Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of α-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of α-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (1H NMR and 13C NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained α-mangostin was 95.6% as determined by HPLC analysis. The binding of native α-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that α-mangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to α-mangostin is 6.4832×105 L/mol and 1.4652×105 L/mol at 298 K and 7.8619×105 L/mol and 1.1582×105 L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between α-mangostin and HSA or TRF were also obtained by virtue of the Förster theory of non-radiation energy transfer. The effect of α-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between α-mangostin and HSA is hydrophobic interactions, while the main interaction between α-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.In order to obtain the biological active compound, α-mangostin, from the traditional native mangosteen ( Garcinia mangostana L.) is a member of the Garcinia genus, which mainly grows in Thailand, Vietnam, Malaysia, Indonesia, Philippines and other Southeast Asian countries. It is also widely cultivated in Guangxi, Hainan and Zhejiang provinces as well as other areas of China. Its rind has been used as traditional medicines in Southeast Asia to treat abdominal pain, diarrhea, dysentery, cholera, infectious wounds, purulence, chronic ulcers and other diseases. In addition, it also exhibits the effect of anti-inflammatory, antibacterial, anti-malarial, lowering blood pressure, anti-oxidation, anti-HIV, immune regulation, and many other pharmacological activities. It has been established that mangosteen contains a variety of active ingredients, including xanthones, phenolic acids, polysaccharides and pigments , 411 [M + 2], 355 [M—C4H6]. UV λmax: 213, 245, 332. IR: νmax: 3620, 3420, 3260: [-OH]; 2920, 2860, 2720: ; 1640, 1610, 1410, 1380: [Ph]; 1280, 1190: [-O-]; 1080: [-C-O-]; 995: [-CH = CH-]; 850, 667: [Ph-H]. 1H-NMR :1. 65 , 1.73 , 3.698 , 3.77 , 5.69 , 5.843 , 6.155 , 6.677 , 13.698 . 13C-NMR : 16.568 (C-15 and C-19), 20.235 (C-16), 24.238 (C-11), 25.064 (C-14 and C-20), 59.307 (7-OMe), 91.383 (C-4), 100.93 (C-9a), 101.601 (C-5), 109.251 (C-2), 109.841 (C-8a), 123.049 , 129.634 , 136.173 (C-8), 142.806 (C-7), 153.932 (C-6), 154.373 (C-10a), 156.085 (C-4a), 159.759 (C-3), 161.663 (C-1), 181.052 (C-9).α-Mangostin was extracted and separated using HPD-400 macroporous resin. GC-MS and NMR spectroscopy was utilized to identify the recrystallized α-mangostin. The results of mass spectra, The analysis results of the IR- and UV-spectra are summarized in Through the analysis, we confirmed that the crystallized sample is α-mangostin of high purity. The IR, UV, MS and NMR spectral results were in accordance with those previously reported ,25. The It can be seen from It can be seen from Fcorr is the value of fluorescence after correction, Fobs is the measured value of fluorescence, A(λexc) is the absorption value at the excitation wavelength, A(λem) is the absorption value at the emission wavelength. All the values of fluorescence intensity were corrected in this study.Mechanisms of fluorescence quenching are usually classified into dynamic quenching and static quenching. The dynamic fluorescence quenching conforms to the Stern-Volmer equation. In order to determine the mechanism of fluorescence quenching between α-mangostin and serum protein, the system mentioned above could be analyzed by the Stern-Volmer equation:F0 and F are fluorescence intensities of the biomacromolecule in the absence and presence of added quencher, respectively; [D] is the concentration of the quencher; Kq is the rate constant of dimolecular quenching; τ0 is the average lifetime of the biomacromolecule without quencher, mol/L; Ksv is the quenching constant of Stern-Volmer, L/mol (Kq = KSV/τ0).Stern-Volmer plots of the fluorescence quenching between HSA or TRF and α-mangostin are shown in Kq dropped significantly with increasing temperature (Kq is three orders of magnitude higher than the maximum scatter collision quenching constant (2.0×1010 L·mol-1s-1), indicating a static quenching mode for α-mangostin upon the binding of HSA and TRF.By the variable temperature experimental method, we could directly determine the fluorescence quenching mechanism. If the temperature effect is negative, the quenching is static quenching; if the temperature effect is positive, the quenching is dynamic –29. It wperature , which pK) between biomacromolecule and quenching molecules may be calculated by Eq  beF0 and F are fluorescence intensities of biomacromolecule in the absence and presence of the quencher, respectively; [Dt] is the concentration of the quencher; K is the binding constant, L/mol; n is the number of binding sites. Combining the experimental data with Eq (Dt] and lg[(F0-F) /F] for each binding reaction system, resulted in plots as shown in  with Eq  by drawiE is dependent on the binding distance r and the critical energy transfer distance R0 as shown in Eq ; N is the refractive index of the medium (N = 1.336); Φ is the light quantum efficiency (Φ = 0.13) of the donor (protein) [J is the overlap integral between fluorescence emission spectrum of the donor and absorption spectrum of the acceptor (drugs), cm3∙L/mol. J can be described as:Where um N = 1.6; Φ is tprotein)  and J isF(λ) is the fluorescence intensity of the fluorescence donor at the wavelength λ and ε(λ) is the molar absorption coefficient of the acceptor at the wavelength λ, L/(mol·cm).E can be calculated by Eq  in the HSA-α-mangostin system than in the TRF-α-mangostin system. The binding distance (r) is smaller in the HSA-α-mangostin system than in the TRF-α-mangostin system, which indicates that the HSA-α-mangostin system is more stable. These results are in accordance to the results of the fluorescence quenching experiment described above. (2) The transfer efficiency (E) of the HAS-α-mangostin and TRF-α-mangostin systems was high and the binding distance of both systems was about 3 nm.The following conclusions may be drawn by an analysis: (1) The differences in molecular structure between HSA and TRF led to a higher energy transfer efficiency , IVH and IVV are the vertically polarized emission intensity and horizontally polarized emission intensity, respectively, under the polarized light of the vertical excitation. IHV and IHH are the vertically polarized emission intensity and horizontally polarized emission intensity, respectively, under the polarized light of the horizontal excitation.Where r may be calculated by the following formula:Anisotropy The fluorescence polarization parameters of α-mangostin-HSA and TRF systems are summarized in P and a small anisotropy r. Due to the differences of the molecular structure of the two proteins, the rotational speed of TRF in buffer solution was faster than that of HSA, which was reflected in a lesser degree of polarization P and anisotropy r of TRF compared to that of HSA. (2) After adding the drug, α-mangostin combined with the serum proteins. If the bonding is tight, the fluorescence molecules will have long relaxation time and the degree of fluorescence polarization P will be high; conversely, if the bonding is weak, the fluorescence molecules will have short relaxation time and the degree of fluorescence polarization P will be low. After complex formation with the drug, the degree of polarization P and anisotropy r of TRF became lower, indicating that the complex generated from the binding of α-mangostin to TRF had short relaxation time and the bonding was weak. While after complex formation with the drug, the degree of polarization P and anisotropy r of HSA became higher, indicating that the complex generated from the binding of α-mangostin to HSA exhibited a long relaxation time and the bonding was tight. Generally speaking, as for the α-mangostin-HSA or -TRF system, the fluorescence polarization experiment reconfirmed that α-mangostin and HSA or TRF could interact with each other, which affected the conformation of the protein. The complex between α-mangostin and HSA was stronger than that of TRF and it was consistent with the results obtained from the fluorescence spectra above.The degree of polarization of phosphor was connected with rotational speed of the fluorescent molecule; the faster the rotational speed, the smaller the fluorescence polarization; the slower the rotational speed, the greater the fluorescence polarization . From TaG < 0; hydrophobic interaction exists in the α-mangostin-HSA/TRF system when ΔH > 0 and ΔS > 0; hydrogen bonds and van der Waals forces exist in the α-mangostin-HSA/TRF system when ΔH < 0 and ΔS < 0; mainly electrostatic attraction exists in the α-mangostin-HSA/TRF when ΔH < 0 and ΔS > 0 [The main interaction between small drug molecules and proteins are hydrophobic interactions, hydrogen bonds and van der Waals forces, electrostatic forces, etc . The intd ΔS > 0 .K that has been calculated, we could calculate the thermodynamic parameters of the binding reaction between α-mangostin and serum proteins and the calculated thermodynamic parameters are listed in According to Eqs , 12), ,  and the G of the two systems is negative, indicating a spontaneous binding process between α-mangostin and HSA/TRF. HSA-α-mangostin has a smaller ΔG, indicating that HSA binds stronger than TRF to α-mangostin. The TRF-α-mangostin complex exhibits negative ΔH- (-162 kJ/mol) and ΔS-values (-442 kJ/mol), suggesting that van der Waals forces and hydrogen bonding play major roles in this binding [H- (12.3 kJ/mol) and ΔS-values (153 kJ/mol), indicating the dominant role of hydrophobic forces during this binding process. The observed distinct different binding of α-mangostin is mainly due to structural differences of HSA and TRF. binding ,42. Howei.e. accurate ligand geometry optimization, energy minimization, charge calculation, docking calculation and protein-ligand complex representation. Thus, the use of molecular docking allows the user to carry out highly efficient and robust docking calculations by integrating a number of popular software used in in silico chemistry into one comprehensive web service. Molecular docking is established based on experimental