to waiting room volunteers in a primary care facility in Albuquerque, New Mexico to assess skin cancer risk reduction behaviors, screening, cancer information seeking and communication, as well as skin cancer beliefs in Hispanics (n=48) and Non-Hispanic Whites (n=36). Results: We found lower levels of sun protection clothing use among Hispanics compared to Non-Hispanic Whites, but comparable use of sunscreen and shade-seeking among these groups. Hispanic ethnicity was the most important predictor of skin cancer misconceptions, with skin cancer information overload and misconceptions reported more often in Hispanics. Conclusions: This study demonstrates the need for culturally relevant information for ethnic minority populations such as Hispanics who have shown an increased risk of presenting with later stage, more aggressive melanoma skin cancer.

The current study was designed to delineate temporal changes in cardiomyocytes and mitochondria at the light and electron microscopic levels in hearts of mice exposed transplacentally to commonly used nucleoside analogs (NRTIs). Pregnant CD-1 mice were given 80 mg AZT/kg, 40 mg 3TC/kg, 80 mg AZT/kg plus 40 mg 3TC/kg, or vehicle alone during the last 7 days of gestation, and hearts from female mouse pups were examined at 13 and 26 weeks postpartum for histopathological or ultrastructural changes in cross-sections of both the ventricles and the interventricular septum. Using light microscopy and special staining techniques, transplacental exposure to AZT, 3TC, or AZT/3TC was shown to induce significant histopathological changes in myofibrils; these changes were more widespread at 13 weeks than at 26 weeks postpartum. While most light microscopic lesions resolved, some became more severe between 13 and 26 weeks postpartum. Transplacental NRTI exposure also resulted in progressive drug-specific changes in the number and ultrastructural integrity of cardiac mitochondria. These light and electron microscopic findings show that a subset of changes in cardiac mitochondria and myofibrils persisted and progressed months after transplacental exposure of an animal model to NRTIs, with combined AZT/3TC exposure yielding additive effects compared with either drug alone.

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)-didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 muM AZT-ddI (equimolar) for 3 days with or without 150 muM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 muM AZT in the absence or presence of 5-1,000 muM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation.

Experiments were performed to investigate the impact of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using cell cloning assays for assessing the effects of individual drugs/drug combinations in (1) TK6 human lymphoblastoid cells exposed in vitro and (2) splenic lymphocytes from male CD-1 mice exposed transplacentally on days 12–18 of gestation. In TK6 cells, dose-related increases in HPRT and TK mutant frequencies were found following 3 days of exposure to AZT or 3TC alone (33, 100, or 300 μM), or to equimolar amounts of AZT-3TC. Compared with single drug exposures, AZT-3TC coexposures generally yielded enhanced elevations in HPRT and TK mutant frequencies. Mutagenicity experiments with ABC alone, or in combination with AZT-3TC, were complicated by the extreme cytotoxicity of ABC. Exposure of cells either to relatively high levels of AZT-3TC short-term (100 μM, 3 days), or to peak plasma-equivalent levels of AZT-3TC for an extended period (10 μM, 30 days), resulted in similar drug-induced mutagenic responses. Among sets of mice necropsied on days 13, 15, or 21 postpartum, Hprt mutant frequencies in T-cells were significantly elevated in the AZT-only (200 mg/kg bw/day) and AZT-3TC (200 mg AZT + 100 mg 3TC/kg bw/day) groups at 13 days of age. These results suggest that the mutagenicity by these nucleoside analogs is driven by cumulative dose, and raises the question of whether AZT-3TC has greater mutagenic effects than AZT alone in perinatally exposed children. Environ. Mol. Mutagen. © 2007 Wiley-Liss, Inc.

