Chromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10 PZP ), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10 PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10 PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10 PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10 PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10 PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10 PZP -dependent CALM-AF10-mediated leukemogenesis.

Over 175,000 diagnoses of estrogen receptor-positive breast cancer are made every year in the US. Of these, >40,000 patients will go on to die of their disease. We recently identified defects in a principal mismatch repair (MMR) gene, MLH1, as a driver of resistance to standard-care treatment and poor patient outcomes. Moreover, we demonstrated increased sensitivity to palbociclib, a CDK4/6 inhibitor, in experimental model systems in vitro and in vivo and in clinical trial data. MLH1 is mutated in 5-10% of treatment-resistant tumors, i.e., 4,000 patient tumors every year. However, most of these mutations are missense and variants of unknown significance (VUS). The impact of these mutations on mismatch repair capability and on treatment resistance remains uncertain. Here, we use experimental model systems (MCF7 cells and a PDX mode, WHIM20) to phenotype MLH1 VUS´ in vitro and in vivo using xenograft systems. Specifically, we use nuclear localization of MLH1 and repair of mismatches through a mismatch repair deficiency assay to test for mismatch repair capability in ER+ breast cancer cells engineered to express mutations in MLH1 that are found in patient tumors associated with poor survival. Second, we assess the ability of variants of MLH1 to induce activation of G1/S cell cycle checkpoints by activating Chk2. Lastly, we directly test the ability of variants in MLH1 to induce resistance to standard-care endocrine therapies and sensitivity to palbociclib, using 2D and 3D growth assays and xenograft experiments. Results from these experiments serve to systematically catalog VUS´ in MLH1 based on their predictive potential. Currently, there are few tests available in the clinic to predict response to therapies early in the timeline of ER+ breast cancer patients. Due to the nature of the disease, this can result in years of overtreatment. Overtreatment comes at great financial, physical, and emotional cost to patients and their families. Simultaneously, a lack of predictive markers can result in missed opportunities of presenting patients likely to relapse on standard care with personalized alternative therapeutic strategies. With the increased feasibility of sequencing patient tumors in the clinic, there is a large gap in knowledge regarding the impact of VUS´ in various oncogenes on disease progression and treatment response. Data presented here address this gap by cataloging the impact of a range of VUS´ from patient tumors on protein localization and function, as well as macrophenotypes of tumor growth and therapeutic response. Note: This abstract was not presented at the conference. Citation Format: Aloran Mazumder, Nindo Punturi, Sujita Khanal, Athena Jimenez, Svasti Haricharan. Using mutations in mismatch repair genes to predict response to palbociclib [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 23.

Background: Human papillomavirus (HPV) etiology has become evident in head and neck cancers (HNCs) and HPV positivity showed a strong association with its malignant progression. Since aberrant DNA methylation is known to drive carcinogenesis and progression in HNCs, we investigated to determine target gene(s) associated with this modification. Methods: We characterized epigenetic changes in tumor-related genes (TRGs) that are known to be associated with HNC development and its progression. Results: The expression levels of 42 candidate HNC-associated genes were analyzed. Of these, 7 TGRs (CHFR, RAR beta, GRB7, EREG, RUNX2, RUNX3, and SMG-1) showed decreased expressions in HPV-positive ((+)) HNC cells compared with HPV-negative ((-)) HNC cells. When gene expression levels were compared corresponding to the DNA methylation conditions, GRB7 and EREG showed significant differential expression between HPV+ and HPV- cells, which suggested these genes as primary targets of epigenetic regulation in HPV-induced carcinogenesis. Furthermore, treatment with a demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dc), caused restoration of EREG expression and was associated with hypomethylation of its promoter in HPV+ cells, while no changes was noted in HPV- cells. EREG promoter hypermethylation in HPV+ cells was confirmed using methylation-specific PCR (MS-PCR). Conclusion: We conclude that EREG is the target of epigenetic regulation in HPV+ HNCs and its suppressed expression through promoter hypermethylation is associated with the development of HPV-associated HNCs.