methods, and the molecular docking method is only one way. The results of molecular docking are verified by experiment [The molecular docking method offers a web-based, easy to use interface that handles all aspects of molecular docking from ligand and protein set-up. Molecular docking integrates a number of computational chemistry software specifically aimed at correctly calculate parameters needed at different steps of the docking procedure, periment . In thisperiment –46 and mperiment . The modE = -8.71 kcal/mol. The distance between α-mangostin and HSA tryptophan residues (Trp214) and phenylalanine residues  is small, which provides convincing evidence that α-mangostin has fluorescence quenching effect on the HSA molecule. In the process of α-mangostin molecule binding to HSA, two hydrogen bonds involving Ser202 with 7-OCH3 of α-mangostin and Ser202 with 6-OH of α-mangostin are formed. In addition, non-covalently bound α-mangostin is at the active site of HSA and a hydrophobic area around its binding pockets is mainly made up of Trp214, Phe206, Phe211, Ala194, Ala210, Ala213, Ala215, Ala217, Ala291, Leu198, Leu203, Leu219, Leu234, Leu238, Val216, Val235, Val241, Val343, and Val455. This indicates that the hydrophobic interaction at the binding site between α-mangostin and HSA is the main driving force of the binding. From the results of molecular simulation, we may conclude that the main interaction between α-mangostin and HSA is hydrophobic interaction but also that hydrogen bonds exist, which is consistent with the results of the thermodynamic experiment.E = -6.94 kcal/mol. The distance between α-mangostin and TRF tryptophan residues (Trp264) is small, which provides convincing evidence that α-mangostin has fluorescence quenching effect on the TRF molecule. In the process of α-mangostin molecule binding to TRF, three hydrogen bonds involving Lys304 with 3-OH of α-mangostin, Asn268 with 6-OH of α-mangostin and Asn268 with 7-OCH3 of α-mangostin are formed. In addition, non-covalently bound α-mangostin is at the active site of TRF and a hydrophobic area around its binding pocket is mainly made up of Trp264, Ala 82, Ala 270, Leu303, Val81, Val305, Pro306, Pro307, and Met309. This indicates that hydrophobic interactions at the binding site exist between α-mangostin and TRF. From the results of molecular simulation, we may conclude that the main interactions between α-mangostin and TRF are hydrogen bonds but also that hydrophobic interaction exists, which is consistent with the results of the thermodynamic experiment.Comparing Figs nm, indicating that an energy transfer exists between the α-mangostin and HSA/TRF. After comprehensively comparing the Ka-value with the Ksv-value, we found that the free binding energy during docking is consistent with the stability of the α-mangostin-HSA complex as determined by spectroscopy. The Ksv-value reflects the strength of drug interaction with the proteins, while the spectroscopically determined Ka-value indicates the binding strength between α-mangostin and HSA/TRF. The determined Ksv-value is consistent with the Ka-value obtained. The Ka-value is closely related to the free binding energy during docking; the greater the Ka value, the smaller the free binding energy, which indicates increased stability of the α-mangostin-HSA/TRF complex. According to current literature, drug binding alters protein conformation. In general, there are three commonly used methods to investigate this, i.e. the detection by the CD spectrum, the attenuated total reflection infrared spectrum (ATR-IR), and the fluorescence spectrum [In this paper, α-mangostin of relatively high purity was extracted successfully using macroporous resin. The extraction process engineering is simple, inexpensive with high yields. The reaction mechanism between α-mangostin and HSA/TRF has been studied by fluorescence and UV spectroscopy, as well as by computer molecular simulation. We calculated binding, energy transfer efficiency and thermodynamic parameters and results of molecular docking of reaction systems. The experimental results show that the fluorescence quenching mechanism between α-mangostin and HSA/TRF were both static quenching and the binding distances between α-mangostin and HSA/TRF were both less than 7 spectrum –46. In tCombining thermodynamic experiments with the molecular modeling studies, we obtained binding domains, binding sites of non-covalent interaction, amino acid residues, mode of interaction information and intuitive combination reaction models of α-mangostin with serum protein. Relevant results can be the theoretical reference to study pharmacological mechanism of α-mangostin.Mangosteen fruits were collected at the Tianmushan nature reserve, Lin’an, Zhejiang province, China. Tianmushan nature reserve is a national nature reserve of China where the plants are state-owned. Zhejiang Agricultural & Forestry University is a state-owned public university of the Zhejiang province, China, and Zhejiang Agricultural & Forestry University has built practice teaching facilities in the Tianmushan nature reserve. The natural resources of the teaching practice base can be used as experimental materials, and mangosteen fruits are available in the reserve. Mangosteen is not an endangered or protected species, so it can be used. The picking of mangosteen fruits in this reserve was approved by the academic committee of Zhejiang Agricultural & Forestry University and our mangosteen experiment conformed with the wild plants protection regulation of the People's Republic of China; α-Mangostin standard (≥98%) was supplied by Shanghai Chunyou Biological Technology Co., Ltd., China; Isopropyl alcohol, methanol and ethanol were all HPLC grade and obtained from Shanghai Xingke High Purity Solvent Co., Ltd., China; Deuterated chloroform (≥99.96%) was from Aladdin reagent, Shanghai Co., Ltd., China and HPD-400 macroporous resin from Zhengzhou Qinshi Technology Co., Ltd., China; Human serum albumin , human transferrin , tris(hydroxmethyl) aminomethane  were all purchased from Shanghai Huamei Biological Engineering Company, China; Hydrochloric acid and sodium chloride were analytical reagent from J&K Scientific Ltd., Shanghai, China. Deionized water with sub-boiling distillation was used in all experiments. The following solutions were prepared: 0.1 M Tris-HCl buffer, pH = 7.3, containing 0.1 M NaCl to maintain ionic strength; HSA, TRF and mixed protein solution (with different volume ratio of HSA and TRF) with the concentration of 10 μM using the buffer solution; 1.0 mM α-mangostin stock solution.The following equipments were used: GC/MS-QP2010SE gas chromatograph-mass spectrometer (GC-MS) ; IR Prestige-21 Fourier transform infrared spectrometer (IR) ; FW-4 tablet press ; AVANCE II /400MHz nuclear magnetic resonance (NMR) ; F-7000 fluorophotometer ; UV-2550 ultraviolet-visible spectrophotometer (UV-VIS) ; Agilent 1100 high performance liquid chromatograph ; ZD-2 precision acidity meter (Shanghai Leici Instrument Factory).The HPD-400 macroporous resin was soaked in 95% ethanol for several hours. Then it was rinsed with a large volume of distilled water. Using the wet method, the cleaned resin was packed into the chromatography column with stirring. After the resin had sedimented, the water was drained off and subsequently the resin was washed with 95% ethanol at the rate of 3 column volumes per hour (V/h) until the effluent had no white turbidity when distilled water was added. Subsequently, the resin was washed with distilled water at the same flow rate.18 chromatographic column  under the following conditions: column temperature: 30°C; mobile phases: methanol (A) and water (B); flow rate: 0.5 mL/min; detection wavelength: 317 nm; sample size: 5 μL; elution conditions: 0–30 min used A of 88%, 30–60 min used A from 88% to 70%, 60–120 min used A from 70% to 80%.HPLC was carried out using an Agilent 1100 Eclipse XDB—CDry native mangosteen pericarps (100 g) were crushed with a shredder. To extract α-mangostin, the pericarp preparation obtained was extracted at 40°C three times with 70% isopropyl alcohol . The combined extract was reduced to 305 g by evaporating isopropyl alcohol. Purified water (2000 ml) was used to disperse the extract. The dispersed extract was passed over the preprocessed HPD-400 macroporous resin. The resin was rinsed with three column volumes of purified water in order to remove polar impurities. α-Mangostin was eluted by a wash of methanol. The eluent was monitored via HPLC-analysis. The fractions with high levels of α-mangostin were combined and evaporated to dryness. Crude α-mangostin (6.3 g) was obtained. The α-mangostin obtained was recrystallized in 100 mL 95% ethanol to obtain the final product. The α-mangostin was identified by GS-MS, NMR, IR and UV-VIS spectroscopy.Fluorescence emission spectra and synchronous spectra of α-mangostin and HSA or TRF solution: HSA or TRF (2.5 mL) was added to a 1 cm quartz cuvette, and then 1.0 mM α-mangostin  was added in potions with a microsyringe into the quartz cuvette for fluorescence titration. The α-mangostin concentrations in the cuvette were 0, 4, 8, 10, 16, 24, 32 or 40 μM. Slit widths of emission and excitation: 2.5 nm; scan rate: 240 nm/min; excitation wavelength: 282 nm. The emission spectra were obtained between 280 and 500 nm and the fluorescence intensities at the maximum emission wavelength were recorded.In order to determine the synchronous fluorescence spectra and fluorescence polarization spectra of α-mangostin binding with HSA or TRF, the wavelength differences (Δλ) of fluorescence emission and excitation were set to 60 nm and 15 nm, respectively, and scan rate to 240 nm/min.UV absorption spectra of α-mangostin and HSA or TRF solution were obtained by setting up 1 cm quartz cuvette with 8 different α-mangostin concentrations  as described above using Tris-HCl buffer as UV blank correction. The ultraviolet absorption spectra were obtained between 200 and 500 nm.Interaction models of drug and proteins were established by molecular simulation via DOCK 4.0 at SGI O2 workstation. The crystal structures of HSA and TRF were obtained from Brookhaven Protein Data Bank . In the process of molecular docking, following some reports –50, we uS1 Fig(TIFF)Click here for additional data file.