Apoptosis and autophagy are physiologically necessary pathways that are vital for cell homeostasis. Apoptosis facilitates type I programmed cell death, while the autophagic survival mechanism counteracts apoptosis. Dysregulation in the homeostatic balance between these two essential cellular pathways has been linked to various diseases. We review relevant Janus molecules and their interactomes, as well as lysosomes which play multiple roles in apoptosis and autophagy, and discuss how targeted interventions can be used in cancer prevention and/or therapy.

Supplemental Digital Content is available in the text, Identification of positive staining is often qualitative and subjective. This is particularly troublesome in pigmented melanoma lesions, because melanin is difficult to distinguish from the brown stain resulting from immunohistochemistry (IHC) using horse radish peroxidase developed with 3,3′-Diaminobenzidine (HRP-DAB). We sought to identify and quantify positive staining, particularly in melanoma lesions. We visualized G-protein coupled estrogen receptor (GPER) expression developed with HRP-DAB and counterstained with Azure B (stains melanin) in melanoma tissue sections (n = 3). Matched sections (n = 3), along with 22 unmatched sections, were stained only with Azure B as a control. Breast tissue (n = 1) was used as a positive HRP-DAB control. Images of the stained tissues were generated using a Nuance Spectral Imaging Camera. Analysis of the images was performed using the Nuance Spectral Imaging software and SlideBook. Data was analyzed using a Kruskal–Wallis one way analysis of variance (ANOVA). We showed that a pigmented melanoma tissue doubly stained with anti-GPER HRP-DAB and Azure B can be unmixed using spectra derived from a matched, Azure B-only section, and an anti-GPER HRP-DAB control. We unmixed each of the melanoma lesions using each of the Azure B spectra, evaluated the mean intensity of positive staining, and examined the distribution of the mean intensities (P = .73; Kruskal–Wallis). These results suggest that this method does not require a matched Azure B-only stained control tissue for every melanoma lesion, allowing precious tissues to be conserved for other studies. Importantly, this quantification method reduces the subjectivity of protein expression analysis, and provides a valuable tool for accurate evaluation, particularly for pigmented tissues.

Although ultraviolet radiation (UV) exposure from indoor tanning has been linked to an increased risk of melanoma, the role of DNA repair genes in this process is unknown. We evaluated the association of 92 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes with the risk of melanoma and indoor tanning among 929 patients with melanoma and 817 controls from the Minnesota Skin Health Study. Significant associations with melanoma risk were identified for SNPs in ERCC4, ERCC6, RFC1, XPC, MGMT, and FBRSL1 genes; with a cutoff of P

Autophagy has been linked with melanoma, but no polymorphisms in autophagy related (ATG) genes have been investigated for association with melanoma prognostic indicators and survival. We examined 5 ATG gene single nucleotide polymorphisms (SNPs) in a large international multicenter population-based case-control study of melanoma. DNA from 911 melanoma patients was genotyped for five SNPs with a known or suspected impact on autophagic flux. While we did not identify an association with survival, a significant association was identified between the minor allele for an ATG16L polymorphism (rs2241880) and a decrease in Breslow thickness (p = 0.03), earlier tumor stage at diagnosis (OR 0.47, 95% CI 0.27-0.81, p = 0.02) and younger age at diagnosis (p = 0.02). In addition, two SNPs in ATG5 (rs2245214 and rs510432) were found to be significantly associated with increased tumor stage of melanoma (OR 1.84 95% CI 1.12-3.02, p = 0.05; OR 1.47 95% CI 1.11-1.94, p = 0.03). Finally, we identified inverse associations between the minor allele of rs2245214 and melanomas on the scalp or neck (OR 0.20, 95% CI 0.05-0.86, p = 0.03); rs1864182 (OR 0.42, 95% CI 0.21-0.88, p = 0.02) and brisk TILs, and rs510432 (OR 0.55 95% CI 0.34-0.87, p = 0.01) with non-brisk TILs, although they were not globally significant. In summary, our data suggests that ATG SNPs, while not associated with survival, may be associated with Breslow thickness, tumor stage, age at diagnosis, and aggressive histopathological factors. These associations could contribute to our current understanding of the significant role of autophagy in melanoma progression. Citation Format: Kirsten A. m. White, Li Luo, Todd A. Thompson, Salina Torres, Chien-An A. Hu, Nancy E. Thomas, Hoda Anton-Culver, Stephen B. Gruber, Lynn From, Klaus J. Busam, Irene Orlow, Peter A. Kanetsky, Lorraine D. Marrett, Richard P. Gallagher, Roberto Zanetti, Stefano Rosso, Terry Dwyer, Anne E. Cust, Allison Venn, Colin B. Begg, Marianne Berwick, Jenna Lillyquist. Variants in autophagy related genes and clinical characteristics in melanoma: a population-based study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1016.