Estrogen receptor (ER) positive breast cancer is a leading cause of cancer-related death among women globally. Metastasis and endocrine treatment resistance are the two principal causes of death from ER+ breast cancer. There are few available targeted therapeutics for metastatic, endocrine treatment resistant disease other than CDK4/6 inhibitors. However, CDK4/6 inhibitors only work in a subset of patients and markers that can identify these patients have not yet been discovered. Considering cost and toxicity associated with chronic use of these inhibitors, it is critical to identify (a) biomarkers of response (b) combinatorial approaches to enhance efficacy. To achieve these goals, we need to understand the biology underlying endocrine treatment resistance and CDK4/6 inhibitor response. We recently identified loss of the MutL complex of mismatch repair (MLH1 and PMS2), as a cause of endocrine treatment resistance in 5-10% of women with primary (non-metastasized) ER+ breast cancer. We also demonstrated that primary ER+ breast cancer cells defective in these genes lacked ATM/Chk2 activation and were, therefore, sensitized to CDK4/6 inhibitors. Here, we present data demonstrating that MutL loss induces metastasis of ER+ breast cancer cells, which can be prevented/treated using ATM/Chk2 activators in combination with CDK4/6 inhibitors. We observed increased migration and invasion in three independent cell line models of MutL loss when treated with endocrine therapies in vitro, and increased lung colonization in vivo. We observe a similar phenotype in a PDX model of MutL defective ER+ disease. We also demonstrate significant inhibition of metastatic phenotypes upon administration of a combination of CDK4/6 inhibitors and ATM/Chk2 activators. Alternative targeted therapies that are effective against metastatic ER+ breast cancer remain elusive, and lethality associated with this diagnosis remains high. Current standard care comprises of endocrine treatment administered with CDK4/6 inhibitors. Even when effective, this combination is costly and associated with side effects as it requires chronic administration to maintain stable disease. The results of this study suggest that (a) MutL loss can be a predictor of increased risk for metastatic disease (b) MutL defective ER+ breast cancer has increased sensitivity to CDK4/6 inhibitors (c) combinatorial use of ATM/Chk2 activators with CDK4/6 inhibitors could significantly benefit ER+ breast cancer patients with defective MutL. These potential outcomes could greatly improve quality of life and decrease cancer-related death in thousands of women every year. Citation Format: Sujita Khanal, Athena Jimenez, Svasti Haricharan. Mismatch repair defects induce metastasis of ER+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-08.

Estrogen receptor (ER) positive breast cancer is a leading cause of cancer-related death among women globally. Metastasis and endocrine treatment resistance are the two principal causes of death from ER+ breast cancer. There are few available targeted therapeutics for metastatic, endocrine treatment resistant disease other than CDK4/6 inhibitors. However, CDK4/6 inhibitors only work in a subset of patients and markers that can identify these patients have not yet been discovered. Considering cost and toxicity associated with chronic use of these inhibitors, it is critical to identify (a) biomarkers of response (b) combinatorial approaches to enhance efficacy. To achieve these goals, we need to understand the biology underlying endocrine treatment resistance and CDK4/6 inhibitor response. We recently identified loss of the MutL complex of mismatch repair (MLH1 and PMS2), as a cause of endocrine treatment resistance in 5-10% of women with primary (non-metastasized) ER+ breast cancer. We also demonstrated that primary ER+ breast cancer cells defective in these genes lacked ATM/Chk2 activation and were, therefore, sensitized to CDK4/6 inhibitors. Here, we present data demonstrating that MutL loss induces metastasis of ER+ breast cancer cells, which can be prevented/treated using ATM/Chk2 activators in combination with CDK4/6 inhibitors. We observed increased migration and invasion in three independent cell line models of MutL loss when treated with endocrine therapies in vitro, and increased lung colonization in vivo. We observe a similar phenotype in a PDX model of MutL defective ER+ disease. We also demonstrate significant inhibition of metastatic phenotypes upon administration of a combination of CDK4/6 inhibitors and ATM/Chk2 activators. Alternative targeted therapies that are effective against metastatic ER+ breast cancer remain elusive, and lethality associated with this diagnosis remains high. Current standard care comprises of endocrine treatment administered with CDK4/6 inhibitors. Even when effective, this combination is costly and associated with side effects as it requires chronic administration to maintain stable disease. The results of this study suggest that (a) MutL loss can be a predictor of increased risk for metastatic disease (b) MutL defective ER+ breast cancer has increased sensitivity to CDK4/6 inhibitors (c) combinatorial use of ATM/Chk2 activators with CDK4/6 inhibitors could significantly benefit ER+ breast cancer patients with defective MutL. These potential outcomes could greatly improve quality of life and decrease cancer-related death in thousands of women every year. Citation Format: Sujita Khanal, Athena Jimenez, Svasti Haricharan. Mismatch repair defects induce metastasis of ER+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-08.