Mortality rate for breast cancer is higher among African American (AA) women than for women of other racial/ethnic groups. Obesity, also higher among AA women, may increase the risk of breast cancer development and recurrence. Lifestyle factors such as healthy nutrition can reduce the rate of obesity and breast cancer. This study examined the determinants of adherence to nutrition-related cancer prevention guidelines among AA breast cancer survivors.AA breast cancer survivors (n=240) were recruited from a breast cancer support group to complete a lifestyle assessment tool for this cross-sectional study. Chi-square test and ordinal logistic regression analysis were used to examine the relationship between adherence to nutrition-related cancer prevention guidelines and potential predictors of adherence.Majority of the survivors met the guideline for red and processed meat , but did not meet the guideline for fruits and vegetables . For survivors with annual household incomes < $25,000, the odds of meeting or partially meeting the guideline for fruits and vegetables was 75.4% less than for participants with incomes > $50,000 . Poor physical functioning , sleep disturbances , and income > $50,000  were associated with meeting the guideline for red and processed meat.Many AA breast cancer survivors are not meeting the nutrition-related cancer prevention guidelines. For this population, more interventions that enhance access to and consumption of healthy diets are needed. Breast cancer is prevalent among African American (AA) women and for this population, the second leading cause of cancer-related mortality . Racial-The National Health and Nutrition Examination Study (NHANES) analyzed trends in obesity between 1999 and 2010, and during this time, obesity in AA women increased . ObesityFactors that may influence diet include health-related quality of life (HR-QoL), age, employment, education, income, and marital status . HR-QoL The present investigation sought to determine, for a sample of AA BCSs, the factors that predict adherence to nutrition-related cancer prevention guidelines. Although previous studies have used diet as a predictor of HR-QoL , we examFollowing IRB approval from the Morehouse School of Medicine, 240 BCSs were recruited for the study by convenience sampling from Survivors Involving Supporters to Take Action in Advancing Health (SISTAAH) Talk, a BCS support group. Following consent, survivors completed a lifestyle assessment tool (LAT), and data were collected from 2013 to 2015.The 30-minute LAT was completed self-administered via email or postal mail; or facilitator-administered in-person or by telephone. The questionnaire consisted of demographic factors, breast cancer diagnosis and treatment history; HR-QoL; weight history; physical activity; dietary intake; overall health; and breast cancer knowledge, attitudes, and beliefs. The present report utilized the HR-QoL and dietary intake components of the LAT.The dietary intake section of the LAT consisted of 25 items. Participants indicated consumption frequencies of various food items per month in terms of days or weeks. The dietary intake section was divided into categories relating to the ACS dietary guidelines of fruits and vegetables and red and processed meat. The ACS recommends 5 daily servings of fruits and vegetables , which wThe present report utilized The World Cancer Research Fund International (WCRFI) defined limited intake of red meat as less than 18 ounces a week . Ounces were changed into daily servings using the conversion: 14oz= 5 servings , which resulted in 6.4 servings per week. “Meeting” was set as 6.39 servings a week and this was doubled to create the cut-off for “partially meeting.” The final cut-offs for red and processed meat daily servings were: meeting=0–0.91, partially meeting=0.92–1.82, and not meeting≥1.83.Open-ended responses for type of cereal were classified as whole or processed grains according to their ingredients. Cereals with “whole grain” on the nutritional label were categorized as whole grain; cereals lacking this ingredient were categorized as processed (refined) grains. The following values were assigned: 1=whole grains and 2=processed grains. Multiple responses from one participant were each assigned a score. A final score of 1 or 2 was assigned depending on the category with more cereals  A value of 2 was assigned when the number for cereal in each category was equal .HR-QoL was measured through the Patient Reported Outcomes Measurement Information System (PROMIS), an assessment measuring survivors’ subjective physical, emotional, social, and cognitive functioning in the context of their breast cancer symptoms and treatment. PROMIS has constructed item banks (a collection of questions measuring the same thing that can be administered in short forms or adaptively through computerized adaptive testing). Short forms require 4–10 items; computerized adaptive testing require 3–7 items for more precise scores. PROMIS item banks and their short forms provide evidence that they are reliable and precise measures of generic symptoms and functional reports comparable to legacy instruments . The HR-QoL section of the LAT consisted of 27 items divided into 8 sub-categories, namely, physical functioning, anxiety, depression, fatigue, sleep disturbance, satisfaction with social role, pain interference, and pain intensity. Cronbach’s alpha was 0.74. All items were scored on a Likert-type scale. Participants rated their physical functioning on a scale of 1=unable to do to 5=without any difficulty; anxiety and depression on a scale of 1=never to 5=always; fatigue, satisfaction with social role, and pain interference on a scale of 1=not at all to 5=very much; sleep disturbance on a scale of 1=very good to 5=very poor; and pain intensity on a scale of 0=no pain to 10=worst imaginable pain. Each HR-QoL section was divided into Good  and Poor . Physical functioning, sleep disturbance, and satisfaction with social role were reverse-scored. Pain intensity was converted into a 5-point scale by halving all responses and assigning a number of 1 through 5 in the following manner: 0–1=1, 1.5–2=2, 2.5–3=3, 3.5–4=4, and 4.5–5=5.2  for continuous variables. To compare participant characteristics across the “fruit and vegetable” and “red and processed meat” outcome categories, chi-square tests were used. To identify factors associated with meeting the dietary guidelines, multivariable logistic regression modeling with purposeful selection of covariates were used with a p-value cut-off of 0.25 . All demMajority (91.3%) of the 240 participants were African American/black, between 50–64 years of age (49.2%), married (40.8%), and had completed some college education or higher (71.7%) . Half  .Bivariate analyses of demographic, clinical, body weight, and HR-QoL variables, with the outcome variable of meeting the guideline for fruit and vegetable consumption guidelines  showed no statistically significant differences among the outcome groups in participant characteristics .2; and the remaining 70% were either overweight or obese. Almost 55% reported gaining approximately 20lbs or more post-diagnosis. Relative to anxiety, depression, fatigue, sleep disturbance, satisfaction with social role, pain interference, and pain intensity, most participants had “good” HR-QoL scores. For most participants, however, their physical functioning score was “poor”.Regardless of the outcome group, most participants (45.7%) reported being diagnosed with breast cancer 5–10 years duration without recurrence (80.3%), and had received surgery (85.3%) and chemotherapy (54.5%). Most (69.1%) had not received hormone treatments or a bone marrow/stem cell transplant (97.9%). Only about a third (30%) of the participants were in the healthy weight category with BMI values less than 25 kg/mContrary to the results for fruit and vegetable consumption, most of the participants met the guideline for consumption of red and processed meat, with only 3.3% (n=8) not meeting the guideline . RegardlIn Although the association for meat consumption and body weight or clinical characteristics were not statistically significant , most ofSimilar to the patterns for fruit and vegetable consumption, regardless of meat consumption, most participants had good HR-QoL scores in terms of anxiety, depression, fatigue, sleep disturbance, satisfaction with social role, and pain interference, whereas their physical functioning score was poor. The proportion of participants who had good pain intensity scores was highest among those that partially met the guideline for consumption of red and processed meat guideline (76.7%) compared to those that met (52.7%) or did not meet the guideline (57.1%).In the ordinal regression model for fruit and vegetable consumption, five demographic variables  and one clinical characteristic variable (surgery) were included. The result of the ordinal logistic regression showed an association between meeting/partially meeting fruit and vegetable guidelines and annual household income .Overall, participants who earned less than $50,000 per year were less likely to meet or partially meet the fruit and vegetable guideline than those who made $50,000 or more per year. Among participants making less than $25,000, the odds of meeting or partially meeting the fruit and vegetable guideline were 0.246  compared to those making more than $50,000. The odds of meeting or partially meeting the fruit and vegetable guideline among participants making between $25,000 and $49,999 was 0.318  compared to those making more than $50,000.In the ordinal regression model for consumption of red and processed meat, five demographic variables, four body weight/clinical characteristics , and three HR-QoL factors  were included. Results of the ordinal logistic regression revealed that poor physical functioning scores ) and poor sleep disturbance score ) were associated with meeting guidelines for consumption of red and processed meat, and lower annual household income was associated with partially meeting or not meeting the guidelines .Among participants making less than < $25,000, the odds of meeting red and processed meat guideline were 0.020  compared to those making more than $50,000 . The oddThe current study examined, for a sample of AA BCSs, socio-demographic, medical and HRQoL factors associated with adherence to dietary guidelines. Overall, for the combined dietary guidelines, most of the participants were not meeting or were partially meeting all of the recommendations. The results however show that most of the participants (80%) were meeting the recommended intake of red/processed meat, but not for fruits and vegetables. Also, annual household income was associated with meeting the recommended intake for fruits and vegetables, and for red/processed meat. Poor physical functioning and sleep disturbance were significantly associated with meeting only the recommendation for red/processed meat. A similar study by This study is among the few to examine adherence to dietary intake guidelines among AA BCSs, a group that is disproportionately disadvantaged by breast cancer morbidity and mortality. The limitations include recall bias resulting from the use of self-reported surveys. The lifestyle assessment tool used for the survey, however was developed from instruments that have been used for very large studies and have high validity and reliability scores. The small sample size of participants does not allow the results of this study to be generalized to other AA populations.Most AA BCSs are not meeting the guidelines on nutrition for cancer prevention, although improved diet quality promotes healthy body weight and survival among BCSs. Additional interventions that enhance access to and consumption of healthy diets among AA BCSs are needed.