The incidence of melanoma is continuing to increase worldwide. UV exposure is a known risk factor for melanoma. Geographic location is known to influence UV exposure and the distribution of the incidence of melanoma. Furthermore, epidemiologic data suggest that gender and genetics may influence the distribution of melanoma on the body surface and histopathologic characteristics of the lesion. This article describes what is known about the impact of gender, ethnicity and geography on the progression of melanoma. Advanced-stage cutaneous melanoma has a median survival time of less than 1 year. Surgical removal, radiotherapy, chemotherapy, targeted therapies and a variety of immunotherapies have been utilized in the treatment of melanoma. Current treatment strategies and the results of recent clinical trials are also discussed in this article.

To delineate temporal changes in the integrity and function of mitochondria/cardiomyocytes in hearts from mice exposed in utero to commonly used nucleoside analogs (NRTIs), CD-1 mice were exposed in utero to 80 mg AZT/kg, 40 mg 3TC/kg, 80 mg AZT/kg plus 40 mg 3TC/kg, or vehicle alone during days 12–18 of gestation and hearts from female mouse offspring were examined at 13 and 26 weeks postpartum. Alterations in cardiac mitochondrial DNA (mtDNA) content, oxidative phosphorylation (OXPHOS) enzyme activities, mtDNA mutations, and echocardiography of NRTI-exposed mice were assessed and compared with findings in vehicle-exposed control mice. A hybrid capture-chemiluminescence assay showed significant twofold increases in mtDNA levels in hearts from AZT- and AZT/3TC-exposed mice at 13 and 26 weeks postpartum, consistent with near doubling in mitochondrial numbers over time compared with vehicle-exposed mice. Echocardiographic measurements at 13 and 26 weeks postpartum indicated progressive thinning of the left ventricular posterior wall in NRTI-exposed mice, relative to controls, with differences becoming statistically significant by 26 weeks. Overall, progressive functional changes occurred in mouse mitochondria and cardiac tissue several months after in utero NRTI exposures; AZT and 3TC acted in concert to cause additive cardiotoxic effects of AZT/3TC compared with either drug alone.

Highly active antiretroviral therapy, which includes nucleoside reverse transcriptase inhibitors (NRTIs) as one of its components, is highly successful in controlling HIV-1 viral burden and reducing the rate of mother-to-child transmission of HIV-1 ( Public Health Task Force, 1998 ). However, experimental models of in utero NRTI exposure have provided evidence that NRTIs act as transplacental carcinogens and mitochondrial toxins. Several large cohort studies have found evidence for a causal relationship between perinatal NRTI exposure and mitochondrial toxicity in children born to HIV-infected women. Despite the varied findings of these studies and the conclusions drawn, it is widely accepted that these antiretroviral drugs have the potential to cause mitochondrial damage albeit the long-term health risks remain uncertain. One of the potential consequences of this NRTI-induced dysfunction is cardiotoxicity, which warrants long-term monitoring of exposed children. This chapter provides a brief review of the mode of action of NRTIs as antiretroviral agents and potential toxins, and evidence of mitochondrial toxicity in cardiac tissue and/or vasculature (endothelium and smooth muscle cells) of perinatally exposed animal models and children.