Heavy alcohol drinking causes alterations in the metabolism of fatty acids and zinc that participate in inflammation and liver injury. HIV infection has been reported to cause dysregulated polyunsaturated fatty acid (PUFA) and zinc metabolism. In this pilot study, we examined the role of dysregulated PUFA metabolism and zinc deficiency in the liver injury occurring in heavy drinkers with early-stage HIV diagnosis. Fourteen heavy drinking alcohol-dependent (AD) patients [seven with treatment-naive HIV diagnosis (AD+HIV) and seven without HIV infection (AD)] participated in this study. Liver injury, serum zinc, PUFAs, viral load, CD4(+) count, and drinking measures using lifetime drinking history (LTDH), and timeline follow-back past 90 days (TLFB90) were evaluated. Liver injury was also assessed in seven age- and gender-matched socially drinking HIV treatment-naive patients who served as disease controls. HIV viral load by itself did not show any correlation with liver injury. Liver enzymes were significantly elevated in both AD+HIV and AD patients, and AD+HIV patients had significantly higher alanine aminotransferase (ALT) levels than did AD patients, even with lower drinking. Serum zinc was significantly lower in AD+HIV patients. Only AD+HIV patients showed a significant elevation in linoleic acid (LA) and alpha-linoleic acid (ALA) levels. Serum zinc and ALT, LA and ALT, and ALA and ALT were significantly associated only in AD+HIV patients. The association between LA and ALT showed a higher effect than did the ALA and ALT association in the AD+HIV patients. Interestingly, AD+HIV subjects (who drank less), nevertheless, showed more liver injury compared with AD patients, who reported heavier drinking. We speculate that the underlying proinflammatory response resulting from zinc deficiency and an elevation in serum LA likely contributed to liver injury in AD+HIV patients, even with a comparatively lower degree of heavy drinking.

Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of the downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was associated with the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies.

This study evaluated the integration and methlyation of human papillomavirus type 16 (HPV16) in head and neck squamous cell carcinoma (HNSCC) and its oral precursor, high-grade oral epithelial dysplasia (hgOED). Archival samples of HPV16-positive hgOED (N = 19) and HNSCC (N = 15) were evaluated, along with three HNSCC (UMSCC-1, -47 and -104) and two cervical cancer (SiHa and CaSki) cell lines. HgOED cases were stratified into three groups with increasing degrees of cytologic changes (mitosis, karyorrhexis and apoptosis). The viral load was higher and the E2/E6 ratio lower (indicating a greater tendency toward viral integration) in group 3 than in groups 1 or 2 (p = 0.002, 0.03). Methylation was not observed in hgOED cases and occurred variably in only three HNSCC cases (26.67%, 60.0% and 93.3%). In HNSCC cell lines, lower E7 expression correlated with higher levels of methylation. HgOED with increased cytologic change, now termed HPV-associated oral epithelial dysplasia (HPV-OED), exhibited an increased viral load and a tendency toward DNA integration, suggesting a potentially increased risk for malignant transformation. More detailed characterization and clinical follow-up of HPV-OED patients is needed to determine whether HPV-OED is a true precursor to HPV-associated HNSCC and to clarify the involvement of HPV in HNSCC carcinogenesis.