Despite the passage of time, a large number of veterans are still affected by injuries acquired during Iran-Iraq war. In addition to their primary injuries, the majority of veterans also experience difficulty with long-term, secondary effects. Studies have shown that the most common of these include a range of disabilities, pain, and dramatic decline in mental health and quality of life. Improving living conditions and providing rehabilitation services to veterans has always been a main priority of authorities. The goal of this study was to explain the methods and materials with which these priorities were explored. It is estimated that 564,108 people nationwide were injured over the 8 years of the Iran-Iraq conflict (1980˗1988), which corresponds to a rate of 0.7% of the Iranian population . A largeThe Janbazan Medical and Engineering Research Center (JMERC), with the support of Veterans and Martyr Affair Foundation (VMAF), has conducted several distinct surveys in which groups of veterans with a particular injury were studied. The key aim of these studies was to promote health and help develop veterans’ abilities to manage their personal and social lives. The current study indicated, unexpectedly, that the health problems of veterans with ankle-foot injuries are more serious than those of other veterans.This health needs assessment study was undertaken by JMERC, with a scientific team that included general practitioners, internists, orthopedists, prosthetic and orthotic specialists, physical therapists and psychologists. Trained experts were responsible for collecting demographic data. The study was carried out in 11 provinces across the country. VMAF invited 2500 veterans with ankle-foot injuries to participate, of whom, 1129 enrolled in this study. Mental health status, quality of life, ability to perform daily living activities, and ability to live independently were evaluated using the Symptom Checklist-90-Revised (SCL-90-R), 36-Item Short Form Survey (SF-36), The Barthel Activities of Daily Living (ADL) Index and the Lawton Instrumental Activities of Daily Living (IADL) Scale survey tools, respectively. Neuromusculoskeletal disorders and neurological lesions were recorded. Any pain in the spine and/or lower limbs was detailed, in addition to defining its primary determinants. Additionally, all types of pain-reducing rehabilitation services received were recorded. The scientific team determined the required rehabilitation services for each case, and appropriate measures were taken at the study site.


Patients with HPV-positive/PD-L1-positive cancer had significantly better event free survival and overall survival compared with patients with HPV-negative/PD-L1-negative cancer. Relative to those patients with HPV-negative/PD-L1-negative disease who had the highest risk of death, patients with HPV-positive/PD-L1-positive cancers had a 2.85 fold lower risk of developing an event  and a 4.5 fold lower risk of death . Our findings will help to guide future clinical trial design in immunotherapy based on PD-L1 expression in tonsillar cancer.In this study, we examined PD-L1 expression by immunohistochemistry in 99 patients with tonsillar cancer and known human papillomavirus (HPV) status to assess its clinical significance. We showed that the pattern of PD-L1 expression is strongly related to HPV status. The PD-L1 positivity rate was 83.3% in HPV-positive cases and 56.9% in HPV-negative cases ( Oropharyngeal squamous cell carcinoma (OSCC) is a clinically and biologically heterogeneous disease. Human papillomavirus (HPV) is implicated in the majority up to 70%) of OSCC in the western world with the tonsillar subsite associated with the highest HPV positivity rate , 2. HPV+0% of OSCProgrammed Death Receptor 1 (PD1) is one of the members of the extended family of T-cell regulators expressed on the surface of activated T-cells, B-cells and macrophages . Its ligIn this study, we analysed PD-L1 expression by immunohistochemistry in a well-defined cohort of tonsillar cancer with known HPV status to assess its clinical significance.p < 0.05). Within the HPV-positive group, there was no significant difference in PD-L1 positivity by smoking status . The number of never smokers within the HPV-negative group was too small for meaningful statistical analysis. In terms of the presence of TILs within the primary tumors, there was no difference between HPV-positive cancer and HPV-negative cancer. However, there was a statistically significant difference in the pattern of TIL distribution, with HPV-positive cancer more likely to have > 25% TIL distribution within the tumor.The baseline characteristics and PD-L1 positivity of the study cohort by HPV status are shown in Table There was no significant difference in age and gender when the study cohort was classified by PD-L1 status Table . PatientThe median follow up time was 56 months (range 1–184 months). A total of 31 patients (31.3%) developed a recurrence. Locoregional recurrence occurred in 25 patients, with failure occurring only at the primary site in 15 patients and only at the regional nodal area in 10 patients. Seven patients developed distant metastases as the first site of recurrence, none of whom had loco-regional failure at the time of the diagnosis of distant metastases. There were 46 events and 38 deaths from any cause, of which 25 patients died from the tonsillar cancer while 13 died from unrelated causes.Univariate associations of patient and disease characteristics with locoregional failure, any event and death from any cause are shown in Table In the multivariate analysis Table , there wp = 0.548). The best event-free survival was observed in HPV-positive/PD-L1-positive cancer and the worst survival was seen in HPV-negative/PD-L1-negative cancer (p = 0.05). Similarly, patients with HPV-positive/PD-L1-positive cancer had the best overall survival compared with patients with HPV-negative/PD-L1-negative cancer (p = 0.006). Relative to those patients with HPV-negative/PD-L1-negative disease who had the highest risk of death, patients with HPV-positive/PD-L1-positive cancers had a 2.85 fold lower risk of developing an event  and a 4.5 fold lower risk of death  (Table p = 0.548).Effects of a combination of HPV and PD-L1 on outcomes are shown in multivariate Kaplan-Meier models Figure . There wp < 0.05). Patients with PD-L1-positive cancer were more likely to be never smokers and non-drinkers. However, there was no statistically significant difference in PD-L1 positivity by smoking status within the HPV-positive group, probably due to the small number of cases. A similar study of 133 OSCC cases using a non-commercial PD-L1 antibody did not show any significant difference in PD-L1 expression by HPV status. [The aim of this study was to analyze the expression of PD-L1 in a large series of tonsillar cancer and to evaluate its clinical relevance. We focused our study on patients with carcinoma of the tonsil, the subsite of oropharyngeal cancer with the highest HPV positivity rate, to limit the variation in prognosis among tumors involving different subsites of the oropharynx . We foun status.  However, status. .We found statistical evidence that gender and HPV status were important prognostic factors in patients with tonsillar cancer. This is consistent with many prior studies showing the better prognosis of HPV-positive OSCC compared to HPV-negative smoking-related OSCC , 16, 17.Since the US Food and Drug Administration approved ipilimumab for treatment of advanced melanoma in 2011, there has been an immense interest in immunotherapy as a cancer treatment. Immunotherapies with checkpoint blockade antibodies that block CTLA-4 and PD-1 (or its ligand PD-L1) can restore and augment cytotoxic T-cell responses against cancers, leading to durable responses and prolonged overall survival with tolerable toxicity. PD-L1 is the most commonly used biomarker in immunotherapy. Upregulation of PD-L1 expression detected by immunohistochemistry has been reported in many different cancer types, e.g. melanoma (40–100%), non-small cell lung cancer (35–95%), and multiple myeloma (93%). A recent meta-analysis demonstrated that PD-L1 expression is significantly associated with response to anti-PD-1/PD-L1 antibodies in patients with non-small cell lung cancer. However,p = 0.0021) [Mutation load has been shown to correlate with response to anti-PD1 immunotherapy . Rizvi e 0.0021) . The undCancers that are densely infiltrated by lymphocytes are considered to reflect host immune response against malignancy . EmerginIn conclusion, our study showed that the pattern of PD-L1 expression in tonsillar cancer is related to HPV and smoking. There was a strong interaction between HPV status and PD-L1 status with the worst outcome seen in patients with HPV-negative/PD-L1-negative cancer. The results of this study will help to guide future clinical trial design in immunotherapy based on PD-L1 expression in tonsillar cancer.th edition. No patient was treated with immunotherapy.The study cohort comprised 99 patients with tonsillar squamous cell cancer treated with curative intent. The study was approved by the ethics committee of Sydney Local Area Health Service (Protocol X12–0141). Demographic and clinicopathological data were obtained from the institutional database. Selection criteria included availability of primary tumor material, availability of baseline clinicopathological data and follow up data. Patients were followed up for the occurrence of an event, defined as recurrence in any form or death from any cause, for a median of 56 months after diagnosis. The study pathologists reviewed the histology and tumor grade in all cases. Cancers were staged using the American Joint Committee in Cancer Staging System 7Evidence that HPV is transcriptionally active is needed to establish oncogenicity in OSCCs. Overexpression of p16 resulting from downregulation of retinoblastoma protein by HPV E7 oncoprotein has been used as a surrogate marker of HPV expression in previous studies. An HPV-pImmunohistochemical staining for PD-L1  was conducted on an Autostainer Plus (Dako - Agilent Technologies) using 4μm-thick tissue sections. Sections were dehydrated for 1 hour at 60°C and heat-induced epitope retrieval was performed using EnVision FLEX target retrieval solution for 20 minutes at 97°C. The sections were then cooled to room temperature in TBST Wash buffer for 5 minutes. PD-L1 staining underwent signal amplification using the Envision flex Mouse linker (K8022) followed by Envision FLEX kit (K8023) with a DAB chromagen (Dako – Agilent technologies) prior to counterstaining with hematoxylin. Scoring of PD-L1 staining was determined as the percentage of tumor cells showing positive membrane staining and any sample displaying any unequivocal tumor staining, however focal, was deemed to be PD-L1 positive.The percentage of the tumor containing infiltrating lymphocytes was assessed semi-quantitatively using a four-tier scale: 0 = 0%, 1 = < 25%, 2 = 25− < 75%, 3 = > 75%. All scoring was conducted blinded to HPV status and clinical outcomes.t-tests for continuous variables and chi-squared tests for categorical variables. The Kaplan-Meier method was used to construct time-to-event curves. Locoregional failure was defined as clinical, radiological and/or pathological evidence of recurrence at the primary site or in the regional nodal area. Times to locoregional failure, any event and death from OSCC or death from any cause were calculated from the date of diagnosis. Patients were censored at last follow-up / distant recurrence / death where applicable, or excluded if they had incomplete information on recurrence. For the analysis of time to death from any cause, patients were censored at last follow-up if they were alive. Univariate and multivariable time-to-event analyses were performed using Cox proportional hazards regression modelling.Comparisons between demographic and clinicopathological characteristics were undertaken using


