PARP inhibition in novel oropharyngeal cancer cell lines

In recent decades many developed countries have seen unprecedented increases in incidence of Human Papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC). HPV-positive OPSCC represents a new disease entity and preclinical assessment of novel therapies is hampered by a lack of relevant in vitro models. It is well-established that HPV-positive OPSCC patients generally survive longer than HPV-negative patients, and this may be partly attributable to defective repair of DNA double-strand-breaks in HPV-positive tumours. This study aimed to develop novel cell line models of HPV-OPSCC and use them, and previously validated lines, to test the hypothesis that defective DNA repair in HPV-positive cells could be exploited by synthetic lethal therapy using the Poly (ADP-ribose) polymerase (PARP) inhibitor Olaparib. Two novel HPV-positive OPSCC cell lines were derived and characterised. mRNA sequencing confirmed expression of the HPV oncogenes and HPV integration state, but did not show consistent differences in transcript levels of genes involved in DNA repair between HPV-positive and negative lines. The effects of Olaparib were assessed in a panel of eight cell lines, including effects on colony formation, cell cycle distribution, DNA double-strand-break persistence and p53 activity. All lines were sensitive to high doses of Olaparib (10 μM), however at doses between 0.5-1 μM, the surviving fractions differed significantly between lines. Two HPV-positive lines were sensitive to Olaparib (Surviving Fraction (SF)80%). Neither HPV-status, nor basal levels of PARP correlated with Olaparib sensitivity. The data were not consistent with the original hypothesis, but did suggest that monotherapy with PARP inhibitors might be useful in some OPSCC patients. The study also included an investigation into the natural history of HPV in the oropharynx. This demonstrated that HPV infection is a rare event in non-malignant tonsil tissue (prevalence of 0%: 95% confidence interval (CI) 0-0.58%)

Sensitivity of human papillomavirus-positive and -negative oropharyngeal cancer cell lines to ionizing irradiation

Human papillomavirus‑positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased in incidence and has a much better prognosis than HPV‑negative (HPV‑) OPSCC with radiotherapy alone, but exactly why is unknown. The present study therefore aimed to further examine the sensitivity and possible changes in gene expression of several HPV+ and HPV‑ OPSCC, including various novel cell lines, upon ionizing irradiation (IR). Previously established HPV+ UM‑SCC‑47, UPCI‑SCC‑90, CU‑OP‑2, CU‑OP‑3 and HPV‑ UM‑SCC‑4, UM‑SCC‑6, UM‑SCC‑74a, UM‑SCC‑19 and newly established CU‑OP‑17 and CU‑OP‑20, characterised here, were subjected to 0‑6 Gy. Surviving fractions of each cell line were tested by clonogenic assays, and irregularities in cell cycle responses were examined by flow cytometry, while changes in gene expression were followed by mRNA sequencing. HPV+ OPSCC cell lines showed greater variation in sensitivity to ionizing irradiation (IR) and tended to be more sensitive than HPV‑ OPSCC cell lines. However, their IR sensitivity was not correlated to the proportion of cells in G2 arrest, and HPV‑ cell lines generally showed lower increases in G2 after IR. Upon IR with 2 Gy, mRNA sequencing revealed an increase in minor HPV integration sites in HPV+ cell lines, and some changes in gene expression in OPSCC cell lines, but not primarily those associated with DNA repair. To conclude, HPV+ OPSCC cell lines showed greater variation in their sensitivity to IR, with some that were radioresistant, but overall the HPV+ OPSCC group still tended to be more sensitive to IR than the HPV‑ OPSCC group. In addition, HPV+ OPSCC lines were more frequently in G2 as compared to HPV‑ cell lines, but the increase in G2 arrest upon IR in HPV+ OPSCC was not correlated to sensitivity to IR. Increases in minor HPV integration sites and changes in gene expression were also demonstrated after irradiation with 2 Gy

Sensitivity to inhibition of DNA repair by Olaparib in novel oropharyngeal cancer cell lines infected with Human Papillomavirus

The incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in the UK. Patients with HPV-positive OPSCC generally show superior clinical responses relative to HPV-negative patients. We hypothesised that these superior responses could be associated with defective repair of DNA double strand breaks (DSB). The study aimed to determine whether defective DNA repair could be associated with sensitivity to inhibition of DNA repair using the PARP inhibitor Olaparib. Sensitivity to Olaparib, and induction and repair of DNA damage, were assessed in a panel of 8 OPSCC cell-lines, including 2 novel HPV-positive lines. Effects on cell cycle distribution and levels of PARP1 and p53 were quantified. RNA-sequencing was used to assess differences in activity of DNA repair pathways. Two HPV-positive OPSCC lines were sensitive to Olaparib at potentially therapeutic doses (0.1–0.5 μM). Two HPV-negative lines were sensitive at an intermediate dose. Four other lines, derived from HPV-positive and HPV-negative tumours, were resistant to PARP inhibition. Only one cell-line, UPCISCC90, showed results consistent with the original hypothesis i.e. that in HPV-positive cells, treatment with Olaparib would cause accumulation of DSB, resulting in cell cycle arrest. There was no evidence that HPV-positive tumours exhibit defective repair of DSB. However, the data suggest that a subset of OPSCC may be susceptible to PARP-inhibitor based therap

Human papillomavirus capsids trigger crosstalk between dendritic and NK cells

The immune system controls, at least partially, human papillomavirus (HPV) infection and subsequent tumour development as demonstrated by a higher tumour prevalence in immunodeficient patients. More than 90% of HPV-infected women will clear the virus within two years. However, it remains unclear which immune cells are implicated in this process and although dendritic cells (DC) and NK cells play a key role in host resistance to virus and tumour, no study has been performed evaluating their crosstalk in this context. Virus-like particles (VLP) formed by the HPV major capsid protein L1 are licensed as vaccine against cervical cancer and we have recently shown that NK cells can directly interact with these HPV-VLP [1]. Here, we investigated the impact of this activation on NK-DC crosstalk. Interestingly, NK cells increase DC maturation induced by HPV-VLP as shown by an up-regulation of HLA-DR and CD86 on DC. Transwell experiments indicated that the expression of HLA-DR is cell-cell contact and soluble factor dependent, whereas only soluble factors seem to be required for CD86 expression. Moreover, in the presence of HPV-VLP and NK cells, DC produce higher amounts of IL12p70, while the production of the immunosuppressive cytokine IL10 remains unchanged. We also demonstrated that DC can up-regulate the expression of NK activation markers (CD69 and HLA-DR) in the presence of HPV-VLP. This up-regulation requires both cell-cell contact and soluble factors. Regarding HLA-DR marker, the increased expression on CD56bright cells is mediated by soluble factors, whereas cell-cell contacts are also important for HLA-DR expression on CD56dim cells. In the presence of DC activated by HPV-VLP, the function of NK cells is also modified since they become more cytotoxic against HPV+ cell line and secrete more IFN-γ. Our results suggest that NK-DC crosstalk could play a role in the immune response induced by HPV-VLP during vaccination protocols against cervical cancer

Neuronal surface antibody-mediated encephalopathy as manifestation of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Although it remained controversial for a long time, central nervous system (CNS) involvement of graft-versus-host disease (GVHD) is now becoming recognized as a real nosological entity. Previous case reports have suggested heterogeneous clinical presentations and it is not excluded that the whole spectrum of manifestations has not yet been fully described. Here, we report the case of a 58-year-old man with chronic GVHD who developed a rapidly ingravescent encephalopathy. There was no evidence for CNS immune-mediated lesions on conventional imaging nor for cellular infiltration in the cerebrospinal fluid. Serum analyses revealed the presence of anti-neuronal antibodies directed against anti-contactin-associated protein 2 (anti-Caspr2), a protein associated with voltage-gated potassium neuronal channels. Functional imaging with 2-deoxy-2-[fluorine-18] fluoro- D-glucose integrated with computed tomography (18F-FDG PET-CT) demonstrated diffuse cortical and subcortical hypometabolism. The patient was treated with a combination of immunosuppressive agents (corticosteroids, cyclophosphamide and rituximab) and progressively recovered normal neurocognitive functions. Taken together, these data suggest that CNS-GVHD may manifest as a reversible antibody-mediated functional encephalopathy. This report suggests for the first time the interest of screening for anti-neuronal antibodies and functional imaging with brain 18F-FDG PET-CT in diagnosing this severe complication of allogeneic hematopoietic cell transplantation (alloHSCT). © 2018 Elsevier B.V