A sensitive vertical denaturing gradient gel electrophoresis (DGGE) method, using 13 unipolar psoralen-clamped PCR primer pairs, was developed for detecting sequence variants in the 22 tRNA genes and flanking regions (together spanning approximately 21%) of the human mitochondrial genome. A study was conducted to determine (i) if mitochondrial DNA (mtDNA) polymorphisms and/or mutations were detectable in healthy newborns and (ii) if prepartum 3'-azido-2',3'-dideoxythymidine (AZT) based HIV-1 prophylaxis was associated with significant increases in mtDNA mutations and changes in the degree of heteroplasmy of sequence variants in uninfected infants born to HIV-1-infected mothers. DGGE analysis of umbilical cord tissue (where vascular endothelium and smooth muscle cells are the major source of mtDNA) showed that mtDNA sequence variants were significantly elevated by threefold in AZT-treated infants compared with unexposed controls (P < 0.001), with 24 changes observed in 19/52 (37%) treated newborns (averaging 0.46 changes/subject) versus only eight changes found in 7/55 (13%) unexposed newborns (averaging 0.15 changes/subject). Six distinct sequence variants occurring in unexposed controls were predominately synonymous and homoplasmic, representing previously reported polymorphisms. Uninfected infants exposed to a combination of AZT and 2',3'-dideoxy-3'-thiacytidine and "maternal HIV-1" had a significant shift in the spectrum of mutations (P = 0.04) driven by increases in nonsynonymous heteroplasmic sequence variants at polymorphic sites (10 distinct variants) and novel sites (four distinct variants). While the weight of evidence suggests that prepartum AZT-based prophylaxis produces mtDNA mutations, additional research is needed to determine the degree to which fetal responses to maternal HIV-1 infection, in the absence of antiretroviral treatment, contribute to prenatal mtDNA mutagenesis.

Experiments were performed to investigate the impact of didanosine (ddI), lamivudine (3TC), and stavudine (d4T) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using a cell cloning assay for assessing the effects of individual nucleoside analogs (NRTIs)/drug combinations in human TK6 B-lymphoblastoid cells. Three-day treatments with 0, 33, 100, or 300 microM ddI, 3TC, or ddI-3TC produced positive trends for increased HPRT and TK mutant frequencies. While dose-related trends were too small to reach significance after treatments with d4T or d4T-3TC, pairwise comparisons with control cells indicated that exposure to 100 microM d4T or d4T-3TC caused significant elevations in HPRT mutants. Measurements of mutagenicity in cells exposed to d4T (or d4T-3TC) were complicated by the cytotoxicity of this NRTI. Enhanced increases in mutagenic responses to combined NRTI treatments, compared with single drug treatments, occurred as additive to synergistic effects in the HPRT gene of cells exposed to 100 microM ddI-3TC or 100 microM d4T-3TC, and in the TK gene of cells exposed to 100 or 300 microM ddI-3TC. Comparisons of these data to mutagenicity studies of other NRTIs in the same system (Meng Q et al. [2000c]: Proc Natl Acad Sci USA 97:12667-126671; Torres SM et al. [2007]: Environ Mol Mutagen) indicate that the relative mutagenic potencies for all drugs tested to date are: AZT-ddI > ddI-3TC > AZT-3TC congruent with AZT-3TC-ABC (abacavir) > AZT >/=ddI > d4T-3TC > 3TC > d4T >/= ABC. These collective data suggest that all NRTIs with antiviral activity against HIV-1 may cause host cell DNA damage and mutations, and impose a cancer risk.