Human papillomaviruses (HPVs) are double-stranded DNA (dsDNA) tumor viruses that are causally associated with human cancers of the anogenital tract, skin, and oral cavity. Despite the availability of prophylactic vaccines, HPVs remain a major global health issue due to inadequate vaccine availability and vaccination coverage. The HPV life cycle is established and completed in the terminally differentiating stratified epithelia, and decades of research using in vitro organotypic raft cultures and in vivo genetically engineered mouse models have contributed to our understanding of the interactions between HPVs and the epithelium. More recently, important and emerging roles for the underlying stroma, or microenvironment, during the HPV life cycle and HPV-induced disease have become clear. This review discusses the current understanding of the bidirectional communication and relationship between HPV-infected epithelia and the surrounding microenvironment. As is the case with other human cancers, evidence suggests that the stroma functions as a significant partner in tumorigenesis and helps facilitate the oncogenic potential of HPVs in the stratified epithelium. Viruses are the etiological agents of approximately 15% of human cancers worldwide . High-riHowever, not all HPV infections cause cancer. HPVs are considered low-risk or high-risk based on their oncogenic potential . Low-risMore than 90% of HPV infections in the cervix are naturally cleared . Thus, tAn increasingly recognized co-factor in many types of cancer is the adjacent stroma, or ‘microenvironment’. The stroma is a supportive scaffold upon which epithelial cells reside, and is composed of connective tissue, vasculature, and various cell types . The rolHPVs are icosahedral, non-enveloped viruses approximately 50–60 nm in diameter. These viruses contain a double-stranded, circular or episomal DNA genome of approximately 8000 base pairs A. A tempThe role of the stroma during initial HPV infection and the life cycle is an underexplored area of research. There is evidence that stromal fibroblasts and/or feeder layers, which are comprised of mitotically inactive fibroblasts, affect various stages of the HPV virus life cycle through both direct and indirect mechanisms B. RheinwThe stromal microenvironment is a critical, if not wholly essential, participant in several facets of both normal and cancer epithelial tissue biology ,42. In tIt is increasingly clear that virus-infected cells communicate with the surrounding microenvironment . In the HPV-positive epithelial cells influence the composition and organization of the microenvironment in both in vitro and in vivo models. This paracrine effect of HPV on the underlying stroma may, in part, be due to the ability of E6 and E7 to alter the secretory profile of epithelial cells, which has been shown to regulate the invasive potential of in vitro organotypic raft epithelial cells via signaling between the epithelium and stroma . For insK14-HPV16 model in which the entire HPV16 early region is expressed [K14E6 [K14E7 [K14E6/E7 mice. In addition to studying cervical cancer, K14E6, K14E7, and K14E6/E7 transgenic mice have been used to study HPV-associated cancers at other anatomical sites, including the skin [The influence of HPV on the stroma in vivo has been largely investigated through the study of genetically engineered HPV transgenic mice. These in vivo models allow observations within a complete biological system, and are therefore especially critical to understanding the interplay between the epithelium and tumor microenvironment. There are two main HPV transgenic murine models, both of which target HPV genes to the basal cells of the stratified epithelium using the basal keratinocyte-specific keratin 14 (K14) promoter. One model is the xpressed . In the d [K14E6  and K14E6 [K14E7  mice, whthe skin , oral cathe skin , and anuthe skin .K14HPV16 transgenic mice, the underlying stromal architecture was extensively remodeled during the course of neoplastic progression [In the skin of gression ,69. Archgression . Much ofK14HPV16 mice, angiogenesis and vascular density increases during progression to cancer [In both the human cervix and the murine cervix of o cancer . Increaso cancer . HPV-medo cancer , conditio cancer . Others o cancer ,74 and to cancer .ADM) and several other pro-angiogenic genes are targets of miR-126 [In addition to the secretion of pro-angiogenic factors from HPV-positive epithelial cells that function in a paracrine manner, there is also evidence that HPV-positive cells can stimulate pro-angiogenic gene expression in cells within the adjacent stroma. For instance, CAFs isolated from the stroma of a cervical cancer secreted more VEGF than cervical cancer epithelial cells under both normal and hypoxic conditions . More re miR-126 . AdrenomK14HPV16 cervical epithelium, which leads to expression of fibroblast growth factors 2 and 7 (FGF-2 and FGF-7) in the stroma to promote angiogenesis and tumor cell growth [K14HPV16 murine skin produce several pro-inflammatory cytokines with well-known roles in angiogenesis [K14HPV16 epidermis [K14HPV16 mice. Therefore, there are several mechanisms by which the HPV-positive epithelium can instruct changes in fibroblasts, endothelial cells, and other stromal cells that enhance angiogenesis and cancer progression.The pro-angiogenic effects of HPV-positive epithelial cells on the stroma have also been observed using in vivo murine models. One such example involves the release of platelet-derived growth factor (PDGF) from the l growth . Fibroblogenesis . When thpidermis . Beginnipidermis . Throughpidermis  and macrpidermis . The masHPVs are notorious for their ability to evade the host immune response during infection, mainly due to the nature of their intraepithelial life cycle that provides ample cover from host detection ,83. HoweK14HPV16 transgenic mice and was induced by epithelial cell-secreted IL-1B [CXCL1, CXCL2, CXCL5), interleukins , the cyclooxygenase-2 gene COX2, and several other genes with well-known pro-inflammatory roles. This group of C-X-C motif ligand chemokines (CXCLs), which are all ligands for the CXCR2 receptor, are considered pro-angiogenic and also function as chemoattractants for neutrophils and macrophages [K14E6/E7 mice [As described in previous sections, a strong pro-inflammatory gene expression signature was measured in fibroblasts isolated from dysplastic skin of ed IL-1B . A promirophages ,87. Inderophages . A similrophages . Our own/E7 mice . It willIn addition to epithelial-derived IL-1B as described above , severalK14HPV16 mice, a significant influx of mast cells that secrete MMP-9 was observed in the microenvironment, which was necessary for promoting angiogenesis and epithelial proliferation in the skin [K14E7 mice is sufficient for leukocyte trafficking to the skin and this function depends on its interaction with the pRb tumor suppressor [K14E7 mouse skin is sufficient to recruit mast cells through elicitation of CCL cytokines, CCL2 and CCL5, and that this contributes to the oncogene driving establishment of an immunosuppressive environment within the skin, in a manner that is dependent upon the induction of epithelial hyperplasia by E7 [Inflammatory cell recruitment by the HPV-positive epithelium was also observed in HPV transgenic mice. In the skin ,96: Inteppressor ,98. An iia by E7 . Therefoia by E7 . FurtherUpon HPV infection, it is clear that the stratified epithelium initiates communication with the underlying stroma. Presumably in response to this communication, the microenvironment reciprocates contact through various stromal-to-epithelial signaling events. While these processes are still not well understood, there is evidence that the stroma plays a role in HPV-positive epithelial cell growth and disease initiation and maintenance.Given its role in normal epithelial cell biology, it is not surprising that the stromal compartment also affects the growth and differentiation of HPV-infected epithelial cells . One sucFibroblast growth factors are a family of secreted factors that are significant players in communication between the stroma and epithelium and are involved in processes ranging from development to cancer . There iVirus-associated carcinogenesis can be studied in vivo using xenografts as well as genetically engineered murine models . As intrSeveral experiments using xenografts have identified positive effects of fibroblasts on HPV-positive epithelial cells. In one set of experiments, xenografts of the HPV-positive cervical cancer cell line ME180 were significantly larger when the epithelial cells were co-inoculated with cervical cancer CAFs or mouse embryonic fibroblasts compared to epithelial cells alone . The posK14HPV16 mice. In a study by Erez and colleagues, dermal fibroblasts were isolated from areas adjacent to normal skin in non-transgenic mice and dysplastic skin lesions spontaneously arising in K14HPV16 transgenic mice, and their gene expression transcriptomes compared [Another set of xenograft experiments analyzed the effect of stromal cells on the growth of PDSC5 cells, which are a murine HPV16-positive squamous cell carcinoma cell line derived from the skin of compared . The CAFcompared . CollectK14HPV16 mice, a paracrine loop between the epithelia and stroma involving PDGF and FGFs was identified [K14HPV16 mice were treated with imatinib to inhibit PDGF signaling, FGF-2 and FGF-7 expression was decreased in the stroma. Imatinib treatment reduced proliferation and increased apoptosis in the epithelium and effectively delayed progression and reduced in vivo cervical cancer growth. These effects were linked to a reduction in angiogenesis driven by fibroblast-derived FGFs. Expression of FGF-2 was similarly increased in the stroma of human cervical cancers. These experiments clearly define a role for the microenvironment in promoting HPV-associated cervical cancer growth through paracrine mechanisms.There is also evidence that the stroma influences de novo cervical cancers in transgenic mouse models of HPV-associated cervical cancer. In estrogen-treated entified . In thisK14E7 mice requires the continuous expression of ERα in the cervical stroma [In our own studies, we have made the important observation that estrogen contributes to cervical carcinogenesis by signaling through the underlying stroma. An extensive body of work by Cunha and colleagues had previously established that stromal–epithelial interactions mediate the effects of estrogen on morphogenesis of the female reproductive tract, and more specifically that stromal estrogen receptor α (ERα) facilitates the proliferative effects of estrogen on the epithelium ,128,129.l stroma . Stroma-K14HPV16 skin [In a parallel study analyzing human cervical tissue samples spanning neoplastic disease progression from normal cervix to cervical cancers, ERα expression became progressively confined to the stromal compartment but was lost in epithelial cancer cells . FibroblV16 skin , human cV16 skin . InteresV16 skin . CollectThe functional consequences of HPV-induced epithelial–stromal crosstalk have been discussed throughout this review, and include influences on the HPV life cycle and virus-induced disease. Many of the factors implicated in the crosstalk are proteins secreted into the extracellular space . In many cases, however, the mechanism for crosstalk has not been elucidated. One emerging mechanism to explain the communication between tissue compartments is the reciprocal secretion and uptake of extracellular vesicles by epithelial and stromal cells. Exosomes are one particular type of vesicle that has been extensively studied in the context of cancer biology . ExosomeMastomys coucha is naturally infected with Mastomys natalensis papillomavirus (MnPV) [In this review, we have outlined the current knowledge of key interactions between human papillomaviruses and the underlying stroma. Established and emerging evidence indicate that bidirectional crosstalk between HPV-infected epithelial cells and the microenvironment contribute to the HPV life cycle , stromals (MnPV) , the excs (MnPV)  will alls (MnPV) , and fut