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

Persistent high-risk genus human Alphapapillomavirus (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from four separate data sets found a significant upregulation of the homologous-recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, and yet both E6 and E7 reduce the ability of the HR pathway to complete double-strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, whereas HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together, these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA.IMPORTANCE This study expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in cells expressing HPV E6 and E7 hinders HR through a distinct mechanism. These observations have broad implications. The impairment of HR by HPV oncogenes may be targeted for treatment of HPV+ malignancies. Further, this attenuation of repair suggests HPV oncogenes may contribute to tumorigenesis by promoting the integration of the HPV genome, a common feature of HPV-transformed cells. Our data support this idea since HPV E6 stimulates the integration of episomes.

OBJECTIVES:Reported cytologic alterations associated with high-risk human papillomavirus (HR-HPV) in oral epithelial dysplasia (HPV-OED) need further characterization. STUDY DESIGN:Archival cases of high-grade oral epithelial dysplasia (hgOED) (N = 38) were assigned a cytologic score (CS) based on the average number of mitotic, karyorrhectic, and apoptotic cells per high-power field. Three groups were then generated on the basis of increasing CS: Focal (group 1, N = 14), Intermediate (group 2, N = 12), and Diffuse (group 3, N = 12). Polymerase chain reaction-based HPV genotyping and p16 immunohistochemistry were performed. RESULTS:HR-HPV was found significantly more in group 3 (83.3%) compared with groups 1 and 2 (group 1&2; 42.9% and 41.7%, respectively; P = .047). HPV16 predominated in HR-HPV-positive cases (90.5%). By location, the tongue or the floor of mouth was associated with all groups (P = .04). Increasing CS was associated with a slightly younger age (P = .04) and increased expression of p16 (P = .005). CS and p16 expression were not sensitive but were highly specific predictors for HR-HPV presence. Based on limited follow-up information, HPV-OED does not differ in clinical aggressiveness compared with conventional OED. CONCLUSIONS:Increased CS in hgOED is strongly associated with HR-HPV (mostly HPV16) and p16 expression. CS and p16 expression are specific predictors of HR-HPV presence. Further molecular study and long-term follow-up of HPV-OED are needed.

Objectives: To compare the outcomes and toxicity of high-dose cisplatin (HDC) versus weekly cisplatin (WC) definitive chemoradiotherapy (CRT) for patients with human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (SCCOPx).Methods: All patients with p16 positive SCCOPx treated with definitive CRT with cisplatin between 2010 and 2014 at a single institution were retrospectively reviewed. CTCAE v 4.03 toxicity criteria were used. The Kaplan-Meier method was used to estimate event-free survival (EFS) and the overall survival (OS).Results: Of the 55 patients included, 22 were patients treated with HDC at dose of 100 mg/m(2) on days 1 and 22; and the remaining 33 patients were treated with WC at 40 mg/m(2). Both cohorts received a median total dose of cisplatin of 200 mg/m(2). At median follow-up of 31 months, there was one local failure and no distant failures in the HDC cohort. In the WC group, there were 6 total failures (2 local, 4 distant). Estimated 2-year EFS was better in HDC cohort as compared to WC (96% vs. 75%; p = 0.04). There was no significant difference in 2-year OS (95% vs. 94%; p = 0.40). Weight loss, gastric tube dependence at six months, acute renal injury and grade 3 or 4 hematological toxicity were all similar between both groups.Conclusions: HPV-related SCCOPx treated with definitive CRT with either HDC or WC had similar toxicity profile. HDC had better EFS when compared with WC and this seems to be driven by increased distant failure rates, although the OS was similar. (C) 2017 Elsevier Ltd. All rights reserved.

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.