The 5-year survival rate for stage IV melanoma is estimated to remain unchanged at only 15%, even with new therapies. One emerging hallmark of cancer is the reprogramming of energy metabolism including the role of autophagy. In cancer, autophagy appears to be an important energy source for nutrient-depleted tumors. Ultraviolet-radiation (UVR) exposure is an established risk factor for melanoma and increases autophagic flux. We hypothesize that UV exposure is independently associated with both levels of autophagy and melanoma survival. To address this hypothesis, we are investigating the association between UVR exposure, autophagy, and development of melanoma. Basal levels of autophagy appear to vary by tumor stage, and higher levels of autophagy are associated with poor clinical prognosis. Melanoma cells contain high levels of autophagosomes, a cellular structure associated with autophagy, as measured by the proxy marker LC3. Also, the expression of the autophagy marker Beclin 1 has been associated with increased risk of early death from melanoma. These findings suggest that autophagy provides metabolic support for tumors as it localizes to hypoxic tumor areas, supports tumor cell survival, and is activated in response to stress. Autophagy can act as a cytoprotective mechanism against UV-induced apoptosis. We have shown that rates of autophagy vary by oncogene status. Autophagy was evaluated in formalin-fixed, paraffin embedded melanoma and normal tissue sections, by staining for LC3 and Beclin 1 using IHC. These markers of autophagy, along with clinical tumor characteristics, were correlated with extensive questionnaire data on UV exposure from the same patients to evaluate the impact of estimated lifetime sun exposure and sunburns on survival. A trend in the expression of Beclin1 was not observed; however, LC3 expression was increased in melanoma sections compared to normal. Our results provide enticing support that lifetime UVR exposure is associated with autophagy protein expression in melanoma. Citation Format: Kirsten A. M. White, Salina Torres, Todd A. Thompson, Chien-An A. Hu, Orrin Myers, Marianne Berwick. LC3 protein expression associates with UV exposure in melanoma histopathology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2909. doi:10.1158/1538-7445.AM2015-2909

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.

One person dies from melanoma every hour worldwide. The American Cancer Society estimates that in 2013 the 5-year survival rate for stage IV melanoma will remain unchanged at about 15%, even with new therapies. Autophagy (self-eating pathway) is a cellular recycling pathway that has been shown to play a role in normal physiological processes including nutrient and/or stress responses, antigen presentation, and aging. In melanoma, basal levels of autophagy appear to vary across tumor types and higher levels of autophagy are associated with increased hypoxia and poor clinical prognosis. Understanding the relationships between autophagy, melanoma and oncogene status will elucidate the molecular mechanisms/pathways contributing to this disease. Our preliminary research indicates that cutaneous malignant melanoma (CMM) cell lines differ in their response to chloroquine (CLQ), a classic inhibitor of autophagy. This study was designed to determine how differences in the autophagy pathway are influenced by oncogene status and whether induction of autophagy is oncogene dependent. Using LC3-II puncta and western blot quantification we evaluated the differential rate of autophagy by oncogene status using CMM cell lines. The rate of autophagy was characterized in 6 melanoma cell lines (e.g. SK-Mel 2, 19, 29, 94,103, and 147) and primary melanocytes to determine the extent to which these cell lines responded to CLQ and determined any decrease in cellular viability caused by treatment. Two common cutaneous melanoma activating mutations, BRAF and NRAS, appear to have opposing roles on the autophagy pathway in our metastatic cell lines. Our data indicate that the targeting of autophagy using CLQ in the BRAF oncogenic cell lines increases apoptosis and leads a significant decrease in viability. In addition, BRAF cell lines have higher basal protein expression of ATG12-ATG5 and LC3-II and a higher number of basal LC3-II punctae. Finally, the induction of autophagy through serum starvation results in differential basal LC3-II kinetics as measured by number of punctae between the BRAF and NRAS cell lines. These results indicate that BRAF is more dependent on autophagy for survival than NRAS mutant melanoma and that oncogene status dictates the sensitivity of melanoma cells to CLQ by altering autophagic flux, subsequently increasing apoptosis. Ongoing experiments include the evaluation of autophagy in patient samples to quantify expression of known autophagy markers as well an evaluation of LC3-II expression correlated with oncogene status. These findings are immediately translatable for patients that would benefit from CLQ co-therapy, which is already approved for clinical use. Citation Format: Kirsten A. M. White, Salina M. Torres, Todd A. Thompson, Chien-An A. Hu, Natalia J. Gurule, Marianne Berwick. Differential inhibition of autophagic pathways in melanoma is oncogene-dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2635. doi:10.1158/1538-7445.AM2014-2635