Here we present the case of a 56-year-old human immunodeficiency virus (HIV)-infected man with multiple anal condylomas and positivity for human papilloma virus (HPV) 18 on anal brushing. Biopsies of the anal mucosa led to the diagnosis of Bowen's disease and a subsequent pelvic magnetic resonance imaging (MRI) scan evidenced multiple reactive lymphoadenopathies and large intra-anal condylomas. The patient was treated with a complete excision of Bowen's lesion and with a 4 months course of supplementation with a high concentration multistrain probiotic formulation administered orally and by rectal instillation with the purpose to reduce local inflammation and to enhance local mucosal immunity.An MRI performed at the end of the supplementation period evidenced the clearance of the anal condylomas previously described and no evidence of residual lymphadenopathies. Trials are therefore required to confirm this therapeutic possibility and for a better understanding of the mechanisms by which this specific probiotic formulation interacts with local epithelium when administered by the anal route. In HIV positive patients, gut dysbiosis is associated with impairment of local immune system –9 and in this scenario any corrective action may contribute to reduce the persistence of HPV infection and subsequently to the onset of epithelial dysplasia.The human immunodeficiency virus (HIV)-infected population shows a higher prevalence of human papilloma virus (HPV) infection and higher rates of HPV related dysplastic lesions in comparison to the general population. Recent studies suggest that the HIV related immune suppression and the interaction between HIV and HPV themselves could be responsible for the increased risk of HPV related malignancies in this population. Data about the effects of local rectal enema with probiotics on anal HPV disease in HIV-infected individuals are unavailable.The use of high doses of oral multistrain probiotics has been investigated as a promising tool for the treatment of gut dysbiosis and the restoration of gut mucosal immunity in HIV-infected people.2Pneumocystis jirovecii, cervical Herpes Zoster, and disseminated nontuberculous mycobacterial infection). During the follow-up, the patient continuously showed poor adherence to antiretrovirals and required numerous therapy switch due to virological failure. His medical records showed other pathologic conditions such as thyroid nodular disease, arterial hypertension, syphilis, recurrent anal warts repeatedly treated with diathermocoagulation and persistent positivity of HPV 18 in anal brushing.We present the case of a 56-year-old homosexual man who was diagnosed with HIV infection in 1998 and which progressed to AIDS in the same year , it was highlighted the presence of multiple intra anal condylomas. Bioptic samples of the anal mucosa led to the diagnosis of squamocellular intraepithelial carcinoma  , with the aim to reduce local inflammation and to rebalance local microbiota, thus possibly restoring in site mucosal immunity.Given the fact that Bowen's disease is considered a high grade dysplasia (AIN3) with a low risk of invasion (around 5%), we adopted a conservative approach undergoing a complete excision of the lesion and, once achieved, proposing to the patient a course of supplementation with a multistrain probiotic with daily endorectal instillation of 900 billion live bacteria and daily oral administration of 3 sachets, each containing 450 billion live bacteria  and higher rates of clearance of HPV associated cytological abnormalities  in comparison to the control group. Other evidences show that a reduced amount of Lactobacillus spp. combined with an increased diversity of the vaginal microbiota could be involved in HPV acquisition and persistence and in the development of dysplastic cervical lesions,13; moreover, in vitro studies demonstrated a direct cytotoxic effect of certain vaginal microbial strains on HPV-infected cells and an inhibition of the expression of HPV E6 and E7 oncogenes, which may prevent malignant transformation in individuals harboring high-risk HPV genotypes.–16This case report suggests a potential role for this specific probiotic formulation in promoting the regression of anal HPV related lesions in HIV-infected individuals. The effects of a number of oral probiotic formulation on HPV associated alterations have been mainly studied in the female genital tract; in a prospective controlled pilot study, women with HPV associated low-grade squamous intraepithelial lesions were randomized for probiotic supplementation: the treated group showed a higher chance of clearing HPV infection  are also possible in the anal district, as observed in this case.Moreover in HIV positive patients, dysbiosis of the gut microbiota largely contributes to the impairment of mucosal immunity and could facilitate HPV persistence and the subsequent epithelial transformation. Given that spontaneous regression of multiple large condylomas, such as those observed in our patient, is not frequently seen among HIV-infected patients, we hypothesize a beneficial role exerted by this specific probiotic formulation in the clearance of HPV related lesions.Finally, data on the effects of probiotic rectal enema on anal HPV infection in HIV positive males are lacking. Given the extremely distal localization of the HPV related lesions of our patient, we decided to use, together with oral administration, rectal enemas of probiotics to maximize the amount of bacterial cells that would reach the anal canal to effectively interact with the local epithelium.4Growing evidences show the potential beneficial effect of probiotics on HPV related disease, also in anal district. In particular, in HIV-infected individuals, due to the impairment of the immune response and the loss of gut intraepithelial lymphocytes, lower rates of HPV clearance are observed and consequently spontaneous resolution of multiple large anal condylomas is rare among this population. For this reason, the positive results of our preliminary experience support the hypothesis of a direct efficacy of oral and rectal multistrain probiotic administration in the treatment of anal condylomatosis in HIV-infected patients.Conceptualization: Giancarlo Ceccarelli, Gabriella d’Ettorre.Data curation: Giancarlo Ceccarelli, Stefano Savinelli, Luigi Bianchi, Alessandra Pierangeli, Francesco Vullo, Antonio Ciardi, Gabriella d’Ettorre.Formal analysis: Francesco Vullo.Investigation: Stefano Savinelli, Luigi Bianchi, Alessandra Pierangeli, Francesco Vullo.Methodology: Giancarlo Ceccarelli, Alessandra Pierangeli.Resources: Stefano Savinelli, Alessandra Pierangeli.Supervision: Antonio Ciardi, Gabriella d’Ettorre.Validation: Alessandra Pierangeli, Gabriella d’Ettorre.Visualization: Luigi Bianchi.Writing – original draft: Giancarlo Ceccarelli, Gabriella d’Ettorre.Writing – review & editing: Giancarlo Ceccarelli, Eugenio Nelson Cavallari, Antonio Ciardi.


Human papillomavirus (HPV) has recently been implicated as a causative agent in a rapidly growing number of oropharyngeal cancers. Emerging literature supports the hypothesis that HPV vaccination may protect against HPV-related head and neck cancer (HNC) in addition to HPV-related cervical and anogenital disease. While the association between HPV infection and cervical cancer is widely understood, its relation to HNC is less well known. The purpose of this study was to better understand HPV counseling practices for infection and vaccination in relation to HNC of primary care physicians (PCPs), Obstetricians/Gynecologists (OBGYNs), and Otolaryngology - Head and Neck Surgeons (OHNSs) in Canada.A Canada-wide electronic questionnaire regarding counseling practices on HPV infection, transmission, and vaccination was designed and distributed to PCPs, OBGYNs, and OHNSs across Canada through electronic and paper-based methods. Basic Descriptive statistics were used to analyze responses.In total, 337 physicians responded . Three out of four PCPs reported routine counseling of their patients regarding HPV infection, transmission, and vaccination. Among this group, 68% reported “never” or “rarely” counseling patients that HPV can cause HNC. The most commonly reported reason that PCPs cited for not counseling was a lack of knowledge. The majority of OHNSs (81%) and OBGYNs (97%) counseled patients regarding HPV infection, transmission, and vaccination. However, very few OHNSs (10%) regularly counseled patients with HPV-related HNC about HPV-related anogenital cancer. Similarly, very few OBGYNs (18%) regularly counseled patients with HPV related cervical/anogenital cancer about HPV related HNC.The rate of counseling on HPV infection, transmission, and vaccination in relation to HNC among PCPs is low. The most common reason is a lack of knowledge. Specialists rarely counsel patients with confirmed HPV-related cancer about other HPV-related malignancies. More research is needed on the relationship between different HPV-related cancers in order to better inform counseling practices. Human papillomavirus (HPV) is a double stranded, non-enveloped DNA virus which has long been implicated in cervical cancer oncogenesis . RecentlIt is estimated that in Canada, 75% of sexually active males and females will contract an HPV infection in their lifetime . All avaMultiple studies have reported that patients with a history of HPV-related malignancies have an increased risk of developing a second primary HPV-related cancer , 15–17. To explore current primary care physician (PCP) and specialist practices regarding counseling on HPV infection, transmission, and vaccination in relation to HNC.To determine gaps in knowledge and/or areas where counseling practices can be improved in the practices of PCPs and those that treat patients with HPV-related cancer.Although the importance of HPV as a causative agent in OPSCC has been increasingly appreciated over the past decade, our understanding of physician counseling practices for HPV infection, transmission, and vaccination with respect to HNC is limited. This study sought to explore this gap in the literature by shedding light on the topic from a Canadian perspective. Our objectives were twofold:We designed a self–administered questionnaire for distribution to Otolaryngologists – Head and Neck surgeons (OHNSs), Family Physicians, Pediatricians, and Obstetricians/Gynecologists (OBGYNs) based on previously published guidelines , 20 RespThe questionnaire was distributed nationally via electronic means through professional association contact lists . We excluded those who provided incomplete responses to the questionnaire, and any non-physician or physician that had not been in practice during the past 5 years. Information was collected anonymously using the online survey software Fluid Surveys  which complies with Canadian privacy standards, as well as hard copy distribution from October 2014 to April 2015. The full questionnaire is included in Appendix A.The questionnaire was divided into two parts. Part I included questions regarding demographic information. Outcomes measured included specialty of practice, current practice setting, current province of practice, years in active practice, patient populations seen in practice, and age groups seen in practice (appendix A).In Part II of the questionnaire, participants were asked to report frequency of HPV counseling related to infection, transmission, and vaccination (appendix A). Physicians were asked to report how often they counsel patients with confirmed HPV-related anogenital cancer about the risk of HNC, as well as how often they counsel patients with confirmed HPV-related HNC about the risk of anogenital cancer.Basic descriptive statistics were used to derive estimates for overall counseling practices for the sample. For ease of interpretation, outcomes of variables with responses ranging from never to always were combined . All analysis were conducted using IBM SPSS 22 .N = 337) were predominantly family physicians 71%, followed by OHNSs 15%, OBGYNs 9%, and primary care Pediatricians 5%  of physicians saw males in their practice setting at least as much as females. However, 54% of physicians counseled females more than males regarding HPV infection, transmission, and vaccination. These results highlight that PCPs are currently more likely to counsel patients regarding HPV if they are female rather than male. In fact, counseling of males is of critical importance in the prevention of both CSCC as well as OPSCC, as males are the primary carriers responsible for the spread of HPV infection .Primary care physicians also report the most common reason as to why HPV counseling was not practiced in relation to HNC was a lack of knowledge, despite the fact that very few (5%) reported any doubt about the evidence surrounding HPV infection and its association with HNC. This may result from uncertainty with regards to the absolute benefit of HPV vaccination in HNC reduction, in addition to how PCPs prioritize HNC counseling in context of multiple competing demands and opportunities in primary care. The results highlight a specific need for further research and national or provincial professional associations with expertise in this area such as the Canadian Society of Otolaryngology-Head and Neck Surgery to educate their PCP colleagues on the changing landscape of this topic.Overall OBGYNs reported a high level of patient counseling with regards to HPV infection, transmission, and vaccination (97%). This is likely due to the widely recognized relationship between HPV and cervical cancer. In fact, 100% of Gynecologist reported that when discussing the HPV vaccine they usually or always counseled patients that the vaccine protects against specific serotypes that cause cervical cancer. However, only 36% usually or always counseled that the vaccine also protects against the same subtypes that cause HPV-related HNC. The majority of OBGYNs also did not counsel patients with confirmed cervical or anogenital cancer on the association between HPV and HNC, although the authors concede that the connection between cervical cancer and HNC remains controversial.OHNSs reported similarly high rates of counseling patients with regards to HPV infection, transmission, and vaccination (81%). However, similar to OBGYNs, the OHNSs reported very low numbers for counseling patients with confirmed HPV-related HNCs on the connection between HPV and anogenital cancer . The low reported levels of counseling may reflect the current debate of actual risk in the literature regarding the development of a second HPV-related malignancy. This highlights the need for more research aimed at filling this gap in order to provide guidelines for day to day practice.Although there are no other studies with which we can compare and contrast self-reported counseling rates, it is clear that there is a paucity of literature on HPV counseling practices for HNC prevention among PCPs. There are likely multiple factors that contribute to the lack of counseling. Nevertheless it is clear that further research is required, and PCPs may benefit from knowledge support on this specific topic as the knowledge base continues to grow. Specialists may benefit from more research and recommendations for referral for additional HPV cancer screening in their patients.Some limitations of this study included the small possibility for some selection bias by overlooking physicians who do not belong to or closely follow emails from professional societies. Also, a small sample size comprised predominantly of clinicians from Alberta, that may not be generalizable to the larger North American population. This is especially important given that different provinces have different recommendations and pubic health policies regarding HPV vaccination. Inherent in the study design (self-reported questionnaire) are a number of biases including recall bias and response bias in which there is likely error in accuracy of recall from respondents. As a result any response bias may have overinflated the true rate of counseling among physicians.The rate of counseling on HPV infection, transmission, and vaccination in relation to HNC among PCPs is low. The most common self reported reason is a lack of knowledge. Specialists rarely counsel patients with confirmed HPV-related cancer about their risk of developing other HPV-related malignancies. More research is needed on the connection between different HPV-related cancers in order to better inform counseling practices and facilitate prevention strategies.


Supplemental Digital Content is available in the text Recently increasing high-risk HPV+ OSCC exhibits unique clinical and molecular characteristics compared to HPV-unrelated (HPV−) counterpart. Genomic copy number variations (CNVs), unique in HPV+ OSCCs, and their role for the prognosis prediction remains poorly studied. Here, we analyzed the distinct genomic copy number variations (CNVs) in human papillomavirus-related (HPV+) oropharyngeal squamous cell carcinoma (OSCC) and their role as a prognosticator after curative resection.For 58 consecutive, Korean OSCC patients that underwent surgery-based treatment with median 10 years of follow-up, HPV-related markers, and genome-wide CNV analysis were analyzed. Clinical associations between the CNV profile and survival analyses were followed.P = 0.0006) better than HPV L1 PCR , smoking, or other variables. Although the overall number of CNVs was not significantly different, 30 loci showed unique CNV patterns between the p16+ and p16− groups. A region containing PRDM2 was amplified only in the p16+ group, whereas EGFR and 11q13.3 showed increased amplification in p16− counterpart. Loss of a locus containing FGF18 led to a worse, but gain of region including CDK10 and RAD18 led to better overall survival (OS) in all OSCC patients. Meanwhile, subgroup analysis of p16+ OSCC revealed that amplification of regions harboring HRAS and loss of locus bearing KDR led to better OS.p16 expression predicted the overall survival (OS) (hazard ratio [HR] = 0.27, confidence interval [CI]: 0.39–0.80, + OSCC exhibit distinct CNV patterns compared with p16− counterpart. Specific patterns of CNVs predict better survival, especially in p16+ OSCC. This might allow better insights of the outcome after curative resection for HPV+ and HPV− OSCC.p16 Interestingly, studies indicate that the incidence of HPV-positive (HPV+) HNSCC has steeply increased in recent years, up to 60% to 85%.3 HPV+ HNSCC usually occur as oropharyngeal squamous cell carcinomas (OSCC), mostly in tonsillar fossa. They usually respond better to treatments, such as ionizing radiation with or without the addition of chemotherapy4 and surgery-based treatments,4 than HPV-negative (HPV−) counterpart. This has led some to propose “de-escalating” contemporary treatment strategies through the guidance of molecular markers; for example, decreasing radiation dosage or adopting less invasive, functional surgeries, such as transoral laser microsurgery5 and transoral robotic surgery.6Head and neck squamous cell carcinoma (HNSCC) is the eighth most common cause of cancer deaths worldwide.8 Thus, a more detailed understanding of these poor responders is necessary to provide better, functional treatment and improve patient outcomes for this expanding disease.In spite of these advances, treatment failures still occur, and the failure rate is increasing due to increased incidence of this disease worldwide. In general, 20% to 40% of HPV+ and 40% to 60% of HPV-HNSCCs still exhibit poor responses to treatment, with patients suffering from disease recurrence.11 Among these CNVs, gains of 3q, 11q, and 12q and losses of 5q, 6q, 8p, 21q, and 22q were reported to predict poor prognosis.12 Also in HNSCC, CNVs of EGFR13 and PIK3CA14 have also been reported to be associated with poor prognosis. Meanwhile, studies of uterine cervical cancers have provided preliminary knowledge on unique CNV signatures, such as gains of 1q in early stages of tumor development, and gains of 3q, 5p, and 8q and losses of 2q, 3p, 4q, 11q, and 19p later in tumor progression.16 Integration of HPV DNA into the host genome causes changes like a gain of 3q, which is related to cancer progression in uterine cervical premalignant lesions.17 As these studies were mostly on uterine cervical cancers, unique CNV patterns of HPV + HNSCCs, especially compared with HPV− counterpart, should be clarified more. Moreover, most data have been obtained from Western populations, whereas little is known about these patterns in Asian countries such as Korea. Finally, these data were mostly analyzed in a heterogeneous treatment group. There has been little evaluation of the clinical and prognostic implications of CNVs in OSCC, especially after surgical resection as a primary treatment.Copy number variations (CNVs), especially those affecting signaling mediators, might represent a critical factor driving cancer development and determining sensitivity to contemporary anticancer treatments. Losses of 4q, 5q, 7q, 8p, 13q, 17p, 18q, 21q, and 22q and gains of 1q, 2q, 3q26, 5p, 7p, 8q, 9q, 11q13, and 20q have been reported in HNSCCs in Western populations.