Background: Current vaccines against Human Papillomavirus (HPV) are highly effective and based on recombinant virus-like particles (VLPs) of the major capsid protein L1. Since these vaccines are HPV type-specific and expensive for global implementation, an alternative, broader-spectrum immunogen would be the N-terminus of the minor capsid protein L2 that induces low titered broadly cross-neutralizing antibodies. Here we analyzed the reactivity of different synthetic L2 peptides containing N-terminus amino acids 17-36 in order to test their antigenicity.Methods: Different synthetic peptides were designed to target the 17-36 amino acid sequences, present in highly antigenic amino-terminus of L2 protein. Six different peptides including Cys22-Cys28 disulfide bonded cyclized L2 peptide were examined for their antigenicity against mouse monoclonal antibody RG-1 and rabbit polyclonal antisera to HPV 12 by enzyme-linked immunosorbent assay (ELISA).Results: Here we report that the cyclized form of synthetic 12 peptide, which is formed through Cys22-Cys28 disulfide bridges, has the highest reactivity to antibodies than other synthetic L2 peptides.Conclusion: A cyclized L2 peptide has potential to be an excellent candidate to formulate a low-cost, broadly protective pan-oncogenic HPV vaccine. (C) 2015 Published by Elsevier Inc.

Background: Human papillomavirus (HPV)-positive oropharyngeal cancer is associated with improved survival and treatment response as compared to HPV-negative cancers. P16 overexpression is widely accepted as a surrogate marker for HPV positivity.Methods: A total of 92 serum samples from 75 head and neck squamous cell carcinoma (HNSCC) patients were examined for HPV16 and 18 E7 antibodies by ELISA. Available tissue was tested for HPV-DNA by PCR, and p16 immunobistochemistry was obtained from a deidentified database.Results: Of 75 HNSCC patients, 25 were HPV E7 seropositive. Seropositivity was strongly associated with cancers of the oropharynx, and correlated with positive p16 immunohistochemistry (IHC) and HPV-DNA. Post-treatment serum was available in a limited subset of patients, revealing a decrease in antibody titers following response to treatmentConclusions: HPV E7 seropositivity correlated with positive tumor HPV-DNA and p16 expression, and was strongly associated with cancers of the oropharynx. E7 serology warrants further study as a potential biomarker in HPVpositive HNSCC. (C) 2015 Elsevier Inc All rights reserved.

We isolated two new manatee papillomavirus (PV) types, TmPV3 and TmPV4, from a Florida manatee (Trichechus manatus latirostris). Two PV types were previously isolated from this species. TmPV1 is widely dispersed amongst manatees and a close-to-root PV; not much is known about TmPV2. The genomes of TmPV3 and TmPV4 were 7622 and 7771 bp in size, respectively. Both PVs had a genomic organization characteristic of all PVs, with one non-coding region and seven ORFs, including the E7 ORF that is absent in other cetacean PVs. Although these PVs were isolated from separate genital lesions of the same manatee, an enlarged E2/E4 ORF was found only in the TmPV4 genome. The full genome and L1 sequence similarities between TmPV3 and TmPV4 were 63.2 and 70.3 %, respectively. These genomes shared only 49.1 and 50.2 % similarity with TmPV1. The pairwise alignment of L1 nucleotide sequences indicated that the two new PVs nested in a monophyletic group of the genus Rhopapillomavirus, together with the cutaneotropic TmPV1 and TmPV2.

Objective. Human papillomavirus (HPV) typing of oral lesions microscopically consistent with multifocal epithelial hyperplasia (MEH) was performed to identify potential novel clinical presentations.Study Design. MEH (N = 22 lesions, 17 patients) and squamous papilloma control samples (N = 9 lesions, 9 patients) were compared by using polymerase chain reaction-based HPV genotyping. Student's t tests were used to compare continuous characteristics.Results. Of the study cases, 86.4% of MEH and only 11% of controls were positive for HPV (P = .0002). In MEH lesions, 45.5% contained HPV32, 36.4% HPV6, and 4.5% HPV40. MEH lesions were mostly multifocal (50%) and occurred in HIV-negative patients (81.3%). They predominated on the labial/buccal mucosa (63.3%), and there were significant differences between groups by anatomic site (P < .0001). HPV32, but not HPV6, was detected in known HIV-positive patients.Conclusions. A novel clinical presentation of MEH associated with HPV32 in HIV-negative, middle-aged to older adults is reported here. One case with HPV40 is the first to be reported. Future detection protocols should include HPV32, as it may be currently overlooked.