Metastatic melanoma offers an ongoing clinical challenge. According to the American Cancer Society, the 5-year survival rate in 2012 for stage IV melanoma is only about 15% even with therapies. To improve outcomes, it is important to investigate new pathways involved in the regulation of metastasis as possible targets for prevention or treatment, such as autophagy. Autophagy (self-eating pathway) destroys a cell's damaged proteins and organelles through lysosomal degradation and has been shown to play roles in a wide variety of normal physiological processes including energy metabolism, nutrient or stress responses, growth regulation, and aging. The role of dysregulated autophagy in melanoma has not been well characterized. Preliminary research found cutaneous malignant melanoma cells display high levels of autophagy suggesting that this cellular process may provide an active metabolic state for invasive and metastatic melanoma. Chloroquine (CLQ), a classic antimalarial agent, is a potent inhibitor of autophagy and has been used safely in human patients for decades. This study is designed to dissect the role of autophagy in melanoma using melanoma cell lines (e.g. SK-Mel 19, 103, and 147), and primary melanocytes as controls to determine whether these cell types respond to CLQ as measured by cell viability . Similar to pancreatic and prostate cancer cell lines, our preliminary results indicate that metastatic melanoma cells require autophagy for growth and show high sensitivity to CLQ at doses as low as 20uM which resulted in greater than 60% decreases in cell viability. These findings suggest that metastatic melanoma cell lines rely on lysosomal degradation to a greater extent than what has been reported for other cell types. In addition, both N-ras mutation and androgen deprivation mitigate the effects of CLQ on melanoma cells. Ongoing experiments will confirm the expression of autophagy related genes, as well as imaging to determine the rate of autophagy in these cell lines. Finally, we will introduce stripped hormonal factors including vitamin D and estrogen, to evaluate whether this addition will progressively restore the original chloroquine sensitive phenotype. Findings from this work have the potential to be immediately translatable to patients as CLQ is already approved for therapeutic use in other human diseases. Citation Format: Kirsten A. White, Salina M. Torres, Natalia J. Gurule, Marianne Berwick, Chien-An A. Hu. Inhibition of autophagy in melanoma: using cell lines to model chloroquine sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2041. doi:10.1158/1538-7445.AM2013-2041