+ and p16− OSCCs. The prognostic role of CNV profile in a consecutive, surgery-treated cohort with OSCC was determined, particularly in the p16+ subset, and we evaluated if the CNV pattern provides more accurate stratification for proper treatment.Here, we report and elucidate the specific patterns of CNVs in HPV+ and HPV− OSCC in a Korean population. We compared these patterns in p16Tissue samples from 58 consecutive, histopathologically confirmed, previously untreated OSCC were obtained during surgical procedures carried out between 2002 and 2007 , at the Department of Otolaryngology—Head and Neck Surgery, Specific Organs Cancer Branch, National Cancer Center, Goyang, Korea. After obtaining informed consent, archives of paraffin-embedded specimens from these patients were constructed. Inclusion criteria for this study were histologically confirmed primary OSCC with primary surgical resection, with or without postoperative radiotherapy. Exclusion criteria included recurrent disease, contraindication of surgery-based treatment, initial presentation with distant metastasis, failure to receive the full course of planned treatment, or cases where formalin-fixed paraffin-embedded (FFPE) specimens lacked sufficient DNA for CNV analysis.18 Histologic grading was performed by a pathologist according to the World Health Organization criteria for squamous cell carcinomas of the oral mucosa.19 All OSCC patients were examined by the head and neck surgeon at each follow-up appointment (every 3 months). Cumulative tobacco and alcohol use were calculated as previously described.8 This study was approved by the Institutional Review Board in accordance with the Center for Clinical Trials at the National Cancer Center, Korea (NCCNCS08200).Clinical data, including tumor staging and details of primary treatment, were obtained from medical, radiology, operative, and pathology reports using the 2002 American Joint Committee on Cancer staging criteria.20 Oropharyngectomy, via a more aggressive transpharyngeal or transmandibular approach, was also performed if indicated. Neck dissection was routinely accompanied, either with elective (cN0) or therapeutic (cN+) intent. Adjuvant radiotherapy was done in patients with positive or close (<2 mm) surgical margins, demonstrated perineural or lymphovascular invasion, any stage N2 neck disease, or neck disease with extracapsular extension after tumor board discussion on oncologic necessity.All patients underwent surgery-based treatment, mostly with transoral lateral oropharyngectomy through en-bloc dissection of the submuscular plane along the buccopharyngeal to the prevertebral fascia, as previously described.21 Immunohistochemistry (IHC) of p16 p16  was performed and scored using modified H-score system, which considers both the intensity and the proportion as follows:23 (1) the staining intensity was defined as 0 for negative, 1+ for weak, 2+ for moderate, and 3+ for strong  dataset among the genes passing the survival test. Then, immunostaining was conducted for the same sample set. Correlation between the CNV pattern and the IHC expression was further tested with Fisher's exact test, followed by survival analysis using the IHC expression.DNA was extracted using the QIAamp Mag Attract DNA Mini M48 Kit . The carcinoma area was punched out from paraffin blocks to obtain the highest percentage of tumors and collected in 1.5-mL Eppendorf tubes for DNA extraction. DNA extraction was carried out using the Qiagen BioRobot M48 workstation. A total of 10 μL of purified total cellular DNA was used in each HPV PCR.8 Fifteen types of high-risk HPV  and 9 types of low-risk HPV  were identifiable with this chip.The presence of HPV DNA was tested simultaneously with genotyping using a PCR-based HPV DNA Chip , as previously described.http://www.macrogen.com) consisted of 4362 human bacterial artificial chromosome (BAC) clones spaced ∼1 Mb across the entire genome. Confirmation of the locus specificity of the chosen clones was performed by fluorescent in situ hybridization (FISH),24 and the labeling and hybridization protocols were used as previously described.25 Test and reference DNA were digested, purified, and labeled by random priming  using Cy3 or Cy5 dCTPs . A Cy3-labeled sample and Cy5-labeled reference DNA were then hybridized, and the arrays were scanned into 2 16-bit TIFF image files and quantitated using the GenePix software .The array used in this study  were defined as consecutive CNVs. To test differences between groups in survival curves, we used Harrington and Fleming's G-rho family test (survdiff) from the R package (www.r-project.org) survival. Statistical P values were obtained with a chi square test. Student's t test was used to compare the overall copy number changes between groups, as well as to rank genetic loci. We also performed average-linkage hierarchical clustering based on the centered correlation measure (Cluster 3.0). To determine how genes affect survival rate, copy number gains, and losses were counted. Among probes with copy number gain or loss in >10 patients, survival analysis was conducted using the Kaplan–Meier (survfit) method and the proportional hazard regression model (coxph). Significance between differences in the survival rate was analyzed by the log-rank test. Results with P values <0.05 were considered significant.A total of 4362 different BAC clones were used, and the log-transformed fluorescent ratios were calculated. Probes in the autosome region were selected for this study. For quality control, probes genotyped with >90% of the samples were used excluding singleton peaks. We defined probes with a logP = 0.0006) and more significantly predicted the 10-year overall survival (OS) than HPV L1 PCR  or smoking  . Both p16+ smokers and p16+ nonsmokers responded better to treatment than p16− OSCC  and in the tonsillar fossa area . Among these, the expression of p16 by IHC had the lowest hazard ratio (HR) .Although we initially hypothesized that p16+ OSCC at 11q13.3, which contains CTTN, PPFIA1, and SHANK2. Close to this region, CCND1 was also amplified. In contrast, various loci at 3p had copy number losses or chromosomal deletions in p16− OSCC. CNTN4, some micro RNAs, and other regulatory elements are located in this area (regulatory elements are not shown). In contrast, 1p36.21, a locus containing PRDM2, was amplified only in the p16+ group. Losses in other chromosomal locations were observed only in the p16− group  compared with other markers, such as HPV- L1 PCR (P = 0.0263) and smoking (P = 0.38). We thus continued to search for post-treatment prognosticators, focusing on p16 expression, speculating that this might stratify our cohort with more clinical and biologic relevance. Despite recent reports suggesting that a combination of HPV DNA and p16 positivity is the most reliable indicator of an active HPV association,26 our data indicated that p16 positivity alone could better predict the OS than HPV L1-PCR. This is in line with some previous reports.27 Although DNA analysis in FFPE is a well-established standard, genomic DNA in an FFPE specimen can become fragmented over time, decreasing the accuracy of HPV L1 PCR. We think that this could lower the accuracy of HPV DNA testing in archival FFPE samples than p16 IHC. In addition, although smoking has occasionally been reported as a prognostic marker for OSCC, smoking history did not predict survival in p16+ patients, in agreement with another previous report.28 It is still unclear whether there is racial disparity in the influence of smoking, which is not well understood in the Asian population.Our initial analysis on primary parameters indicated that p16 was the most potent prognostic marker . However, the loci of CNVs showed different patterns . Cyclin D1 is known to be overexpressed with inactivated p53 in immortalized cells.35.36 TP53 loss or deletion was present in 70% of our current patients, regardless of the p16/infection status  compared to p16ients 14.%. Cyclin41 PFFIA1 in 11q13 is also reported in several earlier studies.43 We also found a copy number gain in 1p36.21. We suspect one of the genes in this locus, PRDM2 , to be involved, as the role is known as a tumor suppressor. Another study on expression patterns based on the p16 status revealed PRDM2 overexpression in p16+ patients, consistent with our result.44Together with CCND1, CTTN is reported to correlate with prognostic tumor stage and contribute to metastasis in HNSCC, often coinciding with resistance to anoikis.+ subset, which has been rarely studied especially after surgical resection. Although EGFR,45 CCND1,39 MYC, FGFR1,47 and PIK3CA amplification have been reported by a number of studies on HNSCC, their role as a prognostic marker with or without the presence of p16 infection is unclear. Among patients in this study, neither of them showed a significant difference in survival: not in all patients, p16+, nor in p16− subgroups . Instead, other CNVs have been suggested to be correlated with better or worse survival. In both p16+ and p16− subsets, higher copy numbers of 5q35.1, a region harboring FGF18, 16q24.3, containing CDK10, and 3p25.3, containing RAD18, typically predicted a better OS. Although the roles of FGF18 and CDK10 in cell cycle progression and proliferation have been well studied, the relationship between their copy number and cancer survival is not fully understood. CDK10 has been reported as a tumor suppressor gene in biliary tract cancer cells.48 RAD18 is involved in telomere maintenance, concordantly showing better survival with copy number gain. Conversely, a higher copy number of 20p12.1, encoding genes such as PCSK2, predicted a poorer OS in our group. PCSK2 is a factor related to tumor development and progression. Recently reported expression analysis and IHC classified PCSK2 as 1 of 3 genetic markers for the classification of benign and malignant thyroid cancer.49We eventually investigated the role of cancer-specific CNVs as prognostic markers in surgically treated OSCCs, especially the p16+ subgroup, we further analyzed the CNV profile. Of note, a higher copy number of 11p15.5, containing HRAS, predicted a better survival outcome. Although HRAS is a known oncogene, it has been reported that activation of HRAS is not sufficient to maintain a neoplastic phenotype.50 This suggests another mechanism for the involvement of HRAS in cancer prognosis and survival. Loss of KDR  predicted a better prognosis among our p16+ patients. Because an increase of KDR promotes angiogenesis, loss of KDR leads to a better OS. Of note, we think these patterns could help guide a “de-escalation” strategy for p16+ OSCC treatment.To better predict the OS within our p16+ group showed a tendency to be correlated between CNV and IHC tests , and the survival of low expression of IHC patients was better than its counterpart , although not significant due to the small number of samples in the IHC low group (n = 5). An extended study might be needed.Meanwhile, the IHC results of genes RAD18, HRAS, KDR did not significantly correlate to the CNV profile and failed to show significance to overall survival (data not shown). Although not significant, HRAS in the p16Our data show that treatment outcome after surgery for OSCC could be more accurately characterized, with the adoption of this array CGH system, and this might help eventually guide optimal treatment. Moreover, our result implicate that less-invasive functional, “de-escalation” surgery would be better performed under stringent guidance from these better prognostic CNV profiles. In addition, considering our data were acquired using “low-resolution” array, reanalyzing the same or independent patient group using the higher resolution array system and further resultant validation would be another enormously important study to be followed soon.+ OSCCs exhibit specific CNV patterns at some loci compared with p16− OSCCs, suggesting distinct carcinogenic events at the gene level. Moreover, after surgery-based treatment for this disease, CNV profiles might help more accurately predict treatment outcome after surgery for OSCC and eventually guide optimal treatment strategy. These implicate that less-invasive functional surgery would be better performed under stringent guidance from these better prognostic CNV profiles. Our data also suggest that more intensive treatment and surgical strategy may be needed for OSCC with nonfavorable CNV profile, necessitating further clinical investigations.In conclusion, p16