Human papillomavirus infection is rare in nonmalignant tonsil tissue in the UK: implications for tonsil cancer precursor lesions

The incidence of human papillomavirus (HPV)-associated tonsil cancer is increasing but the prevalence of HPV, and of premalignant precursors, in tonsil tissue is unknown. We aimed to assess prevalence of HPV infection in nonmalignant tonsillar crypt epithelia and to histopathologically characterise positive samples. Formalin-fixed paraffin-embedded (FFPE) tonsil tissue specimens were obtained from an age- and sex-stratified random sample of patients aged 0–69 years whose paired tonsils were archived following elective tonsillectomy at hospitals throughout England and Southern Scotland from 2004 to 2008. Homogenised fresh-frozen tonsil tissue was also obtained from archive for two random subsets of males aged 25–34 and over 44. HPV status was assessed in all samples for 20 mucosal HPV types by GP5+/6+ polymerase chain reaction (PCR) enzyme immunoassay and by HPV16 type-specific PCR targeting the E6 gene. In the homogenised material, HPV status was also assessed for 44 HPV types by SPF10-PCR enzyme immunoassay. Of 4,095 randomly sampled FFPE specimens, amplifiable DNA was extracted from 3,377 (82.5%) and from 511 of 524 (97.5%) homogenised tonsils. HPV DNA was identified in 0 of 3,377 (0%, 95% CI 0–0.089%) fixed samples and 0 of 511 (0%, 95% CI 0–0.58%) homogenised samples. This suggests HPV infection may be rare in tonsil reticulated crypt epithelia. Furthermore, we found no evidence of HPV-associated premalignant neoplasia. These data suggest that if HPV-associated premalignant lesions do occur, they are likely to be rare and may have a high risk of progression to carcinoma

Overall Survival Rate in Allogeneic Stem Cell Transplanted Patients Requiring Intensive Care Can Be Predicted by the Prognostic Index for Critically Ill Allogeneic Transplantation Patients (PICAT) and the Sequential Organ Failure Assessment (SOFA) Scores

Background. Allogeneic hematopoietic stem cell transplantation (allo-HCT) recipients requiring intensive care unit (ICU) have high mortality rates. Methods. In the current study, we retrospectively assessed whether the Prognostic Index for Critically Ill Allogeneic Transplantation patients (PICAT) score predicted overall survival in a cohort of 111 consecutive allo-HCT recipients requiring ICU. Results. Survival rates at 30 days and 1 year after ICU admission were 57.7% and 31.5%, respectively, and were significantly associated with PICAT scores (p = 0.036). Specifically, survival at 30-day for low, intermediate, and high PICAT scores was 64.1%, 58.1%, and 31.3%, respectively. At one-year, the figures were 37.5%, 29%, and 12.5%, respectively. In multivariate analyses, high PICAT score (HR = 2.23, p = 0.008) and relapse prior to ICU admission (HR = 2.98, p = 0.0001) predicted higher mortality. We next compared the ability of the PICAT and the Sequential Organ Failure Assessment (SOFA) scores to predict mortality in our patients using c-statistics. C statistics for the PICAT and the SOFA scores were 0.5687 and 0.6777, respectively. Conclusions. This study shows that while the PICAT score is associated with early and late mortality in allo-HCT recipients requiring ICU, it is outperformed by the SOFA score to predict their risk of mortality