Veterinary Clinical PathologyVolume 45, Issue 1 p. 8-9 Laboratory Medicine Yesterday · Today ·Tomorrow Is this cell hollow? A Russell Moore, A Russell Moore Colorado State UniversitySearch for more papers by this authorArlene L. Libby, Arlene L. Libby Summit Pathology Group, Loveland, COSearch for more papers by this authorSujita Khanal, Sujita Khanal University of LouisvilleSearch for more papers by this authorE. J. Ehrhart III, E. J. Ehrhart III Colorado State UniversitySearch for more papers by this authorPaul Avery, Paul Avery Colorado State UniversitySearch for more papers by this author A Russell Moore, A Russell Moore Colorado State UniversitySearch for more papers by this authorArlene L. Libby, Arlene L. Libby Summit Pathology Group, Loveland, COSearch for more papers by this authorSujita Khanal, Sujita Khanal University of LouisvilleSearch for more papers by this authorE. J. Ehrhart III, E. J. Ehrhart III Colorado State UniversitySearch for more papers by this authorPaul Avery, Paul Avery Colorado State UniversitySearch for more papers by this author First published: 30 December 2015 https://doi.org/10.1111/vcp.12313Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume45, Issue1March 2016Pages 8-9 RelatedInformation

Head and neck cancers (HNCs) are common causes of cancer-related deaths worldwide. An increasing incidence of subsets of HNCs is due to human papillomavirus (HPV). Other subsets are associated with tobacco and alcohol use. Determination of differential biomarkers and molecular mechanisms distinguishing HPV-associated and unassociated HNCs should improve diagnostic and therapeutic strategies. This study was designed to identify potential biomarkers for HPV detection, and to evaluate viral and host genetic and epigenetic mechanisms involved in HPV-associated HNCs. Overexpression of a cellular protein, p16INK4a, which is widely used as a surrogate marker for HPV-positive HNC, is non-specific because some HPV-negative cancers can overexpress p16INK4a. I found “mitosoid cells” and “HPV E7 serology”, respectively, as histologic and serum biomarkers, which would improve diagnosis of HPV-positive head and neck (HN) tumors. Mitosoid cells were not only observed in HPV-positive benign epithelial hyperplasias but also abundantly present in subsets of pre-malignant tumors (high-grade oral epithelial dysplasia, hgOED). P16INK4a could be used as an HPV-surrogate marker only in Group 1 hgOED (containing diffuse mitosoid cells), but not in Group 2 (with focal mitosoid cells) and Group 3 (lacking these cells). My study revealed that E7 seropositivity complements p16INK4a overexpression in HNCs and a decrease in E7 serology potentially predicts patients’ response to treatment. To evaluate genetic and epigenetic changes in HPV-induced malignancy, I assessed viral DNA- integration and -methylation, and viral-induced methylation of host tumor vii suppressor genes (TSGs). Since viral DNA integration predicts malignancy, my data showing integrated HPV in Group 1 and malignant tumors, suggest greater malignant transforming potential of Group 1 than the other groups. Although viral methylation is another regulatory mechanism for malignancy, the HPV epigenome was mostly unmethylated in both premalignant and malignant HN tumors. Screening 38 host TSGs identified EREG as a candidate gene, which may be epigenetically regulated, specifically in HPV-positive HNC. Overall, the present study found that mitosoid cells and E7 serology in combination with p16INK4a overexpression are significant markers for HPV-associated head and neck malignancy. HPV DNA integration and host EREG gene methylation, but not viral DNA methylation, may play roles in HPV-associated head and neck carcinogenesis.