Dysregulated activation of the epidermal growth factor receptor (ErbB) family (EGFR, ErBB2, ErbB3, and ErbB4) is involved in promoting the cellular growth of human melanoma. EGFR, ErbB3, and ErbB4 are promising targets of malignant melanoma for new molecular drug development. However, the biology of ErbB family signaling pathways indicates that functional redundancy and heterodimerization among ErbB family members can greatly compromise the therapeutic efficacy of single ErbB targeting tyrosine kinase inhibitors (TKIs). In fact, Phase II clinical trials have already shown that EGFR (ErbB1) targeting TKIs provide limited benefits to melanoma patients. Here, this study has compared single vs. multiple ErbB targeting TKIs for inhibiting the growth of melanoma cells with different ErbB family protein expression profiles. We examined the expression of ErbB family members in 14 different human melanoma cell lines (MEL-526, M14, UPCI-MEL 527.1 and 11 SK-MEL cells). EGFR was expressed in 5 of the cell lines examined (35.7 %). ErbB2 was present in 13 lines (92.9 %); ErbB3 was found in 12 lines (85.7 %); and ErbB4 was found in all the cell lines. Furthermore, individual cell lines had a particular dominant form of ErbB protein. This differential expression pattern of ErbB family proteins was also seen in melanoma tissues from patients. Out of 14 melanoma cases examined by immunohistochemistry, 8 samples (61.5 %) were positive for ErbB1 and ErbB4 expression while ErbB2 was detected in 11 samples (84.6 %). We also found that Neuregulin 3 (which ranged from 500 to 76500 pg/ml) and Neuregulin 4 (which ranged from 26-300 pg/ml), which activate ErbB3 and/or ErbB4, are the major ErbB ligands released by the human melanoma cell lines. TGFβ and Amphiregulin, which mainly bind to EGFR, were released in relatively lower levels by the majority of the cell lines, but high levels of these ligands were distinctly found in individual cell lines. Among all the cell lines examined, HB-EGF was barely undetectable. In addition, different sensitivities towards single ErbB targeting tyrosine kinase inhibitors (TKIs) and pan-ErbB targeting TKIs were seen among the melanoma cells. For example, the TKI sensitivities of MEL526 which expressed no EGFR but high ErbB2, 3, and 4 proteins, was found to be: lapatinib (IC50: 6.7 μM) > canertinib (IC50: 12.4 μM) = erlotinib (IC50: 12.9 μM) > gefitinib (IC50: 23 μM). Similarly, M14 and UPCI-MEL527.1, which also have no EGFR expression but express ErbB 2, 3, 4, also responded better to lapatinib and canertinib respectively than to gefitinib or erlotinib. Our study is the first to suggest that the pan-ErbB targeting approach may be more effective in treatment malignant melanoma. Our findings also suggest that future screening of melanoma patients for their dominantly expressed ErbB protein types can potentially serve as a guide for the choice of effective ErbB TKI in treating malignant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3864. doi:1538-7445.AM2012-3864

Progressive myocardial dysfunction, which can lead to cardiomyopathy, congestive heart failure, and sudden cardiac death, is a well-recognized late effect of several agents used in the treatment of childhood cancer. While the mechanism of this damage is not yet fully characterized, mounting data from rodent models implicate treatment-induced mitochondrial DNA (mtDNA) mutations as one potential mechanism of late cardiotoxicity. In the current study, the Dana-Farber Cancer Institute Acute Lymphoblastic Leukemia (ALL) Consortium collected peripheral blood mononuclear cell samples from 93 long-term ALL survivors who were 4 or more years post-doxorubicin and other multi-agent therapy including asparaginase, corticosteroids, vincristine, and methotrexate, as well as radiation therapy. Data from healthy newborn children (n=45) represented a negative control and data from children receiving highly active antiretroviral therapy (HAART) (mean age=11.3 years; SD=2.61; n=51) provided a positive control for mtDNA mutations. Maximum cumulative doxorubicin dose varied among patients by treatment protocol (median=300 mg/m2; range 45 mg/m2 to 470 mg/m2). Median age at treatment was 4.5 years (range=0.5 to 20.8 years) and at mtDNA screening, 14.9 years (range=6.0 to 41.1 years). The mitochondrial tRNA genes and flanking regions were screened via PCR-based denaturing gradient gel electrophoresis (DGGE). Preliminary data showed 47 confirmed polymorphisms or mutations in 39 of 93 ALL survivors screened (42%), occurring in PCR products from 6 of 13 primer sets. The mutation incidence in HIV positive controls receiving HAART was 35 changes in 25 of 51 patients (49%). To date, the negative control population data suggest the occurrence of very few to no polymorphisms or mutations. Most mtDNA changes in ALL survivors were distinct from those in children receiving HAART, and included some sequence variants with suggested pathogenic characteristics. Analysis of cardiac function is ongoing. These data suggest that childhood ALL and its treatment may lead to mutations, over-expression of rare polymorphisms, and the induction of persistent changes that when spontaneously occurring have been associated with clinically significant cardiac effects. Conclusive evidence that doxorubicin-containing multi-agent therapy leads to persistent mtDNA mutations fundamental to cardiac dysfunction might allow for the isolation and prevention of these cardiotoxic effects.